KR20200038411A - Composition for Preventing or Treating Bone Diseases Comprising IF1 - Google Patents

Abstract

The present invention relates to a composition for preventing or treating bone diseases containing ATPase inhibitory factor 1 (IF1). More specifically, the present invention relates to a pharmaceutical composition and food for preventing or treating bone diseases containing IF1 having an effect of bone growth and bone reinforcement as an active component. IF1 according to the present invention exhibits effects of increasing bone length, changing bone structure, and increasing bone strength, thereby being very useful as an agent for preventing, alleviating or treating bone diseases such as osteoporosis.

Description

Pharmaceutical composition for preventing or treating bone disease containing IF1 (ATPase inhibitory factor 1) as an active ingredient {Composition for Preventing or Treating Bone Diseases Comprising IF1}

The present invention relates to a composition for the prevention or treatment of bone diseases containing IF1 (ATPase inhibitory factor 1), and more specifically, contains IF1 having an effect of bone growth and bone strengthening according to an increase in bone length and strength as an active ingredient It relates to a pharmaceutical composition and food for the prevention or treatment of bone diseases.

Bone metabolism is composed of repetition of bone resorption and bone formation, and bone mass is determined by these two processes. However, when a female hormone deficiency appears, it directly and indirectly affects osteoblasts and osteoclasts involved in this bone remodeling process, causing bone loss. When bone resorption is increased compared to bone formation, it causes osteoporosis, which increases the risk of fracture.

Osteoporosis, which has emerged as a major problem in the elderly society among bone diseases, is a skeletal disease that increases the risk of fracture due to weakening of bone strength, and bone strength is determined by bone density and bone quality. Bone density is determined by the highest bone mass value and the amount of bone loss, and can be known from the structure of the bone, the rate of bone replacement, accumulation of micro-damage, and mineralization. Osteoporosis is classified into primary (primary) osteoporosis and secondary (secondary) osteoporosis, and primary osteoporosis is divided into post-menopausal osteoporosis and senile osteoporosis. Secondary osteoporosis occurs when bone formation is impaired or bone loss increases due to a specific disease, surgery, or medication. Representative causes include steroid administration, hypogonadism, and celiac disease (NATIONAL INSTITUTES OF HEALTH: Osteoporosis Prevention, Diagnosis, and Therapy NIH Consensus Statement 17, 1, 2000).

Substances currently being used to treat osteoporosis include estrogens, androgenic anabolic steroids, calcium preparations, phosphates, fluoride preparations, ipriflavone, and vitamin D ₃ . Estrogen inhibits osteoblast cell death, increases cell survival, promotes osteoclast cell death, and reduces cell survival, which is an effective method for treating menopausal symptoms and maintaining bone density, or breast cancer and endometrial hyperplasia. There are side effects that cause back. These, existing osteoporosis treatment drugs cause many side effects when administered for a long time. Therefore, there is a need to develop a safe preventive and therapeutic agent that exhibits a continuous increase in bone density even with long-term administration and has few side effects.

On the other hand, ATPase inhibitory factor 1 (IF1) binds to the mitochondrial enzyme F1Fo ATP synthase (multi-subunit, membrane-bound assembly), which is involved in the synthesis and decomposition of ATP synthesis in mitochondria, thereby inhibiting ATP depletion. It is known to inhibit cell death by blocking depletion (G. Kroemer, et al., Immunol . Today , 18 (1), 44-51, 1997). It is reported that β-F1-ATPase (F1 catalytic subunit of this enzyme) is present in cell membranes of various types of cells, and ATPase is also present in bone cell membranes (Yonally, SK, & Capaldi, RA, Mitochondrion , 6 (6), 305-314, 2006). IF1 induces an intracellular signaling system by binding to β-F1-ATPase at the cell membrane and thereby induces a bioreaction. That is, IF1 inhibits ATP hydrolysis through binding of the plasma membrane to the F1-ATPase subunit, resulting in an increase in the amount of extracellular ATP (extracellular ATP, exATP) in the extracellular fluid, and this increased exATP purine of the cell membrane It reacts with sex receptors to induce various useful intracellular responses through purine signaling. Purine signaling is also known to be important in bone turnover (Wang, Ning, et al., Molecular endocrinology 26.1, 142-152, 2012).

As a result, the present inventors tried hard to develop a bone disease treatment agent that has no side effects and has an excellent effect and can compensate for the limitations of existing treatments. As a result, the bone length and bone strength of mice increased and bone structure changed by administration of IF1 recombinant protein After confirming that, the present invention was completed.

An object of the present invention is to provide a pharmaceutical composition and food for the prevention or treatment of bone disease containing as an active ingredient IF1 (ATPase inhibitory factor 1) exhibiting bone growth and bone strengthening effect according to the increase in bone length and strength.

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

The present invention also provides a food for preventing or improving bone disease containing IF1 (ATPase inhibitory factor 1) as an active ingredient.

IF1 (ATPase inhibitory factor 1) according to the present invention shows an effect of increasing bone length, changing bone structure and increasing bone strength without side effects in obese-induced mice or ovarian resected mice, thus preventing, improving or treating bone diseases such as osteoporosis Very useful as.

1 is a measure of cytotoxicity in osteoblasts by IF1 treatment, the relative cell viability up to 48 hours. * P <0.05, ** p <0.01, *** p <0.001 and *** p <0.0001 compared with each control group.
Figure 2 shows the extracellular ATP release over time by IF1 treatment. * P <0.05, ** p <0.01 compared with each control group.
Figure 3 shows the effect of promoting proliferation and differentiation of osteoblasts and osteoclasts by IF1 treatment, (A) changes in cell morphology, (B) changes in cell number, (C) degree of calcification (Alizarind red S staining) Results). * P <0.05, ** p <0.01, *** p <0.001, **** p <0.0001 compared with each control group normalized by body weight.
Figure 4 is a measurement of the increase in bone length of mice by IF1 treatment by micro-CT imaging, (A) right femoral length (mm), (B) femoral AP (Anterior-posterior) diameter (cm), (C) Femur ML (Medial-lateral) diameter (cm), (D) right tibia length (cm), (E) tibia AP diameter (cm), (F) tibia ML diameter (cm). Each value represents the mean SE value of two groups (n = 15). * P <0.05 compared with each control group normalized by body weight.
FIG. 5 shows changes in bone strength and bone structure of mice treated with IF1 through bone microarchitecture analysis (BMA), (A) total volume (mm ³ ), (B) bone surface area (mm ² ), (C) Endocortical perimeter (mm), (D) Total cross-sectional area (mm ² ). Each value represents the mean SE value of two groups (n = 15). * P <0.05 compared with each control group.
FIG. 6 shows bone structure and mass change in mice by IF1 treatment using Micro-CT, (A) high-fat diet, and (B) high-fat diet + IF1 administration, confirming the bone diameter and cross-sectional area of the experimental group. will be.
7 is an analysis of changes in bone strength and bone structure of ovarian ablation model mice by IF1 treatment through BMA (Bone microarchitecture analysis), (A) total volume (mm ³ ), (B) total cross-sectional area (Total cross) -sectional area) (mm ² ), (C) Endocortical perimeter (mm), (D) fiber line thickness (mm). Each value represents the mean SE value of two groups (n = 8). * P <0.05 compared with each control group.
Figure 8 is a BMA (Bone microarchitecture analysis) analysis of changes in bone density in mice by IF1 treatment, (A) obesity model, (B) ovarian ablation model mouse BMD and cortical BMD values are corrected by weight Shows. Each value represents the mean SE value of two groups (obesity model) (n = 15) and (ovarian ablation model) (n = 8). * P <0.05 compared with each control group.
FIG. 9 shows the effect of preventing bone slit by IF1 treatment through Safranin O & Fast green staining, and shows representative images of each group (Sham, GST control group, IF1 experimental 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 many studies have been conducted as targets 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 changes in bone length and bone strength or changes in bone structure 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 mouse model of ovarian ablation, and the effect of improving bone metabolism indicators such as an increase in bone length and bone strength or a change in bone structure by IF1 was confirmed. That is, the effect of preventing and treating ATPase inhibitory factor 1 (IF1) on bone disease and its effect on bone metabolism was demonstrated.

Accordingly, the present invention relates to a pharmaceutical composition for the prevention or treatment of bone disease containing IF1 (ATPase inhibitory factor 1) as an active ingredient in a consistent sense.

In the present invention, the composition may be characterized by increasing bone length and bone strength.

The term "bone disease" of the present invention means any disease that occurs due to reduced bone density. For example, it refers to a bone metabolism disease that is caused by an imbalance of osteoblasts and osteoclasts and causes a disorder of bone tissue or destroys bone tissue.

 The bone disease is not particularly limited, but osteoporosis (osteoporosis), sluggish growth, fracture, osteoarthritis, Paget disease, periodontal disease, rickets, osteomalacia, osteogenic dysfunction, rickets, osteomalacia ), Scoliosis, pelvic deformity, rheumatoid arthritis, hyperparathyroidism, nephrotic dystrophy in patients with renal failure, inflammatory alveolar bone resorption disease, and inflammatory bone resorption disease. It may be a selected disease.

The term "osteoporosis" of the present invention refers to a state in which the amount of bone is reduced and the strength of bone is weakened due to a qualitative change, so that fracture is likely to occur, and is also called osteoporosis or osteoporosis.

In the present invention, the osteoporosis means primary osteoporosis, primary osteoporosis, secondary osteoporosis, secondary osteoporosis and idiopathic osteoporosis.

Osteoporosis is largely divided into primary and secondary osteoporosis, and primary osteoporosis is divided into type 1 osteoporosis (postmenopausal) and type 2 osteoporosis (senile osteoporosis). Osteoporosis of type 1 is the main cause of estrogen deficiency after menopause, and as bone resorption increases, calcium in the blood increases, and accordingly, the secretion of parathyroid hormone decreases, and as a result, osteoporosis occurs as a result of lowering calcium absorption in the intestines. It occurs mainly in women of the age, and bone loss occurs in the bone, so fractures occur frequently in the spine and wrist. Osteoporosis is a type 2 osteoporosis that occurs in both men and women after 65 years of age, the mechanism of occurrence is decreased calcium absorption in the intestinal tract and increased secretion of parathyroid hormone, which increases cortical bone loss and decreases osteogenic bone formation, resulting in osteoporosis. (Roodman GD et al, Advances in bone biology: the osteoclast, Endocrinol Rev 17, 308-332, 1996) In addition to normal physiological bone loss caused by type 1 and type 2, environmental factors such as genetic predisposition and wrong lifestyle It is a disease caused by a combination of hyperhormonal mismatch and the like, and most of the osteoporosis patients belong to this. On the other hand, secondary osteoporosis refers to osteoporosis caused by other diseases or drugs and refers to osteoporosis caused by various endocrine diseases (Avioli LV et al, The osteoporotic syndrome; Detection, prevention & treatment 3rd ed Wielis, 1993).

Fractures caused by osteoporosis often occur in the elderly after 65 years of age and are caused by a slight trauma to the fall, and the fractures occur mainly in the neck or electronic part of the femur, and as the age increases, more fractures of the electronic part occur. Unlike fractures, it is a fracture that has high morbidity and mortality and is difficult to treat.

In the present invention, "bone growth" is meant to include some or all of the increase in the size, thickness, density, length or function of the tissue of the bone (bone).

In the present invention, the stunted growth may be normal mutation hyponephrosis or hyponephrosis secondary to disease. The normal mutation hyponephrosis may be familial short stature, delayed constitutional growth, and idiopathic short stature in consultation. In addition, hyponephrosis, which is secondary due to disease, may be primary growth disorder (endogenous disorder) or secondary growth disorder (exogenous growth disorder), and the primary growth disorder includes osteochondrosis, chromosomal abnormality (down syndrome or Turner Syndrome), short stature, unreasonable lightweight (delayed growth in the uterus), shortened by Freder-Willi Syndrome, shortened by Russell-Silver Syndrome, and shortened by Nunan Syndrome. There are short stature caused by systemic disease, short stature caused by growth hormone deficiency, short stature caused by hypothyroidism, short stature caused by Cushing's syndrome, and psychosocial dwarfism.

The term "osteoarthritis" of the present invention refers to a joint disease caused by damage to cartilage tissue, particularly a disease involving loss of cartilage function, and has various other names, such as degenerative joint disease, osteoarthritis, hypertrophic arthritis or degenerative arthritis, etc. Is known. The osteoarthritis can be characterized by chronic progressive destruction of cartilage tissue in major joints such as fingers, elbows, knees or ankles.

The term "periodic disease" of the present invention refers to all diseases in which plaque (plaque microflora, plaque) and plaque formed by many bacteria always present in the mouth cause inflammation and destroy periodontal tissue. The periodontal disease is also commonly referred to as qichi, meaning it includes both gingivitis and periodontitis.

According to a specific embodiment of the present invention, when the IF1 of the present invention was administered to a mouse, it was confirmed that an increase in bone length and an increase in bone strength, and also an effect on changes in bone structure and composition. As a result, it was confirmed that IF1 increases bone length and strength, effectively exhibiting bone growth and bone strengthening effects.

The therapeutic effect of IF1 can be equally applied to bone metabolic diseases as well as bone related diseases caused by various causes.

The term "prevention" of the present invention means all actions that suppress or delay the onset of bone-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 bone-related diseases by administering the pharmaceutical composition of the present invention.

The bone disease treatment can be applied to any mammal that can develop a bone-related disease, and includes, for example, humans and primates as well as cattle, pigs, sheep, horses, dogs and cats, and livestock without limitation. It can 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 bone disease, which further comprises 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 for preventing or treating bone disease comprising administering a pharmaceutical composition containing IF1 (ATPase inhibitory factor 1) as an active ingredient to an individual.

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 bone disease.

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 bone disease.

The term "individual" of the present invention means all animals, including humans, who have already developed or are likely to develop bone disease, 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 bone disease containing 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 composition may be characterized by increasing bone length and bone strength.

In the present invention, the bone disease is osteoporosis (osteoporosis), anaphylaxis, fracture, osteoarthritis, Paget disease, periodontal disease, rickets, osteomalacia, osteoplastic dysfunction, rickets, osteomalacia , Scoliosis, pelvic deformity, rheumatoid arthritis, hyperparathyroidism, nephrotic dystrophy in renal failure, inflammatory alveolar bone resorption disease and inflammatory bone resorption disease and inflammatory bone absorption disease It is preferably a disease, but is not limited thereto.

In the present invention, the osteoporosis means primary osteoporosis, primary osteoporosis, secondary osteoporosis, secondary osteoporosis and idiopathic osteoporosis.

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: Measurement of cytotoxicity and extracellular ATP release of IF1

Cells were cultured in mouse-derived MC3T3-E1 cells in an α-MEM medium containing 10% FBS and 1% penicilin / streptomycin in a 37 ° C, 5% carbon dioxide incubator. Osteoblast differentiation induction was 50ug / mL ascorbic acid and 10 mM beta-glycerol phosphate.

Cytotoxicity of IF1 was measured using MTT (3- (4,5-dimethythiazol-2-yl) -2,5diphenyltetrazolium bromide). Each MC3T3-E1 cell cultured in a 96-well plate was treated with IF1 protein by concentration, and then 1 mg / mL MTT was added after 24 hours and 48 hours. After incubation for 3 hours, the medium was removed and the absorbance of formazan at 540 nm was measured using DMSO (FIG. 1).

As a result, it was confirmed that there was no cytotoxicity of IF1.

Next, the extracellular fluid ATP release according to the treatment of IF1 was measured. After treating IF1 1 μM in MC3T3-E1 cells, ATP concentration over time was measured using a CellTiter-Glo Luminescent Assay kit (Promega, Madison, WI, USA) (FIG. 2).

As a result, it was found that the released ATP concentration increased.

Example 2: Effects of IF1 on osteoblast proliferation and differentiation

In order to reveal the function of IF1 related to osteoblast proliferation, MC3T3-E1 cells were treated with IF1, and it was observed that the morphology and number of cells were similar compared to the control group (FIGS. 3A and 3B).

In order to confirm the effect of IF1 on osteoblast differentiation, calcification, the last step of differentiation, was measured by alizarin red s staining. Cells cultured in 12-well plates were differentiated for 3 weeks with differentiation medium and undifferentiated medium. Each PBS, GST, and IF1 were used for material treatment in the differentiation medium. After the differentiation, the cells were fixed with 4% paraformaldehyde, and then stained with alizarin red s solution. The comparison for each group was compared through absorbance measured at 550 nm using a 10% cetylpyridinium chloride solution.

As a result, significantly increased calcification was observed in the IF1 treatment group compared to the control group.

Example 3: Mouse preparation

3-1: Obese animal model

C57BL / 6J males were used as the obese mouse model, and 40 mice of 5 weeks of age 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 one cycle was adjusted through lighting, and free feeding was performed in both the adaptation period and the experiment period.

After the one-week adjustment period, the animals 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.

3-2: Ovariectomized animal model

Bone disease occurs due to female hormone deficiency during ovarian ablation, so female hormone deficiency was induced using the ovarian ablation model, and then the effect of IF1 on bone disease treatment was observed.

As an animal model for ovarian ablation, 16 animals of C57BL / 6J, 8 weeks of age, who underwent ovarian ablation, were pre-sold, and they were provided with a normal diet for 1 week to undergo adaptation. As a breeding environment, the humidity of 18 ~ 24 ℃ and 50 ~ 60% was maintained at all times, and one cycle was adjusted through lighting, and free feeding was performed in both the adaptation period and the experiment period.

After the one-week adjustment period, the animals were divided into two groups and reared under the following conditions.

Group 1: High Fat Diet (HFD) Control (n = 8)

Group 2: High Fat Diet (HFD) experimental group (n = 8)

3-3: Osteoarthritis Animal Model

To confirm the effect of IF1 on osteoarthritis, a mouse model of osteoarthritis induced through surgery was used. As a mouse model, a 15-week-old C57BL / 6J male was used, and DMM (Destabilization of the medial meniscus) and Sham surgery were performed as follows.

The mouse under sufficiently anesthesia was fixed on the dissecting microscope to block movement. First, using a scalpel, the skin of the joint area where the tibia of the left leg and the thigh of the mouse meet was incised about 3 mm. After removing the exposed joint capsule, the fat was removed and the medial meniscus (MM) and ligament (medial meniscotibal ligament) were lifted. Finally, the surgical site was closed using a sealing thread, and the sealing site was removed after observing the sealing area for two days. Sham surgery was performed after the ligament exposure was omitted.

After surgery, mice had a recovery period of 1 week, and were divided into 5 groups and reared under the following conditions.

Group 1: High Fat Diet (HFD) Sham Control (n = 6)

Group 2: High Fat Diet (HFD) DMM Osteoarthritis Prevention Control (n = 8)

Group 3: High Fat Diet (HFD) DMM Osteoarthritis Prevention Experiment Group (n = 8)

Group 4: High Fat Diet (HFD) DMM Osteoarthritis Treatment Control (n = 8)

Group 5: High-fat diet (HFD) DMM osteoarthritis treatment experimental group (n = 8)

Example 4: Increase in bone length following IF1 administration

Obese model mice with 6 weeks of obesity induction were given an intraperitoneal injection of IF1 (5 mg / kg BW) 4 times per week for 4 weeks. In the case of ovarian model mice, IF1 (5 mg / kg BW) was injected intraperitoneally every day for 8 weeks. IF1 produced recombinant protein by separating and purifying DNA data of IF1 including GST-tag through cloning.

The conditions for administering IF1 to the obese and ovarian ablation group of Example 3 are as follows (Table 2 and Table 3).

Obesity model Dietary Injection protein Group 1 control (n = 10) Normal diet (ND) GST Group 2 control group (n = 15) High Fat Diet (HFD) GST Group 3 experimental group (n = 15) High Fat Diet (HFD) IF1 (5mg / kg BW)

Ovarian ablation model Dietary Injection protein Group 1  Control (n = 8) High Fat Diet (HFD) GST Group 2 Experimental group  (n = 8) High Fat Diet (HFD) IF1 (5mg / kg BW)

The mice, which had all the experiments and breeding periods, were fasted for 16 hours for dissection, and then anesthetized with 1.5 g / ml of 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 10 minutes at 8000 rpm to separate the serum, and the blood-collected mice were dissected and bones were quickly removed. The bone separated from the body is a femoral length, femoral AP (Anterior-posterior) diameter, femoral ML (Medial-lateral) diameter, femur ML (Medial-lateral) diameter, tibial length, tibial AP diameter and tibial ML diameter via a digital caliper. The first length measurement was performed. For a more precise measurement, the bones after the 1st measurement were stored immediately in a 10% formalin solution and the 2nd measurement was performed by micro CT measurement.

As a result, it was confirmed that in both the first measurement and the second measurement, the normal diet and the high-fat diet had an increased bone length in the IF1 administration group compared to the control group (FIG. 4). That is, it shows that IF1 is effective in increasing bone length.

Example 5: Bone strength and bone structure change according to IF1 administration

5-1: Obese animal model

The micro CT image obtained in Example 4 was analyzed through BMA (Bone microarchitecture analysis) to confirm changes in bone strength and bone structure.

As a result, in the IF1 administration group, TV (Total volume), BS (Bone surface), Tt.Ar (Total cross-sectional area), Ec.Pm (Endocortical perimeter) were observed. It was found that it increased significantly (Fig. 5). In particular, it was confirmed that the treatment of IF1 showed an effect on bone diameter and cross-sectional area compared to the control group (FIG. 6). This increase in TV and BS values and Tt.Ar values indicate that IF1 functions positively in bone metabolism, thus suggesting an increase in the total volume and size of bone.

In addition, in the case of cortical fraction, Ct.Ar (Cortical area fraction) tended to increase, whereas Ec.Pm (Endocortical perimeter) increased significantly in the IF1 administration group. The increase in Ec.Pm is known to be closely related to bone resorption and periosteal bone formation (Hammond, MA et al., PloS one, 11 (9), e0163273, 2016), purine effect of IF1 It can be seen that in connection with the osteoclasts in the vicinity of the bone cell membrane, it has a beneficial effect on changes in structure and composition due to bone resorption and responses to external stimuli.

Next, in the case of bone mineral density (BMD), as a result of correcting the weight of each mouse, BMD and cortical BMD values were observed to be significantly increased to confirm an increase in bone density of IF1 (FIG. 8A). Osteoblast differentiation (Agrawal, A. et al., Bone, 95: 91-101, 2017) and mineralization of bone marrow cells occur downstream of purinergic receptor responses by extracellular ATP (Xing, Y. et al., PLoS One , 9 (9), e108417, 2014), which is directly related to the increase in bone length, volume elongation and internal volume caused by IF1, and IF1's action on bone includes osteoblasts You can see that

5-2: Ovariectomized animal model

By administering IF1 to the ovarian ablation animal model, changes in bone strength and bone structure were confirmed by the method of Example 5-1.

As a result, in the IF1 administration group, TV (Total volume), Tt.Ar (Total cross-sectional area), Ec.pm (Endocortical perimeter), Tb.Th (Trabecular thickness) ) Was observed to increase significantly (FIG. 7). That is, the effect of increasing bone size and volume of IF1 was confirmed as in Example 5-1 obesity model.

In the case of bone mineral density (BMD), it was observed that BMD and cortical BMD values significantly increased as a result of correcting the weight of each mouse (FIG. 8B). Similarly, as in Example 5-1 obesity model, it was confirmed that the increase in bone density of IF1.

Example 6: Effect of improving osteoarthritis according to IF1 administration

The osteoarthritis-improved mouse group underwent GST and IF1 intraperitoneal administration daily for about 8 weeks after DMM surgery. The concentration of each material was conducted at 7.5 mg / kg BW. IF1 produced recombinant protein by separating and purifying DNA data of IF1 including GST-tag through cloning.

Conditions for administering IF1 to the osteoarthritis mouse group of Example 3 are as follows (Table 4).

Bone fracture  Model Dietary Injection protein Group 1  Sham control (n = 8) High Fat Diet (HFD) GST Group 2  DMM preventive control (n = 8) High Fat Diet (HFD) GST Group 2  DMM prevention Experimental group  (n = 8) High Fat Diet (HFD) IF1 (7.5mg / kg BW)

All experiments and mice after the breeding period were dissected as described in Example 4. However, in the case of osteoarthritis mice, the entire left hind leg bone was immersed in 10% formalin solution for 1 day to fix it. After the bone was fixed for 3 weeks using 10% EDTA, the extent of arthritis was assessed using Safranin O & Fast green.

As a result, it was observed that in the IF1 treatment group, the number of cells increased and the degree of safranin O staining increased compared to the GST control group (FIG. 9). That is, it can be seen that the treatment of IF1 is involved in the improvement of arthritis.

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 Bone Diseases Comprising          IF1 <130> P19-B199 <150> KR 10-2018-0117693 <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 bone disease containing IF1 (ATPase inhibitory factor 1) as an active ingredient.
The pharmaceutical composition of claim 1, wherein the composition increases bone length and bone strength.
The method of claim 1, wherein the bone disease is selected from the group consisting of osteoporosis (osteoporosis), sluggish growth, fracture, osteoarthritis, Paget disease, periodontal disease, rickets, osteomalacia and osteoplastic dysfunction. Pharmaceutical composition.
The pharmaceutical composition according to claim 3, wherein the osteoporosis is primary osteoporosis, secondary osteoporosis or idiopathic osteoporosis.
The pharmaceutical composition of claim 1, further comprising a pharmaceutically acceptable carrier, excipient or diluent.
Food for prevention or improvement of bone disease containing IF1 (ATPase inhibitory factor 1) as an active ingredient.
The food according to claim 6, wherein the composition increases bone length and bone strength.
The method of claim 6, wherein the bone disease is characterized in that it is selected from the group consisting of osteoporosis (osteoporosis), sluggish growth, fracture, osteoarthritis, Paget disease, periodontal disease, rickets, osteomalacia and osteoplastic dysfunction. Food.
The food according to claim 8, wherein the osteoporosis is primary osteoporosis, secondary osteoporosis or idiopathic osteoporosis.
7. The food product of claim 6, further comprising a food additive that is food acceptable.

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