KR101586336B1 - Pharmaceutical composition for preventing or treating muscle weakness diseases comprising Estropipate - Google Patents

Abstract

The present invention relates to a composition for promoting differentiation of a source cell comprising estropipate or a pharmaceutically acceptable salt thereof, a pharmaceutical composition for preventing or treating muscle weakness-related diseases, a composition for preventing or ameliorating muscle weakness-related diseases A food composition, a composition for strengthening muscle strength, and a feedstuff for enhancing strength or a feed additive.
The present invention also relates to a method for promoting differentiation of myoblast cells using estropypate or a pharmaceutically acceptable salt thereof, a method for producing differentiated myocytes, and a method for treating weakness related to muscle weakness.

Description

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

The present invention relates to a composition for promoting differentiation of a source cell comprising estropipate or a pharmaceutically acceptable salt thereof, a pharmaceutical composition for preventing or treating muscle weakness-related diseases, a composition for preventing or ameliorating muscle weakness-related diseases A food composition, a composition for strengthening muscle strength, and a feedstuff for enhancing strength or a feed additive.

The present invention also relates to a method for promoting differentiation of myoblast cells using estropypate or a pharmaceutically acceptable salt thereof, a method for producing differentiated myocytes, and a method for treating weakness related to muscle weakness.

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 addition to the decrease in muscle mass, which is the most important feature of myopenia, changes in the type of muscle fiber are also 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 pysical 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 when starvation, exhaustion (cancer, etc.) and senescence occurs. Myocardial fibers are thin and thin, and 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.

There are three main treatment methods for 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 therapies include DHEA (dehydroepiandrosterone) and growth hormone, which have been reported to be therapeutically feasible at sites that include SARMs (Selective Androgen Receptor Modulators) (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 muscular function. As a result, it has been found that esophosphate 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.

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

In the present invention, "S trophy sulfate (Estropipate)" is the name of the IUPAC (International Union of Pure and Applied Chemistry) [(8 R, 9 S, 13 S, 14 S) -13-Methyl-17-oxo-7, 8,9,11,12,14,15,16-octahydro-6 H -cyclopenta [ a] phenanthren-3-yl] hydrogen sulfate; piperazine, and derivatives thereof as long as they have an effect of promoting the differentiation of the source cells. For example, the formula is C ₂₂ H ₃₂ N ₂ O ₅ S and the molecular weight is 436.56. Estrope is generally known to be a type of estrogen, a type of steroid hormone known to be a female hormone, because it is mainly secreted in the follicles and luteum in the female ovary and secreted in the placenta. Estrogen is the cause of female secondary growth It acts in conjunction with follicle stimulating hormone (FSH), luteinizing hormone (LH), and progesterone to regulate the thickness of the uterine wall and participate in ovulation. It is known to play a role in regulating the reproductive cycle. Estropipate is an agent that increases the solubility and stability by introducing sulfate and piperazine groups into estrone, a type of estrogen, and is used as a therapeutic agent for female diseases such as hormone replacement therapy, vaginal atrophy and infertility However, there is no known association with myoblast differentiation.

The inventors of the present invention have completed the present invention by first confirming that estropeate or a pharmaceutically acceptable salt thereof has a purpose of differentiation of a source cell, and the structure of esropapeate is as shown in the following 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 monocyte myoblasts form myotubes through fusion. Myocyte precursor cells correspond to Pax7 ⁺ markers in self-renewal and Pax7 ⁺ / MyoD ⁺ in proliferation. Cells at the differentiation stage that form the root canal can be distinguished using the Pax7 ^- MyoD ⁺ MyoG ⁺ marker. The cells at the early stage of differentiation to form the canal increase the expression of myogenic transcription factors such as MyoD (D), and increase myosin G (MyoG) in the middle stage. The expression of Myosin heavy chain (MyHC) is increased in the late stage of differentiation.

Specifically, one embodiment of the present invention relates to a composition for promoting the differentiation of myoblasts, which comprises esculpate or a pharmaceutically acceptable salt thereof at a concentration of 0.01 μM to 2.0 μM. 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 the composition, more specifically, esropapate or a pharmaceutically acceptable salt thereof may be contained at a concentration of 0.05 μM to 1.5 μM, most specifically at a concentration of 0.1 μM to 1.0 μM. In addition, the composition may further include additional substances necessary for cell culture or differentiation.

When esophosphate or a pharmaceutically acceptable salt thereof is contained in an amount of less than 0.01 μM, the concentration may be too low and the effect of promoting differentiation may be deteriorated. If it is contained in an amount exceeding 2.0 μM, toxicity may be caused.

In one embodiment of the present invention, estropypate was treated at a concentration of 0.0001, 0.001, 0.01, 0.1, 0.2, 1 and 2 μM in a cell culture medium and DM (DM) DM), even when treated with _2.0 μM esters, the ratio of dead cells was less than 11.13% compared with the value treated with 50 μM H ₂ O ₂ . (FIG. 3), it was confirmed that treatment of esropapeate within the above range can promote differentiation with little toxicity on cells.

Another embodiment of the present invention relates to a method of promoting differentiation of a source cell, comprising the step of treating a source cell with esophosphate or a pharmaceutically acceptable salt thereof. Estropipate or a pharmaceutically acceptable salt thereof is as described above. Specifically, the method of promoting differentiation of myofilaments can promote the differentiation by treating esophosphate or a pharmaceutically acceptable salt thereof with an extracorporeal or intracorporeal stem cell.

Another embodiment of the present invention provides a method of producing differentiated source cells comprising treating the source cells with estrophipate or a pharmaceutically acceptable salt thereof to differentiate the source cells.

Estropipate or a pharmaceutically acceptable salt thereof is as described above. The production method of the present invention can be characterized in that the differentiated stem cells comprising the step of treating the extracellular or intracorporeal stem cells with estrophipate or a pharmaceutically acceptable salt thereof to differentiate the stem cells can be produced.

In one embodiment of the present invention, the degree of differentiation of myoblast cells after treatment with esoprotein at a concentration of 0.5 μM in myoblasts was observed by phase contrast microscopy. As a result, the number of differentiation was higher than that of negative control (DMSO) (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).

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 muscular weakening-related diseases comprising estropypate or a pharmaceutically acceptable salt thereof. The concentration of esophosphate 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 muscles, such as, for example, muscular dystrophy, muscular dystrophy, or dystrophy.

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

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 muscular 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 slows down the onset of muscle weakness associated with 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.

A pharmaceutical composition of the present invention may contain, for administration, a pharmaceutically acceptable carrier, excipient or diluent in addition to the esropiate 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 prepared into pharmaceutical formulations using methods well known in the art so as to provide rapid, sustained or delayed release of esropiate 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, but is preferably prepared 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, esropifate or a pharmaceutically acceptable salt thereof may be formulated into a solution or suspension by mixing in water with a stabilizer or buffer, and then formulated into a unit dose of ampoule or vial.

A pharmaceutical composition comprising esophosphate or a pharmaceutically acceptable salt thereof of the present invention 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 the composition does not interfere with the prevention or treatment of muscular weakening-related diseases, esters or pharmaceutically acceptable salts thereof may contain additional ingredients, for example, substances known to be 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 degree of differentiation of myoblast cells after treatment with esoprotein at a concentration of 0.5 μM in myoblasts was observed by phase contrast microscopy. As a result, the number of differentiation was higher than that of negative control (DMSO) (FIG. 4), and it was confirmed that the effect of promoting myocyte differentiation was very high even through immunocytochemistry and Western blotting (FIGS. 5 and 6). From these results, it was confirmed that esculpate Or a pharmaceutically acceptable salt thereof, is effective for promoting differentiation of myoblast and may be useful for preventive treatment of muscular weakening-related diseases.

Another embodiment of the present invention relates to a food composition for preventing or ameliorating a muscle weakening-related disease comprising esophosphate 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. The concentration of esophosphate or a pharmaceutically acceptable salt thereof is as described above.

The muscle weakness-related disease refers to all diseases that may occur due to weakness of muscle strength, for example, muscle dyspnea, muscular dystrophy, or dystrophy, but the present invention is not limited thereto. 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. In general, the composition of the present invention may be added in an amount of not more than 15% by weight, specifically not more than 10% by weight 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 estrophepate or a pharmaceutically acceptable salt thereof.

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

In the present invention, "feed" means a substance which supplies 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 is not limited to, amino acids, vitamins, enzymes, flavors A silicate, a buffer, an extractant, an oligosaccharide, and the like.

The content of esophosphate or pharmaceutically acceptable salt thereof contained in the feed or feed additive of the present invention is not particularly limited, but may be 0.001 to 1% (w / w), specifically 0.005 To 0.9% (w / w), and most specifically from 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 estropeptide or a pharmaceutically acceptable salt thereof to a subject in need thereof.

The muscle weakness-related disease refers to all diseases that may occur due to weakness of muscle strength, for example, muscle dyspnea, muscular dystrophy, or dystrophy, but the present invention is not limited thereto.

In the present invention, the term "individual" refers to all animals, including humans, who have already developed or are capable of developing a weakness related to muscular weakness. By administering to a subject a composition comprising estrophepate 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. Specifically, the object may be an object other than a human.

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 those skilled 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.

The esropapeate or pharmaceutically acceptable salt thereof according to the present invention can promote root differentiation of root cells to form a root canal, thereby preventing muscular 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 is higher than that of negative control DMSO when treated with esroprotein.
FIG. 2 is a graph showing the effect of accelerating the differentiation of myofibers by concentrations of estropeptide at 0.0001, 0.001, 0.01, 0.1, 0.2, and 1.0 .mu.M, respectively, on primary myocytes.
Fig. 3 shows the result of measuring the toxicity according to the concentration of esropapeate in the differentiation medium (DM).
Fig. 4 shows the result of discrimination of the differentiation of the estrope-treated root cell line C2C12 by a phase contrast microscope.
FIG. 5 shows the results of immunopositive assays for differentiation of escropic-treated root cell line C2C12.
FIG. 6 shows the results of western blot analysis of the expression of myosin heavy chain 3 (MYH3) in estrope-treated root cell line C2C12.

Hereinafter, the present invention will be described in more detail with reference to examples. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

Example  1: source cell main C2C12 Cultivation of

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 1X phosphate buffered saline (PBS) to remove skin and bones with sterilized tweezers, and muscle tissue was collected. Collected muscle tissue was washed 3 times with 1X PBS and minced. 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 and the mixture was incubated at 37 ° C. for 30 minutes Lt; / RTI > The muscle tissue subjected to the enzyme reaction was filtered using a nylon mesh (80 μM) to filter out bones and the like, and cells were obtained by centrifugation at 800 rpm for 5 minutes.

The cells obtained above (primary source cells) were re-dissolved in 2 ml of F10 medium (Invitrogen), transferred to a 100 mm general culture container, and transferred to a culture container coated with 0.1% gelatin at intervals of 1 hour repeatedly until P5 Respectively. 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 main 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. In - Cell ELISA Promotion of differentiation using

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 cultured in 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 μl of paraformaldehyde (3.7%) was treated at room temperature for 15 minutes to fix the cells. After washing with phosphate buffer (1 × PBS), 100 μl of phosphate buffer solution permeabilization buffer containing 0.1% saponin, 3% triton X-100 and 0.009% sodium azide was added to the solution at room temperature For 15 minutes to pierce the cell membrane. The cells were washed with 1X PBS and treated with blocking buffer (100 μl) containing 0.1% bovine serum albumin for 1 hour at room temperature. The cells were washed 3 times with 1 × PBS, and then added with 100 μl of a primary antibody (SC-20641, Santa Cruz Biotechnology) diluted 1: 500 and reacted at 37 ° C for 2 hours. The cells were washed three times with 1X PBS, and then added with 100 μl of a secondary antibody (Goat anti-Rabbit IgG-HRP) diluted 1: 10,000 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. The reaction was terminated by adding 50 μl stop solution (Gen Depot # T3552) Respectively. To analyze the MYH3 protein level of the cells, the absorbance at 485 nm was measured 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 differentiation promoting effect of 0.2 μM esropapeate was similar to that of 0.6 μg / ml insulin (positive control) when compared with DMSO (negative control) and the MYH fold change value was 1.240 n = 2) (Fig. 1).

The results of this in-cell ELISA analysis showed that estropypate promoted 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 as a muscle cell differentiation inducing drug I could confirm.

2-2. Esophosphate  Identification of induction of differentiation by concentration

After confirming that estropypate promoted differentiation in the above 2-1, estropypate was treated with the concentrations of 0.0001, 0.001, 0.01, 0.1, 0.2 and 1.0 μM on the primary myocytes and then the amount of MYH3 was measured (N = 2).

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

As a result, it was confirmed that estropypate showed an abrupt increase in differentiation-promoting effect at a concentration of 0.2 μM, and that the effect of promoting differentiation was increased to 1.0 μM (FIG. 2).

Example  3. Esophosphate  Cytotoxicity measurement of myocytes

To measure cytotoxicity against insulin and estropypate, the Cyto Tox 96 Non-Radioactive Cytotoxicity Assay (Promega) kit was used to measure LDH (lactate dehydrogenase), an enzyme secreted during apoptosis.

5 x 10 ³ primary stem cells per well were cultured on a 96-well plate using a cell culture medium (GM). After 24 hours, the cell culture medium and the differentiation medium (DM) were inoculated with insulin and estropypate (0.0001, 0.001, 0.01, 0.1, 0.2, 1 and 2 [mu] M). 50 μl of the medium containing each sample was transferred to a 96-well flat bottom plate, and 50 μl of a reconstituted substrate mix was added thereto, followed by reaction at room temperature for 30 minutes. As a control for complete cell death was treated with 50μM H ₂ O _2. After 30 minutes of the reaction, 50 stop 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 apoptosis by lysis buffer.

As a result, there was no difference in cytotoxicity of esophosphate according to the concentrations measured in the differentiation medium (DM) of the primary myoblast cell and there was no difference compared to the same concentration of insulin. Even when treated with _2.0 μM of esophosphate, it was confirmed that the proportion of apoptotic cells was less than 11.13% compared to the value of 50 μM H ₂ O ₂ which resulted in complete apoptosis, and that the toxicity by myocardial differentiation was very small ).

Example  4: Esophosphate  Confirmation of promoting effect of source cell differentiation

4-1. Phase contrast microscope Phase contrast microscopy )

In order to confirm the formation of large amounts of myotubes in myoblasts by esophosphate, C2Cl2 cells were treated with 0.1% gelatin-coated coverslips and treated with DMSO and esthropipate (0.5 μM each) And observed with a phase contrast microscope. As a result of this experiment, it was found that the root canal was formed when the estrophipate was treated as compared with the control group, DMSO (x100) (FIG. 4).

4-2. Immunocytochemistry Immunocytochemistry )

C2Cl2 cells were differentiated for 3 days in a cover glass coated with 0.1% gelatin. The cells were washed with 1X PBS, fixed with 3.7% paraformaldehyde at room temperature for 15 minutes, washed three 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 1X PBS, secondary antibody (Goat anti-Rabbit IgG-HRP) diluted 1: 5000 in blocking buffer was added and incubated 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.

In the present invention, differentiation of C2Cl2 cell line was induced while treating DMSO (negative control) and estropypate, respectively, and on day 3, protein expression was confirmed by staining with MYH3 antibody to compare the degree of myocyte differentiation.

As a result, it was confirmed that the expression of MYH3 was very high when 0.5 μM of esroprotein was treated compared with the DMSO-treated group (FIG. 5).

4-3. Western Blat ( Western blot )

C2C12 cells were cultured in a culture medium for 24 hours, and the differentiation was induced by treating DMSO and estropic acid in 0.5 ml of each of the differentiation media, respectively. On the third day of differentiation induction, cells were obtained and centrifuged at 1200 rpm for 3 minutes. After adding 100 μl of lysis buffer to the cells, the cells were sonicated and centrifuged at 3000 rpm for 10 minutes to obtain a water-soluble protein. A 4 × sample buffer was added and reacted in boiling water for 5 minutes. Ten micrograms of the 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 with TTBS (0.03% Tween 20, Tris 2.42 g, NaCl 9 g, pH 7.4, 1 L) for 5 minutes each. 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. 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 greatly increased when the estrophipate was treated (FIG. 6) as compared with the control DMSO treatment.

It was found from the above experimental results that the effect of promoting the differentiation of muscle cells by esophosphate was very high, and it was confirmed that the effect of preventing or treating muscular weakening-related diseases can be demonstrated.

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 above-described embodiments are to be considered in all respects as illustrative 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 extracellular myoblasts comprising estropipate or a pharmaceutically acceptable salt thereof.
2. The composition of claim 1, wherein the concentration of estroppate or a pharmaceutically acceptable salt thereof is from 0.01 to 2 [mu] M.
A method for promoting differentiation of a source cell comprising treating esoprotein or a pharmaceutically acceptable salt thereof with extracorporeal stem cells.
Treating the extracellular source cells with estrope- phate or a pharmaceutically acceptable salt thereof to differentiate the source cells.
A pharmaceutical composition for the prevention or treatment of muscular dystrophy, muscular dystrophy, or ataxia, including esophobia or a pharmaceutically acceptable salt thereof.
delete 6. The composition of claim 5, wherein the composition promotes differentiation of myocytes into myocytes.
A food composition for improving myopenia, muscular dystrophy, muscle dystrophy or cardiac atrophy, comprising escropipate or a pharmaceutically acceptable salt thereof.
delete delete 0.0 > a < / RTI > pharmaceutically acceptable salt thereof.
A dietary or feed additive for strengthening muscle strength, comprising esropiate or a pharmaceutically acceptable salt thereof.

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