KR20180125242A - Pharmaceutical composition for preventing or treating muscle weakness diseases comprising Nitrendipine - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for promoting differentiation of myoflagellates, pharmaceutical compositions for preventing or treating myasthenia-related diseases, a food composition for preventing or ameliorating muscle weakness-related diseases, a composition for strengthening muscle strength, a feed or feed additive for strengthening muscle strength, and a composition for reducing wrinkles by weakening muscle strength, comprising nitrendipine or pharmaceutically acceptable salts thereof. In addition, the present invention relates to a method for promoting differentiation of myoflagellates using the nitrendipine or pharmaceutically acceptable salts thereof, to a method for producing the myoflagellates and to a method for treating the myasthenia-related diseases.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing or treating muscular weakening-related diseases, including Nitrendipine or a pharmaceutically acceptable salt thereof,

The present invention relates to a composition for promoting differentiation of a source cell comprising Nitrendipine or a pharmaceutically acceptable salt thereof, a pharmaceutical composition for preventing or treating a disease associated with weakness in muscle strength, a composition for preventing or ameliorating muscle weakness-related diseases A food composition, a composition for strengthening muscle strength, a feedstuff for enhancing muscle strength or a feed additive, and a composition for improving wrinkles by weakening muscle strength.

The present invention also relates to a method for promoting differentiation of myoblast cells using the nontrendipine or a pharmaceutically acceptable salt thereof, a method for producing differentiated myocytes, and a method for treating a disease associated with weakness in muscle strength.

Diseases that cause weakness of muscle strength include sarcopenia with aging, muscle atrophy caused by imbalance of protein metabolism or muscle use, starvation, wasting disease (such as cancer), aging And acardiotrophy.

Sarcopenia refers to a decrease in muscle strength with a decrease in skeletal muscle mass during aging. In muscular dystrophy, not only the decrease in muscle mass, which is the greatest characteristic, but also the kind of muscle fiber is observed. Type 1 and Type 2 decrease in age at the same rate, whereas Type 2 muscle fibers do not change much in the presence of myopenia, but Type 1 muscle fiber thickness decreases significantly. This myopenic disorder has been reported to cause senescence and dysfunctions among the elderly (Roubenoff R., Can. J. Appl . Physiol . 26, 78-89, 2001).

Although myopenia is caused by various factors, research on each factor is still insignificant. The balance of growth hormone reduction or neurological change, changes in physical activity, changes in metabolism, sex hormone levels or fat or catabolic cytokines, and the synthesis and differentiation of proteins (Roubenoff R. and Hughes VA, J. Gerontol . A. Biol . Sci. Med . Sci . 55, M716-M724, 2000). One of the most important features of myopenia is the decrease in satellite cell activation. Satellite cells are small mononuclear cells located between the basement membrane and the sarcolemma of the myofiber. They are activated by stimulation, such as injury or exercise, and proliferate into myoblasts. When differentiation proceeds, they fuse with other cells to form multinuclear muscle fibers. Thus, as the activity of satellite cells decreases, the ability to regenerate damaged muscle or response to differentiation signals is reduced, resulting in decreased muscle formation.

Muscle atrophy is caused by lack of nutrient deficiency or long-term muscle, which is caused by the breakdown of the normal balance of protein synthesis and degradation.

On the other hand, acardiotrophy is caused by starvation, wasting disease (such as cancer), or senility. The myocardial fibers are thin and thin, and the nucleus is concentrated and floating. Therefore, the muscle fascicle is also reduced in volume and the entire heart is smaller, the adipocyte adipose tissue is markedly decreased, and the coronary arteries are curved. A consumable pigment (lipofuscin) appears as a brown pigment on both sides of the core of the myocardial fiber and the whole heart is brownish with decreasing adipose tissue.

Three methods have been known to treat muscle weakness related diseases such as myopenia. The first is exercise. Exercise has been reported to increase skeletal muscle protein synthesis in the short term and to increase muscular strength and motility of the elderly. However, it is inappropriate for long-term treatment (Timothy J. Doherty, J. Appl . Physiol . 95, 1717-1727, 2003). The second is the use of testosterone or anabolic steroid as a medication, but it induces menstruation in women and side effects such as prostate symptoms in men. Other approved regimens include DHEA (dehydroepiandrosterone) and growth hormone, which have been reported to be therapeutically feasible at sites that include SARMs (DD Thompson, J. Musculoskelet Neuronal Interact 7, 344-345 , 2007). Diet therapy is also known as a treatment, but nutritional assessment shows malnutrition and modern eating habits are inadequate to maintain a reasonable total body mass.

Recently, stem cell therapy (stem cell therapy) which separates satellite cells and introduces them into the body after in vitro differentiation and activates satellite cells directly in the body and promotes myogenesis, (Shihuan Kuang and Michael A. Rudnicki, Trends in Molecular Medicine 14, 82-31, 2008). Therefore, in order to treat muscular weakness related to muscle weakness, it is necessary to develop a material that can promote the differentiation of myofibers.

Accordingly, the present inventors have made intensive efforts to develop a therapeutic agent for muscle weakness-related diseases of muscles, which increases muscle mass by promoting differentiation of myoblasts and effectively restores muscle function. As a result, it has been found that nitrendipine or a pharmaceutically acceptable salt thereof Can be used for the prophylaxis or treatment of muscular weakening-related diseases by promoting the differentiation of the source cells.

It is an object of the present invention to provide a composition for promoting the differentiation of myoblasts.

Another object of the present invention is to provide a method of promoting differentiation of myoblast cells.

It is yet another object of the present invention to provide a method for producing differentiated myocytes.

It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating muscle weakness-related diseases.

It is still another object of the present invention to provide a food composition for preventing or ameliorating muscular weakness-related diseases.

It is still another object of the present invention to provide a composition for strengthening muscular strength.

Yet another object of the present invention is to provide a feedstuff for enhancing strength or a feed additive.

It is still another object of the present invention to provide a composition for improving wrinkles by weakening muscle strength.

This will be described in detail as follows. On the other hand, each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention fall within the scope of the present invention. Further, the scope of the present invention is not limited by the detailed description described below.

In order to accomplish the above object, one embodiment of the present invention relates to a composition for promoting differentiation of myoblast comprising nitrendipine or a pharmaceutically acceptable salt thereof.

In the present invention, " nitrenedipine " means a compound having the name of IUPAC (RS) -3-Ethyl 5-methyl 2,6-dimethyl-4- (m-nitrophenyl) -dihydropyridine-3,5-dicarboxylate, and its derivatives are also included in the scope of the present invention as long as they have the effect of promoting the differentiation of the source cells. For example, the nitrenedipine used in the examples of the present invention is not limited to the compound having the formula C ₁₈ H ₂₀ N ₂ O ₆ and the molecular weight of 360.36. In general, the nystrendipine is a dihydropyridine calcium channel blocker, which is used for the treatment of hypertension. However, there is no known connection with myocardial differentiation. The inventors of the present invention have completed the present invention by first confirming that there is use of nystrendipine or a pharmaceutically acceptable salt thereof for source cell differentiation, and the structure of nystrendipine is as shown in the following chemical formula 1.

[Chemical Formula 1]

The term " pharmaceutically acceptable salt " as used herein means a formulation of a compound that does not cause serious irritation to the organism to which the compound is administered and does not impair the biological activity and properties of the compound. The pharmaceutical salts may be formed with acids which form non-toxic acid addition salts containing a pharmaceutically acceptable anion such as inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid and the like, tartaric acid, formic acid, Organic carboxylic acids such as acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid and salicinic acid, and organic carboxylic acids such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p- Sulfonic acid, sulfonic acid, sulfonic acid, sulfonic acid, sulfonic acid, sulfonic acid, sulfonic acid, For example, pharmaceutically acceptable carboxylic acid salts include metal salts or alkaline earth metal salts formed with lithium, sodium, potassium, calcium, magnesium and the like, amino acid salts such as lysine, arginine and guanidine, dicyclohexylamine, N Organic salts such as methyl-D-glucamine, tris (hydroxymethyl) methylamine, diethanolamine, choline and triethylamine, and the like.

As used herein, the term " myoblast differentiation " refers to a process in which a monocyte myoblast forms a polynuclear myotube through fusion. At the end of the differentiation, the myosin heavy chain (MyHC, Myosin Heavy Chain). Regarding myoblast differentiation, myocyte precursor cells are known to exhibit Pax7 ⁺ / MyoD ⁺ when proliferating, and Pax7 ⁺ markers in self-renewal. Further, the cells in the differentiation stage for forming the root canal can be distinguished, for example, by using the Pax7 ^- MyoD ⁺ MyoG ⁺ marker. Cells at the early stage of differentiation to form the canal may have increased expression of myogenic transcription factors such as MyoD (D), and increased myosin G (MyoG) in the middle stage.

The composition may be a serum-containing DMEM differentiation medium, but may be any medium or composition capable of promoting differentiation of a source cell. In this composition, the nontranedipine or a pharmaceutically acceptable salt thereof may be contained at a concentration of, for example, 0.01 μM to 2.0 μM, and may be contained at a concentration of 0.05 μM to 1.5 μM, or 0.1 μM to 1.0 μM Concentration, but is not limited thereto. In addition, the composition may further include additional substances necessary for cell culture or differentiation.

In a specific example of the present invention, when cytotoxicity was measured after treatment of nitrendipine with DME at concentrations of 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1 and 2 μM, In the case of treating the trending pin at 2.0 μM, it was confirmed that the toxicity of myocardial differentiation was very small within the range of 5% or less of the death cell ratio compared to the treatment with 1.0 mM H ₂ O ₂ (FIG. 3) Fins can promote differentiation with little toxicity to cells.

Another embodiment of the present invention is directed to a method of promoting differentiation of a source cell, comprising the step of treating the source cell with a nitrendipine or a pharmaceutically acceptable salt thereof. Nitrenedipine or a pharmaceutically acceptable salt thereof is as described above. In particular, the method of promoting differentiation of myofilaments can promote the differentiation by treating nitrendipine or a pharmaceutically acceptable salt thereof in vitro or in vivo.

Another embodiment of the present invention provides a method of producing differentiated myocytes comprising the step of differentiating the source cells by treating the source cells with nitrendipine or a pharmaceutically acceptable salt thereof.

Nitrenedipine or a pharmaceutically acceptable salt thereof is as described above. The production method of the present invention may be characterized by producing the differentiated stem cells comprising the step of treating the extracellular or intracorporeal stem cells with nitrendipine or a pharmaceutically acceptable salt thereof to differentiate the stem cells.

In a specific example of the present invention, the nystrendipine was treated with 0.2 μM of the source cells and the degree of differentiation of the source cells was observed by phase contrast microscopy. As a result, (Fig. 4). It was also confirmed that the effect of stimulating myocyte differentiation was very high through immunocytochemistry and Western blotting (Figs. 5 and 6).

Thus, the present invention is capable of forming root canals and differentiating source cells expressing the MYH3 protein in vitro or in vivo.

Another embodiment of the present invention relates to a pharmaceutical composition for the prevention or treatment of muscle weakness-related diseases comprising nitrendipine or a pharmaceutically acceptable salt thereof. The concentration of the nystrendipine or a pharmaceutically acceptable salt thereof is as described above.

As used herein, the term " muscle weakness " means a state in which the force of one or more muscles is reduced. The muscle weakness may be limited to one muscle, one side, upper or lower side of the body, or may appear throughout the body. In addition, subjective muscle weakness symptoms including myopia and myalgia can be quantified in an objective way through physical examination.

In the present invention, the term "muscle weakness-related disease" refers to all diseases that may occur due to weakness of muscle strength, specifically, a decrease in the number of myocytes in the body or a decrease in the activity of satellite cells, Which can be prevented, improved, or treated. In the present invention, muscle weakness diseases include, but are not limited to, for example, muscular dystrophy, muscular dystrophy, muscle dystrophy, or ataxia.

Accordingly, the composition of the present invention can be used for the prevention or treatment of myopenia, muscular dystrophy, or dystrophy through promotion of differentiation of myoblast cells.

Specifically, the myopenia of the present invention means a gradual decrease in the skeletal muscle amount due to aging, which directly leads to a decrease in muscle strength, resulting in a decrease in various bodily functions and a disorder.

In addition, muscular dystrophy of the limbs is progressively diminished symmetrically, resulting in the progressive denaturation of motor nerve fibers and cells in the spinal cord, leading to amyotrophic lateral sclerosis (ALS) and spinal atrophy progressive muscular atrophy, SPMA).

Muscle dystrophy is a disease in which progressive muscle atrophy and muscle weakness develops, which means degenerative myopathy characterized by necrosis of muscle fibers pathologically. Muscle fiber necrosis and degeneration are caused by damage of muscle cell membrane, resulting in muscle weakness and atrophy.

Myocardial dysfunction of the present invention is a condition in which the heart is contracted by an external or internal factor and causes heartburn brown atrophy which causes the myocardial fiber to dry and taper when it is starved, have.

As used herein, the term " prevention " means any action that inhibits or delays the onset of a muscle weakening related disorder by administration of the composition.

The term " treatment " as used in the present invention means all the actions of improving or alleviating symptoms caused by muscle weakness related diseases by the administration of the composition.

The pharmaceutical composition of the present invention may contain, for administration, a pharmaceutically acceptable carrier, excipient or diluent in addition to the above-mentioned nontrendipine or a pharmaceutically acceptable salt thereof. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical compositions of the present invention may be formulated into pharmaceutical formulations using methods well known in the art so as to provide rapid, sustained or delayed release of nontrendipine or a pharmaceutically acceptable salt thereof. In the preparation of the formulations, it is preferred that the active ingredient is mixed with or diluted with the carrier, or enclosed in a carrier in the form of a container.

In addition, the pharmaceutical composition of the present invention can be applied to any formulation, and for example, it can be manufactured for parenteral use. As the parenteral formulation, it may be of the spray type such as injection, application, aerosol, etc.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like.

In order to formulate into a spray-type formulation, the nitrilefin or pharmaceutically acceptable salt thereof may be formulated as a solution or suspension by mixing in water with a stabilizing agent or buffer to prepare a unit dose of ampoule or vial.

The pharmaceutical composition comprising the nystrendipine of the present invention or a pharmaceutically acceptable salt thereof can be directly injected into a site where a muscle weakening-related disease has developed or a site where a potentially susceptible individual is required to be strengthened, After producing differentiated myocytes by applying to the myoblast cells of the body, the differentiated myocytes can be injected into a site where a muscle weakness-related disease has occurred or a site where a potentially susceptible individual needs to be strengthened.

In addition, as long as nontrendipine or a pharmaceutically acceptable salt thereof does not interfere with the prevention or treatment of muscular weakening-related diseases, the composition may contain additional components, for example, substances known as therapeutic agents for weakness related to muscle weakness .

Specifically, the pharmaceutical composition of the present invention may be characterized by promoting differentiation of myoblast. In one embodiment of the present invention, the nystrendipine was treated at a concentration of 0.2 μM to the source cells, and the degree of differentiation of the source cells was observed by phase contrast microscopy. As a result, (FIG. 4), and it was confirmed that the effect of stimulating myocyte differentiation was very high even through immunocytochemistry and Western blotting (FIGS. 5 and 6). From these results, it was found that nystrendipine or a pharmaceutically acceptable salt thereof is effective in promoting differentiation of myoflaginal cells and may be useful in the prevention and treatment of muscular weakening-related diseases.

Another embodiment of the present invention relates to a food composition for preventing or ameliorating muscle weakness-related diseases comprising nitrendipine or a pharmaceutically acceptable salt thereof. The composition of the present invention can be used simultaneously or separately with a medicament for the treatment of a disease before or after the onset of a muscle weakening-related disease in order to prevent or ameliorate a muscle weakening-related disease. Neotrendipine or its pharmaceutically acceptable salts and weakness-related diseases are as described above.

Specifically, the food composition is characterized by promoting the differentiation of the source cells.

The term " improvement " as used in the present invention means all actions that at least reduce the degree of symptom associated with the condition being treated.

In addition, when the food composition of the present invention is used as a food additive, the composition may be added as it is, or may be used together with other food or food ingredients, and suitably used according to a conventional method. Generally, the composition of the present invention may be added in an amount of, for example, 15% by weight or less, or 10% by weight or less, based on the raw material in the production of food or beverage. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, it may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. Examples of the food to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharin and aspartame, and the like . The ratio of the natural carbohydrate can be appropriately determined by a person skilled in the art.

In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, A carbonating agent used in a carbonated beverage, and the like. In addition, the composition of the present invention may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The ratios of these additives can also be appropriately selected by those skilled in the art.

Another embodiment of the present invention is directed to a composition for enhancing muscle strength, comprising a nitrilefin or a pharmaceutically acceptable salt thereof.

Still another embodiment of the present invention relates to a composition for strengthening muscle strength, comprising naltrendipine or a pharmaceutically acceptable salt thereof.

The term " muscle strengthening " as used herein refers to strengthening body performance, strengthening maximum endurance, increasing muscle mass, strengthening muscle recovery, reducing muscle fatigue, improving energy balance, or a combination thereof.

The composition for muscle strengthening comprising the nystrendipine of the present invention or a pharmaceutically or pharmacologically acceptable salt thereof can increase the total muscle mass by increasing the muscle mass through the ability to differentiate the source cells into muscle cells, Is strengthened, thereby enhancing body performance and reducing muscle fatigue. In addition, muscle cells can be quickly replaced because muscle cells can be replaced quickly.

The composition for muscle strengthening of the present invention may contain, for administration, a pharmaceutically acceptable carrier, excipient or diluent in addition to the nontrendipine or a pharmaceutically or pharmacologically acceptable salt thereof. The pharmaceutically acceptable carrier, excipient or diluent is as described above.

In addition, the composition for improving muscle strength of the present invention can be manufactured in the form of a food composition or a food additive, and in particular, can be manufactured in the form of a health food composition. The food composition is as described above. Therefore, the composition for muscle strengthening of the present invention can be used not only as a muscle for aging, but also as an adjuvant for general muscle building and muscle strengthening.

Another embodiment of the present invention relates to a muscle strengthening feed or feed additive comprising a nitrilefin or a pharmaceutically acceptable salt thereof.

In the present invention, the term " feed " means a substance supplying organic or inorganic nutrients necessary for maintaining animal life. The feed includes nutrients such as energy, protein, lipid, vitamins, and minerals required by animals such as livestock, and includes nutrients such as cereals, muscle roots, food processing busines logistics, algae, fibrous materials, But are not limited to, animal feed such as vegetable diets or proteins such as cereal and busan logistics, mineral oils, fats, mineral oils, oils, and single cell proteins.

In the present invention, the term " feed additive " means a substance added to the feed to improve the productivity of an animal or health, and includes, but not limited to, amino acids, vitamins, enzymes, flavors A silicate, a buffer, an extractant, an oligosaccharide, and the like.

The content of nystrendipine or a pharmaceutically acceptable salt thereof contained in the feed or feed additive of the present invention is not particularly limited but may be, for example, 0.001 to 1% (w / w), 0.005 to 0.9% (w / w / w), or 0.01 to 0.5% (w / w).

Another embodiment of the present invention relates to a method for treating muscular weakness related to muscular weakness, comprising administering a nontrendipine or a pharmaceutically acceptable salt thereof to a subject in need thereof.

The weakness-related diseases are as described above.

The term " individual " as used herein refers to all animals, including humans, who have already developed or are capable of developing a muscle weakness-related disorder, and by administering to a subject a composition comprising nystrendipine or a pharmaceutically acceptable salt thereof, The disease can be effectively prevented and treated. The term refers to whole mammals including dogs, cows, horses, rabbits, mice, rats, chickens or humans, but the mammal of the present invention is not limited by these examples. The subject may be an individual other than a human, but is not limited thereto.

The optimal amount and dosage interval of the individual doses of the compositions of the present invention will be determined by the nature and extent of the disease being treated, the dosage form, route and site of administration, and the age and health status of the particular patient being treated, It will be apparent to one of ordinary skill in the art that the appropriate dosage will be determined. Such dosing can be repeated as often as is appropriate. If side effects occur, dosage and frequency can be altered or decreased depending on the usual clinical practice.

The route of administration of the composition may be administered via any conventional route so long as it can reach the target tissue. The composition of the present invention may be administered intraperitoneally, intravenously, subcutaneously, intradermally, or orally, but is not limited thereto. The composition may also be administered by any device capable of transferring the active agent to the target cell.

Another embodiment of the present invention relates to a composition for improving wrinkles by weakening muscle strength, comprising nystrendipine or a pharmaceutically acceptable salt thereof.

Another embodiment of the present invention relates to a method for improving wrinkles by weakening muscle strength, comprising the step of administering or applying to a subject a composition comprising nystrendipine or a pharmaceutically acceptable salt thereof.

In the present invention, " muscle weakness " is as described above.

In the present invention, " wrinkle " means a phenomenon in which skin elasticity is reduced due to skin aging, muscle loss, or the like, and the skin is bent or folded. The bending of the skin forming the wrinkles is affected by the movement of the muscles. Specifically, in the face, the bending down to both sides of the mouth centered on the eyes and nose is caused by the repetitive expression by the movement of the facial muscles In addition, when facial muscles are not used well or the muscle mass or muscle strength decreases due to aging, the skin is worn down by gravity and wrinkles occur.

The wrinkle-improving composition comprising nystrendipine or a pharmaceutically acceptable salt thereof according to the present invention promotes differentiation of muscle cells, thereby inducing cell differentiation, muscle strength and elasticity recovery of facial muscles, The skin wrinkles can be improved.

Specifically, the composition may be a pharmaceutical composition, a health functional food composition, or a cosmetic composition, and the pharmaceutical composition and the health functional food composition are as described above.

The cosmetic composition for improving skin wrinkles according to the present invention can be used as a cream, a lotion, an essence, a gel, an ointment, a foam, a lotion, a pack, a flexible water, a milky lotion, a foundation, a makeup base, a soap, Oil, and the like.

The cosmetic composition for improving skin wrinkles according to the present invention further comprises at least one additive selected from the group consisting of water, a surfactant, a moisturizer, a 16-4 lower alcohol, a chelating agent, a bactericide, an antioxidant, an antiseptic, can do.

The nystrendipine or a pharmaceutically acceptable salt thereof according to the present invention can promote root differentiation and form a root canal, thereby preventing muscle weakness and effectively improving muscle function. Accordingly, the pharmaceutical composition containing the same can be usefully used for the prevention or treatment of muscular weakening-related diseases.

FIG. 1 is a graph showing in-cell ELISA analysis that a change in the myosin heavy chain 3 (MYH3) protein was higher than that of a negative control DMSO when treated with a nitrile pin.
FIG. 2 is a graph showing the effect of stimulating the differentiation of myotenic fibroblasts by concentrations of 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1 and 2 μM, respectively, on primary myocytes.
Fig. 3 shows the result of measuring the toxicity according to the myocardial differentiation in the differentiation medium (DM) of the nystrendipine.
FIG. 4 shows the result of discriminating the differentiation of the root cell line C2C12 treated with the nitrile pin by a phase contrast microscope.
FIG. 5 shows the results of immunocytochemistry for differentiation of the nystrendipine-treated root cell line C2C12.
FIG. 6 shows the results of western blot analysis of the expression of myosin heavy chain 3 (MYH3) in the nystrelin-treated root cell line C2C12.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are intended to illustrate the present invention, and the scope of the present invention is not limited to these examples.

Example  1: Culture of myoblasts

1-1. Primary myoblast cell separation and culture

To isolate primary myoblasts, mice 1-5 days old were washed with 70% ethanol and choked using CO ₂ . The upper part of the hind leg ankle and the knee were cut and immersed in 1 X phosphate buffer (PBS), and skin and bones were removed with sterilized tweezers to collect muscle tissue. The collected muscle tissue was washed 3 times with 1 X PBS and finely chopped. An enzyme solution prepared by mixing 1 ml of collagenase (1.5 U / ml), 1 ml of dispase (2.4 U / ml) and 5 μl of CaCl ₂ (1 M) was added to the fragmented muscle tissue at 37 ° C And reacted for 30 minutes. The muscle tissue reacted with the enzyme was filtered with a nylon mesh (80 [mu] M) to filter off bones, etc., and centrifuged at 800 rpm for 5 minutes to obtain cells.

The cells obtained above (primary source cells) were re-dissolved in 2 ml of F10 medium (Invitrogen) and transferred to a 100-mm general culture container. The transfer was carried out at intervals of 1 hour to a culture container coated with 0.1% . The cells were cultured in an incubator containing 5% CO ₂ at 37 ° C and replaced with fresh F10 medium every 2 days. For cell passage, the cells were separated in a culture vessel using 0.005% trypsin. To induce differentiation into muscle cells, DMEM (Dulbecco's Modified Eagle Medium, Invitrogen) supplemented with 5% horse serum was used .

1-2. Source cell line Of C2Cl2 culture

C2Cl2 is a parental cell line obtained from a mouse of C3H species, and is widely used in research on myotube differentiation.

The C2C12 cells were cultured in a general cell culture medium and a differentiation medium, respectively. DMEM supplemented with 10% fetal bovine serum was used as a normal cell culture medium (DM), and DMEM containing 2% horse serum was used as a differentiation medium Respectively.

Example  2: source cell ( myoblast ) Induction of differentiation induction

2-1. Promotion of Differentiation by In-Cell ELISA

In-cell ELISA was performed to compare the amount of myosin heavy 3 (MYH3) protein expressed in primary myoblasts.

5 x 10 ³ primary stem cells were inoculated into a 96-well plate coated with 0.1% gelatin, and after 24 hours, differentiation was induced by DMEM supplemented with 5% horse serum. Was replaced with fresh medium supplemented with DMSO (5%) or treatment concentration of chemicals, or DMSO (5%) with insulin every 24 hours. On the third day after the differentiation, the medium was removed and 100 쨉 l paraformaldehyde (3.7%) was treated at room temperature for 15 minutes to fix the cells. After washing with phosphate buffer (1 × PBS), 100 μl permeabilization buffer containing 0.1% saponin, 3% triton X-100 and 0.009% sodium azide was added to the wells for 15 min at room temperature The pores were drilled in the cell membrane. The cells were washed with 1X PBS and treated with 100 μl blocking buffer containing 0.1% albumin (bovine serum albumin) for 1 hour at room temperature. The cells were washed three times with 1 × PBS, and then 100 μl of a primary antibody diluted 1: 500 (SC-20641, Santa Cruz Biotechnology) was added and reacted at 37 ° C. for 2 hours. After the reaction, the cells were washed 3 times with 1X PBS, 100 μl of a secondary antibody (Goat anti-Rabbit IgG-HRP) diluted 1: 10,000 was added and reacted at 37 ° C for 1 hour. The cells were washed three times with 1 × PBS, and then 50 μl of TMB solution (Gen Depot # T3551) was added thereto for 20 minutes, and 50 μl of stop solution (Gen Depot # T3552) was added to stop the reaction Respectively. To analyze the level of MYH3 protein in the cells, absorbance was measured at 485 nm wavelength and the results were analyzed.

In particular, the differentiation process specifically induced the differentiation into muscle cells by replacing the primary myoblasts with the differentiation medium and then changing the medium with fresh differentiation medium daily for 3 days. After 3 days, the levels of MYH3 protein were compared by in-cell ELISA.

As a result, the effect of promoting the differentiation of 0.2 μM nitrendipine was markedly increased when the MYH fold change value was 1.228 (n = 2) as compared with the DMSO treatment group (negative control), and 0.6 μg / (Positive control group) (Fig. 1).

The results of this in-cell ELISA analysis show that the nitrendipine promotes differentiation by higher MYH drainage change than DMSO, which is a drug carrier, and its degree of promotion is similar to that of insulin promoted already as a muscle cell differentiation inducing drug I could confirm.

2-2. Ny trendy pin  Identification of induction of differentiation by concentration

In the above 2-1, nitrendipine was treated with 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1 and 2 μM of each of the primary myocytes The amount of MYH3 was measured (n = 2).

The nandrinetic pin treatment was carried out in the same manner as described in 2-1 above.

As a result, it was confirmed that nystrendipine showed an effect of promoting rapid differentiation at a concentration of 0.01 μM and an effect of promoting differentiation to 1.0 μM (FIG. 2).

Example  3. Ny trendy pin  Cytotoxicity measurement of myocytes

To determine the cytotoxicity of nystreendipine, the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega) kit was used to measure LDH (lactate dehydrogenase), an enzyme secreted during apoptosis.

The cells were inoculated with 5 x 10 ³ primary stem cells per well in a 96-well plate using a cell culture medium (GM). After 24 hours, the concentration of nitrendipine in the differentiation medium (DM, 0.01, 0.02, 0.05 , 0.1, 0.2, 0.5, 1 and 2 μM), respectively, and cultured for 24 hours. 50 μl of the medium containing each sample was transferred to a 96-well flat bottom plate, and a 50 μl reconstituted substrate mix was added thereto, followed by reaction at room temperature for 30 minutes. And treated with 1 mM H ₂ O ₂ as a control for complete cell death. After 30 minutes of the reaction, 50 ㎕ of stop solution was added to the cells, and the absorbance at 490 nm was measured and expressed as a ratio with respect to the LDH value at the time of cell death by lysis buffer.

As a result, there was no difference in the cytotoxicity of nitrendipine in the differentiation medium (DM) of primary myoblast cells, and even when treated with 2.0 μM of nitrendipine, 1 mM H ₂ O ₂ , the toxicity of myocardial differentiation was very small (Fig. 3).

Example  4: Ny trendy pin  Confirmation of promoting effect of source cell differentiation

4-1. Phase difference microscope  (Phase contrast microscopy)

To confirm the formation of large amounts of myotubes in the nystrin-fused myocytes, C2Cl2 cells were incubated in 0.1% gelatin-coated coverslips for 3 days with 0.2 μM each of DMSO and nystrendipine, After differentiation, they were observed with a phase contrast microscope. As a result of this experiment, the root canal was formed when nitrendipine was treated as compared with the control group, DMSO. As a result, it was confirmed that the effect of promoting root differentiation of nystrendipine was confirmed (x 100) (FIG.

4-2. Immunocytochemistry Immunocytochemistry )

DMSO (negative control) and nystrendipine were induced to induce the differentiation of C2Cl2 cell line. On the third day, protein expression was confirmed by staining with MYH3 antibody to compare the degree of myocyte differentiation.

Specifically, C2Cl2 cells were differentiated in a cover glass coated with 0.1% gelatin for 3 days. The cells were washed with 1X PBS, fixed with 3.7% paraformaldehyde at room temperature for 15 minutes, washed 3 times with 1X PBS, permeabilized with a permeabilization buffer, and allowed to react at room temperature for 15 minutes. After washing three times with 1X PBS, the cells were reacted with PBST containing 1% BSA (blocking buffer, PBS containing 0.5% Tween 20) for 30 minutes to inhibit unspecific antibody binding. The primary antibody (SC-20641, Santa Cruz Biotechnology) for MYH3 was diluted 1: 500 in blocking buffer and reacted at room temperature for 1 hour. After washing three times with 1 × PBS, secondary antibody (Goat anti-Rabbit IgG-HRP) diluted 1: 5000 in blocking buffer was added and reacted at room temperature for 1 hour. And washed. The cover glass was placed on a slide glass and photographed with a fluorescence microscope to analyze the results.

As a result, it was confirmed that the expression of MYH3 was very high when 0.2 μM of nitrendipine was treated as compared with the group treated with DMSO (FIG. 5).

4-3. Western Blat  (Western blot)

C2C12 cells were cultured in a culture medium for 24 hours, and differentiation was induced by 0.2 .mu.M each of DMSO and nitrendipine in the differentiation medium, respectively. On the third day of differentiation induction, cells were obtained and centrifuged at 1200 rpm for 3 minutes. The cells were subjected to sonication by sonication, followed by centrifugation at 3000 rpm for 10 minutes to obtain a water-soluble protein. A 4X sample buffer was added to the cells, followed by reaction in boiling water for 5 minutes. 10 [mu] g of protein was loaded on a 12% SDS-PAGE gel, developed and transferred to a Watman membrane. The membrane was blocked with 5% skim milk for 1 hour at room temperature and then washed five times for 5 minutes with TTBS (0.03% Tween 20, Tris 2.42 g, NaCl 9 g, pH 7.4, 1 L). The primary antibody was diluted 1: 500 in TTBS containing 5% skim milk, incubated at room temperature for 2 hours, and then washed 5 times with TTBS for 5 minutes each. Then, the secondary antibody was diluted 1: 5000 in TTBS supplemented with 5% skim milk, and then reacted at room temperature for 2 hours, washed 5 times with TTBS for 5 minutes, and then ECL (Enhanced Chemiluminescent solution, Pierce) was added. Then, the membrane was exposed to an X-ray film to confirm the amount of protein.

As a result of the above experiment, it was confirmed that the amount of MYH3 protein contained in the same amount of protein was significantly increased when treated with nitrendipine (FIG. 6) compared with the control DMSO treatment.

As a result of the above experiment, it was found that the effect of nystrendipine on the promotion of myocyte differentiation was very high, and it was confirmed that the use of the nystrendipine could prevent or treat weakness related to muscle weakness.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all aspects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (12)

A composition for promoting the differentiation of myoblasts comprising Nitrendipine or a pharmaceutically acceptable salt thereof.
2. The composition of claim 1, wherein the concentration of the nystrendipine or a pharmaceutically acceptable salt thereof is from 0.01 to 2 [mu] M.
A method of promoting differentiation of a source cell, comprising the step of treating extracorporeal cells with Nitrendipine or a pharmaceutically acceptable salt thereof.
A method for the production of differentiated source cells, comprising treating the extracorporeal cells with Nitrendipine or a pharmaceutically acceptable salt thereof to differentiate the source cells.
A pharmaceutical composition for preventing or treating muscular weakness related to muscular weakness comprising Nitrendipine or a pharmaceutically acceptable salt thereof.
6. The composition of claim 5, wherein the muscular weakness-related disorder is myopenia, muscular dystrophy, or ataxia.
6. The composition of claim 5, wherein the composition promotes differentiation of myocytes into myocytes.
WHAT IS CLAIMED IS: 1. A food composition for preventing or ameliorating muscular weakness related to muscle comprising Nitrendipine or a pharmaceutically acceptable salt thereof.
9. The composition of claim 8, wherein the disease is myopenia, muscular dystrophy, or ataxia.
A composition for strengthening a muscle comprising Nitrendipine or a pharmaceutically acceptable salt thereof.
Strengthening feed or feed additive comprising nitrendipine or a pharmaceutically acceptable salt thereof.
A composition for improving wrinkles by weakening muscle strength, comprising nitrendipine or a pharmaceutically acceptable salt thereof.

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