KR20130060850A - Pharmaceutical composition of cordycepin showing an inhibitory effect of osteoclastogenesis - Google Patents

Abstract

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition containing cordycepin as an active ingredient, and is a bone disease caused by an imbalance caused by an increase in bone resorption to bone formation in bone remodeling. The present invention relates to a pharmaceutical composition that can effectively suppress an increase in osteoclast activity, which is a major cause of the disease, and thus can help to improve bone diseases caused by bone diseases in the elderly or menopause in women. More specifically, osteoporosis improvement pharmaceuticals including osteoporosis through inhibition of osteoclast differentiation, inhibition of osteoclast activity including inhibition of tartarate resistance acid phosphatase (TRAP), inhibition of MITF expression, and inhibition of p-38 expression. It relates to a composition.

Description

Pharmaceutical composition of cordycepin showing an inhibitory effect of osteoclastogenesis}

The present invention relates to a pharmaceutical composition having an osteoporosis inhibitory effect containing cordycepin as an active ingredient.

Kumamoto H, Ooya K. ExpreASion of parathyroid hormone-related protein (PTHrP), osteoclast differentiation factor (ODF) / receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoclastogenesis inhibitory factor (OCIF) / osteoprotegerin (OPG) in ameloblastomas. J Oral Pathol Med. 2004; 33 (1): 46-52.

2. Reddy SV. Regulatory mechanisms operative in osteoclasts. Crit Rev Eukaryot Gene Expr. 2004; 14 (4): 255-70.

3.Pang M, Martinez AF, Fernandez I, Balkan W, Troen BR. AP-1 stimulates the cathepsin K promoter in RAW 264.7 cells. Gene. 2007; 403 (1-2): 151-8.

Boyce BF, Yamashita T, Yao Z, Zhang Q, Li F, Xing L. Roles for NF-kappa B and c-Fos in osteoclasts. J Bone Miner Metab. 2005; 23 Suppl: 11-5.

Khosla S. Minireview: the OPG / RANKL / RANK system. Endocrinology. 2001; 142 (12): 5050-5.

6.Han SY, Lee NK, Kim KH, Jang IW, Yim M, Kim JH, Lee WJ, Lee SY. Transcriptional induction of cyclooxygenase-2 in osteoclast precursors is involved in RANKL-induced osteoclastogenesis. Blood. 2005; 106 (4): 1240-5.

7.Yamashita M, Otsuka F, Mukai T, Yamanaka R, Otani H, Matsumoto Y, Nakamura E, Takano M, Sada KE, Makino H. Simvastatin inhibits osteoclast differentiation induced by bone morphogenetic protein-2 and RANKL through regulating MAPK, AKT and Src signaling. Regul Pept. 2010; 162 (1-3): 99-108

Murakami A, Song M, Ohigashi H. Phenethyl isothiocyanate suppresses receptor activator of NF-kappaB ligand (RANKL) -induced osteoclastogenesis by blocking activation of ERK1 / 2 and p38 MAPK in RAW264.7 macrophages. Biofactors. 2007; 30 (1): 1-11.

9.H Choi HJ, Park YR, Nepal M, Choi BY, Cho NP, Choi SH, Heo SR, Kim HS, Yang MS, Soh Y. Inhibition of osteoclastogenic differentiation by Ikarisoside A in RAW 264.7 cells via JNK and NF-kappaB signaling pathways. Eur J Pharmacol. 2010; 636 (1-3): 28-35

10. Park SE, Kim J, Lee YW, Yoo HS, Cho CK. Antitumor activity of water extracts from Cordyceps militaris in NCI-H460 cell xenografted nude mice. J Acupunct Meridian Stud. 2009; 2 (4): 294-300.

Lee JS, Hong EK. Immunostimulating activity of the polysaccharides isolated from Cordyceps militaris. Int Immunopharmacol. 2011; 11 (9): 1226-33

12. Oh JY, Choi WS, Lee CH, Park HJ. The ethyl acetate extract of Cordyceps militaris inhibits IgE-mediated allergic responses in mast cells and passive cutaneous anaphylaxis reaction in mice. J Ethnopharmacol. 2011; 135 (2): 422-9.

Bone homeostasis is shown by balancing the action of osteoblasts responsible for bone formation and osteoclasts responsible for bone resorption. However, factors such as menopause in women, hyperthyroidism, diabetes, stress, smoking and lack of exercise, physical aging and taking glucocorticoid drugs are known to cause osteoporosis, most of which inhibit osteoblast function or osteoclasts. Increased activity results in osteoporosis (1-3). That is, when osteoblast activity is decreased or osteoclast activity is increased, bone density may be reduced to cause osteoporosis. Therefore, inhibiting osteoclast function may treat or prevent osteoporosis.

Osteoclasts are differentiated by various differentiation-causing factors in macrophage progenitor cells (4-5). In particular, RANKL (receptor activator of nuclear factor kappa B ligand) secreted from osteoblasts binds to osteoclast precursor cells and RANK (receptor activator of nuclear factor kappa B) present on the surface of osteoclasts. Causes differentiation and activation (6). Differentiated osteoclasts were activated by NF-B, c-Fos, c-jun, AP-1, NFATc1, MAPK, ERK, JNK, p38 activation process, MITF, Src, Akt activation, etc. Induces bone resorption by promoting the expression of osteoclast-specific proteins such as cathepsin K and calcitonin receptor (7-9). When osteoclast differentiation and function are activated and absorption is increased, various bone diseases including osteoporosis can occur.

Cordycepin is known as the main component of Militaris cordyceps (or silkworm cordyceps), which contains about 2% of cordyceps militaris, and is mainly anticancer (10), immune enhancer (11), and antiallergic (12). It is reported that there is. However, none of the literature teaches or discloses the osteocytic or osteoclast inhibitory effects and anti-osteoporosis of cordycepin.

Accordingly, the present inventors have completed the present invention by confirming the osteoporosis inhibitory effect by inhibiting gene expression of cordycepin osteoclast differentiation, osteoclast differentiation and osteoclast activity related factors such as TRAP, MITF, p-38, etc. .

In order to achieve the above object, the present invention provides a pharmaceutical composition for the treatment and prevention of bone metabolic diseases containing the cordycepin (cordycepin) of the following structural formula 1 showing the osteoclast inhibitory effect.

(One)

The bone metabolic disease as defined herein includes osteoprosis, paget disease, periodontal disease, metastatic cancer or rheumatoid arthiritis, preferably osteoporosis. Characterized in that.

Cordycepin of the present invention is commercially available but can be extracted and separated from Cordyceps sinensis by well-known extraction and separation methods in the art.

Cordycepin according to the present invention has the effect of inhibiting osteoclast differentiation, osteoclast differentiation remarkably, and significantly inhibiting the expression of genes such as TRAP, MITF, p-38, etc. It has been found useful as a pharmaceutical composition.

The compound of the present invention comprises 0.01 to 99% by weight of the compound relative to the total weight of the composition.

However, the composition is not limited thereto, and may vary depending on the condition of the patient, the type of disease, and the progress of the disease.

The pharmaceutical compositions containing the compounds according to the present invention may be prepared in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Can be formulated and used.

Carriers, excipients and diluents that may be included in the compositions comprising the compounds of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate , Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc are also used.

Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester 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.

The preferred dosage of the compound of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art.

However, for the desired effect, the composition of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg per day. The administration may be carried out once a day or divided into several doses. Accordingly, the dosage is not limited in any way to the scope of the present invention.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

Cordycepin according to the present invention exhibits inhibitory effect on osteoclast differentiation (TRAP (+) multinucleated cell production), gene expression inhibitory factors such as TRAP, MITF, p-38, etc. It can be usefully used as a pharmaceutical composition.

1 is a diagram showing the effect on the formation of TRAP (+) multinucleated cells from RANKL treated RAW264.7 cells according to cordycepin concentration,
Figure 2 is a diagram showing the effect on TRAP expression according to the cordycepin concentration,
Figure 3 is a diagram showing the effect on MITF expression according to cordycepin concentration,
Figure 4 is a diagram showing the effect on p-38 expression according to the cordycepin concentration.

Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples.

However, the following examples and experimental examples are illustrative of the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

Example 1. Sample Preparation

Cordicepin used in this experiment was purchased from Sigma-Aldrich (C3394) and used in the following experimental example.

Experimental Example 1. Cell Culture and Drug Treatment

The RAW 264.7 cells used in the experiment were cultured in a CO2 cell incubator using DMEM (Dulbecco's modified eagle medium) / 10% fetal bovine serum (FBS) / PC-SM medium, and the number of cells was 96 well plate at 5x10 ³ cells / well. And cultured using. After culturing for 24 hours, the culture medium was discarded, and the cells were cultured by exchanging with a-MEM to which 10% FBS, 50 ng / ml RANKL, and 1 ng / ml TGFb were added. Different concentrations of AS were added to the culture. It was incubated for 6 days while changing to the same medium every two days. The experimental group was (1) RANKL-treated control group (N) (2) RANKL-induced control group (C), (3) RANKL-induced group + 0.1ug / ml dose of cordycepin (0.1), (4) RANKL induction Group + 0.5ug / ml cordycepin group (0.5), (5) RANKL induction group + 1ug / ml cordycepin group (1.0). (6) It was set as the group (2.0) which administered RANKL induction group + 2ug / ml cordycepin.

Experimental Example 2. Measurement of osteoclast generation capacity

In order to determine the effect on the osteoclast-producing ability of the cordycepin was carried out by applying the method disclosed in the literature as follows.

After inducing RAW 264.7 cells to osteoclasts with RANKL (receptor activator for nuclear factor B ligand), TRAP-positive multinucleated cells (TRAP (+) MNCs) were identified by staining TRAP, a marker for expression of mature osteoclasts. . Differentiated cells were washed twice with PBS, fixed with 3.7% formaldehyde-citrate-acetone solution for 10 minutes and washed twice with distilled water. Treat the cells prepared by mixing 2% TRAP fast garnet GBC base solution with NaNO3 solution in the same ratio and a solution containing 5% naphtha AS-BI phosphoric acid, 4% acetic acid and 2% tartaric acid. It was left for more than a minute. Observed by light microscopy, TRAP-positive multinucleated cells (TRAP (+) MNCs) with three or more nuclei were counted and used as an index for generating osteoclasts (15).

Experimental Example  3. Impact on Gene Expression

In order to determine the effect on the gene expression of the osteoclast differentiation and proliferation-related factors of cordycepin, such as TRAP, MITF, p-38, experiments were performed by applying the method disclosed in the literature as follows.

3-1. gun RNA  detach

Total RNA was isolated from TRAP (+) multinuclear cells differentiated into osteoclasts by treatment with 1 ml Trizol solution (Invitrogen, USA). 100 phenol and 100 chloroform: isoamyl alcohol (24: 1) are added to the isolated RNA, and the mixture is mixed well and centrifuged twice to separate the supernatant. RNA is precipitated using 0.5 ml isopropyl alcohol, washed with 70% ethanol and dried naturally. RNA was thawed in RNAase free water (RNA) free water (Promega, USA), followed by the addition of ALNAISE-free DNase (RNase-free DNase, Promega, USA) and stored at -70 ^° C.

3-2. cDNA  Produce

Oligo dT 1 was added to the total RNA solution (containing 13 ug RNA) of each of the control and experimental groups isolated in Experimental Example 2-1, mixed carefully, and then incubated at 70 ^° C. for 5 minutes. Leave primers at room temperature for about 10 minutes to release the primers, then add cyscript buffer, 0.1 M DTT, dUTP nucleotides, dUTP seeded-labeled nucleotides, Cyscript reverse transcriptase, and then add Mix carefully. Thereafter, the cells were incubated at 42 ^° C. for 90 minutes and then left on ice. 2.5 M sodium hydroxide was added thereto, followed by incubation at 37 ^° C. for 15 minutes, and neutralized by adding 2 M HEPES buffer to prepare cDNA.

3 -3. Real time RT - PCR

First, 5 ug of RNA, 50 ng / random hexamer (random hexamer) 3, 10 mM dNTP 1, were added to the test tube, and DEPC-H ₂ O was added to make an RNA / primer mixture of 10. The experimental sample was incubated at 65 ° C. for 5 minutes and then left on ice for at least 1 minute. The reaction mixture was prepared by mixing 10-fold RT buffer 2, 25 mM magnesium chloride 4, 0.1 M DTT 2, RNAase (Promega, USA) 1. The reaction mixture was added to the RNA / primer mixture, left to stand at room temperature for 2 minutes, then SuperScript II RT (Promega, USA) 1 (50 units) was added and incubated at 25 ^° C. for 10 minutes. Incubated at 42 ^o C for 50 min, then inactivated by heating at 70 ^o C for 15 min and cooled on ice. RNase (Promega, USA) 1 was added and incubated again at 37 ^o C for 20 min, then stored at -20 ^o C until use. Into each optical tube (optical tube, Gibco, USA) add 2x Cyber Green Mix (SYBR Green Mix, Takara, Japan) 12.5, cDNA 0.2, 5 pmol / primer pair mix 1, 11.3 H ₂ O, 50 ^o Amplification with C 2 min 1 cycle, 95 ^° C 10 min 1 cycle, 95 ^° C 15 sec, 60 ^° C 30 sec, 72 ^° C 30 sec 40 cycles, 72 ^° C 10 min 1 cycle. After completing the PCR, the tube was taken out, and PCR specificity was measured on a 3% agarose gel using 5 ul of the reaction solution. Real time PCR results were analyzed using SDS 7000 software.

Treatment of receptor activator for nuclear factor B ligand (RANKL) in RAW264.7 cells increased the expression of TRAP (+) multinucleated cells (MNCs) to promote osteoclast differentiation. It was shown to inhibit the receptor activator for nuclear factor B ligand (RANKL) -induced osteoclast differentiation at the concentration of / ml, (FIG. 1). , The cordycepin was confirmed to significantly inhibit the gene expression increased at 0.1 ug / ml, 1 ug / ml, concentration (see Fig. 2, 3 and 4). Thus, it was found that cordycepin has an excellent effect on suppressing TRAP, JNK, and AKT1 gene expression.

Statistical treatments were compared individually using Student's t-test and determined that there was a significant difference when the p-value was less than 0.05.

Hereinafter, formulation examples of the composition containing the compound of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

Formulation example  One. Sanje  Produce

Cordycepin 200 mg

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation example  2. Preparation of tablets

Cordycepin 200 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation example  3. Preparation of capsules

Cordycepin 200 mg

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed in accordance with a conventional method for producing a capsule, and filled in a gelatin capsule to prepare a capsule.

Formulation example  4. Preparation of injections

Cordycepin 200 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO _{4 ,} 12H ₂ O 26 mg

(2 ml) per 1 ampoule according to the usual injection preparation method.

Formulation example  5. Liquid  Produce

Cordycepin 200 mg

10 g per isomer

5 g mannitol

Purified water

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.

Claims (4)

A pharmaceutical composition for treating and preventing bone metabolic diseases, comprising cordycepin as an active ingredient, which exhibits an osteoclast inhibitory effect. The method of claim 1,
The bone metabolic disease is osteopharmaceuticals (osteoprosis), Paget disease (paget disease), periodontal disease (periodontal disease), metastatic cancer (rheumatoid arthiritis) pharmaceutical composition (rheumatoid arthiritis). The method of claim 1,
Pharmaceutical composition comprising 0.01 to 99% by weight relative to the total weight of the composition, The method of claim 1,
The composition is a pharmaceutical composition in the form of a powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injectable solutions.

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