KR20200038408A - Composition for Preventing or Treating Sarcopenia Comprising IF1 - Google Patents

Abstract

The present invention relates to a composition for preventing or treating sarcopenia containing ATPase inhibitory factor 1 (IF1). More specifically, the present invention relates to a pharmaceutical composition and food for preventing or treating sarcopenia, containing, as an active ingredient, IF1 having an effect of increasing muscle mass and muscle production. IF1 according to the present invention exhibits effects of increasing muscle mass, increasing muscle protein, and muscle production without side effects in obesity-induced or aged mouse, thereby being able to be very useful as an agent for preventing, alleviating or treating muscle-loss-related diseases such as sarcopenia, etc.

Description

Pharmaceutical composition for the prevention or treatment of muscular dystrophy containing IF1 (ATPase inhibitory factor 1) as an active ingredient {Composition for Preventing or Treating Sarcopenia Comprising IF1}

The present invention relates to a composition for the prevention or treatment of muscular dystrophy containing IF1 (ATPase inhibitory factor 1), more specifically for the prevention or treatment of muscular dystrophy containing IF1 as an active ingredient having an increase in muscle mass and muscle growth It relates to a pharmaceutical composition and food.

Skeletal muscle is the largest organ in the human body, accounting for 40-50% of the total weight, and plays an important role in various metabolic functions in the body, including energy homeostasis and heat generation. A person's muscles decrease by 1% or more each year since the age of 40, and by age 80, a level of 50% of the maximum muscle mass decreases, and muscle loss in old age is recognized as the most important factor that decreases overall physical function. The decrease in muscle strength due to a decrease in skeletal muscle mass during aging is called sarcopenia.

Myopathy is caused by a variety of factors, but research on each factor has not been done. Decreased growth hormone or neurological changes, changes in physiological activity, changes in metabolism, the amount of sex hormones or fat or catabolic cytokines, and the balance of protein synthesis and differentiation Induced by change (Roubenoff R and Hughes VA, J Gerontol A Biol Sci Med Sci 55: M716-M724, 2000).

In addition, muscle atrophy (muscular atrophy), muscle tissue damage due to the absence of mechanical stimulation, such as reduced muscle use, muscle injury caused by direct injury or physical factors, impaired resilience of muscle cells due to aging, and muscle function It is caused by factors such as impairment of muscle use due to damage to the nerves that regulate it (Booth FW et al., J Appl Physiol Respir Environ Exerc Physiol, 1982). In general, disuse atrophy, which gradually progresses to muscle atrophy due to loss of muscle strength due to the loss of muscle strength due to disability or accident, does not use the muscles in the affected area and the periphery for a long time, and myasthenia caused by disease of the muscle itself gravis, dystrophy, inflammation in the muscles themselves, spinal muscular amyotrophy, amyotrophic lateral sclerosis (ALS), spinal cord It also occurs in the form of bulbous muscular atrophy (sphinobulbar muscular atrophy).

Various studies have been conducted on how to effectively control such hypokinesia, and as a method of slowing the progression of myopathy, a method of suppressing muscular dystrophy caused by degeneration or progressive mutation of muscle cells, which is mainly a type of myopathy. Is being used. For example, WO 2007/088123 discloses a therapeutic agent for muscular dystrophy, which includes a nitroxy derivative as an active ingredient, and WO 2006/081997 discloses a therapeutic agent for muscular dystrophy, which includes atrachic acid or a derivative thereof as an active ingredient. However, these therapeutic agents containing the compound as an active ingredient act not only on skeletal muscles with muscular dystrophy, but also on visceral muscles or myocardium not associated with muscular dystrophy, and various side effects, such as large and small, may be caused, and thus have not been used for practical treatment. . In addition, although treatment with growth hormone (GH) has been shown to increase muscle mass, this treatment has the disadvantage of being very expensive.

Therefore, there is an urgent need to develop an effective drug and technology for the treatment of muscular dystrophy that can delay the progression of muscular dystrophy without side effects even in elderly patients or patients lying in bed.

On the other hand, ATPase inhibitory factor 1 (IF1) binds to F1Fo ATP synthase (multi-subunit, membrane-bound assembly), which is involved in the synthesis and degradation of ATP synthesis in mitochondria, that is, IF1 binds to β-F1-ATPase in the cell membrane. It induces an intracellular signaling system such as the PI3K-Akt pathway, and thereby a bioreaction. In addition, the present inventors have confirmed that if L1 myoblast is treated with IF1, the ATP concentration in the medium is significantly increased, and phosphorylation of Akt in muscle cells is increased by the triggered cell signaling system. Recent studies have reported that Akt phosphorylation in muscle cells and subsequent mTORC pathway activation delay protein degradation and promote protein synthesis (Bodine et al., Nat Cell Biol . 3 (11): 1014-9, 2001). It is also known that an increase in myostatin expression, known as a negative regulator for muscle growth, causes muscle atrophy and muscle loss by regulating the expression of myogenic marker genes (Rodriguez et al., Cell Mol Life) Sci. 71 (22): 4361-71, 2014). Therefore, it can be seen that IF1 causes an increase in the extracellular fluid ATP concentration through interaction with the F1-ATPase subunit of the muscle cell membrane, which promotes muscle production through subsequent biometabolism signaling and inhibits muscle atrophy and reduction. This is the basis for judging that you can.

Thus, the present inventors confirmed that the muscle mass and the amount of muscle protein in the mouse increased by administration of the recombinant protein IF1 as a result of diligent efforts to develop a therapeutic agent for muscular dystrophy having no side effect and excellent effect, and completed the present invention.

An object of the present invention is to provide a pharmaceutical composition and food for the prevention or treatment of muscular dystrophy, which contains IF1 (ATPase inhibitory factor 1) that exhibits an increase in muscle mass and an effect of muscle formation as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of muscular dystrophy containing IF1 (ATPase inhibitory factor 1) as an active ingredient.

The present invention also provides a food for preventing or improving muscular dystrophy, which contains IF1 (ATPase inhibitory factor 1) as an active ingredient.

IF1 (ATPase inhibitory factor 1) according to the present invention shows an increase in muscle mass, an increase in muscle protein, and an angiogenesis effect without side effects in obese-induced mice or aging mice, thereby preventing, improving or improving muscle-related diseases such as muscular dystrophy or It is very useful as a treatment.

Figure 1 shows the increase in muscle mass and muscle fiber density in mice by IF1 treatment, (A) quadriceps, (B) gastrocnemius, (C) sum of quadriceps and gastrocnemius muscle weights, (D) It shows the results of tissue immunostaining of the quadriceps muscle. Each data represents the mean ± SE value for each group. * P <0.05 compared with HF-GST group
2 shows an increase in the amount of gastrocnemius protein in mice by IF1 treatment, and shows that the amount of gastrocnemius protein decreased in obese mice was increased by administration of IF1. Each data represents the mean (relative to control,%) of each group. * P <0.05 compared with HF-GST group
Figure 3 shows the expression change of muscle-related genes in mice by IF1 treatment, and decreases the expression of the myostatin gene, which is known as a negative regulator for muscle growth, and increases the expression of myogenin, a gene involved in muscle production. Shows. Each data represents the mean (relative to control,%) of each group. * P <0.05 compared with HF-GST group
Figure 4 shows the strengthening and recovery of muscle function in mice by IF1 treatment, including (A) obesity model, (B) hanging time in the aging model and correcting it by weight (impulse). (C) Swimming time (log ₂ ) of the obese model mouse through an improved FST (Forced swimming test) is shown. Each data represents the mean (relative to control,%) of each group. * P <0.05, ** P <0.01, *** P <0.001 compared with HF-GST group

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known in the art and commonly used.

ATPase inhibitory factor 1 (hereinafter referred to as IF1) is a 9.6-kDa basic protein composed of 84 amino acids and is encoded by the ATP5IF1 gene. ATPase is composed of F0, F1 domain and central, pheripheral stalk, and is subdivided into several subunits. IF1 is a major protein that interferes with the function of ATPase and is naturally produced in the human body, and has been studied as a target for controlling ATP production and degradation by mitochondria (Campanella et al., Cell Metab , 8: 13-25, 2008). The inhibition of IF1 begins by interfering with the rotational motion by binding to the α subunit and β subunit of ATPase. IF1 binds to the F1-ATPase subunit located on the plasma membrane, and at this time, ATP hydrolysis is suppressed by controlling F1-ATPase activity, thereby causing extracellular ATP (extracellular ATP, exATP) increase, and exATP is the cell membrane After reacting with the purinergic receptor of, it produces various useful intracellular reactions through purine signaling.

However, there are no known mechanisms related to muscle mass, muscle protein increase, or muscle growth in IF1.

Thus, in the present invention, based on the entire mouse IF1 mRNA sequence (NCBI No. NM_007512.3), recombinant DNA (SEQ ID NO: 1) is produced by cloning DNA data of IF1 including GST-tag, and recombinant IF1 is an obese mouse. And it was administered to the aging mouse model to confirm the effects of muscle mass, muscle protein and muscle growth by IF1. That is, the effect of preventing and treating ATPase inhibitory factor 1 (IF1) was observed.

Accordingly, the present invention, in one aspect, relates to a pharmaceutical composition for the prevention or treatment of muscular dystrophy containing IF1 (ATPase inhibitory factor 1) as an active ingredient.

In the present invention, the composition may be characterized by increasing muscle mass or preventing muscle loss.

In the present invention, the muscular dystrophy may be characterized by aging or obesity.

The term "muscular reduction by aging" of the present invention refers to a gradual decrease in skeletal muscle mass or a gradual decrease in muscle density and function due to aging, which directly leads to a decrease in muscle strength and, as a result, various physical functions. Refers to a condition that can cause reduction and disability.

The muscle aging-related disease may be, but is not limited to, age-related sarcopenia, motion neuron disease, metabolic muscle disease, inflammatory myopathy, neuromuscular junction disease, endocrine myopathy.

In the present invention, the myopathy is muscle atrophy (muscular atrophy), inuse atrophy (disuse atrophy), spinal muscular atrophy (spinal muscular amyotrophy), muscular dystrophy (dystrophy), muscle spasticity, muscular dystonia (muscular hypotonia), muscle weakness , Muscular dystrophy, amyotrophic lateral sclerosis, spinal bulb muscular atrophy, and myasthenia gravis are preferably selected from the group consisting of, but not limited to .

As used herein, the term "muscular hypothyroidism" refers to a disease in which muscle mass is reduced, and a disease in which muscle volume and muscle strength gradually declines. In particular, the muscles of the extremity are almost symmetrically, which means a general term of atrophy, which can accompany cancer, aging, kidney disease, hereditary disease, and various chronic diseases, and amyotrophic lateral sclerosis (ALS) ), Including spinal muscular atrophy, etc., and muscular dystrophy.

The term "muscular atrophy" of the present invention is preferably muscle atrophy due to loss of muscle tissue caused by not using muscle, muscle atrophy due to disease of the muscle itself, or muscle atrophy due to damage to the nerves that govern the muscle. . Muscular atrophy due to loss of muscle tissue caused by not using the muscle is disuse atrophy, muscle atrophy due to disease of the muscle itself is myasthenia gravis or dystrophy: progressive muscular dystrophy , Myotonic muscular dystrophy, Dusen's type, Becker's type, zone type, facial scapular brachial type, inflammation occurring in the muscle itself, and muscular atrophy due to damage to the nerves that dominate the muscles are spinal muscular amyotrophy: Verardny Hi-Hoffman's type, Kugelberg's, Bellander's disease, amyotrophic lateral sclerosis: Lou Gehrig's disease, or sphinobulbar muscular atrophy: Kennedy's disease is more preferred, but not limited thereto.

Diseases related to muscle aging, including aging muscular dystrophy, are distinguished from muscle dystrophy or muscle atrophy.

Specifically, muscular dystrophy patients show muscle necrosis on muscle biopsy, muscle fiber size is uneven and various, and the muscle fibers are replaced by fat and fibrous tissue in the necrotic spot, and the muscular dystrophy includes Becker muscular dystrophy, Duchenne muscular dystrophy. , Congenital muscular dystrophy, Emery-Dreifuss muscular dystrophy, etc. (Alan EH Emery, Lancet 23; 359 (9307): 687-95, 2002).

In addition, muscular dystrophy means that the muscles of the limbs are atrophic, and amyotrophic lateral sclerosis and spinal progressive amyotrophy are typical, which are known as diseases caused by progressive degeneration of motor nerve fibers and cells in the spinal cord.

Specifically, the spinal muscular atrophy is a genetic disorder caused by degeneration of the motor neurons of the spinal cord, and is known as a neuromuscular disease in infants and young children. In addition, amyotrophic lateral sclerosis is characterized by refractory and irreversible neurodegenerative changes due to the death of upper and lower motor neurons of the cerebral and spinal cord, and is known to be the main cause of the lack of nerve growth factor and neuroinflammation.

Currently, there are treatment methods for muscular dystrophy caused by aging, such as using eurocortin II, hormone replacement therapy, etc., but some side effects have been reported, thus fundamental treatment for muscle aging-related diseases. There is a lacking aspect.

According to a specific embodiment of the present invention, when the IF1 of the present invention is administered to a mouse, it was confirmed that an increase in muscle weight and an increase in the amount of muscle protein were found, and that it also influenced the expression of genes related to muscle production and degradation. Did. As a result, it was confirmed that IF1 increases muscle mass and prevents muscle loss, thereby effectively restoring and improving muscle function.

The therapeutic effect of IF1 can be equally applied to muscular dystrophy as well as muscular dystrophy caused by various causes such as cancer and aging.

The term "prevention" of the present invention means any action that suppresses or delays the onset of myopathy or muscle wasting related diseases by administration of the pharmaceutical composition according to the present invention.

The term "treatment" of the present invention means any action that improves or beneficially alters the symptoms of myopathy or muscle wasting related diseases by administering the pharmaceutical composition of the present invention.

The myopathy treatment can be applied to any mammal that can develop myopathy or muscle wasting related diseases, for example, humans and primates, as well as cattle, pigs, sheep, horses, dogs, and cats. Includes, but may preferably be human.

In the present invention, "administration" means introducing a predetermined substance to a patient in any suitable way, and the route of administration of the compositions can be administered through any general route as long as the drug can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, and the like, but are not limited thereto.

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount, the term "pharmaceutically effective amount" of the present invention to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention By sufficient amount, the effective dose level refers to the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, patient's sensitivity to the drug, the time of administration, route of administration and rate of excretion of the composition of the invention used. The duration of treatment, factors including drugs used in combination or coincidental with the composition of the present invention used, and other factors well known in the medical field.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered single or multiple. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect in a minimal amount without side effects.

The dosage of the pharmaceutical composition of the present invention can be determined by a person skilled in the art in consideration of the purpose of use, the degree of poisoning of the disease, the age, weight, sex, history of the patient, or the type of substance used as an active ingredient. For example, the pharmaceutical composition of the present invention may be administered to mammals including humans for 10 to 100 mg / kg, more preferably 10 to 30 mg / kg for one day, and the frequency of administration of the composition of the present invention is It is not particularly limited, but may be administered once to three times a day or divided into doses and administered several times.

The pharmaceutical composition of the present invention may be prepared in the form of a pharmaceutical composition for the treatment or prevention of muscular dystrophy, further comprising a suitable carrier, excipient or diluent commonly used in the manufacture of the pharmaceutical composition, the carrier being unnatural It may include a carrier (non-naturally occuring carrier).

Specifically, the pharmaceutical composition is formulated in the form of an oral dosage form such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, external preparation, suppository, and sterile injection solution, respectively, according to a conventional method. You can.

In the present invention, the carrier, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose. It is prepared by mixing (lactose) and gelatin. In addition, lubricants such as magnesium stearate and talc are used in addition to simple excipients.

Liquid preparations for oral use may include various excipients, such as wetting agents, humectants, sweeteners, fragrances, and preservatives, in addition to water and liquid paraffin, which are simple diluents commonly used for suspending agents, liquid solutions, emulsions, syrups, etc. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized 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.

The pharmaceutical composition may be in the form of a sterile injectable preparation as an aqueous or oily suspension for sterile injectable use. This suspension can be formulated according to techniques known in the art using suitable dispersants or wetting agents (eg Tween 80) and suspending agents. Sterile injectable preparations may also be non-toxic parenterally acceptable diluents or sterile injectable solutions or suspensions in solvents (eg solutions in 1,3-butanediol). Vehicles and solvents that may be acceptable are mannitol, water, Ringel's solution and isotonic sodium chloride solution. In addition, sterile, non-volatile oils are commonly used as solvents or suspending media. For this purpose, any non-volatile oil with less irritation can be used, including synthetic mono or diglycerides. Fatty acids such as oleic acid and glyceride derivatives thereof are useful in injection formulations, such as pharmaceutically acceptable natural oils (eg olive oil or castor oil), especially their polyoxyethylated.

The pharmaceutical composition of the present invention can also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing the compounds of the present invention with suitable non-irritating excipients that are solid at room temperature but liquid at rectal temperatures. Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycol.

Parenteral administration of a pharmaceutical composition according to the present invention is particularly useful when the desired treatment involves a site or organ that is accessible by topical application. When applied topically to the skin, the pharmaceutical composition should be formulated with a suitable ointment containing the active ingredient suspended or dissolved in a carrier. Carriers for topical administration of the compounds of the present invention include, but are not limited to, mineral oil, liquid paraffin, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax and water. Alternatively, the pharmaceutical composition may be formulated as a suitable lotion or cream containing the active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical composition of the present invention can also be topically applied to the lower intestinal tract by rectal suppositories and also with suitable enema. Topical applied transdermal patches are also included in the present invention.

The pharmaceutical composition of the present invention can be administered by intranasal aerosol or inhalation. These compositions are prepared according to techniques well known in the field of medicament and are used in benzyl alcohol or other suitable preservatives, absorption accelerators to enhance bioavailability, fluorocarbons and / or other solubilizers or dispersants known in the art in saline. It can be prepared as a solution.

The content of the formulation contained in the pharmaceutical composition of the present invention is not particularly limited, but may be included in an amount of 0.0001 to 50% by weight, more preferably 0.01 to 10% by weight based on the total weight of the final composition.

In another aspect, the present invention relates to a method of preventing or treating muscular dystrophy, comprising administering to a subject a pharmaceutical composition containing IF1 (ATPase inhibitory factor 1) as an active ingredient.

In another aspect, the present invention relates to the use of a pharmaceutical composition containing IF1 (ATPase inhibitory factor 1) as an active ingredient for the prevention or treatment of muscular dystrophy.

In another aspect, the present invention relates to the use of a pharmaceutical composition containing IF1 (ATPase inhibitory factor 1) as an active ingredient for the manufacture of a medicament for the prevention or treatment of muscular dystrophy.

The term "individual" of the present invention means all animals, including humans, who have already developed or are likely to develop myopathy, and by administering the composition of the present invention to an individual, the disease can be effectively prevented and treated.

In another aspect, the present invention relates to a food for preventing or improving muscular dystrophy, which contains IF1 (ATPase inhibitory factor 1) as an active ingredient.

The term "improvement" of the present invention means any action that at least reduces the severity of the parameters associated with the condition being treated, for example symptoms.

In the present invention, the food may be characterized by increasing muscle mass or preventing muscle loss.

In the present invention, the muscular dystrophy may be characterized by aging or obesity.

In the present invention, the myopathy is muscle atrophy (muscular atrophy), inuse atrophy (disuse atrophy), spinal muscular atrophy (spinal muscular amyotrophy), muscular dystrophy (dystrophy), muscle spasticity, muscular dystonia (muscular hypotonia), muscle weakness , Muscular dystrophy, amyotrophic lateral sclerosis, spinal bulb muscular atrophy, and myasthenia gravis are preferably selected from the group consisting of, but not limited to .

When the food composition of the present invention is used as a food additive, the composition may be added as it is or used with other foods or food ingredients, and may be suitably used according to conventional methods. Generally, the composition of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, with respect to the raw materials in the production of food or beverage. However, in the case of long-term intake for the purpose of health and hygiene or for health control, it may be below the above range, and since there is no problem in terms of safety, the active ingredient may also be used in an amount above the above range.

The food of the present invention can be prepared in any form, such as functional food, nutritional supplement, health food, and food additives. For example, as a health food, the composition of the present invention may be prepared in the form of tea, juice, and drink to be consumed or granulated, encapsulated, and powdered. In addition, functional foods include beverages (including alcoholic beverages), fruits and processed foods (e.g. canned fruits, canned foods, jams, marmalade, etc.), fish, meat, and processed foods (e.g. ham, sausage, corn beef, etc.) , Breads and noodles (e.g. udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), juice, various drinks, cookies, syrup, dairy products (e.g. butter, cheats, etc.), edible vegetable oils, margarine, vegetable protein, retort foods , Frozen food, various seasonings (eg, miso, soy sauce, sauce, etc.) can be prepared by adding the composition of the present invention.

The health functional food also includes various forms such as functional foods, nutritional supplements, health foods, and food additives as food compositions, and various forms according to conventional methods known in the art, for example, the present invention mentioned above. Prepare the composition in the form of tea, juice, drink, granulate, encapsulate, powder, or drink these compounds or extracts, fruit and processed foods, fish oil, meat and processed foods thereof, breads, noodles, seasonings, etc. It can be provided by manufacturing in addition to.

The health drink composition of the present invention may contain various flavoring agents or natural carbohydrates, etc., as additional components, like a conventional beverage. The natural carbohydrates described above may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharin and aspartame. . The proportion of the natural carbohydrate can be appropriately determined by the choice of those skilled in the art.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, And carbonated agents used in carbonated beverages. In addition, the composition of the present invention may contain flesh for the preparation of natural fruit juice, fruit juice beverages and vegetable beverages. These ingredients can be used independently or in combination. The proportions of these additives can also be appropriately selected by those skilled in the art.

[ Example ]

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example  1: Mouse preparation

1-1: Obese animal model

C57BL / 6J males were used as the obese mouse model, and 5 weeks old mice were pre-saled from JOONGAH BIO to provide a normal diet for 1 week, and then went through an adaptation period. As a breeding environment, the humidity of 18 ~ 24 ℃ and 50 ~ 60% was maintained at all times, and free feeding was performed in both the adaptation period and the experimental period.

After the one-week adaptation period, they were divided into three groups and reared for 6 weeks under the following conditions.

Group 1: Normal diet (ND) control (n = 10)

Group 2: High fat diet (HFD) control (n = 15)

Group 3: High Fat Diet (HFD) experimental group (n = 15)

Normal (ND; Normal Diet) and high fat diet (HFD; 40% High Fat Diet) composition table Ingredients ND (g) HFD (g) corn starch 15 15 Casein 20 20 Sucrose 50 34 corn oil 5 3 Mineral mix 3.5 3.5 Vitamin mix One One Cellulose 5 5 DL-methionine 0.3 0.3 choline bitartrate 0.2 0.2 Lard 17 Cholesterol One BHT 0.001 0.001 Total 100 (g) 100 (g)

After the 1 week adaptation period, mice were induced obesity for 6 weeks in a high-fat diet, and then administered with IF1.

1-2: aging animal model

As an aging animal model, an average 8-month-old C57BL / 6J female entering adulthood is used, and the aging process is progressed over time for the next 2 months. Times). After a rest period of 4 months, the animals were divided into the following two groups through a randomization process, and each GST and IF1 (5 mg / kg BW, once daily) were treated with a high-fat diet. As a breeding environment, humidity of 18 ~ 24 ℃ and 50 ~ 60% was maintained at all times.

Group 1: High fat diet (HFD) control + GST intraperitoneal injection (n = 4)

Group 2: High Fat Diet (HFD) experimental group + IF1 intraperitoneal injection (n = 4) (5mg / kg BW)

Example  2: Increased muscle mass with IF1 administration

Mice inducing persistent obesity for 6 weeks after 1 week of adaptation period were intraperitoneally injected 4 times per week for 4 weeks after IF1 (5 mg / kg BW). IF1 produced recombinant protein by separating and purifying DNA data of IF1 including GST-tag through cloning.

The conditions for administering IF1 to the three groups of mice in Example 1 are as follows.

Group 1: Normal diet (ND) control + GST intraperitoneal injection (n = 10)

Group 2: High fat diet (HFD) control group + GST intraperitoneal injection (n = 15)

Group 3: High Fat Diet (HFD) experimental group + IF1 intraperitoneal injection (n = 15) (5mg / kg BW)

Mice that had all experiments and breeding periods up to 4 weeks after IF1 administration were fasted for 16 hours for dissection, and then anesthetized 1.5 g / ml urethane the next day to completely anesthetize. Anesthesia was verified by the reflex action of the leg, and when anesthesia was achieved, blood was collected through an insulin syringe. The blood obtained by blood collection was immediately centrifuged at 4 ° C for 30 minutes at 2500 rpm to separate serum, and the blood-collected mice were dissected to separate the muscles and quickly soaked in PBS to remove foreign substances on the surface. The weights of the quadriceps (Quadriceps) and gastrocnemius (gastrocnemius) were measured, respectively.

As a result, it was confirmed that the weights of quadriceps (quadriceps) and gastrocnemius (gastrocnemius) were significantly increased in the IF1 administration group compared to the control group in the normal diet and high fat diet (FIGS. 1A to 1C). That is, it shows that IF1 is effective in increasing muscle mass.

Additionally, in order to observe changes in muscle cells, the muscle specimen quadriceps and gastrocnemius muscles were subjected to immunostaining (Immunohistochemistry) using a solution of hematoxylin and eosin. As a result, the muscle fiber density of the muscle tissue of the high-fat diet mice treated with IF1 showed a similar level to that of the mice fed the normal diet (FIG. 1D).

Example  3: Increased muscle protein with IF1 administration

The total amount of cellular protein in the quadriceps and gastrocnemius isolated in Example 2 was measured.

Specifically, after dissolving RIPA Lysis buffer containing protease inhibitor in gastrocnemius tissue 0.05g, and dissolving it, the amount of protein was calculated by quantifying the supernatant centrifuged at 4 ℃ and 13000rpm for 20 minutes.

As a result, although the amount of protein in the gastrocnemius muscle was decreased in mice in which obesity was induced by the high-fat diet, it was confirmed that the protein amount in the gastrocnemius muscle was significantly increased by IF1 administration (FIG. 2). That is, it shows that IF1 is effective in increasing protein in muscle.

Example 4: Muscle-related gene expression changes according to IF1 administration

The expression levels of genes involved in muscle production and degradation in the quadriceps and gastrocnemius isolated in Example 2 were compared through qPCR (quantitative polymerase chain reaction) analysis.

Specifically, RNA of gastrocnemius tissue was extracted using QIAzol Lysis reagent RNeasy Lipid Tissue Mini Kit (Qiagen, USA), and 1 μg RNA was raised and cDNA with oligo-dT (oligo-dT) and superscript II reverse transcriptase (Invitrogen, USA) Synthesized. Gene expression of the synthesized 1000ng cDNA was compared through real-time quantitative PCR amplification. Data were obtained using a comparative-cycle threshold method and expressed as a fold change, and beta-actin was used as a control in the comparative CT method.

As a result, it was found that the high-fat diet had a significantly reduced expression level of the myostatin gene, which is known to inhibit muscle production, in the case of the IF1 administration group compared to the control group. In addition, it was found that the expression level of the myogenin gene, which is known to be involved in muscle production, and the expression of the IGF-1 and MYF5 genes also increased (FIG. 3). That is, these results suggest that the extracellular fluid ATP produces purine signaling, which leads to an increase in protein synthesis and inhibition of protein degradation by the subsequently triggered cellular signaling system.

Example 5: Increased muscle strength in animals following administration of IF1

The muscle strength was measured for the obese model mouse and the aging model mouse of Example 1, and it was confirmed that the muscles were recovered and strengthened (FIG. 4). At this time, the measurement of the strength was carried by improving the hanging test (Figs. 4A and 4 B) and FST (Forced swimming test) (Fig. 4C and 4D).

The Hanging test measures the time until a 1x1cm mouse hangs upside down on an iron cage and falls to the floor. The impulse is an indicator corrected by multiplying the weight. Both the obesity model (Figure 4A) and the aging model (Figure 4B) showed a significant difference compared to the control group, which showed the same trend regardless of before and after weight correction.

The FST (forced swimming test) was conducted by changing each obese model mouse to 5 L / min after all individuals were adapted to the experiment by training at 34 ° C. and a flow rate of 2 L / min. Only one mouse was tested in each lane, and the experiment was stopped when it did not appear for more than 7 seconds in water. In the case of IF1 treatment, a log- ₂ value was taken, and the swimming time was significantly increased compared to the control group (FIG. 4C). This represents the same meaning as the hanging test in FIGS. 4A and 4B, indicating an increase in muscle strength in the IF1 treatment group.

As the specific parts of the present invention have been described in detail above, it will be apparent to those of ordinary skill in the art that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Korea University Research and Business Foundation <120> Composition for Preventing or Treating Sarcopenia Comprising IF1 <130> P19-B198 <150> KR 10-2018-0117692 <151> 2018-10-02 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> Recombinant IF1 <400> 1 Val Ser Asp Ser Ser Asp Ser Met Asp Thr Gly Ala Gly Ser Ile Arg   1 5 10 15 Glu Ala Gly Gly Ala Phe Gly Lys Arg Glu Lys Ala Glu Glu Asp Arg              20 25 30 Tyr Phe Arg Glu Lys Thr Lys Glu Gln Leu Ala Ala Leu Arg Lys His          35 40 45 His Glu Asp Glu Ile Asp His His Ser Lys Glu Ile Glu Arg Leu Gln      50 55 60 Lys Gln Ile Glu Arg His Lys Lys Lys Ile Gln Gln Leu Lys Asn Asn  65 70 75 80 His    

Claims (10)

A pharmaceutical composition for the prevention or treatment of muscular dystrophy, which contains IF1 (ATPase inhibitory factor 1) as an active ingredient.
The pharmaceutical composition of claim 1, wherein the composition increases muscle mass or prevents muscle loss.
The pharmaceutical composition according to claim 1, wherein the muscular dystrophy is caused by aging or obesity.
The method of claim 1, wherein the muscular dystrophy is muscular atrophy, disuse atrophy, spinal muscular amyotrophy, dystrophy, muscular spasticity, muscular hypotonia, muscular strength. Pharmaceutical composition, characterized in that it is selected from the group consisting of weakening, muscular dystrophy, amyotrophic lateral sclerosis, sphinobulbar muscular atrophy and myasthenia gravis.
The pharmaceutical composition of claim 1, further comprising a pharmaceutically acceptable carrier, excipient or diluent.
Food for preventing or improving muscular dystrophy, which contains IF1 (ATPase inhibitory factor 1) as an active ingredient.
The food according to claim 6, wherein the food increases muscle mass or prevents muscle loss.
The food according to claim 6, wherein the muscular dystrophy is caused by aging or obesity.
The method of claim 6, wherein the muscular dystrophy is muscular atrophy, disuse atrophy, spinal muscular amyotrophy, dystrophy, muscular spasticity, muscular hypotonia, muscular strength Food characterized in that it is selected from the group consisting of weakening, muscle dystrophy, amyotrophic lateral sclerosis, spinal bulb muscular atrophy and myasthenia gravis.
7. The food product of claim 6, further comprising a food additive that is food acceptable.

Images