Muscle atrophy inhibitor and method for inhibiting muscle atrophy

Abstract

This muscular atrophy inhibitor contains a pyrimidine nucleotide or a precursor thereof as an active ingredient. This method is for inhibiting muscular atrophy through administration of a pyrimidine nucleotide or a precursor thereof.

Description

Technical Field

[0001] The present invention relates to a muscle atrophy inhibitor and a method for suppressing muscle atrophy.

Background Art

[0002] Skeletal muscle is the largest organ, accounting for about 40% of an adult's body weight. Skeletal muscle plays an important role not only in maintaining movement and posture, but also in maintaining body temperature through heat production and regulating blood glucose levels as a target organ for insulin.

[0003] The decline in motor function due to skeletal muscle atrophy causes falls, fractures, etc. This leads to a vicious cycle of further decline in motor function and muscle atrophy, ultimately causing defects in daily movements and reducing the quality of life (QOL). Therefore, countermeasures against muscle atrophy are an important issue for maintaining and improving QOL.

[0004] Exercise is an effective means of increasing skeletal muscle both quantitatively and qualitatively, but it is often difficult for the elderly and others to continuously perform exercise of sufficient intensity. Therefore, research is being conducted to enhance skeletal muscle function through nutritional approaches.

[0005] Skeletal muscle atrophy is caused by various factors and can be classified into several types depending on the cause of the symptoms, etc. It is known that skeletal muscle atrophies with aging, and such age-related muscle atrophy is called sarcopenia (age-related muscle wasting). Also, it is known that skeletal muscle atrophies not only due to aging, but also due to long-term disuse of muscles, such as in bedridden, overly sedentary, or microgravity environments, and this is called disuse muscle atrophy.

[0006] In recent years, it has become clear that enhanced ubiquitin-proteasome protein degradation is involved in muscle atrophy. Atrogin-1 and MuRF1 are ubiquitin-proteasome protein degradation genes whose expression increases during various types of muscle atrophy, including age-related muscle mass loss, and they play an important role in muscle atrophy (Non-Patent Literature 1).

[0007] Furthermore, it has been revealed that glucocorticoids are involved in the enhancement of the ubiquitin-proteasome protein degradation system. Dexamethasone, a type of synthetic glucocorticoid, induces protein degradation and muscle atrophy in muscle tissue by increasing the expression of Atrogin-1 and / or MuRF1, which are part of the ubiquitin-proteasome system, after binding to glucocorticoid receptors. These metabolic changes caused by dexamethasone are similar to those in muscle atrophy in animals or humans, and the dexamethasone-induced muscle atrophy model is widely used in muscle atrophy research (Non-Patent Literature 2). Therefore, materials that suppress the expression of Atrogin-1 and other ubiquitin-proteasome systems in the dexamethasone-induced muscle atrophy model are considered to be effective in suppressing muscle atrophy.

[0008] Recently, muscle atrophy inhibitors using food materials have been reported. For example, Patent Document 1 discloses a peptide that exhibits ubiquitin ligase inhibitory activity in vitro. Patent Document 2 also discloses a peptide that suppresses myotubular atrophy induced by dexamethasone treatment of myotubular cells derived from a mouse skeletal muscle cell line (C2C12), and that suppresses Atrogin-1 expression in dexamethasone-administered mouse models.

[0009] Cytidylic acid and uridylic acid are types of nucleotides, and are substances that are widely found in living organisms and in food, or added to food. They are extremely safe and are ideal materials for food additives. Patent document 3 describes an agent for improving emotional disorders that is characterized by containing nucleotides.

[0010] Patent Document 4 discloses an anti-fatigue agent or physical fitness enhancer containing uridine, uracil, uridylic acid, or a uridine derivative or a pharmaceutically acceptable salt thereof, and describes that in mice orally administered uridine, the limit running time on a treadmill was extended. Non-Patent Document 2 describes that rats administered a mixture of cytidylic acid and uridylic acid were able to withstand prolonged treadmill exercise.

[0011] However, the improvement in physical fitness described in Patent Document 4 and Non-Patent Document 2 refers to an improvement in the ability to continuously perform a certain state of exercise, such as on a treadmill, and it can be said that this evaluates a phenomenon different from the suppression of muscle atrophy due to aging or disuse of muscles.

[0012] Patent Document 5 describes a composition comprising uridine and / or uridylic acid and various other components, used to prevent or treat weakness in mammals, and states that one specific example of preventing or treating weakness is an increase in muscle mass.

[0013] However, the verification in the examples of Patent Document 5 is limited to stating that when Alzheimer's disease model mice were given a diet containing UMP and DHA, they gained more weight compared to mice given a control diet, and that when elderly human patients with cognitive impairment were given a beverage fortified with UMP, EPA, DHA, lecithin, choline, vitamin E, vitamin C, selenium, and B vitamins, their BMI increased compared to patients given a control beverage, and in particular, patients with a BMI of less than 26 showed an improvement in ADL (ADCS score).

[0014] In other words, while Patent Document 5 mentions muscle mass as one of the various symptoms included in "weakness," it only evaluates weight gain in both mouse and human studies, and does not directly measure muscle mass or examine related gene expression. Furthermore, Patent Document 5 only includes examples that assume Alzheimer's disease, and does not investigate the inhibitory effect on muscle atrophy in general.

[0015] Furthermore, the beverage administered to elderly patients contained a variety of ingredients other than UMP, and it cannot be concluded from this study that UMP itself has an effect on improving weight loss. ADL is defined as the ability to independently perform daily living activities such as bathing, getting dressed, and moving around, and is by no means an indicator that focuses on muscle strength.

[0016] Therefore, it has not been previously investigated whether nucleotides such as cytidylic acid and uridylic acid, or nucleosides such as cytidine and uridine, actually have an inhibitory effect on muscle atrophy, and it was completely unclear. [Prior art documents] [Patent Documents]

[0017] [Patent Document 1] Japanese Patent Publication No. 2016-160183 [Patent Document 2] Re-tabled publication No. 2018-105550 [Patent Document 3] Japanese Patent Application Publication No. 10-203989 [Patent Document 4] Japanese Patent Publication No. 2010-248161 [Patent Document 5] Japanese Patent Publication No. 2017-061466 [Non-patent literature]

[0018] [Non-Patent Document 1] Stewart H. Lecker and 8 others, “Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression,” FASEB J. 18:39-51, 2004 [Non-Patent Document 2] Michael Menconi et al., "Dexamethasone and corticosterone include similar, but not identical, muscle wasting responses in cultured L6 and C2C12 myotubes", J Cell. Biochem., 105, 353-364, 2008 SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0019] An object of the present invention is to provide a novel and highly safe muscle atrophy inhibitor that exhibits a muscle atrophy inhibitory effect when used as food, feed, pharmaceuticals, quasi-drugs, etc. MEANS FOR SOLVING THE PROBLEMS

[0020] As a result of intensive studies to achieve the above object, the inventors of the present application have first found that pyrimidine nucleotides or their precursors have not only the conventionally known effect of improving physical strength but also a clear effect in suppressing muscle atrophy, and have thus completed the present invention. That is, the present invention One or more selected from the group consisting of cytidylic acid and uridylic acid. containing as an active ingredient do is a muscle atrophy inhibitor. Further, the present invention One or more selected from the group consisting of cytidylic acid and uridylic acid. by administering )in is a method for suppressing muscle atrophy (excluding humans as the application target). EFFECTS OF THE INVENTION

[0021] The muscle atrophy inhibitor containing pyrimidine nucleotides or their precursors as an active ingredient according to the present invention leads to providing a new means for improving the QOL of diseased and elderly people caused by muscle atrophy. Further, the muscle atrophy inhibitor of the present invention can also be used for prevention by using it before the onset of muscle atrophy. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] [Figure 1] Figure 1 shows a 100x magnification photograph illustrating the inhibitory effect of uridylic acid on dexamethasone-induced muscle atrophy in C2C12 cells of Example 1-1. In the figure, Dex refers to dexamethasone, and UMP refers to disodium uridylate. [Figure 2] Figure 2 shows the inhibitory effect of uridylic acid on dexamethasone-induced muscle atrophy in myotube diameter in Example 1-1. In the figure, Dex: dexamethasone, UMP: disodium uridylate, error bars: standard error, *: p < 0.05. [Figure 3] Figure 3 shows the inhibitory effect of cytidylic acid, uridylic acid, cytidine, and uridine treatment on dexamethasone-induced muscle atrophy in C2C12 cells of Examples 1-2, as shown in 100x magnification photographs. In the figure, Dex(-) means untreated with dexamethasone, Dex(+) means treated with dexamethasone, CMP means disodium cytidylic acid, and UMP means disodium uridylic acid. [Figure 4] Figure 4 shows the inhibitory effect of cytidylic acid and cytidine treatment on dexamethasone-induced muscle atrophy in C2C12 cells of Examples 1-2. In the figure, Dex: dexamethasone, Cyd: cytidine, CMP: disodium cytidylate, n=6, error bars: standard error, *: p < 0.05. [Figure 5] Figure 5 shows the inhibitory effect of uridylic acid treatment on dexamethasone-induced muscle atrophy in C2C12 cells of Examples 1-2. In the figure, Dex: dexamethasone, UMP: disodium uridylate, n=6, error bars: standard error, *: p < 0.05. [Figure 6] Figure 6 shows the inhibitory effect of uridine treatment on dexamethasone-induced muscle atrophy in C2C12 cells of Examples 1-2. In the figure, Dex: dexamethasone, n=6, error bars: standard error, *: p < 0.05. [Figure 7]Figure 7 shows the inhibitory effect of the combined use of cytidylic acid and uridylic acid in Example 2 on dexamethasone-induced muscle atrophy. In the figure, Dex: dexamethasone, CMP: disodium cytidylic acid, UMP: disodium uridylic acid, n=6, error bars: standard error, *: p < 0.05. [Figure 8] Figure 8 shows the inhibitory effect on dexamethasone-induced muscle atrophy of the combined use of cytidylic acid and uridylic acid, and cytidine and uridine, in Example 3. In the figure, Dex: dexamethasone, CMP: disodium cytidylic acid, UMP: disodium uridylic acid, Cyd: cytidine, Urd: uridine, n=6, error bars: standard error, *: p < 0.05. 1mM CMP+UMP means that a total of 2mM of cytidylic acid + uridyl acid was added, and 1mM Cyd+Urd means that a total of 2mM of cytidine + uridine was added. [Figure 9] Figure 9 shows the effect of uridylic acid on suppressing Atrogin-1 gene expression in Example 4. In the figure, Dex: dexamethasone, UMP: disodium uridylate, n=6, error bars: standard error, *: p < 0.05. [Figure 10] Figure 10 shows the effect of uridine on suppressing Atrogin-1 gene expression in Example 4. In the figure, Dex means dexamethasone, n=6, error bars mean standard error, and * means p < 0.05. [Figure 11] Figure 11 shows the effect of uridylic acid on suppressing MuRF1 gene expression in Example 4. In the figure, Dex: dexamethasone, UMP: disodium uridylate, n=6, error bars: standard error, *: p < 0.05. [Figure 12] Figure 12 shows the MuRF1 gene expression suppression effect of uridine in Example 4. In the figure, Dex: dexamethasone, n=6, error bars: standard error, *: p < 0.05. [Modes for carrying out the invention]

[0023] The present invention relates to a muscle atrophy inhibitor containing pyrimidine nucleotide or its precursor as an active ingredient.

[0024] In the present invention, muscle atrophy refers to the reduction in muscle mass and muscle wasting that occurs when skeletal muscle, which makes up the muscle, is broken down.

[0025] In the present invention, the suppression of muscle atrophy refers to preventing, delaying, reversing, or preventing the progression of the aforementioned muscle atrophy.

[0026] Furthermore, the suppression of muscle atrophy in the present invention may also be the suppression of the expression of genes related to the ubiquitin-proteasome protein degradation system, and as a gene related to the ubiquitin-proteasome protein degradation system, it may be the suppression of the expression of the Atrogin-1 gene. The fact that the suppression of the expression of genes related to the ubiquitin-proteasome protein degradation system is involved in the suppression of muscle atrophy is known, for example, from Non-Patent Documents 1 and 2.

[0027] The muscle atrophy inhibitor of the present invention contains pyrimidine nucleotides or their precursors as active ingredients. Pyrimidine nucleotides or their precursors are suitable as active ingredients of the present invention in terms of safety as a food product and ease of absorption into the body.

[0028] In this specification, pyrimidine nucleotides mean cytidylic acid and uridylic acid.

[0029] Cytidylic acid (cytidine monophosphate, CMP) is a compound represented by CAS registry number 63-37-6. In this specification, the term "cytidylic acid" includes salts of cytidylic acid.

[0030] In this specification, when the mass of cytidylic acid is stated, it shall be the mass converted to disodium cytidylic acid salt (CMP,2Na). When the concentration (%) of cytidylic acid is stated, unless otherwise specified, it shall be the mass / volume percentage concentration (w / v%), and the mass of cytidylic acid shall be the mass converted to CMP,2Na. When a salt other than the disodium salt is selected, or in the case of a free acid that does not form a salt, the amount of cytidylic acid shall be used as the basis, and the mass shall be converted to CMP,2Na.

[0031] Uridylic acid (uridine monophosphate, UMP) is a compound represented by CAS registry number 58-97-9. In this specification, the term "uridylic acid" includes salts of uridylic acid.

[0032] In this specification, when the mass of uridylic acid is stated, it shall be the mass converted to disodium uridylate (UMP,2Na). When the concentration (%) of uridylic acid is stated, unless otherwise specified, it shall be the mass-volume percentage concentration (w / v%), and the mass of uridylic acid shall be the UMP,2Na converted mass. When a salt other than the disodium salt is selected, or in the case of a free acid that does not form a salt, the amount of uridylic acid shall be used as the basis, and the mass shall be converted to UMP,2Na.

[0033] In this specification, a pyrimidine nucleotide precursor means a compound that can be metabolized to pyrimidine nucleotides, i.e., cytidylic acid and / or uridylic acid. Whether a compound is included as a pyrimidine nucleotide precursor is determined by whether or not it is known to be converted to a pyrimidine nucleotide. Specifically, cytidine diphosphate, cytidine triphosphate, uridine diphosphate, and uridine triphosphate (Isao Matsuoka, "Ectonucleotidase in the Nervous System", Clinical Chemistry 33:11-18, 2004), which are known to be broken down to cytidylic acid and / or uridylic acid by the action of ectonucleotidases, etc., and cytidine, cytosine, uridine, and uracil (A. Orengo, "Regulation of enzymic activity by metabolites. I. Uridine-cytidine kinase of Novikoff ascites rat tumor", J Biol Chem. 1969 Apr 25;244(8):2204-9.), which are known to be phosphorylated to cytidylic acid and / or uridylic acid by the action of kinases, are exemplified as pyrimidine nucleotide precursors in this specification.

[0034] Examples of pyrimidine nucleotides or their precursors in the present invention include, as mentioned above, cytidine, cytosine, cytidylic acid, cytidine diphosphate, cytidine triphosphate, uridine, uracil, uridylic acid, uridylic acid, and uridylic triphosphate. Among these, cytidylic acid, uridylic acid, cytidine, and uridine are preferred.

[0035] As mentioned above, the concept of cytidylic acid in the present invention includes salts. Examples of salts of cytidylic acid include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt, magnesium salt and barium salt; basic amino acid salts such as arginine and lysine; ammonium salts such as ammonium salt and tricyclohexylammonium salt; and various alkanolamine salts such as monoethanolamine salt, diethanolamine salt, triethanolamine salt, monoisopropanolamine salt, diisopropanolamine salt and triisopropanolamine. Preferably, it is an alkali metal salt such as sodium salt. Specifically, examples of such alkali metal salts include monosodium cytidylate and disodium cytidylate, and disodium cytidylate is preferred from the standpoint of ease of handling.

[0036] As mentioned above, the concept of uridylic acid in the present invention includes salts. Examples of uridylic acid salts include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt, magnesium salt and barium salt; basic amino acid salts such as arginine and lysine; ammonium salts such as ammonium salt and tricyclohexylammonium salt; and various alkanolamine salts such as monoethanolamine salt, diethanolamine salt, triethanolamine salt, monoisopropanolamine salt, diisopropanolamine salt and triisopropanolamine. Preferably, it is an alkali metal salt such as sodium salt. Specifically, examples of such alkali metal salts include monosodium uridylate and disodium uridylate, and disodium uridylate is preferred from the standpoint of ease of handling.

[0037] The aforementioned active ingredients may be used alone or in combination of two or more.

[0038] There are no particular restrictions on the origin of the active ingredients, but those derived from natural products such as yeast, bacteria, fish and shellfish, animals, and plants are preferred.

[0039] The muscle atrophy inhibitor of the present invention can be put into practical use as a composition of foods and beverages, supplements, powdered milk, enteral nutrition formulas, health foods and beverages (including foods for specified health uses and foods with functional claims), additives for animal feed, pharmaceuticals for humans or non-human animals, etc.

[0040] When the agent of the present invention is used as food or beverage, health food or powdered milk, etc., the active ingredient can be appropriately added to known food or beverages to create a food or beverage that has a muscle atrophy-inhibiting effect. Examples of target food and beverages include milk and dairy products, seasonings, beverages, confectionery, bread, noodles, oils and fats, processed meat products, processed seafood products, processed agricultural products, frozen foods, instant foods, etc.

[0041] Furthermore, it is possible to manufacture new food and beverage products that have a muscle atrophy-inhibiting effect by mixing the active ingredient into the raw materials of the food and beverage product. The target food and beverage product can be in various forms, such as tablets, granules, capsules, powders, solutions, syrups, milk, pastes, etc. In the manufacture of these food and beverage products, in addition to the active ingredient of the present invention, various excipients and flavorings that can be used as food should be added as appropriate.

[0042] The aforementioned food and beverage may be provided and sold as a food or beverage labeled with a health claim for inhibiting muscle atrophy. The act of "labeling" includes all acts to inform consumers of the aforementioned use, and any expression that can evoke or infer the aforementioned use, regardless of the purpose of the labeling, the content of the labeling, or the object or medium to which it is displayed, falls under the "labeling" act of this technology.

[0043] The aforementioned "display" is preferably made in a manner that allows consumers to directly recognize the above-mentioned use. Specifically, this includes acts such as transferring, delivering, displaying for transfer or delivery, or importing food and beverage products or product packaging on which the above-mentioned use is described; displaying or distributing advertisements, price lists, or transaction documents related to products that describe the above-mentioned use; or providing information containing such information by electromagnetic means (such as the Internet).

[0044] The content of the display should preferably be a display approved by the government or other administrative body (for example, a display approved under various systems established by the government and made in accordance with such approval). Furthermore, it is preferable to attach such display content to packaging, containers, catalogs, brochures, point-of-sale (POP) materials and other documents used at sales sites.

[0045] Furthermore, when the muscle atrophy inhibitor of the present invention is put into practical use as a pharmaceutical, supplement, enteral nutritional supplement, etc., the active ingredient can be formulated alone or in combination with formulation aids, etc. The method of administration of the formulation may be orally or parenterally, but oral or enteral administration is preferred.

[0046] The form of the aforementioned preparation can be, if administered orally, tablets, granules, capsules, powders, solutions, syrups, emulsions, etc., or if administered parenterally, injections, sprays, ointments, patches, etc.

[0047] In the aforementioned formulation, in addition to the active ingredient of the present invention, any formulation aids such as excipients, binders, disintegrants, lubricants, flavoring and odor-correcting agents, solubilizers, suspending agents, and coating agents may be used in appropriate combinations according to their respective delivery forms.

[0048] The amount of the active ingredient in the muscle atrophy inhibitor of the present invention may be appropriately selected from the range of 0.1 to 30% (w / w) depending on the purpose of use (prevention, health care, or symptom relief, etc.), the age of the target person, the method of administration or intake, the dosage form, etc.

[0049] The dosage of the muscle atrophy inhibitor of the present invention may vary depending on the subject's age, weight, degree of muscle atrophy, and method of administration or intake, but it can be appropriately selected from a range of approximately 1 mg to 800 g per day. [Examples]

[0050] The present invention will be described below with reference to examples, but the present invention is not limited in any way by these examples. (Example 1) Investigation of the effect of inhibiting myotubular atrophy To evaluate the inhibitory effect of pyrimidine nucleotides or their precursors on myotubular atrophy, we investigated the extent to which pyrimidine nucleotides or their precursors suppress dexamethasone-induced muscle atrophy.

[0051] (Example 1-1) Mouse myoblast cell line C2C12 cells (RIKEN BRC, RCB0987) were suspended in growth medium (Dulbecco's modified Eagle's Medium containing 10% FBS, 100 units / ml penicillin, and 100 μg / ml streptomycin), seeded in a 24-well plate, and cultured in an incubator at 37°C and 5% CO2 until the cell density reached 70-90%.

[0052] The growth medium was removed and replaced with differentiation induction medium (Dulbecco's modified Eagle's Medium containing 2% adult bovine serum, penicillin 100 units / ml, and streptomycin 100 μg / ml). The cells were cultured for 4 days, changing the medium every two days, to differentiate into myotubes. The medium was removed and replaced with the following four types of media.

[0053] (1) Differentiation induction medium that does not contain dexamethasone or disodium uridylate [Dex(-), UMP(-)] (2) Differentiation induction medium containing 100 μM dexamethasone but not disodium uridylate [Dex(+), UMP(-)] (3) Differentiation induction medium containing 100 μM dexamethasone and 2 mM disodium uridylate [Dex(+), 2 mM UMP] (4) Differentiation induction medium containing 100 μM dexamethasone and 5 mM disodium uridylate [Dex(+), 5 mM UMP].

[0054] The culture medium was changed again after 24 hours. After 48 hours, photographs were taken at 100x magnification at five locations near the center of each well. Using the image analysis software ImageJ, the diameters of 10 myotubes were measured for each photograph, in descending order of diameter, and the average of 50 measurements was taken as the myotube diameter of each well. For statistical analysis, Dunnett's multiple comparison test was performed using the control group (differentiation induction medium containing 100 μM dexamethasone but no uridylic acid) as a control. The threshold for statistical significance was set at 5%. Representative photographs of each group are shown in Figure 1. Graphs showing the statistically processed myotube diameters for each group are shown in Figure 2.

[0055] As shown in Figures 1 and 2, the diameter of myotubes decreased with the addition of dexamethasone, but this decrease was suppressed in cells treated with uridylic acid. These results indicate that uridylic acid has an excellent inhibitory effect on myotube atrophy.

[0056] (Examples 1-2) Mouse myoblast cell line C2C12 cells (RIKEN BRC, RCB0987) were suspended in growth medium (Dulbecco's modified Eagle's Medium containing 10% FBS, 100 units / ml penicillin, and 100 μg / ml streptomycin), seeded in a 24-well plate, and cultured in an incubator at 37°C and 5% CO2 until the cell density reached 70-90%.

[0057] The growth medium was removed and replaced with differentiation induction medium (Dulbecco's modified Eagle's Medium containing 2% adult bovine serum, penicillin 100 units / ml, and streptomycin 100 μg / ml). The cells were cultured for 4 days, changing the medium every two days, to differentiate into myotubes. The medium was removed and replaced with the following medium. (1) Differentiation induction medium without dexamethasone [Dex(-)] (2) Differentiation induction medium containing 100 μM dexamethasone [Dex(+)] (3) Differentiation induction medium containing 100 μM dexamethasone and a specified amount of disodium cytidylate [Dex(+), specified amount of CMP] (4) Differentiation induction medium containing 100 μM dexamethasone and a specified amount of cytidine [Dex(+), specified amount of cytidine (sometimes abbreviated as "Cyd")], (5) Differentiation induction medium containing 100 μM dexamethasone and a specified amount of disodium uridylate [Dex(+), specified amount of UMP], (6) Differentiation induction medium containing 100 μM dexamethasone and a specified amount of uridine [Dex(+), specified amount of Uridine].

[0058] After culturing for 3 days, photographs were taken at 100x magnification at five locations near the center of each well. Using the image analysis software ImageJ, the diameters of 10 myotubes were measured for each photograph, in descending order of diameter, and the average of these 50 measurements was taken as the myotube diameter for each well. For statistical analysis, a t-test or Dunnett's multiple comparison test was performed using the differentiation induction medium treatment group, which was supplemented with 100 μM dexamethasone alone, as the control group. The threshold for statistical significance was set at 5%. Representative photographs of each group are shown in Figure 3.

[0059] As shown in Figure 3, dexamethasone treatment reduced the diameter of myotubes, but treatment with cytidylic acid, uridylic acid, cytidine, and uridine significantly increased the diameter.

[0060] Graphs showing the statistically processed myotube cell diameter for each group are shown in Figures 4 to 6.

[0061] As shown in Figure 4, dexamethasone treatment reduced the diameter of myotubes, while cytidylic acid and cytidine treatment increased the diameter of myotubes.

[0062] As shown in Figure 5, dexamethasone treatment reduced the diameter of myotubes, while uridylic acid treatment increased the diameter of myotubes.

[0063] As shown in Figure 6, dexamethasone treatment reduced the diameter of myotubes, while uridine treatment increased the diameter of myotubes.

[0064] Based on these results, it was found that cytidylic acid, uridylic acid, cytidine, and uridine have an inhibitory effect on myotubular atrophy.

[0065] (Example 2) Investigation of the inhibitory effect of combined use of cytidylic acid and uridylic acid on myotubular cell atrophy. To investigate whether a stronger inhibitory effect on myotubular atrophy is achieved by combining cytidylic acid and uridylic acid, the inhibitory effect on myotubular atrophy by combining cytidylic acid and uridylic acid was evaluated using the same method as in Examples 1-2.

[0066] Cytidylic acid and uridylic acid were added to the culture medium at the concentrations shown in Figure 7, so that the total concentration of cytidylic acid and uridylic acid was 1000 μM. The inhibitory effect on myotubular cell atrophy of the sample was evaluated using the same procedure as in Example 1-2. The results are shown in Figure 7.

[0067] As shown in Figure 7, dexamethasone treatment reduced the diameter of myotubes, but cell diameter increased in all samples treated with either cytidylic acid or uridylic acid. All samples showed an inhibitory effect on muscle atrophy, but a particularly significant inhibitory effect was observed in samples using cytidylic acid in amounts equal to or greater than that of uridylic acid.

[0068] (Example 3) Investigation of the inhibitory effect of combination therapy with cytidylic acid and uridylic acid, and combination therapy with cytidine and uridine on myotubular cell atrophy. To investigate whether a stronger inhibitory effect on myotubular atrophy is achieved by combining cytidylic acid and uridylic acid, the inhibitory effect on myotubular atrophy by combining cytidylic acid and uridylic acid was evaluated using the same method as in Examples 1-2.

[0069] In Example 2, the total concentration of cytidylic acid / uridylic acid added to the culture medium was standardized to 1000 μM, and the cytidylic acid / uridylic acid ratio was varied. In Example 3, the inhibitory effect on myotubular atrophy was evaluated when 1 mM (1000 μM) of cytidylic acid + 1 mM (1000 μM) of uridylic acid was used, for a total concentration of 2000 μM. Similarly, the combined effects of cytidine and uridine were also evaluated. These results are shown in Figure 8.

[0070] As shown in Figure 8, dexamethasone treatment reduced myotubular diameter, but treatment with cytidylic acid, uridylic acid, cytidine, and uridine significantly increased it. Furthermore, the combined use of cytidylic acid and uridylic acid, and the combined use of cytidine and uridine, significantly increased myotubular diameter.

[0071] Based on these results, additive inhibitory effects on myotubular atrophy were observed with the combined use of cytidylic acid and uridylic acid, and with the combined use of cytidine and uridine.

[0072] (Example 4) Effect of suppressing the expression of muscle atrophy-related genes (Atrogin-1, MuRF1) Mouse myoblast cell line C2C12 cells were suspended in growth medium, seeded in a 24-well plate, and cultured in a 37°C, 5% CO2 incubator until the cell density reached 70-90%. The growth medium was removed and replaced with differentiation induction medium. The cells were cultured for 6 days, changing the medium every two days, to differentiate them into myotubes. The medium was removed and replaced with differentiation induction medium or differentiation induction medium containing the test substance. After 48 hours, the medium was removed and replaced with differentiation induction medium, differentiation induction medium supplemented with 1 μM dexamethasone only, or medium supplemented with 1 μM dexamethasone and the test substance (specified amount of UMP (disodium uridylate), specified amount of Urd (uridine), specified amount of UMP (disodium uridylate)).

[0073] After 24 hours, the culture medium was removed, and total RNA was extracted from the cells using either the RNeasy Mini kit (QIAGEN) or NucleoSpin RNA (Takara Bio).

[0074] Using this total RNA as a template, a reverse transcription reaction mixture was prepared using the ReverTra Ace(R) qPCR RT Kit (Toyobo). The mRNA expression levels of Atrogin-1, MuRF1, and β-actin (as an internal standard) were measured using the reverse transcription reaction mixture, GoTaq(R) qPCR Master Mix (Promega), and a real-time PCR instrument, the Thermal Cycler Dice Real Time System (Takara Bio). Analysis was performed by relative quantification, and the mRNA expression level was corrected using β-actin mRNA expression as an endogenous control. For statistical analysis, Dunnett's multiple comparison test was performed using a differentiation induction medium treatment group treated with only 100 μM dexamethasone as a control. The threshold for statistical significance was set at 5%. These results are shown in Figures 9-12.

[0075] As shown in Figure 9, dexamethasone treatment increased Atrogin-1 mRNA expression, while uridylic acid treatment decreased Atrogin-1 mRNA expression.

[0076] As shown in Figure 10, dexamethasone treatment increased Atrogin-1 mRNA expression, while uridine treatment decreased Atrogin-1 mRNA expression.

[0077] As shown in Figure 11, dexamethasone treatment increased MuRF1 mRNA expression, while uridylic acid treatment decreased MuRF1 mRNA expression.

[0078] As shown in Figure 12, dexamethasone treatment increased MuRF1 mRNA expression, while uridine treatment decreased MuRF1 mRNA expression.

[0079] Based on these results, it was found that uridylic acid and uridine have excellent inhibitory effects on the expression of muscle atrophy-related genes (Atrogin-1, MuRF1).

[0080] It should be noted that the suppressive effect on the expression of muscle atrophy-related genes demonstrated in this embodiment does not necessarily explain the entire mechanism of action of the muscle atrophy-inhibiting effect of the present invention. As described in Example 3, the additive effect achieved by the combined use of cytidylic acid and uridylic acid suggests that the muscle atrophy-inhibiting effect of the present invention may not be due to a single mechanism of action, but rather to multiple mechanisms of action.

Claims

1. A muscle atrophy inhibitor containing one or more selected from the group consisting of cytidylic acid and uridylic acid as an active ingredient.

2. The muscle atrophy inhibitor according to claim 1, wherein the inhibition of muscle atrophy is the inhibition of the expression of Atrogin-1 and / or MuRF1.

3. A method for suppressing muscle atrophy by administering one or more substances selected from the group consisting of cytidylic acid and uridylic acid (however, the target of application is not humans).

4. The method according to claim 3, wherein the method for suppressing muscle atrophy is by suppressing the expression of Atrogin-1 and / or MuRF1.

Images