KR20130060834A - Pharmaceutical composition of brazilin showing an inhibitory effect of osteoclastogenesis - Google Patents

Abstract

PURPOSE: Brazilin or a pharmaceutically acceptable salt thereof is provided to suppress differentiation of osteoclast and expression of genes such as TRAP, cathepsin K, and MMP-9, and to be used as a pharmaceutical composition for suppressing osteoporosis. CONSTITUTION: A pharmaceutical composition for preventing and treating metabolic bone diseases contains brazilin or a pharmaceutically acceptable salt thereof as an active ingredient. The pharmaceutical composition contains a Caesalpinia sappan L. extract as an active ingredient, which suppresses osteoporosis. The metabolic bone diseases are osteoporosis, Paget's disease, periodontal disease, metastatic cancer, or rheumatoid arthritis. The pharmaceutical composition is manufactured in the form of powder, a tablet, a capsule, an injection, and liquid.

Description

Pharmaceutical composition of brazilin showing an inhibitory effect of osteoclastogenesis}

The present invention relates to a pharmaceutical composition having an inhibitory effect on osteoporosis containing brazil or a pharmaceutically acceptable salt thereof 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. C.K. Moon, S.H. Lee, M.O. Lee and S.G. Kim, Effects of brazilin on glucose oxidation, lipogenesis and therein involved enzymes in adipose tissues from diabetic KK-mice, Life Sci. 53 (1993), pp. 12911297

11.G.S. Hwang, J.Y. Kim, T.S. Chang, S.D. Jeon, D.S. So and C.K. Moon, Effects of brazilin on the phospholipase A2 activity and changes of intracellular free calcium concentration in rat platelets, Arch. Pharm. Res. 21 (1998), pp. 774778.

12. C.K. Moon, K.S. Park, S.G. Kim, H.S. Won and J.H. Chung, Brazil in protects cultured rat hepatocytes from BrCCl3-induced toxicity, Drug Chem. Toxicol. 15 (1992), pp. 8191

13.MS Mok, SD Jeon, KM Yang, DS So and CK Moon, Effects of brazilin on induction of immunological tolerance by sheep red blood cells in C57BL / 6 female mice, Arch. Pharm. Res. 21 (1998), pp. 769773.

Bone metabolism is composed of osteoblasts responsible for bone formation and osteoclasts responsible for bone resorption, and the function of these cells is balanced to maintain homeostasis. However, when osteoblast activity decreases or osteoclast activity increases, bone density decreases and osteoporosis may be induced. Factors causing osteoporosis include menopause, hyperthyroidism, diabetes, stress, lack of smoking and exercise, physical aging, and the use of glucocorticoid drugs in women (1-3).

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 catarsin TRAP (tartarate resistant acid phosphatase) through activation of NF-B, c-Fos, c-jun, AP-1, NFATc1, MAPK, ERK, JNK, p38 activation process, Src, Akt activation It promotes the expression of osteoclast specific proteins such as K and calcitonin receptor (7-9).

The main component of joinery, brazilian, is known to have antidiabetic effects (10) and has been reported to inhibit coagulation by regulating calcium concentration and prostaglandin in platelets (11). In addition, hepatoprotective action (12) and immunomodulatory action have been reported.

Accordingly, the present inventors have completed the present invention by confirming the effect of inhibiting osteoclast differentiation of osteoclasts, osteoclast differentiation and osteoclast activity by inhibiting gene expression such as osteoclast differentiation factors such as TRAP, Cathepsin K, and MMP-9.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the treatment and prophylaxis of bone metabolic disease, which contains as an active ingredient a braziline or a pharmaceutically acceptable salt thereof showing an osteoclast inhibitory effect.

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

The brazil of the present invention can be extracted or purchased from joiner.

The present invention also provides a pharmaceutical composition for treating and preventing bone metabolic diseases, which contains brazilin or a pharmaceutically acceptable salt thereof as an active ingredient.

The brazilians of the present invention are commercially available or obtainable from plants such as joiner by extraction and separation methods well known in the art.

In another aspect, the present invention provides a pharmaceutical composition for the treatment and prevention of bone metabolic diseases containing brazilin or a pharmaceutically acceptable salt thereof prepared by the above method.

Since brazil according to the present invention exhibits inhibitory effect on osteoclast differentiation, osteoclast differentiation and proliferation-related factors, such as TRAP, Cathepsin K, MMP-9, etc., it was confirmed that it is useful as a pharmaceutical composition for inhibiting osteoporosis.

Brasins of the present invention comprise from 0.01 to 99% by weight of the compound relative to the total weight.

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.

Compositions comprising a compound of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Compositions comprising compounds and extracts according to the invention are in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. Carriers, excipients and diluents that may be included in the formulation 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, 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, Or 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.

Preferred dosages of brazilins of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the compound and extract is preferably administered at 0.01 mg / kg to 10 g / kg, preferably 1 mg / kg to 1 g / kg per day. The administration may be carried out once a day or divided into several doses. Therefore, the dose is not intended to limit the scope of the present invention in any aspect.

The compounds and extracts of the present invention can be administered to mammals such as mice, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example by oral and rectal or intravenous methods.

Since extracts containing brazil or pharmaceutically acceptable salts thereof according to the present invention exhibit an inhibitory effect on osteoclast differentiation, osteoclast differentiation and proliferation, TRAP, Cathesin K, MMp-9, etc. It can be usefully used as a pharmaceutical composition for inhibiting osteoporosis.

1 is a diagram showing ¹ H-NMR spectra (DMSO-d ₆ , δ 0-12 ppm) of Brazilin;
FIG. 2 is a diagram showing ¹ H-NMR spectra (DMSO-d ₆ , δ 6.1 to 7.2 ppm) of Brazilin; FIG.
3 is a diagram showing ¹ H-NMR spectra (DMSO-d ₆ , δ 2.4 to 4.0 ppm) of Brazilin;
4 is a diagram showing ¹³ C-NMR spectra (DMSO-d ₆ , δ 0 to 220 ppm) of Brazilin;
5 shows a low-resolution FAB-MS spectra of Brazilin;
6 is a diagram showing the molecular weight of Brazilin measured by high-resolution FAB-MS;
7 is a diagram showing the effect on the formation of TRAP (+) multinucleated cells from RANKL treated RAW264.7 cells according to Brazilin concentration;
8 is a diagram showing the effect on TRAP expression according to Brazilin concentration;
9 is a diagram showing the effect on MMP-9 expression according to Brazilin concentration;
10 is a diagram showing the effect on Cathepsin K expression according to Brazilin concentration;
11 is a diagram showing the effect on MITF expression according to Brazilin 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 Isolation of Brazilin from the Joiner Extract

100 g of saplings were made into a powder and placed in a flask, and 500 ml of MeOH was added thereto, followed by heating at reflux for 6 hours or more. After filtering using filter paper, the filtrate was concentrated under reduced pressure using an Evaporator (EYELA, Japan), and then suspended in distilled water. The suspension was extracted with EtOAc, and the rest of the solution was extracted with BuOH to concentrate each extract, and the concentrate was separated and purified using a silica gel column to obtain brazilian compound of formula 1 having physical properties as described below. It was.

¹ H-NMR spectrum; 1 to 3, see Table 1

¹³ C-NMR Spectrum: See FIGS. 4-6 and Table 1

FAB-MS Spectrum: See FIGS. 5-6.

Position dH  ( ppm ) dC  ( ppm ) One 7.11, 1H, d, J = 8.3 130.8 1a - 114.3 2 6.39, 1H, dd , J = 8.3,2.35 108.6 3 - 154.0 4 6.19, 1 H, d, J = 2.35 102.7 4a - 156.4 6 3.55, 1H, d, J = 11.3
3.82, 1 H, d, J = 11.3 69.6 6a - 76.3 7 2.67, 1 H, d, J = 15.65
2.85, 1 H, d, J = 15.65 41.9 7a - 129.7 8 6.52, 1H, s 112.0 9 - 144.2 10 - 143.9 11 6.62, 1H, s 111.6 11a - 135.5 12 3.83, 1H, s 49.5

Experimental Example  1. Evaluation of osteoclast inhibition effect

1-1. 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), (2) RANKL-induced group + 1 / group of braziline of Example 1 (Br 0.1), (3 ) RANKL induction group + 5 / of the brazilin administration of Example 1 (Br 5).

Experimental Example  2. Osteoclast Generation  Measure

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. Effect on Gene Expression

In order to determine the effect on the gene expression of the osteoclast differentiation and proliferation-related factors, such as TRAP, JNK, AKT1 of Example 1 was performed 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 3-1, 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 Amplified 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 (see Table 2). 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.

Recombinant protein Primer sequence TRAP Sense: SEQ ID NO: 1 5′-ACACAGTGATGCTGTGTGGCAACTC-3 ′ Antisense: SEQ ID NO: 2 5′-CCAGAGGCTTCCACATATATGATGG-3 ′ MMP-9 Sense: SEQ ID NO: 3 5′- CGTCGTGATCCCCACTTACT-3 ′ Antisense: SEQ ID NO: 4 5′- AGAGTACTGCTTGCCCAGGA-3 ′ Cathepsin k Sense: SEQ ID NO: 5 5’- AGGCGGCTATATGACCACTG-3 ’ Antisense: SEQ ID NO: 6 5’- CCGAGCCAAGAGAGCATATC-3 ’ MITF Sense: SEQ ID NO: 7 5'-TTC AAA TGA GAT TGT GGG AAA AT-3 Antisense: SEQ ID NO: 8 5'-AGA TCA TCT CTG CCT GAG TAT CTT-3 '

Treatment of receptor activator for nuclear factor B ligand (RANKL) in RAW264.7 cells increased the expression of TRAP (+) multinucleated cells (MNCs), thereby promoting osteoclast differentiation. In this example, brazilin is 0.1 ug / ml, 1 It has been shown to inhibit the receptor activator for nuclear factor B ligand (RANKL) -induced osteoclast differentiation at ug / ml and 5 ug / ml (FIG. 7) .TAP (tartarate resistance Acid Phosphatase), JNK, and AKT1 were expressed during RANKL treatment. It was confirmed that the brazilin significantly inhibited gene expression increased at 0.1 ug / ml, 1 ug / ml, and concentration (see FIGS. 8, 9, 10, and 11). Thus, brazilin inhibited osteoclast differentiation by inhibiting TRAP, MMP-9, Cathepsin K, and MITF 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.

Preparation Example 1. Preparation of powder

Brazilian 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

Brazilian 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

Brazilian 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

Brazilian 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. Preparation of a liquid preparation

Brazilian 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, which contains brazirin or a pharmaceutically acceptable salt thereof as an active ingredient that exhibits osteoporosis inhibition efficacy. A pharmaceutical composition for treating and preventing bone metabolic diseases, comprising as an active ingredient a joiner extract containing brazil, which has an effect of inhibiting osteoporosis. The method according to claim 1 and 2,
The bone metabolic disease is osteoprosis (osteoprosis), Paget disease (paget disease), periodontal disease (periodontal disease), metastatic cancer (rheumatoid arthiritis) pharmaceutical composition (rheumatoid arthiritis). The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition is a powder, tablet, capsule, injection, liquid formulation.

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