Composition for preventing or treating neurodegenerative or motor neuron diseases containing halofuginone 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 Halofuginone 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, and multiple sclerosis are representative degenerative neurological 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 the clinical course and prognosis are heterogeneous due to various pathophysiological mechanisms, including genetic mutations, impaired protein homeostasis, 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. Following the emergence of a link between TGF-β and ALS, the role of TGF-β in ALS progression has begun to emerge. In skeletal muscle, TGF-β plays a role in repairing damaged muscle and generally regulates myogenesis, growth, and differentiation. However, sustained increases in TGF-β decrease myogenesis and promote muscle fibrosis and atrophy. Previous studies using a murine model of ALS demonstrated enhanced TGF-β signaling pathways during the symptomatic stage, resulting in abundant extracellular matrix (ECM) production due to increased fibrogenic and adipogenic precursors. Similarly, in the normal nervous system, TGF-β is known to protect neurons from excitotoxicity and oxidative damage and play an important role in neurogenesis. However, sustained increases in TGF-β have 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, we have been working 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 demonstrate therapeutic effects. As a result, we have demonstrated that this agent can effectively treat ALS in ALS cell models and animal models. Halofuginone It was confirmed that (Halofuginone) exhibits dual effects of inhibiting fibrosis caused by elevated TGF-β, improving joint structure by increasing skeletal muscle production, and inhibiting inflammatory responses and neuronal death in the central nervous system, thereby delaying the progression of ALS symptoms and improving performance and survival. Halofuginone The present application was made by clarifying that the compound can be usefully used as an active ingredient in a composition for preventing or treating neurodegenerative or motor neuron diseases, including ALS. [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] The object of the present invention is to Halofuginone The present invention provides a composition for preventing or treating neurodegenerative or motor neuron diseases, which comprises (Halofuginone) or a pharmaceutically acceptable salt thereof as an active ingredient. [Means for solving the problem]

[0009] In order to achieve the object of the present invention, the present invention Halofuginone The present invention provides a pharmaceutical composition for preventing or treating neurodegenerative or motor neuron diseases, which contains (Halofuginone) or a pharmaceutically acceptable salt thereof as an active ingredient.

[0010] The present invention also provides Halofuginoneor a pharmaceutically acceptable salt thereof as an active ingredient, and a functional health food composition for preventing or ameliorating neurodegenerative or motor neuron diseases is provided.

[0011] The present invention also provides Halofuginone or a pharmaceutically acceptable salt thereof to an individual.

[0012] The present invention also provides a method for preventing or treating neurodegenerative or motor neuron diseases. Halofuginone or a pharmaceutically acceptable salt thereof.

[0013] The present invention also provides a method for preventing or ameliorating neurodegenerative or motor neuron diseases. Halofuginone or a pharmaceutically acceptable salt thereof.

[0014] The present invention also provides a method for producing a drug for preventing or treating neurodegenerative or motor neuron diseases. Halofuginone or a pharmaceutically acceptable salt thereof.

[0015] In addition, the present invention provides a functional health food composition for preventing or improving neurodegenerative or motor neuron diseases. Halofuginone or a pharmaceutically acceptable salt thereof.

[0016] [Effects of the Invention]

[0017] The present inventors have confirmed that halofuginone exhibits dual effects in cell and animal models of amyotrophic lateral sclerosis (ALS), including suppressing fibrosis, 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. Halofuginonecan be usefully used as an active ingredient in a composition for preventing or treating neurodegenerative or motor neuron diseases, including ALS. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 is a schematic diagram illustrating a method for 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 (Fig. 2a) and the protein expression of TGF-β1, α-SMA, and MyoD (Fig. 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 (Fig. 2a) and the protein expression of TGF-β1, α-SMA, and MyoD (Fig. 2b) after stimulation of myoblasts with TGF-β1 (Transforming growth factor-β1). [Figure 3a] Figure 3a shows the cell viability of myoblasts after treatment with various concentrations of halofuginone (Figure 3a) or with TGF-β1 and various concentrations of halofuginone (Figure 3b). The p-Smad2 / Samd2 ratio, and the protein expression of TGF-β1, α-SMA, MyoD, and collagen I were also examined (Figure 3c). [Figure 3b] Figure 3a shows the cell viability of myoblasts after treatment with various concentrations of halofuginone (Figure 3a) or with TGF-β1 and various concentrations of halofuginone (Figure 3b). The p-Smad2 / Samd2 ratio, and the protein expression of TGF-β1, α-SMA, MyoD, and collagen I were also examined (Figure 3c). [Figure 3c]Figure 3a shows the cell viability of myoblasts after treatment with various concentrations of halofuginone (Figure 3a) or with TGF-β1 and various concentrations of halofuginone (Figure 3b). The p-Smad2 / Samd2 ratio, and the protein expression of TGF-β1, α-SMA, MyoD, and collagen I were also examined (Figure 3c). [Figure 4a] After myoblast treatment with TGF-β1 and halofuginone, TGF-β1, α-SMA, MyoD, and collagen I mRNA expression (Figure 4a) and TGF-β1, α-SMA, MyoD, and collagen I protein expression (Figure 4b) were confirmed, and α-SMA and MyoD expression was confirmed by immunocytochemical analysis (Figure 4c). [Figure 4b] After myoblast treatment with TGF-β1 and halofuginone, TGF-β1, α-SMA, MyoD, and collagen I mRNA expression (Figure 4a) and TGF-β1, α-SMA, MyoD, and collagen I protein expression (Figure 4b) were confirmed, and α-SMA and MyoD expression was confirmed by immunocytochemical analysis (Figure 4c). [Figure 4c] After myoblast treatment with TGF-β1 and halofuginone, TGF-β1, α-SMA, MyoD, and collagen I mRNA expression (Figure 4a) and TGF-β1, α-SMA, MyoD, and collagen I protein expression (Figure 4b) were confirmed, and α-SMA and MyoD expression was confirmed by immunocytochemical analysis (Figure 4c). [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 halofuginone. [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 halofuginone. [Figure 6a]FIG. 6 shows changes in motor function and body weight (FIG. 6a) and changes in symptom onset (FIGS. 6b and 6c) after administration of halofuginone to an ALS animal model. [Figure 6b] FIG. 6 shows changes in motor function and body weight (FIG. 6a) and changes in symptom onset (FIGS. 6b and 6c) after administration of halofuginone to an ALS animal model. [Figure 6c] FIG. 6 shows changes in motor function and body weight (FIG. 6a) and changes in symptom onset (FIGS. 6b and 6c) after administration of halofuginone to an ALS animal model. [Figure 7a] Halofuginone 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 (Fig. 7a) and males (Fig. 7b), and in 120-day-old females (Fig. 7c) and males (Fig. 7d). [Figure 7b] Halofuginone 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 (Fig. 7a) and males (Fig. 7b), and in 120-day-old females (Fig. 7c) and males (Fig. 7d). [Figure 7c] Halofuginone 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 (Fig. 7a) and males (Fig. 7b), and in 120-day-old females (Fig. 7c) and males (Fig. 7d). [Figure 7d] Halofuginone 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 (Fig. 7a) and males (Fig. 7b), and in 120-day-old females (Fig. 7c) and males (Fig. 7d). [Figure 8a] Halofuginone was administered to an ALS animal model, and the range of motion (ROM) was measured in the synovial cavity of the knee joint at 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]Halofuginone was administered to an ALS animal model, and the range of motion (ROM) was measured in the synovial cavity of the knee joint at 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] Halofuginone was administered to an ALS animal model, and microglial activity (Fig. 9a), astrocyte activity (Fig. 9b), IL-1β expression (Fig. 9c), the number of ChAT-positive motor neurons (Fig. 9d), ChAT mRNA expression (Fig. 9e), and ChAT protein expression (Fig. 9f) in the lumbar spinal cord were confirmed at 120 days after birth. [Figure 9b] Halofuginone was administered to an ALS animal model, and microglial activity (Fig. 9a), astrocyte activity (Fig. 9b), IL-1β expression (Fig. 9c), the number of ChAT-positive motor neurons (Fig. 9d), ChAT mRNA expression (Fig. 9e), and ChAT protein expression (Fig. 9f) in the lumbar spinal cord were confirmed at 120 days after birth. [Figure 9b] Halofuginone was administered to an ALS animal model, and microglial activity (Fig. 9a), astrocyte activity (Fig. 9b), IL-1β expression (Fig. 9c), the number of ChAT-positive motor neurons (Fig. 9d), ChAT mRNA expression (Fig. 9e), and ChAT protein expression (Fig. 9f) in the lumbar spinal cord were confirmed at 120 days after birth. [Figure 9c] Halofuginone was administered to an ALS animal model, and microglial activity (Fig. 9a), astrocyte activity (Fig. 9b), IL-1β expression (Fig. 9c), the number of ChAT-positive motor neurons (Fig. 9d), ChAT mRNA expression (Fig. 9e), and ChAT protein expression (Fig. 9f) in the lumbar spinal cord were confirmed at 120 days after birth. [Figure 9d] Halofuginone was administered to an ALS animal model, and microglial activity (Fig. 9a), astrocyte activity (Fig. 9b), IL-1β expression (Fig. 9c), the number of ChAT-positive motor neurons (Fig. 9d), ChAT mRNA expression (Fig. 9e), and ChAT protein expression (Fig. 9f) in the lumbar spinal cord were confirmed at 120 days after birth. [Figure 9e]Halofuginone was administered to an ALS animal model, and microglial activity (Fig. 9a), astrocyte activity (Fig. 9b), IL-1β expression (Fig. 9c), the number of ChAT-positive motor neurons (Fig. 9d), ChAT mRNA expression (Fig. 9e), and ChAT protein expression (Fig. 9f) in the lumbar spinal cord were confirmed at 120 days after birth. [Figure 9f] Halofuginone was administered to an ALS animal model, and microglial activity (Fig. 9a), astrocyte activity (Fig. 9b), IL-1β expression (Fig. 9c), the number of ChAT-positive motor neurons (Fig. 9d), ChAT mRNA expression (Fig. 9e), and ChAT protein expression (Fig. 9f) in the lumbar spinal cord were confirmed at 120 days after birth. [Figure 10a] Halofuginone was administered to an ALS animal model, and microglial cell activity (FIG. 10a) and the number of ChAT-positive motor neurons (FIG. 10b) were confirmed in the lumbar spinal cord on postnatal day 90. [Figure 10b] Halofuginone was administered to an ALS animal model, and microglial cell activity (FIG. 10a) and the number of ChAT-positive motor neurons (FIG. 10b) were confirmed in the lumbar spinal cord on postnatal day 90. [Figure 11a] Halofuginone was administered to an ALS animal model, and TGF-β, iNOS, CD86, arginase 1, IFN-α, IL-6, IL-1β, TNF-α, caspase-3, bcl2, and bax mRNA expression (Figure 11a), as well as cleaved caspase-3, bcl2, and bax protein expression (Figure 11a) were confirmed in the lumbar spinal cord at 120 days after birth. [Figure 11b] Halofuginone was administered to an ALS animal model, and TGF-β, iNOS, CD86, arginase 1, IFN-α, IL-6, IL-1β, TNF-α, caspase-3, bcl2, and bax mRNA expression (Figure 11a), as well as cleaved caspase-3, bcl2, and bax protein expression (Figure 11a) were confirmed in the lumbar spinal cord at 120 days after birth. Best Mode for Carrying Out the Invention

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

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

[0021] In the present invention, the term "administration" means providing a predetermined 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. Furthermore, the composition can be administered by any device that can transport the active substance to the target cells.

[0022] The present invention Halofuginone The present invention provides a pharmaceutical composition for preventing or treating neurodegenerative or motor neuron diseases, which contains (Halofuginone) or a pharmaceutically acceptable salt thereof as an active ingredient.

[0023] The present invention also provides Halofuginone or a pharmaceutically acceptable salt thereof to an individual.

[0024] The present invention also provides a method for preventing or treating neurodegenerative or motor neuron diseases. Halofuginone or a pharmaceutically acceptable salt thereof.

[0025] The present invention also provides a method for producing a drug for preventing or treating neurodegenerative or motor neuron diseases. Halofuginone or a pharmaceutically acceptable salt thereof.

[0026] In the present invention, Halofuginone or a pharmaceutically acceptable salt thereof is a TGF-β inhibitor and has the chemical structure set forth below in Formula 1: [Chemical formula 1] JPEG0007812929000001.jpg3083.

[0027] In addition, the above Halofuginone can be used without regard to whether it is commercially available or synthetic.

[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, dysregulation of the muscular system, spinal muscular atrophy, or inflammatory neurological disease.

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

[0030] In addition, in the above-mentioned neurodegenerative or motor neuron disease, Halofuginone can suppress the inflammatory response and neuronal death in the central nervous system caused by TGF-β stimulation, and can prevent or treat diseases. Halofuginone increases the expression of anti-inflammatory factors in the central nervous system, such as arginase 1, and promotes the expression of pro-inflammatory factors, such as iNOS, CD86, IFN-α, and TNF The expression of IL-1α, IL-1β, or IL-6 can be reduced to prevent or treat diseases. can prevent or treat diseases by increasing the expression of anti-neuron death factors, such as bcl-2, and decreasing the expression of neuron death factors, such as caspase-3 and bax, in the central nervous system.

[0031] In addition, in the above-mentioned neurodegenerative or motor neuron disease, Halofuginone can delay progression and improve functioning and survival.

[0032] In a specific embodiment of the present invention, the inventors have demonstrated that TGF-β1 stimulation induces fibrosis in myoblasts and reduces myogenesis, while Halofuginone It was confirmed that treatment with β-glucan suppressed the induction of fibrosis and the reduction of myogenesis induced by TGF-β1 stimulation.

[0033] In addition, the inventors isolated fibroblasts from an amyotrophic lateral sclerosis (ALS) animal model, Halofuginone Treatment with α-glucan suppressed fibrosis and the decrease in muscle transcription factors in fibroblasts.

[0034] In addition, the inventors have investigated the effects of ALS on animal models. Halofuginone As a result of administering this drug, it was confirmed that the onset of ALS was delayed and performance and survival time were improved. Halofuginone In an ALS animal model administered with , it was confirmed that fibrosis in the synovial cavity of the joints was reduced, skeletal muscle production was improved, and joint structure was improved. Halofuginone It was confirmed that administration of this compound resulted in anti-inflammatory and neuronal death inhibitory effects in the central nervous system (CNS) of ALS animal models.

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

[0036] The present invention Halofuginone includes all pharmaceutically acceptable salts, possible solvates, hydrates, racemates or stereoisomers that can be produced therefrom.

[0037] of the invention Halofuginonecan be used in the form of a pharmaceutically acceptable salt, and useful salts are acid addition salts formed with pharmaceutically acceptable free acids. Acid addition salts can be obtained 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, and aliphatic and aromatic sulfonic acids. Such pharmaceutically non-toxic salts include sorbate, pyrosorbate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, iodide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, pomate, isobutyrate, caprate, 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 of 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 using a water-miscible organic solvent such as methanol, ethanol, acetone, or acetonitrile. Alternatively, the mixture can be dried by evaporating the solvent and excess acid, or the precipitated salt can be filtered by suction.

[0039] Pharmaceutically acceptable metal salts can also be prepared using bases. Alkali metal or alkaline earth metal salts can be obtained, for example, by dissolving a compound in an excess amount of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the compound salt, and evaporating and drying the remaining solution. In this case, sodium, potassium, or calcium salts are suitable for certain metal salts. Corresponding silver salts can also be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

[0040] The present invention Halofuginone or a pharmaceutically acceptable salt thereof, in a therapeutically effective amount and a pharmaceutically 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 pharmaceutically 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 additionally contain stabilizers and preservatives. 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 pharmaceutically acceptable carriers may be found 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 oral and parenteral administration.

[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 injectable dosage form and administered by lightly pricking the skin with a 30-gauge thin needle, or by directly applying or adhering 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 routes as described above.

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

[0047] For parenteral administration, the formulations may be formulated in the form of injections, creams, lotions, topical ointments, oils, moisturizers, gels, patches, aerosols, and nasal inhalants according to methods known in the art, and are described in Remington's Pharmaceutical Sciences, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a formulary commonly known to pharmaceutical scientists.

[0048] The total dose of the pharmaceutical composition of the present invention can be administered to a patient in a single dose, more specifically, in multiple single doses administered 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 vary in the content of the active ingredient depending on the symptoms of the disease. Specifically, the total dose of the composition of the present invention may be about 0.01 μg to 1,000 mg per kg of patient body weight per day, 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 can be determined by a person skilled in the art, taking into account various factors such as the patient's age, body weight, health condition, sex, severity of the disease, diet, and excretion rate, as well as the route of administration and frequency of treatment. The pharmaceutical composition of the present invention is not particularly limited in its dosage form, route of administration, or method of administration, as long as it exhibits the effects of the present invention.

[0049] Furthermore, the pharmaceutical composition of the present invention can 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 can be administered simultaneously, separately, or sequentially. In this case, the other therapeutic agents may be substances 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 can be separately formulated in separate containers or can be combined in the same dosage form.

[0050] The present invention also provides Halofuginone or a pharmaceutically acceptable salt thereof as an active ingredient, and a functional health food composition for preventing or ameliorating neurodegenerative or motor neuron diseases is provided.

[0051] The present invention also provides a functional health food composition for preventing or improving neurodegenerative or motor neuron diseases. Halofuginone or a pharmaceutically acceptable salt thereof.

[0052] In addition, the present invention provides a functional health food composition for preventing or improving neurodegenerative or motor neuron diseases. Halofuginone or a pharmaceutically acceptable salt thereof.

[0053] In the present invention, Halofuginone The details regarding the composition of the present invention, or its pharmaceutically acceptable salts, and neurodegenerative or motor neuron diseases have been described above, so the above details will be used for specific explanations, and only the unique composition 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, improving joint structure by increasing skeletal muscle production, 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, halofuginone can be usefully used as an active ingredient in health functional food compositions for the prevention or amelioration of 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, but not limited to, powder, granules, rings, refined powder, capsules, candies, syrups and beverages.

[0056] The functional health food composition of the present invention is not particularly limited as long as it can be ingested to prevent or improve neurodegenerative or motor neuron diseases. When used as a food additive, the functional health food composition of the present invention can be added directly or in combination with other foods or food ingredients, and can be used appropriately in a conventional manner. The active ingredient can be appropriately used depending on its intended use (prevention or improvement). Generally, the functional health 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 functional health food composition of the present invention during the production of the food or beverage. However, for long-term intake for health regulation purposes, the amount is within the above range, and there is no safety issue, so the active ingredient can be used in an amount greater than the above range. There are no particular limitations on the type of food. Examples of foods to which the functional health food composition can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, drinking water, tea drinks, alcoholic beverages, and vitamin complexes, including all health foods in the conventional 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 contains ingredients commonly added during food production, such as proteins, carbohydrates, fats, nutrients, and seasonings. For example, when produced as a drinkable supplement, 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., maltos, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrin, cyclodextrin, etc.), or sugar alcohols (e.g., xylitol, sorbitol, erythritol, etc.). The flavorings may include natural flavorings (e.g., tau marthin, stevia extract, etc.) and 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, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, 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] The effect of TGF-β (Transforming growth factor-β) on myoblasts and Halofuginone To investigate the effect of halofuginone, mouse myoblasts were cultured and the cells were treated with TGF-β1 and Halofuginone was processed.

[0062] Specifically, mouse myoblast C2C12 cells (CRL-1772) were purchased 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 changed to serum-free medium and stimulated with 5 ng / ml rhTGF-β1 (recombinant human TGF-β1; R&D Systems). After stimulation, the cells were incubated for 24 hours in serum-free medium at various concentrations (0 [control] or 1, 2.5, 5 (low concentration), 10 (high concentration), 20, 50, 100 ng / ml). Halofuginone (halofuginone; Sigma) was used.

[0063] Example 2: Cell viability analysis

[0064] C2C12 cells were assayed for rhTGF-β1 or rhTGF-β1 in serum-free conditions using the Cell Counting Kit-8 (CCK-8 kit) assay (Enzo Life Sciences, ALX-850-039). Halofuginone Cell viability was measured after treatment.

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

[0068] Specifically, C2C12 cells were plated on a 6-well plate with a cover slip and stimulated with rhTGF-β1 in serum-free medium. After 24 hours of stimulation, the cells were treated with low or high concentrations of rhTGF-β1 in serum-free medium for 24 hours. Halofuginone Cells were washed three times with PBS and fixed with 4% paraformaldehyde for 15 minutes at room temperature. Subsequently, fixed cells were washed three times with PBS, treated with 0.5% Triton X-100 for 5 minutes, and then blocked with 5% antiserum albumin in 0.3% PBS-T for 1 hour at room temperature. Cells were then incubated with anti-α-SMA antibody (1:200; Abcam, cat#ab7817) or anti-MyoD antibody (1:200; Santa Cruz, cat#sc377460) in blocking buffer at 4°C for 24 hours. After washing three times with PBS, cells were incubated with Alexa Fluor secondary antibody for 1 hour at room temperature. Nuclear DNA was stained with DAPI (4,6-diamino-2-phenylindole, 1:1,000; Sigma) and washed twice with PBS. After that, 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 40x objective. Nuclei were quantified in a blinded fashion using Leica Application Suite X software (Leica Microsystems).

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

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

[0071] Specifically, all animal experiments were conducted in accordance with the guidelines of the Seoul National University Animal Hospital Animal Care and Use Committee (IACUC, approval number SNU-200220-1-2). Transgenic mice expressing the human G93A mutant SOD1 gene (B6SJL-Tg(SOD1-G93A)1Gur / J) as an ALS animal model were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Male transgenic ALS mice of the mtSOD1(G93A)H1 high-expressor strain (Jackson Laboratories, Bar Harbor, ME, USA) were mated with female B6 / SJLF1 mice. 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 listed below in Table 1, and the copy number of the transgene was confirmed. The G93A mutant SOD1 transgenic mice were then divided into three groups based on time point: 60 days (asymptomatic), 90 days (early symptomatic), and 120 days (late symptomatic). A total of 96 mice were used in the experiment. Non-transgenic mice served as a comparison group.

[0072] [Table 1] Example 5: Fibroblast culture from ALS animal model and Halofuginone process

[0073] In ALS Halofuginone To investigate the efficacy of the present invention, fibroblasts were isolated from the ALS animal model in which the G93A mutant SOD1 gene was identified in Example 4, and the cells were then treated with 100 mg of ... Halofuginone was processed.

[0074] Specifically, primary fibroblasts were isolated from skeletal muscle of 120-day-old non-transgenic or G93A mutant SOD1 transgenic mice (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 then cut from an ice block. The tissue was then cut into 1 mm slices and enzymatically reacted with 0.2% type IV collagenase (Sigma) at 37°C for 1 hour. The enzymatic reaction was terminated 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 0.2% gelatin (Sigma)-coated plates 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: ALS animal model Halofuginone Administration

[0076] In ALS Halofuginone In order to investigate the efficacy of the compound, the G93A mutant SOD1 gene was confirmed in the ALS animal model in Example 4. Halofuginone was administered.

[0077] Specifically, as shown in the schematic diagram of FIG. 1, the sex- and age-matched mice in Example 4 were randomly divided into three groups. As shown in Table 2 below, the DMSOTG group and the HalTG group were treated with a vehicle (DMSO, Duchefa Biochemie, D1370) and the G93A mutant SOD1 transgenic mice. Halofuginone(Sigma, S8144) injections were performed for 10 weeks or more at 10 weeks of age (early stage). As a comparison group, non-transgenic mice were intraperitoneally (ip) injected with vehicle (DMSO) in the same manner for the same period.

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

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

[0080] In ALS Halofuginone In order to examine the efficacy of Halofuginone After administration, behavioral experiments were performed in a single-blind manner.

[0081] Specifically, the clinical condition and weight of the mice in Example 6 were evaluated three times a week starting from postnatal day 83. The onset of the disease was defined as the trembling of the mouse's limbs when the tail was suspended in the air, which has been reported to be clinically related to 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 when the animal was unable to right itself within 30 seconds after being placed on its side on a flat surface. At this point, the mice were considered dead [C. Zheng, I. Nennesmo, B. Fadeel, J.I. Henter, Vascular endothelial growth factor prolongs survival in a transgenic mouse model of ALS, Ann Neurol, 56 (2004), pp. 564-567]. The motor function of mice was assessed using a rotaload device (JD-A-07M, JEUNG DO BIO & PLANT CO., LTD.). Mice were trained for one week before recording began. The mice were placed on a rotaload device that began at a speed of 4 rpm and accelerated to 40 rpm over 300 seconds, increasing by 1 rpm approximately every 8.3 seconds. The rotaload test was performed from day 83 of age, three times a week for an average of three measurements, until the mice could no longer remain on the rotaload device for more than 10 seconds three times in a row.

[0082] Example 8: Immunohistochemical analysis

[0083] In ALS Halofuginone In order to examine the efficacy of HalofuginoneAfter administration, immunohistochemical analysis was performed using lumbar spinal cords and knee joint tissues.

[0084] Specifically, the mice from Example 6 were perfused with 4% paraformaldehyde (PFA) on days 90 and 120 of age, and the lumbar spinal cord and knee joint were isolated. After fixation in PFA for 24 hours, the knee joints were 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 fresh solution every day until the decalcification process was complete, as indicated by easy penetration of the bone with a needle. The decalcified knee joints were then rinsed with running tap water for 24 hours. The spinal cord and knee joints were then 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 minutes, and then blocked with 5% antiserum albumin in 0.3% PBS-T for 1 hour 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 24 hours at 4°C. Sections were washed three times with PBS and then incubated with Alexa Fluor secondary antibody for 1 hour at room temperature in the dark.Stained and washed spinal cord sections were stained with DAPI (DAPI; 1:1,000; Sigma) for 10 minutes at room temperature, then rinsed twice with PBS. Coverslips were mounted with Dako fluorescent mounting medium (Dako) and images were 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. Images were analyzed using Leica Application Suite X software (Leica Microsystems).

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

[0086] In ALS Halofuginone In order to examine the efficacy of Halofuginone After administration, the lumbar spinal cord was isolated and the motor neurons were counted.

[0087] Specifically, mice were sacrificed at various time points (90 and 120 days) according to their clinical condition in "Example 6." Each mouse was transcardially infused with cold PBS followed by cold 4% paraformaldehyde (PFA) to isolate the lumbar spinal cord. Samples were post-fixed in 4% paraformaldehyde, treated with cold 30% glucose solution, cryoprotected, and serially harvested at 14 μm thickness. Tissue sections were washed with PBS and immersed in PBS containing 0.3% hydrogen peroxide (H2O2) for 15 minutes to remove resistant peroxidase activity. Sections were washed with PBS, permeabilized with 0.5% Triton X-100 for 5 minutes, and blocked with 5% serum albumin in 0.3% PBS-T for 1 hour at room temperature. 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. After washing with PBS, the sections were 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 incubated with diaminobenzidine (DAB, ImmPACT® DAB Vector Laboratories, cat#SK-4105) to visualize peroxidase staining. Sections were dehydrated, air-dried, and mounted with toluene-soluble Permount mounting medium (Fisher Scientific, cat#SP15-500). Motor neurons were counted in ChAT-stained sections using a computer-assisted microscope (Olympus BX53) and software (cellSens software) at 4x magnification. Counts were performed in the per ventral horn of the spinal cord for a total of three mice per group. The analyzed field 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] In ALS Halofuginone In order to examine the efficacy of Halofuginone After administration, the range of motion (ROM) of the knee joint was measured.

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

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

[0092] After stimulation with TGF-β1 in Example 1, Halofuginone The myoblasts treated with the above-mentioned Halofuginone or in an ALS animal model as in Example 6 above. Halofuginone After administration, real-time qRT-PCR analysis was performed using isolated lumbar spinal cords.

[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 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 using ReverTra Ace-α- TM 1 μg of total RNA was synthesized using SYBR green Excel Taq (Toyobo). TM Quantitative RT-PCR was performed using the primers listed in Table 3 below in a thermocycler CFX Connect Real-Time PCR Detection System (BIO-RAD) with 2X Fast Q-PCR Master Mix (TQ1200, Smobio). Fluorescence data were analyzed using Bio-Rad CFX Manager 3.1 software. (-ΔΔCT) Relative mRNA expression was calculated relative to the control group using the methods. All samples were run in quadruplicate. Primers were designed using Primer3 online software, and GAPDH was used as a control.

[0094] [Table 3]

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

[0096] After stimulation with TGF-β1 in Example 1, Halofuginone The myoblasts treated with the above-mentioned Halofuginone fibroblasts treated with α-glucan or an ALS animal model as in Example 6 Halofuginone After administration, Western blot analysis was performed using isolated lumbar spinal cords or knee joints.

[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, C2C12 cells in Example 1, or fibroblasts in Example 5 were lysed in RIPA buffer (Thermo, MA, USA) with protease inhibitors and phosphatase inhibitors (Roche, IN, USA) and incubated on ice for 30 minutes. The lysates were centrifuged at 4°C for 20 minutes at 13,000 rpm, and insoluble material was removed. Protein concentration was measured by BCA analysis (Pierce Biotechnology). Equal amounts of cell lysates were loaded onto an SDS-PAGE gel for electrophoresis and transferred to a nitrocellulose membrane (nitrocellulose membrane, Amersham Protran 0.2 μm NC, Amersham Pharmacia Biotech, Piscataway, NJ, USA). The membrane was then blocked with 5% (w / v) skim milk in 1X TBST for 1 hour 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), and anti-ChAT antibody (1:1000; Millipore, cat#AB144P) were used for the treatment of 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 at room temperature (RT) for 1 hour. The membranes were then washed and briefly incubated with SuperSignal West Pico Plus Chemiluminescent Substrate (Pierce Biotechnology) according to the manufacturer's protocol. The membranes were then quantified using an image analyzer (Amersham Pharmacia Biotech, Piscataway, NJ, USA). β-actin was used as an internal control. Protein intensity densitometry was quantified using Image J (National Institutes of Health).

[0098] Example 13: Statistical analysis

[0099] All data are presented as means and standard errors of the means (SEM) based on at least three independent experiments and expressed as percentages 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 of <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 effects of TGF-β1 on myoblasts, we performed real-time qRT-PCR and Western blot analysis on the C2C12 mouse myoblast cell line after TGF-β1 stimulation.

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

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

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

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

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

[0107] In myoblasts Halofuginone To examine the cytotoxicity of halofuginone, cell viability assay and Western blot analysis were performed.

[0108] Specifically, C2C12 cells were stimulated with or without TGF-β1 using the same method as described in Example 2 above. HalofuginoneThe cells were treated with PBS and subjected to CCK-8 assay to analyze cell viability (Figures 3a and 3b). Western blot analysis was performed as described in Example 12 to confirm the expression of p-Smad2, Smad2, TGF-β1, MyoD, collagen I protein, and α-SMA (Figure 3c).

[0109] As a result, as shown in Figures 3a and 3b, the concentration of 1 ng / ml to 10 ng / ml Halofuginone It was confirmed that the cell viability did not change significantly depending on the concentration (Fig. 3a and Fig. 3b).

[0110] Furthermore, as shown in Figure 3c, the concentrations of 5 ng / ml and 10 ng / ml Halofuginone At this concentration, 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 (Fig. 3c).

[0111] This resulted in the Halofuginone Based on the optimal concentration, the low concentration was determined to be 5 ng / ml and the high concentration was determined to be 10 ng / ml.

[0112] <Experimental Example 3> In TGF-β1 stimulated myoblasts Halofuginone Confirmed that fibrosis was promoted and myogenesis was suppressed by

[0113] TGF-β1-induced changes in myoblasts act as a TGF-β inhibitor Halofuginone To investigate whether TGF-β1 suppresses the expression of β-glucan in C2C12 mouse myoblast cell line, Halofuginone were processed and subjected to real-time qRT-PCR analysis, Western blot analysis, and immunocytochemistry analysis.

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

[0115] As shown in Figure 4a and Figure 4b, in the groups treated with low or high concentrations of halofuginone, the TGF-β1-stimulated increases in TGF-β1, α-SMA, and collagen I mRNA levels were significantly reduced, and the TGF-β1-stimulated decrease in MyoD mRNA levels was significantly restored (Figure 4a). Protein expression also showed similar patterns (Figure 4b).

[0116] Moreover, as shown in Figure 4c, using immunofluorescence staining and confocal microscopy, Halofuginone The intensity of α-SMA reduction and MyoD restoration by low- or high-concentration treatment was confirmed (Fig. 4c).

[0117] Based on the above results, it is possible to promote fibrosis and inhibit myogenesis by TGF-β1 stimulation in myoblasts using TGF-β inhibitors. Halofuginone It is known that it can be prevented by using

[0118] <Experimental Example 4> Halofuginone inhibits fibrosis in fibroblasts derived from an ALS animal model

[0119] Changes in TGF-β1 and TGF-β1 in fibroblasts derived from ALS animal models Halofuginone To investigate the effect of Halofuginone After processing, real-time qRT-PCR analysis and Western blot analysis were performed.

[0120] Specifically, fibroblasts were isolated from the ALS animal model using the same method as described in Example 5 above, Halofuginone After treatment, real-time qRT-PCR was performed using the same method as described in Example 11 to confirm the expression of TGF-β1, α-SMA, and collagen I (Col-I) mRNA (Figure 5a). Western blot analysis was performed using the same method as described in Example 12 to confirm the expression of p-Smad2, Smad2, TGF-β1, α-SMA, and collagen I protein (Figure 5b). Fibroblasts isolated from non-transgenic mice were used as a control.

[0121] As shown in Figure 5a and Figure 5b, the ratio of p-Smad2 / Smad2, and the protein expression as well as 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-transgenic mice (Non-TG). These changes were observed regardless of whether the treatment was at low or high concentrations for 24 hours. Halofuginone It was confirmed that the effect was attenuated by treatment (Fig. 5a and Fig. 5b).

[0122] These results demonstrate that fibrosis is enhanced in fibroblasts from ALS mouse models and that halofuginone treatment can suppress fibrosis.

[0123] <Experimental Example 5> ALS animal model Halofuginone Administration confirmed delay in onset of ALS and improvement in performance

[0124] In vivo Halofuginone To evaluate the effects of Halofuginone and disease progression, survival, and motor function analyses were performed.

[0125] Specifically, the same method as described in Example 6 was used to generate an ALS animal model. Halofuginone The disease progression, survival, and motor function analyses were performed in the same manner as described in Example 7 above.

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

[0127] As shown in Figures 6b and 6c, there was a difference in the onset of symptoms between the DMSO TG and Hal TG groups, with the onset of symptoms being significantly delayed in the Hal TG group compared with the DMSO TG group (mean ± standard deviation [SD], 113 ± 3.6 vs. 94.2 ± 5.7, p < 0.5, respectively). Furthermore, the age at which rotarod failure was achieved was delayed in the Hal TG group compared with 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, Figures 6b and 6c).

[0128] <Experimental Example 6> Early stage of disease in ALS animal model Halofuginone Administration of this drug reduced fibrosis in the synovial cavity and improved skeletal muscle production.

[0129] In ALS pathogenesis Halofuginone To evaluate the efficacy of this drug, we conducted an ALS animal model Halofuginone The changes in the synovial cavity of the knee joint were confirmed by immunohistochemical analysis (IHC) in the early and late stages of the disease.

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

[0131] As shown in Figures 7a and 7b, the DMSO TG group showed a significant increase in TGF-β1 in the synovial cavity of knee joints at the early stage of disease compared with the DMSO non-TG group, regardless of gender. Furthermore, compared with the DMSO non-TG group, the DMSO TG group showed an increase in TGF-β1 in the synovial cavity, along with an increase in α-SMA and Col-I, and a decrease in MyoD. Meanwhile, in the HalTG group, the increase in TGF-β was suppressed at the early stage of disease, and the expression of α-SMA and Col-I was also significantly lower than in the DMSO TG group (Figures 7a and 7b).

[0132] Furthermore, as shown in Figures 7c and 7d, the Hal TG group maintained significantly lower TGF-β1, α-SMA, and Col-I levels and higher MyoD levels in the synovial cavity compared with the DMSO TG group, even in the later stages of the disease. Furthermore, the levels of TGF-β1, MyoD, and Col-I in the female Hal TG group were similar to those in the DMSO non-TG group, whereas the MyoD level in the male Hal TG group was slightly increased compared with the DMSO non-TG group (Figures 7c and 7d).

[0133] These results suggest that increased TGF-β activates myofibroblasts and enhances fibrosis in the synovial cavity of ALS mice. Halofuginone The results also show that administration of ATP can improve skeletal muscle myogenesis, which is reduced in the synovial cavity of ALS mice. Halofuginone Furthermore, the administration of α-glucan can prevent the disease in the early stages of ALS in mice. Halofuginone It can be seen that administration of significantly reduces fibrosis in the synovial cavity and improves muscle formation in both sexes.

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

[0135] In ALS, functional resistance due to fibrosis and its response Halofuginone To investigate the effects of HalofuginoneAfter administration, the range of motion (ROM) of the knee joint was measured and Western blot analysis was performed.

[0136] Specifically, the same method as described in Example 6 was used to induce the G93A mutant SOD1 mice. Halofuginone The ROM of the knee joint was measured using the same method as described in Example 10 (Fig. 8a). Western blot analysis was also performed using knee joint tissue using the same method as described in Example 12 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 Figure 8a, it was confirmed that the ROM was significantly reduced in the DMS OTG group compared with the DMSONon-TG group on day 120, while the ROM remained unchanged in the Hal TG group (Figure 8a).

[0138] Furthermore, as shown in Figure 8b, the p-Smad2 / Smad2 ratio, TGF-β, α-SMA, and Col-I expression levels were significantly increased and MyoD was decreased in the DMSO TG group compared with the DMSO non-TG group. Furthermore, 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 Col-I expression levels were actually decreased (Figure 8b).

[0139] The above results suggest that increased TGF-β in ALS mice enhances fibrosis in the knee joints and induces joint contraction. Halofuginone It is clear that this can be improved by administration.

[0140] Experimental Example 8: In the central nervous system (CNS) tissue of an ALS animal model Halofuginone Confirmation of anti-inflammatory and neuronal cell death suppression effects by administration

[0141] In ALS Halofuginone To investigate the effects of administration within the central nervous system (CNS), we used the drug in an ALS animal model. HalofuginoneAfter administration, immunohistochemical analysis, motor neuron count in the lumbar spinal cord, and Western blot analysis were performed using the lumbar spinal cord.

[0142] Specifically, the same method as described in Example 6 was used to induce the G93A mutant SOD1 mice. Halofuginone After administering the drug to the mice, the mice were sacrificed at 120 days of age. Immunohistochemical analysis was performed to confirm TGF-β1 and glial cells in the lumbar spinal cord using the same method as described in Example 8. Given that astrocyte-derived TGF-β has been reported to accelerate disease in ALS mice, the spinal cords of each group were co-stained for TGF-β and GFAP during immunohistochemical analysis. IBA1 staining was also performed to confirm microglial activity (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, the same method as described in Example 6 was used to generate G93A mutant SOD1 mice. Halofuginone After 90 days of age, the mice were sacrificed and then stained with Iba1 to confirm the activity of microglial cells (Fig. 10a) and observed and counted the number of motor neurons (Fig. 10b) using the same method as above.

[0146] As shown in Figures 9a and 9b, GFAP intensity was significantly increased in the DMSO TG group of 120-day-old mice compared with the DMSO non-TG group, and TGF-β1 was also increased at the same site. This increase in astrocyte activity and TGF-β1 was significantly reduced in the Hal TG group (Figure 9b). Meanwhile, microglial cells were significantly increased in the DMSO TG group, and a sustained increase in microglial cells was also confirmed in the Hal TG group (Figure 9a).

[0147] Furthermore, as shown in Figure 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 (Figure 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 in the DMSO non-TG group, whereas in the Hal TG group, they were preserved to the same extent as 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 suggest that increased TGF-β in the CNS of ALS mice is associated with increased inflammatory responses and the associated loss of motor neurons. Halofuginone It can be seen that the effect is suppressed by administration.

[0151] <Experimental Example 9> In ALS animal models Halofuginone Administration of this drug was confirmed to suppress chronic inflammation and neuronal death in the CNS.

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

[0153] Specifically, the same method as described in Example 6 was used to induce the G93A mutant SOD1 mice. Halofuginone Mice were then administered 120 days after birth, sacrificed, and their lumbar spinal cords were isolated. The mRNA expression of CNS inflammation-related factors and neuronal death-related factors was then confirmed by real-time qRT-PCR analysis using the same method as described in Example 11 (Figure 11a). The protein expression of neuronal death-related factors was confirmed by Western blot analysis using the same method as described in Example 12 (Figure 11b). Because the roles (inflammatory or anti-inflammatory effects) of microglial cells differ depending on whether they are M1 or M2 subtypes, the expression of CNS inflammation-related factors was confirmed, including M1 markers (iNOS, CD86), M2 markers (arginase 1), and neuronal death factors (IFN-α, TNF-α, IL-1b, IL-6). The expression of neuronal death-related factors, caspase-3, bax, and bcl-2, was also confirmed.

[0154] As a result, as shown in Figure 11a, the mRNA levels of M1 markers and infectious factors significantly increased in response to the mRNA level of TGF-β in the DMSOTG group, which was Halofuginone Furthermore, the mRNA level of bcl-2, an anti-neuronal cell death factor, was reduced in the DMSO-TG group compared to the DMSO-On-TG group, but was preserved in the Hal-TG group (Fig. 11a).

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

[0156] From the above results, HalofuginoneIt was found that this compound can block persistently elevated TGF-β in ALS mice, exert anti-inflammatory effects in the CNS, and suppress neuronal death.

[0157] Based on the results of the above <Experimental Example 1> to <Experimental Example 9> Halofuginone It was confirmed that the compound exhibits dual therapeutic effects in ALS, improving joint construction by increasing TGF-β and suppressing chronic inflammation and neuronal death in the CNS. Halofuginone can be used to prevent or treat neurodegenerative or motor neuron diseases caused by increased TGF-β, including ALS. [Industrial Applicability]

[0158] In this invention, it was confirmed that halofuginone inhibits fibrosis, improves skeletal muscle production, improves joint structure, and exhibits dual effects of inhibiting inflammatory responses and neuronal death in the central nervous system in cell and animal models of amyotrophic lateral sclerosis (ALS), thereby delaying the progression of ALS symptoms and improving functional ability and survival time. Halofuginone or a pharmaceutically acceptable salt thereof can be usefully utilized 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 pharmaceutically acceptable salt thereof as an active ingredient, A pharmaceutical composition for preventing or treating neurodegenerative or motor neuron diseases, wherein the neurodegenerative or motor neuron disease is amyotrophic lateral sclerosis.

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

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

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

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

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

7. A health functional food composition for preventing or improving neurodegenerative or motor neuron diseases, comprising halofuginone or a pharmaceutically acceptable salt thereof as an active ingredient, The health functional food composition for preventing or improving neurodegenerative or motor neuron disease, wherein the neurodegenerative or motor neuron disease is amyotrophic lateral sclerosis.

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