WO2008126974A1 - Composition for preventing and treating osseous metabolic disease - Google Patents

Composition for preventing and treating osseous metabolic disease Download PDF

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WO2008126974A1
WO2008126974A1 PCT/KR2007/006532 KR2007006532W WO2008126974A1 WO 2008126974 A1 WO2008126974 A1 WO 2008126974A1 KR 2007006532 W KR2007006532 W KR 2007006532W WO 2008126974 A1 WO2008126974 A1 WO 2008126974A1
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bone
formula
compound
pharmaceutical composition
disease
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PCT/KR2007/006532
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French (fr)
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Zang-Hee Lee
Hyun-Mo Ryoo
Suk-Chul Bae
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Biorunx Co., Ltd.
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00Drugs for skeletal disorders
    • A61P19/02Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00Drugs for skeletal disorders
    • A61P19/08Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis

Definitions

  • the present invention relates to a pharmaceutical composition for preventing or treating metabolic bone diseases, comprising a pharmaceutically effective amount of a compound represented by the following Formula 1 or a pharmaceutically acceptable salt thereof.
  • the skeleton consists of highly specialized bone cells including osteocytes, osteoclasts and osteoblasts, bone matrix including hydroxyapatite crystal, collagenous fibers and glycosaminoglycans, and spaces including bone marrow cavities, vascular canals, canaliculi and lacunae (Stavros C. M., Endocrine Reveiws, 21(2), 115-137 (2000)). Bone functions to mechanically support the body, protect mapr organs, supply microenvironment required for hemopoiesis, and store calcium and several minerals.
  • BMU Basic Multicellular Units
  • Osteoclasts remove the bone matrix such as hydroxyapaptite crystal or collagenous fibers, which constitute bone, by adhering to the bone surface and secreting hydrochloric acid and proteases. Osteoblasts synthesize and secrete the bone matrix, and regulate the local concentration of calciun and phosphate to form skeleton (Stavros C. M., Endocrine Reviews, 21(2), 115-137 (2000)).
  • Metabolic bone diseases are caused by breakdown of the balance between osteoclasts and osteoblasts in the body.
  • a representative example of metabolic bone diseases is osteoporosis. Osteoporosis occurs due to reduction of total bone mass, resulting from both the excessive osteoclast activity and insufficient osteoblast activity.
  • osteoporosis With of cortical bone is reduced, bone marrow cavity is enlarged, and thickness of trabecular bone is lowered, causing bone to be continuously porous. With progress of osteoporosis, physical strength of bone decreases, and thus lurbago and arthralgia are induced, and bone is easily fractured even by weak impact.
  • metabolic bone diseases include metastatic bone lesions caused by metastasis of breast and prostate carcinomas to bone, primary tumors of bone (e. g., multiple myeloma), rheumatoid or degenerative arthritis, periodontal disease accompanying destruction of alveolar bone by periodontal disease-causing bacteria, inflammatory periodontal disease with alveolar bone destruction generated after surgical application of dental implant, inflammatory bone resorption disease caused by implant implanted to fix bone by plastic surgery, and Paget's disease induced by various genetic factors.
  • primary tumors of bone e. g., multiple myeloma
  • rheumatoid or degenerative arthritis e.g., multiple myeloma
  • periodontal disease accompanying destruction of alveolar bone by periodontal disease-causing bacteria
  • inflammatory periodontal disease with alveolar bone destruction generated after surgical application of dental implant inflammatory bone resorption disease caused by implant implanted to fix bone by plastic surgery
  • Paget's disease induced by various genetic factors
  • Myeloma is a bone disease featured by fragile bone accompanying severe pain, and caused by osteoclast activity increased by carcinomas. Breast and prostate carcinomas easily metastasize to bone, and stimulate osteoclast activity, resulting in destruction of bone. In the case of rheunatoid or degenerative arthritis, tumor necrosis factor (TNF), interleukin- 1 and interleukin-6, which are produced by the immune response, stimulate osteoclast activity present at the pint space, causing local destruction of bone at the pint.
  • TNF tumor necrosis factor
  • interleukin- 1 and interleukin-6 which are produced by the immune response
  • inflammatory cytokines including TNF, interleukin- 1 and interleukin-6 are produced by the immune response to the pathogenic bacterial infection to stimulate differentiation of osteoclasts, leading to destruction of alveolar bone supporting teeth.
  • bone formation- stimulating factors include fluoride, parathyroid hormone, TGF- ⁇ , bone morphogenetic protein, and insulin-like growth factor.
  • Osteoclast-suppressing factors include estrogen, calcitonin, vitamin D and its analogues, and bisphosphonates (Jardine et al., Annual Reports in Medicinal Chemistry, 31, 211 (1996)).
  • estrogen which is most frequently used for treating osteoporosis, has disadvantages as follows: it is still not demonstrated to be practically effective in treating osteoporosis, it should be administered throughout the patient's life, and it has side effects of increasing the incidence of breast cancer or cervical cancer when administered for a long period of time.
  • Alendronate is also problematic in terms of being not clearly identified for its therapeutic efficacy for osteoporosis, being slowly absorbed by the gastrointestinal tract, and causing inflammation in the stomach, intestine and mucosa of the esophagus.
  • Calciun preparations are known to have mild side effects and good efficacy, but are a preventive agent rather than a therapeutic agent.
  • vitamin D and calcitonin are not sufficiently studied for their preventive or therapeutic efficacy and side effects.
  • the compound of Formula 1 inhibits the expression of osteoclast differentiation-related genes, c-Fos and NFATcI, promotes the activity of osteoblast differentiation-inducing gene, Runx-2, and has excellent efficacies of suppressing osteoporosis in mouse model of metabolic bone disease and bone loss caused by inflammation, and treating collagen-induced arthritis, thereby completing the present invention.
  • Fig. 1 is a graph showing the effect of the compound of Formula 1 on the differentiation of bone marrow-derived osteoclast progenitor into osteoclast;
  • Fig. 2 is the result of RT-PCR and Western blot, which shows the effect of the compound of Formula 1 on the expression of master genes for osteoclast differentiation, c-Fos and NFATcI;
  • Fig. 3 shows the effect of the compound of Formula 1 on the activity of master genes for osteoblast differentiation, Runx-2;
  • Fig. 4 is the result of micro-computed tomography, which shows the effect of the compound of Formula 1 on osteoporosis mouse model induced by ovariectomy;
  • Fig. 5 is the result of micro-computed tomography, which shows the effect of the compound of Formula 1 on inflammatory bone loss mouse model induced by LPS (lipopolysaccharide) ;
  • Fig. 6 is a graph showing the effect of the compound of Formula 1 on arthritis score in CIA mouse model (CIA: collagen-induced arthritis);
  • Fig. 7 is the result of micro-computed tomography, which shows the effect of the compound of Formula 1 on bone loss in CIA mouse model;
  • Fig. 8 is the result of H-E staining(hematoxylin-eosin staining) of histological sections, which shows the effect of the compound of Formula 1 on bone loss in CIA mouse model;
  • Fig. 9 is a microscopic photograph showing the effect of the compound of Formula 1 on in vivo bone formation.
  • the present invention provides a pharmaceutical composition for preventing or treating metabolic bone diseases, comprising a pharmaceutically effective amount of a compound represented by the following Formula 1 or a pharmaceutically acceptable salt thereof.
  • the compound of Formula 1 used in the pharmaceutical composition of the present invention is known as an MHC class- 1 expression promoting factor, disclosed in Japanese Patent No. 349462, but there is no mention of its functions such as promoting osteoblast differentiation, or suppressing osteoclast differentiation or therapeutic efficacy for metabolic bone diseases.
  • the active ingredient used in the pharmaceutical composition of the present invention comprises "pharmaceutically acceptable salt” of the compound of Formula 1.
  • pharmaceutically acceptable refers to compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset and dizziness, when administered to himans.
  • the salt is prepared by reaction with a stoichiometric amount of a suitable base or acid in water or an organic solvent, or in a mixture thereof.
  • the pharmaceutically acceptable salt according to the present invention includes inorganic base salts, organic base salts, inorganic acid salts, organic acid salts, and basic or acidic amino acid salts.
  • Examples of the inorganic base salts include alkali metal salts such as sodiun salts or potassium salts, alkali earth metal salts such as calciun salts or magnesium salts, aluninum salts, and ammoniun salts.
  • Examples of the organic base salts include salts of trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, di- ethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine and N, N'-dibenzylethylenediamine.
  • Examples of the inorganic acid salts include salts of hydrochloric acid, boric acid, funaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzensulfonic acid and p- toluenesulfonic acid.
  • Examples of the basic amino acid salts include salts of arginine, lysine and ornithine.
  • Examples of the acidic amino acid salts include salts of aspartic acid and glutamic acid.
  • the salts of the present invention may be prepared by conventional methods such as ion exchange.
  • the compound of the present invention and pharmaceutically acceptable salt thereof can be used individually or as a mixture of two or more thereof for the prevention and treatment of metabolic bone diseases.
  • metabolic bone diseases refers to a disease accompanying a physiopathological state caused by excessive destruction and resorption of bone.
  • metabolic bone diseases include osteoporosis, metastatic bone lesions caused by metastasis of breast or prostate carcinomas to bone, primary bone turors, rheumatoid or degenerative arthritis, periodontal disease, inflammatory periodontal disease with alveolar bone destruction, inflammatory bone resorption disease, and Paget's disease, preferably osteoporosis, inflammatory periodontal disease with alveolar bone destruction, and rheumatoid arthritis, but are not limited thereto.
  • the compound of Formula 1 of the present invention suppresses the mRNA and protein expression of c-Fos and NFATcI, which are master regulators for osteoclast differentiation, to suppress bone resorption by osteoclast.
  • c-Fos and NFATcI are master regulators for osteoclast differentiation, to suppress bone resorption by osteoclast.
  • transcription factors including NF-kB (nuclear factor kB), c-Fos, NFATcI (nuclear factor of activated T cells cl), PUl, and MITF, are known to play an important role in osteoclast differentiation by the treatment of RANKL (receptor activator of NK ligand) (Teitelbaun et al., Nature Review of Genetics 4 :638-649, 2003).
  • RANKL receptor activator of NK ligand
  • RANKL-induced expression of c-Fos is known to play an essential role in the initiation of osteoclast differentiation.
  • the c-Fos expression causes the expression of NFATcI (Matsuo et al., Journal of Biological Chemistry 279:26475-26480, 2004), and NFATcI can induce osteoclast differentiation in the absence of RANKL.
  • NFATcI is known as an essential factor in osteoclast differentiation (Takayanagi et al., Developmental Cell 3:889-901, 2002).
  • the present inventors performed RT-PCR and Western blot to confirm the effect of the compound of Formula 1 on the expression of c-Fos and NFATcI. As a result, when the compound of Formula 1 was added upon culturing osteoclast, the expression of c-Fos and NFATcI was found to be significantly suppressed (see Fig. 2).
  • Runx-2 which is a master gene for osteoblast differentiation, to induce new bone formation, thereby suppressing bone resorption by osteoclast.
  • the present inventors treated mouse C2C12 cells with the compound of Formula 1. As a result, the activity of Runx-2 was found to be markedly increased, compared to the cell treated with FGF2 (fibroblast growth factor-2) (see Fig. 3).
  • the present inventors performed an in vivo test of the compound of Formula 1.
  • An estrogen-deficient mouse due to ovariectomy was administered with the compound of the present invention, and bone volune of femur was measured using micro-computed tomography.
  • bone volune significantly increased to inhibit osteoporosis see Fig. 4
  • an LPS (lipopolysaccharide)-induced bone loss mouse model was administered with the compound of the present invention, and bone volune of femur was measured using micro-computed tomography.
  • Fig. 5 it was found that bone volune significantly increased to inhibit inflammatory bone loss
  • a collagen-induced arthritis mouse model was administered with the compound of the present invention, and micro-computed tomography and histological analysis were performed. As a result, it was found that bone loss was significantly inhibited to prevent and treat arthritis (see Figs. 6 to 8). In addition, the compound of the present invention exhibited the effect of promoting bone formation (see Fig. 9).
  • the compound of Formula 1 of the present invention can be used as an osteoclast differentiation inhibitor, osteoblast differentiation promoter, or bone formation promoter, and as a pharmaceutical composition for preventing or treating metabolic bone diseases, comprising the compound as an active ingredient.
  • composition of the present invention may include one or more active ingredients having the same or similar function, for example, BMP-2,4,7 (bone morphogenetic protein-2,4,7) which is known to promote osteoblast differentiation.
  • BMP-2,4,7 bone morphogenetic protein-2,4,7
  • the composition of the present invention may include at least one pharmaceutically acceptable carrier, in addition to the above described active ingredients.
  • the pharmaceutically acceptable carrier include a saline solution, sterile water, a Ringer's solution, a buffered saline solution, a dextrose solution, a maltodextrin solution, glycerol, ethanol and a mixture of one or more thereof.
  • the composition may also include other conventional additives such as antioxidants, buffers, and bacteriostatic agents.
  • the composition may additionally include diluents, dispersants, surfactants, binders, and lubricants to formulate it into injectable formulations such as aqueous solution, suspension, and emulsion, pills, capsules, granules and tablets.
  • injectable formulations such as aqueous solution, suspension, and emulsion, pills, capsules, granules and tablets.
  • the composition may be preferably formulated depending on particular diseases and its components, using a suitable method in the relevant field or the method described in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA.
  • the pharmaceutical composition of the present invention may be administered by oral or parenteral route (e.g., intravenous, subcutaneous, intraperitoneal, or topical administration) depending on its purpose.
  • An effective dosage of the present composition may be determined depending on the patient's body weight, age, gender, health state, and diet, administration time, administration routes, excretion rates, and severity of the diseases.
  • the composition may be preferably administered at a daily dosage of about 0.1 mg/kg to 1 mg/kg one time or several times.
  • the present composition may be used singly or in combination with surgical operation, hormone therapy, drug therapy and biological response regulators in order to prevent and treat metabolic bone diseases.
  • Example 1 Isolation of bone marrow cell and induction of osteoclast progenitors
  • tibias were isolated aseptically.
  • the isolated tibias were placed into 3 x HBSS (Gibco BRL), and soft tissue was completely removed. Both ends of tibias were cut, and 1 x ⁇ -MEM (Gibco BRL) was injected into bone marrow and then sucked up using a 1 cc syringe, collecting bone marrow cells. After sufficient suspending by pipetting, the collected bone marrow cells were centrifuged(1600 rpm, 5 min, 4 0 C), and the supernatant was discarded to harvest cells. The resulting pellet (bone marrow cells and erythrocytes) was suspended in ACK buffer (155 mM NH Cl, 11 mM KHCO , 0.01 mM EDTA).
  • phosphate buffer was added to the cell suspension to minimize damage to bone marrow cells and lyse erythrocytes. Thereafter, the cell suspension was centrifuged (1600 rpm, 5 min, 4 0 C), and the resulting cell pellet was suspended in ⁇ -MEM containing 10% (v/v) FBS (fetal bovine serxrn, Gibco BRL), 100 U/ml of penicillin, 100 ⁇ g/ml of streptomycin, 10 ng/ml of macrophage-colony stimulating factor (M-CSF; Peprotech EC, London, England).
  • FBS fetal bovine serxrn, Gibco BRL
  • osteoclast progenitors After culturing overnight, non- adherent cells were collected and cultured in a culture plate containing 30 ng/ml of M-CSF for 3 days. The cultured cells were designated as osteoclast progenitors, and used to evaluate the effect of the compound of the present invention on their differentiation into osteoclasts.
  • the osteoclast progenitors prepared in Example 1 into osteoclasts were cultured in 1 X ⁇ -MEM supplemented with 30 ng/ml of macrophage-colony stimulating factor (M-CSF) and 50 ng/ml of RANKL (Peprotech EC, London, England).
  • M-CSF macrophage-colony stimulating factor
  • RANKL RANKL
  • the compound of Formula 1 was added to the cells at various concentrations (10 nM, 25 nM, 50 nM, 75 nM, 100 nM).
  • the exclusive patent license for the compound of Formula 1 was assigned by Professor Nishino at Kyushu national University (Japan).
  • the completely differentiated osteoclasts were stained with TRAP(tartrate resistant acid phosphatase).
  • TRAP staining was carried out using a Leukocyte Acid Phosphatase Kit (Sigma, cat. No. 387-A). After removing culture media, the completely differentiated osteoclasts were fixed with 10% formalin for 5 min. After removing formalin, the fixed osteoclasts were treated with 0.1% Triton X- 100 for 10 sec. After removing Triton X-100, the cells were stained with TRAP for about 5 min. After eliminating the TRAP staining solution, the cells were washed with distilled water twice, and dried. TRAP-positive osteoclasts were counted under an optical microscope (x 100).
  • the compound of Formula 1 inhibited osteoclast differentiation in a concentration-dependent manner, compared to a control. Accordingly, these results indicate that the compound of Formula 1 inhibits the differentiation of bone marrow- derived osteoclast progenitor into osteoclast in a dose-dependent manner.
  • PCR conditions included 22 cycles of denaturation at 94 0 C for 30 sec, annealing at
  • Formula 1 inhibits protein expression levels of c-Fos and NFATcI.
  • the prepared cells were lysed in a buffer solution containing 20 mM Tris-HCl, 150 mM NaCl, 1% Triton X-IOO, protease and phosphatase inhibitors. Proteins in cell lysate (30 ⁇ g) were separated on a 10% SDS-PAGE, and transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA). After blocking with 5% skim milk, the membrane was analyzed with anti-Fos, NFATcI, and TRAF6 (Cell Signaling Technology, everly, MA).
  • the membrane was reanalyzed with an anti-actin antibody (Cell Signaling Technology). As shown in Fig. 2 (lower), the compound of Formula 1 was found to significantly inhibit the protein expressions of c-Fos and NFATcI. Accordingly, it can be seen that the compound of Formula 1 significantly inhibited the expression of c-Fos and NFATcI, which are master genes for osteoclast differentiation.
  • Runx-2 can be influenced by fetal bovine serum (FBS) which is generally used for cell culture
  • FBS fetal bovine serum
  • change in the activity of Runx-2 was analyzed according to the presence of serum. As a result, it was found that the activity was decreased in the presence of serum, but the activity was still higher, compared to the control group and FGF2 group. Accordingly, it can be seen that the compound of formula 1 increased the activity of Runx-2 which is a master gene for osteoblast differentiation, thereby promoting osteoblast differentiation.
  • Osteoporosis is a disease caused by increase in osteoclast formation due to estrogen deficiency after menopause (J Clin Invest 112:915-923, Lean et al., 2003).
  • the effect of inhibiting osteoclast differentiation of the compound of Formula 1 was examined in an ovariectomized mouse as an animal model for osteoporosis. Both ovaries were excised from 16 week-old female mice and after 4 weeks, bone resorption was induced. Then, the compound of Formula 1 was intraperitoneally administered to the mice three times per week at a dose of 0.1 ⁇ g per 1 g of body weight for 4 weeks. For comparison, one group was administered with a phosphate buffer solution instead of the compound of Formula 1.
  • mice were sacrificed by cervical dislocation. Femurs were isolated, and bone volumes in horizontal and vertical cross- sections were measured by micro-computed tomography scan (Skyscan 1072 micro- CT system; SkyScan, Aartselaar, Belgium). For quantification, the ratio of bone volume to total tissue volume was calculated. As shown in Fig. 4, the bone volume was significantly increased and virtually normal in the group treated with the compound of formula 1, as compared to the group treated with the phosphate buffer solution only. Accordingly, it can be seen that osteoporosis induced by ovariectomy can be treated with the compound of Formula 1.
  • the compound of Formula 1 was intraperitoneally administered to the mice at a dose of 0.1 ⁇ g per 1 g of body weight on day -1, 1, 3, 5, and 7.
  • a phosphate buffer solution was intraperitoneally administered.
  • the mice were sacrificed. Femurs were isolated, and bone volumes in horizontal and vertical cross- sections were measured by micro-computed tomography scan (Skyscan 1072 micro- CT system; SkyScan, Aartselaar, Belgium). For quantification, the ratio of bone volume to total tissue volume was calculated.
  • Rheumatoid arthritis is a chronic inflammatory disease, and accompanied by bone loss around the pint by increased osteoclast activity.
  • a collagen-induced arthritis mouse model was used. Type II collagen, which is freeze-dried and extracted from bovine (Chondrex, Redmond, WA, USA, Cat. No. 2002-1), was slowly mixed with 0.05 M acetic acid at 4 0 C overnight to a concentration of 2.0 mg/ml. The next day, the solubilized collagen solution was mixed with an equal volume of complete Freund's adjuvant (Chondrex, Redmond, WA, USA, Cat. No. 7001), and suspended using a homogenizer.
  • the suspension was put in a 1.0 ml tuberculin syringe. DBA/1 mouse was restrained, and then the hair on the back near the tail base was shaved. 0.1 ml of suspension was intradermally injected with caution (not deep into the subcutaneous tissue).
  • secondary immunization was performed. Upon secondary immunization, an incomplete Freund's adjuvant (Chondrex, Redmond, WA, USA, Cat. No. 7002) was used instead of the complete Freund's adjuvant used in primary immunization, and injected in the same manner as in primary immunization.
  • the compound of Formula 1 was intraperitoneally administered to the mice at a dose of 0.1 ⁇ g per 1 g (body weight) three times per week at two weeks after primary immunization.
  • the compound of Formula 1 was administered in the same manner at three weeks after primary immunization.
  • the mice were sacrificed, and micro-computed tomography scan and histological analysis were performed. Beginning at three weeks, arthritis score observed with the naked eye was monitored weekly, according to the following criteria of American College of Rheunatology.
  • 0 point was given for normal pint of paw, 1 point for swelling of toe, 2 points for swelling of paw pad, and 3 points for extensive swelling of the entire paw.
  • the scores ranged from 0 to 12 points.
  • Forefoot of arthritis- induced mouse was imaged by micro-computed tomography scan (Skyscan 1072 micro-CT system; SkyScan, Aartselaar, Belgium) for three-dimensional analysis of bone loss and changes in bone tissue, and then reconstructed by V- works (Cyberland, Seoul, Korea).
  • the collected bones were fixed in 4% paraformaldehyde, decalcified with 12% EDTA, and then embedded in paraffin.
  • H-E hematoxylin-Eosin
  • the arthritis score was significantly decreased in the groups treated with the compound of Formula 1 for the purpose of prevention and treatment, as compared to the group of collagen-induced arthritis.
  • bone loss was significantly inhibited in the groups treated with compound of Formula 1, as compared to the group of collagen-induced arthritis. Further, from the result of histological analysis, it was found that bone loss was significantly inhibited to virtually normal level in the groups treated with the compound of Formula 1, as compared to the group of collagen-induced arthritis.
  • Example 8 Effect of compound of Formula 1 on mouse periosteal bone formation
  • Freeze-dried collagen sponge was cut into pieces suitable for implantation, and then impregnated with the compound of Formula 1 which was diluted with the phosphate buffer solution in an amount of 1.25 ⁇ g per mouse.
  • the collagen sponge was impregnated with BMP-2 in an amount of 2 ⁇ g per mouse.
  • the collagen sponge was impregnated with DMSO and phosphate buffer solution.
  • the scalps of mice were incised, and then the treated collagen sponges were implanted to contact with the surface of skull, and sutured. After 14 to 21 days, the mice were sacrificed to obtain their skulls.
  • the skulls were washed with the phosphate buffer solution three to four times, and then fixed in 4% paraformaldehyde for 24 hrs. Then, the skulls were decalcified with 10% formic acid for 7 days, and embedded in paraffin. Histological sections were prepared and stained with H-E (hematoxylin-Eosin), followed by observation under an optical microscope.
  • H-E hematoxylin-Eosin
  • the compound of Formula 1 inhibits the expression of osteoclast differentiation- related genes, c-Fos and NFATcI, promotes the activity of osteoblast differentiation- inducing gene, Runx-2, and has excellent efficacy of promoting bone formation.
  • the pharmaceutical composition of the present invention is useful for suppressing osteoporosis induced by ovariectomy and bone loss caused by inflammation, and treating collagen-induced arthritis.

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Abstract

The present invention relates to a pharmaceutical composition for preventing or treating metabolic bone diseases, comprising a pharmaceutically effective amount of a compound represented by the following Formula 1 or a pharmaceutically acceptable salt thereof.

Description

Description
COMPOSITION FOR PREVENTING AND TREATING OSSEOUS METABOLIC DISEASE
Technical Field
[1] The present invention relates to a pharmaceutical composition for preventing or treating metabolic bone diseases, comprising a pharmaceutically effective amount of a compound represented by the following Formula 1 or a pharmaceutically acceptable salt thereof.
[2]
[3] [Formula 1]
[4]
Figure imgf000002_0001
[5]
Background Art
[6] The skeleton consists of highly specialized bone cells including osteocytes, osteoclasts and osteoblasts, bone matrix including hydroxyapatite crystal, collagenous fibers and glycosaminoglycans, and spaces including bone marrow cavities, vascular canals, canaliculi and lacunae (Stavros C. M., Endocrine Reveiws, 21(2), 115-137 (2000)). Bone functions to mechanically support the body, protect mapr organs, supply microenvironment required for hemopoiesis, and store calcium and several minerals.
[7] Growth, development and maintenance of bone continue throughout life. Old bone is destroyed and replaced with new bone by regeneration. Such bone turnover mainly occurs at BMU (Basic Multicellular Units) consisting of osteoclasts and osteoblasts, which serves to repair fine damage by growth and stress, and maintain function of bone. Destruction or resorption of old bone is accomplished by osteoclasts. In contrast, osteoblasts are responsible for new bone formation. Osteoclasts remove the bone matrix such as hydroxyapaptite crystal or collagenous fibers, which constitute bone, by adhering to the bone surface and secreting hydrochloric acid and proteases. Osteoblasts synthesize and secrete the bone matrix, and regulate the local concentration of calciun and phosphate to form skeleton (Stavros C. M., Endocrine Reviews, 21(2), 115-137 (2000)).
[8]
[9] Metabolic bone diseases are caused by breakdown of the balance between osteoclasts and osteoblasts in the body. A representative example of metabolic bone diseases is osteoporosis. Osteoporosis occurs due to reduction of total bone mass, resulting from both the excessive osteoclast activity and insufficient osteoblast activity. In osteoporosis, with of cortical bone is reduced, bone marrow cavity is enlarged, and thickness of trabecular bone is lowered, causing bone to be continuously porous. With progress of osteoporosis, physical strength of bone decreases, and thus lurbago and arthralgia are induced, and bone is easily fractured even by weak impact. In addition to osteoporosis, metabolic bone diseases include metastatic bone lesions caused by metastasis of breast and prostate carcinomas to bone, primary tumors of bone (e. g., multiple myeloma), rheumatoid or degenerative arthritis, periodontal disease accompanying destruction of alveolar bone by periodontal disease-causing bacteria, inflammatory periodontal disease with alveolar bone destruction generated after surgical application of dental implant, inflammatory bone resorption disease caused by implant implanted to fix bone by plastic surgery, and Paget's disease induced by various genetic factors.
[10] Myeloma is a bone disease featured by fragile bone accompanying severe pain, and caused by osteoclast activity increased by carcinomas. Breast and prostate carcinomas easily metastasize to bone, and stimulate osteoclast activity, resulting in destruction of bone. In the case of rheunatoid or degenerative arthritis, tumor necrosis factor (TNF), interleukin- 1 and interleukin-6, which are produced by the immune response, stimulate osteoclast activity present at the pint space, causing local destruction of bone at the pint. When inflammation is induced by infection with periodontal disease-causing bacteria, inflammatory cytokines including TNF, interleukin- 1 and interleukin-6 are produced by the immune response to the pathogenic bacterial infection to stimulate differentiation of osteoclasts, leading to destruction of alveolar bone supporting teeth.
[11] With recently active molecular biological research related to therapy of metabolic bone diseases including osteoporosis, bone formation- stimulating factors and osteoclast-suppressing factors were developed. The bone formation- stimulating agents include fluoride, parathyroid hormone, TGF-β, bone morphogenetic protein, and insulin-like growth factor. Osteoclast-suppressing factors include estrogen, calcitonin, vitamin D and its analogues, and bisphosphonates (Jardine et al., Annual Reports in Medicinal Chemistry, 31, 211 (1996)).
[12]
[13] Until now, several therapeutic agents for osteoporosis have been developed. Among them, estrogen, which is most frequently used for treating osteoporosis, has disadvantages as follows: it is still not demonstrated to be practically effective in treating osteoporosis, it should be administered throughout the patient's life, and it has side effects of increasing the incidence of breast cancer or cervical cancer when administered for a long period of time.
[14] Alendronate is also problematic in terms of being not clearly identified for its therapeutic efficacy for osteoporosis, being slowly absorbed by the gastrointestinal tract, and causing inflammation in the stomach, intestine and mucosa of the esophagus. Calciun preparations are known to have mild side effects and good efficacy, but are a preventive agent rather than a therapeutic agent. In addition, vitamin D and calcitonin are not sufficiently studied for their preventive or therapeutic efficacy and side effects.
[15]
Disclosure of Invention Technical Problem
[16] Therefore, there is a need for development of new therapeutic agents for metabolic bone diseases, having few side effects and excellent therapeutic efficacy. The present inventors found that the compound of Formula 1 inhibits the expression of osteoclast differentiation-related genes, c-Fos and NFATcI, promotes the activity of osteoblast differentiation-inducing gene, Runx-2, and has excellent efficacies of suppressing osteoporosis in mouse model of metabolic bone disease and bone loss caused by inflammation, and treating collagen-induced arthritis, thereby completing the present invention.
[17]
Technical Solution
[18] It is an object of the present invention to provide a pharmaceutical composition for preventing or treating metabolic bone diseases, comprising a pharmaceutically effective amount of a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.
[19] Brief Description of the Drawings
[20] Fig. 1 is a graph showing the effect of the compound of Formula 1 on the differentiation of bone marrow-derived osteoclast progenitor into osteoclast;
[21] Fig. 2 is the result of RT-PCR and Western blot, which shows the effect of the compound of Formula 1 on the expression of master genes for osteoclast differentiation, c-Fos and NFATcI;
[22] Fig. 3 shows the effect of the compound of Formula 1 on the activity of master genes for osteoblast differentiation, Runx-2;
[23] Fig. 4 is the result of micro-computed tomography, which shows the effect of the compound of Formula 1 on osteoporosis mouse model induced by ovariectomy;
[24] Fig. 5 is the result of micro-computed tomography, which shows the effect of the compound of Formula 1 on inflammatory bone loss mouse model induced by LPS (lipopolysaccharide) ;
[25] Fig. 6 is a graph showing the effect of the compound of Formula 1 on arthritis score in CIA mouse model (CIA: collagen-induced arthritis);
[26] Fig. 7 is the result of micro-computed tomography, which shows the effect of the compound of Formula 1 on bone loss in CIA mouse model;
[27] Fig. 8 is the result of H-E staining(hematoxylin-eosin staining) of histological sections, which shows the effect of the compound of Formula 1 on bone loss in CIA mouse model; and
[28] Fig. 9 is a microscopic photograph showing the effect of the compound of Formula 1 on in vivo bone formation.
[29]
Best Mode for Carrying Out the Invention
[30] In order to achieve the object, the present invention provides a pharmaceutical composition for preventing or treating metabolic bone diseases, comprising a pharmaceutically effective amount of a compound represented by the following Formula 1 or a pharmaceutically acceptable salt thereof.
[31]
[32] [Formula 1] [33]
Figure imgf000006_0001
[34]
[35] The compound of Formula 1 used in the pharmaceutical composition of the present invention is known as an MHC class- 1 expression promoting factor, disclosed in Japanese Patent No. 349462, but there is no mention of its functions such as promoting osteoblast differentiation, or suppressing osteoclast differentiation or therapeutic efficacy for metabolic bone diseases.
[36] The active ingredient used in the pharmaceutical composition of the present invention comprises "pharmaceutically acceptable salt" of the compound of Formula 1. The phrase "pharmaceutically acceptable" refers to compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset and dizziness, when administered to himans. The salt is prepared by reaction with a stoichiometric amount of a suitable base or acid in water or an organic solvent, or in a mixture thereof. Typically, the pharmaceutically acceptable salt according to the present invention includes inorganic base salts, organic base salts, inorganic acid salts, organic acid salts, and basic or acidic amino acid salts. Examples of the inorganic base salts include alkali metal salts such as sodiun salts or potassium salts, alkali earth metal salts such as calciun salts or magnesium salts, aluninum salts, and ammoniun salts. Examples of the organic base salts include salts of trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, di- ethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine and N, N'-dibenzylethylenediamine. Examples of the inorganic acid salts include salts of hydrochloric acid, boric acid, funaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzensulfonic acid and p- toluenesulfonic acid. Examples of the basic amino acid salts include salts of arginine, lysine and ornithine. Examples of the acidic amino acid salts include salts of aspartic acid and glutamic acid. The salts of the present invention may be prepared by conventional methods such as ion exchange. [37] The compound of the present invention and pharmaceutically acceptable salt thereof can be used individually or as a mixture of two or more thereof for the prevention and treatment of metabolic bone diseases. As used herein, the term "metabolic bone diseases" refers to a disease accompanying a physiopathological state caused by excessive destruction and resorption of bone. Examples of the metabolic bone diseases include osteoporosis, metastatic bone lesions caused by metastasis of breast or prostate carcinomas to bone, primary bone turors, rheumatoid or degenerative arthritis, periodontal disease, inflammatory periodontal disease with alveolar bone destruction, inflammatory bone resorption disease, and Paget's disease, preferably osteoporosis, inflammatory periodontal disease with alveolar bone destruction, and rheumatoid arthritis, but are not limited thereto.
[38] The compound of Formula 1 of the present invention suppresses the mRNA and protein expression of c-Fos and NFATcI, which are master regulators for osteoclast differentiation, to suppress bone resorption by osteoclast. Several transcription factors, including NF-kB (nuclear factor kB), c-Fos, NFATcI (nuclear factor of activated T cells cl), PUl, and MITF, are known to play an important role in osteoclast differentiation by the treatment of RANKL (receptor activator of NK ligand) (Teitelbaun et al., Nature Review of Genetics 4 :638-649, 2003). In particular, RANKL-induced expression of c-Fos is known to play an essential role in the initiation of osteoclast differentiation. In addition, the c-Fos expression causes the expression of NFATcI (Matsuo et al., Journal of Biological Chemistry 279:26475-26480, 2004), and NFATcI can induce osteoclast differentiation in the absence of RANKL. Thus, NFATcI is known as an essential factor in osteoclast differentiation (Takayanagi et al., Developmental Cell 3:889-901, 2002). In a specific embodiment, the present inventors performed RT-PCR and Western blot to confirm the effect of the compound of Formula 1 on the expression of c-Fos and NFATcI. As a result, when the compound of Formula 1 was added upon culturing osteoclast, the expression of c-Fos and NFATcI was found to be significantly suppressed (see Fig. 2).
[39] Further, the compound of Formula 1 of the present invention promotes the activity of
Runx-2, which is a master gene for osteoblast differentiation, to induce new bone formation, thereby suppressing bone resorption by osteoclast. In a specific embodiment, the present inventors treated mouse C2C12 cells with the compound of Formula 1. As a result, the activity of Runx-2 was found to be markedly increased, compared to the cell treated with FGF2 (fibroblast growth factor-2) (see Fig. 3).
[40] Further, the present inventors performed an in vivo test of the compound of Formula 1. An estrogen-deficient mouse due to ovariectomy was administered with the compound of the present invention, and bone volune of femur was measured using micro-computed tomography. As a result, it was found that bone volune significantly increased to inhibit osteoporosis (see Fig. 4). In addition, an LPS (lipopolysaccharide)-induced bone loss mouse model was administered with the compound of the present invention, and bone volune of femur was measured using micro-computed tomography. As a result, it was found that bone volune significantly increased to inhibit inflammatory bone loss (see Fig. 5). A collagen-induced arthritis mouse model was administered with the compound of the present invention, and micro-computed tomography and histological analysis were performed. As a result, it was found that bone loss was significantly inhibited to prevent and treat arthritis (see Figs. 6 to 8). In addition, the compound of the present invention exhibited the effect of promoting bone formation (see Fig. 9).
[41] Accordingly, the compound of Formula 1 of the present invention can be used as an osteoclast differentiation inhibitor, osteoblast differentiation promoter, or bone formation promoter, and as a pharmaceutical composition for preventing or treating metabolic bone diseases, comprising the compound as an active ingredient.
[42]
[43] In addition to the compound of Formula 1 or pharmaceutical acceptable salt thereof, the composition of the present invention may include one or more active ingredients having the same or similar function, for example, BMP-2,4,7 (bone morphogenetic protein-2,4,7) which is known to promote osteoblast differentiation.
[44] For administration, the composition of the present invention may include at least one pharmaceutically acceptable carrier, in addition to the above described active ingredients. Examples of the pharmaceutically acceptable carrier include a saline solution, sterile water, a Ringer's solution, a buffered saline solution, a dextrose solution, a maltodextrin solution, glycerol, ethanol and a mixture of one or more thereof. If necessary, the composition may also include other conventional additives such as antioxidants, buffers, and bacteriostatic agents. Moreover, the composition may additionally include diluents, dispersants, surfactants, binders, and lubricants to formulate it into injectable formulations such as aqueous solution, suspension, and emulsion, pills, capsules, granules and tablets. Furthermore, the composition may be preferably formulated depending on particular diseases and its components, using a suitable method in the relevant field or the method described in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA. [45] The pharmaceutical composition of the present invention may be administered by oral or parenteral route (e.g., intravenous, subcutaneous, intraperitoneal, or topical administration) depending on its purpose. An effective dosage of the present composition may be determined depending on the patient's body weight, age, gender, health state, and diet, administration time, administration routes, excretion rates, and severity of the diseases. The composition may be preferably administered at a daily dosage of about 0.1 mg/kg to 1 mg/kg one time or several times.
[46] The present composition may be used singly or in combination with surgical operation, hormone therapy, drug therapy and biological response regulators in order to prevent and treat metabolic bone diseases.
[47]
Mode for the Invention
[48] Hereinafter, the preferred Examples are provided for better understanding. However, these Examples are for illustrative purposes only, and the invention is not intended to be limited by these Examples.
[49]
[50] Example 1. Isolation of bone marrow cell and induction of osteoclast progenitors
[51] After sacrificing 6 to 7 week ICR (Institute of Cancer Research) female mice by cervical dislocation and disinfecting their rear legs with 70 % ethanol, tibias were isolated aseptically. The isolated tibias were placed into 3 x HBSS (Gibco BRL), and soft tissue was completely removed. Both ends of tibias were cut, and 1 x α-MEM (Gibco BRL) was injected into bone marrow and then sucked up using a 1 cc syringe, collecting bone marrow cells. After sufficient suspending by pipetting, the collected bone marrow cells were centrifuged(1600 rpm, 5 min, 40C), and the supernatant was discarded to harvest cells. The resulting pellet (bone marrow cells and erythrocytes) was suspended in ACK buffer (155 mM NH Cl, 11 mM KHCO , 0.01 mM EDTA).
4 3
After incubation for 2 min, phosphate buffer was added to the cell suspension to minimize damage to bone marrow cells and lyse erythrocytes. Thereafter, the cell suspension was centrifuged (1600 rpm, 5 min, 40C), and the resulting cell pellet was suspended in α-MEM containing 10% (v/v) FBS (fetal bovine serxrn, Gibco BRL), 100 U/ml of penicillin, 100 μg/ml of streptomycin, 10 ng/ml of macrophage-colony stimulating factor (M-CSF; Peprotech EC, London, England). After culturing overnight, non- adherent cells were collected and cultured in a culture plate containing 30 ng/ml of M-CSF for 3 days. The cultured cells were designated as osteoclast progenitors, and used to evaluate the effect of the compound of the present invention on their differentiation into osteoclasts.
[52]
[53] Example 2. Effect of compound of Formula 1 on differentiation of osteoclast progenitor
[54] To differentiate the osteoclast progenitors prepared in Example 1 into osteoclasts, the osteoclast progenitors were cultured in 1 X α-MEM supplemented with 30 ng/ml of macrophage-colony stimulating factor (M-CSF) and 50 ng/ml of RANKL (Peprotech EC, London, England). At this time, to evaluate the effect of the compound of Formula 1 on osteoclast differentiation, the compound of Formula 1 was added to the cells at various concentrations (10 nM, 25 nM, 50 nM, 75 nM, 100 nM). The exclusive patent license for the compound of Formula 1 was assigned by Professor Nishino at Kyushu national University (Japan). The completely differentiated osteoclasts were stained with TRAP(tartrate resistant acid phosphatase). TRAP staining was carried out using a Leukocyte Acid Phosphatase Kit (Sigma, cat. No. 387-A). After removing culture media, the completely differentiated osteoclasts were fixed with 10% formalin for 5 min. After removing formalin, the fixed osteoclasts were treated with 0.1% Triton X- 100 for 10 sec. After removing Triton X-100, the cells were stained with TRAP for about 5 min. After eliminating the TRAP staining solution, the cells were washed with distilled water twice, and dried. TRAP-positive osteoclasts were counted under an optical microscope (x 100).
[55] As shown in Fig. 1, the compound of Formula 1 inhibited osteoclast differentiation in a concentration-dependent manner, compared to a control. Accordingly, these results indicate that the compound of Formula 1 inhibits the differentiation of bone marrow- derived osteoclast progenitor into osteoclast in a dose-dependent manner.
[56]
[57] Example 3. Effect of compound of Formula 1 on c-Fos and
NFATcI
[5S] To identify the mechanism by which the compound of formula 1 inhibits osteoclast differentiation, the expression of c-Fos or NFATcI, which is an essential gene for osteoclast differentiation, was confirmed by RT-PCR and Western blot. Total RNA was prepared using TRI reagent(Gibco BRL), and RT-PCR was performed with 2 μg of total RNA using Superscript II reverse transcriptase (Gibco BRL). 10% of the reverse-transcribed cDNA was amplified by PCR. Sequences of the used primers are as follows: [59]
[60] 5'-CTGGTGCAGCCCACTCTGGTC-S' (SEQ ID NO. 1)
[61] 5'-CTTTCAGCAGATTGGCAATCTC-3'(SEQ ID NO. 2)
[62] 5'-CAACGCCCTGACCACCGATAG-S' (SEQ ID NO. 3)
[63] 5'-GGCTGCCTTCCGTCTCATAGT-S' (SEQ ID NO. 4)
[64] 5'-ACTTTGTCAAGCTCATTTCC-S' (SEQ ID NO. 5)
[65] 5'-TGCAGCGAACTTTATTGATG-S' (SEQ ID NO. 6)
[66]
[67] PCR conditions included 22 cycles of denaturation at 940C for 30 sec, annealing at
580C for 30 sec and extension at 720C for 30 sec. The PCR product was subjected to electrophoresis on a 1.2% agarose gel, and stained with ethidiun bromide. As shown in Fig. 2 (upper), when the compound of formula 1 (100 nM) was added, the expression of c-Fos and NFATcI in the presence of RANKL was markedly inhibited. That is, it can be seen that the compound of formula 1 significantly inhibited mRNA expressions of c-Fos and NFATcI, which are master genes for osteoclast differentiation.
[68] In addition, Western blot was performed to confirm whether the compound of
Formula 1 inhibits protein expression levels of c-Fos and NFATcI. For Western blot, the prepared cells were lysed in a buffer solution containing 20 mM Tris-HCl, 150 mM NaCl, 1% Triton X-IOO, protease and phosphatase inhibitors. Proteins in cell lysate (30 βg) were separated on a 10% SDS-PAGE, and transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA). After blocking with 5% skim milk, the membrane was analyzed with anti-Fos, NFATcI, and TRAF6 (Cell Signaling Technology, everly, MA). The membrane was reanalyzed with an anti-actin antibody (Cell Signaling Technology). As shown in Fig. 2 (lower), the compound of Formula 1 was found to significantly inhibit the protein expressions of c-Fos and NFATcI. Accordingly, it can be seen that the compound of Formula 1 significantly inhibited the expression of c-Fos and NFATcI, which are master genes for osteoclast differentiation.
[69]
[70] Example 4. Effect of compound of Formula 1 on Runx-2
[71] To confirm whether the compound of formula 1 increases the activity of Runx-2 which is a master gene for osteoblast differentiation, the following experiment was performed using C2C12 cells transfected with a 6xOSE2-Luc reporter vector. OSE2 (osteoblast specific element-2) is the binding site for Runx-2 in the promoter region of bone marker, osteocalcin. C2C12 cells are cells transfected with a reporter vector, prepared by cloning the pGL3 promoter vector with six tandem copies of the OSE2 motif. As shown in Fig. 3, the compound of formula 1 increased the activity of Runx-2 more than a positive control, FGF2, which is known to increase the activity of Runx-2. In addition, since the activity of Runx-2 can be influenced by fetal bovine serum (FBS) which is generally used for cell culture, change in the activity of Runx-2 was analyzed according to the presence of serum. As a result, it was found that the activity was decreased in the presence of serum, but the activity was still higher, compared to the control group and FGF2 group. Accordingly, it can be seen that the compound of formula 1 increased the activity of Runx-2 which is a master gene for osteoblast differentiation, thereby promoting osteoblast differentiation.
[72]
[73] Example 5. Effect of compound of Formula 1 on osteoporosis mouse model
[74] Osteoporosis is a disease caused by increase in osteoclast formation due to estrogen deficiency after menopause (J Clin Invest 112:915-923, Lean et al., 2003). The effect of inhibiting osteoclast differentiation of the compound of Formula 1 was examined in an ovariectomized mouse as an animal model for osteoporosis. Both ovaries were excised from 16 week-old female mice and after 4 weeks, bone resorption was induced. Then, the compound of Formula 1 was intraperitoneally administered to the mice three times per week at a dose of 0.1 βg per 1 g of body weight for 4 weeks. For comparison, one group was administered with a phosphate buffer solution instead of the compound of Formula 1. After 4 weeks, the mice were sacrificed by cervical dislocation. Femurs were isolated, and bone volumes in horizontal and vertical cross- sections were measured by micro-computed tomography scan (Skyscan 1072 micro- CT system; SkyScan, Aartselaar, Belgium). For quantification, the ratio of bone volume to total tissue volume was calculated. As shown in Fig. 4, the bone volume was significantly increased and virtually normal in the group treated with the compound of formula 1, as compared to the group treated with the phosphate buffer solution only. Accordingly, it can be seen that osteoporosis induced by ovariectomy can be treated with the compound of Formula 1.
[75]
[76] Example 6. Effect of compound of Formula 1 on inflammatory bone loss mouse model
[77] In general, inflammation causes bone loss, which was confirmed by intraperitoneally administering mice with LPS (lipopolysaccharide, E.coli O55:B5, Sigma) and measuring bone loss of femurs (Miyaura et al., J Exp Med 197:1303-1310, 2003). To confirm the effect of the compound of Formula 1 in the bone loss mouse model induced by LPS, mice were intraperitoneally administered with 5 μg of LPS (per body weight) on day 0 and day 4 to induce bone resorption of femur. At this time, to analyze the effect of the compound of Formula 1, the compound of Formula 1 was intraperitoneally administered to the mice at a dose of 0.1 μg per 1 g of body weight on day -1, 1, 3, 5, and 7. As a control group, a phosphate buffer solution was intraperitoneally administered. On day 8 after LPS administration, the mice were sacrificed. Femurs were isolated, and bone volumes in horizontal and vertical cross- sections were measured by micro-computed tomography scan (Skyscan 1072 micro- CT system; SkyScan, Aartselaar, Belgium). For quantification, the ratio of bone volume to total tissue volume was calculated.
[78] As shown in Fig. 5, the bone volume was significantly increased and virtually normal in the group treated with the compound of formula 1, as compared to the group treated with the phosphate buffer solution only. Accordingly, it can be seen that inflammatory bone loss induced by LPS can be inhibited by the compound of Formula 1.
[79]
[80] Example 7. Effect of compound of Formula 1 on arthritis mouse model
[81] Rheumatoid arthritis is a chronic inflammatory disease, and accompanied by bone loss around the pint by increased osteoclast activity. To analyze the effect of the compound of Formula 1 on bone loss in arthritis mouse model, a collagen-induced arthritis mouse model was used. Type II collagen, which is freeze-dried and extracted from bovine (Chondrex, Redmond, WA, USA, Cat. No. 2002-1), was slowly mixed with 0.05 M acetic acid at 40C overnight to a concentration of 2.0 mg/ml. The next day, the solubilized collagen solution was mixed with an equal volume of complete Freund's adjuvant (Chondrex, Redmond, WA, USA, Cat. No. 7001), and suspended using a homogenizer. The suspension was put in a 1.0 ml tuberculin syringe. DBA/1 mouse was restrained, and then the hair on the back near the tail base was shaved. 0.1 ml of suspension was intradermally injected with caution (not deep into the subcutaneous tissue). At two weeks after primary immunization, secondary immunization was performed. Upon secondary immunization, an incomplete Freund's adjuvant (Chondrex, Redmond, WA, USA, Cat. No. 7002) was used instead of the complete Freund's adjuvant used in primary immunization, and injected in the same manner as in primary immunization. At this time, to analyze the preventive effect of the compound of Formula 1 on arthritis, the compound of Formula 1 was intraperitoneally administered to the mice at a dose of 0.1 μg per 1 g (body weight) three times per week at two weeks after primary immunization. In addition, to analyze the therapeutic effect of the compound of Formula 1 on arthritis, the compound of Formula 1 was administered in the same manner at three weeks after primary immunization. At seven weeks, the mice were sacrificed, and micro-computed tomography scan and histological analysis were performed. Beginning at three weeks, arthritis score observed with the naked eye was monitored weekly, according to the following criteria of American College of Rheunatology. In regard to the criteria, 0 point was given for normal pint of paw, 1 point for swelling of toe, 2 points for swelling of paw pad, and 3 points for extensive swelling of the entire paw. The scores ranged from 0 to 12 points. Forefoot of arthritis- induced mouse was imaged by micro-computed tomography scan (Skyscan 1072 micro-CT system; SkyScan, Aartselaar, Belgium) for three-dimensional analysis of bone loss and changes in bone tissue, and then reconstructed by V- works (Cyberland, Seoul, Korea). In addition, for histological analysis, the collected bones were fixed in 4% paraformaldehyde, decalcified with 12% EDTA, and then embedded in paraffin. Histological sections were prepared and stained with H-E (hematoxylin-Eosin), followed by observation under an optical microscope. As shown in Fig. 8, typical features of rheunatoid arthritis such as bone loss and deformity were observed in the arthritis-induced mice.
[82] As shown Fig. 6, the arthritis score was significantly decreased in the groups treated with the compound of Formula 1 for the purpose of prevention and treatment, as compared to the group of collagen-induced arthritis. As shown Fig. 7, bone loss was significantly inhibited in the groups treated with compound of Formula 1, as compared to the group of collagen-induced arthritis. Further, from the result of histological analysis, it was found that bone loss was significantly inhibited to virtually normal level in the groups treated with the compound of Formula 1, as compared to the group of collagen-induced arthritis.
[83]
[84] Example 8. Effect of compound of Formula 1 on mouse periosteal bone formation
[85] To confirm the function of the compound of Formula 1 in bone formation, its effect on periosteal bone formation in vivo was analyzed. Specifically, a predetermined amount (5 ml) of collagen (Cellmatrix type I- A, Wako co., Japan, Cat. No. 637-00653) was put into a Petri dish (60 x 15 mm), and freeze-dried. The compound of Formula 1 was dissolved in DMSO, and then diluted with a phosphate buffer solution to a volume of 10 βi. As a positive control, BMP-2 was dissolved in the phosphate buffer solution. Freeze-dried collagen sponge was cut into pieces suitable for implantation, and then impregnated with the compound of Formula 1 which was diluted with the phosphate buffer solution in an amount of 1.25 βg per mouse. As a positive control, the collagen sponge was impregnated with BMP-2 in an amount of 2 βg per mouse. As a negative control (vehicle-treated group), the collagen sponge was impregnated with DMSO and phosphate buffer solution. The scalps of mice were incised, and then the treated collagen sponges were implanted to contact with the surface of skull, and sutured. After 14 to 21 days, the mice were sacrificed to obtain their skulls. The skulls were washed with the phosphate buffer solution three to four times, and then fixed in 4% paraformaldehyde for 24 hrs. Then, the skulls were decalcified with 10% formic acid for 7 days, and embedded in paraffin. Histological sections were prepared and stained with H-E (hematoxylin-Eosin), followed by observation under an optical microscope.
[86] As shown Fig. 9, new bone formation (arrow) was observed in the group treated with the compound of Formula 1 and the positive control group treated with BMP-2, as compared to the negative control group treated with vehicle. Accordingly, it can be seen that new bone formation was promoted by the compound of Formula 1.
[87]
Industrial Applicability
[88] The compound of Formula 1 inhibits the expression of osteoclast differentiation- related genes, c-Fos and NFATcI, promotes the activity of osteoblast differentiation- inducing gene, Runx-2, and has excellent efficacy of promoting bone formation. The pharmaceutical composition of the present invention is useful for suppressing osteoporosis induced by ovariectomy and bone loss caused by inflammation, and treating collagen-induced arthritis.
[89]

Claims

Claims
[1] A pharmaceutical composition for preventing or treating metabolic bone diseases, comprising a pharmaceutically effective amount of a compound represented by the following Formula 1 or a pharmaceutically acceptable salt thereof: [Formula 1]
Figure imgf000016_0001
[2] The pharmaceutical composition according to claim 1, wherein the metabolic bone disease is selected from the group consisting of osteoporosis, bone metastatic lesion, primary bone tumors, rheumatoid or degenerative arthritis, periodontal disease, inflammatory periodontal disease with alveolar bone destruction, inflammatory bone resorption disease, and Paget's disease.
[3] The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is a pharmaceutical composition for treating rheumatoid arthritis.
[4] The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is a pharmaceutical composition for treating osteoporosis.
[5] The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is a pharmaceutical composition for treating inflammatory bone resorption disease.
[6] An osteoclast differentiation inhibitor, comprising the compound of Formula 1 according to claim 1 as an active ingredient.
[7] The osteoclast differentiation inhibitor according to claim 6, wherein activities of c-Fos and NFATcI are suppressed by the compound of Formula 1 according to claim 1.
[8] An osteoblast differentiation promoter, comprising the compound of Formula 1 according to claim 1 as an active ingredient.
[9] The osteoblast differentiation promoter according to claim 8, wherein activity of
Runx-2 is promoted by the compound of Formula 1 according to claim 1.
[10] A bone formation promoter, comprising the compound of Formula 1 according to claim 1 as an active ingredient.
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