Composition for preventing or treating neurodegenerative or motor neuron diseases containing halopudinone as an active ingredient

Abstract

The present invention relates to a composition for preventing or treating neurodegenerative or motor neuron diseases, comprising halofuginone as an active ingredient. Specifically, it has been confirmed in cell and animal models of amyotrophic lateral sclerosis (ALS) that halofuginone exhibits dual effects of suppressing fibrosis, improving skeletal muscle production, and improving joint structure, as well as suppressing inflammatory responses and neuronal death in the central nervous system, thereby delaying the progression of ALS symptoms and improving performance and survival time. Therefore, the halofuginone can be usefully used as an active ingredient in a composition for preventing or treating neurodegenerative or motor neuron diseases, including ALS.

Description

[Technical field]

[0001] The present invention relates to a composition for preventing or treating neurodegenerative or motor neuron diseases, which contains halofuginone as an active ingredient. [Background technology]

[0002] Amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, and the like are representative degenerative neurological diseases or motor neuron diseases, and the therapeutic agents currently in clinical use are extremely limited.

[0003] ALS reduces quality of life and death through progressive muscle atrophy, joint contracture and pain, and has heterogeneous clinical courses and prognoses due to various pathophysiological mechanisms including genetic mutations, impaired proteostasis, mitochondrial dysfunction, neuronal dysfunction and neuroinflammation.

[0004] Transforming growth factor-β (TGF-β) is a multifunctional cytokine involved in cellular regulation such as cell growth, differentiation, and cell death. After the association between TGF-β and ALS emerged, the role of TGF-β in ALS progression began to become clear. In skeletal muscle, TGF-β plays a role in repairing damaged muscles and generally regulates myogenesis, growth, and differentiation. However, persistently elevated TGF-β reduces myogenesis and promotes muscle fibrosis and atrophy. Previous studies using a murine model of ALS showed enhanced TGF-β signaling pathways at symptomatic stages, with abundant extracellular matrix (ECM) production due to increased fibrogenic / adipogenic precursors. Similarly, in the normal nervous system, TGF-β is known to protect neurons from excitotoxic and oxidative damage and play an important role in neurogenesis. However, persistently elevated TGF-β has been reported to be associated with accelerated ALS disease progression.

[0005] It has also been reported that the TGF-β signaling pathway is associated not only with ALS, but also with Alzheimer's disease, Parkinson's disease, Huntington's disease, and multiple sclerosis.

[0006] Therefore, the company made efforts to develop a therapeutic agent that can prevent the acceleration of neurodegenerative or motor neuron diseases, including ALS, caused by persistent TGF-β and can exhibit therapeutic effects. As a result, it was confirmed in ALS cell models and animal models that Halofuginone exhibits dual effects of suppressing fibrosis caused by elevated TGF-β, improving joint structure by increasing skeletal muscle production, and suppressing inflammatory responses and neuronal death in the central nervous system, thereby delaying the progression of ALS symptoms and improving performance and survival. The company has demonstrated that Halofuginone can be usefully used as an active ingredient in a composition for preventing or treating neurodegenerative or motor neuron diseases, including ALS, which led to the present application. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Katsuno, M. et al. Transforming growth factor-β signaling in motor neuron diseases. Current molecular medicine 11,48-56,2011. [Non-Patent Document 2] Kashima, R. & Hata, A. The role of TGF-βsuperfamily signaling in neurological disorders. Acta biochimica et biophysica Sinica 50,106-120,2018. [Non-Patent Document 3] Peters, S. et al. The TGF-β System As a Potential Pathogenic Player in Disease Modulation of Amyotrophic Lateral Sclerosis. Frontiers in neurology 8,669,2017. Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a composition for preventing or treating neurodegenerative or motor neuron diseases, which comprises halofuginone or a pharma- ceutical acceptable salt thereof as an active ingredient. [Means for solving the problem]

[0009] To achieve the object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating neurodegenerative or motor neuron diseases, comprising halofuginone or a pharma- ceutical acceptable salt thereof as an active ingredient.

[0010] The present invention also provides a functional health food composition for preventing or improving neurodegenerative or motor neuron diseases, which contains halopudinone or a pharma- ceutical acceptable salt thereof as an active ingredient.

[0011] The present invention also provides a method for preventing or treating a neurodegenerative or motor neuron disease, comprising the step of administering halopudinone or a pharma- ceutically acceptable salt thereof to an individual.

[0012] The present invention also provides a pharmaceutical composition comprising halopudinone or a pharma- ceutically acceptable salt thereof for use in the prevention or treatment of neurodegenerative or motor neuron diseases.

[0013] The present invention also provides a health functional food composition containing halopudinone or a pharma- ceutical acceptable salt thereof for use in the prevention or amelioration of neurodegenerative or motor neuron diseases.

[0014] The present invention also provides a use of halopudinone or a pharma- ceutically acceptable salt thereof for the manufacture of a medicament for the prophylaxis or treatment of neurodegenerative or motor neuron diseases.

[0015] In addition, the present invention provides a use of halopudinone or a pharma- ceutical acceptable salt thereof for producing a health functional food composition for preventing or improving neurodegenerative or motor neuron diseases.

[0016] Effect of the Invention

[0017] The present inventors have confirmed that halofuginone exhibits dual effects of suppressing fibrosis, improving joint structure by enhancing skeletal muscle production, and suppressing inflammatory responses in the central nervous system and neuronal death in cell and animal models of amyotrophic lateral sclerosis (ALS), thereby delaying the progression of ALS symptoms and improving performance and survival time. Therefore, halofuginone can be usefully used as an active ingredient in a composition for preventing or treating neurodegenerative or motor neuron diseases, including ALS. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 is a schematic diagram illustrating a method of administering Halofuginone to an animal model of amyotrophic lateral sclerosis (ALS) according to one embodiment of the present invention. [Figure 2a] This figure shows the mRNA expression of TGF-β1, TGF-β2, TGF-β3, α-SMA, and MyoD (Figure 2a), and the protein expression of TGF-β1, α-SMA, and MyoD (Figure 2b) after stimulation of myoblasts with TGF-β1 (Transforming growth factor-β1). [Figure 2b] This figure shows the mRNA expression of TGF-β1, TGF-β2, TGF-β3, α-SMA, and MyoD (Figure 2a), and the protein expression of TGF-β1, α-SMA, and MyoD (Figure 2b) after stimulation of myoblasts with TGF-β1 (Transforming growth factor-β1). [Figure 3a] FIG. 3A shows the cell viability after myoblast treatment with various concentrations of halopudinone (FIG. 3A), or with TGF-β1 and various concentrations of halopudinone (FIG. 3B), and the p-Smad2 / Samd2 ratio, TGF-β1, α-SMA, MyoD, and collagen I protein expression (FIG. 3C). [Figure 3b] FIG. 3A shows the cell viability after myoblast treatment with various concentrations of halopudinone (FIG. 3A), or with TGF-β1 and various concentrations of halopudinone (FIG. 3B), and the p-Smad2 / Samd2 ratio, TGF-β1, α-SMA, MyoD, and collagen I protein expression (FIG. 3C). [Figure 3c] FIG. 3A shows the cell viability after myoblast treatment with various concentrations of halopudinone (FIG. 3A), or with TGF-β1 and various concentrations of halopudinone (FIG. 3B), and the p-Smad2 / Samd2 ratio, TGF-β1, α-SMA, MyoD, and collagen I protein expression (FIG. 3C). [Figure 4a]After treating myoblasts with TGF-β1 and halopuginone, the mRNA expression of TGF-β1, α-SMA, MyoD, and collagen I (Figure 4a) and the protein expression of TGF-β1, α-SMA, MyoD, and collagen I (Figure 4b) were confirmed, and the expression of α-SMA and MyoD (Figure 4c) was confirmed by immunocytochemical analysis. [Figure 4b] After treating myoblasts with TGF-β1 and halopuginone, the mRNA expression of TGF-β1, α-SMA, MyoD, and collagen I (Figure 4a) and the protein expression of TGF-β1, α-SMA, MyoD, and collagen I (Figure 4b) were confirmed, and the expression of α-SMA and MyoD (Figure 4c) was confirmed by immunocytochemical analysis. [Figure 4c] After treating myoblasts with TGF-β1 and halopuginone, the mRNA expression of TGF-β1, α-SMA, MyoD, and collagen I (Figure 4a) and the protein expression of TGF-β1, α-SMA, MyoD, and collagen I (Figure 4b) were confirmed, and the expression of α-SMA and MyoD (Figure 4c) was confirmed by immunocytochemical analysis. [Figure 5a] This figure shows the mRNA expression of TGF-β1, α-SMA, and collagen I (FIG. 5a), the ratio of p-Smad2 / Samd2, and the protein expression of TGF-β1, α-SMA, and collagen I (FIG. 5b) after treatment of fibroblasts isolated from an ALS animal model with halopuginone. [Figure 5b] This figure shows the mRNA expression of TGF-β1, α-SMA, and collagen I (FIG. 5a), the ratio of p-Smad2 / Samd2, and the protein expression of TGF-β1, α-SMA, and collagen I (FIG. 5b) after treatment of fibroblasts isolated from an ALS animal model with halopuginone. [Figure 6a] FIG. 6A shows changes in motor function and body weight, and changes in symptom onset (FIGS. 6B and 6C) after administration of halopuginone to an ALS animal model. [Figure 6b] FIG. 6A shows changes in motor function and body weight, and changes in symptom onset (FIGS. 6B and 6C) after administration of halopuginone to an ALS animal model. [Figure 6c]FIG. 6A shows changes in motor function and body weight, and changes in symptom onset (FIGS. 6B and 6C) after administration of halopuginone to an ALS animal model. [Figure 7a] Halopudinone was administered to an ALS animal model, and the expression of TGF-β1, α-SMA, MyoD, and collagen I was confirmed by immunohistochemical analysis in 90-day-old females (Figure 7a) and males (Figure 7b), and in 120-day-old females (Figure 7c) and males (Figure 7d). [Figure 7b] Halopudinone was administered to an ALS animal model, and the expression of TGF-β1, α-SMA, MyoD, and collagen I was confirmed by immunohistochemical analysis in 90-day-old females (Figure 7a) and males (Figure 7b), and in 120-day-old females (Figure 7c) and males (Figure 7d). [Figure 7c] Halopudinone was administered to an ALS animal model, and the expression of TGF-β1, α-SMA, MyoD, and collagen I was confirmed by immunohistochemical analysis in 90-day-old females (Figure 7a) and males (Figure 7b), and in 120-day-old females (Figure 7c) and males (Figure 7d). [Figure 7d] Halopudinone was administered to an ALS animal model, and the expression of TGF-β1, α-SMA, MyoD, and collagen I was confirmed by immunohistochemical analysis in 90-day-old females (Figure 7a) and males (Figure 7b), and in 120-day-old females (Figure 7c) and males (Figure 7d). [Figure 8a] Halopuzinone was administered to an ALS animal model, and the range of motion (ROM) was confirmed in the synovial cavity of the knee joint 120 days after birth (Figure 8a), as well as the p-Smad2 / Samd2 ratio, and the expression of TGF-β1, α-SMA, MyoD, and collagen I proteins (Figure 8b). [Figure 8b] Halopuzinone was administered to an ALS animal model, and the range of motion (ROM) was confirmed in the synovial cavity of the knee joint 120 days after birth (Figure 8a), as well as the p-Smad2 / Samd2 ratio, and the expression of TGF-β1, α-SMA, MyoD, and collagen I proteins (Figure 8b). [Figure 9a]Halopudinone was administered to an ALS animal model, and microglial cell activity (Figure 9a), astroglial cell activity (Figure 9b), IL-1β expression (Figure 9c), the number of ChAT-positive motor neurons (Figure 9d), ChAT mRNA expression (Figure 9e), and ChAT protein expression (Figure 9f) in the lumbar spinal cord were confirmed on postnatal day 120. [Figure 9b] Halopudinone was administered to an ALS animal model, and microglial cell activity (Figure 9a), astroglial cell activity (Figure 9b), IL-1β expression (Figure 9c), the number of ChAT-positive motor neurons (Figure 9d), ChAT mRNA expression (Figure 9e), and ChAT protein expression (Figure 9f) in the lumbar spinal cord were confirmed on postnatal day 120. [Figure 9b] Halopudinone was administered to an ALS animal model, and microglial cell activity (Figure 9a), astroglial cell activity (Figure 9b), IL-1β expression (Figure 9c), the number of ChAT-positive motor neurons (Figure 9d), ChAT mRNA expression (Figure 9e), and ChAT protein expression (Figure 9f) in the lumbar spinal cord were confirmed on postnatal day 120. [Figure 9c] Halopudinone was administered to an ALS animal model, and microglial cell activity (Figure 9a), astroglial cell activity (Figure 9b), IL-1β expression (Figure 9c), the number of ChAT-positive motor neurons (Figure 9d), ChAT mRNA expression (Figure 9e), and ChAT protein expression (Figure 9f) in the lumbar spinal cord were confirmed on postnatal day 120. [Figure 9d] Halopudinone was administered to an ALS animal model, and microglial cell activity (Figure 9a), astroglial cell activity (Figure 9b), IL-1β expression (Figure 9c), the number of ChAT-positive motor neurons (Figure 9d), ChAT mRNA expression (Figure 9e), and ChAT protein expression (Figure 9f) in the lumbar spinal cord were confirmed on postnatal day 120. [Figure 9e] Halopudinone was administered to an ALS animal model, and microglial cell activity (Figure 9a), astroglial cell activity (Figure 9b), IL-1β expression (Figure 9c), the number of ChAT-positive motor neurons (Figure 9d), ChAT mRNA expression (Figure 9e), and ChAT protein expression (Figure 9f) in the lumbar spinal cord were confirmed on postnatal day 120. [Figure 9f]Halopudinone was administered to an ALS animal model, and microglial cell activity (Figure 9a), astroglial cell activity (Figure 9b), IL-1β expression (Figure 9c), the number of ChAT-positive motor neurons (Figure 9d), ChAT mRNA expression (Figure 9e), and ChAT protein expression (Figure 9f) in the lumbar spinal cord were confirmed on postnatal day 120. [Figure 10a] FIG. 10A shows the activity of microglial cells (FIG. 10A) and the number of ChAT-positive motor neurons (FIG. 10B) in the lumbar spinal cord 90 days after birth when halopuginone was administered to an ALS animal model. [Figure 10b] FIG. 10A shows the activity of microglial cells (FIG. 10A) and the number of ChAT-positive motor neurons (FIG. 10B) in the lumbar spinal cord 90 days after birth when halopuginone was administered to an ALS animal model. [Figure 11a] Halopudinone was administered to an ALS animal model, and the expression of TGF-β, iNOS, CD86, arginase 1, IFN-α, IL-6, IL-1β, TNF-α, caspase-3, bcl2, and bax mRNA (Figure 11a), as well as the expression of cleaved caspase 3, bcl2, and bax proteins (Figure 11a) were confirmed in the lumbar spinal cord 120 days after birth. [Figure 11b] Halopudinone was administered to an ALS animal model, and the expression of TGF-β, iNOS, CD86, arginase 1, IFN-α, IL-6, IL-1β, TNF-α, caspase-3, bcl2, and bax mRNA (Figure 11a), as well as the expression of cleaved caspase 3, bcl2, and bax proteins (Figure 11a) were confirmed in the lumbar spinal cord 120 days after birth. Best Mode for Carrying Out the Invention

[0019] The present invention will now be described in more detail.

[0020] In the present invention, the term "prevention" refers to any action that inhibits or delays the onset and acceleration of a disease, the spread of symptoms, and recurrence of the disease by administering a composition of the present invention, and the term "treatment" refers to any action that improves or favorably changes the symptoms of the disease by administering a composition of the present invention.

[0021] In the present invention, the term "administration" means providing a given substance to a patient by any suitable method, and the administration route of the composition of the present invention can be oral or parenteral administration through any common route as long as it can reach the target tissue. In addition, the composition can be administered by any device that can transfer the active substance to the target cell.

[0022] The present invention provides a pharmaceutical composition for preventing or treating neurodegenerative or motor neuron diseases, which comprises halofuginone or a pharma- ceutical acceptable salt thereof as an active ingredient.

[0023] The present invention also provides a method for preventing or treating a neurodegenerative or motor neuron disease, comprising the step of administering halopudinone or a pharma- ceutically acceptable salt thereof to an individual.

[0024] The present invention also provides a pharmaceutical composition comprising halopudinone or a pharma- ceutically acceptable salt thereof for use in the prevention or treatment of neurodegenerative or motor neuron diseases.

[0025] The present invention also provides a use of halopudinone or a pharma- ceutically acceptable salt thereof for the manufacture of a medicament for the prophylaxis or treatment of neurodegenerative or motor neuron diseases.

[0026] In the present invention, the halopudinone or a pharma- ceutically acceptable salt thereof is a TGF-β inhibitor and has the chemical structure represented by the following formula 1: [Chemical formula 1] JPEG2025503765000002.jpg3083.

[0027] Moreover, the above-mentioned halopuginone can be used without any concern as to whether it is commercially available or synthesized.

[0028] In the present invention, the neurodegenerative or motor neuron disease may be, but is not limited to, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, dystonia, spinal muscular atrophy, or inflammatory neurological disease.

[0029] Specifically, in the above-mentioned neurodegenerative or motor neuron diseases, halopuginone can alleviate TGF-β-stimulated fibrosis in the synovial cavity of joints, improve skeletal muscle production, and prevent or treat the diseases.

[0030] In addition, in the above-mentioned neurodegenerative or motor neuron diseases, halopudinone can prevent or treat the disease by suppressing the inflammatory response and neuronal death in the central nervous system due to TGF-β stimulation. More specifically, the halopudinone can increase the expression of anti-inflammatory factors, such as arginase 1, in the central nervous system, and decrease the expression of proinflammatory factors, such as iNOS, CD86, IFN-α, TNF-α, IL-1β, or IL-6, thereby preventing or treating the disease. In addition, the halopudinone can increase the expression of anti-neuron death factors, such as bcl-2, in the central nervous system, and decrease the expression of neuronal death factors, such as caspase-3 and bax, thereby preventing or treating the disease.

[0031] Also, in the aforementioned neurodegenerative or motor neuron diseases, halopuginone can slow the progression of symptoms and improve performance and survival.

[0032] In a specific embodiment of the present invention, the inventors confirmed that TGF-β1 stimulation induces fibrosis and reduces myogenesis in myoblasts, whereas treatment with halopuginone inhibits the induction of fibrosis and the reduction in myogenesis caused by TGF-β1 stimulation.

[0033] The inventors also isolated fibroblasts from an animal model of amyotrophic lateral sclerosis (ALS) and treated them with halopuginone, and confirmed that fibrosis and the decline of muscle transcription factors were suppressed in the fibroblasts.

[0034] The inventors also confirmed that administration of halopudinone to an ALS animal model delayed the onset of ALS and improved performance and survival. In addition, the inventors confirmed that fibrosis in the synovial cavity of the joints was reduced and skeletal muscle production was improved in the ALS animal model administered with halopudinone, improving joint structure. In addition, the inventors confirmed that anti-inflammatory and neuronal death inhibitory effects were observed in the central nervous system (CNS) of the ALS animal model administered with halopudinone.

[0035] Therefore, the present inventors have confirmed in ALS cell models and animal models that halopudinone exhibits dual effects of inhibiting fibrosis, enhancing skeletal muscle production, improving joint structure, and inhibiting inflammatory responses and neuronal death in the central nervous system, thereby delaying the progression of ALS symptoms and improving performance and survival. Therefore, the halopudinone can be usefully used as an active ingredient in a pharmaceutical composition for the prevention or treatment of neurodegenerative or motor neuron diseases, including ALS.

[0036] The halopudinone of the present invention includes all pharma- ceutically acceptable salts, possible solvates, hydrates, racemates, or stereoisomers which can be prepared therefrom.

[0037] The halopudinone of the invention may be used in the form of a pharma- ceutically acceptable salt, and useful salts are acid addition salts formed with pharma- ceutically acceptable free acids, which may be derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and non-toxic organic acids such as aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates and alkanedioates, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharma- ceutically non-toxic salts include, but are not limited to, sorbate, pyrosorbate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, iodide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, pomate, isobutyrate, caffrate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, pumalate, malate, butyrate, 1,4-dioate, , hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

[0038] The acid addition salts according to the present invention can be prepared by a conventional method, for example by dissolving the compound of the present invention in an excess amount of aqueous acid and precipitating the salt with a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile, or by evaporating the solvent and excess acid from the mixture to dryness, or by suction filtering the precipitated salt.

[0039] Also, a pharma- ceutically acceptable metal salt can be prepared using a base. An alkali metal or alkaline earth metal salt can be obtained, for example, by dissolving a compound in an excess amount of an alkali metal hydroxide or an alkaline earth metal hydroxide solution, filtering the compound salt without dissolving it, and evaporating and drying the remaining liquid. In this case, it is suitable to prepare a sodium, potassium or calcium salt as the metal salt under certain restrictions. Also, the corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

[0040] The halopudinone or a pharma- ceutically acceptable salt thereof of the present invention may be present in a therapeutically effective amount and in a pharma- ceutically acceptable carrier.

[0041] As used herein, "effective amount or effective dose" refers to an amount of a compound of the present invention sufficient to slow or minimize neurodegenerative and / or motor neuron disorders; or to provide a therapeutic benefit in the treatment or management of neurodegenerative and / or motor neuron disorders.

[0042] The pharma- ceutically acceptable carrier may be, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. Carriers for parenteral administration may include water, a suitable oil, saline, aqueous glucose, glycol, etc., and may further include a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Other pharma- ceutically acceptable carriers may be referred to in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

[0043] The pharmaceutical composition of the present invention can be administered to mammals, including humans, by any route, including orally or parenterally.

[0044] Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal administration. For example, the pharmaceutical composition of the present invention may be prepared in an injection form and administered by lightly pricking the skin with a 30-gauge thin needle, or by directly applying or attaching to the skin.

[0045] The pharmaceutical composition of the present invention can be formulated as a preparation for oral or parenteral administration by the administration route as described above.

[0046] In the case of oral preparations, the composition of the present invention can be formulated into powder, granules, pellets, sugar-coated pellets, capsules, liquids, gels, syrups, slurries, suspensions, etc., using methods publicly known in the art. For example, oral preparations can be prepared by blending the active ingredient with a solid excipient, grinding it, adding suitable auxiliary agents, and processing it into a granular mixture. Examples of suitable excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, etc., starches including corn starch, wheat starch, rice starch, potato starch, etc., celluloses including cellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethyl-cellulose, etc., gelatin, polyvinylpyrrolidone, and other charging agents. In addition, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate can be added as a disintegrant. Furthermore, the pharmaceutical composition of the present invention may additionally contain an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, and the like.

[0047] For parenteral administration, the formulations can be formulated in the form of injections, creams, lotions, external ointments, oils, moisturizers, gels, patches, aerosols and nasal inhalants by methods known in the art. These formulations are described in the literature (Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour), which is a formulary commonly known to all pharmaceutical chemists.

[0048] The total dosage of the pharmaceutical composition of the present invention may be administered to a patient in a single dose, more specifically, in a single dose administered multiple times over a long period of time, or in a fractionated treatment protocol in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention may have a different content of active ingredient depending on the symptoms of the disease. Specifically, the total dosage of the composition of the present invention may be about 0.01 μg to 1,000 mg, more specifically, 0.1 μg to 100 mg per kg of patient body weight per day. However, the dosage of the pharmaceutical composition of the present invention may be determined by a person having ordinary skill in the art in consideration of various factors such as the age, body weight, health condition, sex, severity of the disease, diet, excretion rate, as well as the administration route and number of treatments. The pharmaceutical composition of the present invention is not particularly limited in its dosage form, administration route, and administration method, as long as it exhibits the effects of the present invention.

[0049] In addition, the pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents. When administered in combination with other therapeutic agents, the composition of the present invention and the other therapeutic agents may be administered simultaneously, separately or sequentially. In this case, the other therapeutic agents may be substances that are already known to have therapeutic or ameliorating effects on neurodegenerative and / or motor neuron diseases. When the pharmaceutical composition of the present invention is administered in combination with other therapeutic agents, the composition of the present invention and the other therapeutic agents may be separately formulated in separate containers or may be combined in the same dosage form.

[0050] The present invention also provides a functional health food composition for preventing or improving neurodegenerative or motor neuron diseases, which contains halopudinone or a pharma- ceutical acceptable salt thereof as an active ingredient.

[0051] The present invention also provides a use of halopudinone or a pharma- ceutical acceptable salt thereof as a health functional food composition for preventing or improving neurodegenerative or motor neuron diseases.

[0052] In addition, the present invention provides a use of halopudinone or a pharma- ceutical acceptable salt thereof for producing a health functional food composition for preventing or improving neurodegenerative or motor neuron diseases.

[0053] In the present invention, the contents regarding the halopuginone or its pharma- ceutical acceptable salt, and neurodegenerative or motor neuron diseases are as described above, so the above contents are incorporated for specific explanations, and only the unique components of the health functional food composition will be described below.

[0054] Meanwhile, the present inventors have confirmed in ALS cell models and animal models that halofuginone exhibits dual effects of inhibiting fibrosis, enhancing skeletal muscle production, improving joint structure, and inhibiting inflammatory responses and neuronal death in the central nervous system, thereby delaying the progression of ALS symptoms and improving performance and survival time. Therefore, halofuginone can be usefully used as an active ingredient in health functional food compositions for preventing or improving neurodegenerative or motor neuron diseases, including ALS.

[0055] The health functional food composition of the present invention can be prepared in any dosage form selected from the group consisting of powder, granule, capsule, candy, syrup and beverage, but is not limited thereto.

[0056] The health functional food composition is not particularly limited as long as it can be taken to prevent or improve neurodegenerative or motor neuron diseases. When the health functional food composition of the present invention is used as a food additive, the health functional food composition can be added as it is or together with other foods or food ingredients, and can be used appropriately by a conventional method. The active ingredient can be used appropriately according to its intended use (prevention or improvement). In general, the health functional food composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, to the health functional food composition of the present invention during the production of the food or beverage. However, in the case of long-term intake for the purpose of health regulation, the amount is within the above range, and there is no problem in terms of safety, so the active ingredient can be used in an amount above the above range. There is no particular limit to the type of the food. Examples of foods to which the health functional food composition can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, drinking water, tea drinks, alcoholic beverages, and vitamin complexes, including all health foods in the usual sense. The functional health food composition of the present invention can also be produced as a food, particularly a functional food.

[0057] The functional food of the present invention includes ingredients that are usually added during food production, such as proteins, carbohydrates, fats, nutrients, and seasonings. For example, when produced as a drink, natural carbohydrates or flavorings may be added as additional ingredients in addition to the active ingredients. The natural carbohydrates are preferably monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrin, cyclodextrin, etc.), or sugar alcohols (e.g., xylitol, sorbitol, erythritol, etc.). The flavorings may be natural flavorings (e.g., tau martini, stevia extract, etc.) or synthetic flavorings (e.g., saccharin, aspartame, etc.). In addition to the above-mentioned health functional food composition, it may further contain various nutrients, vitamins, electrolytes, flavorings, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated drinks, etc.

[0058] The present invention will be described in detail below with reference to examples and experimental examples.

[0059] However, the following examples and experimental examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0060] Example 1: Myoblast culture and treatment with TGF-β1 (Transforming growth factor-β1) and halofuginone

[0061] To investigate the effect of transforming growth factor-β (TGF-β) on myoblasts and the effect of halopuginone, mouse myoblasts were cultured and treated with TGF-β1 and halopuginone.

[0062] Specifically, C2C12 cells (CRL-1772) were purchased as mouse myoblasts from ATCC (American Type Culture Collection) and maintained in DEM medium (Welgene) containing 10% (v / v) fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (P / S; Gibco) at 37°C and 5% CO2. C2C12 cells were cultured in a 6-well plate (3 × 10 5 After 24 hours, the medium was replaced with serum-free medium and stimulated with 5 ng / ml rhTGF-β1 (recombinant human TGF-β1; R&D Systems). After stimulation, the cells were treated with various concentrations of halopuginone (Sigma) (0 [control] or 1, 2.5, 5 (low concentration), 10 (high concentration), 20, 50, 100 ng / ml) in serum-free medium for 24 hours.

[0063] Example 2: Cell viability analysis

[0064] Cell counting kit-8 (CCK-8 kit) assay (Enzo Life Sciences, ALX-850-039) was used to measure cell viability following rhTGF-β1 or halopuginone treatment in serum-free conditions for C2C12 cells.

[0065] Specifically, the C2C12 cells of Example 1 were cultured in a 96-well plate (10 4 The cells were cultured in 1000 x 1000 cells / well for 24 hours and stimulated with 5 ng / ml rhTGF-β1 for 24 hours. After stimulation, the cells were treated with various concentrations of halopuginone (0 [control] or 1, 2.5, 5, 10, 20, 50, 100 ng / ml) for 24 hours. After 24 hours, 10 μl of CCK-8 solution was added to each well and incubated at 37°C for 1 hour. The absorbance was then measured at 450 nm using a VersaMax microplate reader (Molecular Devices). The cell viability was expressed as the viability of the control (untreated) cells. For each concentration of halopuginone, the average absorbance obtained from six wells was calculated.

[0066] Example 3: Immunocytochemical analysis

[0067] As in Example 1, C2C12 cells were treated with rhTGF-β1 or halopuginone under serum-free conditions and then subjected to immunocytochemical analysis.

[0068] Specifically, C2C12 cells were plated on 6-well plates with cover slips and stimulated with rhTGF-β1 in serum-free medium. After 24 hours of stimulation, the cells were treated with low or high concentrations of halopuginone in serum-free medium for 24 hours. The cells were washed three times with PBS and fixed with 4% paraformaldehyde for 15 minutes at room temperature. The fixed cells were then washed three times with PBS, treated with 0.5% Triton X-100 for 5 minutes, and blocked with 5% antiserum albumin in 0.3% PBS-T for 1 hour at room temperature. The cells were incubated with anti-α-SMA antibody (1:200; abcam cat#ab7817) and anti-MyoD antibody (1:200; Santa Cruz, cat#sc377460) in blocking buffer at 4°C for 24 hours, washed three times with PBS, and then incubated with Alexa Fluor secondary antibody for 1 hour at room temperature. After staining nuclear DNA with DAPI (4,6-diamino-2-phenylindole, 1:1,000; Sigma) and washing twice with PBS, coverslips were mounted on glass slides using Dako fluorescent mounting medium (Dako). Images were viewed with a Leica TCS SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) using a 40× objective and quantified in a blinded fashion using Leica Application Suite X software (Leica Microsystems).

[0069] Example 4: Genetic testing of animal models of amyotrophic lateral sclerosis (ALS)

[0070] To investigate the efficacy of halopudinone in amyotrophic lateral sclerosis (ALS), an ALS animal model was used.

[0071] Specifically, all animal experiments were performed in accordance with the guidelines of the Animal Care Committee of Seoul National University Animal Hospital (IACUC, approval number SNU-200220-1-2). Transgenic mice expressing the human G93A mutant SOD1 gene (B6SJL-Tg(SOD1-G93A)1Gur / J), an ALS animal model, were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Male transgenic ALS mice of the mtSOD1(G93A)H1 high expresser strain (Jackson Laboratories, Bar Harbor, ME, USA) were mated with female mice (B6 / SJLF1). The mice were housed under standard conditions of constant temperature (22±1°C), relative humidity (40%), and a 12-hour light-dark cycle, with free access to food and water. Genotypes were confirmed by polymerase chain reaction (PCR) using the primers in Table 1 below, and the copy number of the transferred gene was confirmed. The G93A mutant SOD1 transgenic mice were then divided into three groups according to time point: 60 days (asymptomatic), 90 days (early symptoms), and 120 days (late symptoms). A total of 96 mice were used in the experiment. Non-transgenic mice were used as a comparison group.

[0072] [Table 1] Example 5: Fibroblast culture from ALS animal model and halopudinone treatment

[0073] To examine the efficacy of halopudinone in ALS, fibroblasts were isolated from the ALS animal model in which the G93A mutant SOD1 gene was confirmed in <Example 4> above, and the cells were treated with halopudinone.

[0074] Specifically, primary fibroblasts were isolated from skeletal muscle of 120-day-old non-transformed or G93A mutant SOD1 transgenic mice of Example 4. The excised tissue was placed in a 100 mm culture dish containing calcium- and magnesium-free HBSS (Hanks' Balanced Salt Solution; Gibco, cat#14175095) and penicillin-streptomycin (Cat#15070-063, Thermo Fisher Scientific). The muscle tissue was cut from the ice block. The tissue was then cut into 1 mm slices and enzymatically reacted with 0.2% collagenase type IV (Sigma) at 37°C for 1 hour. The enzymatic reaction was stopped by adding 10 ml of FBS (Gibco). The tissue mixture was centrifuged at 1,800 rpm at 4°C for 5 minutes and reconstituted in DMEM medium (Welgene) containing 10% FBS. The cells were filtered through a 70 μm cell strainer (BD Biosciences, San Jose, CA) and cultured on a 0.2% gelatin (Sigma)-coated plate in DMEM medium containing 10% FBS and 1% penicillin-streptomycin, and then treated with halofuginone in the same manner as described in Example 1.

[0075] Example 6: Administration of halopuginone to an ALS animal model

[0076] To examine the efficacy of halopudinone in ALS, halopudinone was administered to an ALS animal model in which the G93A mutant SOD1 gene was confirmed in <Example 4> above.

[0077] Specifically, as shown in the schematic diagram of Figure 1, the sex- and age-matched mice of <Example 4> were randomly divided into three groups, and as shown in Table 2 below, the DMSOTG group and the HalTG group were injected with vehicle (DMSO, Duchefa Biochemie, D1370) and halopuginone (Sigma, S8144), respectively, at 10 weeks of age (early stage) for 10 weeks or more. As a comparison group, the DMSOOn-TG group was injected intraperitoneally (ip) with vehicle (DMSO) for the same period and in the same manner as non-transgenic mice.

[0078] [Table 2] Meanwhile, G93A mutant SOD1 transgenic mice or non-transgenic mice used for biochemical analysis were sacrificed on postnatal days 90 and 120, and all experiments were performed in triplicate.

[0079] Example 7: Disease progression, survival and motor function analysis of ALS animal models

[0080] To investigate the efficacy of halopudinone in ALS, halopudinone was administered to an ALS animal model as in Example 6 above, and behavioral experiments were then performed in a single-blind manner.

[0081] Specifically, the clinical condition and body weight of the mice of <Example 6> were evaluated three times a week from the 83rd day after birth. The onset of the disease was defined as the shaking of the limbs of the mice when the tail was hung in the air, which has been reported to be due to the clinical involvement of the upper motor neuron system [S. Nagano, Y. Fujii, T. Yamamoto, M. Taniyama K. Fukada, T. Yanagihara, et al. The efficacy of trientine or ascorbate alone compared to that of the combined treatment with these two agents in familial amyotrophic lateral sclerosis model mice Exp Neurol, 179 (2003), pp. 176-180]. The terminal age was determined as the inability of the animals to right themselves within 30 seconds after being laid on a flat surface. At this point, the mouse was considered dead [C. Zheng, I. Nennesmo, B. Fadeel, JI Hanter Vascular endothelial growth factor prolongs survival in a transgenic mouse model of ALS Ann Neurol, 56 (2004), pp. 564-567]. The motor function of the mouse was examined using a rotaload apparatus (JD-A-07M, JEUNG DO BIO & PLANT CO., LTD.). The mouse was trained for one week before recording began. The mouse was placed on a load that started at a speed of 4 rpm for 300 s and accelerated to 40 rpm, increasing by 1 rpm every approximately 8.3 s. The rotaload test was performed from 83 days after birth, three times a week, on average, until the mouse could not stay on the rotaload for more than 10 s for three consecutive times.

[0082] Example 8: Immunohistochemical analysis

[0083] To investigate the efficacy of halopuginone in ALS, halopuginone was administered to an ALS animal model as described in Example 6 above, and immunohistochemical analysis was performed using lumbar spinal cords and knee joint tissues.

[0084] Specifically, the mice of <Example 6> were perfused with 4% paraformaldehyde (PFA) on days 90 and 120 after birth, and the lumbar spinal cord and knee joint were isolated. After fixing in PFA for 24 hours, the knee joint was rinsed with running tap water for 24 hours and incubated with decalcifying solution (Calci-Clear, HS-104, National Diagnostics) at 4°C with constant shaking. The decalcifying solution was replaced with a new solution every day until the decalcification process was completed, when the bone was easily penetrated with a needle. The decalcified knee joint was then rinsed with running tap water for 24 hours, and the spinal cord and knee joint were treated with 30% glucose until the samples sank, then cryoprotected and serially sectioned (14 μm). Lumbar spinal cord and knee joint tissue sections were washed three times with PBS, permeabilized with 0.5% Triton X-100 for 5 min, and then blocked with 5% antiserum albumin in 0.3% PBS-T for 1 h at room temperature. Knee joint tissue sections were treated with anti-TGF-β1 antibody (1:200;Santa Cruz, cat#sc130348), anti-α-SMA antibody (1:200;abcam cat#ab7817), anti-MyoD antibody (1:200;SantaCruz, cat#sc377460), and anti-collagen I antibody (1:200;abcam cat#ab21286) and lumbar spinal cord sections were treated with anti-GFAP antibody (1:200; Dakocat#Z0334), anti-Iba1 antibody (1:200; Wakocat#016)-20001), and anti-TGF-β1 antibody (1:200; Santa Cruz, cat#sc130348), anti-TGF-β1 antibody (1:200;abcam,cat#ab92486), anti- -IL-1β antibody (1:200; R&D Systems cat#AF-401-NA) for an additional 24 h at 4° C. Sections were washed three times with PBS and then incubated with Alexa Fluor secondary antibody for 1 h at room temperature in the dark.Stained and washed spinal cord sections were stained with DAPI (DAPI; 1:1,000; Sigma) for 10 min at room temperature, rinsed twice with PBS, coverslip mounted with Dako fluorescent mounting medium (Dako), and observed under a Leica TCS SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) using a 7.5x objective for the knee joint and a 9.75x objective for the spinal cord, and analyzed using Leica Application Suite X software (Leica Microsystems).

[0085] <Example 9> Motor neuron count in the lumbar spinal cord

[0086] To investigate the efficacy of halopudinone in ALS, halopudinone was administered to an ALS animal model as described in Example 6 above, and the lumbar spinal cord was isolated and motor neurons were counted.

[0087] Specifically, mice were sacrificed at various time points (90 days and 120 days) according to the clinical condition of the mice in "Example 6". Each mouse was perfused transcardially with cold PBS followed by cold 4% paraformaldehyde (PFA) to isolate the lumbar spinal cord. The samples were post-fixed in 4% paraformaldehyde, treated with cold 30% autocrine solution, cryoprotected, and then serially harvested in 14 μm-thick transverse sections. The tissue sections were washed with PBS and immersed in PBS containing 0.3% hydrogen peroxide (H2O2) for 15 minutes to remove resistant peroxidase activity. The sections were washed with PBS, permeabilized with 0.5% Triton X-100 for 5 minutes, and blocked with 5% antiserum albumin in 0.3% PBS-T at room temperature for 1 hour. Each set of tissue sections was incubated with anti-ChAT antibody (1:400; Millipore, cat#AB144P) as the primary antibody for 24 hours. After washing with PBS, the sections were incubated with biotinylated anti-ChAT IgG(H+L) antibody (1:200; Vector Laboratories, cat#BA-9500) for 1 hour, washed with PBS, and then incubated with peroxidase-conjugated avidin-biotin complex (ABC kit; 1:200; Vector Laboratories, cat#PK4000) for 1 hour at room temperature. After extensive washing, the sections were stained with diaminobenzidine (DAB, ImmPACT®) for 1 hour. Peroxidase staining was visualized by incubating sections with DAB Vector Laboratories, cat#SK-4105. Sections were dehydrated, air-dried, and mounted with toluene-soluble Permount mounting medium (Fisher Scientific, cat#SP15-500). Motor neurons were calculated in ChAT-stained sections using a computer-assisted microscope (Olympus BX53) and software (cellSens software) at 4x magnification. Coefficients were performed in the per ventral horn for a total of 3 mice per group. The field analyzed was all ChAT+ profiles located in the dorsal half of the immunostained sections that were clearly visible in the gray matter of each hemisphere.

[0088] <Example 10> Measurement of joint range of motion (ROM)

[0089] To investigate the efficacy of halopuginone in ALS, halopuginone was administered to an ALS animal model as in Example 6 above, and the range of motion (ROM) of the knee joint was then measured.

[0090] Specifically, anatomical landmarks were used to determine knee extension angles. At the end of disease age (120 days), manual knee ROM was measured using a 2D angle analysis system, Random Two-line. The mice from Example 6 were anesthetized by inhaling 1% isoflurane, placed on an acrylic plate, and the skin of the hind legs was shaved. The femur was fixed to the plate, and a constant force extension moment was applied to the knee joint per mouse. Then, markers were attached to the greater trochanter, the lateral side of the knee, and the lateral malleolus to obtain markers for the photographed images. The angle between the axis of the femur (greater trochanter relative to the lateral joint space of the knee) and the fibula (from the lateral joint space of the knee to the lateral malleolus) was measured as knee extension ROM. 16 mice were analyzed in each group, and the knees of non-transgenic mice injected with DMSO on day 120 were used as a control group.

[0091] Example 11: Real-time qRT-PCR analysis

[0092] Real-time qRT-PCR analysis was performed using myoblasts stimulated with TGF-β1 and then treated with halopudinone in <Example 1>, fibroblasts isolated from an ALS animal model and treated with halopudinone in <Example 5>, or lumbar spinal cord isolated after administering halopudinone to an ALS animal model as in <Example 6>.

[0093] Specifically, total RNA was FavorPrep TMUsing Tri-RNA Reagent (Favorgen), RNA was isolated from the C2C12 cells in <Example 1>, the fibroblasts in <Example 5>, and the lumbar spinal cord of the mouse in <Example 6>. The lumbar spinal cord was isolated from the mouse in <Example 6> and then frozen and stored at -80°C until the experiment. The total RNA concentration was measured at 260 nm absorbance using a spectrophotometer (NanoDrop Spectrophotometer ND-2000, Thermo Scientific). cDNA was prepared by chromatin synthesis using ReverTra Ace-α- TM The primers were synthesized using 1 μg of total RNA using SYBR green Excel Taq (Toyobo). TM Quantitative RT-PCR was performed using the primers in Table 3 below in a thermocycler CFX Connect Real-Time PCR Detection System (BIO-RAD) using 2X Fast Q-PCR Master Mix (TQ1200, Smobio). Fluorescence data were analyzed with Bio-Rad CFX Manager 3.1 software and 2 (-ΔΔCT) Relative mRNA expression was calculated versus the control group in the methods. All samples were run in quadruplicate. All primers were designed using Primer3 online software, and GAPDH was used as the control group.

[0094] [Table 3]

[0095] Example 12: Protein extraction and Western blot analysis

[0096] Western blot analysis was performed using myoblasts stimulated with TGF-β1 and then treated with halopudinone in <Example 1>, fibroblasts isolated from an ALS animal model and treated with halopudinone in <Example 5>, or lumbar spinal cord or knee joint isolated after administering halopudinone to an ALS animal model as in <Example 6>.

[0097] Specifically, the lumbar spinal cord or knee joint of the mouse in <Example 6> was isolated and immediately frozen at -80°C. Then, each lumbar spinal cord or knee joint was homogenized and chemically treated to obtain cells. The chemically treated cells, the C2C12 cells in <Example 1>, or the fibroblasts in <Example 5> were dissolved in RIPA buffer (Thermo, MA, USA) with protease inhibitor and phosphatase inhibitor (Roche, IN, USA) and incubated on ice for 30 minutes. The lysates were centrifuged at 4°C and 13,000 rpm for 20 minutes, and insoluble materials were removed. Protein concentration was measured by BCA analysis (Pierce Biotechnology). The same amount of cell lysates was loaded onto an SDS-PAGE gel for electrophoresis and transferred to a nitrocellulose membrane (nitrocellulose membrane, Amersham Protran 0.2um NC, Amersham Pharmacia Biotech, Piscataway, NJ, USA). The membrane was then blocked with 5% (w / v) skim milk in 1× TBST for 1 h at room temperature. After blocking, the primary antibodies on the membrane were anti-β-actin antibody (1:5000; Santa Cruz, cat#sc47778), anti-Smad2 antibody (1:1000; Cell Signaling, cat#5339), and anti-p-Smad2 antibody (1:1000; Cell Signaling, cat#3108), anti-TGF-β1 antibody (1:1000;Santa Cruz,cat#sc130348), anti-α-SMA antibody (1:10000;abcam cat#ab7817), anti-MyoD antibody (1:1000;Santa Cruz,cat#sc377460), anti-collagen I antibody (1:1000;abcam cat#ab21286), anti-cleaved caspase 3 antibody (1:1000;Cell Signaling, cat#9661), anti-bax antibody (1:500; Santa Cruz, cat#sc493), anti-bcl2 antibody (1:1000; Novus Biologicals, cat#NB100-56098), or anti-ChAT antibody (1:1000; Millipore, cat#AB144P) were then used to treat the cells and incubated overnight at 4°C.After several washes with 1X TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (anti-mouse, rabbit or goat, 1:5000; GE Healthcare) in blocking buffer for 1 h at room temperature (RT). The membranes were then washed and briefly incubated with SuperSignal West Pico Plus Chemiluminescent Substrate (Pierce Biotechnology) according to the manufacturer's protocol and quantified on an image analyzer (Amersham Pharmacia Biotech, Piscataway, NJ, USA). β-actin was used as an internal control. Densitometry of protein intensity was quantified using Image J (National Institutes of Health).

[0098] Example 13: Statistical analysis

[0099] All data are presented as the mean and standard error of the mean (SEM) based on at least three independent experiments and expressed as percentage of control values. The significance of differences between groups was analyzed by Student's t-test or one-way ANOVA followed by Tukey's post hoc comparison. A p-value <0.05 was considered statistically significant. Kaplan-Meier curves were used to analyze the cumulative probability of symptom onset, rotarod failure, and disease endpoints. All analyses were performed using GraphPad Prism.

[0100] <Experimental Example 1> Confirmation of promotion of fibrosis and inhibition of myogenesis by TGF-β1 stimulation in myoblasts

[0101] To investigate the effect of TGF-β1 on myoblasts, real-time qRT-PCR and Western blot analysis were performed on C2C12 mouse myoblast cell line after stimulation with TGF-β1.

[0102] Specifically, C2C12 cells were treated with TGF-β1 for 24 hours as in <Example 1>, and then real-time qRT-PCR was performed in the same manner as described in <Example 11> to confirm the expression of TGF-β1, 2, and 3, α-SMA, and MyoD mRNA (Figure 2a), and Western blot analysis was performed in the same manner as described in <Example 12> to confirm the autocrine expression of TGF-β1, Myo protein, and α-SMA (Figure 2b).

[0103] As a result, as shown in Figure 2a, 24 hours after TGF-β1 stimulation in C2C12 cell line, the mRNA levels of TGF-β1, 2, and 3 were significantly increased compared to the control group. In addition, the activation of myoblasts into myofibroblasts by TGF-β stimulation was confirmed by a significant increase in the mRNA expression of α-SMA. In contrast, the mRNA level of MyoD was significantly decreased after TGF-β stimulation (Figure 2a).

[0104] Furthermore, as shown in Fig. 2b, TGF-β1 and α-SMA protein expression was significantly increased 24 hours after TGF-β1 stimulation in C2C12 cells. In contrast, MyoD protein expression was significantly decreased after TGF-β stimulation (Fig. 2b).

[0105] These results indicate that TGF-β1 stimulation promotes fibrosis and inhibits myogenesis in myoblasts.

[0106] <Experimental Example 2> Confirmation of cytotoxicity of halofuginone

[0107] To examine the cytotoxicity of halofuginone in myoblasts, cell viability assays and Western blot assays were performed.

[0108] Specifically, C2C12 cells were treated with or without TGF-β1 stimulation and then treated with halopuginone in the same manner as described in <Example 2>, and cell viability was analyzed using CCK-8 assay (FIGS. 3a and 3b). Western blot analysis was also performed in the same manner as described in <Example 12> to confirm the expression of p-Smad2, Smad2, TGF-β1, MyoD, collagen I protein, and α-SMA (FIG. 3c).

[0109] As a result, as shown in Figures 3a and 3b, it was confirmed that the cell viability did not change significantly at halopudinone concentrations of 1 ng / ml to 10 ng / ml (Figures 3a and 3b).

[0110] Furthermore, as shown in Figure 3c, at halopudinone concentrations of 5 ng / ml and 10 ng / ml, the TGF-β1-stimulated increases in the p-Smad2 / Smad2 ratio, TGF-β1, α-SMA, and collagen I protein levels were significantly reduced, and the TGF-β1-stimulated decrease in MyoD protein levels was significantly restored (Figure 3c).

[0111] Thus, the optimal concentration of halopudinone for C2C12 cells was determined to be 5 ng / ml for the low concentration and 10 ng / ml for the high concentration.

[0112] <Experimental Example 3> Confirmation of the promotion of fibrosis and the alleviation of myogenesis inhibition by halopuginone in TGF-β1 stimulated myoblasts

[0113] To investigate whether TGF-β1-induced changes in myoblasts could be inhibited by halopudinone as a TGF-β inhibitor, we treated C2C12 mouse myoblast cell line with halopudinone after TGF-β1 stimulation and performed real-time qRT-PCR, Western blot, and immunocytochemical analyses.

[0114] Specifically, as in <Example 1>, C2C12 cells were stimulated with TGF-β1 for 24 hours, and then treated with halopuginone for 24 hours. Real-time qRT-PCR was performed in the same manner as in <Example 11> to confirm the expression of TGF-β1, α-SMA, MyoD, and collagen I (Col-I) mRNA (FIG. 4a). Western blot analysis was performed in the same manner as in <Example 12> to confirm the expression of TGF-β1, α-SMA, MyoD, and collagen I protein (FIG. 4b). Immunocytochemistry was also performed in the same manner as in <Example 3> to confirm the expression of α-SMA and MyoD (FIG. 4c).

[0115] As a result, as shown in Figure 4a and Figure 4b, in the groups treated with low or high concentrations of halofuginone, the increase in TGF-β1, α-SMA and collagen I mRNA levels due to TGF-β1 stimulation was significantly reduced, and the decrease in MyoD mRNA level due to TGF-β1 stimulation was significantly restored (Figure 4a). Protein expression also showed a similar pattern (Figure 4b).

[0116] Furthermore, as shown in FIG. 4c, immunofluorescence staining and confocal microscopy were used to confirm the intensity of the decrease in α-SMA and the restoration of MyoD by treatment with low or high concentrations of halopudinone (FIG. 4c).

[0117] These results suggest that the promotion of fibrosis and inhibition of myogenesis caused by TGF-β1 stimulation in myoblasts can be prevented by using halopuginone as a TGF-β inhibitor.

[0118] <Experimental Example 4> Fibrosis inhibition by halofuginone confirmed in fibroblasts derived from an ALS animal model

[0119] To investigate the changes in TGF-β1 and the effect of halopudinone in fibroblasts derived from an ALS animal model, real-time qRT-PCR analysis and Western blot analysis were performed after treating fibroblasts derived from an ALS animal model with halopudinone.

[0120] Specifically, fibroblasts were isolated from the ALS animal model in the same manner as described in <Example 5>, treated with halopuginone, and then real-time qRT-PCR was performed in the same manner as described in <Example 11> to confirm the expression of TGF-β1, α-SMA, and collagen I (Col-I) mRNA (FIG. 5a). Western blot analysis was performed in the same manner as described in <Example 12> to confirm the expression of p-Smad2, Smad2, TGF-β1, α-SMA, and collagen I protein (FIG. 5b). Fibroblasts isolated from non-transgenic mice were used as a control group.

[0121] As a result, as shown in Figures 5a and 5b, the ratio of p-Smad2 / Smad2, and the protein expression as well as the mRNA levels of TGF-β, α-SMA, and Col-I were significantly increased in fibroblasts derived from G93A mutant SOD1 mice compared to fibroblasts derived from non-transformed mice (Non-TG). It was confirmed that such changes were attenuated by treatment with low or high concentrations of halopuginone for 24 hours (Figures 5a and 5b).

[0122] These results demonstrate that fibrosis is enhanced in fibroblasts from an ALS mouse model and that fibrosis can be inhibited by treatment with halofuginone.

[0123] <Experimental Example 5> Confirmation of delay in onset of ALS and improvement of performance by administration of halopuginone in ALS animal model

[0124] To evaluate the effects of halopudinone in vivo, we administered it to an ALS animal model and performed disease progression, survival, and motor function analyses.

[0125] Specifically, halopuginone was administered to an ALS animal model in the same manner as described in <Example 6>, and disease progression, survival and motor function analyses were performed in the same manner as described in <Example 7>.

[0126] As a result, as shown in Figure 6a, each TG group in the symptom stage showed a significantly shorter time on the rotarod compared to the non-TG group. On the other hand, among the TG groups, the halopuginone-administered TG group (Hal TG group) showed a significantly longer time on the rotarod compared to the DMSO-administered TG group (DMSO TG group). Body weight was significantly reduced in the TG group compared to the non-TG group, and no significant difference was observed between the DMSO TG group and the Hal TG group (Figure 6a).

[0127] As shown in Fig. 6b and Fig. 6c, there was a difference in the onset of symptoms between the DMSO TG group and the Hal TG group, and the onset of symptoms in the Hal TG group was significantly delayed compared to the DMSO TG group (mean ± standard deviation [SD], 113 ± 3.6 vs. 94.2 ± 5.7, p < 0.5 for each). The age at which rotarod failure was reached was also delayed in the Hal TG group compared to the DMSO TG group (124.3 ± 3.5 vs. 116.5 ± 3.1, respectively, p < 0.001), and the age at termination was also significantly increased (141.8 ± 9.8 vs. 129.3 ± 7.6, respectively, Fig. 6b and Fig. 6c).

[0128] <Experimental Example 6> Confirmation of reduction in synovial fibrosis and improvement in skeletal muscle formation by administration of halopuginone at the early stage of disease in ALS animal models

[0129] To evaluate the effect of halopuginone on the pathogenesis of ALS, halopuginone was administered to an ALS animal model, and changes in the synovial cavity of the knee joint were examined by immunohistochemistry (IHC) at early and late stages of the disease.

[0130] Specifically, halopudinone was administered to G93A mutant SOD1 mice from the early stage of ALS onset (day 73) using the same method as described in Example 6 above, and immunohistochemical analysis was performed using knee joints from female and male mice with early disease (90 days old, Figures 7a and 7b) and female and male mice with late disease (120 days old, Figures 7c and 7d) using the same method as described in Example 8 above.

[0131] As a result, as shown in Figures 7a and 7b, the DMSO TG group showed a significant increase in TGF-β1 in the synovial cavity of the knee joint at the early stage of the disease, regardless of gender, compared to the DMSO Non-TG group. In addition, it was confirmed that, in addition to the increase in TGF-β1 in the synovial cavity of the DMSO TG group, α-SMA and Col-I increased, and MyoD decreased, compared to the DMSO Non-TG group. On the other hand, in the HalTG group, the increase in TGF-β was suppressed at the early stage of the disease, and the expression of α-SMA and Col-I was also significantly lower than that of the DMSOTG group (Figures 7a and 7b).

[0132] As shown in Figures 7c and 7d, the Hal TG group maintained significantly lower TGF-β1, α-SMA, Col-I, and higher MyoD in the synovial cavity compared to the DMSO TG group at the later stage of the disease. The intensities of TGF-β1, MyoD, and Col-1 in the female Hal TG group were not different from those in the DMSO non-TG group, while the intensity of MyoD in the male Hal TG group was slightly increased compared to the DMSO non-TG group (Figures 7c and 7d).

[0133] The above results indicate that the activation of myofibroblasts and enhanced fibrosis in the synovial cavity of ALS mice caused by increased TGF-β can be improved by administration of halopudinone. It is also shown that the decreased skeletal muscle myogenesis in the synovial cavity of ALS mice can be prevented by administration of halopudinone. Furthermore, it is shown that administration of halopudinone at the early stage of disease in ALS mice significantly reduces fibrosis in the synovial cavity and improves muscle formation, regardless of gender.

[0134] <Experimental Example 7> Confirmation of improvement in joint structure in ALS animal models by administration of halopuginone

[0135] To investigate functional resistance due to fibrosis in ALS and the effect of halopuginone on this, we administered halopuginone to an ALS animal model, measured the range of motion (ROM) of the knee joint, and performed Western blot analysis.

[0136] Specifically, halopuginone was administered to G93A mutant SOD1 mice in the same manner as described in <Example 6>, and the ROM of the knee joint was measured in the same manner as described in <Example 10> (FIG. 8a). In addition, Western blot analysis was performed in the same manner as described in <Example 12> using knee joint tissue to confirm the expression of p-Smad2, Smad2, TGF-β1, α-SMA, MyoD, and collagen I protein (FIG. 8b).

[0137] As a result, as shown in Fig. 8a, it was confirmed that ROM was significantly decreased in the DMS OTG group compared with the DMSONon-TG group on day 120, whereas ROM was unchanged in the Hal TG group (Fig. 8a).

[0138] As shown in Fig. 8b, the p-Smad2 / Smad2 ratio, TGF-β, α-SMA, and Col-I ratio, and MyoD ratio were significantly increased and decreased in the DMSO TG group compared to the DMSO non-TG group. The p-Smad2 / Smad2 ratio and the expression levels of TGF-β1, α-SMA, and MyoD in the HalTG group were similar to those in DMSO non-TG mice, whereas the expression level of Col-1 was decreased (Fig. 8b).

[0139] These results suggest that increased TGF-β in ALS mice enhances fibrosis in the knee joints and induces joint contraction, but that this can be improved by administration of halopidine.

[0140] <Experimental Example 8> Confirmation of anti-inflammatory and neuronal death inhibitory effects by halopuginone administration in central nervous system (CNS) tissue of ALS animal models

[0141] To investigate the effects of halopudinone administration in the central nervous system (CNS) in ALS, we administered halopudinone to ALS animal models and then performed immunohistochemistry, motor neuron counts in the lumbar spinal cord, and Western blot analysis.

[0142] Specifically, halopuginone was administered to G93A mutant SOD1 mice in the same manner as described in <Example 6>, and the mice were sacrificed at 120 days after birth. Immunohistochemical analysis was performed in the same manner as described in <Example 8> to confirm TGF-β1 and glial cells in the lumbar spinal cord. Considering that astrocyte-derived TGF-β has been reported to accelerate the disease in ALS mice, the spinal cord of each group was co-stained for TGF-β and GFAP during immunohistochemical analysis. In addition, IBA1 staining was performed to confirm the activity of microglia (Figures 9a and 9b).

[0143] In addition, to examine the effects of TGF-β and CNS neuroglial changes on inflammation, IL-1β, a representative infectious cytokine, was co-stained with GFAP during immunohistochemical analysis (Fig. 9c).

[0144] In addition, to investigate the source of motor nerves in the spinal cord, motor neurons in the lumbar spinal cord were observed and counted using the same method as described in <Example 9> (Figure 9d), and real-time qRT-PCR and Western blot analysis were performed using the same methods as described in <Example 11> and <Example 12> to quantitatively analyze ChAT mRNA (Figure 9e) and ChAT protein (Figure 9f) expression.

[0145] In addition, halopudinone was administered to G93A mutant SOD1 mice in the same manner as described in Example 6 above, and the mice were sacrificed 90 days after birth. Iba1 staining was then performed to confirm the activity of microglial cells in the same manner as described above (Figure 10a), and motor neurons were observed and counted (Figure 10b).

[0146] As a result, as shown in Figures 9a and 9b, GFAP intensity was significantly increased in the DMSO TG group of 120-day-old mice compared to the DMSO Non-TG group, and TGF-β1 was also increased at the same site. This increase in astrocyte activity and TGF-β1 was confirmed to be significantly decreased in the HalTG group (Figure 9b). On the other hand, microglial cells showed a significant increase in the DMSO TG group, and it was confirmed that microglial cells also continued to increase in the Hal TG group (Figure 9a).

[0147] Furthermore, as shown in Fig. 9c, compared with the DMSO Non-TG group in 120-day-old mice, the DMSO TG group showed increased IL-1β along with GFAP activity, indicating activated inflammation, which was suppressed in the Hal TG group (Fig. 9c).

[0148] Furthermore, as shown in Figures 9d to 9f, in the DMSO TG group of 120-day-old mice, all ChAT mRNA levels, the number of ChAT-positive motor neurons, and ChAT expression were significantly lower than those in the DMSO non-TG group, whereas in the Hal TG group, they were preserved to the same extent as those in the DMSO non-TG group (Figures 9d to 9f).

[0149] Additionally, as shown in Figures 10a and 10b, the above-mentioned changes were similar in 90-day-old mice (Figures 10a and 10b).

[0150] These results indicate that increased TGF-β in the CNS of ALS mice is associated with increased inflammatory responses and associated loss of motor neurons, a process that is inhibited by halopuginone administration.

[0151] <Experimental Example 9> Halopudinone administration inhibits chronic inflammation and neuronal death in the CNS in an ALS animal model

[0152] To investigate the effect of halopudinone administration on the inflammatory response of the CNS in ALS, real-time qRT-PCR analysis and Western blot analysis were performed using the lumbar spinal cord after administration of halopudinone to an ALS animal model.

[0153] Specifically, halopuginone was administered to G93A mutant SOD1 mice in the same manner as described in <Example 6>, and the mice were sacrificed at 120 days after birth, and the lumbar spinal cord was isolated. Next, the mRNA expression of CNS inflammation-related factors and neuronal death-related factors was confirmed by real-time qRT-PCT analysis in the same manner as described in <Example 11> (FIG. 11a), and the protein expression of neuronal death-related factors was confirmed by Western blot analysis in the same manner as described in <Example 12> (FIG. 11b). Since the role (inflammatory or anti-inflammatory effect) differs depending on the M1 or M2 subtype of microglial cells, the expression of M1 markers (iNOS, CD86), M2 markers (arginase1), and infectious factors (IFN-a, TNF-a, IL-1b, IL-6) was confirmed as CNS inflammation-related factors. In addition, the expression of caspase-3, bax, and bcl-2 was confirmed as neuronal death-related factors.

[0154] As a result, as shown in Figure 11a, the mRNA levels of M1 markers and infectious factors were significantly increased in the DMSOTG group according to the mRNA level of TGF-β, and this was confirmed to be suppressed by halopuginone. In addition, the mRNA level of bcl-2, an anti-neuron death factor, was confirmed to be decreased in the DMSO TG group compared to the DMSO-TG group, and was preserved in the Hal TG group (Figure 11a).

[0155] Furthermore, as shown in Figure 11b, the protein expression of cleaved caspase-3 and bax, which are neuronal death factors, increased and the anti-neuronal death factor bcl-2 decreased in the DMSOTG group compared to the DMSO Non-TG group, and it was confirmed that these changes were improved in the Hal TG group (Figure 11b).

[0156] These results indicate that halopuginone can block persistently increased TGF-β in ALS mice, exert anti-inflammatory effects in the CNS, and inhibit neuronal cell death.

[0157] From the results of <Experimental Example 1> to <Experimental Example 9>, it was confirmed that halopudinone exhibits dual therapeutic effects of improving joint structure by increasing TGF-β in ALS and inhibiting chronic inflammation and neuronal death in the CNS. Therefore, halopudinone can be used to prevent or treat neurodegenerative or motor neuron diseases caused by increased TGF-β, including ALS. [Industrial Applicability]

[0158] In the present invention, it has been confirmed that halofuginone exhibits dual effects of suppressing fibrosis, improving skeletal muscle production, improving joint structure, and suppressing inflammatory responses and neuronal death in the central nervous system in cell models and animal models of amyotrophic lateral sclerosis (ALS), thereby delaying the progression of ALS symptoms and improving performance and survival time. Therefore, the halofuginone or a pharma- ceutically acceptable salt thereof can be usefully used as an active ingredient in a composition for preventing or treating neurodegenerative or motor neuron diseases, including ALS.

Claims

1. A pharmaceutical composition for preventing or treating neurodegenerative or motor neuron diseases, comprising halofuginone or a pharma- ceutical acceptable salt thereof as an active ingredient.

2. The pharmaceutical composition for preventing or treating neurodegenerative or motor neuron diseases according to claim 1, wherein said halopudinone or a pharma- ceutical acceptable salt thereof is a TGF-β inhibitor.

3. The pharmaceutical composition for preventing or treating neurodegenerative or motor neuron diseases according to claim 1, wherein the halopuginone is a compound represented by the following formula 1: [Chemical formula 1] .

4. The pharmaceutical composition for preventing or treating neurodegenerative or motor neuron diseases according to claim 1, wherein said halopuginone alleviates fibrosis in synovial cavity of joints and enhances skeletal muscle production.

5. The pharmaceutical composition for preventing or treating neurodegenerative or motor neuron diseases according to claim 1, wherein said halopuginone inhibits inflammatory reactions and neuronal cell death in the central nervous system.

6. The pharmaceutical composition for preventing or treating neurodegenerative or motor neuron diseases according to claim 1, wherein the halopuginone delays the progression of symptoms of neurodegenerative or motor neuron diseases and improves performance and survival time.

7. The pharmaceutical composition for preventing or treating neurodegenerative or motor neuron diseases according to claim 1, wherein the neurodegenerative or motor neuron diseases are amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, dysregulation of muscular tone, spinal muscular atrophy or inflammatory neurological diseases.

8. A functional health food composition for preventing or improving neurodegenerative or motor neuron diseases, comprising halopudinone or a pharma- ceutical acceptable salt thereof as an active ingredient.

9. 20. A method for preventing or treating a neurodegenerative or motor neuron disease, comprising the step of administering to an individual halopudinone or a pharma- ceutically acceptable salt thereof.

10. Use of halopudinone or a pharma- ceutically acceptable salt thereof for the manufacture of a medicament for the prophylaxis or treatment of a neurodegenerative or motor neuron disease.

11. Use of halopudinone or a pharma- ceutically acceptable salt thereof for producing a health functional food composition for preventing or ameliorating neurodegenerative or motor neuron diseases.

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