MXPA98004449A - Agents to promote training or - Google Patents

Abstract

The present invention relates to providing a promoting agent for bone formation, a therapeutic method for bone formation and a process for preparing a bone formation promoting agent. An agent that promotes or promotes the formation is that which comprises at least one cistrin, echstatin, a peptide or polypeptide that contains the amino acid sequence ArgGlyAsp in the molecule such as a peptide represented by Gly-Asp-Gly-Asp-Ser or a compound represented by the general formula (IX) and so on, as well as a biologically acceptable salt thereof, wherein R16 represents -N (R20) 2 (R20 represents a hydrogen atom or an alkyl group with 1 to 4 carbon atoms), R17 represents a hydrogen atom or an alkyl group of 1 to 4 carbon atoms, R18 represents a hydrogen atom, an alkyl group of 1 to 4 carbon atoms and assiduously, R19 represents OH, NH2, and asystically, Y represents -NH-, -O- or a direct link, a represents 1-3, b represents 1 or 2, c represents 0 or 1 and represents 0

Description

AGENTS TO PROMOTE OSEA TRAINING FIELD OF THE INVENTION This invention relates to a promoter agent for bone formation comprising a peptide or polypeptide containing the amino acid sequence consisting of ArgGlyAsp (referred to below as a sequence of RGD) in the molecule. In addition, the invention relates to a prophylactic and therapeutic method for bone fracture using a promoter agent for bone formation comprising the peptide or polypeptide containing the RGD sequence. Also, the invention relates to a novel cyclic peptide, which contains the sequence of RGD. And further, it relates to a promoter agent for bone formation which comprises a compound represented by the general formulas (IX), (X), (XI) and (XII).

BACKGROUND OF THE INVENTION The skeleton is composed of external cortical bones and internal trabecular bones. The function of the skeleton in the living body is to maintain a given form as a skeleton and to store several REF: 27522 inorganic substances such as calcium, phosphoric acid and the like. The bone may manifest itself as a tissue with less variability, but the currently old bone is absorbed and new bone is formed instead. This is usually referred to as a bone reformation. The bone can be reformed by coupling osteoclasts that control bone absorption with osteoblasts that control bone formation, both of which can participate mainly in it. It has now been elucidated that the function of osteoblasts is not limited only to the formation of bones, but is related to the differentiation and activation of osteoclasts and osteoblasts that can play a role or function as a control center in the reconstruction of the cellular bone. . These diseases generally referred to as bone metabolism diseases may include osteoporosis, Behcet's disease, osteocia, hyperostosis and osteopetrosis. Among these, osteoporosis is the most frequently developed disease and often its appearance occurs to increase with old age so that the diagnosis and effective therapy of it have been intensely desired. > V Diseases of bone metabolism mean those diseases in which the bone cells have specific metabolic abnorties in any bone tissue. The present invention has made intensive studies to find a promoter factor for bone formation using a culture assay and this invention is finally completed. Integrin can participate in the interaction between cells and cells or between cells and matrices extracellular and plays an important role in wound healing, development, immunization, hemostasis or metastasis. The integrin superfamily is an α, β-heterodimer group found on the cell surface and can combine ligands extracellular and cytoskeleton. All integrins are heterodimers and each subunit is 90% extracellular and may have a broad domain or membrane permeable region and a short intracellular domain. The extracellular region joins the matrices extracellular or ligands of the cell surface, while the intracellular region is bound with cytoskeleton proteins. Bone matrices such as osteopontin, bone sialoglycoproteins, thrombospondin, fibronectin and vitronectin are found in the bone and all proteins have been found to have the sequence of RGD. Currently, osteoclasts have been found to have aVß3 and a2ßl integrin on the surface of the cell membrane (Davies J. et al., J. Cell Biol., Vol.l, 109, p.1817, 1989 and Za bonin ZA et al. al., Connect, Tissue Res., Vol. 20, p.143, 1989). From the facts that bone absorption by osteoclasts can be inhibited by the action of an antibody to integrins (Davies J. et al., J. Cell Biol., Vol.109, p.1117, 1989), that bone absorption by osteoclasts of the rat can be inhibited by the synthetic GRGDSP peptide (GlyArgGlyAspSerPro) (Horton MA et al., Exp. Cell Res. ., Vol.195, p.368, 1991), and additionally that echstatin, a protein derivative of the snake venom and having the sequence of RGD and an activity that inhibits platelet aggregation, a synthetic GdRGDSP peptide and A cyclic synthetic GPenGRGDSPCA peptide can inhibit bone absorption by mouse osteoclasts and the GdRGDSP peptide can inhibit the formation of multinuclear osteoclasts resistant to tartaric acid and positive to phosphatase (Gabri VDP et al., J. Bone Miner, Res. , Vol.9, p.1021, 1994), it is suggested that the recognition and adhesion of bone matrices by integrin and cytoskeleton referred to participate intensively in the development of osteoclast osseous absorption function. It can then be considered that the adhesion of cellular matrices between osteoblasts and bone matrices should be caused by the mechanism of adhesion via collagen and fibronectin in bone matrices and ßl integrins of osteoblasts. Also, it should be possible in the adhesion mechanism between heterocytes, that the cell adhesion of osteoblasts with osteoclasts can be effected via fibronectin that both the ß3 integrin of osteoclasts and ßl integrin in osteoblasts can be a receptor for fibronectin. However, it has not yet been suggested that the disintegrin family that includes echstatin or cistrin (William RG et al., Protein Science, Vol.2, p.1749, 1993) and the RGD peptide show a promoter action for the formation of bones .

DESCRIPTION OF THE INVENTION It is the object of this invention to provide a bone-building promoter, a therapeutic method for bone formation and a process for preparing a bone-building promoter. Bone or bone formation can be promoted by administering a peptide or polypeptide containing the RGD sequence in the molecule or a biologically acceptable salt thereof to patients. Peptides or polypeptides containing the sequence of RGD in the molecule can illustratively include cistrine, echstatin, a peptide represented by Gly-Arg-Gly-Asp-ser (referred to below as GRGDS), a compound represented by the general formula (I) wherein Ri, R2, R3, R, R5 and Rβ may be the same or different and each represents one selected from the group consisting of a hydrogen atom; a group *" alkyl of 1-8 carbon atoms optionally substituted with one selected from the group consisting of a hydroxy group, a carboxy group, a cycloalkyl group of 3-10 carbon atoms optionally substituted by a hydroxy group, and an aryl group of 6 12 carbon atoms optionally substituted with a hydroxy group; a cycloalkyl group of 3-10 carbon atoms optionally substituted with the hydroxy group and an aryl group of 6-12 carbon atoms The carbon optionally substituted with a hydroxy group, R7 and R8 may be the same or different and each represents a group selected from the group consisting of a hydroxy group, an alkoxy group of 1-8 carbon atoms, an alkenyloxy group of 2-12 atoms carbon, a cycloalkyloxy group of 3-10 carbon atoms and an aryloxy group of 6-12 carbon atoms, and X represents S or S02 and a compound represented by the general formula (II) wherein R9, Rio, Rp and R12 may be the same or different and each represents one selected from the group consisting of a hydrogen atom, an alkyl group of 1-8 carbon atoms, a cycloalkyl group of 3-10 atoms of carbon and an aryl group of 6-12 carbon atoms optionally substituted with a hydroxy group, R13, R? and R15 may be the same or different and each represents a group selected from the group consisting of a hydroxy group, an alkoxy group of 1-8 carbon atoms, an alkenyloxy group of 2-12 carbon atoms, a cycloalkyloxy group of 3-10 carbon atoms and an aryloxy group of 6-12 carbon atoms, and X represents S or SO. And, this invention provides a new compound represented by the formula (II). Also, this invention is directed to a therapeutic method for promoting bone formation comprising administering to patients a compound represented by the general formula (IX) wherein Ri6 represents -N (R2o) 2, -C (= NH) -NH2, -NH-C (= NH) -NH2 or -CO-NH-C (= NH) -NH2 (in which R2o independently represents a hydrogen atom or an alkyl group of 1-4 carbon atoms optionally substituted with a phenyl group), R 17 represents a hydrogen atom or an alkyl group of 1-4 carbon atoms optionally substituted with a phenyl group, Ris represents a hydrogen atom, an alkyl group of 1-4 carbon atoms, a phenyl group optionally substituted with a methoxy group or -COR2? (in which R2? represents -OH, -NH2, -NH- (CH2) 2-phenyl, an alkoxy group of 1-3 carbon atoms, a benzyloxy group, Pro or Aoc), R19 represents an alkyl group of 1 -5 carbon atoms optionally substituted with a substituent selected from the group consisting of OH, -NH2, -CONH2 cyclohexyl, phenyl, naphthyl, indolyl or adamantyl, a methyl group substituted with -COOH and -NHCOOCH2-phenyl, a cyclohexyl group optionally substituted with a methoxy group or an aryl group of 6-10 carbon atoms optionally substituted with a methoxy group, provided that Ris and R19 together with the carbon atoms to which they are attached can form adamantyl, naphthyl or fluorenyl, and represents -NH-, -O- or a direct bond, a represents 1, 2, or 3, b represents 1 or 2, c represents 0 or 1 and d represents 0 or 1, a compound represented by the general formula (X) wherein R22 represents -N (R23) 2, -C (= NH) NH2, -NH-C (= NH) -NH2 or -CO-NH-C (= NH) -NH2 (in which R23 independently represents a hydrogen atom or an alkyl group of 1-4 carbon atoms optionally substituted with a phenyl group) and represents 2-6, a compound of the formula (XI) a compound of the formula (XII) or a biologically acceptable salt thereof. In the previous formulas, Pro and Aoc represent respectively Cistrine and echstatin are proteins having molecular weights of about 7,300 and about 5,400, respectively, and they are described, for example, in Protein Science (1993), 2, 1749-1755. The GRGDS can be easily synthesized according to a conventional peptide synthesis. The compounds of the general formula (I) are described in US Pat. No. 5,384,309 and their representative compounds can be illustrated by the formulas (III) - (VI).

(III) (V) (I V) (VI) The compounds of the general formula (II) are new and can be prepared according to the method described in Barker et al., J. Med. Chem., 1992, 35,2040. Their representative compounds can be illustrated by formulas (VII) and (VIII).

Illustrative compounds of the general formula (IX) are as mentioned below.

Compound no. 1 C-om not.

Compound no. 3 Compound no. 4 Compound no. "5 Compound Compound no. 7 Compound no.

Compound no. 9 Compound no. 10 CS tax no. 11 Compound no. 12 Compound no. 13 Compound no. 14 Compound no. 15 Compound and Compound no. 17 Compound no. 18 Compound no. 19 Compound no. 20 'N Compound no-, 21 Compound no. 23 Comp Compound no. 25 Compound no. 26 Compound no.2X Compound no. 28 Compound no. 29 Compound no. 30 Compound no. 31 Compound no. 32 Compound no. 33 o Compound no. 3. 4 Compound no. 35 Compound no. 36 Compound no. 37 Compound no.38 Compound no. 39 Compound no. 40 Compound no. 42 Compound no. 43 Compound no. 44 Compound no. 45 Compound no. 46 Compound no. 48 Compound no. 49 Cl Compound no. 50 Compound "No. 51 N Compound No. 52 Ci The illustrative compounds of the general formula (X) on as cited below. Compound no. 53 Cl Compound no. 55 Compound no. 56 Compound no. 57 Compound no. 58 Compound no. 60 -s -o Compound no. 61 Compound no. 62 Compound no. 63 The illustrative compound of the general formula (XI) is as cited below.

Compound no. 64 The illustrative compound of the general formula (XII) is as cited below.

Compound n ° 65 a The compounds of the general formulas (IX), (X), (XI) and (XII) are known and can be synthesized according to the methods described in EP-A0499079, EP-A0530505, EP-A0566919, WO 95 / 14008, EP-A0528586 and WO 93/19046. When cistrine or echstatin are to be administered to the human as a promoter for bone formation, a daily dose of 0.001-100 μg / kg per body weight, preferably 0.01-10 μg / kg per body weight is used. In the case of the GRGDS, a daily dose of 0.001 -10 mg / kg per body weight, preferably 0.01-1 mg / kg per body weight is used. In the case of the compound represented by the general formula (I), (II), (IX), (X), (XI) or (XII), a daily dose of 0.001 -10 mg / kg per body weight is used, preferably 0.01 - 1 mg / kg per body weight. The present drug can be administered systemically by intravenous injection, intramuscular injection, intraperitoneal injection, oral administration, parenteral administration such as suppositories or any other means of the prior art. As pharmaceutical preparations, injectable or oral pharmaceutical preparations can be attempted. Injectable pharmaceutical preparations may include, for example, injectable powders. In 33 In this case, one or more of the suitable water-soluble excipients such as mannitol, sucrose, maltose, glucose and fructose can be added and dissolved in water and the solution is poured into prudent portions in ampoules and freeze-dried and sealed to form pharmaceutical preparations. Pharmaceutical preparations can include conventional tablets, capsules, fine granules, powders as well as enteric coated preparations. In the treatment of bone fracture, it can be administered systemically or locally provided by injection and others. For local administration, a carrier containing the present drug can be implanted more preferably in the closed area or near the fractured portion. In this case, natural polymeric substances such as collagen or fibrin glue, synthetic polymeric substances capable of being dispersed in the body in life such as poly-acylated glycolic acid can be used by carriers. In plastic surgery, cosmetic surgery, bone transplantation or dental transplantation, the present agent can be applied by coating the surface of the bone or tooth to be transplanted with an adhesive substance such as collagen paste or fibrin glue. And, it can be applied to the tissue, bone or alveolar bone in the portion to which the bone or tooth is to be transplanted. In the transplantation of bone or tooth, the artificial bone or root of an artificial tooth can be used, which is composed of, for example, metals, ceramics, glass and other natural or artificial inorganic substances such as hydroxyapatite. In this case, it may also be easy to form a core portion with a dense material and form a portion of the surface with a porous material such as hydroxyapatite and penetrate the present agent into the porous portion. Also, it is possible that the artificial bone surface composed of denser materials is rough to maintain the present agent on the surface.

Example of Synthesis of Compounds GH 4 and GH 5 Compounds GH 4 and GH 5 were prepared according to the standard FMOC method described in P.L. Barker et al., J. Med. Chem., 35, p. 2040-2048, 1992. The FMOC-S-trityl-cysteine bound to the Wang resin was used as a starting material and the amino acid of FMOC having a suitable side-chain protecting group, D-aspartic acid (Ot- butyl); L-aspartic acid (O-t-butyl); glycine; L-arginine (N-2, 2, 5, 7, 8-pentamethylchroman-6-sulfonyl) was used again in 3-mole portions. FMOC at the N-terminus was removed with piperidine (in the form of a 20% solution in dimethyl acetamide) and racemic 2-bromo-2-phenylacetic acid (4 equivalents) activated with diisopropylcarbodii ida (2 equivalents) were added to the terminal of N free. The S-trityl group in the cysteine side chain was removed with a chloro-methane solution containing dilute trifluoroacetic acid (2%) and then the resulting peptide was crystallized by the addition of a solution of diisopropylethylamine (2 equivalents) in dimethylacetamide. The deprotection and selection of the cyclized peptide was carried out by treatment with trifluoroacetic acid containing triethylsilane (2%). The removal of triethyl fluoroacetic acid produced a crude peptide, which was then purified using the reverse phase HPLC column [Vydac, C-18, at 0.1% aqueous trifluoroacetic acid solution, acetonitrile] with linear gradient (acetonitrile: 0- 40%, 80 min). The compounds involved in those fractions obtained after approximately 22 minutes and approximately 24 minutes, respectively, are named GH 4 and GH 5. The peptides were isolated as white powders by drying with freezing in water. Both compounds were detected for molecular ion peaks (M + H) + = 680.7 in the electroaspersion mass spectrum. Both compounds evidently showed different NMR spectra of 0-10 ppm in water at pH 4-5. Coupling constancy (NH-CH, Hz) Amino acid GH 4 GH 5 Arginine 8.05 4.9 D-aspartic acid 8.02 8.1 L-aspartic acid 3.14 7.7 Cysteine 6.08 7.6 It was confirmed that GH 4 and GH 5 have the formula (VII) u (VIII ), but they may not be identified to any of them.

Preparation Example 1 Preparation of injections 10 μg of echstatin were dissolved in 1 ml of water and a water-soluble excipient, sucrose, was thus added to be 1.25-40% w / v. It was sterile filtered by means of a 0.22 μm filter (available from Milipore Inc.). It was added in portions to containers, dried by freezing and sealed to form preparations. The promoter action of bone formation by the present agent will be illustrated by means of the following test results.

Test Result 1 Calvary was excised from the fetus of the 20-day-old rat and osteoblast-like cells were prepared by enzymatic treatment. The osteoblast-like cells were incubated in the MEM (+) medium containing 100 μl / ml of ascorbic acid, 2mM of β-glycerol phosphate and 10% of fetal bovine serum to form the tubercle of the bone. For bone tuber formation by primary culture osteoblasts, cistrin and echstatin promote calcification and tuber number of the bone at 10 ~ 7 and 10_8M. The promoted alkaline phosphatase activity was observed at 10 ~ 8M. Synthetic cyclic peptides, compounds of formulas (IV), (VII), (VIII) and (V) promoted calcification and bone tuber number at 10"6 and 10" 8M. The compound of the formula (III) promoted the calcification at 10 ~ 8 and 10"10M, and the number of tubers of the bone at 10" 6M. The GRGDS promoted calcification at 10 ~ 6M, but completely prevents calcification at 10"4 M. Test Result 2 Formation of the tubercle of the bone by calvarium osteoblasts of the fetus of the rat 1) Preparation of the enzymatic solution 100 mg of collagenase (0.2%) and 50 mg of hyaluronidase (0.1%) were weighed and added to 50 ml of bovine fetal serum free medium F12 and shaken with an agitator, filtered sterile with a 0.22 μm filter and then 2) Preparation and culture of osteoblasts Fetuses of 20 days of age (approximately 14 fetuses) were extracted from the pregnant rat and immersed in 70% ethanol.The calvary was exposed by removing the scalp using forceps or curved forceps in the The calvary was cut without removing the periosteum and was immersed in a medium free of fetal bovine serum, the connective tissues that traverse to the front and back in the Central area and surrounding soft or soft tissues were removed by cutting using a scalpel. All pieces of the bone thus prepared were placed in a 50 ml centrifuge tube. 10 ml of the enzyme solution were added and the tube was stirred in a thermostat at 37 ° C for 5 minutes. The liquid treated with dispersed cells was recovered and centrifuged at 1200 rpm for 5 minutes followed by stirring a supernatant. 15 ml of F12, 10% of the FCS medium were added and the cells were detached from the cavity and then spread on a 10 cm petri dish, which was defined as Fraction 1. To the bone pieces, after the recovery of the treated liquid of Fraction 1, 10 ml of the enzyme solution was added and stirred in a thermostat at 37 ° C for 10 minutes. After centrifugation, the resulting cells were defined as Fraction 2. Subsequently, the cells in those fractions up to Fraction 5, were obtained in the similar enzymatic treatment as above and the cells of Fraction 3-5 were incubated in an incubator of carbon dioxide for 2-3 days using the F12 medium containing 10% fetal bovine serum. After incubation, the cells were recovered by treatment with 0.25% trypsin (EDTAlibre). After counting the number of cells, the cells were incubated in a 4-well incubation plate (Nunc) at 1900 cells / 300 μl (1000 cells / cm 2) and the incubation was continued for an additional 2-3 days.

After incubation, all the cultured liquids were removed, 300 μl of the medium containing the active compound or dexamethasone (10 ~ 8M) instead of being added and then the incubation was continued for 14 days. For the incubation of this point, MEMa (+) medium containing 100 μl / ml of ascorbic acid, 2mM of β-glycerol phosphate and 10% of fetal bovine serum optimal for calcification was used. The medium was exchanged every 2 days and the active compound was added at each change. The area of the calcified region and the tuber number of the bone was used as an index for the formation of the tubercle of the bone. After incubation, the cells were fixed with physiological saline buffered 10% formalin / phosphate for 60 minutes and then washed three times with distilled water. Subsequently, calcium phosphate in the tubercle of the bone is stained or stained by treating with a red coloring solution of 1% alizarin for 10 minutes and washed three times with distilled water. The tubercle area of the stained bone was measured using the Nikonruzex 3U image analysis device, while the tuber tuber number of the bone was visually counted under the microscope. 3) Alkaline phosphatase activity The cells incubated in the same manner as in 2) above, were washed twice with 500 μl of physiological saline buffered with phosphate and 300 μl of the eluent (1% Triton X-100, 0.5 -mM magnesium chloride, 10 mM Tris, pH 7.2) was added and the mixture was allowed to stand on ice for 3 minutes. It was then transferred into an Eppendorf tube, homogenized by means of a homogenizer and allowed to remain in a thermostat at 37 ° C overnight to elute the alkaline phosphatase. Subsequently, extracellular stromal and cell residues were removed by centrifugation at 12,000 rpm for 10 minutes and 10 μl of a supernatant was recovered in another tube, 190 μl of an alkaline phosphatase substrate solution (100 mM 2-amino-2). -methyl-l-propanol, 2 mM magnesium chloride and 2 mM sodium p-nitrophenyl phosphate) were added and allowed to react in a thermostat at 37 ° C for 10 minutes. The reaction was discontinued by the addition of 800 μl of an IN sodium hydroxide solution and the absorbance was measured at a wavelength of 405 nm.

The activity of alkaline phosphatase was calculated according to the following equation: Alkaline phosphatase activity (μmoles / min / cavity) = (-absorbency of the sample - absorbance of the blank) x 0.05714 X 300 10 (minutes) 10 The results of the effect of bone tuber formation observed in the osteoblasts cultured at the beginning are shown in Table 1.

Table 1 No. of Activity of tubercle area of phosphatase calcified alkaline bone Treatment (mm2 / cavity) (per cavity) (μM / min / cav.) Not treated 2.37 ± 0.98 146.5 ± 10.9 0.044 ± 0.014 Dexamethasone 8.00 ± 0.20 307.3 ± 34.1 0.181 ± 0.018 Cistrine 10"6M 0.01 + 0.02 147.8 ± 14.2 0.040 ± 0.003 "7M 3.95 ± 0.82 189.5 ± 16.3 0.051 ± 0.007 "8M 4.03 ± 0.67 161.8 ± 19.2 0.067 ± 0.005 "9M 3.14 + 0.95 153.8 ± 12.3 0.051 ± 0.008 Echstatin 10"6M 0.00 ± 0.00 179.0 ± 17.9 0.042 ± 0.007 "7M 3.89 ± 0.54 195.3 ± 16.0 0.046 ± 0.006 "8M 4.82 ± 0.64 180.5 ± 13.7 0.067 + 0.001 10" 9M 3.13 ± 0.51 153.8 ± 12.3 0.053 ± 0.010 mean ± standard deviation (n = 4) In a positive control, the group treated with dexamethasone drug, remarkable promoter effects were observed throughout the area of the calcined region, the tuber number of the bone and the alkaline phosphatase activity, as compared to the untreated group. Cistrine and echstatin promoted calcification and the number of bone tuber at 10"7 and 10 ~ 8M, while they completely prevented calcification at 10 ~ 6M, but it was not observed that it affected the number of bone tubercle. which promote the alkaline phosphatase activity at 10 ~ 8 M. The effect results for the compounds of the formulas (III), (IV), GH 4, GH 5 and (V) are shown in Table 2.

Table 2 Do not . of tubercle area of calcified bone Treatment (mm2 / cavity) (per cavity) Not treated 2.37 ± 0.98 146.5 ± 10.9 Dexamethasone 8.00 ± 0.20 307.3 ± 34.1 Formula (III) 10"4M 0.00 ± 0.00 164.5 ± 12.0 "6M 1.82 ± 0.49 177.5 ± 11.4 "8M 3.67 ± 0.54 162.8 ± 31.0 "10M 4.39 ± 0.65 158.0 ± 28.7 Formula (IV) 10"4M 1.82 ± 0.46 182.8 ± 22.8 "6M 4.99 + 0.84 194.3 ± 8.1 "8M 5.58 ± 0.95 194.8 ± 19.3 "10M 3.13 ± 0.67 157.0 ± 17.6 GH4 10"4M 0.00 ± 0.00 152.3 ± 8.5 "6M 4.24 ± 0.55 196.0 ± 16.3 "8M 5.64 ± 0.81 184.8 ± 23.7 '10M 3.78 ± 0.47 166.0 ± 17.0 GH5 10"4M 0.00 ± 0.00 207.5 ± 18.9 "6M 4.00 ± 0.81 211.8 ± 14.5 "8M 5.88 ± 0.78 204.8 ± 22.9 10_10M 4.51 ± 0.57 155.0 ± 13.5 Formula (V) 10"4M 0.00 ± 0.00 148.8 ± 15.2 "6M 3.37 ± 0.48 182.8 ± 22.8 "8M 3.47 ± 0.75 174.8 ± 23.7 10" 10M 2.41 ± 0.99 158.2 ± 23.6 or 10"" 4M 0.00 ± 0.00 168.3 ± 17.1 10"'6M 3.74 ± 0.95 166.5 ± 10.6 mean ± standard deviation (n = 4) The compounds of the formula (IV), GH 4, GH 5 and the formula (V) promoted the calcification and the tuber number of the bone at 10"6 and 10" 8M. The compounds of the formula (III), GH 4, GH 5 and the formula (V) completely prevent calcification at 10"4M, but it was observed that they do not affect the number of bone tuber The compound of the formula (III) promotes calcification at 10 ~ 8 and 10"10M and promotes the number of bone tuber at 10 ~ 6M. The GRGDS promoted calcification at 10"6M, but completely prevented calcification at 10 ~ 4 M. The tuber tuber number of the bone was observed to show a tendency to be increased at these concentrations.Cutrin and Compound No. 43 were tested using the bone tuber formation test as described above The results are shown in Table 3.

Table 3 No. of Tuber area Calcified bone Treatment (mm2 / cavity) (per cavity) Not treated 4.46 ± 0.83 0.084 ± 0.008 Dexamethasone 10"" 8M 13.82 ± 1.20 0.143 ± 0.015 Cistrina 10 '' 5M 4.54 ± 0.88 0.085 ± 0.009 10 '"6M 7.93 ± 1.37 0.119 ± 0.008 10" "7M 11.25 ± 1.04 0.133 ± 0.006 10"" 8M 9.87 ± 1.04 0.126 ± 0.006 Comp. '. No. 43 10"" 5M .4.83 ± 0.87 0.088 ± 0.008 10"" 6M 7.31 ± 0.69 0.108 ± 0.005 10' "7M 8.45 ± 0.66 0.099 ± 0.004 10 ' "8M 6.29 ± 0.43 0.100 ± 0.003 mean ± standard deviation (n = 4) Compound No. 63 was tested using the bone tuber formation test as described above. The results are shown in Table 4.

Table 4 Calcified Treatment Area (mm2 / cavity) Not treated 2.56 ± 0.11 Dexamethasone 10"" 8M 12.17 ± 1.05 Comp. No. 63 * 10"" 5M 1.62 ± 0.55 10 '"6M 4.26 ± 2.14 10'" 7M 5.14 ± 1.29 10"~ 8M 3.84 ± 1.06 mean ± standard deviation (n = 4) Effect of the Invention An agent for promoting bone formation, which comprises as an active ingredient a peptide or polypeptide having the amino acid sequence composed of ArgGlyAsp in the molecule such as cistrin, echstatin, a peptide represented by Gly-Arg- Gly-Asp-Ser, a compound of the above formula (I) or (II), or a compound of the formula (IX), (X), (XI) or (XII), can be administered to a 5Q body human or implanted in the area near the fractured bone to effect effective prophylaxis and therapy of bone fracture.

It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, the content of the following is claimed as property

Claims (25)

1. An agent for promoting bone formation, characterized in that it comprises as an active ingredient at least some of the compound selected from the group consisting of cistrin, echstatin, a peptide represented by Gly-Arg-Gly-Asp-Ser, a compound represented by the general formula (I) wherein Ri, R2, R3, R4, R5 and Re may be the same or different and each represents a group selected from the group consisting of a hydrogen atom; an alkyl group of 1-8 carbon atoms optionally substituted with one selected from the group consisting of a hydroxy group, a carboxy group, a cycloalkyl group of 3-10 carbon atoms optionally substituted by a hydroxy group, and an aryl group of 6-12 carbon atoms optionally substituted with a hydroxy group; a cycloalkyl group of 3-10 carbon atoms optionally substituted with a hydroxy group and an aryl group of 6-12 carbon atoms optionally substituted with a hydroxy group, R7 and R8 may be the same or different and each represents a selected group from the group consisting of a hydroxy group, an alkoxy group of 1-8 carbon atoms, an alkenyloxy group of 2-12 carbon atoms, a cycloalkyloxy group of 3-10 carbon atoms and an aryloxy group of 6 12 carbon atoms, and X represents S or SO, a compound represented by the general formula (II) wherein R9, Rio, p and R12 may be the same or different and each represents a group selected from the group consisting of a hydrogen atom, an alkyl group of 1-8 carbon atoms, a cycloalkyl group of 3-10 atoms of carbon and an aryl group of 6-12 carbon atoms optionally substituted with a hydroxy group, R13, R14 and R15 can be the same or different and each represents a group selected from the group consisting of a hydroxy group, an alkoxy group of 1-8 carbon atoms, an alkenyloxy group of 2-12 carbon atoms, a cycloalkyloxy group of 3-10 carbon atoms and an aryloxy group of 6-12 carbon atoms, and X represents S or SO, a compound represented by the general formula (IX) where Rie represents -N (R20) 2, -C (= NH) -NH2, -NH-C (= NH) -NH2 or -CO-NH-C (= NH) -NH2 (in which R20 independently represents a hydrogen atom or an alkyl group of 1-4 carbon atoms optionally substituted with a phenyl group), Ri represents a $ 4 hydrogen atom or an alkyl group of 1-4 carbon atoms optionally substituted with a phenyl group, Rie represents a hydrogen atom, an alkyl group of 1-4 carbon atoms, a phenyl group optionally substituted with a methoxy group or - COR2? (in which R2? represents -OH, -NH2, -NH- (CH2) 2-phenyl, an alkoxy group of 1-3 carbon atoms, a benzyloxy group, Pro or Aoc), R19 represents an alkyl group of 1 -5 carbon atoms optionally substituted with a substituent selected from the group consisting of OH, -NH2, -CONH2, cyclohexyl, phenyl, naphthyl, indolyl or adamantyl, a methyl group substituted with -COOH and -NHCOOCH2-phenyl, a cyclohexyl group optionally substituted with a methoxy group or an aryl group of 6-10 carbon atoms optionally substituted with a methoxy group, provided that R 8 and 19 together with the carbon atoms to which they are attached can form adamantyl, naphthyl or fluorenyl , Y represents -NH-, -0- or a direct bond, a represents 1, 2, or 3, b represents 1 or 2, c represents 0 or 1 and d represents 0 or 1, a compound represented by the general formula (X ) wherein R22 represents -N (R23) 2 -C (= NH) NH2, -NH-C (= NH) -NH2 or -CO-NH-C (= NH) -NH2 (in which R23 independently represents an atom of hydrogen or an alkyl group of 1-4 carbon atoms optionally substituted with a phenyl group) and represents 2-6, a compound of the formula (XI) a compound of the formula (XII or a biologically acceptable salt thereof 2. The agent for promoting bone formation according to claim 1, characterized in that cistrine is contained as an active ingredient. 3. The agent for promoting bone formation according to claim 1, characterized in that echstatin is contained as an active ingredient. 4. The agent for promoting bone formation according to claim 1, characterized in that a peptide represented by Gly-Arg-Gly-Asp-Ser is contained as an active ingredient. 5. The agent for promoting bone formation according to claim 1, characterized in that a compound represented by the following formula (III) is contained as an active ingredient. 6. The agent for promoting bone formation according to claim 1, characterized in that a compound represented by the following formula (IV) is contained as an active ingredient. (IV) 7. The agent for promoting bone formation according to claim 1, characterized in that a compound represented by the following formula (V) is contained as an active ingredient. 8. The agent for promoting bone formation according to claim 1, characterized in that a compound represented by the following formula (VI) is contained as an active ingredient. 9. The agent for promoting bone formation according to claim 1, characterized in that a compound represented by the following formula (VII) is contained as an active ingredient. 10. The agent for promoting bone formation according to claim 1, characterized in that a compound represented by the following formula (VIII) is contained as an active ingredient. (VIII) 11. The agent for promoting bone formation according to claim 1, characterized in that a compound represented by the following formula (XIII) is contained as an active ingredient. H 12. The agent for promoting bone formation according to claim 1, characterized in that a compound represented by the following formula (XIV) is contained as an active ingredient. 13. A compound represented by the following formula (II) 6 wherein R9, Rio, Rp and R12 may be the same or different and each represents a group selected from the group consisting of a hydrogen atom, an alkyl group of 1-8 carbon atoms, a cycloalkyl group of 3-10 carbon atoms and an aryl group of 6-12 carbon atoms optionally substituted with a hydroxy group, R13, Ri4 and R15 can be the same or different and each represents a group selected from the group consisting of a hydroxy group, a group alkoxy of 1-8 carbon atoms, an alkenyloxy group of 2-12 carbon atoms, a cycloalkyloxy group of 3-10 carbon atoms and an aryloxy group of 6-12 carbon atoms, and X represents S or SO. 14. The use of a compound selected from the group consisting of cistrin, echstatin, a peptide represented by Gly-Arg-Gly-Asp-Ser and a compound represented, eg: - the general formula (I), (II), (IX), (X) or < XII > A salt of the is or biologically acceptable to make an agent to promote bone formation. fifteen . The use of cistrin to produce an agent to promote bone formation or bone formation. 16 The use of echstatin to produce an agent to promote bone formation. . The use of a peptide represented by Gly-Arg-Gly-Asp-Ser to produce an agent to promote bone formation. 18 The use of a compound represented by the formula (III) to manufacture an agent to promote bone formation. 19 The use of a compound represented by the formula (IV) to manufacture an agent to promote bone formation. twenty . The use of a compound represented by the formula (V) to make an agent to promote bone formation. twenty-one . The use of a compound represented by the formula (VI) to manufacture an agent to promote bone formation. 22 The use of a compound represented by the formula (VII) to manufacture an agent to promote bone formation.

2.

3 . The use of a compound represented by the formula (VIII) to manufacture an agent to promote bone formation. 24 The use of a compound represented by the formula (XIII) to manufacture an agent to promote bone formation. 25 The use of a compound represented by the formula (XIV) to manufacture an agent to promote bone formation.