KR20150014307A - Pharmaceutical composition for preventing or treating muscle weakness diseases comprising Orotic Acid - Google Patents

Abstract

The present invention relates to: a composition for promoting differentiation of myoblast comprising orotic acid or a pharmaceutically acceptable salt thereof; a method for promoting differentiation of the myoblast comprising a step of treating the orotic acid or a pharmaceutically acceptable salt thereof to the myoblasts; a method for preparing differentiated myoblasts comprising a step of differentiating the myoblast by treating the orotic acid or a pharmaceutically acceptable salt thereof to the myoblasts; a pharmaceutical composition for preventing or treating muscle weakness related diseases comprising the orotic acid or a pharmaceutically acceptable salt thereof; a food composition for preventing or alleviating the muscle weakness related diseases; and a composition for strengthening muscle. The orotic acid according to the present invention promotes differentiation of the myoblast and forms root cap, thereby preventing muscle weakness and effectively improving muscle function. Therefore, the pharmaceutical composition comprising the same can be useful for preventing or treating the muscle weakness related diseases.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing or treating muscle weakness-related diseases,

The present invention provides a method for promoting the differentiation of myoblasts comprising orotic acid or a pharmaceutically acceptable salt thereof, comprising treating ovarian or ophthalmic acid or a pharmaceutically acceptable salt thereof with extracorporeal cells A method for promoting the differentiation of myoblast cells, a method for treating differentiated myocytes with orotroic acid or a pharmaceutically acceptable salt thereof to treat extracorporeal stem cells, a method for producing differentiated myocytes, A pharmaceutical composition for preventing or treating muscular weakness related to muscle weakness including an acceptable salt, a food composition for preventing or ameliorating muscle weakness-related diseases, and a composition for strengthening muscle strength.

Diseases that cause weakness in muscle strength include sarcopenia associated 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 by starvation, consuming disease (such as cancer) and senility. 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 muscular weakness, it is necessary to develop a material that can promote the differentiation of myoblast cells.

Under these circumstances, the present inventors have made intensive efforts to develop a therapeutic agent for muscular weakness related to muscles that increases muscle mass by promoting differentiation of myoblasts and effectively restores muscle function. As a result, it has been found that a composition containing orotic acid And thus can be used for the prevention or treatment of diseases associated with weakness in muscle strength, thereby completing the present invention.

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 method for preventing or treating muscular weakness related to muscular weakness.

Another object of the present invention is a food composition for preventing or ameliorating muscle weakness-related diseases.

Still another object of the present invention relates to a composition for strengthening muscular strength.

In one aspect of the present invention, the present invention provides a composition for promoting the differentiation of myoblasts comprising orotic acid or a pharmaceutically acceptable salt thereof.

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, for example inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid and the like, Organic carboxylic acids such as acetic acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicinic acid and the like; organic carboxylic acids such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p- Sulfonic acid, and the like. 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.

In the present invention, "Orotic Acid" refers to 2,4-dioxo-1H-pyrimidine-6-carboxylic acid as IUPAC (International Union of Pure and Applied Chemistry), and C ₅ H ₄ N ₂ O ₄ and a molecular weight of 156.09. In general, orotic acid is a precursor of pyrimidine nucleotides required for the biosynthesis of nucleic acids, and is known to be involved in nucleic acid synthesis and cell regeneration processes. In addition, although it is generally known as a therapeutic agent for heart diseases including hypertension and congestive heart failure, there is no known relation with myoblast differentiation. The present inventors have accomplished the present invention by first identifying the use of orotactic acid or a pharmaceutically acceptable salt thereof for the purpose of source cell differentiation.

Orotic acid is known as a substance that is biosynthesized in the human body as a constituent of a vitamin B complex produced in the human body and is known to be a substance that is biosynthesized in humans (Engelbrecht C et al .: Experientia 33 (3): 302-4 (1977) et al; Psychopharmacology (Berl) 73 (4): 399-401 (1981)). Since it is well known that no toxicity is shown in the experiments, it is advantageous to confirm the efficacy through other clinical tests City is advantageous.

As used herein, "source cell differentiation" is 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 is increased in the late stage of differentiation

The promotion of source cell differentiation of the present invention can be performed by using a medium in which DMEM differentiation medium containing serum is treated with Orotic Acid or a pharmaceutically acceptable salt thereof, but the present invention is not limited to this. Orotate or a pharmaceutically acceptable salt thereof may be included, and the medium capable of promoting the source cell differentiation may be included without limitation. Preferably, 0.01 to 2 μM orotic acid can be used for the differentiation medium, and more preferably 0.01 to 1.0 μM orotic acid can be used.

In one embodiment of the present invention, when Orotic acid was treated at a concentration of 0.2 μM in myoblasts, in-cell ELISA was used to search for promotion of differentiation. As a result, the MYH3 multiples were higher than the negative control (DMSO) , And a positive control (insulin 0.6 μg / mL), indicating that orotic acid promotes myocardial differentiation (FIG. 1).

In one embodiment of the present invention, the orotic acid was treated with different concentrations of 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, and 1.0 μM in the differentiation medium of the extracorporeal cells, and the rapid differentiation promoting effect was observed at the concentration of 0.01 μM I could. In addition, it was confirmed that the concentration of insulin could promote myocardial differentiation at a lower concentration than the positive control group (Fig. 2).

Therefore, the orotic acid of the present invention or a pharmaceutically acceptable salt thereof can be usefully used for promoting differentiation of a source cell. Further, the composition may contain additional components as long as it does not interfere with the promotion of source cell differentiation.

In another embodiment, the present invention provides a method of promoting differentiation of a source cell comprising treating the source cell with the orotic acid or a pharmaceutically acceptable salt thereof.

The orotic acid or its pharmaceutically acceptable salt is as described above.

The method of promoting the differentiation of myoblast cells of the present invention is characterized in that orotic acid or its pharmaceutically acceptable salt is treated to extracorporeal or intracorporeal stem cells to promote differentiation.

In another aspect, the present invention provides a method of producing differentiated parental cells comprising treating parent cells with orotic acid or a pharmaceutically acceptable salt thereof to differentiate the parental cells.

The concentrations of orotic acid or its pharmaceutically acceptable salts and salts are as described above.

The method of the present invention is characterized in that the differentiated source cells are produced by treating orotrophic acid or a pharmaceutically acceptable salt thereof with an extracorporeal or intracorporeal source cell to differentiate the source cells.

In one embodiment of the present invention, the orotic acid was treated with a differentiation culture solution (0.5 μM) of myocytes for 3 days and subjected to phase contrast microscopy and immunocytochemistry. As a result, the myocytes ) And that the expression of protein MYH3 was very high (FIGS. 4 and 5). In addition, the composition for promoting differentiation was treated with myoblasts, followed by Western blotting, and the amount of MYH3 protein was confirmed to be higher than that of negative control (FIG. 6).

Thus, the present invention is able to produce differentiated, source cells that can form canals and express the MYH3 protein in vitro or in vivo.

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating muscle weakness-related diseases comprising orotic acid or a pharmaceutically acceptable salt thereof.

The concentrations of orotic acid or its pharmaceutically acceptable salts and salts are 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, muscle weakness related to muscular weakness refers to all diseases that may occur due to weakness of muscle strength, such as, for example, muscular dystrophy, muscular dystrophy or acardiotrophia.

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

Specifically, the myopenia of the present invention means a gradual decrease in skeletal muscle mass 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).

Myocardial dysfunction of the present invention is a condition in which the heart is contracted by external factors or internal factors, which may cause a brown atrophy of the heart causing starvation, wasting disease, have.

As used herein, the term "prophylactic " 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 any action that improves or alleviates symptoms due to muscle weakness-related diseases by administering the composition.

The pharmaceutical composition of the present invention may contain, for administration, a pharmaceutically acceptable carrier, excipient or diluent in addition to the orotic acid or its pharmaceutically acceptable salt. 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 orotic acid or a pharmaceutically acceptable salt thereof. , It is preferable 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, orotic acid or a pharmaceutically acceptable salt thereof may be formulated as a solution or suspension by mixing in water with a stabilizer or a buffer to prepare a unit dose of ampoule or vial.

The composition comprising orotic acid of the present invention or a pharmaceutically acceptable salt thereof can be directly injected into a site where a muscle weakening-related disease has occurred or a site where a potentially susceptible individual is required to be strengthened. Or orotate or a pharmaceutically acceptable salt thereof may be applied to in vitro or in vivo endogenous cells to produce differentiated endogenous cells and then the differentiated endothelial cells may be administered to a subject suffering from, Can be injected into the site where muscle strengthening is required.

The composition of the present invention is also known as a therapeutic agent for an additive component, for example, a muscle weakening-related disease, so long as orotic acid or a pharmaceutically acceptable salt thereof does not interfere with the prevention or treatment of muscle weakness- Substances may be included.

In the composition of the present invention, orotic acid may be used at a concentration of 0.01 to 2 μM orotic acid or a pharmaceutically acceptable salt thereof, more preferably 0.01 to 1.0 μM of orotic acid or a pharmaceutically acceptable salt thereof .

Preferably, the pharmaceutical composition of the present invention is characterized by promoting differentiation of the source cells.

In one embodiment of the present invention, when Orotic acid was treated at a concentration of 0.2 μM in myoblasts, in-cell ELISA was used to search for promotion of differentiation. As a result, the MYH3 multiples were higher than the negative control (DMSO) , And a positive control (insulin 0.6 μg / mL), indicating that orotic acid promotes myocardial differentiation (FIG. 1).

In one embodiment of the present invention, the orotic acid or its pharmaceutically acceptable salt, orotic acid, was treated with different concentrations of 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, , And at the concentration of 0.01 μM, the rapid differentiation promoting effect was confirmed. In addition, it was confirmed that the concentration of insulin could promote myocardial differentiation at a lower concentration than the positive control group (Fig. 2).

From these results, it was confirmed that the orotic acid or its pharmaceutically acceptable salt has an effect of promoting muscle cell differentiation at a level equal to or higher than that of insulin, which is known as an accelerator for differentiation of muscle cells, It is effective in promoting differentiation of cells and may be useful for preventive treatment of muscular weakening related diseases.

In another aspect, the present invention provides a method of treating muscular weakness-related diseases by administering orotic acid or a pharmaceutically acceptable salt thereof to an individual.

In another aspect, the present invention provides a food composition for preventing or ameliorating muscular weakness related to muscular weakness comprising orotic acid or a pharmaceutically acceptable salt thereof. That is, the composition of the present invention can be used either simultaneously with or separately from the agent for treating a disease before or after the onset of the muscle weakening-related disease to prevent or ameliorate the muscle weakening-related disease.

The concentration of orotic acid or its pharmaceutically acceptable salt and salt is similar to that described above for the concentration of orotic acid or its pharmaceutically acceptable salt and salt.

In the food composition of the present invention, the concentration of orotic acid or its pharmaceutically acceptable salt may be 0.01 to 2 μM, more preferably 0.01 to 1.0 μM.

Preferably, the composition of the present invention may be used for the prevention or amelioration of myopenia, muscular atrophy or cardiac atrophy. Preferably, the food composition is characterized by promoting differentiation of myoblasts.

As used herein, the term " improvement " means any action that at least reduces 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 is added in an amount of not more than 15% by weight, preferably 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. Such natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharine 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.

In another aspect, the present invention provides a composition for strengthening muscle strength comprising orotic acid or a pharmaceutically or pharmacologically acceptable salt thereof.

The concentrations of orotic acid or its pharmaceutically or pharmacologically acceptable salts and salts are as described above.

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 orotic acid or its pharmaceutically acceptable salt of the present invention can increase the total muscle mass by increasing the muscle mass through the ability to differentiate the source cells into muscle cells, , 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 orotic acid or its pharmaceutically acceptable salt. 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.

In one embodiment of the present invention, the orotic acid is treated with a mouse to perform muscular recovery ability test, exercise ability test (grip test, equilibrium ability test, endurance test) Or a pharmaceutically acceptable salt thereof, can be used for strength-enhancing purposes.

Orotic Acid or a pharmaceutically acceptable salt thereof according to the present invention can promote root differentiation and form 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.

1 is a graph showing the effect of stimulating myocardial differentiation in primary myoblasts of Orotic Acid.
2 is a graph showing the effect of promoting differentiation of myocytes according to the concentration of orotic acid (Orotic Acid).
Fig. 3 is a diagram showing cytotoxicity in the differentiation medium (DM) of orotic acid (Orotic Acid).
FIG. 4 is a microscopic photograph showing the effect of orotic acid on promoting the differentiation of muscle cells.
FIG. 5 is a graph showing the effect of stimulating the differentiation of orogenic myotubes of orotic acid by immunocytochemistry. FIG.
Figure 6 compares the amount of myosin heavy chain 3 (MYH3) expression in primary myoblasts.

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: myocyte ( myoblast ) Culture

Example  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) to remove skin and bones with sterilized tweezers. The collected muscle tissue was washed 3 times with 1 X PBS and finely chopped. An enzyme solution containing 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 incubated at 37 ° C. for 30 minutes Lt; / RTI > The muscle tissue subjected to the enzymatic reaction was filtered using a nylon mesh (80 [mu] M) to filter out bones and the like, and centrifuged at 800 rpm for 5 minutes to obtain cells.

The cells (primary source cells) obtained above were re-dissolved in 2 ml of F10 medium (Invitrogen) and transferred to a 100 mm general culture container. Then, in order to enrich only primary myoblasts, the culture medium transferred to the culture vessel and the suspended primary myoblasts contained therein were incubated at 0.1 hour intervals for 1 hour, using the difference in the adherence ability between primary stem cells and other cells such as Fibroblast % Gelatin-coated culture container was repeated until P5. 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 .

Example  1-2. Source cell main C2Cl2 culture

C2Cl2 is a parental cell line obtained from mice of C3H species and is widely used for studying myoblast differentiation.

The C2C12 cells are 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

Example  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 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 of a permeabilization buffer containing 0.1% saponin, 3% tiriton X-100 and 0.009% sodium azide was treated at room temperature for 15 minutes And punched holes in the cell membrane. The cells were washed with 1X PBS and treated with blocking buffer (100 μl) 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 reacted for 20 minutes with 50 μl of TMB solution (Gen Depot # T3551). Stop reaction by adding 50 μl stop solution (Gen Depot # T3552) Respectively. Absorbance was measured at 485 nm wavelength to analyze MYH3 protein levels of the cells.

Specifically, 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 for 3 days each day. 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 orotic acid was similar to that of 0.6 μg / ml insulin (positive control) when compared to DMSO (negative control) and MYH fold change Value was 1.207 (n = 2) (Fig. 1).

The results of this in-cell ELISA analysis showed that orotic acid promoted differentiation by higher MYH fold change value than DMSO, which is a drug carrier, and its promoted degree was the degree of stimulation of insulin that was already reported as a muscle cell differentiation inducing drug Similarity was confirmed.

Example  2-2. Mount Oratory  Induced differentiation induction

The amount of MYH3 (n = 2) after treatment with orotous acid at concentrations of 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, and 1.0 μM on primary myocytes was significantly And increased to 1.0 μM (FIG. 2).

Example  3: Origin cells of orotic acid ( myoblast ) Toxic effect

To determine the cytotoxicity of insulin and aurotic acid, the Cytotox 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 were cultured on a 96-well plate using a cell culture medium (GM). After 24 hours, insulin and orotic acid were added to the cell culture medium and the differentiation medium (DM) (0.001, 0.01, 0.1, 0.2, 1, 2 μM). 50 μl of 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. 50 μM H ₂ O ₂ was treated 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 to the LDH value in cell death by lysis buffer.

As a result, there was no difference in the cytotoxicity of the aortic acid concentration measured in the primary myoblast cell differentiation medium (DM) and there was no difference compared to the same concentration of insulin. In the case of treatment with 2.0 μM of orotic acid, it was confirmed that the ratio of apoptotic cells was less than 10.32%, compared with the value obtained by treatment with complete lysis buffer leading to complete apoptosis, and thus the toxicity by myocardial differentiation was very small (FIG. 3).

Example  4: Origin cells of orotic acid ( myoblast ) Identification Promotion of Differentiation

Example  4-1. Phase contrast microscope Phase contrast microscopy )

To confirm the formation of large amounts of myotubes in myoblasts by orotic acid (0.5 μM), C2Cl2 cells were treated with 0.1% gelatin-coated coverslips and treated with vehicle DMSO and orotic acid for 3 days Were observed with a phase contrast microscope.

As a result of this experiment, it was confirmed that the myotube was formed more when the orotic acid (0.5 μM) was treated than the control group, DMSO (X100) (FIG. 4).

Example  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 1 × PBS, fixed with 3.7% paraformaldehyde for 15 min at room temperature, washed three times with 1 × PBS, permeabilized with permeabilization buffer, and incubated for 15 min at room temperature . After washing 3 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, DMSO (negative control) and orotic acid were each treated to induce the differentiation of C2Cl2 cell line. On the third day, in order to compare the degree of myocyte differentiation, the protein expression was confirmed by staining with MYH3 antibody. As a result, The expression of MYH3 was found to be very high when treated with orotic acid (0.5 μM) (FIG. 5).

Example  4-3. Western  Blat ( Western blot )

After the cells were cultured for 24 hours in a culture medium, differentiation was induced by treating DMSO and 0.5 μM orotic acid each day in the differentiation medium. 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, sonication was performed and centrifuged at 3000 rpm for 10 minutes to obtain a water-soluble protein. A 4 × sample buffer was added to the cells, The reaction was carried out for 5 minutes. 10 [mu] g of protein was loaded on a 12% SDS-PAGE gel and developed and transferred to a Watman membrane. The membrane was blocked with 5% skim milk for 1 hour at room temperature and then washed 5 times with TTBS (0.03% Tween 20, 2.42 g of Tris, 9 g of NaCl, 1 L of pH 7.4) 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. After the addition of TTBS containing 5% skim milk, the secondary antibody was added at a ratio of 1: 5000, followed by reaction at room temperature for 2 hours, washing with TTBS 5 times for 5 minutes, and ECL (Enhanced Chemiluminescent solution, Pierce) . The membrane was exposed to X-ray film to confirm the amount of protein.

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

These results show that the amount of myosin protein in the same amount of protein is rapidly increased by orotic acid, suggesting that the effect of orotic acid on promoting differentiation of myocytes is very high.

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 (13)

A composition for promoting the differentiation of myoblasts comprising orotic acid or a pharmaceutically acceptable salt thereof.
2. The composition of claim 1, wherein the concentration of the orotic acid or its pharmaceutically acceptable salt is from 0.01 to 2 [mu] M.
A method of promoting differentiation of myoblast cells comprising treating orotic acid or a pharmaceutically acceptable salt thereof with extracellular myoblasts.
Treating oolithic acid or a pharmaceutically acceptable salt thereof with an extracellular myoblast to differentiate a source cell.
A pharmaceutical composition for preventing or treating muscular weakness related to muscular weakness, comprising orotic acid or a pharmaceutically acceptable salt thereof.
6. The pharmaceutical composition according to claim 5, wherein the disease is myopenia, muscular atrophy or ataxia.
6. The pharmaceutical composition according to claim 5, wherein the concentration of the orotic acid or its pharmaceutically acceptable salt is 0.01 to 2 [mu] M.
6. The pharmaceutical composition according to claim 5, wherein the pharmaceutical composition promotes differentiation of myoblasts into myocytes.
A food composition for preventing or ameliorating muscular weakness related to muscles comprising orotic acid or a pharmaceutically acceptable salt thereof.
10. The food composition according to claim 9, wherein the disease is myopenia, muscular atrophy or cardiac atrophy.
10. The food composition according to claim 9, wherein the concentration of orotic acid or its pharmaceutically acceptable salt is 0.01 to 2 [mu] M.
A composition for strengthening muscle strength comprising orotic acid or a pharmaceutically acceptable salt thereof.
Orroctic acid or a pharmaceutically acceptable salt thereof.

Images