WO2018038502A1 - Pharmaceutical composition for preventing and treating bone diseases including arteannuin b as active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition, which is for preventing and treating bone diseases, and includes arteannuin B as an active ingredient. The pharmaceutical composition including arteannuin B of the present invention has low side effects and toxicity, and exhibits a remarkably effective osteoclastogenesis inhibiting function compared to artemisinin, which is another major component of Artemisia annua L. Thus, the pharmaceutical composition is expected to be widely utilized in the medical field.

Description

Pharmaceutical composition for the prevention and treatment of bone diseases comprising Arteanuine B as an active ingredient

The present invention relates to a composition for the prevention and treatment of bone diseases comprising Arteanuin B as an active ingredient.

Osteooclast, a multinucleated large cell, has the function of destroying and absorbing bone tissue, and is known to play a role in destroying bone matrix and degrading bone minerals. Activated osteoclasts have three or more nuclei, which require different hormones and factors to differentiate from osteoclast progenitor cells to mature multinucleated osteoclasts. The two most important factors are the macrophage colony stimulating factor (M-CSF) and the receptor activator of nuclear factor-kappa B ligand (RANKL) produced by osteoblasts (Mojtaba A., et al., Cancer Biol Ther., 7: 1,3-9; 1 (2008)). M-CSF is a cytokine expressed by osteoblasts and stromal cells and plays an important role in osteoclast formation, and is known to play an important role in cell proliferation, survival, and cytoskeleton formation (Kim SY., Et al., J Korean Orthop Assoc., 44: 151-158 (2009)). Another important factor, RANKL, is expressed by osteoblasts and attaches to RANK receptors in osteoclast progenitor cells to induce growth and differentiation of osteoclasts (Mojtaba A., Cancer biology & Therapy, 7: 1,3-9 ; 1 (2008)). In addition, RANKL promotes the differentiation of osteoclasts by activating transcription factors such as c-fos, nuclear factor of activated T-Cells, cytoplasmic, calcineurin-dependent 1 (NFATk1), NF-kB, and PI-3K, or ERK. It is also known to play a role in promoting the survival and function of osteoclasts by activating a signaling system such as (LEE ZH., Et al., Biochem Biophys Res Commun., 305: 211-213 (2003)).

These osteoclasts cause abnormal bone tissue destruction and absorption due to imbalance with osteoblasts in the bone, thereby causing osteoporosis, which causes bone mass and bone density to decrease. Osteomalacia in which lime is lost, fibrous ostitis in which the bone marrow becomes fibrous, periodontitis in which the alveolar bone is lost, and rheumatoid arthritis, which causes joint destruction and deformation. Known. Imbalance between osteoclasts and osteoblasts can be caused by a variety of causes, including bone metastasis of cancer cells, endocrine disorders, and hyperparathyroidism. Therefore, if it is possible to effectively inhibit the destruction and absorption of bone tissue by osteoclasts, it is expected to be able to treat a variety of bone diseases caused by this, various drugs and treatments for osteoclasts are being actively studied. Recently, bisphosphonate-based therapeutic agents such as Fosamax (component name: aledronate), Actonel (component name: risedronate), and Zometa (component name: zoledronate) have been used to treat bone damage caused by osteoclasts such as osteoporosis. It is widely used. Most of these bisphosphonate preparations act to attenuate the function of osteoclasts that destroy bone and to induce the death of osteoclasts, thereby delaying or inhibiting bone loss. However, the number of side effects such as jawbone necrosis, severe atrial fibrillation, incapacitation of bones or joints, and musculoskeletal pain has been increasing yearly in patients taking bisphosphonate-based drugs (Coleman RE., Br J Cancer, 98). : 1736-1740 (2008). In addition, the formation of osteoclasts is also promoted by cancer cells that have metastasized to bone in breast cancer and prostate cancer, and serious bone diseases occur. However, no drug has been developed to treat them.

As such, there is a need for the development of drugs that supplement the disadvantages of existing bisphosphonate formulations, are nontoxic, and can effectively inhibit bone resorption by osteoclasts.

On the other hand, Arteannuin B (Arteannuin B) is a major component contained in the firewood, which is biosynthesized through a different metabolic pathway from Artemisinin, another major component of the firewood. Recently, as the anti-cancer and osteoclast inhibitory effects of artemisinin, which is one of the components contained in the firewood, are known, the interest in firewood is increasing. However, very little research has been carried out on Arteanu, B.

Therefore, the present invention relates to a pharmaceutical composition for preventing and treating bone diseases comprising Arteanuin B as an active ingredient, the pharmaceutical composition of the present invention has less side effects and toxicity, and has a significant effect of inhibiting osteoclast formation than artemisinin. As it seems, it is expected to be widely used in the medical field.

The present invention has been made to solve the above-mentioned problems in the prior art, an object of the present invention to provide a pharmaceutical composition for preventing and treating bone disease containing Arteanuin B as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

Hereinafter, various embodiments described herein are described with reference to the drawings. In the following description, for a thorough understanding of the present invention, various specific details are described, such as specific forms, compositions, processes and the like. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and forms. In other instances, well known processes and manufacturing techniques have not been described in particular detail in order to not unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "embodiment" means that a particular feature, form, composition or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Thus, the context of “in one embodiment” or “embodiment” expressed at various places throughout this specification does not necessarily represent the same embodiment of the invention. In addition, particular features, forms, compositions, or properties may be combined in any suitable manner in one or more embodiments.

As used herein, "bone disease" refers to a disease caused by an imbalance between osteoclasts and osteoblasts in bone. Preferably, bone damage caused by bone metastasis of cancer cells, osteoporosis, osteomalacia, fibrous ostitis, periodontitis, rheumatoid arthritis, metabolic bone disease, etc. However, if the disease caused by the bone resorption of osteoclasts is not limited thereto.

In the present specification, "Artemisia annua" is a perennial plant of the dicotyledon plant Campanula Asteraceae. It is also called as a leafy mugwort, or cauliflower, and is commonly found on roadsides, glades, and rivers. It is about 1 m high and has a hairy, peculiar smell throughout the grass. Stems are green with many branches and leaves are alternately divided into two or three times thinly and deeply. The leaves are 4 ~ 7 cm long and the shape is bar-shaped, and the outer hairs have fine hairs and occupancy. The middle of the leaf is comb-shaped and the upper leaf is small. Flowers bloom greenish yellow in June-September, with small heads running like ears, forming a whole cone. The head is ball-shaped and 1.5 cm in diameter. The sill is exposed. The piece of gun is hairless and arranged in two to three lines. The piece of shell is long oval and green. Fruits are achenes, about 0.7 mm long.

Pharmacologically, it has been used in ancient China to treat symptoms caused by infection with Plasmodium falciparum, such as malaria (Kooy and Sullivan, J Ethnophamacol., 150: 1-13 (2013)), and fever in Chinese medicine It has been used to treat colds, mucus, childhood, indigestion and dysentery.

According to the Korea Food and Drug Administration, fireflies are allowed to use only young leaves for food, and all of the currently available pill or essence are processed into young leaves. However, the roots, leaves, and stems of firewood, which are known to be effective as medicinal, are being studied.

In the present specification, "artemisinin" is a kind of firewood extract, represented by the following formula (1).

Artemisinin is a sesquiterpene trioxane lactone-based compound isolated from firefly, which is known to have an excellent therapeutic effect against malaria infection by eliminating malaria parasites through the generation of free radicals, oral or intramuscular injection It is used as. Also toxoplasma gondii. It is also known to be effective against other infectious diseases such as Schistosoma, Pneumocystis carinii, Human Cytomegalovirus, Herpes simplex viruses, Hepatitis B, and Hepatitis C. In order to enhance the efficacy of artemisinin, studies have been made on the synthesis of various derivatives such as Dihydroartemisinin, Arteether, Artemether, Artesunate, etc. Dihydroartemisinin has stronger antimalarial efficacy than artemisinin and is known as the final metabolite of atethers, atamers and atesunates (Li QG., Et al., J Pharm Pharmacol., 50: 173-182 (1998). In addition, recent studies have shown that artemisinin and dihydroartemisinin exhibit excellent anticancer effects by inducing apoptosis of cancer and inhibiting invasiveness in lung cancer, breast cancer and prostate cancer.

In addition, artemisinin-glycolipid and the like have been developed as derivatives of artemisinin. A representative artemisinin-glycolipid complex derivative is a complex derivative prepared by combining artemisinin with a glycolipid called daumone isolated from Caenorhabditis elegans. Damon is known as a substance that induces nematodes to dormant and delays aging and inhibits cancer angiogenesis (Jung M., et al., Eur J Med Chem., 44: 3120-3129 (2009) )). These artemisinin-glycolipid complex derivatives have been reported to have higher anticancer effects in oral cancer, lung cancer and breast cancer than artemisinin or daumone (Ricci J., et al., Chem Pharm Bull., 59: 1471-1475 (2011)).

[Formula 1]

Artemisinin (AN) first converts the precursor artemisinic aldehyde (AAA) to dehydroartisinic aldehyde (DHAAA) and converts the dihydroartemisinic aldehyde to dehydroartemisi After secondary conversion to dehydroartemisinic acid (DHAA), the dehydroartemisinic acid is biosynthesized by the process of conversion to artemisinin through photooxidation.

In the present specification, "Arteannuin B" is a kind of firewood extract, which is represented by the following formula (2).

Arteanuin B is converted to arteminic acid (AA) from the precursor artemisinic aldehyde (AAA), and then biosynthesized into arteanuine B through photooxidation.

Unlike artemisinin, which is a component of the same firewood extract, studies on the physiological function of arteanuin B are very poor.

[Formula 2]

As used herein, the term "pharmaceutical composition" means a composition to be administered for a specific purpose. For the purposes of the present invention, the pharmaceutical composition of the present invention is a composition for preventing and treating bone diseases comprising Arteanuin B as an active ingredient, and may include a protein and a pharmaceutically acceptable carrier, excipient or diluent involved therein. have. Said "pharmaceutically acceptable" carrier or excipient means that which has been approved by the governmental regulatory authority, or listed in government or other generally approved pharmacopoeia for use in vertebrates, and more particularly in humans. do.

For parenteral administration, the pharmaceutical compositions of the invention may be in the form of suspensions, solutions or emulsions in oily or aqueous carriers, and may be prepared in the form of solids or semisolids. In addition, the pharmaceutical compositions of the present invention may include formulating agents such as suspending, stabilizing, dissolving and / or dispersing agents and may be sterilized. The pharmaceutical composition may be stable under the conditions of manufacture and storage, and may be preserved against the contaminating action of microorganisms such as bacteria or fungi. Alternatively, the pharmaceutical compositions of the present invention may be in sterile powder form for reconstitution with a suitable carrier prior to use. The pharmaceutical compositions may be in unit-dose form, in microneedle patches, in ampoules, or in other unit-dose containers, or in multi-dose containers. Alternatively, the pharmaceutical composition may be stored in a freeze-dried (freeze-dried) state, which requires the addition of a sterile liquid carrier, eg, water for injection just before use. Immediately injectable solutions and suspensions may be prepared as sterile powders, granules or tablets.

In some non-limiting embodiments, the pharmaceutical compositions of the present invention can be formulated or included in the form of microspheres in a liquid. In certain non-limiting embodiments, the pharmaceutical compositions of the present invention may comprise their pharmaceutically acceptable compounds and / or mixtures at concentrations between 0.001 and 100,000 U / kg. Also in certain non-limiting embodiments, the pharmaceutical compositions of the present invention may include suitable excipients, preservatives, suspending agents, additional stabilizers, dyes, buffers, antibacterial agents, antifungal agents, and isotonic agents, for example, sugars or sodium chloride. Can be. As used herein, the term "stabilizer" refers to a compound that is optionally used in the pharmaceutical compositions of the present invention to increase shelf life. In a non-limiting implementation, the stabilizer can be a sugar, an amino acid, or a polymer. In addition, the pharmaceutical composition of the present invention may include one or more pharmaceutically acceptable carriers, and the carrier may be a solvent or a dispersion medium. Non-limiting examples of pharmaceutically acceptable carriers include water, saline, ethanol, polyols (eg glycerol, propylene glycol and liquid polyethylene glycols), oils, and suitable mixtures thereof. Non-limiting examples of sterilization techniques applied to the pharmaceutical compositions of the present invention include filtration through bacteria-inhibiting filters, terminal sterilization, incorporation of sterile preparations, irradiation, sterile gas irradiation, heating, vacuum drying and freeze drying. do.

As used herein, the "food composition" is used in various ways for improving and preventing bone diseases, the food composition comprising the composition of the present invention as an active ingredient is a variety of foods, for example, beverages, gum, tea, vitamins It may be prepared in the form of a combination, powder, granules, tablets, capsules, sweets, rice cakes, bread and the like. Since the food composition of the present invention is configured and improved from the existing food intake which has little toxicity and side effects, it can be used with confidence even when taken for long periods of time. When the composition of the present invention is included in the food composition, the amount may be added at a ratio of 0.1 to 100% of the total weight. Herein, when the food composition is prepared in the form of a beverage, there is no particular limitation other than the food composition in the proportion indicated, and may include various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. That is, natural carbohydrates include monosaccharides such as glucose, disaccharides such as fructose, sucrose and the like, and common sugars such as polysaccharides, dextrins and cyclodextrins, and sugar alcohols such as xylitol, sorbitol, and erythritol. can do. Examples of the flavourant include natural flavourant (tautin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition is a variety of nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, Stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, etc. These components may be used independently or in combination The ratio of such additives is typically per 100 parts by weight of the composition of the present invention. It is generally selected from the range of 0.1 to 100 parts by weight, but is not limited thereto.

As used herein, "administration" means introducing the composition of the present invention to a patient in any suitable manner, and the route of administration of the composition of the present invention may be administered via any general route as long as it can reach the desired tissue. have. Oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, pulmonary administration, rectal administration, intraluminal administration, intraperitoneal administration, intradural administration, The composition is most preferably provided as a sibling of oral administration, but is not limited thereto.

The method of treatment of the present invention may comprise administering the pharmaceutical composition in a pharmaceutically effective amount. In the present invention, the effective amount is defined as the type of disease, the severity of the disease, the type and amount of the active ingredient and other ingredients contained in the composition, the type and formulation of the patient and the age, body weight, general health condition, sex and diet, time of administration, route of administration And various factors, including the rate of secretion of the composition, the duration of treatment, and the drugs used concurrently.

In one embodiment of the present invention, there is provided a pharmaceutical composition for preventing and treating bone diseases comprising Arteanuin B as an active ingredient, wherein the bone diseases are osteoporosis, osteomalacia, fibrous ostitis ), Periodontitis (periodontitis), rheumatoid arthritis and metabolic bone disease provides a pharmaceutical composition for the prevention and treatment of bone disease, wherein the bone disease is due to bone absorption of osteoclasts It provides a pharmaceutical composition for the prevention and treatment of bone diseases, characterized in that induced, the bone disease provides a pharmaceutical composition for the prevention and treatment of bone diseases, characterized in that caused by bone metastasis of cancer cells.

In another embodiment of the present invention, there is provided a food composition for improving and alleviating bone disease comprising arteanuin B as an active ingredient, wherein the bone disease is osteoporosis, osteomalacia, fibrous ostitis ), Periodontitis (periodontitis), rheumatoid arthritis and metabolic bone disease provides a food composition for improving and alleviating bone diseases, wherein the bone diseases are caused by bone resorption of osteoclasts Provided is a food composition for improving and alleviating bone disease, characterized in that induced, the bone disease provides a food composition for alleviating and alleviating bone disease, characterized in that caused by bone metastasis of cancer cells.

In another embodiment of the present invention, there is provided a method for preventing or treating bone disease, characterized in that the administration of Arteanuin B as an active ingredient, wherein the bone disease is osteoporosis, osteomalacia, fibrotic It provides a method for preventing or treating bone diseases, characterized in that selected from the group consisting of fibrous ostitis, periodontitis, rheumatoid arthritis and metabolic bone disease.

In another embodiment of the present invention, there is provided a use of the prophylaxis or treatment of bone disease of arteanuin B, wherein the bone disease is osteoporosis, osteomalacia, fibrous ostitis, periodontitis ), Rheumatoid arthritis and metabolic bone disease.

Hereinafter, the present invention will be described in detail step by step.

The present invention relates to a pharmaceutical composition for preventing and treating bone diseases comprising Arteanuin B as an active ingredient. The pharmaceutical composition according to the present invention has a low side effect and toxicity, and can effectively prevent bone loss. It is expected to be utilized.

1 is a diagram showing the biosynthetic pathway and structure of artemisonic acid (AA), artemisinin (AN), or arteanuine B (AB) according to an embodiment of the present invention.

Figure 2 is a diagram confirming the inhibitory effect of osteoclast formation of artemisinic acid (AA), artemisinin (AN), or arteanuine B (AB) according to an embodiment of the present invention.

Figure 3 c-Fos, NAFTc1 and GAPDH expression in mouse bone marrow macrophages treated with artemisonic acid (AA), artemisinin (AN), or arteanuine B (AB) according to an embodiment of the present invention It is a figure confirming the quantity change.

Figure 4 is a view showing the result of converting the c-Fos expression amount of Figure 3 based on GAPDH according to an embodiment of the present invention.

5 is a view showing the results of converting the NAFTc1 expression amount of Figure 3 based on GAPDH according to an embodiment of the present invention.

Figure 6 is a diagram showing the cytotoxicity measurement results of the extract of the firewood in the rat macrophage stimulated with periodontal bacteria-derived lipopolysaccharide according to an embodiment of the present invention.

Figure 7 is a diagram showing the results of cytotoxicity measurements of the active ingredients and synthetic derivatives of the firewood in the macrophages stimulated with periodontal bacteria-derived lipopolysaccharide according to an embodiment of the present invention. In detail, FIG. 7A shows Arteanuin B, FIG. 7B shows Artemisinin, FIG. 7C shows Dihydroartemisinin, FIG. 7D shows Artemisinic Acid, and FIG. 7E shows Artemisinin-Glycolipids. to be.

8 is a view showing the results of measuring the inhibition of TNF-α production of the firewood extract from mouse macrophage stimulated with periodontal bacteria-derived lipopolysaccharide according to an embodiment of the present invention.

9 is a diagram showing the results of measuring the inhibitory activity of TNF-α production of the active ingredients and synthetic derivatives of firewood in mouse macrophages stimulated with periodontitis-derived lipopolysaccharide according to an embodiment of the present invention.

FIG. 10 is a diagram showing weight change during 12 weeks of administration of Ganoderma lucidum extract (AaE), Artemisinin (ART), or Arteanewin B (AB) in an ovarian isolated mouse, according to an embodiment of the present invention. . In detail, Figure 10a and Figure 10b is the result of the administration of the Artemisia extract, Figure 10c and Figure 10d is the Artemisinin or Arteanewin B.

FIG. 11 is a microcomputer tomography of the femur bone after 12 weeks of administration of Artemisia extract (AaE), Artemisinin (ART), or Arteanewin B (AB) in an ovarian isolated mouse, according to an embodiment of the present invention. The figure shows the results of the photographing. In detail, FIG. 11A shows the results of the administration of the firefly extract, and FIG. 11B shows the administration of Artemisinin or Arteanuin B. FIG.

FIG. 12 shows bone morphology of the femur after 12 weeks of administration of Ganoderma lucidum extract (AaE), Artemisinin (ART), or Arteanewin B (AB) in an ovarian isolated mouse, according to an embodiment of the present invention. A diagram showing the results of the analysis. In detail, FIG. 12A shows the bone volume coefficient (BV / TV), FIG. 12B shows the number of bone strains (Tb.N), FIG. 12C shows the thickness of the bone strains (Tb.Th), and FIG. Tb.Sp), and FIG. 12E is a result of analyzing the structural shape index (SMI).

FIG. 13 shows bone metabolism markers in serum after 12 weeks of administration of Ganoderma lucidum extract (AaE), Artemisinin (ART), or Arteanewin B (AB) in ovarian isolated mice, according to an embodiment of the present invention. Figures showing the results of analyzing their activity. In detail, FIG. 13A shows basic dephosphoryase (ALP) activity, FIG. 13B shows osteocalcin, FIG. 13C shows calcium, FIG. 13D shows tartrate resistant acidic phosphatase (TRAP5b) activity, and FIG. 13E shows type 1 Collagen's C-terminal telopeptide (CTX) is analyzed.

FIG. 14 is a 12-week administration of firewood extract (AaE), artemisinin (ART), or arteanuine B (AB) in ovarian isolated mice, according to one embodiment of the present invention. Figure showing the results of analyzing the activity. In detail, FIG. 14A shows tumor necrosis factor-alpha (TNF-α), which is an inflammatory factor, and FIG. 14B shows the result of analyzing interleukin-1 beta.

FIG. 15 is a 12-week administration of Ganoderma lucidum extract (AaE), Artemisinin (ART), or Arteanewin B (AB) in an ovarian isolated mouse according to one embodiment of the present invention. The results are shown. In detail, FIG. 15A shows the results of analysis of alanine aminotransferase (ALT) and FIG. 15B of aspartate aminotransferase (AST).

FIG. 16 is a 12-week administration of the wormwood extract (AaE), artemisinin (ART), or arteanuine B (AB) in ovarian extraction mice according to an embodiment of the present invention, and evaluated for renal toxicity. Figure 1 shows the results. In detail, FIG. 16A shows blood urea nitrogen (BUN), and FIG. 16B shows creatinine.

Artemisia annua extract (AaE), Artemisianine (artemisinin: ART), and Arteanuine B (arteannuin B :) were extracted from ovarian-extracted ICR mice (Central Experimental Animal, Korea). AB) was confirmed to inhibit the osteoporosis. As a result of measuring the changes in bone metabolism index, the ALP activity, osteocalcin, TRAP5b activity and CTX amount decreased by ovarian extraction in the control group of 10 mg / kg Artemisinin 20 mg / kg group significantly decreased the amount of ALP activity, calcium, TRAP5b activity and CTX increased by ovarian extraction, and Arteanuin B increased osteocalcin and TRAP5b among the levels increased by ovarian extraction. It was confirmed that the activity and the amount of CTX significantly inhibited.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example  1: using mouse bone marrow macrophages Artean  B, Artemisonic Acid , Artemisinin Inhibits Osteoclast Formation

Example  1-1. Preparation of Mouse Bone Marrow Macrophages

For the preparation of mouse bone marrow macrophages, three weeks-old male ICR mice (Narabiotech, South Korea) were dislocated from the cervical spine, and the hind limbs were removed using a forceps, and the hind limbs were cut with surgical scissors. Immersion was performed in no added α-MEM (minimum essential medium alpha; Gibco, USA). The bones in the muscles were separated using tweezers and transferred to a new α-MEM, and 600 μl of α-MEM was placed in a syringe. The bone marrow cells were extracted by injection into the central spinal cord of the separated leg bone and sprayed 2 or 3 times. . The extracted bone marrow cells were removed from the supernatant by centrifugation, mixed with new α-MEM, and the bone marrow macrophages were separated from the bone marrow cells using a separation medium histopark (Sigma-Aldrich, USA). . And 1% by volume of antibiotic-antimycotic solution in α-MEM (Gibco, USA), 10% by volume fetal bovine serum (FBS); Gibco, USA), M-CSF (macrophage-colony) stimulating factor (R & D system Inc., USA) 30 ng / ml was added, and then isolated mouse bone marrow macrophages were cultured.

Example  1-2. Arteinyuin  B, Artemisonic Acid , Artemisinin  Osteoclast formation Inhibitory ability  Confirmation experiment

To determine whether arteignin B, artemisinic acid, and artemisinin inhibit osteoclast formation induced by receptor activator of nuclear factor kappa-B ligand (RANKL), “Park EK. et al., Biochem Biophys Res Commun., 325 (4): 1472-1480 (2004). And 40 or 80 μM of artemisinic acid, Arteanuin B and artemisinin prepared 1 or 5 μM, respectively. To each well of a 96-well plate was added 5 x 10 ⁴ mouse bone marrow macrophages prepared by the method of Example 1-1, followed by α-MEM (1% by volume of antibiotic-) 200 μl of antibacterial solution, 10% by volume of FBS, 30 ng / ml of M-CSF, and 100 ng / ml of RANKL) were added, and the mouse bone marrow macrophages were cultured in a cell incubator at 37 ° C. and 5% CO ₂ . Incubated. Every two days, the cells were exchanged with a new α-MEM medium containing each extract and incubated for 5 days. Subsequently, multinucleated osteoclasts were stained using a TRAP assay kit (tartrate resistant acid phosphatase assay kit; Sigma-Aldrich, USA), and the number of osteoclasts having three or more nuclei was counted using an optical microscope. The results are shown in Table 1 and FIG.

Ingredient Name	Component concentration (μM)	Number of osteoclasts induced by RANKL	Osteoclast formation inhibition rate (%)
-	-	0	-
	RANKL	426 ± 5	0
Artemisonic Acid	RANKL + 40	399 ± 7	7
	RANKL + 80	60 ± 8	86
Artaigne B	RANKL + 1	198 ± 12	54
	RANKL + 5	8 ± 1	98
Artemisinin	RANKL + 1	300 ± 11	30
	RANKL + 5	53 ± 4	76

As shown in the above results, osteoclasts were formed when 100 ng / ml of RANKL was treated, and osteoclasts were formed when artemisinic acid, arteanuine B, or artemisinin were treated with RANKL, respectively. It was confirmed that the decrease with the concentration. In particular, Arteanuin B inhibited the differentiation of osteoclasts by 54% and 98% at 1 or 5μM concentration, respectively, it was confirmed that the effect is better than Artemisinin.

Example  1-3. Artemisonic Acid , Arteinyuin  B, Artemisinin  Experiment to confirm c-Fos and NFATc1 expression in osteoclasts

When RANKL is treated to osteoclasts, RANKL binds to RANK of osteoclasts and transmits signals into cells, and activates transcription factors c-Fos and NFATc1 in the nucleus to differentiate osteoclasts (Asagiri M., et al., Bone, 40: 251-264 (2007)). The present inventors investigated whether the osteoclast formation inhibitory effects of artemisinic acid, arteanuine B, or artemisinin are related to these transcription factors. 10 ⁶ mouse bone marrow macrophages prepared by the method of Example 1-1 were treated with arteanin B (1, or 5 μM), artemisinic acid (40, or 80 μM), or artemisinin (in a 60 mm ² dish. 1, or 5 μM), and further incubated for 48 hours in medium containing RANKL (50 ng / ml) and M-CSF (30 ng / ml) and cells were obtained using a scraper. Protein lysis solution (150 mM NaCl, 0.5% Triton X-100, 50 mM Tris-HCl, pH 7.4, 20 mM EGTA, 1 mM DTT, 1 mM Na ₃ VO ₄ , protease inhibitor cocktail tablet ) 40 μl) was added and left on ice for 1 hour, followed by centrifugation (12,000 rpm, 10 min) to obtain a supernatant. Supernatant proteins were quantified using the BCA method (bicinchoninic acid method). The supernatant was then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the separated proteins were transferred to PVDF membranes. PVDP membranes were immersed in 5% skim milk powder dissolved in 0.1% TBST (Tris-buffered saline Tween 20) for 3 hours and then reacted with primary antibody against c-Fos, NAFTc1 or GAPDH diluted 1: 1,000 at 4 ° C for 24 hours. And a secondary antibody that specifically reacts with the primary antibody. At the end of each step, the nonspecific antibody was terminated with 0.1% TBST, reacted with ECL (Enhanced chemiluminesence) solution, and exposed to x-ray film to confirm changes in c-Fos, NAFTc1 and GAPDH. The results are shown in FIG. 3, and the results of converting the c-Fos or NAFTc1 expression levels based on GAPDH are shown in FIGS. 4 and 5, respectively.

As a result, the expression of c-Fos and NFATc1 was increased by 2.8 and 1.6 times in osteoclasts treated only with RANKL, but RANKL in osteoclasts treated with Artea nuine B (1 or 5 μM) with RANKL. Induced expression of c-Fos and NFATc1 was significantly inhibited. On the other hand, the osteoclasts treated with artemisinin or artemisinic acid with RANKL did not significantly inhibit c-Fos expression. Only in significant cases. As a result, Arteanuin B at the same concentration inhibited the expression of c-Fos and NFATc1 by RANKL, which was superior to Artemisinin.

Example  2. Breast cancer cell line Artean  B, Artemisonic Acid , Arte Check for cytotoxicity of mycinin

Example  2-1. Culture of Breast Cancer Cell Lines

Human luminal breast cancer cell lines (MCF-7 and ZR-75-1) and human basal-like / triple-negative breast cancer cell lines (MDA) -MB-231, BT549, and HCC38) were purchased from the American Cell Line Bank (American Type Culture Collection, Manassas, VA, USA) and the Korean Cell Line Bank (Seoul, Korea). MCF-7 and MDA-MB-231 cell lines were cultured using 10% by volume FBS-DMEM, and HCC38 and ZR-75-1 cell lines were cultured using 10% by volume FBS-RPMI 1640. The BT549 cell line was cultured in RPMI 1640 medium containing 0.023 IU / ml insulin and 10 vol% FBS. All breast cancer cells were cultured in a 37 ° C., 5% CO ₂ incubator.

Example  2-2. For breast cancer cell lines Artean  B, Artemisonic Acid , Arte Cytotoxicity Test of Misinin

Breast cancer cell lines cultured by the method of Example 2-1 were treated with arteanuine B, artemisinic acid, or artemisinin to 80 μM and cultured for 72 hours. MTT (3- (4,5-dimethythiazol-2-yl) -2,5-diphenyl tetrazolium bromide; Sigma, USA) solution was added to a concentration of 0.5 mg / ml for each well, and 37 ° C for 4 hours. After incubation in, washed with PBS to completely remove the supernatant, 200 μl of DMSO was added to react for 30 minutes, the absorbance was measured at 570nm. Cell viability was calculated as the percentage of the absorbance of the experimental group (well treated with arteinuine B, artemisonic acid, or artemisinin) relative to the absorbance of the control group (experimentally treated experimental group). From the results, the concentration (IC50) for inhibiting cell survival rate by 50% was calculated and shown in Table 2 below. As a result, it was found that the IC ₅₀ value of Arteanewin B was significantly lower than Artemisinin and Artemisinic acid.

Breast cancer cell line	IC 50 (mM)
	Artemisinin	Artemisonic Acid	Artaigne B
MDA-MB-231	> 80	> 80	1.6
BT549	> 80	> 80	5.78
HCC38	> 80	> 80	13.4
MCF-7	> 80	> 80	25.0
ZR-75-1	> 80	> 80	20.0

Example  3. Periodontal infection  origin As a lipopolysaccharide  Cytotoxicity Test of Extracts from Stimulated Mouse Macrophages

Artemisia annua extract, Artemisia annua extract, Artemisia annulus, an active ingredient of the genus Artemisia, using RAW264.7 mouse macrophages stimulated with lipopolysaccharide (LPS) obtained from the periodontal bacterium Porphyromonas gingivalis The cytotoxicity of B (arteannuin B), artemisinin, artemisinic acid, and synthetic derivatives, dihydroartemisinin, artemisinin-glycolipid, was confirmed. . The artemisinin-glycolipids were synthesized with Daumone. The culture medium was DMEM (Dulbecco's Modified Eagle's medium, Gibco, USA) containing 1% antibiotic-antibacterial solution and 10% FBS. 1 × 10 ⁴ mouse macrophages were dispensed into each well of a 96-well plate and incubated for 24 hours at 37 ° C., 5% CO ₂ conditions. Each extract was added with LPS 1 μg / ml in 96-well plates in which mouse macrophages were divided and incubated for 24 hours. After 24 hours, 20 μl of 5 mg / ml MTT (Thiazolyl Blue Tetrazolium Bromide; Sigma Aldrich, USA) solution was added to each well in 37 ° C., 5% CO ₂ cell incubator, and reacted for 4 hours. After removing the medium, 100 μl of DMSO was added, and dissolved in a shaker for 20 minutes, and the absorbance was measured at 570 nm using an ELISA reader to confirm cell viability and toxicity of the sample. Cell viability was calculated as a percentage of the survival of the experimental group (each well treated with each extract) relative to the survival of the control (well treated with medium only). The results of measuring cytotoxicity of the firewood extracts are shown in Table 3 and FIG. 6, and the results of the active ingredients and the synthetic derivatives are shown in Tables 4 and 7.

Mugwort Extract Concentration (μg / ml)	Absorbance	Cell survival rate (%)
0	3.512 ± 0.181	100
LPS	3.381 ± 0.107	96
LPS + 1	3.588 ± 0.160	102
LPS + 3	3.514 ± 0.184	100
LPS + 5	3.521 ± 0.115	100
LPS + 10	2.98 ± 0.007	85
LPS + 20	2.578 ± 0.271	73

compound	Concentration (μM)	Absorbance	Cell survival rate (%)
-	Control	3.512 ± 0.181	100
-	LPS	3.381 ± 0.107	96
Arteannuin B	LPS + 1	3.227 ± 0.168	92
	LPS + 3	3.073 ± 0.280	88
	LPS + 5	2.867 ± 0.262	82
	LPS + 10	2.828 ± 0.130	81
	LPS + 20	1.111 ± 0.112	32
	LPS + 40	0.658 ± 0.042	19
	LPS + 80	0.276 ± 0.047	8
Artemisinin	LPS + 1	3.526 ± 0.279	100
	LPS + 3	3.489 ± 0.308	99
	LPS + 5	3.495 ± 0.283	100
	LPS + 10	3.342 ± 0.205	95
	LPS + 20	3.353 ± 0.074	95
	LPS + 40	3.167 ± 0.233	90
	LPS + 80	3.162 ± 0.220	90
Artemisinic acid	LPS + 1	3.605 ± 0.07	103
	LPS + 3	3.499 ± 0.142	100
	LPS + 5	3.403 ± 0.062	97
	LPS + 10	3.288 ± 0.213	94
	LPS + 20	3.105 ± 0.225	88
	LPS + 40	2.897 ± 0.202	82
	LPS + 80	2.813 ± 0.284	80
Dihydroartemisinin	LPS + 0.01	3.453 ± 0.038	102
	LPS + 0.05	3.536 ± 0.028	101
	LPS + 0.1	3.543 ± 0.032	101
	LPS + 0.5	3.588 ± 0.005	98
Artemisinin-glycolipid	LPS + 1	3.488 ± 0.190	99
	LPS + 3	2.313 ± 0.199	66
	LPS + 5	2.249 ± 0.116	64
	LPS + 10	2.080 ± 0.164	59
	LPS + 20	2.031 ± 0.234	58
	LPS + 40	0.887 ± 0.122	25
	LPS + 80	0.134 ± 0.016	4

As a result, 20 μg / ml of extract from the firewood wormwood was stimulated with LPS-derived lipopolysaccharide (LPS), 10 μM of Arteanuine B, 80 μM of Artemisinin, 80 μM of Artemisinic Acid, Hydroartemisinin was 0.5 μM and artemisinin-glycolipids showed a cell survival rate of 70% or more up to a concentration of 1 μM, indicating no cytotoxicity.

Example  4. Periodontitis As a lipopolysaccharide  In stimulated mouse macrophages TNF -α production inhibitory activity confirmation experiment

Artemisia annua extract and Artemisia annua extract, the active ingredient of Artemisia annua extract, using RAW264.7 mouse macrophages stimulated with lipopolysaccharide (LPS) obtained from the periodontal bacterium Porphyromonas gingivalis Inhibition of TNF-α production by artenunuin B, artemisinin, artemisinic acid, and synthetic derivatives, dihydroartemisinin and artemisinin-glycolipid It was confirmed. The artemisinin-glycolipids were synthesized with Daumone. 1 × 10 ⁴ mouse macrophages were dispensed into each well of a 96-well plate and 200 μl of DMEM medium was added and incubated at 37 ° C., 5% CO ₂ for 24 hours. In a 96-well plate in which mouse macrophages were cultured, each extract was added at a concentration of 1 μg / ml with LPS and incubated for 24 hours. After 24 hours, the supernatants were collected and the concentration of TNF-α was measured using a mouse TNF-α ELISA kit (R & D systems, USA). The results of the inhibitory activity of TNF-α production of the firewood extract are shown in Tables 5 and 8, and the results of the active ingredients and the synthetic derivatives are shown in Tables 6 and 9.

Mugwort Extract Concentration (μg / ml)	TNF-α production amount (pg / ml protein)	TNF-α production inhibition rate (%)
Control	99.4 ± 1.4	-
LPS	704.4 ± 33	0
LPS + 5	1126.5 ± 54.1	0
LPS + 10	984.8 ± 21.5	0
LPS + 20	879.8 ± 71.4	0

compound	Concentration (μM)	TNF-α production amount (pg / ml protein)	TNF-α production inhibition rate (%)
	Control	117.3 ± 6.3	-
	LPS	1094.4 ± 18.1	0
Arteannuin B	LPS + 1	789.4 ± 48.0	28
	LPS + 5	567.8 ± 25.5	48
	LPS + 10	393.6 ± 18.0	64
Artemisinin	LPS + 10	1478.6 ± 4.0	0
	LPS + 40	1418.2 ± 79.9	0
	LPS + 80	1317.8 ± 53.3	0
Artemisinic acid	LPS + 10	1075.3 ± 49.1	2
	LPS + 40	1011.1 ± 51.8	8
	LPS + 80	829.0 ± 30.8	24
Dihydroartemisinin	LPS + 0.1	1104.8 ± 45.0	0
	LPS + 0.5	1355.3 ± 54.4	0
Artemisinin-glycolipid	LPS + 0.5	936.5 ± 37.3	14
	LPS + 1	1176.5 ± 13.1	0
	LPS + 3	1555.3 ± 49.9	0

As a result, 28% of Arteanewin B treated with macrophages treated with periodontitis-derived lipopolysaccharide (LPS) treated with 1 μM, and 48% with 10 μM treated with 5 μM 64% of TNF-α production was inhibited. Through the above results, it was confirmed that Arteanuin B has the ability to inhibit the production of the inflammatory cytokine TNF-α. Therefore, Arteanuin B can suppress alveolar bone damage in periodontitis and periodontitis by inhibiting the production of inflammatory cytokines by periodontitis and inhibiting osteoclast formation. On the other hand, artemisinin, artemisinic acid, dihydroartemisinin, artemisinin-glycolipids did not inhibit the production of TNF-α by periodontal bacterium-derived lipopolysaccharides.

Example  5. Osteoporosis in Ovariectomized Mice Inhibitory ability  Confirm

Artemisia annua extract (AaE), Artemisianine (artemisinin: ART), and Arteanuine B (arteannuin B :) were extracted from ovarian-extracted ICR mice (Central Experimental Animal, Korea). AB) was confirmed to inhibit the osteoporosis. Sham-operating (Sham) mice, which were surgically removed but not ovarian, were used as controls for ovarian extraction mice. Divine ovary mice were divided into 10 rats in each group, and the firewood extract (1 mg / kg, or 10 mg / kg), artemisinin (10 mg / kg, or 20 mg / kg), or arteanuine B (20). mg / kg) was administered orally 5 times a week for 12 weeks. The control group was administered orally only vehicle (phosphate buffered saline containing 1% DMSO, 1% Tween-20) at the same number and frequency as the experimental group, and the positive control group was 17β-estradiol (17β estradiol: E2, sigma-). aldrich, USA) was orally administered at a dose of 0.1 mg / kg. Mice were weighed at intervals of two weeks during the material administration period, and the results are shown in FIG. 10. No significant weight loss was observed in the firewood extract group. In the case of Artemisinin and Arteanuine B, active ingredients of the firewood extract, the weight of mice increased by ovarian extraction was significantly increased in Artemisinin 20 mg / kg or Arteanuine B 20 mg / kg. It was confirmed that the decrease.

Twelve weeks after substance administration, the mice were sacrificed and the femoral bones of the mice were isolated, followed by micro computed tomography (micro-CT, SkyScan, Belgium) to determine the bone volume coefficient (bone volume, BV / TV) and bone thickness (trabecular thickness, Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), and structure model index (SMI) were analyzed. The results are shown in FIGS. 11 and 12. When oral administration of 1, 10 mg / kg of Artemisia paniculata extract, 10, 20 mg / kg of Artemisinin, and 20 mg / kg of Arteanewin B were detected by ovarian extraction on a three-dimensional reconstructed image using micro-CT. It was observed that the loss of the induced osteoblasts was significantly reduced. Bone morphological index analysis showed that the reduction of BV / TV and Tb.N induced by ovarian extraction and the increase of Tb.Sp were inhibited by oral administration of 10 mg / kg of the extract from Tungs. There was no significant effect on Th and SMI. Oral administration of artemisinin and arteanuine B at 20 mg / kg significantly inhibited the decrease of BV / TV and Tb.N induced by ovarian extraction and the increase of SMI.

Blood was collected immediately before sacrifice in mice to separate serum, followed by bone metabolism markers such as calcium, alkaline phosphatase (ALP) activity, osteocalcin, and tartrate-resistant acid phosphatase. acid phosphatase 5b: TRAP5b) activity, C-terminal crosslinking telopeptide of type I (CTX) of type 1 collagen assay kit (calcium assay kit, BioAssay Systems, USA) ), ALP assay kit (ALP assay kit, BioAssay Systems, USA), osteocalcin assay kit (Biomedical Technologies, USA), TRAP5b assay kit (TRAP5b assay kit, Immunodiagnostic Systems, UK), and CTX assay It was confirmed using a Seit kit (CTX assay kit, Immunodiagnostic Systems, UK), the results are shown in FIG. As a result of measuring the changes in bone metabolism index, the ALP activity, osteocalcin, TRAP5b activity and CTX amount decreased by ovarian extraction in the control group of 10 mg / kg Artemisinin 20 mg / kg group significantly decreased the amount of ALP activity, calcium, TRAP5b activity and CTX increased by ovarian extraction, and Arteanuin B increased osteocalcin and TRAP5b among the levels increased by ovarian extraction. It was confirmed that the activity and the amount of CTX significantly inhibited.

Tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β), which are inflammatory factors in the serum of the blood, were obtained from the respective ELISA kits (R & D Systems, USA). TNF-α and IL-1β, which were increased by ovarian extraction, were significantly decreased in 10 mg / kg of Artemisia japonica extract and 20 mg / kg of Artemisinin. The results are shown in FIG.

To assess hepatotoxicity following substance administration, the activity of alanine aminotransferase (ALT) and aspartate transaminase (AST), and blood urea nitrogen (BUN) ) And creatinine concentrations were measured using respective measurement kits (BioAssay Systems, USA). As a result of evaluating hepatotoxicity and nephrotoxicity, it was confirmed that the wormwood extract 1, 10mg / kg, Artemisinin 10, 20 mg / kg and Arteanewin B 20 mg / kg administration group showed no toxicity in liver and kidney. The hepatotoxicity results are shown in FIG. 15, and the kidney toxicity results are shown in FIG. 16.

The present invention relates to a pharmaceutical composition for preventing and treating bone diseases comprising Arteanuin B as an active ingredient, the pharmaceutical composition of the present invention has less side effects and toxicity, and shows an osteoclast formation inhibitory effect than the artemisinin. Therefore, it is expected to be greatly utilized in the medical field.

Claims (12)

1. A pharmaceutical composition for preventing and treating bone diseases comprising Arteanuine B as an active ingredient.
2. The method of claim 1,

   The bone disease is characterized in that selected from the group consisting of osteoporosis, osteomalacia, fibrous ostitis, periodontitis, periodontitis, rheumatoid arthritis and metabolic bone disease Prophylactic and therapeutic pharmaceutical compositions.
3. The method of claim 1,

   The bone disease is characterized in that caused by the bone absorption of osteoclast, bone disease prevention and treatment pharmaceutical composition.
4. The method of claim 1,

   The bone disease is characterized in that caused by bone metastasis of cancer cells, a pharmaceutical composition for preventing and treating bone diseases.
5. Food composition for improving and mitigating bone disease comprising Arteanuine B as an active ingredient.
6. The method of claim 5, wherein

   The bone disease is characterized in that selected from the group consisting of osteoporosis, osteomalacia, fibrous ostitis, periodontitis, periodontitis, rheumatoid arthritis and metabolic bone disease Food compositions for improvement and mitigation.
7. The method of claim 5, wherein

   The bone disease is characterized in that caused by bone absorption of osteoclasts, food composition for bone disease improvement and alleviation.
8. The method of claim 5, wherein

   The bone disease is characterized in that caused by bone metastasis of cancer cells, food composition for bone disease improvement and alleviation.
9. Arteanuin B is administered as an active ingredient, characterized in that the prevention or treatment of bone diseases.
10. The bone disease is characterized in that selected from the group consisting of osteoporosis, osteomalacia, fibrous ostitis, periodontitis, periodontitis, rheumatoid arthritis and metabolic bone disease Methods of prevention or treatment.
11. Use for the prophylaxis or treatment of bone disease of arteanuine B.
12. The bone disease is characterized in that selected from the group consisting of osteoporosis, osteomalacia, fibrous ostitis, periodontitis, periodontitis, rheumatoid arthritis and metabolic bone disease For prophylactic or therapeutic purposes.

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