WO2013048145A2 - Composition for preventing or treating inflammatory diseases or diseases related to bone mass reduction, containing 6-(3-hydroxyphenyl)-2-naphthol or pharmaceutically acceptable salt thereof as active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing or treating inflammatory diseases or diseases related to bone mass reduction, containing 6-(3-hydroxyphenyl)-2-naphthol or a pharmaceutically acceptable salt thereof as an active ingredient. The 6-(3-hydroxyphenyl)-2-naphthol of the present invention inhibits the generation of proinflammatory cytokine in synoviocytes, reduces the rate of arthritis progression and the clinical disease index of arthritis in an animal model with arthritis, suppresses the loss and deformation of cartilage and bone, and alleviates the degree of inflammation of synovial tissue, the extent of bone erosion, the extent of chondrocyte destruction and the infiltration of immunocytes, and thus can be used as a composition for preventing or treating inflammatory diseases including arthritis. In addition, the 6-(3-hydroxyphenyl)-2-naphthol of the present invention inhibits the formation of osteoclasts, reduces bone resorption, suppresses the bone loss in an animal model with LPS-induced bone resorption, and improves trabecular bone volume, trabecular bone thickness, trabecular bone number, trabecular space area and the like in an ovariectomized animal model, and thus can be used as a composition for preventing or treating diseases related to bone mass reduction including osteoporosis.

Description

6- (3-hydroxyphenyl) -2-naphthol or a pharmaceutically acceptable salt thereof as an active ingredient A composition for preventing or treating inflammatory diseases or bone loss-related diseases

The present invention is an inflammatory disease or bone loss-related diseases comprising 6- (3-hydroxyphenyl) -2-naphthol (6- (3-Hydroxyphenyl) -2-naphthol) or a pharmaceutically acceptable salt thereof as an active ingredient It relates to a composition for the prophylaxis or treatment of.

Inflammation is one of tissue damage, an external stimulus, or a defense response of biological tissues to various infectious agents. Inflammation is the activation of enzymes, inflammatory mediators, and cell infiltration due to organic interactions between various inflammatory mediators and various immune cells in blood vessels and body fluids. A series of complex pathologies, including fluid exudation, circulatory disorders, tissue degeneration and hyperproliferation. Inflammatory reactions occur in the early stages when macrophages gather into the wound and attack the invading bacteria, then plasma builds up in the wound and increases blood flow resulting in external symptoms such as fever, erythema, swelling and pain. . If these inflammatory reactions occur continuously or excessively, they proceed to the main pathology of the disease (sensitized allergic disease, chronic inflammatory disease), which leads to serious abnormal disorders.

Inflammation in vivo causes various biochemical phenomena. Macrophage is a multifunctional cell that plays an important role in the inflammatory response by generating various cytokines and nitric oxides (NO) by chemical stimulation. Inducible nitric acidase, especially expressed by cytokine stimulation such as lipopolysaccharide (LPS), interferon-γ, and tumor necrosis factor-α (TNF-α) in macrophages oxide synthase (iNOS) produces large amounts of NO over long periods of time. This oxidative stress is known to promote NFκB activity, a transcription factor of the inflammatory response inhibited by IκB. Activated NFκB is known to migrate to the nucleus and promote gene expression that induces several inflammatory responses, such as iNOS, COX-2 and interleukin-1β, IL-1β, or TNF-α. Inhibition of factors is known to inhibit inflammatory responses (Baeuerle et al., Annu. Rev. Immunol., 12: 141-179, 1994). Therefore, the search for a substance that inhibits the production of TNF-α, NO, prostaglandin or IL-1β may prove to be an effective substance for inflammatory diseases such as edema or dermatitis.

A non-steroidal anti-inflammatory drug (NSAID _S ), a widely used drug for the treatment of most inflammatory diseases, is a prostaglandin from arachidonic acid called cyclooxygenase (COX). Inhibition of enzymatic activity involved in biosynthesis results in anti-inflammatory action, which causes serious side effects such as gastrointestinal disorders, liver disorders, and renal disorders, in addition to the main therapeutic effects, resulting in restrictions on long-term use (Rajakariar R et al. 2006).

Therefore, there is little side effect, so there is a need for the development of a new anti-inflammatory analgesic that is excellent in anti-inflammatory efficacy without any difficulty for long-term use.

On the other hand, the bone repeats the cycle for about 120 to 150 days, including bone resorption by osteoclasts, bone formation and osteostasis by osteoblasts. In healthy adults, bone resorption and bone formation are precisely regulated, resulting in little change in overall bone mass. However, in diseases related to bone loss, the balance of both is broken, resulting in a decrease in bone mass and deterioration of bone tissue. Because of this, fractures occur easily, which causes the disease to lie down, deformation of the body, and death depending on the fracture site.

A typical example of a bone loss related disease is osteoporosis. Osteoporosis is a systemic disease whose main symptoms are a decrease in bone mass and deterioration of bone tissue. Osteoporosis is a condition in which bone mineralization is reduced by thinning the bone tissue and the bone marrow cavity is widened. As a result, the bone weakens as the symptoms progress, so it is easy to fracture even in a small impact. Bone mass is influenced by many factors, including genetic factors, nutrition, hormone changes, differences in exercise and lifestyle, and the causes of osteoporosis include old age, lack of exercise, low weight, smoking, low calcium diet, menopause, Ovarian ablation and the like are known. On the other hand, although there are individual differences, blacks have lower bone resorption levels than whites, resulting in higher bone mass. Usually, bone mass is highest at 14-18 years of age and decreases by about 1% per year in old age. Especially in women, bone reduction continues after 30 years of age, and when menopause reaches bone growth rapidly due to hormonal changes.

As described above, osteoporosis is unavoidable for the elderly, especially postmenopausal women, and as the population ages in developed countries, interest in osteoporosis and its therapeutics is gradually increasing. It is also known that there is a market of about $ 130 billion related to the treatment of bone diseases around the world, and it is expected to increase further, so that various research institutes and pharmaceutical companies around the world are investing in the development of the treatment for bone diseases. .

The treatment of osteoporosis includes general therapies and pharmacotherapy. General therapies require changes in calcium and vitamin D intake, exercise and lifestyle changes. Medications include hormonal supplementation, alendronate, calcitonin, raloxifene, Na-F, calcitriol, and bisphosphonate preparations. Osteoporosis treatment requires a long-term treatment period, it is necessary to develop a new osteoporosis treatment agent that does not have side effects due to side effects such as urolithiasis, endometrial cancer, breast cancer when long-term administration of conventional drugs.

Materials currently used to treat osteoporosis include estrogens, androgen anabolic steroids, calcium preparations, phosphates, fluorides, ifriflavones, and vitamin D3. In 1995, Merck, USA, developed aminobisphosphonate, and in 1997, Lilly, USA, raloxifene, which acts as a selective estrogen receptor regulator, was developed as a new drug for osteoporosis. Conventional osteoporosis therapeutic agents are mostly estrogen-based substances, and estrogen-based substances are known to exhibit side effects such as cancer, gallstones, and thrombosis when administered for a long time. However, osteoporosis cannot be treated with short-term administration of drugs, and long-term administration of drugs is essential. Therefore, long-term administration of drugs does not have such side effects, and there is an urgent need for the development of new substances with superior efficacy to replace estrogen. Situation.

Accordingly, the present inventors have endeavored to develop a composition for preventing or treating inflammatory diseases or bone loss-related diseases without toxicity and side effects, resulting in 6- (3-hydroxyphenyl) -2-naphthol (6- (3-Hydroxyphenyl). The present invention was completed by confirming that) -2-naphthol) showed an excellent inflammatory disease or a prophylactic or therapeutic effect of a disease related to bone loss.

An object of the present invention to provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases or bone loss-related diseases comprising 6- (3-hydroxyphenyl) -2-naphthol or a pharmaceutically acceptable salt thereof as an active ingredient. will be.

Another object of the present invention to provide a food composition for the prevention or improvement of inflammatory diseases or bone loss-related diseases comprising 6- (3-hydroxyphenyl) -2-naphthol or a pharmaceutically acceptable salt thereof as an active ingredient. It is.

In order to achieve the above object, the present invention is to prevent or treat inflammatory diseases or bone loss-related diseases comprising 6- (3-hydroxyphenyl) -2-naphthol or a pharmaceutically acceptable salt thereof as an active ingredient It provides a pharmaceutical composition.

The present invention also provides a food composition for the prevention or improvement of inflammatory diseases or bone loss-related diseases comprising 6- (3-hydroxyphenyl) -2-naphthol or a pharmaceutically acceptable salt thereof as an active ingredient.

6- (3-hydroxyphenyl) -2-naphthol of the present invention inhibits the production of proinflammatory cytokines in synovial cells, reduces the incidence of arthritis and clinical disease index of arthritis in arthritis animal models, It has an excellent effect of suppressing loss and deformation, reducing the degree of inflammation of the synovial tissue, the degree of bone erosion, the degree of cartilage cell destruction and the infiltration of immune cells, and thus can be used as a composition for preventing or treating inflammatory diseases including arthritis. have. In addition, the 6- (3-hydroxyphenyl) -2-naphthol of the present invention inhibits the formation of osteoclasts, decreases bone resorption, inhibits bone loss and ovarian extraction in LPS-induced bone resorption animal models In the animal model has an excellent effect of improving the striatal bone mass, striatal bone thickness, striatal bone marrow, aerospace space, etc., can be used as a composition for the prevention or treatment of diseases related to bone loss, including osteoporosis.

1 shows mRNA expression of pro-inflammatory cytokines (IL-6, IL-8, MCP-1) after treatment with TNF-α and 6- (3-hydroxyphenyl) -2-naphthol in synovial cells. It is also.

Figure 2 shows the amount of pro-inflammatory cytokines (IL-6, IL-8, MCP-1) after treatment with TNF-α and 6- (3-hydroxyphenyl) -2-naphthol in synovial cells. to be.

3 is a diagram showing the incidence of arthritis according to administration of 6- (3-hydroxyphenyl) -2-naphthol in the collagen-induced arthritis mouse model.

4 and 5 are diagrams showing the effect of treating foot edema following administration of 6- (3-hydroxyphenyl) -2-naphthol in the collagen-induced arthritis mouse model as an arthritis clinical disease index.

FIG. 6 is a diagram illustrating three-dimensional reconstruction of knee joint and hind paw tissue of the collagen-induced arthritis mouse model through micro-computed tomography.

7 is a diagram showing the knee cross section of the collagen-induced arthritis mouse model through trichrome and safranin-O staining.

8 is a diagram showing the degree of inflammation of the knee tissue (the degree of inflammation of the synovial tissue, the degree of bone erosion, the degree of chondrocyte destruction and immune cell infiltration) of the collagen-induced arthritis mouse model.

9 is a diagram showing the concentration of inflammatory cytokines (TNF-α, IL-1β, IL-6, INF-γ, MCP-1 and IL-17) in the serum of the collagen-induced arthritis mouse model.

FIG. 10 is treated with 6- (3-hydroxyphenyl) -2-naphthol with concentrations of osteoclasts (M-CSF and RANKL) in macrophages obtained from mouse bone marrow and cultured for 9 days. The number of osteoclasts is shown through TRAP staining.

11 is treated with 6- (3-hydroxyphenyl) -2-naphthol with concentrations of osteoclasts (M-CSF and RANKL) in macrophages obtained from mouse bone marrow and cultured for 9 days. Figure shows the degree of differentiation into (A: osteoclast number, B: quantification by measuring TRAP activity).

FIG. 12 shows hydroxyapatite for 9 days by treating 6- (3-hydroxyphenyl) -2-naphthol with concentrations of osteoclasts (M-CSF and RANKL) in macrophages obtained from mouse bone marrow. After culturing in a well coated plate, the bone absorbed area is shown (A: micrograph, B: pit area, C: number of absorbed feet).

Figure 13 is a macrophage obtained from the mouse bone marrow treated with osteoclasts differentiator (M-CSF and RANKL) with 6- (3-hydroxyphenyl) -2-naphthol concentrations in the cells obtained after 9 days incubation It is a diagram showing the mRNA expression of osteoclast target genes (A: TRAP, B: calcitonin receptor, C: cathepsin K related mRNA expression).

FIG. 14 is a three-dimensional image of a skull obtained after daily injection of 6- (3-hydroxyphenyl) -2-naphthol at 10 mg / kg for 6 days in an LPS-induced bone resorption animal model. Reconstructed diagram.

FIG. 15 is a photograph (A) of TRAP staining of a cross section of a skull obtained after daily injection of 6- (3-hydroxyphenyl) -2-naphthol at 10 mg / kg for 6 days in an LPS-induced bone resorption animal model; The graph (B) which quantified the osteoclast staining site area is shown.

FIG. 16 is a graph illustrating bone analysis of the femur obtained after 24 injections of 6- (3-hydroxyphenyl) -2-naphthol at 10 mg / kg into the abdominal cavity at 2 days intervals in an ovarian isolated animal model (A A: holding bone mass, B: holding bone thickness, C: holding bone marrow, D: holding space area.

The present invention provides a composition for the prevention and treatment of inflammatory diseases or bone loss-related diseases comprising 6- (3-hydroxyphenyl) -2-naphthol or a pharmaceutically acceptable salt thereof as an active ingredient represented by the following formula (1) to provide.

Formula 1

The composition comprises a pharmaceutical composition or a food composition.

6- (3-hydroxyphenyl) -2-naphthol of Chemical Formula 1 is a resveratrol derivative having a molecular formula of C ₁₆ H ₂ O ₂ and a molecular weight of 236.0837.

In the present invention, "resveratrol" is a natural substance present in herbs, grape skins, nuts and red wine.

Pharmaceutically acceptable salts of 6- (3-hydroxyphenyl) -2-naphthol in the present invention include all salts of acidic or basic groups which may be present in the compounds of the structure shown in formula (I). Examples include the sodium, calcium and potassium salts of the hydroxy group and include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandel Latex, methanesulfonate (mesylate) and p-toluenesulfonate (tosylate), and the like, and the like, but are not limited thereto as long as they can be prepared through a method or a process for preparing a salt known in the art.

6- (3-hydroxyphenyl) -2-naphthol of the present invention inhibits the production of proinflammatory cytokines in synovial cells, reduces the incidence of arthritis and clinical disease index of arthritis in arthritis animal models, It has an excellent effect of suppressing loss and deformation, relieving synovial tissue inflammation, bone erosion, cartilage destruction and immune cell infiltration. In addition, the 6- (3-hydroxyphenyl) -2-naphthol of the present invention inhibits the formation of osteoclasts, decreases bone resorption, inhibits bone loss and ovarian extraction in LPS-induced bone resorption animal models In animal model, it has excellent effect to improve strut bone mass, strut bone thickness, strut bone marrow, and strut space area.

As described above, since 6- (3-hydroxyphenyl) -2-naphthol according to the present invention has an excellent therapeutic effect against inflammatory diseases or bone loss-related diseases, it is useful for the prevention or treatment of inflammatory diseases or bone loss-related diseases. It can be used as a useful medicine or dietary supplement.

In the present invention, "inflammatory disease" is a dermatitis, allergy, atopic, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, lupus , Fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendonitis, hay salt, periarthritis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis, acute inflammation and chronic inflammation, Preferably osteoarthritis or rheumatoid arthritis, most preferably rheumatoid arthritis.

In the present invention, the "bone loss-related disease" refers to a disease in which bone loss occurs with symptoms such as a decrease in bone density and deterioration of bone tissue. For example, 1) primary osteoporosis (eg, primary osteoporosis with age, primary osteoporosis with menopause, primary osteoporosis with ovarian extraction, etc.), 2) secondary osteoporosis (eg, glucocorticoid-induced osteoporosis, thyroid function) Anti-osteoporosis, fixed-induced osteoporosis, heparin-induced osteoporosis, immunosuppressive osteoporosis, osteoporosis following renal failure, inflammatory osteoporosis, osteoporosis according to Cushing syndrome, rheumatoid osteoporosis, etc.), 3) cancer metastasis, hypercalcemia, Bone diseases such as jet disease, bone defects (alveolar bone defects, mandibular bone defects, childhood sudden bone defects, etc.), bone necrosis, and the like.

The composition of the present invention may further contain at least one known active ingredient having 6- (3-hydroxyphenyl) -2-naphthol and a prophylactic or therapeutic effect for inflammatory diseases or bone loss-related diseases.

In addition, the composition of the present invention may further include a component that does not increase the efficacy, but may improve the odor, taste, time, etc. that are commonly used in pharmaceutical compositions. In addition, the composition of the present invention is an inorganic or organic additive such as vitamins B1, B2, B6, C, E, niacin, carnitine, betaine, folate pantothenic acid, biotin, zinc, iron, calcium, chromium, magnesium, and mixtures thereof. These may further include.

The composition of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. It may also be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like in the form of conventional formulations, external preparations, suppositories, and sterile injectable solutions. Suitable formulations known in the art are preferably those disclosed in Remington's Pharmaceutical Science, recently, Mack Publishing Company, Easton PA. Carriers, excipients and diluents that may be included in the 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, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. In formulating the composition, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations are prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin and the like in the composition. It is prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin, may be used. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. In addition, non-aqueous solvents and suspending agents may be used propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

6- (3-hydroxyphenyl) -2-naphthol of the present invention may be included in 0.001 to 50% by weight, preferably 0.01 to 20% by weight in the total pharmaceutical composition. In addition, the 6- (3-hydroxyphenyl) -2-naphthol can be adjusted in an amount such that an appropriate amount can be administered per kg of the individual to which the pharmaceutical composition is administered. For example, when the individual to which the 6- (3-hydroxyphenyl) -2-naphthol is administered is a human, the 6- (3-hydroxyphenyl) -2-naphthol is 0.0001 to 500 mg / kg, preferably The content included in the pharmaceutical composition may be adjusted to be administered in an amount of 0.001 to 500 mg / kg, more preferably 0.001 to 300 mg / kg.

The composition of the present invention can be administered to a subject to prevent or treat inflammatory diseases or diseases related to bone loss.

In the present invention, "individual" refers to a mammal, such as horses, sheep, pigs, goats, dogs, etc., including a human having a disease that can improve symptoms by administering the pharmaceutical composition of the present invention.

By “administration” in the present invention is meant to provide the subject with the composition of the present invention in any suitable way. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections.

The composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the sex, age, severity, drug activity, drug of the patient. Sensitivity to, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses.

The compositions of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention or treatment of inflammatory diseases or bone loss associated diseases.

6- (3-hydroxyphenyl) -2-naphthol of the present invention may be added to a dietary supplement for the purpose of preventing or ameliorating inflammatory diseases or diseases related to bone loss.

There is no particular limitation on the kind of food. Examples include meat products, sausages, breads, chocolates, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, teas, drinks, alcoholic beverages and vitamin complexes. It includes all dietary supplements in the usual sense.

Food of the present invention can be used in the form of capsules, tablets, powders, liquid suspensions, pills and granules, but is not limited thereto.

The dietary supplement in tablet form may be prepared as it is or by adding excipients, binders, disintegrants or other additives into granules in a suitable manner and then compressed and molded by adding a lubricant or the like. Either by direct compression molding of an even mixture of excipients, binders, disintegrants or other suitable additives, or by mixing the nutraceuticals as they are or by adding the appropriate additives and mixing them evenly and then compression molding or excipients in the nutraceuticals, A binder or other suitable additive is added to evenly mix the powder with a solvent, the wet powder is molded into a mold at low pressure, and then dried and prepared by a suitable method. In addition, the nutraceutical can be added to the health functional food in the form of tablets, if necessary, can be avoided with a suitable epidermis.

Among the dietary supplements in the form of capsules, the hard capsules are usually a mixture of a dietary supplement or an excipient suitable for a dietary supplement, or granulated by a suitable method, or granulated by a suitable epidermal, as it is or Soft capsules are prepared by filling them, and soft capsules are usually packed in capsules containing glycerin or sorbitol in a suitable capsule base such as gelatin, with appropriate excipients for health functional food or health functional food. It is prepared by molding into a shape, and if necessary, a coloring preservative or the like may be added to the capsule base.

Supplements, binders, disintegrants, etc. are usually mixed into a dietary supplement and then molded into a spherical form by appropriate methods. You can also be greeted with a suitable substance.

Functional foods in granule formulations are usually made by adding functional foods or excipients, binders, disintegrants, etc., and mixing them evenly and then granulating them in a proper way to make the particles as even as possible. , Mating agent, etc. can be added.

The nutraceuticals of the beverage formulations may contain various flavors or natural carbohydrates, etc. as additional ingredients, like conventional beverages. Examples of the above-mentioned natural carbohydrates are sugars such as glucose, monosaccharides such as fructose, maltose, disaccharides such as sucrose, dextrins, polysaccharides such as cyclodextrins, xylitol, sorbitol, and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. Functional ingredients such as deer antler extract, fructooligosaccharide, acacia honey, which help to absorb bowel movements and calcium absorption, complex golden extract, which is a natural preservative, and gellan gum, which are sediment improving agents, can be added, but are not limited thereto. Suitable components can be used as appropriate. The proportion of the natural carbohydrate is generally about 0.001 to 0.4 g, preferably about 0.002 to 0.03 g per 100 ml of the food composition of the present invention.

When the composition of the present invention is used as a food additive, the composition may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). Generally, in the preparation of food or beverages the compositions of the invention are added in amounts of up to 15% by weight, preferably up to 10% by weight relative to the raw materials. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, it may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

Hereinafter, preferred examples and preparation examples are provided to aid in understanding the present invention. However, the following examples and preparations are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples and preparations.

Example. Preparation of 6- (3-hydroxyphenyl) -2-naphthol injection solution for mouse intraperitoneal administration

6- (3-hydroxyphenyl) -2-naphthol is a resveratrol derivative having the molecular formula C ₁₆ H ₂ O ₂ and a molecular weight of 236.0837. In order to prepare an injection solution for mouse intraperitoneal administration of the compound, 6- (3-hydroxyphenyl) -2-naphthol was quantified using an electronic balance, dissolved in DMSO, and diluted in sterile PBS to 10% DMSO. Prepared.

Experimental Example 1. Verification of proinflammatory cytokine inhibitory effect

To determine the effect of 6- (3-hydroxyphenyl) -2-naphthol on the expression of proinflammatory cytokines, TNF- was obtained after culturing and culturing fibroblast-like synoviocytes obtained from surgery in arthritis patients. Stimulation with α increased the production of proinflammatory cytokine, followed by treatment with 6- (3-hydroxyphenyl) -2-naphthol. Thereafter, mRNA changes of each proinflammatory cytokine were confirmed by reverse transcriptase PCR, and the production amount of each proinflammatory cytokine was confirmed by ELISA. The results are shown in FIGS. 1 and 2, respectively.

As shown in Fig. 1 and Fig. 2, the mRNA and the amount of proinflammatory cytokines such as IL-6, IL-8 and MCP-1 were produced by treatment with 6- (3-hydroxyphenyl) -2-naphthol. It was confirmed that the decrease.

Experimental Example 2 Effect Verification in Arthritis Animal Model

2-1. Animal model design

In order to confirm the effects of 6- (3-hydroxyphenyl) -2-naphthol on the treatment of arthritis, experiments were conducted using a collagen-induced arthritis mouse model. More specifically, bovine type 2 collagen was homogeneously emulsified using a T-connector connected to a 3 ml syringe while cooling with Freund's adjuvant on ice. The emulsified bovine type 2 collagen solution was injected intradermally into the tail tip of 6-week-old DBA1 / J mice to administer the immunogen. On day 21 after the first immunization, the emulsified bovine type 2 collagen solution was injected intradermally into the tail of the mouse to induce a secondary immune response. Symptoms of arthritis gradually after secondary immunity. After the induction of the second immune response, the 6- (3-hydroxyphenyl) -2-naphthol injection prepared in Example was injected into the abdominal cavity of mice at a total of 12 injections for 3 weeks at a time of 2 days at 5 mg / kg. It was. The control vehicle was injected intraperitoneally with 10% DMSO only. During the experimental period 6- (3-hydroxyphenyl) -2-naphthol showed no toxic signal and no lethality of mice was observed.

2-2. Finding Arthritis Incidence

During the experimental period, the incidence of arthritis was determined based on the degree of joint swelling of the mouse joint region. The results are shown in FIG.

As shown in Figure 3, in the case of the control group, the incidence of arthritis was 100%, while the 6- (3-hydroxyphenyl) -2-naphthol-administered group of the present invention was confirmed to show the incidence of arthritis of 50-60%. It was confirmed that 6- (3-hydroxyphenyl) -2-naphthol has an excellent effect of reducing the onset of arthritis.

2-3. Confirmation of Arthritis Clinical Disease Index

The clinical arthritic score index of the arthritis of the mice was checked during the experiment. The clinical disease index of arthritis was scored as follows.

0: no swelling and redness, 1: slight redness and swelling of the ankle joint, 2: ankle joint redness and swelling prominent, 3: redness, severe swelling, including finger joints, 4: all areas of the joint Severe swelling throughout.

The best score for the joint lesion was 4 per leg, and the best score was 16 for the left and right forefoot and hind foot per mouse.

The results are shown in FIGS. 4 and 5.

As shown in Figures 4 and 5, the 6- (3-hydroxyphenyl) -2-naphthol administration group of the present invention compared to the control group was confirmed that the clinical disease index of arthritis significantly reduced.

2-4. Cartilage and Bone Tissue 3D Analysis

In order to analyze the cartilage and bone tissue of the mouse three-dimensionally, the knee joints and hind feet of the mouse were photographed by a micro-computed tomography scan device and then reconstructed three-dimensionally by a software program. The results are shown in FIG.

As shown in FIG. 6, the control group showed typical arthritis findings of cartilage and bone loss and deformation in the knee joint and hind paw, and the 6- (3-hydroxyphenyl) -2-naphthol group of the present invention was normal mice. Similar findings were observed, confirming the excellent therapeutic effect for arthritis.

2-5. Histopathological Analysis

Knee tissues of the mouse were separated, fixed in 10% NBF (neutral buffered formalin) solution, and subjected to calcium decalcification (decalcification) using 10% EDTA solution, and then embedded in paraffin. After 4 μm tissue sections were made, trichrome staining for bone staining and safranin-O staining for cartilage staining were performed. In addition, researchers trained using the tissue staining photographs may not know which animals have been given a vehicle or drug, and thus the incidence and severity of arthritis may be associated with synovial inflammation and bone erosion. ), Four factors including cartilage damage and leukocyte infiltration were determined according to the evaluation criteria of 0 to 4. The results are shown in FIGS. 7 and 8, respectively.

As shown in Figure 7 and 8, compared to the control group 6- (3-hydroxyphenyl) -2-naphthol of the tissue of the present invention, the degree of inflammation, bone erosion, cartilage cell destruction and immune cells of the synovial tissue It was confirmed that infiltration and the like were significantly alleviated.

2-6. Molecular Biological Analysis

The concentration of inflammatory cytokines (TNF-α, IL-1β, IL-6, INF-ν, MCP-1, and IL-17) in mouse blood was measured using ELISA. The results are shown in FIG.

As shown in FIG. 9, it was confirmed that the concentration of inflammatory cytokines in the blood of the 6- (3-hydroxyphenyl) -2-naphthol-administered group of the present invention was significantly reduced compared to the control group.

Through the above experimental results, it was confirmed that 6- (3-hydroxyphenyl) -2-naphthol of the present invention is very excellent in preventing and treating arthritis.

Experimental Example 3. Verification of inhibition of osteoclast formation

3-1. Bone marrow cell culture and differentiation into osteoclasts

Muscle tissue around the leg bone (femur, tibia) of the 6-8 week old Balb / c mouse was removed cleanly, cut at both ends of the femur, plugged with a syringe needle, and washed down with medium until the bone marrow completely drained. Bone marrow was collected and centrifuged at 2000 rpm for 5 minutes to obtain cells. The cells were resuspended in alpha-MEM medium containing 10% FBS, aliquoted into a culture dish, and then in a 37 ° C. and 5% CO ₂ incubator. Incubated. After 24 hours, non-attached cells were collected and centrifuged at 2000 rpm for 5 minutes to collect the cells, which were incubated for 3 days in alpha-MM medium containing M-CSF (50 ng / ml) and 10% FBS for macrophages. Differentiated to After 3 days, the adherent cells were collected and cultured in alpha-M medium containing M-CSF (50 ng / ml), RANKL (50 ng / ml) and 10% FBS to induce differentiation into osteoclasts.

3-2. Identify the effect on the formation of osteoclasts

After inoculating the macrophages obtained in Experimental Example 3-1 into a 48-well plate, 6- (3-hydroxyphenyl) -2-naphthol was added to the well at each concentration (0, 5, 10 μM) After 9 days incubation. The cells were fixed with 10% formaldehyde and stained with tartrate-resistant acidic phosphatase (TRAP), a cytochemical marker that can identify osteoclasts. The number of osteoclasts was measured by counting the number of multinucleated cells expressing TRAP, and the number of TRAP-positive cells and TRAP activity with three or more nuclei. The results are shown in FIGS. 10 and 11.

As shown in Figure 10 and 11, it was confirmed that the formation of osteoclasts in a concentration-dependent manner by the treatment of 6- (3-hydroxyphenyl) -2-naphthol of the present invention.

3-3. Identify the impact on bone resorption (pit formation)

The macrophages obtained in Experimental Example 3-1 were inoculated into a well plate (Biologic, BD) coated with hydroxyapatite (hydroxyapatite), and then 6- (3-hydroxyphenyl) -2-naphthol was inoculated. Concentration (0, 5, 10 μM) was added to the wells and incubated for 9 days. The area of the pit formed on the hydroxyapatite slide was measured. The results are shown in FIG.

As shown in Figure 12, the treatment of 6- (3-hydroxyphenyl) -2-naphthol of the present invention significantly reduced the number of resorbed areas (resorbed area) and pit, confirmed that the bone absorption is reduced It was.

3-4. Confirmation of mRNA expression of osteoclast target gene

The effect of extracting RNA from the cells obtained in Experimental Example 3-1 on osteoclast target gene-associated mRNA expression was confirmed using reverse transcriptase PCR. The results are shown in FIG.

As shown in FIG. 13, the expression of TRAP-related mRNA was about 4-5 fold and the expression of calcitonin receptor-related mRNA was about 2-3 by treatment with 6- (3-hydroxyphenyl) -2-naphthol of the present invention. It was confirmed that the expression of embryo, cathepsin K-related mRNA was inhibited about five-fold, through which the expression of osteoclast differentiation-related genes was greatly reduced.

Experimental Example 4. Verification of osteoporosis treatment effect in LPS-induced bone resorption animal model

4-1. Animal model design

In order to confirm the osteoporosis treatment effect of 6- (3-hydroxyphenyl) -2-naphthol, experiments were conducted using an LPS-induced bone resorption mouse model with characteristics similar to human osteoporosis. More specifically, LPS (lipopolysaccharide) was dissolved in PBS and injected into the head of the head of C57BL / 6 female mice at a concentration of 12.5 mg / kg. 6- (3-hydroxyphenyl) -2-naphthol was dissolved in PBS at 10 mg / kg and injected into the head of the mouse once daily for a total of six times for 6 days. Normal group was injected with PBS only and control group with LPS only. During the experimental period 6- (3-hydroxyphenyl) -2-naphthol showed no toxic signal and no lethality of mice was observed.

4-2. Skull 3D Analysis

In order to extract the skull of the mouse and to analyze it three-dimensionally, the extracted skull was photographed with a micro-computed tomography scan, and then three-dimensional reconstruction by a software program. The results are shown in FIG.

As shown in FIG. 14, typical osteoporosis findings of bone loss in the skull of the control group were observed, and similar findings were observed in the 6- (3-hydroxyphenyl) -2-naphthol-administered group of the present invention. Through this, the therapeutic effect of osteoporosis of 6- (3-hydroxyphenyl) -2-naphthol was visually confirmed.

4-3. Histological analysis

The skull of the mouse was isolated and fixed in 10% neutral buffered formalin (NBF) fixative, and then decalcified using 10% EDTA solution and embedded in paraffin. Thereafter, tissue sections of 4 μm were made and TRAP staining was performed. The results are shown in FIG.

As shown in FIG. 15, the 6- (3-hydroxyphenyl) -2-naphthol-administered group of the present invention was found to have about 2 to 3 times more bone area than the control group administered with LPS only. .

Experimental Example 5. Verification of osteoporosis treatment effect in ovarian harvested animal model

5-1. Animal model design

In order to confirm the osteoporosis treatment effect of 6- (3-hydroxyphenyl) -2-naphthol, experiments were conducted using an ovarian isolated animal model. More specifically, bilateral ovarian extraction was performed in C57BL / 6 female mice. The hair of the lower abdomen of the anesthetized mouse was removed, the surgical site was disinfected, and the operation was performed under aseptic manipulation. The skin, abs and peritoneum were incised, the ovaries were exposed with sterile tweezers and the fallopian tubes were ligated with silk threads, and the left and right ovaries were extracted. Antibiotics were injected intraperitoneally to prevent infection, and peritoneal, abs and skin sutured with absorbent sutures. After recovery from ovaries, 6- (3-hydroxyphenyl) -2-naphthol (10 mg / kg) was intraperitoneally injected 24 times for 8 weeks at a time interval of 2 days. PBS was administered to the normal group (sham) without ovarian extraction and the control group (OVX) with ovarian extraction.

5-2. Bone analysis

The femurs of the mice were separated and bone analyzes were performed (abutment bone mass (BT / TV (%), striatal bone thickness (Tb.Th), striatal bone marrow (Tb.Sp), strut space area measurement (Tb.N)). The results are shown in FIG.

As shown in Fig. 16, the 6- (3-hydroxyphenyl) -2-naphthol-administered group of the present invention compared to the control group (OVX) to which only PBS was administered, 4 of the strut bone mass, strut bone thickness, strut bone marrow, and strut space area. It was confirmed that about 20% to 60% of all the indicators were effective in treating osteoporosis.

Through the above experimental results, it was confirmed that 6- (3-hydroxyphenyl) -2-naphthol of the present invention is very excellent in inhibiting the occurrence or treatment effect of bone loss-related diseases.

Hereinafter, the preparation examples of the pharmaceutical composition and the food composition comprising the composition of the present invention, but are not intended to limit the present invention only intended to be described in detail.

Formulation Example 1 Preparation of Pharmaceutical Composition

1-1. Manufacture of powder

6- (3-hydroxyphenyl) -2-naphthol 20 mg

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

1-2. Manufacture of tablets

6- (3-hydroxyphenyl) -2-naphthol 10 mg

Corn starch 100 mg Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

1-3. Preparation of Capsules

6- (3-hydroxyphenyl) -2-naphthol 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

1-4. Preparation of Injectables

6- (3-hydroxyphenyl) -2-naphthol 10 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO ₄ 2H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

1-5. Preparation of liquid

6- (3-hydroxyphenyl) -2-naphthol 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding a proper amount of lemon aroma, and then mixing the above components, adding purified water and adjusting the whole to 100 ml by adding purified water and filling into a brown bottle. The solution is prepared by sterilization.

Formulation Example 2 Preparation of Food Composition

2-1. Manufacture of health food

6- (3-hydroxyphenyl) -2-naphthol 100 mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

Biotin 10 μg

Nicotinamide 1.7 mg

Folic acid 50 μg

Calcium Pantothenate 0.5 mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

15 mg potassium monophosphate

Dicalcium Phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

2-2. Manufacture of health drinks

6- (3-hydroxyphenyl) -2-naphthol 100 mg

15 g of vitamin C

100 g of vitamin E (powder)

Iron lactate 19.75 g

3.5 g of zinc oxide

Nicotinamide 3.5 g

0.2 g of vitamin A

0.25 g of vitamin B1

0.3 g of vitamin B2

Water quantification

After mixing the above components in accordance with the conventional healthy beverage manufacturing method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 l container, sealed sterilization and refrigerated Used to prepare the healthy beverage composition of the invention.

Although the composition ratio is a composition suitable for a preferred beverage in a preferred embodiment, the compounding ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

6- (3-hydroxyphenyl) -2-naphthol of the present invention inhibits the production of proinflammatory cytokines in synovial cells, reduces the incidence of arthritis and clinical disease index of arthritis in arthritis animal models, It has an excellent effect of suppressing loss and deformation, reducing the degree of inflammation of the synovial tissue, the degree of bone erosion, the degree of cartilage cell destruction and the infiltration of immune cells, and thus can be used as a composition for preventing or treating inflammatory diseases including arthritis. have. In addition, the 6- (3-hydroxyphenyl) -2-naphthol of the present invention inhibits the formation of osteoclasts, decreases bone resorption, inhibits bone loss and ovarian extraction in LPS-induced bone resorption animal models In the animal model has an excellent effect of improving the striatal bone mass, striatal bone thickness, striatal bone marrow, aerospace space, etc., can be used as a composition for the prevention or treatment of diseases related to bone loss, including osteoporosis.

Claims (7)

Inflammatory disease or bone mass including 6- (3-hydroxyphenyl) -2-naphthol (6- (3-Hydroxyphenyl) -2-naphthol) or a pharmaceutically acceptable salt thereof represented by Formula 1 as an active ingredient Pharmaceutical compositions for the prevention and treatment of lowering related diseases. [Formula 1]

 The method of claim 1, wherein the inflammatory disease is dermatitis, allergy, atopic dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever Group consisting of lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, hay salt, peritonitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis, acute inflammation and chronic inflammation A pharmaceutical composition for the prevention and treatment of inflammatory diseases or diseases related to bone loss, characterized in that at least one selected from.  According to claim 2, wherein the inflammatory disease is osteoarthritis or rheumatoid arthritis pharmaceutical composition for the prevention and treatment of inflammatory diseases or bone loss associated diseases. The disease according to claim 1, wherein the disease related to bone loss is at least one selected from the group consisting of primary osteoporosis with age, primary osteoporosis with menopause, and primary osteoporosis with ovarian extraction. Pharmaceutical compositions for the prevention and treatment of related diseases. According to claim 1, wherein the disease associated with bone loss is glucocorticoid-induced osteoporosis, hyperthyroidism osteoporosis, fixed-induced osteoporosis, heparin-induced osteoporosis, immunosuppressive osteoporosis, osteoporosis following renal failure, inflammatory osteoporosis, Cushing syndrome According to claim 1, characterized in that at least one selected from the group consisting of osteoporosis and rheumatoid osteoporosis, pharmaceutical composition for the prevention and treatment of inflammatory diseases or bone loss related diseases. According to claim 1, wherein the disease related to bone loss is characterized in that one or more selected from the group consisting of cancer metastasis, hypercalcemia, Beget's disease, bone defects and bone necrosis, prevention of inflammatory disease or bone loss related diseases And therapeutic pharmaceutical compositions. Inflammatory disease or bone mass including 6- (3-hydroxyphenyl) -2-naphthol (6- (3-Hydroxyphenyl) -2-naphthol) or a pharmaceutically acceptable salt thereof represented by Formula 1 as an active ingredient Food composition for preventing and improving degradation-related diseases. [Formula 1]

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