Composition for preventing or treating muscle diseases containing an oxicam compound

Abstract

The present invention relates to a composition for preventing, improving or treating muscle diseases, which contains an oxicam compound, and the composition increases muscle mass and strengthens muscle strength through the effects of promoting myoblast differentiation and increasing muscle fibers, thereby having a therapeutic effect on various muscle diseases and having an effect of strengthening muscle strength or increasing athletic ability.

Description

[Technical Field]

[0001] The present invention relates to a composition containing an oxicam compound for preventing, ameliorating or treating a muscle disease. [Background technology]

[0002] Muscles play an important role in bodily functions such as energy metabolism and athletic performance, and can be damaged or weakened by a variety of factors, including sarcopenia due to aging, muscle atrophy due to nutritional imbalance or lack of exercise, other diseases such as other cancers, and aging.

[0003] Sarcopenia, a major muscle-damaging disease, is a condition in which muscle strength declines as skeletal muscle mass decreases with aging. The most notable feature of sarcopenia is a decrease in muscle mass, which can also lead to a change in muscle fiber type. While type 1 and type 2 muscle fibers decrease at similar rates with aging, type 1 muscle fibers decrease in thickness more significantly in sarcopenic patients. Sarcopenia has been reported to cause muscle weakness and functional impairment in the elderly (Roubenoff R., Can. J. Appl. Physiol. 26, 78-89, 2001).

[0004] Muscle atrophy is induced by malnutrition or prolonged muscle disuse, and occurs when the balance between normal protein synthesis and breakdown is disrupted, causing proteins in the muscles to break down.

[0005] Various treatment methods are being developed to fundamentally treat these muscle diseases, and in particular, treatment methods using mechanisms that promote muscle strengthening or muscle regeneration by promoting differentiation of stem cells into muscle cells have been proposed. These methods can fundamentally treat muscle diseases, so research into various therapeutic substances that make this possible is currently needed.

[0006] The present inventors have conducted research into various compounds that have the effect of improving muscle diseases through the above-mentioned effects, and have thus completed the present invention. Summary of the Invention [Problem to be solved by the invention]

[0007] The present inventors have discovered that oxicam compounds promote the differentiation of myoblasts, improve muscle energy metabolism, and strengthen muscle strength, and are particularly effective in treating muscle diseases such as muscular atrophy, and have thus completed the present invention.

[0008] Therefore, an object of the present invention is to provide a pharmaceutical composition for preventing or treating a muscle disease, comprising an oxicam compound or a pharmaceutically acceptable salt thereof.

[0009] Another object of the present invention is to provide a functional health food composition for preventing or improving muscle diseases, which comprises an oxicam compound or a nutrient-based acceptable salt thereof.

[0010] It is yet another object of the present invention to provide a muscle strengthening composition comprising an oxicam compound.

[0011] It is yet another object of the present invention to provide a composition for enhancing athletic performance comprising an oxicam compound.

[0012] A further object of the present invention is to provide a composition for promoting muscle stem cell differentiation, comprising an oxicam compound.

[0013] It is yet another object of the present invention to provide a composition for muscle regeneration comprising an oxicam compound.

[0014] It is yet another object of the present invention to provide a composition for increasing muscle mass, comprising an oxicam compound.

[0015] It is yet another object of the present invention to provide a feed additive composition comprising an oxicam compound. [Means for solving the problem]

[0016] To achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating a muscle disease, comprising an oxicam compound or a pharmaceutically acceptable salt thereof.

[0017] In order to achieve another object of the present invention, the present invention provides a functional health food composition for preventing or improving a muscle disease, which comprises an oxicam compound or a nutrient-acceptable salt thereof.

[0018] In order to achieve still another object of the present invention, the present invention provides a composition for strengthening muscle, which comprises an oxicam compound.

[0019] In order to achieve yet another object of the present invention, the present invention provides a composition for enhancing exercise performance, which comprises an oxicam compound.

[0020] In order to achieve yet another object of the present invention, the present invention provides a composition for promoting muscle stem cell differentiation, comprising an oxicam compound.

[0021] In order to achieve still another object of the present invention, the present invention provides a composition for increasing muscle mass, which comprises an oxicam compound.

[0022] In order to achieve still another object of the present invention, the present invention provides a feed additive composition containing an oxicam compound. [Effects of the Invention]

[0023] The present invention relates to a composition for preventing, improving, or treating muscle diseases, which contains an oxicam compound. The composition increases muscle mass and strengthens muscle strength through the effects of promoting myoblast differentiation and regenerating muscle fibers, and has therapeutic effects on various muscle diseases, so it may be used as a pharmaceutical or health functional food composition for these diseases. [Brief explanation of the drawings]

[0024] [Figure 1] FIG. 1 shows the effect of oxicam compounds on myoblast differentiation, comparing the relative expression levels of Myo and eMHC when myoblasts were treated with each compound. [Figure 2] FIG. 2 shows the results showing the degree of differentiation of myoblasts (C2C12) depending on the administration concentration of meloxicam. [Figure 3] FIG. 3 shows the results of confirming the expression level of MHC, a myoblast differentiation marker, depending on the administration concentration of meloxicam. [Figures 4a-4b. 5a-5b] Figures 4a and 4b show the results of confirming the expression level of MHC, a myoblast differentiation marker, following administration of meloxicam in human muscle stem cells from 17 years old, and Figures 5a and 5b show the results of confirming the expression level of MHC, a myoblast differentiation marker, in human muscle stem cells from 66 years old. [Figure 6a-6b] FIG. 6a shows the change in body weight after administration of meloxicam, and FIG. 6b shows the muscle mass increasing effect confirmed through the change in weight of hind leg muscles (TA, EDL, SOL, GAS) after administration of meloxicam. [Figure 7a-7b] FIG. 7a shows the change in body weight due to meloxicam administration, and FIG. 7b shows the change in weight of organs other than skeletal muscle. [Figure 8a-8b] Figures 8a and 8b show the muscle regeneration and muscle fiber size confirmed by H&E staining when meloxicam was administered to a mouse model with CTX-induced muscle damage on days 4, 7, and 21, respectively. [Figures 9a-9c] Figure 9a shows the relative expression levels of muscle fiber types when meloxicam was administered to a mouse model with muscle damage induced by CTX. Figure 9b shows the size of the muscle fiber cross-sectional area, and Figure 9c shows the area of ​​the expression type (MHC II type). [Figure 10] Figure 10 shows the cross-sectional area (Fig. 10a) and relative proportion (Fig. 10b) of muscle fibers in which GPDH, a glycolytic enzyme, was activated when meloxicam was administered to a mouse model of muscle damage induced by CTX. [Figure 11]FIG. 11 shows the results of grip strength test and motor performance test when meloxicam was administered to a mouse model in which muscle damage was induced by CTX. [Figure 12] FIG. 12 shows the results of comparing changes in body weight and blood glucose (FIG. 12a) and changes in hind leg muscle weight (FIG. 12b) when meloxicam was administered to 28-month-old aged mice. [Figure 13] FIG. 13 shows the results of comparing the relative expression levels of muscle fiber types when 28-month-old aged mice were administered meloxicam. [Figure 14] FIG. 14 shows the results of comparing the changes in motor performance in 28-month-old aged mice administered with meloxicam through the rotarod test. [Figure 15] Figure 15 shows the results of comparing changes in body weight and grip strength (Figure 15a) and changes in motor performance (Figure 15b) when meloxicam was administered to 14-month-old middle-aged mice. [Figure 16] Figure 16 compares the changes in body weight (Figure 16a) and intake (Figure 16b) when meloxicam was administered to 4-month-old young mice, and Figure 17 compares the changes in grip strength and motor performance (Figure 17a) and muscle mass relative to body weight (Figure 17b). DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention relates to a pharmaceutical composition for preventing or treating a muscle disease, comprising an oxicam compound or a pharmaceutically acceptable salt thereof.

[0026] The present invention relates to a functional health food composition for preventing or improving muscle diseases, which comprises an oxicam compound or a nutrient-acceptable salt thereof.

[0027] The present invention relates to a muscle strengthening composition containing an oxicam compound.

[0028] The present invention relates to a composition for enhancing exercise performance that contains an oxicam compound.

[0029] The present invention relates to a composition for promoting muscle stem cell differentiation, which comprises an oxicam compound.

[0030] The present invention relates to a composition for muscle regeneration containing an oxicam compound.

[0031] The present invention relates to a composition for increasing muscle mass, which comprises an oxicam compound.

[0032] The present invention relates to a feed additive composition containing an oxicam compound.

[0033] The present invention will be described in detail below.

[0034] As one aspect of the present invention, the present invention may include the oxicam compound and its pharmaceutically acceptable salts. The oxicam compound (oxicam compound) of the present invention is a compound widely used as a non-steroidal anti-inflammatory agent, and more specifically, may include lornoxicam, piroxicam, tenoxicam, ampiroxicam, droxicam, meloxicam, clonotexicam, tenoxicam, and piroxicam, and more preferably, may be meloxicam (Chemical Formula 1), piroxicam (Chemical Formula 2), tenoxicam (Chemical Formula 3), or lornoxicam (Chemical Formula 4), but is not limited thereto. The oxicam compound is represented by the following chemical formula:

[0035] [ka]

[0036] [ka]

[0037] [ka]

[0038] [ka]

[0039] In one embodiment of the present invention, the oxicam compound of the present invention has an effect of preventing or treating muscle diseases induced by aging, muscle decline, muscle wasting, muscle degeneration, or muscle damage. The term "muscle disease" refers to a state in which muscle strength is weakened due to muscle damage or loss caused by aging or disease, and can be caused by a variety of causes, including genetic predisposition, age-related diseases such as hypertension, impaired glucose tolerance, diabetes, obesity, dyslipidemia, atherosclerosis, or cardiovascular disease, chronic diseases such as cancer, autoimmune diseases, infectious diseases, AIDS, chronic inflammatory diseases, arthritis, malnutrition, kidney disease, chronic obstructive pulmonary disease, emphysema, rickets, chronic lower spinal pain, peripheral nerve damage, central nervous system damage, and chemical damage, fractures, trauma, or loss of movement due to long-term bed rest, or aging.

[0040] The muscle disease may include, but is not limited to, at least one muscle disease selected from the group consisting of atony, muscle atrophy, muscle degeneration, muscle rigidity, amyotrophic lateral sclerosis, myasthenia, cachexia, and senile sarcopenia, specifically muscle-related diseases caused by senile muscular atrophy or cancer, more specifically muscle atrophy, muscle degeneration, muscle rigidity, amyotrophic lateral sclerosis, myasthenia, cachexia, senile sarcopenia, and muscle wasting caused by senile muscular atrophy or cancer. That is, in one embodiment, the muscle disease means a disease selected from the group including atony, muscular atrophy, muscle degeneration, muscular rigidity, amyotrophic lateral sclerosis, myasthenia, cachexia, and sarcopenia.

[0041] In the present invention, the preventive, therapeutic, or ameliorative effect on muscle diseases may be due to the promotion of myoblast differentiation. In one embodiment of the present invention, it was confirmed that oxicam compounds promote myoblast differentiation. The term "myoblast differentiation" refers to the process in which mononuclear myoblasts fuse to form multinuclear myotubes. Cells in the differentiation stage that form myotubes may be distinguished using markers such as Pax7-, MyoD+, and MyoG. Cells in the early differentiation stage that form myotubes show increased expression of myogenic transcription factors such as myosin D (MyoD), and in the intermediate stage, myosin G (MyoG) shows increased expression. In the late stage, when differentiation is almost complete, the expression of myosin heavy chain (MHC) increases. In one embodiment of the present invention, it was confirmed that treatment of myoblasts with meloxicam, an oxicam compound, increased expression of MHC and myogenin.

[0042] The oxicam compounds of the present invention can improve athletic performance. "Athletic performance" refers to the ability to exercise using muscle strength, and muscle strength is improved by increasing muscle mass, muscle endurance, and oxidative muscle mass, improving muscle recovery and intramuscular energy balance, and also by reducing intramuscular fatigue substances. The oxicam compounds of the present invention have the effect of improving muscle-based athletic performance through their effects on the various muscles listed above. In relation to the above effect, in one example of the present invention, it was confirmed that actual muscle strength and athletic performance were increased in the group administered with meloxicam.

[0043] In other words, the oxicam compound of the present invention has the effect of improving muscle functions such as increasing muscle mass, improving muscle strength, and increasing muscle recovery ability, and can have preventive, therapeutic and ameliorative effects on muscle-related diseases.

[0044] The oxicam compounds of the present invention may have preventive, therapeutic, or ameliorative effects on muscle diseases through muscle regeneration in injured mice. In one embodiment of the present invention, the effects of meloxicam were examined when administered to a mouse model of muscle injury (CTX-injury). As a result, an increase in muscle mass was observed compared to the control group. Furthermore, the degree of muscle and muscle fiber regeneration observed with meloxicam administration was confirmed to be more detailed. Furthermore, the administration of oxicam compounds such as meloxicam significantly increased the proportion of type II muscle fibers (MHC type II) compared to the control group. Furthermore, the compounds may be effective in improving muscle strength and athletic performance by promoting muscle stem cell differentiation, increasing muscle fiber size, and increasing muscle mass not only in muscles damaged by CTX but also in muscle tissue of mice damaged or weakened by aging.

[0045] In addition, the oxicam compounds of the present invention can improve muscle strength and function by not only regenerating muscles damaged by muscle diseases or aging, but also by regenerating and increasing muscle mass in normal muscles. In one example of the present invention, the effects of muscle stem cell differentiation and muscle mass increase in young mice (14 months and 4 months) were confirmed, and it was confirmed that long-term administration of meloxicam can increase muscle strength and improve exercise performance even in young muscles.

[0046] The composition of the present invention may be used for various purposes such as pharmaceuticals, functional health foods, functional foods, animal feed, and cell culture media compositions, and has the effect of promoting myoblast differentiation or increasing mitochondrial activity in muscles, thereby preventing, improving, or treating muscle diseases.

[0047] As used herein, the term "prevention" refers to any action that can suppress or delay the onset of a muscular disorder by administering the pharmaceutical composition according to the present invention.

[0048] As used herein, the term "treatment" refers to any action in which symptoms are ameliorated or beneficially altered by administration of the pharmaceutical composition according to the present invention.

[0049] The pharmaceutical composition of the present invention may be formulated by conventional methods into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, topical preparations, suppositories, and sterile injections, and may further contain carriers or excipients necessary for the formulation. Pharmaceutically acceptable carriers, excipients, and diluents that may be further contained in the active ingredient include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, and mineral oil. When formulated, the composition may be prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0050] For example, solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations are prepared by mixing the extract or compound with at least one excipient, such as menthol, starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, liquid preparations, emulsions, syrups, etc., and may contain various excipients, such as wetting agents, sweeteners, flavoring agents, preservatives, etc., in addition to commonly used simple diluents such as water and liquid paraffin.

[0051] Preparations 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, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, tween 61, cocoa butter, laurin butter, and glycerol gelatin.

[0052] The pharmaceutical composition of the present invention may be administered orally or parenterally (by intravenous injection, subcutaneously, intraperitoneally, or topically) according to a desired method, and the dosage varies depending on the condition and weight of the patient, the degree of the disease, the drug form, the administration route, and the time, and may be selected as an appropriate form by a person skilled in the art.

[0053] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, a "pharmaceutically effective amount" refers to a reasonable amount applicable to medical treatment and sufficient to treat a disease. The criteria for this amount may be determined based on the patient's disease, severity, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration, co-administered ingredients, and other factors. The pharmaceutical composition of the present invention may be administered in combination with an individual therapeutic agent or other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. Taking all of these factors into consideration, a dosage that minimizes side effects may be determined, which can be easily determined by those skilled in the art. Specifically, the dosage of the pharmaceutical composition varies depending on the patient's age, body weight, severity, sex, etc., and is generally 0.001 to 150 mg per kg of body weight, preferably 0.01 to 50 mg, and more preferably 0.01 to 5 mg, administered daily or every other day, 1 to 3 times a day. However, this is merely an example, and the dosage may be adjusted as needed.

[0054] Furthermore, the composition of the present invention may be a food or a functional health food, and in particular, the term "functional health food" refers to a food manufactured and processed using raw materials or ingredients that have functional properties useful to the human body in accordance with Act No. 6727 on Functional Health Foods, and "functional" refers to the food being ingested for the purpose of regulating nutrients for the structure and function of the human body or obtaining beneficial effects for health purposes such as physiological actions.

[0055] The food or health functional food of the present invention can be manufactured and processed as a pharmaceutical dosage form such as powder, granules, tablets, capsules, pills, suspensions, emulsions, syrups, etc., or as a health functional food such as tea bags, infused tea, beverages, candies, jellies, gums, etc., for the purpose of preventing and improving muscle diseases.

[0056] The food or health functional food composition of the present invention may be used as a food additive or may be manufactured alone or in combination with other ingredients. It may also contain nutrients, vitamins, electrolytes, flavors, colorants and enhancers, pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, etc. The above ingredients may be used alone or in combination, and may be combined in appropriate amounts.

[0057] In one embodiment, the present invention relates to a muscle strengthening composition comprising an oxicam compound.

[0058] The term "muscle strengthening" refers to the enhancement of physical performance, enhancement of maximum endurance, increase in muscle mass, enhancement of muscle recovery, reduction of muscle fatigue, improvement of energy balance, or a combination thereof. The composition can increase muscle mass through its ability to differentiate myoblasts into muscle cells, thereby increasing overall muscle mass, enhancing maximum endurance, thereby enhancing physical performance, and reducing muscle fatigue. Furthermore, the composition can rapidly replace muscle cells, allowing for rapid recovery from muscle damage. The muscle strengthening composition of the present invention may be prepared in the form of a food composition or food additive, particularly a health food composition. The food composition is as described above. Therefore, the muscle strengthening composition of the present invention may be used not only for muscle loss due to aging, but also as a supplement for muscle building and muscle strengthening in the general population.

[0059] In another embodiment, the present invention relates to a feed or feed additive composition comprising an oxicam compound.

[0060] In the present invention, the term "feed" refers to a substance that provides organic or inorganic nutrients necessary to sustain the life of an animal. The feed contains nutrients such as energy, protein, lipids, vitamins, and minerals required by animals such as livestock, and includes, but is not limited to, plant-based feeds such as grains, roots and fruits, food processing by-products, algae, fibers, oils and fats, starches, butterbur, and grain by-products, and animal-based feeds such as proteins, minerals, oils and fats, minerals, oils and fats, and single-cell proteins.

[0061] In the present invention, the term "feed additive" refers to a substance added to feed to improve the productivity and health of animals, and may further include, but is not limited to, amino acid supplements, vitamin supplements, enzyme preparations, flavoring agents, silicate preparations, buffering agents, extractants, oligosaccharides, etc. for growth promotion, disease prevention, etc.

[0062] MODE FOR CARRYING OUT THE INVENTION

[0063] Hereinafter, the present specification will be described in detail with reference to examples in order to specifically explain the present specification. However, the embodiments according to the present specification may be modified into various other forms, and the scope of the present specification should not be construed as being limited to the examples described below. The examples of the present specification are provided to more completely explain the present specification to those skilled in the art.

[0064] Example 1: Isolation and culture of muscle cells C2Cl2 is a myoblast cell line derived from a C3H mouse and is widely used in muscle cell differentiation research. The C2C12 cells were cultured in standard cell culture medium and differentiation medium. The growth medium (GM) was DMEM supplemented with 10% fetal bovine serum, and the differentiation medium (DM) was DMEM supplemented with 2% horse serum.

[0065] In addition, human muscle stem cells (skMDC Human Skeletal Muscle Cells (Standard Donors), Cat NO: SK-1111) isolated from the rectus abdominis muscles of 17-year-old and 66-year-old Caucasian men were purchased from MyoSite and used.

[0066] Example 2: Creation of a mouse model of muscle injury (CTX-injury) Thirty 5-month-old C57BL / 6 male mice were used for the experiment. The animals were assigned to groups of five mice of similar weight and divided into a control group that did not receive meloxicam and an experimental group that received meloxicam. Meloxicam was orally administered to the experimental group at a dose of 0.02 mg / kg for 7 days. Then, 50 μL of 20 μM cardiotoxin (CTX) was directly injected into the tibialis anterior (TA) muscle to induce muscle damage, and the mice were orally administered meloxicam (0.02 mg / kg) for 21 days.

[0067] <Experimental Example 1> Confirmation of myoblast differentiation enhancement effect 1-1. Enhancement of differentiation of mouse muscle stem cells The C2Cl2 cell line from Example 1 was dispensed into cell culture medium and cultured in DMEM medium for 24 hours. The differentiation medium was then treated with the oxicam compounds meloxicam (F03), lornoxicam (F03-1), piroxicam (F03-2), and tenoxicam (F03-3), and the relative expression levels of the differentiation markers myogenin and myosin heavy chain (MHC) were analyzed. C2C12 cells induced to differentiate in Example 1 were lysed in lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X, proteinase inhibitor). The lysate was quantified, and the same amount of protein was subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk and washed with TTBS (0.03% Tween 20, Tris 2.42 g, NaCl 9 g, pH 7.4 1 L). A primary antibody against MHC (myosin heavy chain), a differentiation marker, was diluted 1:500 in TTBS containing 5% BSA and incubated overnight at 4°C. A secondary antibody was then diluted 1:5000 in TTBS containing 5% skim milk and incubated at room temperature. Enhanced Chemiluminescent solution (ECL, Pierce) was then added. The membrane was then exposed to X-ray film to confirm protein expression.

[0068] As a result, as shown in FIG. 1, it was confirmed that the relative expression level of myoblast differentiation markers increased when treated with oxicam compounds compared to the control group (DMSO).

[0069] Additionally, C2C12 cells were treated with meloxicam at concentrations of 1, 10, 100, 1,000, and 10,000 nM, respectively, and differentiation was induced for 3 days with the medium changed every other day. After differentiation, cells were examined under a microscope, disrupted, and subjected to Western blot analysis using differentiation markers, myogenin and MHC antibodies. C2C12 cells were washed with 1X PBS, fixed with 4% paraformaldehyde at room temperature, and then incubated in permeabilization buffer at room temperature. Nonspecific antibody binding was inhibited by incubation in PBST (blocking buffer) containing 5% goat serum or PBS containing 0.05% Tween 20. Primary antibodies against myosin heavy chain (MHC) were added and incubated at room temperature. Secondary antibodies diluted 1:5000 in blocking buffer were added and reacted at room temperature, and then the sections were fixed with mounting solution (40% glycerol in DW) and photographed under a fluorescent microscope to analyze the results.

[0070] As a result, as shown in Figure 2, MHC (myosin heavy chain) was clearly observed in myoblasts treated with a high concentration of meloxicam, and it was confirmed that the expression level was also increased, as shown in Figure 3.

[0071] 1-2. Enhancement of human muscle stem cell differentiation An experiment was conducted to determine whether meloxicam also has the effect of promoting differentiation in human muscle stem cells.

[0072] Human muscle stem cells (skMDC Human Skeletal Muscle Cells (Standard Donors), Cat. No.: SK-1111) isolated from the rectus abdominis muscles of 17- and 66-year-old Caucasian men were purchased from MyoSite. Human muscle stem cells were cultured for 24 hours in cell culture medium, and then treated with meloxicam (1 μM), an oxicam-based compound, in differentiation medium to induce differentiation into muscle fibers for 4 days. The degree of differentiation into muscle fibers was measured by immunostaining for the muscle fiber marker MHC (myosin heavy chain). Differentiation was analyzed by myofiber diameter.

[0073] As a result, as shown in Figure 4, it was confirmed that the muscle stem cells extracted from the 17-year-old subject showed an increase in myotube diameter, and as shown in Figure 5, it was confirmed that the muscle stem cells extracted from the 66-year-old subject showed an increase in both expression level and myotube diameter.

[0074] <Experimental Example 2> Confirmation of muscle recovery effect in muscle injury mouse model

[0075] 2-1. Increased muscle mass in muscle-damaged mouse models We conducted an experiment to confirm the muscle recovery effect using a CTX-injured mouse model. 21 days after CTX-injury, meloxicam was administered. The weights of the hindlimb (hindlimb) muscles, including the tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL), and gastrocnemius (GAS), were measured and compared. Results showed that muscle mass increased by 5.8% in the TA, 14.1% in the EDL, 5.4% in the GAS, and 3.4% in the GAS compared to the control group (Figure 6). This result, compared with the minimal changes in body weight and other organ weights over the same period (Figure 7), indicates that meloxicam specifically increases skeletal muscle mass.

[0076] 2-2. Muscle regeneration and muscle fiber size increase in muscle injury mouse models To confirm the effects of meloxicam on muscle regeneration and myofiber size in a CTX-injured mouse model, H&E staining (hematoxylin and eosin staining) was performed on TA muscles isolated from experimental animal tissue. Frozen sections fixed in 4% paraformaldehyde were stained with hematoxylin and eosin for control staining, fixed in mounting solution, and observed under a light microscope. Increased muscle regeneration and myofiber size were confirmed on days 4, 7, and 21 of recovery after muscle injury, as shown in Figure 8.

[0077] To further confirm the increased muscle type, we compared the relative expression levels of mRNA related to muscle type. As shown in Figure 9, the meloxicam-treated group showed a significant increase in the number of Type II muscle fibers compared to the control group, especially on day 21 after injury.

[0078] Furthermore, to analyze muscle fiber size, TA muscle tissue was immunohistochemically stained using a laminin antibody and observed under a fluorescence microscope. As shown in the bottom photograph of Figure 9, laminin staining confirmed an increase in the cross-sectional area (CSA) of muscle fibers, indicating that more cross-sectional area of ​​metabolic muscle fibers expressing MHC was observed. This confirms that the increased muscle fibers and resulting increase in muscle mass are due to an increase in the diameter and proportion of metabolic muscle fibers.

[0079] 2-3. Increased activity of metabolic enzymes in muscle of a mouse model of muscle injury Meloxicam was administered to a CTX-injured mouse model 21 days after injury, and the effect of increasing glycolytic enzyme activity in muscle mitochondria was confirmed in the treatment group.

[0080] α-glycerol phosphate, a substrate for the enzyme GPDH (Glycerol-3-phosphate dehydrogenase), was added to TA muscle sections isolated from the tissues of experimental animals, and the color change of the muscle fibers was confirmed in response to GPDH activity. The changed muscle sections were fixed in Merck's Aquatex and observed under a microscope. As a result, as shown in Figure 10, in the meloxicam-treated group, the size of metabolic muscle fibers increased (left), and it was confirmed that the proportion of muscle fibers in which GPDH was activated was higher than in the control group. These results indicate that meloxicam administration increases the activity of intramuscular metabolic enzymes.

[0081] 2-4. Increased muscle strength and exercise capacity in mouse models with muscle injury On day 21 after CTX-injury induction, experiments were conducted to assess muscle strength and motor ability in the meloxicam-treated and control groups. A grip strength test was conducted to assess muscle strength in the treated and control groups. Grip strength tests were performed using a mouse grip strength meter manufactured by BIOSEB. Mice were placed on a wire mesh attached to a monitor panel, and the force with which the mice gripped the mesh was measured by pulling their tails. The average of four consecutive measurements was divided by body weight to determine the strength. As shown in Figure 11, muscle strength increased by approximately 21.9% in the meloxicam-treated group compared to the control group. In the motor ability evaluation experiment, the meloxicam-treated group demonstrated a maximum increase in motor ability of 72.3%. A rotarod test was also conducted to assess the motor ability of the treated and control groups. The rotarod test was performed using a mouse rotarod device manufactured by Ugo Basile. The distance taken by the mouse to fall from a rotating rotor was measured. The rotarod test was performed after adaptation training, which consisted of 5 minutes at 8 rpm, 3 minutes at 10 rpm, and 1 minute at 13 rpm for two days. The constant rotarod test was performed at 13 rpm until the mouse fell, and the accelerated rotarod test was performed with an acceleration rate of 5 rpm to 18 rpm over 40 seconds. In both experiments, measurements were taken at the maximum value up to 500 seconds, at which point the experiment was stopped and measurements were taken.

[0082] <Experimental Example 3> Confirmation of age-dependent muscle mass and athletic ability increases with long-term administration of meloxicam 3-1. Changes in muscle mass, muscle type, and exercise capacity in aging mice After administering meloxicam to 24-month-old mice for four months, the weights of the hindlimb and skeletal muscle were measured and compared. As a result, as shown in Figure 12, an increase in muscle mass was observed in the hindlimb muscles and EDL muscles (bottom), which indicates that the increase in muscle mass occurred at a relatively high rate compared to the increase in total body weight (top).

[0083] In addition, the muscle types expressed were confirmed through the relative expression levels of mRNA. As a result, the expression rate of Myh Type II muscle fibers was particularly increased in the meloxicam-treated group, as shown in Figure 13. In other words, it was confirmed that muscle mass and the rate of metabolic muscle fibers increased in aged mice treated with meloxicam.

[0084] Furthermore, to confirm the changes in motor ability between the meloxicam-treated and control groups, a rota-rod experiment was conducted. As a result, as shown in Figure 14, it was confirmed that the meloxicam-treated group had a motor ability increase of more than 50% compared to the control group. Furthermore, the motor ability-increasing effect was confirmed not only in aged mice but also in 7-month-old mice. In other words, it was confirmed that the motor ability-improving effect of meloxicam is not only observed in aged muscles, but also in normal muscles.

[0085] 3-2. Changes in muscle strength and motor ability in mice (4 months old) Experiments were also conducted to measure changes in muscle strength and motor ability due to meloxicam administration in relatively young mice. On day 21 after CTX injury, a grip strength test was conducted to measure muscle strength and motor ability in the meloxicam-treated and control groups. Grip strength tests were performed using a BIOSEB mouse grip strength meter. Mice were placed on a wire mesh attached to a monitor panel, which monitors strength. The force with which the mice gripped the mesh was measured by pulling their tails. Measurements were repeated four times consecutively, and the average value was divided by body weight to calculate the strength. As a result, as shown in Figure 15, muscle strength increased by approximately 21.9% in the meloxicam-treated group compared to the control group. Experiments to evaluate motor ability also confirmed an increase in motor ability of up to 72.3% in the meloxicam-treated group.

[0086] 3-3. Changes in muscle strength and motor ability in normal mice (4 months old) after long-term administration We conducted an experiment to observe changes in muscle strength and exercise capacity during long-term administration of meloxicam to 4-month-old normal mice. First, we administered meloxicam to normal mice for two months and compared their weight changes with those of a control group. No significant changes were observed (Figure 16a), and no changes in dietary intake were observed (Figure 16b). To assess the exercise capacity of the treated and control groups, we performed a grip test and a treadmill test. The treadmill test was performed using a Columbus Instruments mouse treadmill. Measurements began with a 10% gradient and a speed of 8 m / min, which was increased by 1 m / min every 2 minutes until the mice fatigued. The results of the grip test confirmed that muscle strength was improved in the meloxicam-treated group, and exercise duration was also improved compared to the control group (Figure 17a). Furthermore, muscle mass relative to body weight was significantly increased in the meloxicam-treated group compared to the control group (Figure 17b). That is, the above experimental results show that administration of meloxicam to normal young mice improves muscle function, muscle strength, and motor performance.

[0087] The present invention has been described above with reference to preferred embodiments. Those skilled in the art will recognize that the present invention may be embodied in various modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is defined by the claims, not the foregoing description, and all variations within the scope of the claims should be construed as being within the scope of the present invention.

Claims

1. A composition for increasing muscle mass, improving muscle function, increasing exercise capacity, promoting muscle stem cell differentiation, regenerating muscle, or increasing muscle mass in patients with sarcopenia, comprising an oxicam compound and a pharmaceutically acceptable salt thereof as active ingredients, The composition, wherein the oxicam compound is meloxicam.

2. The composition according to claim 1 , wherein the composition is at least one selected from the group consisting of food, functional food, health functional food, pharmaceutical, animal feed, and feed additive.

3. Use of the composition described in claim 1 in the manufacture of a medicine for increasing muscle mass, improving muscle function, increasing athletic ability, promoting muscle stem cell differentiation, muscle regeneration, and increasing muscle mass in patients with sarcopenia.

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