Composition for preventing or treating muscle diseases comprising xanthine oxidase inhibitor and nitric oxide donor

Abstract

The present invention relates to a composition for preventing or treating muscle diseases, comprising a xanthine oxidase inhibitor and a nitric oxide donor and, more specifically, to a composition for preventing or treating muscle diseases, comprising a xanthine oxidase inhibitor and a nitric oxide donor, which exhibit synergistic effects in inhibiting muscular atrophy and promoting muscle cell differentiation.

Description

Composition for preventing or treating muscle disease comprising a xanthine oxidase inhibitor and a nitric oxide donor

[0001] This application claims priority to Republic of Korea Patent Application No. 10-2024-0024891, filed February 21, 2024, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a composition for preventing or treating muscle disease, comprising a xanthine oxidase inhibitor and a nitric oxide donor, and more particularly, to a composition for preventing or treating muscle disease, comprising a xanthine oxidase inhibitor and a nitric oxide donor that exhibit a synergistic effect in suppressing muscle atrophy and promoting muscle cell differentiation.

[0003]

[0004] For muscle to be maintained in living organisms, muscle cells must maintain a balance between protein anabolism and catabolism. An imbalance between protein anabolism and catabolism leads to decreased protein synthesis and increased protein breakdown, resulting in decreased muscle mass and impaired exercise capacity. Conversely, when the signaling responses that induce protein synthesis in muscle cells increase, protein synthesis in muscle cells is promoted, resulting in hypertrophy.

[0005]

[0006] Meanwhile, aging is known to progressively slow skeletal muscle regeneration, leading to a decrease in muscle mass, strength, and function. Furthermore, this loss of muscle mass leads to decreased strength and function, and the loss of muscle function is a major public health problem that worsens the quality of life of the ever-increasing aging population.

[0007]

[0008] In addition to sarcopenia, which is a decrease in muscle mass, muscle strength, and muscle function due to aging, there are various muscle diseases that cause muscle damage, muscle mass loss, muscle dysfunction, and muscle atrophy as symptoms, such as amyotrophic lateral sclerosis and spinal muscular atrophy, which cause muscle atrophy due to damage to motor neurons caused by genetic abnormalities, muscular dystrophy, a degenerative muscle disease characterized by necrosis of muscle fibers caused by defects in genes encoding skeletal muscle structural proteins, myasthenia gravis, an autoimmune disease with muscle weakness as its main symptom, and myopathy and myositis, which are caused by various intrinsic or extrinsic causes such as inflammation, metabolism, and drugs.

[0009]

[0010] Genetic mutations that cause these muscle diseases are associated with various cellular processes, such as autophagy, protein aggregation, mitochondrial stress, and RNA metabolism.

[0011]

[0012] Exercise therapy, medication, and / or surgery can be used to increase muscle mass and / or improve muscle function in individuals suffering from aging or muscle disease. Exercise therapy is known to promote myogenic differentiation within skeletal muscle in the short term, increase protein synthesis, and increase muscle strength and individual mobility. However, it has the disadvantage of not being applicable or having limited use depending on the individual's physical condition. Furthermore, immunosuppressants, testosterone, or anabolic steroids can be used as pharmacological treatments. Testosterone or steroid medications exhibit rapid effects, but can induce masculinization in women and cause serious side effects such as prostate symptoms in men. Immunosuppressants have the disadvantage of causing changes in immune system function. Pharmacological treatments that prescribe dehydroepiandrosterone (DHEA) and growth hormone are also available. Several companies are developing SARMs (Selective Androgen Receptor Modulators) as improved hormones that minimize the androgen characteristics of androgens and maximize their anabolic properties.

[0013]

[0014] However, because these drug therapies can cause problems with side effects, there is an increasing demand for novel muscle disease treatments that show excellent therapeutic effects even at low doses without causing side effects.

[0015]

[0016] Accordingly, the inventor of the present invention has conducted repeated research to develop a new therapeutic agent capable of inducing differentiation of muscle cells, suppressing muscle atrophy, and improving muscle function in individuals suffering from symptoms such as muscle damage, muscle mass loss, muscle dysfunction, and muscle atrophy due to aging and / or muscle disease, and as a result, has discovered that a xanthine oxidase inhibitor and a nitric oxide donor exhibit significantly improved muscle disease treatment effects compared to each drug alone, thereby completing the present invention.

[0017]

[0018] Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating muscle disease, which comprises a xanthine oxidase inhibitor and a nitric oxide donor as active ingredients.

[0019]

[0020] Another object of the present invention is to provide a food composition for preventing or improving muscle disease, comprising a xanthine oxidase inhibitor and a nitric oxide donor as active ingredients.

[0021]

[0022] Another object of the present invention is to provide a method for treating muscle disease in an animal other than a human, comprising the step of administering a xanthine oxidase inhibitor and a nitric oxide donor simultaneously, separately or sequentially.

[0023]

[0024] Another object of the present invention is to provide a use of a xanthine oxidase inhibitor and a nitric oxide donor for preparing a pharmaceutical composition for treating muscle diseases.

[0025]

[0026] Another object of the present invention is to provide a method for treating muscle disease, which comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a xanthine oxide inhibitor and a nitric oxide donor as active ingredients.

[0027]

[0028] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating muscle disease comprising a xanthine oxidase inhibitor and a nitric oxide donor.

[0029]

[0030] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating muscle disease, which essentially consists of a xanthine oxidase inhibitor and a nitric oxide donor.

[0031]

[0032] Another object of the present invention is to provide a food composition for preventing or improving muscle disease, comprising a xanthine oxidase inhibitor and a nitric oxide donor.

[0033]

[0034] Another object of the present invention is to provide a food composition for preventing or improving muscle disease, which essentially consists of a xanthine oxidase inhibitor and a nitric oxide donor.

[0035]

[0036] In order to achieve the above-described object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating muscle disease, which comprises a xanthine oxidase inhibitor and a nitric oxide donor as active ingredients.

[0037]

[0038] In order to achieve another object of the present invention, the present invention provides a food composition for preventing or improving muscle disease, which comprises a xanthine oxidase inhibitor and a nitric oxide donor as active ingredients.

[0039]

[0040] In order to achieve another object of the present invention, the present invention provides a method for treating a muscle disease in an animal other than a human, comprising a step of administering a xanthine oxidase inhibitor and a nitric oxide donor simultaneously, separately or sequentially.

[0041]

[0042] In order to achieve another object of the present invention, the present invention provides the use of a xanthine oxidase inhibitor and a nitric oxide donor for preparing a pharmaceutical composition for treating muscle diseases.

[0043]

[0044] In order to achieve another object of the present invention, the present invention provides a method for treating muscle disease, which comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a xanthine oxide inhibitor and a nitric oxide donor as active ingredients.

[0045]

[0046] In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating muscle disease, comprising a xanthine oxidase inhibitor and a nitric oxide donor.

[0047]

[0048] In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating muscle disease, which essentially consists of a xanthine oxidase inhibitor and a nitric oxide donor.

[0049]

[0050] In order to achieve another object of the present invention, the present invention provides a food composition for preventing or improving muscle disease, comprising a xanthine oxidase inhibitor and a nitric oxide donor.

[0051]

[0052] In order to achieve another object of the present invention, the present invention provides a food composition for preventing or improving muscle disease, which essentially comprises a xanthine oxidase inhibitor and a nitric oxide donor.

[0053]

[0054] Hereinafter, the present invention will be described in detail.

[0055]

[0056] The present invention provides a pharmaceutical composition for preventing or treating muscle disease, comprising a xanthine oxidase inhibitor and a nitric oxide donor as active ingredients.

[0057]

[0058] In this specification, the term “comprising” is used with the same meaning as “including” or “characterized by,” and does not exclude additional components or method steps, etc. that are not specifically mentioned in the composition or method according to the present invention. In addition, the term “consisting of” means excluding additional elements, steps, or components, etc. that are not separately described. The term “essentially consisting of” means that, in the scope of the composition or method, it may include materials or steps, etc. that do not substantially affect the basic characteristics thereof, in addition to the materials or steps described.

[0059]

[0060] In the present invention, the content of the composition is not particularly limited depending on the purpose or aspect of use, and may be, for example, 0.01 to 99 wt%, preferably 0.5 to 50 wt%, and more preferably 1 to 30 wt%, based on the total weight of the composition. In addition, the pharmaceutical composition according to the present invention may further include additives such as pharmaceutically acceptable carriers, excipients, or diluents in addition to the active ingredient. The pharmaceutical composition of the present invention may include 0.1 to 99.9 wt% of the xanthine oxidase inhibitor and nitric oxide donor of the present invention, and 99.9% to 0.1 wt% of the carrier.

[0061]

[0062] In the present invention, the "xanthine oxidase inhibitor" refers to a substance capable of inhibiting the activity or expression of xanthine oxidase, which contributes to the metabolism of purine into uric acid in the body. There are various types of xanthine oxidase inhibitors, and it will be understood by those skilled in the art that the present invention can be performed using any class of pharmaceutically acceptable xanthine oxidase inhibitor.

[0063]

[0064] In the present invention, the xanthine oxidase inhibitor may include functional derivatives of a xanthine oxidase inhibitor. A "functional derivative" refers to a substance that possesses a biological activity (functional or structural) substantially similar to the biological activity of a xanthine oxidase inhibitor. The term "functional derivative" includes a "variant," "analog," or "chemical derivative" of a xanthine oxidase inhibitor. The term "variant" refers to a molecule that is substantially similar in structure and function to a xanthine oxidase inhibitor or a portion thereof. If two molecules have a substantially similar structure or two molecules have similar biological activity, the molecule is substantially similar to a xanthine oxidase inhibitor and may be included in the present invention. The term "analog" refers to a molecule that is substantially similar in function to a xanthine oxidase inhibitor. The term "chemical derivative" generally refers to a molecule that includes an additional chemical moiety that is not part of the basic molecule. Derivatives include, but are not limited to, bioprecursors or prodrugs that can be converted to xanthine oxidase inhibitors.

[0065]

[0066] Non-limiting examples of the xanthine oxidase inhibitor in the present invention may include oxypurinol, allopurinol, febuxostat, baicalein, benzbromarone, topiroxostat, inositol, TMX-049, DNHB (3,4-Dihydroxy-5-nitrobenzaldehyde), niflumic acid, and pharmaceutically acceptable salts thereof. Preferably, the xanthine oxidase inhibitor in the present invention may be oxypurinol, allopurinol, or a combination thereof.

[0067]

[0068] In the present invention, the nitric oxide donor may be a substance capable of storing nitric oxide within its molecule and releasing nitric oxide under specific conditions, i.e., a substance capable of supplying exogenous nitric oxide, or a substance capable of promoting the production of nitric oxide in a living body, i.e., a substance capable of supplying endogenous nitric oxide, and may be included in the present invention without limitation.

[0069]

[0070] Non-limiting examples of the nitric oxide donor in the present invention include arginine, homoarginine, N-hydroxy-arginine, citrulline, isosorbide, organic nitrites, organic nitrates, nitrosothiols, C-nitroso compounds, N-hydroxyl nitrosamine, diazetine dioxides, oxatriazole-5-imine, N-nitrosamines, sydnonimines, oximes, hydroxylamines, N-hydroxyguanidines, hydroxyurea, Nitrosiamine, N-hydroxyl nitrosamines, NO-metal complexes, N-diazeniumdiolates (NONOates), and pharmaceutically acceptable salts thereof may be included. Preferably, the nitric oxide donor in the present invention may be arginine, citrulline, or a combination thereof.

[0071]

[0072] In a preferred embodiment of the present invention, the composition may be characterized by comprising (i) oxypurinol, allopurinol or a combination thereof and (ii) arginine, citrulline or a combination thereof as active ingredients.

[0073]

[0074] In another preferred embodiment of the present invention, the composition may be characterized by comprising a combination of allopurinol and citrulline, or oxypurinol and arginine as active ingredients.

[0075]

[0076] In the present invention, the pharmaceutically acceptable salt may be an acid addition salt formed by a pharmaceutically acceptable free acid. The acid addition salt is obtained from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, or phosphorous acid, and non-toxic organic acids such as aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, and alkanedioates, aromatic acids, and aliphatic and aromatic sulfonic acids. These pharmaceutically non-toxic salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphate chlorides, bromides, iodides, fluorides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caprates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyn-1,4-dioate, hexane-1,6-dioate, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, Examples of suitable esters include, but are not limited to, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, or mandelate.

[0077]

[0078] According to one embodiment of the present invention, it was confirmed that the complex of a xanthine oxidase inhibitor and a nitric oxide donor has a significantly superior effect on suppressing atrophy of muscle cells and promoting differentiation compared to each single substance.

[0079]

[0080] Therefore, the combination of a xanthine oxidase inhibitor and a nitric oxide donor has the advantage of a synergic effect, that is, it can show improved effects at a lower dose compared to using each drug alone, thereby reducing side effects and improving the effectiveness of treating muscle diseases.

[0081]

[0082] The Bliss independence combined response C for two single compounds with effects A and B is C = A + B - A*B, where each effect is expressed as a fractional inhibition value between 0 and 1. The Bliss value, defined as the difference between the experimental response and the calculated Bliss independence value, indicates whether the effects of the two components in combination are additive or synergistic.

[0083]

[0084] A Bliss value of zero (0) is considered additive. The term "additive" means that the result of combining two target agents is the sum of the individual drugs.

[0085]

[0086] The term "synergy" or "synergistic" is used to mean that the response of a combination of two agents is greater than the sum of the responses of each of the individual agents. More specifically, in an in vitro setting, one measure of synergy is known as "Bliss synergy." Bliss synergy means "exceeding the Bliss independent value," as determined by the previously defined Bliss value. A Bliss value greater than zero (0) is considered an indicator of synergy. Of course, the concept of "synergy" as used herein also includes in vitro synergy measured by additional and / or alternative methods.

[0087]

[0088] In the present invention, the in vitro biological effect of the combination of a xanthine oxidase inhibitor and a nitric oxide donor may be correlated with the Bliss value, indicating that the combination exhibits an activity equal to or greater than the sum of its individual components. Furthermore, the "synergistic effect," which encompasses the case where the combination of components used in the present invention exhibits an activity equal to or greater than the sum of its individual components, may be measured by additional and / or alternative methods.

[0089]

[0090] In one aspect of the present invention, the composition of the present invention is for preventing or treating muscle disease, and may be combined with an effective amount of a xanthine oxidase inhibitor or a pharmaceutically effective salt, derivative or metabolite thereof in an amount sufficient to achieve a synergistic effect with an effective amount of a nitric oxide donor or a pharmaceutically effective salt thereof.

[0091]

[0092] In one aspect of the present invention, the xanthine oxidase inhibitor and the nitric oxide donor may be combined in a molar ratio of 1:1 to 3000, preferably in a molar ratio of 1:1 to 2000, more preferably in a molar ratio of 1:1 to 1500, and most preferably in a molar ratio of 1:1 to 1000.

[0093]

[0094] In the present invention, the muscle disease may be included without limitation as long as it is a disease caused by muscle damage, loss, atrophy or a combination thereof, and for example, Duchenne Muscular Dystrophy, Spinal Muscular Atrophy, Amyotrophic Lateral Sclerosis, Becker Muscular Dystrophy, Distal Muscular Dystrophy, Congenital Muscular Dystrophy, Emery-Dreifuss Muscular Dystrophy, Central Cord Disease, Nemaline Myopathy, Myotonic Dystrophy, Age-Related Sarcopenia, Inflammatory Myopathy, Metabolic Muscle Disease, Neuromuscular Junction Disease, Endocrine Myopathy, This may include, but is not limited to, muscle atrophy.

[0095]

[0096] The term "prevention" above refers to any action that reduces the frequency or severity of a pathological phenomenon. Prevention may be complete or partial. In this case, it may refer to a reduction in symptoms such as muscle damage, loss, and atrophy within the subject compared to when the composition was not used.

[0097]

[0098] The term "treatment" as used herein refers to any clinical intervention intended to alter the natural processes of a subject or cell to be treated, and may be performed during the progression of a clinical pathological condition or to prevent it. Furthermore, "treatment" broadly refers to improving symptoms caused by a muscle disorder, which may include curing, substantially preventing, or improving the condition of the disorder, including, but not limited to, alleviating, curing, or preventing one or most of the symptoms resulting from the disorder.

[0099]

[0100] The desired therapeutic effect may include preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, slowing the progression of the disease, alleviating or temporarily alleviating the disease state, or improving the prognosis.

[0101]

[0102] In the composition of the present invention, the xanthine oxidase inhibitor and the nitric oxide donor can be administered simultaneously, separately, or sequentially, and the xanthine oxidase inhibitor and the nitric oxide donor can exhibit muscle disease prevention or treatment activity through synergy in the body.

[0103]

[0104] Typically, the xanthine oxidase inhibitor and the nitric oxide donor are administered simultaneously as a composition, but even if each of the above active ingredients is administered to the human body at different times, each of the individually administered active ingredients can simultaneously act in the body, thereby achieving an equivalent level of therapeutic activity.

[0105]

[0106] Specifically, 'simultaneous administration' means administering the two active ingredients together through the same administration route, or administering them through the same or different administration routes at substantially the same time (e.g., an administration time interval of 15 minutes or less). The separate administration means administering the two active ingredients through the same or different administration routes at a certain time interval (e.g., a 3-day interval). The sequential administration means administering the two active ingredients through the same or different administration routes according to a certain order rule depending on the patient's disease condition.

[0107]

[0108] Administration can be oral or parenteral. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraneural, intracerebroventricular (subventricular region), intracerebrovascular, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal administration.

[0109]

[0110] The pharmaceutical composition according to the present invention may contain only a pharmaceutically effective amount of a xanthine oxidase inhibitor and a nitric oxide donor, or may additionally contain a pharmaceutically acceptable carrier. The term "pharmaceutically effective amount" refers to an amount that exhibits a greater response than a negative control group, and preferably refers to an amount sufficient to exhibit the effects of increasing lifespan, improving motility, suppressing muscle atrophy, suppressing muscle inflammation, suppressing muscle cell death, and promoting muscle cell differentiation by co-administering the two effective ingredients in treating or preventing muscle diseases.

[0111]

[0112] The pharmaceutical composition of the present invention can be formulated in various ways according to the route of administration by a method known in the art, together with a pharmaceutically acceptable carrier, in order to exhibit a synergistic effect by using a xanthine oxidase inhibitor and a nitric oxide donor in combination. The term "pharmaceutically acceptable" refers to a non-toxic composition that is physiologically acceptable and does not inhibit the action of the active ingredient when administered to a human and does not typically cause allergic reactions such as gastrointestinal disorders or dizziness or similar reactions. The carrier includes all kinds of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads, and microsomes. Pharmaceutically acceptable carriers included in the above composition are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0113]

[0114] The pharmaceutical composition may further include, in addition to the above ingredients, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc. Specifically, in the case of oral administration, a binder, a lubricant, a disintegrating agent, an excipient, a solubilizer, a dispersing agent, a stabilizer, a suspending agent, a coloring agent, or a flavoring agent, etc. may be used, and in the case of injections, a buffer, a preservative, an analgesic, a solubilizer, an isotonic agent, a stabilizer, etc. may be mixed and used, and in the case of topical administration, a base, an excipient, a lubricant, a preservative, etc. may be used.

[0115]

[0116] In addition, the composition of the present invention can be used in the form of general pharmaceutical preparations. Parenteral preparations may be prepared in the form of sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions or lyophilized preparations, injections, transdermal preparations, nasal inhalers, etc., and oral administration may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups or wafers. Injections may be prepared in the form of unit dose ampoules or multiple doses. Injections must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. Suitable carriers for injections include, but are not limited to, solvents or dispersion media including water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol), mixtures thereof and / or vegetable oils. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol, and 5% dextrose. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal may be additionally included. In addition, the injection may in most cases additionally contain isotonic agents such as sugars or sodium chloride.

[0117]

[0118] In addition, the pharmaceutical composition of the present invention can be administered by any device that allows the active ingredient to move to the target site. Preferred administration methods and formulations include intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, or drip injection. The injection can be prepared using an aqueous solvent such as saline solution or Ringer's solution, a non-aqueous solvent such as vegetable oil, higher fatty acid ester (e.g., ethyl oleate, etc.), alcohol (e.g., ethanol, benzyl alcohol, propylene glycol, or glycerin, etc.), and may include a pharmaceutical carrier such as a stabilizer to prevent deterioration (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), an emulsifier, a buffer to adjust pH, a preservative to inhibit microbial growth (e.g., phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.). A method for treating or preventing a neurodegenerative disease using the composition of the present invention comprises administering an effective amount (pharmaceutically effective amount) of the therapeutic composition of the present invention to a subject in need thereof. The pharmaceutically effective amount can be readily determined by those skilled in the art based on factors well known in the medical field, such as the type of disease, the patient's age, weight, health, sex, the patient's sensitivity to drugs, the route of administration, the method of administration, the number of administrations, the duration of treatment, and drugs used in combination or concurrently.

[0119]

[0120] Additionally, the pharmaceutical composition of the present invention can be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.

[0121]

[0122] The total effective amount of the composition of the present invention may be administered to a patient as a single dose, or may be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time, and refers to an amount that, when administered, exhibits an effect of improving, treating, detecting, diagnosing, or inhibiting or reducing a muscle disease. The 'subject' may be an animal, preferably a mammal, particularly an animal including a human, or may be a cell, tissue, organ, etc. derived from an animal, and the subject may be a patient in need of the effect. The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the severity of the disease. Preferably, the preferred total dose of the pharmaceutical composition of the present invention may be about 0.01 ㎍ to 10,000 mg, most preferably 0.1 ㎍ to 5,000 mg per 1 kg of patient body weight per day. However, since the dosage of the pharmaceutical composition is determined by taking into account various factors such as the formulation method, administration route, and number of treatments, as well as the patient's age, weight, health condition, sex, severity of disease, diet, and excretion rate, a person having ordinary skill in the art will be able to determine the appropriate effective dosage of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route, and administration method as long as it exhibits the effects of the present invention.

[0123]

[0124] The composition of the present invention may be formulated so that the components, a xanthine oxidase inhibitor and a nitric oxide donor, are simultaneously included in a single formulation, depending on the administration method and route, or each component may be individually formulated and included in a single package according to a daily or single-dose dosage unit. The individually formulated xanthine oxidase inhibitor and nitric oxide donor may or may not be the same. The specific formulation method of the pharmaceutical composition of the present invention and the pharmaceutically acceptable carrier that may be included in the formulation are as described above in the pharmaceutical composition.

[0125]

[0126] The present invention also provides a food composition for preventing or improving muscle disease, comprising a xanthine oxidase inhibitor and a nitric oxide donor as active ingredients.

[0127]

[0128] The food composition of the present invention includes all forms such as functional food, nutritional supplement, health food, and food additives.

[0129]

[0130] The above-mentioned type of food composition can be manufactured in various forms according to conventional methods known in the art. For example, but not limited to, as a health food, the xanthine oxidase inhibitor and the nitric oxide donor can be manufactured in the form of tea, juice, and drinks, and can be liquefied, granulated, encapsulated, or powdered for consumption. Furthermore, the xanthine oxidase inhibitor and the nitric oxide donor can be mixed with a known active ingredient known to be effective against infectious diseases to manufacture a composition. In addition, functional foods include, but are not limited to, beverages (including alcoholic beverages), fruits and their processed foods (e.g., canned fruits, bottled fruits, jams, marmalades, etc.), fish, meats and their processed foods (e.g., ham, sausages, corned beef, etc.), breads and noodles (e.g., udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, taffy, dairy products (e.g., butter, cheese, etc.), edible vegetable oils, margarine, vegetable proteins, retort foods, frozen foods, various seasonings (e.g., soybean paste, soy sauce, sauces, etc.), etc., which can be manufactured by adding a xanthine oxidase inhibitor and a nitric oxide donor. In addition, in order to use the xanthine oxidase inhibitor and the nitric oxide donor in the form of an additive, they can be manufactured and used in the form of a powder or concentrate.

[0131]

[0132] The preferred content of the xanthine oxidase inhibitor and the nitric oxide donor in the food composition of the present invention is not limited thereto, but is preferably 0.1 to 90 wt% of the final manufactured food. More preferably, the food composition containing the xanthine oxidase inhibitor and the nitric oxide donor of the present invention as active ingredients can be manufactured in the form of a health functional food or dietary supplement, particularly by mixing them with an active ingredient known to be effective against infectious diseases.

[0133]

[0134] The present invention also provides a method for treating a muscle disease in an animal other than a human, comprising administering a xanthine oxidase inhibitor and a nitric oxide donor simultaneously, separately, or sequentially.

[0135]

[0136] The present invention also provides the use of a xanthine oxidase inhibitor and a nitric oxide donor for preparing a pharmaceutical composition for treating muscle diseases.

[0137]

[0138] The present invention also provides a method for treating a muscle disease, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a xanthine oxide inhibitor and a nitric oxide donor as active ingredients.

[0139]

[0140] The composition according to the present invention can exhibit an enhanced preventive and therapeutic effect on muscle disease compared to single administration by co-administering a xanthine oxidase inhibitor and a nitric oxide donor, and has the effect of alleviating side effects that may be caused by overdose or long-term administration of each drug.

[0141]

[0142] Figure 1 is an immunostaining image of a test to confirm the effect of inhibiting muscle atrophy. It is a representative image of myotube cells stained with MHC after 72 hours of treatment with L-citrulline and allopurinol, alone or in combination with TNF-α, a muscle atrophy-inducing substance, in differentiated C2C12 myotube cells.

[0143]

[0144] Figures 2a to 2d show the results of evaluating the muscle atrophy inhibitory effect by quantifying the area ratio of the muscle cell region stained with MHC after 72 hours of treatment with differentiated C2C12 myotube cells alone or in combination with L-citrulline and allopurinol, which are muscle atrophy-inducing substances, along with TNF-α.

[0145]

[0146] Figure 3 is an immunostaining image of a test to confirm the effect of inhibiting muscle atrophy. It is a representative image of myotube cells stained with MHC after 72 hours of treatment with L-arginine and oxypurinol, alone or in combination with TNF-α, a muscle atrophy-inducing substance, to differentiated C2C12 myotube cells.

[0147]

[0148] Figures 4a to 4c show the results of evaluating the muscle atrophy inhibitory effect by quantifying the area ratio of the muscle cell region stained with MHC after 72 hours of treatment with differentiated C2C12 myotube cells alone or in combination with L-arginine and oxypurinol, which are muscle atrophy-inducing substances, along with TNF-α.

[0149]

[0150] Figures 5a to 5c show the results of qPCR analysis of mRNA expression changes of Atrogin-1, a marker gene of the protein degradation pathway in Figure 5a, and IGF-1, a marker gene of the protein synthesis pathway in Figure 5b, after 72 hours of treatment with L-citrulline and allopurinol, alone or in combination, together with TNF-α, a muscle atrophy inducer, in differentiated C2C12 myotube cells.

[0151]

[0152] Figures 6a to 6d show the results of qPCR analysis of mRNA expression changes of Atrogin-1, a marker gene of the protein degradation pathway (Figure 6a), IGF-1, a marker gene of the protein synthesis pathway (Figure 6b), and IL-6, an inflammatory cytokine (Figure 6c), after 72 hours of treatment with L-arginine and oxypurinol, alone or in combination, together with TNF-α, a muscle atrophy inducer, in differentiated C2C12 myotube cells.

[0153]

[0154] Figures 7a and 7b show the results of evaluating the differentiation promotion effect by measuring the expression level of MHC protein, a myotube cell marker, by In-Cell Western blot 96 hours after treating C2C12 cell line, a mouse-derived myoblast, with L-arginine and oxypurinol alone or in combination.

[0155]

[0156] Figures 8a to 8c show the results of qPCR analysis of the mRNA expression changes of MyoG, a muscle cell differentiation regulatory gene, and MHC, a myotube cell marker, in Figure 8a, 24 hours after L-arginine and oxypurinol were treated alone or in combination in C2C12 cell lines, which are mouse-derived myogenic cells.

[0157]

[0158] Figures 9a and 9b show the results of confirming the degree of decreased mobility and recovery through the Rotarod Test by administering L-citrulline and Allopurinol alone or in combination for 12 weeks to the animal model B10.mdx mouse (DMD disease animal model).

[0159]

[0160] Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended only to illustrate the present invention and the present invention is not limited thereto.

[0161]

[0162] Example 1: Confirmation of the inhibitory effect on muscle atrophy in myotube cells

[0163] Muscle atrophy is a hallmark pathological feature of many muscle diseases, including muscular dystrophy and sarcopenia, leading to a decrease in muscle mass and strength and a decline in motor function. Causes of muscle atrophy include chronic inflammation, oxidative stress, and nutritional imbalances. In particular, increased cytokine production due to persistent inflammation is known to directly induce muscle atrophy by promoting ubiquitin-dependent protein degradation in muscle cells and reducing muscle protein synthesis.

[0164] In the present invention, the muscle atrophy inhibitory effect of a combination of L-citrulline and allopurinol, and L-arginine and oxypurinol, and the synergy of each combination were confirmed in vitro. C2C12 myoblasts were differentiated into myotubes using a low-serum myocyte differentiation medium, and then the myotubes were treated with the inflammatory cytokine TNF-α at a concentration of 10 or 20 ng / ml for 72 hours to form a muscle atrophy-inducing model. The muscle atrophy inhibitory effect of drug treatment was confirmed by treating the single and combined agents of L-citrulline and allopurinol, and the single and combined agents of L-arginine and oxypurinol simultaneously with TNF-α treatment. The muscle atrophy inhibitory effect was evaluated by quantifying the proportion of myotube cells stained with MHC in each test group using immunostaining. (Fig. 1, Fig. 2a to Fig. 2d, Fig. 3 and Fig. 4a to Fig. 4c).

[0165] As a result of the combination experiment of L-citrulline and allopurinol, in Fig. 1 and Fig. 2a to Fig. 2d, it was confirmed that in the TNF-α (20 ng / ml) single treatment group, the MHC area ratio was reduced by approximately 75% compared to the untreated control group. In the case of single treatment with L-citrulline and allopurinol, a significant muscle atrophy inhibitory effect was confirmed in the high-concentration treatment group of each drug (20 mM and 100 μM, respectively), and the drug did not show a significant effect in the low-concentration treatment group (4 mM and 10 μM, respectively). The combined treatment group of the two drugs showed a significant muscle atrophy inhibitory effect regardless of the treatment concentration (**P< 0.01, ****P< 0.0001, one-way ANOVA). In order to verify the synergy of the two drugs, the Bliss independence model was used, and the actual efficacy of the combination treatment was significantly higher than the Bliss predicted efficacy predicted from the results of the single experiment of the two drugs, confirming the remarkable synergy of the two drugs in the muscle atrophy inhibition efficacy.

[0166] As a result of the combination experiment of L-arginine and oxypurinol, as shown in Figs. 3 and 4a to 4c, in the case of the TNF-α (10 ng / ml) single treatment group, the MHC area ratio was confirmed to decrease by approximately 40% compared to the untreated control group. The single treatment groups of L-arginine and oxypurinol did not show a significant muscle atrophy inhibitory effect, and only the combination of the two drugs showed a significant muscle atrophy inhibitory effect (**P< 0.01, ****P< 0.0001, one-way ANOVA). As a result of using the Bliss independence model to verify the synergy of the two drugs, the actual effect of the combination treatment was significantly higher than the Bliss predicted effect predicted from the results of the single experiment of the two drugs, confirming the remarkable synergy of the two drugs in the muscle atrophy inhibitory effect.

[0167]

[0168] Example 2: Confirmation of changes in muscle atrophy-related gene expression in myotube cells

[0169] Muscle atrophy is associated with the regulation of protein synthesis and degradation within muscle. Protein balance within muscle is regulated by the IGF-1 / PI3K / Akt signaling pathway, with increased IGF-1 inducing activation of the protein synthesis pathway and inhibiting protein degradation. Protein degradation in muscle is regulated by genes such as Atrogin-1 and MuRF1, which are muscle-specific E3 ubiquitin ligases. Oxidative stress and inflammatory cytokines are known to induce protein degradation and muscle atrophy by increasing the expression of these genes.

[0170] In the present invention, the gene expression changes of IGF-1 and Atrogin-1 by single and combined treatment of L-citrulline and allopurinol, and single and combined treatment of L-arginine and oxypurinol under the muscle atrophy induction conditions used in Example 1 were analyzed by q-PCR.

[0171] As a result, in the case of the combination test of L-citrulline and allopurinol, a significant increase in Atrogin-1 mRNA and a decrease in IGF-1 mRNA were confirmed in the TNF-α alone treatment group compared to the untreated control group, as shown in Figures 5a to 5c. L-citrulline alone treatment did not show a significant improvement effect, and allopurinol alone treatment showed a significant increase in IGF-1 mRNA. As a result of the combined treatment of the two drugs, a tendency to decrease Atrogin-1 mRNA was confirmed, and a significant increase in IGF-1 mRNA was confirmed (**P< 0.01, ***P< 0.001, ****P< 0.0001, one-way ANOVA). In order to verify the synergy of the two drugs, the Bliss independence model was used, and the actual efficacy of the combination treatment was significantly higher than the Bliss predicted efficacy predicted from the results of the single experiment of the two drugs, confirming the remarkable synergy of the two drugs in the changes in gene expression related to muscle protein regulation.

[0172] In the case of the combination test of L-arginine and oxypurinol, as shown in Figures 6a to 6d, a significant increase in Atrogin-1 mRNA, a decrease in IGF-1 mRNA, and an increase in IL-6 mRNA as an additional inflammatory indicator were confirmed in the TNF-α alone treatment group compared to the untreated control group. L-arginine alone treatment showed a significant increase in IGF-1 mRNA and a significant decrease in IL-6 mRNA, while oxypurinol alone treatment showed only a significant decrease in IL-6 mRNA. As a result of the combined treatment of the two drugs, a decreasing trend in Atrogin-1 mRNA was confirmed, and a significant increase in IGF-1 mRNA and a significant decrease in IL-6 mRNA were confirmed (*P< 0.05, **P< 0.01, ****P< 0.0001, one-way ANOVA). In order to verify the synergy of the two drugs, the Bliss independence model was used, and the actual efficacy of the combination treatment was significantly higher than the Bliss predicted efficacy predicted from the results of the single experiment of the two drugs, confirming the remarkable synergy of the two drugs in the changes in gene expression related to muscle protein regulation.

[0173]

[0174] Example 3: Confirmation of the effect of promoting differentiation of myogenic cells

[0175] The increase in muscle fibers through differentiation of muscle stem cells can supplement muscle mass deficiency that occurs in various muscle diseases and alleviate muscle strength loss and motor function deterioration, thereby mediating the preventive and therapeutic effects of muscle diseases.

[0176] In the present invention, mouse-derived myogenic cells, C2C12, were cultured for 4 days under differentiation conditions of a low-serum culture medium composition, and treated with L-arginine and oxypurinol alone or in combination at 24-hour intervals. Then, the expression level of MHC protein, a marker of myotube differentiation, was measured by In-Cell Western blot to analyze the effect of drug-induced differentiation promotion.

[0177] As a result of the experiment, as shown in Fig. 7a and Fig. 7b, a significant increase in MHC expression was confirmed in the single treatment group of L-arginine and oxypurinol and the combined treatment group compared to the untreated control group (*P< 0.05, ***P< 0.001, ****P< 0.0001, one-way ANOVA). More specifically, it was confirmed that each single drug increased the differentiation of myogenic cells by approximately 1.2-1.4 times compared to the untreated control group, and the combined treatment group increased it by approximately 1.6 times. As a result of using the Bliss independence model to verify the synergy of the two drugs, the actual efficacy of the combined treatment was significantly higher than the Bliss predicted efficacy predicted from the results of the single experiment of the two drugs, confirming the remarkable synergy of the two drugs in the efficacy of promoting myogenic cell differentiation.

[0178]

[0179] Example 4: Confirmation of changes in differentiation-regulating gene expression in myogenic cells

[0180] Through the process of myogenesis, myogenic cells fuse with surrounding myogenic cells to differentiate into myotubes, multinucleated cells with a tubular shape. Muscle differentiation is regulated by myogenic regulatory factors (MRFs), such as MyoD, MRF 5, and Myogenin (MyoG). It is known that the expression of myosin heavy chain (MHC), a major structural protein of myotubes, increases in the later stages of differentiation.

[0181] In the present invention, mouse-derived myogenic cells, C2C12, were treated with L-arginine and oxypurinol alone or in combination under differentiation medium conditions composed of a low serum culture medium. After 24 hours, the expression levels of MyoG, a muscle cell differentiation regulatory gene, and MHC, a myotube cell marker, were analyzed by q-PCR to confirm the differentiation promoting effect of the drug.

[0182] As a result of the experiment, as shown in Figures 8a to 8c, it was confirmed that the expression of MyoG and MHC was significantly increased in the L-arginine only treatment group and the L-arginine and oxypurinol combination treatment group compared to the untreated control group. In the oxypurinol only treatment group, a significant increase was confirmed only in the expression of MHC compared to the untreated control group (*P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001, one-way ANOVA). The degree of increase in the expression of MyoG and MHC was greater in the combination treatment group of the two drugs. As a result of using the Bliss independence model to verify the synergy of the two drugs, the actual effect of the combination treatment was significantly higher than the Bliss predicted effect predicted from the results of the single experiment of the two drugs, confirming the remarkable synergy of the two drugs in increasing the expression of genes related to muscle differentiation.

[0183]

[0184] Example 5: Determination of decreased mobility and degree of recovery using an animal model

[0185] In the present invention, the Rotarod Test, a test to determine the degree of motor decline and recovery, was conducted using an animal model B10.mdx mouse (an animal model of DMD disease) by placing the animal on a rotating rod and measuring the time taken for it to fall. The drug for the experiment was administered orally once a day for a total of 12 weeks, and the Rotarod was measured at weekly intervals. Each test group consisted of 5 mice, and the composition of the test groups is as shown in Table 1 below.

[0186]

[0187] [Table 1]

[0188]

[0189]

[0190] As shown in Figures 9a and 9b, the latency to decline increased in the L-citrulline only treatment group and the Allopurinol only treatment group compared to the untreated control group, and it was confirmed that the L-citrulline and Allopurinol combination treatment group also showed significant efficacy (*P< 0.05, one-way ANOVA). As a result of using the Bliss independence model to verify the synergy of the two drugs, the actual efficacy of the combination treatment was significantly higher than the Bliss predicted efficacy predicted from the results of the two drugs' single experiments, confirming the remarkable synergy of the two drugs in the degree of motor decline and recovery.

[0191]

[0192] The composition according to the present invention can exhibit an enhanced preventive and therapeutic effect on muscle disease by co-administering a xanthine oxidase inhibitor and a nitric oxide donor compared to single administration, and has the effect of alleviating side effects that may be caused by overdose or long-term administration of each drug, so it has very high potential for industrial use.

Claims

1. A pharmaceutical composition for preventing or treating muscle disease, comprising a xanthine oxidase inhibitor and a nitric oxide donor as active ingredients.

2. A pharmaceutical composition according to claim 1, wherein the xanthine oxidase inhibitor is selected from the group consisting of oxypurinol, allopurinol, febuxostat, baicalein, benzbromarone, topiroxostat, inositol, TMX-049, DNHB (3,4-Dihydroxy-5-nitrobenzaldehyde), niflumic acid, and pharmaceutically acceptable salts thereof.

3. A pharmaceutical composition according to claim 1, wherein the nitric oxide donor supplies exogenous nitric oxide or promotes the production of endogenous nitric oxide.

4. A pharmaceutical composition according to claim 1, characterized in that the nitric oxide donor is selected from the group consisting of arginine, homoarginine, N-hydroxyarginine, citrulline, isosorbide, and pharmaceutically acceptable salts thereof.

5. A pharmaceutical composition according to claim 1, wherein the muscle disease is selected from the group consisting of Duchenne muscular dystrophy, spinal muscular atrophy, amyotrophic lateral sclerosis, Becker muscular dystrophy, distal muscular dystrophy, congenital muscular dystrophy, Emery-Dreifuss muscular dystrophy, central cord disease, nemaline myopathy, myotonic dystrophy, age-related sarcopenia, inflammatory myopathy, metabolic muscle disease, neuromuscular junction disease, endocrine myopathy, and muscle atrophy.

6. A pharmaceutical composition according to claim 1, characterized in that the composition exhibits effects of inhibiting muscle atrophy and promoting muscle cell differentiation.

7. A pharmaceutical composition according to claim 1, characterized in that the xanthine oxidase inhibitor and the nitric oxide donor are included in a molar ratio of 1:1 to 3000.

8. A pharmaceutical composition according to any one of claims 1 to 7, characterized in that the xanthine oxidase inhibitor and the nitric oxide donor are administered simultaneously, separately, or sequentially.

9. A food composition for preventing or improving muscle disease, comprising a xanthine oxidase inhibitor and a nitric oxide donor as active ingredients.

10. A food composition according to claim 9, characterized in that the food is a health functional food.

11. A method for treating a muscle disease in an animal other than a human, comprising a step of administering a xanthine oxidase inhibitor and a nitric oxide donor simultaneously, separately, or sequentially.

12. Use of a xanthine oxidase inhibitor and a nitric oxide donor for preparing a pharmaceutical composition for treating muscle disease.

13. A method for treating muscle disease, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a xanthine oxidase inhibitor and a nitric oxide donor as active ingredients.

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