TW200820980A - A sustained-release preparation having osteoconductive activity - Google Patents

Abstract

The present invention provides a sustained-release preparation of the osteoclasts formation control factor OCIF/OPG having excellent osteoconductive activity, which is characterized by that being a sustained-release preparation including the osteoclasts formation control factor, the analogues or the mutant thereof, or those modification bodies, and the porous particle contsisting of calcium phosphate Nano crystals and polysaccharides. Above-mentioned osteoclasts formation control factor, the analogues or the mutant or those modification bodies are combined with whole hole inside of above mentioned porous particle by mediate of two value metal ion.

Description

200820980 IX. Description of the Invention: [Technical Field] The present invention relates to the combination of the osteoclast formation inhibitor, its congeners or variants or the modifications thereof on the inner surface of the pores of the porous particles. preparation. [Prior Art] A method in which a physiologically active substance is directly embedded in a polymer by a ubiquitous preparation of a physiologically active substance such as a protein. ® In the past, there was a report that apatite using one of calcium phosphate was a release preparation (Non-Patent Documents 1 and 2). Non-Patent Document 1 reports that the antibiotic is adsorbed and released. In other respects, Non-Patent Document 2 reports that the adsorbed protein is released by dissolution of calcium depending on the size of the crystal. Further, Non-Patent Document 3 reports that human growth hormone is released by using apatite particles having a specific surface area of 3 m 2 /g to 22 m 2 /g and a particle diameter of 200 nm to 500 nm. Further, Non-Patent Document 4 reports that apatite particles having a particle diameter of 40/zm to 80/zm adsorb 1% of human growth hormone and are released. However, the use of apatite in these reports is not a product of the product. Further, W. Paul et al. have reported a large number of proteins based on calcium phosphate particles (Non-Patent Documents 5 to 8). In these reports, Xu Fanghua was coated with a biodegradable polymer such as polylactic acid (PLA) or polyvinyl acetate (PEVA). The particles used were of a size of 2 0 0 /z m~1 0 0 μ m and formed into a dense body at 1100 ° C. Further, for example, Patent Documents 1 to 2 and Non-Patent Document 9 show a reproducible preparation for the injection of a possible protein using apatite particles (excluding bulk materials) 200820980. Patent Document 1 discloses a nucleating composition characterized by inclusion of a porous active hydroxyapatite microparticle, a biologically active agent, a human serum protein, a mucopolysaccharide, and a divalent metal ion. Further, Patent Document 2 discloses a nucleating microparticle preparation of human growth hormone characterized by containing a human growth hormone and a water-soluble divalent metal compound from a porous apatite derivative and a porous apatite derivative. In Non-Patent Document 9, the porous apatite particles are impregnated with a protein such as interferon α, and zinc ions are added thereto. Patent Document 3 discloses a composite particle in which a calcium compound and a glycosaminoglycan are produced by a spray drying method and a process for producing the same. Patent Document 3 describes that the composite particles are useful as materials for bone filling materials, materials for cells, and materials for chromatography, but are not described for use in a sputum preparation. His side's physiologically active protein containing osteoclastogenesis inhibitory factor (〇ste 〇c 1 ast 〇genesis inhibitory factor: 〇CIF) (also known as osteoprotegerin: 〇PG); hereinafter referred to as "OCIF/OPG") is used in Xu The technique of putting it on is shown in Patent Document 4. The present technology relates to a releasable composition which can be used for physiologically active proteins using alginate gel beads. However, in Patent Document 4, there is no report on the optimization of the release of 0CIF/0PG, and there is no description of the example of the Xu Fang test. Further, it is also disclosed in Patent Documents 5 to 8 by a formulation technique for improving the maintenance of the blood concentration of 0CIF/0PG and the biological activity in the living body. These patent documents are not complexes of CIF / 0 PG and polysaccharides and their derivatives or 0CIF/0PG and polyPEG (copolymer of polyethylene glycol allyl methyl ether and sodium maleate) A complex of these complexes that enhances the biological activity of OCIF/OPG by improving the retention of 200820980 in the blood. However, these techniques do not point to the partial administration of 0CIF/0PG, nor do they consider the suitability for bone. Patent Document 1 JP-A-2004-75662, JP-A-2005-8545, JP-A-2004-236895, JP-A-2004-236895, Patent Document 4, International Publication No. 98/4621, No. 1 Bulletin No. 160601

Patent Document 6 US Patent Application Publication No. 2003/0045456 Patent Document No. 2005-206569 Patent Document No. US Patent Application Publication No. 2006/0062754 Non-Patent Document 1 K. Yamamura, "Journal of Biomedical Materials Research 1992, Vol. 26, p.1053-1064 Non-Patent Document 2 T. Matsumoto, "Biomaterials", 2004, Vol. 25, p. 3807-3 8 1 2 Non-Patent Document 3 H. Gautier, " Journal of Biomedical

Materials Research", 998, Vol. 40, p. 606-61 3 Non-Patent Literature 4 J. Guicheux, "Journal of Biomedical

Materials Research", 1997, Vol. 34, p. 16 5-170 Non-Patent Literature 5 W. Paul and CP. Sharma, "Journal of

Materials Science Letters, 1997, Vol. 16, pp. 2050-205 1 Non-Patent Document 6 W. Paul and CP. Sharma, "Journal of materials Science-materials in medicine", 1999, Vol. 10, p. 3 83-388 200820980 Non-Patent Document 7 W. Paul, "Journal of Biomedical Materials Research", 2002, Vol. 61, p. 660-662 Non-Patent Document 8 W. Paul and CP. Sharma, "Journal of

Biomaterials Applications, 2003, Vol. 17, p. 25 3-264 Non-Patent Document 9 Y. Mizushima, "Journal of Controlled Release", 2006, Vol. 110, No. 2, p. 260-265 [Invention In the present invention, the present invention aims to provide a OCIF/OPG preparation having excellent bone conduction energy. The results of a review of the method of formulating a composite porous particle obtained by the method of releasing the calcium phosphate nanocrystals and the polysaccharides produced by the method of the above-mentioned Patent Document 3, and the like, found that the substrate of the OCIF/OPG was placed in the composite porous. The plasmid is most suitable, and the present invention is finally completed by using divalent metal ions to achieve zero release of the initial release of control. The present invention comprises the following: (1) A releasable preparation characterized by containing OCIF/OPG, its equivalents® or variants or modifications thereof, and porous particles containing calcium phosphate nanocrystals and polysaccharides. The OCIF/OPG, its congener or variant or a modification thereof is bound to the entire inner surface of the pore of the porous particle by a secondary divalent metal ion. (2) The releasable preparation according to (1), wherein the calcium phosphate nanocrystals and the polysaccharide are uniformly dispersed in the porous particles. (3) The releasable preparation according to (1) or (2), wherein the calcium phosphate is partially substituted with calcium or a part of calcium and contains a group selected from the group consisting of magnesium, zinc, lanthanum and cerium 200820980. metal. (4) The releasable preparation according to any one of (1) to (3) wherein the molar ratio of calcium to phosphorus in the calcium phosphate is from 1.3 to 3.4. (5) The releasable preparation according to any one of (1) to (4) wherein the calcium phosphate contains a carbonate group. (6) The releasable preparation according to any one of (1) to (5), wherein the long diameter of the calcium phosphate crystal is lxl 〇 2 nm or less. (7) The releasable preparation according to (6), wherein the long-diameter is Inm~lxl02nm (8), wherein the polysaccharide is selected as described in any one of (1) to (7). (9) The sputum preparation according to (8), wherein the polysaccharide is chondroitin sulfate or hyaluronic acid. (10) The releasable preparation according to any one of (1) to (9), wherein the content of the polysaccharide is 4 x 10% by weight or less. (11) The releasable preparation according to (10), wherein the content is 4 x 10% by weight to 1 x 10% by weight. (1) The releasable preparation according to any one of (1) to (1), wherein the diameter of the porous particles is 1 x 10 〇 2 # m or less. (1) The releasable preparation according to (12), wherein the diameter is 1 A melon to 1 X 1 Ο 2 μ m 〇 (14), and the releasable preparation according to any one of (1) to (13) The specific surface area of the aforementioned porous particles is 5 x l 〇 m 2 /g 2 2 x 10 2 m 2 /g. The porous preparation according to any one of (1) to (14), wherein the porous particles have a porosity of from 3 x 10% to 8 x 10%. (16) The releasable preparation according to any one of (1) to (15), wherein the in vivo absorption of the porous particles is within 6 months. (17) The releasable preparation according to any one of (1) to (16), wherein the porous particles are produced by spray drying. The above-mentioned divalent metal ion is a group selected from the group consisting of a zinc ion, a magnesium ion, a calcium ion, a strontium ion, a strontium ion, and a copper ion, in the releasable preparation according to any one of (1) to (17). (19) The releasable preparation according to (18), wherein the divalent metal ion is a zinc ion. (20) The sputum preparation according to any one of (1) to (19), which is a therapeutic agent for bone metabolism disorders. (21) The prolonged preparation according to any one of (1) to (19), which is a fracture healing accelerator. (22) A bone-filling material containing a sputum material according to any one of (1) to (19). (23) A method for preparing a sputum release preparation, which comprises the steps of: spray-drying a suspension containing calcium phosphate nanocrystals and polysaccharides to obtain porous particles containing calcium phosphate nanocrystals and polysaccharides, The porous particles are impregnated with a solution containing OCIF/OPG, its congeners or variants or modifications thereof, and OCIF/OPG, its congeners or variants or modifications thereof are carried on the aforementioned porous For the entire inner surface of the pores of the plasmid, the above-mentioned OCIF/OPG, its congener or variant or a modification thereof is loaded with a solution containing a divalent metal ion in a porous particle of -10-200820980. The above-mentioned OCIF/OPG, its congeners or variants or modifications thereof and the entire inner surface of the pores of the porous particles are combined with the above-mentioned divalent metal ions. The present specification contains the priority of Japan in this case. The contents of the specification and/or drawing of the patent application No. 2006-271102. [Embodiment] BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below. The release preparation of the present invention is a porous particle containing OCIF/OPG, a congener or variant thereof or a modification thereof, a calcium phosphate nanocrystal and a polysaccharide. In the prolonged preparation of the present invention, the OCIF/OPG, its congener or variant or a modification thereof is carried on the entire inner surface of the pore of the porous particle. Further, the prolonged preparation of the present invention is a secondary divalent metal ion and OCIF/OPG, its congener or variant or a modification thereof is combined with the entire inner surface of the pore of the porous particle. ® OCIF/OPG is a protein which inhibits the differentiation/maturation of osteoclasts by using osteoclastogenesis inhibitory activity as an indicator (International Publication No. 96/262, and U.S. Patent No. 6,855,808). Specifically, OCIF/OPG is a protein having the physicochemical properties described in the following (a) to (d) and having activity for inhibiting differentiation and/or maturation of osteoclasts (International Publication No. 96/26217 and USA) Patent No. 6,855,808) 〇

(a) Molecular weight (by SDS-PAGE): about 60 kD (under reducing conditions), about 60 kD -11 - 200820980, and about 120 kD (under non-reducing conditions). (b) Affinity: affinity for cation exchangers and heparin. (c) Thermal stability: heat treatment at 70 ° C, 10 minutes or 56 ° C, 30 minutes, and the differentiation and maturation inhibitory activity of osteoclasts is reduced, and the bone is broken by heat treatment at 90 ° C for 10 minutes. Differentiation of cells ♦ The maturation inhibitory activity disappears. (d) Internal amino acid sequence: The amino acid sequence having the sequence numbers 1 to 3 is listed as an internal amino acid sequence. In the amino acid sequence described in SEQ ID NOs: 1 to 3, Xaa may take any amine #基酸, preferably 1 in the sequence number 1 &&&&> is 1'111:, Xaa in the sequence number 2 is 1 Arg, Xaa of 5 is Ser, Xaa of 13 is Leu, and Xaa of 1 of SEQ ID NO: 3 is Thr. From the physicochemical properties of the above (a), OCIF/OPG is in the form of a monomer and a dimer (International Publication No. 96/26217 and U.S. Patent No. 6,855,808). The monomer and the dimer are usually mixed in a culture solution containing a cell expressing OCIF/OPG (for example, a human fetal lung line bud cell IMR-90 (ATCC registered-registered number CCL1 8 6)). Any form of OCIF/OPG can be refined by the usual method of separation, and used in the present invention. Human OCIF/OPG was synthesized with the precursor of the information peptide (cDNA: SEQ ID NO: 4 and amino acid sequence: SEQ ID NO: 5), and the information peptide was cleaved into a mature protein (amino acid sequence shown in SEQ ID NO: 5). In the present invention, the OCIF/OPG is a precursor and a mature type including the above-mentioned human-derived information peptide. Any of the proteins, only mature proteins. -12- 200820980 〇CIF/〇PG congener is encoded by DNA hybridized with DNA encoding the base sequence complementary to OCIF/OPG, and has osteoclast differentiation and/or maturation inhibition. Active protein. For example, a cDNA library derived from animal cells, body fluids or tissues is used as a template, and a DNA obtained by hybridizing a base sequence complementary to human OCIF/OPG cDNA (SEQ ID NO: 4) is used as a probe to encode DNA hybridized under severe conditions. a protein having osteoclast differentiation and/or maturation inhibitory activity. Severe conditions refer to the conditions under which the so-called special hybrids are formed. Such harsh conditions are well known to the practitioner and can be expressed, for example, in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1 9 8 9), 6.3.1-6.3.6. Suitable non-limiting conditions for harsh hybridization conditions can be washed in 6 X sodium chloride/sodium citrate (SSC) at about 45 ° C, and washed at 0.2 ° SSC, 0.1% SDS at 50 ° C ~ 65 ° C. 1 or more. The specific 0CIF/0PG congener can be, for example, CIF2 (cDNA: SEQ ID NO: 6 and amino acid sequence: SEQ ID NO: 7), 〇CIF3, as shown in International Publication No. 96/26217 and U.S. Patent No. 6,85 5,808. (cDNA: SEQ ID NO: 8 and amino acid sequence: SEQ ID NO: 9), 〇CIF4 (cDNA: SEQ ID NO: 10 and amino acid sequence: SEQ ID NO: 11) and 〇CIF5 (cDNA: SEQ ID NO: 12 and amino acid sequence: Sequence number 13). The amino acid sequence of the information peptide in 0CIF2 to 5 and the amino acid sequence of the mature protein are shown in Table 1 below. -13- 200820980 Table 1 OCIF/OPG base sequence amino acid sequence information peptide mature protein human 〇 CIF / 〇 PG SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 5 - 21 - 1 - amino acid sequence SEQ ID NO: 5 in the 1st to the 380th amino acid sequence 0CIF2 SEQ ID NO: 6 SEQ ID NO: 7 SEQ ID NO: 7 - 21 - 1 -1 amino acid sequence number 7 in the middle 1 to 373 amino acid Sequence 0CIF3 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 9 to 1-1 amino acid sequence SEQ ID NO: 9 in the first to the 341th amino acid sequence 0CIF4 SEQ ID NO: 10 SEQ ID NO: 11 21 to 1 amino acid sequence number 11 in the first to 133th amino acid sequence 0CIF5 SEQ ID NO: 12 SEQ ID NO: 13 SEQ ID NO: 13 in the 21st to the -1 amino acid sequence number 13 The amino acid sequence of the 1st to the 124th OCIF/OPG variant is an amine group substituted, deleted, inserted and/or added to the above OCIF/OPG or OCIF/OPG congener, 1 or a plurality of amino acids. a protein sequence that has osteoclast differentiation and/or maturation inhibitory activityThe plurality of amino acids may be plural (for example, 1 to 10, preferably 1 to 5, particularly preferably 1 to 3). In human OCIF/OPG (amino acid sequence of SEQ ID NO: 5), osteoclast differentiation and/or maturation inhibitory activity was also confirmed in C-terminal deletion variants in which C-terminal to 204 amino acid was deleted, and biological activity was mainly Due to the field of the N-terminal side (International Publication No. 9 6 / 2 6 2 1 7 , U.S. Patent No. 6, 8 5 5, 8 8 and Yamaguchi K. et al., The Journal of Biological Chemistry (1 998), Vol. 273, No. 9, pp. 5117-5123). A suitable aspect of the OCIF/OPG variant may be the substitution, deletion, insertion and/or replacement of several amino acids in human 〇CIF/〇P(} (Ordinary J! J No. 5 amino acid sequence). The amino acid sequence is added, and the protein having osteoclast differentiation -14-200820980 and/or maturation inhibitory activity, and the first Glu of human OCIF/OPG (amino acid sequence of SEQ ID NO: 5) Any protein having an N-terminal, any amino acid of 176 to 379 which is a C-terminal protein and having osteoclast differentiation and/or maturation inhibitory activity. Furthermore, the 0CIF/0PG variant includes the above-mentioned 0CIF/0PG. Or the nucleotide sequence of the CIF/〇PG congener encoded by at least about 60% to about 65%, preferably from about 70% to about 75%, more preferably from about 80% to about 85%, particularly preferably about 90%. ~ about 95% of the same nucleotide sequence encoded by the same nucleotide sequence and having osteoclast fractionation and/or maturation inhibitory activity. The OCIF/OPG modification is, for example, to the above 0CIF/0PG, its analog or variant. Enzymes or chemical modifications of peptides and sugar chains, or polymer compounds such as polymers or modified polysaccharides. OCIF/OPG modification These may be, for example, post-translational modifications of proteins (eg, addition of sugars, phosphorylation by protein kinases, and dephosphorylation by phosphatases), water-soluble polymers (polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxylic acids). OCIF/OPG modified by chemical modification such as methyl cellulose, polyvinyl alcohol, polyPEG (copolymer of polyethylene glycol allyl methyl ether and sodium maleate), heparin®, polydextrose sulfate, etc. And its congeners or variants, including, for example, OCIF/OPG, its congeners or variants, and Fc (Fc domain derived from immunoglobulin such as IgG) and protein (albumin), or fusion proteins with proteins, 〇CIF/〇PG, a complex of its analogs or variants with polysaccharides (heparin, polydextrose sulphate, etc.), and a complex of OCIF/OPG, its congeners or variants and polyPEG are also included in OCIF/OPG Specifically, the OCIF/OPG, its congener or variant of the modification of the polyethylene di-15-200820980 alcohol can be produced according to the method described in, for example, International Publication No. 97/236 14. Further OCIF/OPG Repair of its congeners or variants from polysaccharides In the body, the polysaccharide may be polyglucose sulphate, particularly sodium polyglucose sulfate 5 (DS5), and the modified body may be produced according to the methods described in, for example, Patent Documents 5 and 6. Further, OCIF/OPG, its congener or The modified form of the polyPEG can be produced by the methods described in, for example, Patent Documents 7 and 8. Animals other than humans (for example, rats, mice, rabbits, dogs, cats, cows, pigs, sheep, goats, etc.) OCIF/OPG from ®, and birds such as chickens, geese, turkeys, etc., as long as they are within the above definition, are included in _OCIF/OPG congeners, variants or modifications. OCIF/OPG, its congeners or variants or their modifications (hereinafter referred to as "OCIF/OPG substances") are natural extracts and refined from proteins such as animal tissues or body fluids, cultures of animal cells, etc. a type of protein, or a DNA fragment encoded by these OCIF/OPG substances or a gene-recombinant type (hereinafter sometimes referred to as "rh") protein produced by transforming a host such as an animal cell or a coliform with a vector containing the DNA fragment. These modifications can be obtained. For example, the method of OCIF/OPG, which is shown in the International Publication No. 96/26217 (and U.S. Patent No. 6,855,808), separates and refines 〇CIF/〇PG substances from OCIF/OPG substance-producing cells. Specifically, OCIF/OPG substance-producing cells were cultured, and the culture solution was applied to a heparin column (heparin-sepharose CL-6B, Pharmacia), and dissolved in 10 mM Tris-HCl buffer (pH 7.5) containing 2 M sodium chloride. . The obtained heparin-adsorbing OCIF/OPG substance was fractionated for Q · anion exchange column (HiLoad-Q/FF, Pharmacia), and its non-adsorbed fraction was collected. Thus, the adsorption of heparin can be alkaline -16-200820980 OCIF/OPG substance. The obtained OCIF/OPG substance activity is divided into S • cation exchange column (HiLoad-S/HP, Pharmacia), heparin column (heparin-5PW, Tosoh Corporation), Ciba colonization blue column (blue-5PW, east) Cao Company), reverse phase column (BU-300 C4, Bajin Aima Company), can separate and refine OCIF/OPG substances. Osteoclast differentiation and/or maturation inhibition of OCIF/OPG substances, such as methods such as Kumikawa (protein, nucleic acid, enzyme, 1 989, Vol. 34, ρ·999) and Takahashi N. (Endocrinology) , ® 1 9 8 8, Vol. 122, p. 1 37 3) Determination. That is, for example, using the bone marrow cells of the mouse which is about π days after birth, the inhibition of the formation of osteoclasts in the presence of active vitamin D3 (Calcitriol) can be inhibited as the inhibition of the activity of tartaric acid-resistant acid phosphatase. . Among the OCIF/OPG substances, OCIF/OPG or a modified form thereof is preferred, and 〇CIF/〇PG or its Fc fusion protein is more preferred. In other respects, the calcium phosphate nanocrystal is a general term for crystals of tricalcium phosphate (Ca3(P〇4)2), octacalcium phosphate (Ca8H2(PO〇6,5H2〇), apatite compound, etc., which are small in size. The apatite compound is represented by the formula CaMPChXhY: (wherein, X is a carbonic acid group or a defect, and Y is a hydroxyl group, a carbonic acid group, a halogen group or a defect), hydroxyapatite, apatite, fluoroapatite, Chlorophosphate, etc. Calcium phosphate may be substituted with calcium or partially substituted calcium containing one or more kinds of divalent metals such as magnesium, zinc, silver and strontium. The molar ratio of calcium to phosphorus in calcium phosphate is, for example, 1.3 to 3.4. It is preferably 1.5 to 3.0. For the production of calcium phosphate nanocrystals, for example, when hydroxyapatite nanocrystals are produced, they can be produced by a wet method and can be carried out according to the following formula (H. Aoki, " -17- 200820980

Medical Applications of hydroxyapatite", 19 94, Ishiyaku

EuroAmerica, Inc.,Tokyo, St.Lousi)0 lOCa (OH) 2+6H3 (P04) 3^Ca10 (P04) 6 (OH) 2 The long diameter of the calcium phosphate nanocrystal thus produced is, for example, 1 x 1 02 nm The following, should be Inm ~ lxl02nm, especially YixlOnm ~ 5xl0nm. Further, the polysaccharide in the present invention may be, for example, chondroitin sulfate, hyaluronic acid, heparin, and heparin sulfate, and chondroitin sulfate and hyaluronic acid are preferred. The polysaccharide content in the releasable preparation of the present invention is, for example, 4 x 10% by weight or less, preferably 4 x 1 〇 _ 重量% to 1 x 10 · 1% by weight, particularly preferably 1% by weight to 2 x 10% by weight. When the chondroitin sulfate is used as the polysaccharide, for example, a molecular weight of l〇k Da to 40 kDa, preferably 15 kDa to 35 kDa is used. The releasable preparation of the present invention is produced from the above-described OCIF/OPG substance, calcium phosphate nanocrystal, and polysaccharide. In the method for producing a sputum release preparation of the present invention (hereinafter referred to as "the present method"), a suspension containing calcium phosphate nanocrystals and a polysaccharide is spray-dried to produce a porous body containing the calcium phosphate crystals and polysaccharides. Plasmid. Among the porous particles, it is preferred to uniformly disperse the calcium phosphate crystal and the polysaccharide. The amount of the polysaccharide of the calcium phosphate nanocrystals may be, for example, 1% by weight to 2 x 10% by weight, preferably 2% by weight to 1% by weight, based on the crystals of the calcium phosphate nanocrystals. Further, the pH of the suspension is adjusted to, for example, 5 to 9, preferably 6 to 8. The suspension containing the calcium phosphate nanocrystals and the polysaccharide is promoted to be composited while continuing to stir, for example, after 0 hours to 12 hours (preferably 1 hour to 4 hours), and then spray-dried. Here, the ripening means that the final pH is kept constant in the range of 7.5 to 8.0, and the surface state of the crystal obtained by the combination is maintained at the same pH as that of the living body of almost -18-200820980 to stabilize. Specifically, the ripening (strong calcium hydroxide) and the strong acid (phosphoric acid) are neutralized to set the final pH to carry out the ripening. Spray drying can be carried out according to the method of using a commercially available device such as Buchi Co., Ltd., Daiwa Scientific Co., Ltd., Okawara Kogyo Co., Ltd., and a four-fluid nozzle. To increase the surface area, the suspension is made to a degree of 1 // m to 5 x 10 2 gm. The fine droplets are sprayed and dried in a hot air of lxl02 ° C to 3 x 10 ° ° C. The diameter of the porous particles obtained after the spray drying is, for example, 1 X 1 02 // m _ or less, preferably 1//m to 1×10 2 /m, and particularly preferably 1//m to 3 x 10 //m. Further, the porous particles are, for example, a specific surface area of 5 x 10 m 2 /g to 2 x 10 2 m 2 /g, and a porosity of 3 x 10% to 8 x 10%. When the specific surface area of the porous particles is smaller than 5x10 m2/g, a sufficient amount of OCIF/OPG substance cannot be carried, and it is not possible to maintain the release during the target release period. On the other hand, if the porosity of the porous particles is less than 3x10%, the OCIF/OPG substance cannot be uniformly supported inside the particles, and if it exceeds 8x10%, the porous particles are destroyed during the formulation. The in vivo absorption of the obtained porous particles after spray drying is, for example, 6 months or less, preferably 1/2 month to 3 months, particularly preferably 1 month to 2 months. After being spray-dried according to the method, the obtained porous particles are impregnated with a solution containing an OCIF/OPG substance (for example, an aqueous solution containing an OCIF/OPG substance) to be uniformly dispersed, and the inside of the pores of the porous particles are carried. Hold OCIF/OPG substances. The amount of the OCIF/OPG substance added is, for example, a porous particle of lxlO·1/^ to 2xl02/zg per lmg, preferably l#g to lxl〇2/zg. The mixing of the porous particles with the OCIF/OPG substance can be carried out, for example, by stirring for 1 hour to 12 hours (preferably 3 hours to 6 hours). -19- 200820980 After the end of the mixing, the mixture containing the porous particles and the 0CIF/〇PG substance was centrifuged to remove the supernatant. At this stage, it is also possible to carry out the combination treatment with divalent metal ions without centrifugation. The evaluation of the OCIF/OPG substance carried on the inner surface of the pores of the porous particle can be carried out by centrifuging the supernatant, and the supernatant is used for immunological manipulation of the OCIF/OPG substance by a specific antibody (for example, ELISA), and the residue remains in the supernatant. The OCIF/OPG quality of the liquid is applied. From the evaluation, it was confirmed that only a small amount of the OCIF/OPG substance remained in the supernatant, and it was judged that the OCIF/OPG substance was sufficiently carried in the pores of the porous particles. The mixture containing the divalent metal ions is added to the mixture containing the porous particles supported by the obtained OCIF/OPG substance, and subjected to a combination treatment. From this work, the combination of the OCIF/OPG substance and the porous particles supported by this is carried out. In the present invention, the combination includes carrying and binding the surface of the calcium phosphate and the OCIF/OPG substance by the coordination, ion, and compatibility of the intermetallic metal ions, or the combination of the functional groups of the polysaccharide and the OCIF/OPG substance. , or the combination of calcium phosphate surface and OCIF/OPG substances and polysaccharides. According to this combination, the OCIF/OPG substance is combined with the entire inner surface of the pores of the porous particles to form a divalent metal ion. The divalent metal ions used in combination may be zinc ions, magnesium ions, calcium ions, strontium ions, strontium ions and copper ions, but zinc ions are most suitable. For the porous particles carrying the OCIF/OPG substance, the amount of the divalent metal ion is, for example, a porous particle of 0CIF/QPG substance per img of ixi〇-3 mg~img, preferably 1 X 1 0 ·2 mg 〜1 X 1 (Γ1 mg 〇-20- 200820980 The binding treatment is to add a solution containing a divalent metal ion to a mixture containing porous particles carrying the OCIF/OPG substance, and then stir it upside down. The mixture is centrifuged to remove the supernatant, and the resulting residue is washed with, for example, purified water, and the precipitate is subjected to freeze-drying or vacuuming for, for example, 1 hour to 24 hours (preferably 1 hour to 12 hours). Drying. The Xu-release preparation of the present invention can be used for the long-term release of the 0CIF/0PG substance carried by the present invention for at least 7 days. The plasmid has the same composition as the natural bone, and the prolonged preparation of the present invention is caused by the bone itself having bone conduction energy. Here, the bone conduction energy refers to the fact that the bone formation is not hindered at the bone defect site, and the bone formation is promoted. Bone by bone bud cells The bone conduction energy of the Xu-release preparation of the present invention is, for example, after applying the Xu-release preparation of the present invention to the defect of the tibia, it can be dyed according to Haematoxylin & Eosii UHE, toluidine cyanine staining, alkali phosphatidase (ALP). It was evaluated by staining, tartaric acid-resistant acid phosphatase (TRAP) staining, bone morphometry such as bone morphology measurement by Vi 11 aneuva-G ο 1 dner staining. Further, the present invention has the effect of inhibiting the reduction of bone mass by carrying the OCIF/OPG substance. Therefore, the prolonged preparation of the present invention can be used alone or in combination with a pharmaceutically acceptable additive. The disease to be treated by the releasable preparation of the present invention may be, for example, a bone metabolic abnormality. That is, the Xu-release preparation of the present invention can be used as a therapeutic agent for abnormal bone metabolism. The bone metabolic disorder is a disease characterized by a decrease in the amount of substantial bone, which is intended to inhibit the bone resorption or the rate of bone resorption. Bone Metabolism Treated or Prevented by the Xu-Release Formulation of the Present Invention - 200820980 Symptoms include disposable osteoporosis (elderly osteoporosis, postmenopausal osteoporosis and idiopathic adolescent osteoporosis), Endocrine osteoporosis (hyperthyroidism, hyperthyroidism, Cushing's syndrome, and terminal hypertrophy), osteoporosis associated with decreased sexual function (pituitary hypofunction, Klinefelter syndrome, and Turner syndrome), hereditary And congenital osteoporosis (bone dysplasia, hypercysteinemia, Menhes disease 'Ruey-Day syndrome and adolescent Pagett disease and hyperphosphatemia caused by OCIF gene defects), by gravity load Reduced or four-limb fixed and immobile bone reduction, bone Pagett disease, osteomyelitis, infectious disease nests caused by bone loss, high calcium caused by solid tumors (breast cancer, lung cancer, renal pelvis cancer, prostate cancer, etc.) Hemorrhagic, hematological malignancies (multiple myeloma, lymphadenopathy, and leukemia), idiopathic hypercalcemia, hyperthyroidism Or hypercalcemia associated with renal failure, osteopenia caused by steroid administration, due to administration of other drugs (Methotrexate and cyclosporine A and other immunosuppressive agents, Leuplin and other anti-testis Osteopenia with ninth agent, heparin and anti-epileptic drugs, osteopenia associated with renal failure®, accompanying surgery, internal organ disease (small bowel disorder, large bowel disorder, chronic hepatitis, gastrectomy, primary disease) Bone reduction in biliary cirrhosis and cirrhosis, various rheumatoid bone reductions such as joint rheumatism, various rheumatoid bone destruction such as limb-deficient joint rheumatism, joint destruction, deformed joint disease, periodontal disease Bone loss, bone metastasis (osteolytic metastasis) of cancer, relaxation of artificial joints (osteolysis around artificial joints), calcification of arteries, osteopenia associated with infections such as periodontal disease, and traumatic Injury, Gaucher disease, sickle red blood cell anemia, systemic erythema 22 - 200820980 Lumbar or non-traumatic traumatic osteonecrosis or bone cell death, kidney disease and other bone nutrition Lower fatty acid alkali hyperamylasemia dish, with a sugar thrombocytopenia, thrombocytopenia accompanying nutritional disorder in the bone, or disorders of reduced feeding psychosis. Further, the abnormality of bone metabolism in the present invention also includes bone metastasis (osteolytic transfer) of a tumor or cancer, or hematological dyscrasia (refer to Japanese Laid-Open Patent Publication No. 2000-1780200). The prolonged preparation of the present invention is used for embedding in the affected part, bone loss of the periodontal bone, bone metastasis of the cancer (osteolytic transfer), artificial® relaxation (osteogenesis around the artificial joint), calcification of the artery, and complication A disease such as osteopenia of an infectious disease, a bone necrosis accompanied by traumatic or non-injured bone necrosis or bone cell death, etc., can also be expected to have an effect on a systemic disease. Further, due to the characteristics and effects of the above-described Xu-release preparation of the present invention, the release preparation is particularly suitable for use in a fracture healing accelerator. The releasable preparation of the present invention can be formulated into various dosage forms for oral or systemic or topical administration. The sustained release preparation of the present invention is orally administered into tablets, capsules, granules, powders, nine doses, internal suspensions, emulsions, syrups, etc., or reconstituted at the time of use. When the preparation of the present invention is administered orally, the intra-injection preparation (including drip), the intramuscular injection, the intraperitoneal injection, the sitting agent, etc., and the dosage of the preparation for injection are multi-injected in units. The status of the container is provided. These various preparations can be specially adapted to the joints, binders, humectants, disintegrating agents, lubricants, bones of interfacial osteogenesis, and other bone-shaped solid diseases. The pine is negatively affected by periodontal trauma. However, in the present invention, when the invention is not oral, it can be prepared into a water-based agent, dried to form a venous injection, a skin-amplifier or a bulking agent, 1. a dispersing agent -23-200820980, a buffering agent, a preservative, Suitable preparations such as dissolution aids, preservatives, flavoring and deodorizing agents, painless agents, stabilizers, isotonic agents, etc. can be produced by conventional methods. The administration route of the releasable preparation of the present invention can be appropriately determined depending on the use, for example, oral, intraperitoneal, intravenous, intraarterial, transdermal, subcutaneous, intramuscular, and the like. The investment route can be directly administered near the affected part. It is preferable to directly implant in the affected part of the bone or in the vicinity of the affected part. The administration amount of the Xu-release preparation of the present invention can be appropriately determined depending on the age, gender, symptoms, administration route, number of administrations, and the like of the subject. For example, the effective amount of the OCIF/OPG substance may be selected from the dose of 1 kg per body weight in the range of lxl0_2 mg to lxl02 mg, preferably from the number of times per day to the frequency of one month. When it is directly administered by embedding or the like in the vicinity of the affected part or the affected part, as an effective amount of the OCIF/OPG substance to be contained, it is possible to select a dose of lxl (T2mg to lxl02mg per 1 part of the administration site) per More preferably, the frequency of the first release is preferably as described above. The porous preparation of the present invention has the same composition as the natural bone and has bone conduction energy, and is inhibited by carrying the OCIF/OPG substance. The effect of reducing the amount of bone mass can be used as a main component of the bone defect filling material. The bone defect filling material can contain, for example, a biodegradable or livingly soluble polymer, which is high in addition to the releasable preparation of the present invention. Molecules (polylactic acid, lactic acid·glycolic acid copolymer, lactic acid-caprolactone copolymer, polyanhydride, poly-o-ester, poly-ε-caprolactone, polyacrylic acid acryl cyanide, polyalkanoic acid ester, polyphosphate, Polyamino acid, methyl propyl acid copolymer, polyethyl alcohol, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose acetate, hydroxy-24- 200820980 ethyl cellulose, carboxymethyl Cellulose, methylcellulose, polyvinylpyrrolidone, polyethyl Alkenyl diol, poly N-alkyl acrylamide, etc., natural polymer (gelatin, collagen, polydextrose, starch, sodium alginate, agar, trisaccharide, protein, carrageenan, pectin , Dixian gum, gelluin gum, casein, chitin, blood fibrinogen, etc., and porous embedding (hydroxyapatite, tricalcium phosphate, apatite/collagen complex, etc.). Further, the content of the Xu-release preparation of the present invention in the bone-defective filling material can be appropriately determined according to the description of the effective amount of the OCIF/OPG substance contained in the above-mentioned Xu-release preparation of the present invention, as described above, according to the present invention. Sexual preparations (for example, therapeutic agents for bone metabolism disorders, fracture healing enhancers) or bone defects filled with sputum, γ. Since OCIF/OPG substances are placed in a stable period for a certain period of time, the reduction of bone mass can be stably suppressed. In the invention, the porous plasmid used as a carrier contains calcium phosphate and a polysaccharide of a main component of bone, and activates bone blast cells to grow crystals of apatite with a carrier as a core, and is a main component. a polysaccharide having an outer matrix of bone cells, Internal cartilage ossification can be promoted to promote bone formation. Thus, according to the present invention, inhibition of bone resorption inhibited by osteoclast differentiation of OCIF/OPG substance and bone formation with calcium phosphate nanocrystals as core can be simultaneously obtained. The two effects of the promoting action are expected to be excellent in the reduction of the bone mass or the effect of the fracture healing. The Xu-release preparation of the present invention is different from the Xu-like composition described in Patent Document 1, and does not contain human serum protein, and is not required. The mucopolysaccharide or the like is filled in the pores of the particles. The present invention will be described in detail below by way of examples, but the technical scope of the present invention is not limited to those of the examples. [Example 1] Hydroxyapatite/chondral sulfate Synthesis of a composite (hereinafter referred to as "HAp/ChS") porous particle (corresponding to the porous particle of the present invention) and measurement of the particle diameter of the particle by 0.25 of the dispersion of chondroitin sulfate (hereinafter referred to as "ChS"; molecular weight 15 kDa) A mol/l calcium hydroxide suspension was dropped with a 0.15 mol/l phosphoric acid aqueous solution to adjust the final pH to 7.5 to 8.0 to prepare a HAp/ChS gel. The amount of ChS added is preferably 2, 5, 10, 20% by weight of the obtained hydroxyapatite (hereinafter referred to as "Η Ap"). After 12 hours of aging, HAp/ChS porous particles were obtained by a spray dryer (trade name: Mini Spray Dryer, manufactured by Buchi). The obtained HAp/ChS porous and the plasmid were subjected to particle size distribution measurement. As a result, the HAp/ChS porous particles were distributed in a range of 1/m to 20/zm, and the average particle diameter was 4 /z m. [Example 2] Production of OCIF/OPG-loaded HAp/ChS porous particles (corresponding to the preparation of the present invention) OCIF/OPG The recombinant OCIF/OPG-Fc expressed as Sf21 cells (hereinafter referred to as "rh" -〇CIF-Fc"; a fusion protein of the full-length human OCIF protein and the Fc domain of human IgG, manufactured by R&D Corporation). The full-length human 0CIF protein is a mature protein having an amino acid sequence other than the information peptide (the -1 1 to -1 amino acid sequence) in the amino acid sequence of SEQ ID NO: 5. The rh-〇CIF-Fc was dissolved in 5 ml of 10% PBS to 5 #g/m. The HAp/ChS (2% by weight) porous particles prepared in Example 1 were added to 250 mg, respectively, -26-200820980 (〇.1) //运/11112;仏-〇(:1?邛(:/1^?/(:1^ porous particles). The mixture was stirred at room temperature for 4 hours. After stirring for 4 hours, The combined treatment, adding 3 ml of an aqueous solution of zinc chloride (10 mg/ml, pH 5.5), and stirring, was carried out. According to the combination treatment, all the intermediate zinc ions of the inner surface of the pores of the rh-〇CIF-Fc and the porous particles were treated. After 2 hours of stirring, the mixture was centrifuged (3500 rpm, 10 minutes) to separate the extracted supernatant, and the residue was washed with purified water, and then the precipitate was freeze-dried for one night®. Thus, the OCIF/OPG carrier was produced. The HAp/ChS porous plasmid was prepared in the same manner without the combination of the processors. The rh-OCIF-Fc remaining in the supernatant after the above centrifugation was subjected to ELISA (Human Os teoprotegerin ELISA, Bio vendor Laboratory Medicine, Inc. Quantitatively, it was found that 99% of rh-〇CIF-Fc was adsorbed on HAp/ChS porous particles in any of the lines. [Example 3] Zinc ion Combination treatment effect - Xu radioactivity 1 The 0CIF/0PG prepared in Example 2 was loaded with HAp/ChS porous particles, and 10 mg was dispersed in 5 ml of PBS, and the mixture was stirred at 37 ° C. The test was released for 1 hour. It was carried out in the period of ～7. The HAp/ChS (5 wt%) porous plasmid prepared in Example 1 was loaded with rh-〇CIF-Fc, and the 0CIF/0PG-loaded HAp produced by the combination treatment of zinc ions was carried out. The release test was also carried out in the same manner as in Example 2. The sample was stirred and centrifuged in the same manner as in Example 2, and the amount of the rh-〇CIF-Fc of the obtained supernatant was measured by ELISA. The results are shown in Fig. 1. The figure shows that the rh-〇CIF-Fc of the HAp/ChS porous particle, -27-2720980, is carried by each of the 0CIF/0PG. In the first figure, the abbreviations are as follows. "HAp": hydroxyapatite only , "HAp/ChS2%": 〇CIF/〇PG carries HAp/ChS (2% by weight) porous particles (no binding treatment), "HAp/ChS2% + Zn": 〇CIF/OPG carries HAp/ChS (2% by weight) porous particles (with binding treatment), "HAp/ChS5% + Zn": OCIF/OPG carrying H Ap/ChS (5 wt%) porous particles (with binding treatment) ® As shown in Figure 1, the HAp/ChS porous plasmid is carried by 〇ciF/OPG in combination with the unbound (no binding treatment) OCIF/OPG-loaded HAp/ChS porous particles and zinc ions. Although the release of the linear rh-〇CIF-Fc was observed, the addition of zinc ions significantly controlled the amount of release. [Example 4] The release behavior of the amount of HAp/ChS porous particles supported by 0CIF/0PG-relaxation 2 The 0CIF/0PG prepared in Example 2 was loaded with HAp/ChS porous particles in an amount of 10 mg or 20 mg. The method of Example 3 was used to review the release property. Result 0 is shown in Figure 2. Fig. 2 is a graph showing the passage of rh-〇CIF-Fc from 10 mg or 20 mg of each OCIF/OPG-loaded HAp/ChS-poly-L-particle. In the second figure, the abbreviations are as follows. "HAp/ChS2% (10mg)": l〇mg of OCIF/OPG carries HAp/ChS (2% by weight) porous particles (no binding treatment), "HAp/ChS2% (20mg)": 20mg OCIF/ OPG carries HAp/ChS (2% by weight) porous particles (no binding treatment), -28- 200820980 r HAp/ChS2% + Zn (10 mg)": 10 mg of OCIF/OPG carries HAp/ChS (2% by weight) Porous plasmid (with binding treatment), "HAp/ChS2% + Zn (20 mg)": 2 mg of OCIF/OPG carried HAp/ChS (2% by weight) porous particles (with binding treatment). As shown in Fig. 2, the HAp/ChS porous particles were supported by OCIF/OPG in 5 ml of PBS to be linear. When HAp/ChS (2% by weight) porous particles were not bound to OCIF/OPG, it was 2.5 ng/ml (weight 10 mg) and 5.〇1^/!111 (weight 2〇1112) after 7 days. On the other hand, when OCIF/OPG treated with zinc ions was used to carry HAp/ChS (2% by weight) porous particles, it was 0.7 ng / m 1 (weight 10 mg) and 1.4 ng / m 1 after 7 days. 2 0 mg), which was found to be positively correlated with the amount of added particles. [Example 5] The OCIF/OPG using the OCIF/OPG which produces different cells and amino acid sequences differs. The OCIF/OPG is a commercially available rh-〇CIF-Fc (expressed as Sf21 cells) of Example 1 and an international publication. The method of No. 96/26217 and U.S. Patent No. 6,855,808 refines rh-OCIF by CH0 cell expression. The rh-OCIF protein expressed by CHO cells is a mature protein obtained from the amino acid sequence except the information peptide (the amino acid sequence of the 21st to the -1) in the amino acid sequence of SEQ ID NO: 5. . Rh-〇CIF-Fc and rh-〇CIF were dispersed in 10 ml of 10% PBS at 10 g/ml, respectively, and 125 mg of HAp/ChS (2 wt%) porous particles prepared in Example 1 were added separately (0.4/ Zg/lmg; rh-〇CIF-Fc or rh-〇CIF / HAp/ChS porous particles). The mixture was stirred at room temperature for 4 hours and stirred. -29- 200820980 After stirring for 4 hours, the combination treatment was carried out, and 3 ml of an aqueous zinc chloride solution (10 mg/ml, pH 5.5) was added thereto, and the mixture was further stirred and stirred. According to this combination treatment, rh-〇CIF-Fc or rh-OCIF binds to the inner surface of the pores of the porous particles. After stirring for 2 hours, the supernatant was separated by centrifugation (3500 rpm, 10 minutes), washed with purified water, and the precipitate was freeze-dried overnight. Thus, 0CIF/0PG, which produces a different cell and amino acid sequence, is loaded with ΗΑρ/ChS porous particles. In the same manner, the 0CIF/0PG-produced HAp/ChS porous particles were weighed and dissolved in 5 ml of PBS, and the mixture was stirred at 37 ° C. The release test is carried out during the period of 1 hour to 1 week. The stirred sample was centrifuged under the same conditions as in Example 2, and the obtained supernatant was quantified by ELISA using rh-OCIF-Fc and rh-OCIF. The result is shown in Figure 3. Fig. 3 is a graph showing the passage of rh-〇CIF-Fc and rh-〇CIF of HAp/ChS porous plasmids carried by 〇CIF/0PG which are different in production cell and amino acid sequence. Figure 3 (in the case of 3丨2 cells, 仏-0 (:1? 〇 and Figure 3B (rh-OCIF expressed by CH0 cells), each abbreviation is as follows. "HAp/ChS2%" : 〇CIF/〇PG carries HAp/ChS (2% by weight) porous particles (no binding treatment), "HAp/ChS2% + Zn": 〇CIF/〇PG carries HAp/ChS (2% by weight) porous Plasmids (with binding treatment). As shown in Figure 3, HAp/ChS (2 -30 - 200820980% by volume) porous plasmids were sold on unconjugated 〇ciF/〇PG PG's commercially available rh-OCIF-Fc (Expressed by Sf2 cells) showed a high release amount of rh-OCIF produced by CH0 cells, and showed unsettled release when not combined. On the other hand, 0CIF/0PG treated with zinc ion combined with HAp/ChS (2 weight) %) A large increase was observed in the porous particles, and the amount of release was a commercially available rh-〇CIF-Fc (expressed as Sf2 cells) showing a number of higher 値. [Example 6] 0CIF/0PG carrying HAp The precipitation of the /ChS porous particles is as shown in Examples 2 to 5, and it is found that the cleavage is effective at the zinc ion binding, so that the HA p / C h S porous particles are more than 1 mg. CIF / 0 PG carrying capacity The experiment was carried out by adding a rhyme test. The test used the rh-〇CIF produced by CH0 cells in Example 5. The rh-〇CIF was dispersed in 5 ml of 10% PBS to form 80/zg/ml, and the addition of 50 mg was carried out. HAp/ChS (2% by weight) porous plasmid (8# g/lmg; rh-OCIF / HAp/ChS porous particle) prepared in Example 1. The mixture was stirred at room temperature for 4 hours and stirred for 4 hours. After the mixture was stirred for agitation, a combination of 3 mi of zinc chloride solution (10 mg/ml, pH 5.5) was added and the mixture was further stirred and stirred. According to the combination, the pores of rh-OCIF and porous particles were treated. The whole surface is combined with zinc ions. After 2 hours of stirring, the supernatant is separated by centrifugation (3,500 rpm, 10 minutes), washed with purified water, and the precipitate is freeze-dried for one night. In this way, 〇ClF/QPG which increases the amount of 0CIF/0PG is added to carry the HAp/ChS porous particles, and the same method is also used in the same method. The 0CIF/0PG produced by the second method carries the HAp/ChS porous material. The amount of the particles was -31-200820980 5 mg, dispersed in 5 ml of PBS and stirred at 37 ° C. The release test was carried out for 1 hour to 1 week. The same centrifugation conditions and stirring the sample in Example 2, the resulting supernatant was quantified by ELISA imitation of the rh-OCIF purposes. The result is shown in Figure 4. Fig. 4 is a graph showing the passage of rh-OCIF in which the HAC/ChS porous plasmid was carried by 0CIF/0PG which was increased by the amount of 0CIF/OPG. In Fig. 4, the abbreviations are as follows. "HAp/ChS2%": 〇CIF/〇PG carries H Ap/ChS (2% by weight) porous particles (no binding treatment), "HAp/ChS2% + Zn": 〇CIF/〇PG carries HAp /ChS (2% by weight) porous particles (with binding treatment) As shown in Fig. 4, when zinc ion binding treatment was not performed as in Example 5, a large amount of release (release) of the drug (rh-OCIF) was observed initially. However, when zinc ion binding treatment is applied, there is almost no initial release. From this experiment, it was found that the concentration range in which the effect of 0CIF/0PG was fully exerted (ED5 〇 8 ng/ml to 2 4 ng/ml per day) was obtained, 〇01?/〇? 0 carrying 11 eight? / (: 113 porous particles are sufficient for 30 mg. [Example 7] Evaluation of bone formation energy of carrier material (HAp/ChS porous particles) 18 Japanese white male rabbits 10 weeks after birth, in rabbits In the lower extremity bone hole (32 limbs, 32 holes), HAp/ChS porous particles prepared in Example 1 with physiological saline (ChS weight: 2% by weight, 10% by weight, 20% by weight) The four types were buried and compared. The rabbit was fixed in the supine position on the operating table, and the periosteum was peeled off from the medial center of the posterior thigh bone -32-200820980 and the proximal medial aspect of the humerus. Hole to the contralateral cortical bone. After suturing the periosteum, soft tissue, and skin with 4 - 0 nylon, the physiological saline or each ChS concentration of HAp/ChS porous particles were buried in a syringe with a 23G needle (1 ml). Into the hole. After 2 weeks or 4 weeks of burying, the rabbit is slaughtered. Thereafter, the bone of the drilled part is divided into 2 (horizontal cut), the proximal side is fixed with formalin, and the distal side is fixed with 70% ethanol. After fixing, the sample is given separately, and Haemat ο X y 1 i & E 〇si η (Η E) staining, toluidine blue staining, alkali phosphatidase (ALP) staining, tartaric acid-resistant acid phosphatase (TRAP) staining, Villaneuva-Goldner staining, and preparation of specimens. Bone volume measurement (Bone volume / Total volume, BV/TV) and Osteoblast surface / Bone surface (Ob. S/BS) were calculated by bone morphology measurement. The results are shown in Fig. 5.

Fig. 5 shows the results of calculation of BV/TV and 〇b.S/BS after 2 weeks of W or 4 weeks of HAp/ChS porous particles embedded in each ChS concentration. Fig. 5A and B show the calculation results of BV/TV and 〇b.S/BS after being buried for 2 weeks. Further, Fig. 5C and D are calculation results of BV/TV and 〇b.S/BS after being buried for 4 weeks, respectively. In the fifth drawing A to D, the abbreviations are as follows. "Saline": physiological saline, "2% ChS": HAp/ChS (2% by weight) porous particles, "10% ChS": HAp/ChS (10% by weight) porous particles, "20% ChS": HAp/ChS (20% by weight) porous particles. -33- 200820980 As shown in Fig. 5, BV/TV and 〇b.S/BS increased in the HAp/ChS porous particle-administered group than in the physiological saline-administered group. HAp/ChS (20% by weight) The porous plasmid was administered to the Ob.S/BS for 2 weeks and then increased. After BV/TV was buried for 4 weeks, it was enlarged, suggesting that it was promoted by the activation of bone bud cells. The possibility of bone formation. [Example 8] Evaluation of osteoinduction of HAp/ChS porous particles in bone elongation 14 Japanese white rabbits (1.9 kg to 2.2 kg) were divided into control groups (n = 7) and HAp/ChS porous plasmids. Experiment with 2 groups of groups (n = 7). • An external fixation device was placed on the right tibia of the whole rabbit under general anesthesia for tibia osteotomy. The bone length of 1 mm was performed on the 1st of the 1st week after the osteotomy. On the 7th day after the start of bone elongation, 0.2 ml of physiological saline was injected into the control group, and HAp/ChS (2% by weight) porous plasmid 100 mg and physiological saline prepared in Example 1 were injected into the HAp/ChS porous plasmid. 0.2 ml of the mixture 〇. 2 ml. The rabbit was euthanized after a fixed period of 6 weeks. ^ Osteoinduction evaluation by TRAP staining, Villaneuva-Goldner staining and ALP staining, sequentially calculated the number of osteoclasts (N.Oc/BS) and relative osteoid volume (Osteoid volume / Bone volume), bone mass (Bone volume), bone The number of bud cells. Further, HE staining and toluidine green dyeing were carried out in the same manner as in Example 7. The // CT image analysis was performed by TRI/3D-BON (RATOC Systems Engineering, Tokyo). Further, DCS-600 (ALOKA, Tokyo) was used to perform bone density measurement according to the DXA (Dual -34-200820980 energy X-ray absorptiometry device) method. The results are shown in Figures 6 to 10. Figure 6 shows the results of /z CT image analysis. From Fig. 6, it was found that both the control group and the HAp/ChS porous particle-administered group had good bone formation. The HAp/ChS porous particle-administered group observed the formation of intra-marrow sponge bone which was better than the control group. Figure 7 shows the results of Villaneuva-Goldnei* staining. Figure 8 shows the results of HE staining and toluidine blue staining. In Figures 7 and 8, staining with ® Villaneuva-Goldner and HE staining showed that the HAp/ChS porous plasmid-administered group formed a good sponge bone than the control group. As also shown in Fig. 8, the control group produced internal cartilage ossification (thick arrow) in the bone marrow, and activating the activated bone bud cells (thin arrows) in the lacunae. On the other hand, as shown in Fig. 8, the HAp/ChS porous particle-administered group also produces internal cartilage ossification (thick arrow) in the bone marrow. In some of the individuals, the intramedullary cells surrounded by several layers of fibroblasts and the particles of the HAp/ChS porous particles were also confirmed. Figure 9 shows the quantitative ^ results for bone mass (Fig. 9A) and relative bone mass (Fig. 9B). In Fig. 9A and B, "control" is the result of the control group, and "HAp/ChS" is the result of the HAp/ChS porous particle-administered group. As shown in Fig. 9, the HAp/ChS porous particle-administered group has a tendency to have a large amount of bone and a relatively small amount of bone. This is shown in the HAp/ChS porous particle-administered group to promote new bone formation and its maturation. Figure 10 shows the results of bone density measured by the DXA method. In the first diagram, "control" is the result of the control group, and "HAp/ChS" is the result of the HAp/ChS porous particle administration group. As shown in Fig. 10, the bone density measured by the DXA method -35-200820980 also has a tendency to cast HAp/ChS porous particles, and together with the tissue image, the bone formation promoting energy of the HAp/ChS porous particles is confirmed. [Example 9] Evaluation of releasability in rats Rh-〇CIF (CHO cell producer shown in Example 5) was dispersed in 12.5 ml of 10% PBS to 800//g/ml, and the examples were added. 1 Preparation of HAp/ChS (2% by weight) porous particles 125 mg (80//g/mg; rh-〇CIF / HAp/ChS porous particles). The mixture was stirred at room temperature for 4 hours and stirred. Non-binding processors are also produced in the same way. The particles referred to as Unbonded ® are hereinafter referred to as "〇CIF-HAp/ChS". After stirring for 4 hours, the mixture was combined and treated, and an aqueous zinc chloride solution (10 mg/ml, pH 5.5) of 0,75 ml was added thereto, and the mixture was further stirred for 2 hours. According to the combination treatment, rh-OCIF binds to all the intermediate zinc ions on the inner surface of the pores of the porous particles. Hereinafter, the particles to be combined are referred to as "OCIF-HAp/ChS-Zn". In the case of OCIF-HAp/ChS-Ζη, after stirring for 2 hours, the supernatant was separated by centrifugation (350 〇 rPm, 10 minutes), and washed with purified water. This ® wash is performed 3 times. The amount of adsorption was calculated by quantifying the rh-QCIF concentration of the supernatant solution after separation for 2 hours, and extracting the supernatant solution and the supernatant solution after washing for 3 times with ADV〇1 (Advanced Protein Assay Reagent). Similarly, OCIF-HAp/ChS was also used to quantify the rh-〇CIF concentration of the supernatant solution, and the amount of adsorption was calculated. As a result, the rh-〇CIF adsorption amounts of 〇CIF-HAp/ChS and 〇CIF-HAp/ChS-Zn were 79·60±0.09 μg/mg and 7 9.7 6±0.02/ig/mg, respectively. The theoretical amount is almost the same as the amount of adsorption. The OCIF-HAp/ChS and -36-200820980 〇CIF-HAp/ChS-Zn were used in the evaluation of the relaxation in the following rats. OCIF-HAp/ChS m 〇CIF-HAp/ChS-Zn was administered intraperitoneally (ip) or subcutaneously (sc) in F344 rats (12 weeks old female, Charles River, Japan) at a dose of 200 mg/kg. ) to vote. There were 5 rats in each group. Before the administration of OCIF-HAp/ChS or OCIF-HAp/ChS-Zn, and after 1, 2, 3, 4, and 7 days, 0.2 ml of blood was collected from each rat, and serum was prepared from the blood samples. The rh-〇CIF concentration was determined by 〇CIF-ELISA (K. Yano et a 1 , J · Β ο M M ine r. R e s., 14 4, p 5 18, 1 9 9 9). The results of intraperitoneal administration were as shown in Figure 11, and the results of subcutaneous administration are shown in Figure 12. In the samples of Figs. 1 and 12, the x-axis is the concentration of rh-OCIF in the serum, and the horizontal axis indicates the number of days after administration. On the other hand, as a control experiment, rh-〇CIF diluted with PBS (pH 6.0) was administered intraperitoneally (ip) in F344 rats (12-week-old female, Charles River, Japan) at a dose of 16.0 mg/kg. With or on the back subcutaneous (sc). Rats were 5 in each group. Rh-OCIF was administered before and after 0.5, 1, 2, 4, 6, and 24 hours after φ. 0.2 ml of blood was collected from each rat, and rh in the serum prepared from the respective blood samples was measured by OCIF-ELISA. -OCIF concentration. The results of intraperitoneal administration are shown in Fig. 13, and the results of subcutaneous administration are shown in Fig. 14. In the samples of Figures 13 and 14, the x-axis is the concentration of rh-OCIF in the serum, and the horizontal axis is the elapsed time after administration. As shown in Figures 13 and 14, the unformulated rh-OCIF is also peaked by any administration route for 2 to 6 hours, and the serum concentration is rapidly reduced. 〇-37-200820980 As shown in Fig. 1, when rh-〇CIF was carried on the HAp/ChS porous particles, the intraperitoneal administration was maintained at 1000 ng/ml or more after 1 day of administration (in OCIF-HAp/ChS-Zn was 1). Rh-〇CIF concentration in high serum of 300 ng/ml or more. Furthermore, in OCIF-HAp/ChS-Zn, the rh-〇CIF concentration in the serum of nearly 500 ng/ml was maintained after 2 days, showing an excellent effect of releasing. In addition, as shown in Fig. 12, CIF-HAp/ChS-Zn (when combined with zinc ions) was administered subcutaneously, and the serum was maintained at 1 Q0 ng/m 1 ® or more after administration for 4 days. -OCIF concentration, showing a very excellent effect. When administered subcutaneously with CIF-HAp/ChS (when combined with zinc ions), the duration of maintenance was shorter than that of CIF-HAp/ChS-Zn, but it was maintained in serum of 100 ng/ml or more after 2 days. -OCIF concentration, this preparation also has an excellent effect. In any of the routes and the HAp/ChS porous particles carried by the rh-〇CIF which is combined with the treatment of the zinc ions, the effect of the zinc ion-binding treatment is remarkably promoted. The effect is also subcutaneously • Significantly presented in the cast. Thus, it has been confirmed in the administration of experimental animals that the formulation technique of the present invention is applied to OCIF, and the blood concentration of 〇CIF can be maintained for a long period of time. INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a stable and large amount of a susceptibility agent for carrying OCIF/OPG. The Xu-release preparation of the present invention improves the binding of the porous particles to OCIF/OPG by divalent metal ions, and has almost no initial release, and can stably release OCIF/OPG for a certain period of time. Further, the Xu-Release preparation of the present invention uses a calcium phosphate compound containing a component of bone-38-200820980 and a porous plasmid of a polysaccharide as a carrier, activates bone bud cells, and grows crystals of apatite with a carrier as a core. Since the main component contains a polysaccharide of the extracellular matrix of bone, internal cartilage ossification occurs to promote bone formation, and it has the characteristics of the conventional Xuanfang preparation. All publications, patents and patent applications cited in this specification are hereby incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a diagram showing the rh-OCIF-Fc in which HAp/ChS porous _ particles were placed on each of 〇CIF/〇PG by time. Fig. 2 is a graph showing the rh-OCIF-Fc of the HAp/ChS porous plasmid supported by 10 mg or 20 mg of each of 0CIF/0PG over time. Fig. 3 is a graph showing the rh-〇CIF-Fc and rh-〇CIF of the HAp/ChS porous plasmids carrying different 0CIF/OPG of the production cell and the amino acid sequence over time. Figure 4 is a graph showing the rh-OCIF of HAp/ChS porous plasmids by OCIF/OPG with increasing OCIF/OPG loading. Fig. 5 is a graph showing the results of calculation of BV/TV and Ob.S/BS after 2 weeks or 4 weeks of HAP/ChS porous particles embedded in each ChS concentration. Fig. 6 is a graph showing the results of analysis of v CT images in the eighth embodiment. Fig. 7 is a graph showing the results of Villaneuva-Goldner staining in Example 8. Fig. 8 is a graph showing the results of HE staining and toluidine blue staining in Example 8. Fig. 9 is a graph showing the quantitative results of bone mass and relative bone mass in Example 8. -39- 200820980 Fig. 10 is a graph showing the results of bone density measured by the DXA method in Example 8. Figure 11 is a graph showing the change in rh-OCIF concentration in the serum of CIF-HAp/ChS and OCIF-HAp/ChS-Zn administered intraperitoneally in rats in Example 9. Fig. 12 is a graph showing the change in the concentration of rh-〇CIF in the serum of OCIF-HAp/ChS and OCIF-HAp/ChS-Zn in the case of subcutaneous administration in rats in Example 9. Fig. 13 is a graph showing the change in the concentration of rh-OCIF in the serum of the case where rh-OCIF dissolved in PBS was administered intraperitoneally in the rat in Example 9. Fig. 14 is a graph showing the temporal change of the concentration of rh-〇CIF in the serum of the case where rh-〇CIF dissolved in PBS was administered subcutaneously in rats in Example 9. All publications, patents and patent applications cited in this specification are hereby incorporated by reference.

-40- 200820980 Sequence Listing <110> Independent Administrative Corporation Material and Materials Research Institute (Independent Administrative Corporation Material and Materials Research Institute) National University Corporation Tokyo Medical and Dental University (National University Corporation Tokyo Medical University) Co., Ltd. <120>>130>PH-3319-PCT<150>PH-3319-PCT<150> JP 2006-271102 <151> 2006-10-02 <160> 13

<170> Patent version 2.1 <210> 1 <211>6 <212> PRT <213> Human <220><221> Undetermined <222> 1 <223> Xaa is any amine Acid <400>1

Xaa Tyr His Phe Pro Lys 1 5

<210>2 <211> 14 <212> PRT <213> Human <220><221> Undetermined <222>1,5 and 13<223> Xaa is any amino acid <;400> 2

Xaa Gin His Ser Xaa Gin Glu Gin Thr Phe Gin Leu Xaa Lys 1 5 10 <210>3 <211>12 -41- 200820980 <212>PRT <213>Human<220><221> It is determined that <222> 1 <223>Xaa is any amino acid <400>

Xaa lie Arg Phe Leu His Ser Phe Thr Met Tyr Lys 1 5 10

<210>4 <211> 1206 <212> DNA <213> Human <220><221> Information peptide <222>(1)..(63) <220> <221> Mature peptide <222>(64)..(1206)<220><221> CDS <222>(1)..(1206) <400> 4 atg aac aac ttg ctg tgc tgc gcg Ett gtg ttt ctg gac ate tcc att 48 Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp lie Ser lie -20 -15 -10 aag tgg acc acc cag gaa aeg ttt cct cca aag tac ett cat tat gac 96 Lys Trp Thr Thr Gin Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp -5 ·1 1 5 10 gaa gaa acc tet cat cag ctg ttg tgt gac aaa tgt cct cct ggt acc 144 Glu Glu Thr Ser His Gin Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr 15 20 25 tac eta aaa caa cac tgt aca gca aag tgg aag acc gtg tgc gcc cct 192 Tyr Leu Lys Gin His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro 30 35 40 tgc cct gac cac tac tac aca Gac age tgg cac acc agt gac gag tgt 240 Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 45 50 55 -42- 200820980 eta tac tgc age ccc gtg tgc Aag gag ctg cag tac gtc aag cag gag 288 Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu Gin Tyr Val Lys Gin Glu 60 65 70 75 tgc aat ege acc cac aac ege gtg tgc gaa tgc aag gaa ggg ege tac 336 Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr 80 85 90 ett gag ata gag ttc tgc ttg aaa cat agg age tgc cct cct gga ttt 384 Leu Glu lie Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe 95 100 105 gga gtg gtg caa get gga acc cca gag ega aat aca gtt tgc aaa aga 432 Gly Val Val Gin Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Arg 110 115 120

Tgt cca gat ggg ttc ttc tea aat gag aeg tea tet aaa gca ccc tgt 480 Cys Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys 125 130 135 aga aaa cac aca aat tgc agt gtc ttt ggt etc ctg eta act Cag aaa 528

Arg Lys His Thr Asn Cys Ser Val Phe Gly Leu Leu Leu Thr Gin Lys 140 145 150 155 gga aat gca aca cac gac aac ata tgt tee gga aac agt gaa tea act 576 Gly Asn Ala Thr His Asp Asn lie Cys Ser Gly Asn Ser Glu Ser Thr 160 165 170 caa aaa tgt gga ata gat gtt acc ctg tgt gag gag gca ttc ttc agg 624 Gin Lys Cys Gly He Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg 175 180 185 ttt get gtt cct aca aag ttt aeg Cct aac tgg ett agt gtc ttg gta 672 Phe Ala Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val 190 195 200 gac aat ttg cct ggc acc aaa gta aac gca gag agt gta gag agg ata 720 Asp Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg lie 205 210 215 aaa egg caa cac age tea caa gaa cag act ttc cag ctg ctg aag tta 768

Lys Arg Gin His Ser Ser Gin Glu Gin Thr Phe Gin Leu Leu Lys Leu 220 225 230 235 tgg aaa cat caa aac aaa gac caa gat ata gtc aag aag ate ate caa 816 Trp Lys His Gin Asn Lys Asp Gin Asp lie Val Lys Lys Lie lie Gin 240 245 250 gat att gac etc tgt gaa aac age gtg cag egg cac att gga cat get 864 Asp lie Asp Leu Cys Glu Asn Ser Val Gin Arg His lie Gly His Ala 255 260 265 -43- 200820980 aac etc acc ttc Gag cag ett cgt age ttg atg gaa age tta ccg gga 912 Asn Leu Thr Phe Glu Gin Leu Arg Ser Leu Met Glu Ser Leu Pro Gly 270 275 280 aag aaa gtg gga gca gaa gac att gaa aaa aca ata aag gca tgc aaa 960 Lys Lys Val Gly Ala Glu Asp lie Glu Lys Thr lie Lys Ala Cys Lys 285 290 295 ccc agt gac cag ate ctg aag ctg etc agt ttg tgg ega ata aaa aat 1008

Pro Ser Asp Gin lie Leu Lys Leu Leu Ser Leu Trp Arg lie Lys Asn 300 305 310 315 ggc gac caa gac acc ttg aag ggc eta atg cac gca eta aag cac tea 1056 Gly Asp Gin Asp Thr Leu Lys Gly Leu Met His Ala Leu Lys His Ser 320 325 330

Aag aeg tac cac ttt ccc aaa act gtc act cag agt eta aag aag acc 1104 Lys Thr Tyr His Phe Pro Lys Thr Val Thr Gin Ser Leu Lys Lys Thr 335 340 345 ate agg ttc ett cac age ttc aca atg tac aaa ttg tat cag Aag tta 1152 He Arg Phe Leu His Ser Phe Thr Met Tyr Lys Leu Tyr Gin Lys Leu 350 355 360 ttt tta gaa atg ata ggt aac cag gtc caa tea gta aaa ata age tgc 1200 Phe Leu Glu Met He Gly Asn Gin Val Gin Ser Val Lys lie Ser Cys 365 370 375 1206 tta taa

Leu 380 <210>5 <211>401 <212> PRT <213> Human <400>5

Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp lie Ser lie -20 -15 -10

Lys Trp Thr Thr Gin Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp -5 -11 5 10

Glu Glu Thr Ser His Gin Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr 15 20 25

Tyr Leu Lys Gin His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro 30 35 40

Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 45 50 55

Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu Gin Tyr Val Lys Gin Glu 60 65 70 75 -44- 200820980

Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr 80 85 90 Leu Glu lie Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe 95 100 105 Gly Val Val Gin Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Arg 110 115 120 Cys Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ly Lys Ala Pro Cys 125 130 135 Arg Lys His Thr Asn Cys Ser Val Phe Gly Leu Leu Leu Thr Gin Lys 140 145 150 Gly Asn Ala Thr His Asp Asn He Cys Ser Gly Asn Ser Glu Ser Thr 160 165 170 Gin Lys Cys Gly He Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg 175 180 185 Phe Ala Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val 190 195 200 Asp Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg lie 205 210 215 Lys Arg Gin His Ser Ser Gin Glu Gin Thr Phe Gin Leu Leu Lys Leu 220 225 230 Trp Lys His Gin Asn Lys Asp Gin Asp lie Val Lys Lys lie lie Gin 240 245 250 Asp lie Asp Leu Cys Glu Asn Ser Val Gin Arg His lie Gly His Ala 255 260 265 Asn Leu Thr Phe Glu Gin Leu Arg Ser Leu Met Glu Ser Leu Pro Gly 270 275 280 Lys Lys Val Gly Al a Glu Asp lie Glu Lys Thr lie Lys Ala Cys Lys 285 290 295 Pro Ser Asp Gin lie Leu Lys Leu Leu Ser Leu Trp Arg lie Lys Asn 300 305 310 Gly Asp Gin Asp Thr Leu Lys Gly Leu Met His Ala Leu Lys His Ser 320 325 330 Lys Thr Tyr His Phe Pro Lys Thr Val Thr Gin Ser Leu Lys Lys Thr 335 340 345 lie Arg Phe Leu His Ser Phe Thr Met Tyr Lys Leu Tyr Gin Lys Leu 350 355 360 Phe Leu Glu Met lie Gly Asn Gin Val Gin Ser Val Lys lie Ser Cys 365 370 375 Leu 380 155 235 315 <210>6 <211> 1185 <212> DNA <213> Human <220><221> Information peptide <222> (1).(63) 45-200820980 <220> <221> Mature peptide <222> (64)..(1185) <220> <221>CDS <222> (1) ..(1185) <400> 6 atg aac aac ttg ctg tgc tgc gcg etc gtg ttt ctg gac ate tee att 48 Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp lie Ser lie -20 -15 -10 aag Tgg acc acc ca ca gaa aeg ttt cct cca aag tac ett cat tat gac 96 Lys Trp Thr Thr Gin Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp -5 -11 5 10

Gaa gaa acc tet cat cag ctg ttg tgt gac aaa tgt cct cct ggt acc 144 Glu Glu Thr Ser Gin Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr 15 20 25 tac eta aaa caa cac tgt aca gca aag tgg aag acc gtg tgc Gee cct 192 Tyr Leu Lys Gin His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro 30 35 40 tgc cct gac cac tac tac aca gac age tgg cac acc agt gac gag tgt 240 Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 45 50 55 75 eta tac tgc age ccc gtg tgc aag gag tgc aat ege acc cac aac ege 288 Leu Tyr Cys Ser Pro Val Cys Lys Glu Cys Asn Arg Thr His Asn Arg 60 65 70 gtg tgc gaa tgc aag Gaa ggg ege tac ett gag ata gag ttc tgc ttg 336 Val Cys Glu Cys Lys Glu Gly Arg Tyr Leu Glu lie Glu Phe Cys Leu 80 85 90 aaa cat agg age tgc cct cct gga ttt gga gtg gtg caa get gga acc 384 Lys His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gin Ala Gly Thr 95 100 105 cca gag ega aat aca gtt tgc aaa aga tgt cca gat ggg ttc ttc tea 432 Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe Ser 110 115 120 aat g Ag aeg tea tet aaa gca ccc tgt aga aaa cac aca aat tgc agt 480 Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn Cys Ser 125 130 135 gtc ttt ggt etc ctg eta act cag aaa gga aat gca aca cac gac Aac 528 Val Phe Gly Leu Leu Leu Thr Gin Lys Gly Asn Ala Thr His Asp Asn -46- 200820980 155 140 145 150 ata tgt tcc gga aac agt gaa tea act caa aaa tgt gga ata gat gtt 576 He Cys Ser Gly Asn Ser Glu Ser Thr Gin Lys Cys Gly He Asp Val 160 165 170 acc ctg tgt gag gag gca ttc ttc agg ttt get gtt cct aca aag ttt 624 Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala Val Pro Thr Lys Phe 175 180 185 aeg cct Aac tgg ett agt gtc ttg gta gac aat ttg cct ggc acc aaa 672 Thr Pro Asn Trp Leu Ser Val Leu Val Asp Asn Leu Pro Gly Thr Lys 190 195 200 gta aac gca gag agt gta gag agg ata aaa egg caa cac age tea caa 720 Val Asn Ala Glu Ser Val Glu Arg lie Lys Arg Gin His Ser Ser Gin 205 210 215

235 gaa cag act ttc cag ctg ctg aag tta tgg aaa cat caa aac aaa gac 768 Glu Gin Thr Phe Gin Leu Leu Lys Leu Trp Lys His Gin Asn Lys Asp 220 225 230 caa gat ata gtc aag agate ate caa gat att gac etc Tgt gaa aac 816 Gin Asp lie Val Lys Lys lie lie Gin Asp lie Asp Leu Cys Glu Asn 240 245 250 age gtg cag egg cac att gga cat get aac etc acc ttc gag cag ett 864 Ser Val Gin Arg His He Gly His Ala Asn Leu Thr Phe Glu Gin Leu 255 260 265 cgt age ttg atg gaa age tta ccg gga aag aaa gtg gga gca gaa gac 912 Arg Ser Leu Met Glu Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp 270 275 280 att gaa aaa aca ata Aag gca tgc aaa ccc agt gac cag ate ctg aag 960 lie Glu Lys Thr lie Lys Ala Cys Lys Pro Ser Asp Gin lie Leu Lys 285 290 295 315 ctg etc agt ttg tgg ega ata aaa aat ggc gac caa gac acc ttg aag 1008 Leu Leu Ser Leu Trp Arg lie Lys Asn Gly Asp Gin Asp Thr Leu Lys 300 305 310 ggc eta atg cac gca eta aag cac tea aag aeg tac cac ttt ccc aaa 1056 Gly Leu Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys 320 325 330 act gtc act cag agt eta aag aag acc ate agg ttc ett cac age ttc 1104 Thr Val Thr Gin Ser Leu Lys Lys Thr He Arg Phe Leu His Ser Phe 335 340 345 aca atg tac aaa ttg tat cag aag tta ttt Tta gaa atg ata ggt aac 1152 Thr Met Tyr Lys Leu Tyr Gin Lys Leu Phe Leu Glu Met lie Gly Asn -47- 200820980 350 355 360 cag gtc caa tea gta aaa ata age tgc tta taa 1185

Gin Val Gin Ser Val Lys lie Ser Cys Leu 365 370 <210>7 <211>394 <212>PRT <213>Human <400〉 7

Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp lie Ser lie -20 -15 -10

Lys Trp Thr Thr Gin Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp -5 -11 5 10

Glu Glu Thr Ser His Gin Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr 15 20 25

Tyr Leu Lys Gin His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro 30 35 40

Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 45 50 55

Leu Tyr Cys Ser Pro Val Cys Lys Glu Cys Asn Arg Thr His Asn Arg 60 65 70 75

Val Cys Glu Cys Lys Glu Gly Arg Tyr Leu Glu lie Glu Phe Cys Leu 80 85 90

Lys His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gin Ala Gly Thr 95 100 105

Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe Ser 110 115 120

Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn Cys Ser 125 130 135

Val Phe Gly Leu Leu Leu Thr Gin Lys Gly Asn Ala Thr His Asp Asn 140 145 150 155

He Cys Ser Gly Asn Ser Glu Ser Thr Gin Lys Cys Gly He Asp Val 160 165 170

Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala Val Pro Thr Lys Phe 175 180 185

Thr Pro Asn Trp Leu Ser Val Leu Val Asp Asn Leu Pro Gly Thr Lys 190 195 200

Val Asn Ala Glu Ser Val Glu Arg lie Lys Arg Gin His Ser Ser Gin 205 210 215

Glu Gin Thr Phe Gin Leu Leu Lys Leu Trp Lys His Gin Asn Lys Asp 220 225 230 235

Gin Asp lie Val Lys Lys He He Gin Asp lie Asp Leu Cys Glu Asn 240 245 250

Ser Val Gin Arg His lie Gly His Ala Asn Leu Thr Phe Glu Gin Leu 255 260 265

Arg Ser Leu Met Glu Ser Leu Pro Gly Lys Lys Val Gly Ala Glu Asp 270 275 280 -48 - 315 200820980 lie Glu Lys Thr He Lys Ala Cys Lys Pro Ser Asp Gin lie Leu Lys 285 290 295

Leu Leu Ser Leu Trp Arg lie Lys Asn Gly Asp Gin Asp Thr Leu Lys 300 305 310

Gly Leu Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys 320 325 330

Thr Val Thr Gin Ser Leu Lys Lys Thr lie Arg Phe Leu His Ser Phe 335 340 345

Thr Met Tyr Lys Leu Tyr Gin Lys Leu Phe Leu Glu Met lie Gly Asn 350 355 360

Gin Val Gin Ser Val Lys lie Ser Cys Leu 365 370

<210>8 <211> 1089 <212> DNA <213> Human <220><221> Information peptide <222>(1)..(63) <220><221> Mature peptide <222> (64)..(1089) <220> <221>CDS <222>(1)..(1089) <400> 8 atg aac aag ttg ctg tgc tgc gcg Etgt gtg ttt ctg gac ate tcc att 48 Met Asn Lys Leu Leu Cys Cys Ala Leu Val Phe Leu Asp lie Ser lie -20 -15 -10 aag tgg acc acc cag gaa aeg ttt cct cca aag tac ett cat tat gac 96 Lys Trp Thr Thr Gin Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp -5 -11 5 10 gaa gaa acc tet cat cag ctg ttg tgt gac aaa tgt cct cct ggt acc 144 Glu Glu Thr Ser His Gin Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr 15 20 25 tac eta aaa caa cac tgt aca gca aag tgg aag acc gtg tgc gcc cct 192 Tyr Leu Lys Gin His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro 30 35 40 tgc cct gac cac tac tac aca gac Age tgg cac acc agt gac gag tgt 240 Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 45 50 55 -49- 200820980 eta tac tgc age ccc gtg tgc aa g gag ctg cag tac gtc aag cag gag 288 Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu Gin Tyr Val Lys Gin Glu 60 65 70 75 tgc aat ege acc cac aac ege gtg tgc gaa tgc aag gaa ggg ege tac 336 Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr 80 85 90 ett gag ata gag ttc tgc ttg aaa cat agg age tgc cct cct gga ttt 384 Leu Glu lie Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe 95 100 105 gga gtg gtg caa get gga acc cca gag ega aat aca gtt tgc aaa aga 432 Gly Val Val Gin Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Arg 110 115 120

Tgt cca gat ggg ttc ttc tea aat gag aeg tea tet aaa gca ccc tgt 480 Cys Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys 125 130 135 aga aaa cac aca aat tgc agt gtc ttt ggt etc ctg eta act Cag aaa 528

Arg Lys His Thr Asn Cys Ser Val Phe Gly Leu Leu Leu Thr Gin Lys 140 145 150 155 gga aat gca aca cac gac aac ata tgt tee gga aac agt gaa tea act 576 Gly Asn Ala Thr His Asp Asn He Cys Ser Gly Asn Ser Glu Ser Thr 160 165 170 caa aaa tgt gga ata gat gtt acc ctg tgt gag gag gca ttc ttc agg 624 Gin Lys Cys Gly lie Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg 175 180 185 ttt get gtt cct aca aag ttt aeg Cct aac tgg ett agt gtc ttg gta 672 Phe Ala Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val 190 195 200 gac aat ttg cct ggc acc aaa gta aac gca gag agt gta gag agg ata 720 Asp Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg lie 205 210 215 aaa egg caa cac age tea caa gaa cag act ttc cag ctg ctg aag tta 768

Lys Arg Gin His Ser Ser Gin Glu Gin Thr Phe Gltf Leu Leu Lys Leu 220 225 230 235 tgg aaa cat caa aac aaa gac caa gat ata gtc aag aag ate ate caa 816 Trp Lys His Gin Asn Lys Asp Gin Asp lie Val Lys Lys Lie lie Gin 240 245 250 gat att gac etc tgt gaa aac age gtg cag egg cac att gga cat get 864 Asp lie Asp Leu Cys Glu Asn Ser Val Gin Arg His lie Gly His Ala 255 260 265 •50- 200820980 aac etc agt ttg Tgg ega ata aaa aat ggc gac caa gac ace ttg aag 912 Asn Leu Ser Leu Trp Arg lie Lys Asn Gly Asp Gin Asp Thr Leu Lys 270 275 280 ggc eta atg cac gca eta aag cac tea aag aeg tac cac ttt ccc aaa 960 Gly Leu Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys 285 290 295 315 act gtc act cag agt eta aag aag acc ate agg ttc ett cac age ttc 1008 Thr Val Thr Gin Ser Leu Lys Lys Thr lie Arg Phe Leu His Ser Phe 300 305 310 aca atg tac aaa ttg tat cag aag tta ttt tta gaa atg ata ggt aac 1056 Thr Met Tyr Lys Leu Tyr Gin Lys Leu Phe Leu Glu Met lie Gly Asn 320 325 330

1089 cag gtc caa tea gta aaa ata age tgc tta taa Gin Val Gin Ser Val Lys lie Ser Cys Leu 335 340 <210>9 <211>362 <212> PRT <213>Human <400>

Met Asn Lys Leu Leu Cys Cys Ala Leu Val Phe Leu Asp lie Ser lie -20 -15 -10

Lys Trp Thr Thr Gin Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp -5 -11 5 10

Glu Glu Thr Ser His Gin Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr 15 20 25

Tyr Leu Lys Gin His Cys Thr Ala Lys Tip Lys Thr Val Cys Ala Pro 30 35 40

Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 45 50 55 75

Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu Gin Tyr Val Lys Gin Glu 60 65 70

Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr 80 85 90

Leu Glu lie Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe 95 100 105

Gly Val Val Gin Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Arg 110 115 120

Cys Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys 125 130 135

Arg Lys His Thr Asn Cys Ser Val Phe Gly Leu Leu Leu Thr Gin Lys 140 145 150

Gly Asn Ala Thr His Asp Asn lie Cys Ser Gly Asn Ser Glu Ser Thr -51- 155 235 200820980 160 165 170

Gin Lys Cys Gly lie Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg 175 180 185

Phe Ala Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val 190 195 200

Asp Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg lie 205 210 215

Lys Arg Gin His Ser Ser Gin Glu Gin Thr Phe Gin Leu Leu Lys Leu 220 225 230

Trp Lys His Gin Asn Lys Asp Gin Asp lie Val Lys Lys lie lie Gin 240 245 250

Asp lie Asp Leu Cys Glu Asn Ser Val Gin Arg His lie Gly His Ala 255 260 265

Asn Leu Ser Leu Trp Arg He Lys Asn Gly Asp Gin Asp Thr Leu Lys 270 275 280

Gly Leu Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe Pro Lys 285 290 295

315

Thr Val Thr Gin Ser Leu Lys Lys Thr He Arg Phe Leu His Ser Phe 300 305 310

Thr Met Tyr Lys Leu Tyr Gin Lys Leu Phe Leu Glu Met He Gly Asn 320 325 330

Gin Val Gin Ser Val Lys lie Ser Cys Leu 335 340 <210>10 <211>465 <212> DN A <213>Human<220><221> Information peptide <222> )..(63) <220><221> Mature peptide <222>(64)..(465) <220> <221>CDS <222>(1)..(465) <400>10 atg aac aag ttg ctg tgc tgc teg etc gtg ttt ctg gac ate tcc att 48 Met Asn Lys Leu Leu Cys Cys Ser Leu Val Phe Leu Asp lie Ser lie -20 -15 -10 aag tgg acc acc cag gaa Aeg ttt cct cca ag tac ett cat tat gac 96 Lys Trp Thr Thr Gin Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp -5 ·1 1 5 10 -52- 200820980 gaa gaa acc tct cat cag ctg ttg tgt gac aaa tgt Cct cct ggt acc 144 Glu Glu Thr Ser Gin Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr 15 20 25 tac eta aaa caa cac tgt aca gca aag tgg aag acc gtg tgc gcc cct 192 Tyr Leu Lys Gin His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro 30 35 40 tgc cct gac cac tac tac aca gac age tgg cac acc agt gac gag tgt 240 Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu C Ys 45 50 55 eta tac tgc age ccc gtg tgc aag gag ctg cag tac gtc aag cag gag 288 Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu Gin Tyr Val Lys Gin Glu 60 65 70 75

Tgc aat ege acc cac aac ege gtg tgc gaa tgc aag gaa ggg ege tac 336 Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr 80 85 90 ett gag ata gag ttc tgc ttg aaa cat agg age tgc cct cct Gga ttt 384 Leu Glu lie Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe 95 100 105 gga gtg gtg caa get ggt aeg tgt caa tgt gca gca aaa tta att agg 432 Gly Val Val Gin Ala Gly Thr Cys Gin Cys Ala Ala Lys Leu lie Arg 110 115 120 ate atg caa agt cag ata gtt gtg aca gtt tag 465 lie Met Gin Ser Gin lie Val Val Thr Val 125 130 <210 11 <211>154 <212> PRT <213> Human <400> 11

Met Asn Lys Leu Leu Cys Cys Ser Leu Val Phe Leu Asp lie Ser lie -20 -15 -10

Lys Trp Thr Thr Gin Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp -5 -11 5 10

Glu Glu Thr Ser His Gin Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr 15 20 25

Tyr Leu Lys Gin His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro 30 35 40

Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 45 50 55

Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu Gin Tyr Val Lys Gin Glu 60 65 70 75 -53- 200820980

Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr 80 85 90

Leu Glu lie Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe 95 100 105

Gly Val Val Gin Ala Gly Thr Cys Gin Cys Ala Ala Lys Leu lie Arg 110 115 120 lie Met Gin Ser Gin lie Val Val Thr Val 125 130 <210> 12 <211>438 <212>DN A <213 〉Human<220>

<221> Information peptide <222>(1)..(63) <220><221> Mature peptide <222> (64)..(438) <220> <221> CDS <222> (1)..(438) <400> 12 atg aac aag ttg ctg tgc tgc gcg etc gtg ttt ctg gac ate tcc att 48 Met Asn Lys Leu Leu Cys Cys Ala Leu Val Phe Leu Asp lie Ser Lie -20 -15 -10 aag tgg acc acc cag gaa aeg ttt cct cca aag tac ett cat tat gac 96 Lys Trp Thr Thr Gin Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp 5 -11 5 10 gaa gaa acc tet cat C g g g g g g g g g g g g g g g g g g g g g g g g g g g g G Lys Gin His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro 30 35 40 tgc cct gac cac tac tac aca gac age tgg cac acc agt gac gag tgt 240 Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 45 50 55 eta tac tgc age ccc gtg tgc aag gag ctg cag tac gtc aag cag gag 288 Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu Gin Tyr Val Ly s Gin Glu 60 65 70 -54- 75 200820980 tgc aat cgc acc cac aac cgc gtg tgc gaa tgc aag gaa ggg cgc tac 336 Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr 80 85 90 ctt gag ata Gag ttc tgc ttg aaa cat agg age tgc cct cct gga ttt 384 Leu Glu lie Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe 95 100 105 gga gtg gtg caa get gga tgc agg aga aga ccc aag cca cag ata tgt 432 Gly Val Val Gin Ala Gly Cys Arg Arg Arg Pro Lys Pro Gin lie Cys 110 115 120 438 ate tga lie 125

<210> 13 <211>145 <212>PRT <213> Human <400> 13

Met Asn Lys Leu Leu Cys Cys Ala Leu Val Phe Leu Asp lie Ser lie -20 -15 -10 *

Lys Trp Thr Thr Gin Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp -5 -11 5 10

Glu Glu Thr Ser His Gin Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr 15 20 25

Tyr Leu Lys Gin His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro 30 35 40

Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 45 50 55

Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu Gin Tyr Val Lys Gin Glu 60 65 70 75

Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr 80 85 90

Leu Glu lie Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe 95 100 105

Gly Val Val Gin Ala Gly Cys Arg Arg Arg Pro Lys Pro Gin lie Cys 110 115 120 lie 125 -55-

Claims (1)

200820980 X. Patent application scope: 1. A Xufang preparation characterized by containing osteoclast formation inhibitor, its congeners or variants or modifications thereof, and containing calcium phosphate nanocrystals and polysaccharides. The porous preparation of the porous particles, and the osteoclast formation inhibitor, the homologous or variant thereof or the modifications thereof are combined with the entire inner surface of the pores of the porous particles as a secondary divalent metal ion. 2. The releasable preparation according to claim 1, wherein the porous particles uniformly disperse the calcium phosphate nanocrystals and the polysaccharide. 3. The releasable preparation according to claim 1 or 2, wherein the calcium phosphate is a divalent metal selected from the group consisting of magnesium, zinc, lanthanum and cerium, in combination with calcium or a part of calcium. 4. The releasable preparation according to any one of claims 1 to 3, wherein the molar ratio of calcium to phosphorus in the calcium phosphate is from 1.3 to 3.4. 5. The releasable preparation according to any one of claims 1 to 4, wherein the calcium phosphate in φ contains a carbonate group. 6. The releasable preparation according to any one of claims 1 to 5, wherein the long diameter of the calcium phosphate nanocrystal is 1 x 1 〇 2 nm or less. 7. The releasable preparation according to claim 6, wherein the long diameter is from 1 nm to 1 X 1 02 nm °. The releasable preparation according to any one of claims 1 to 7 wherein the polysaccharide is It is selected from the group consisting of chondroitin sulfate, hyaluronic acid, heparin and heparin sulfate. 9. The releasable preparation according to item 8 of the patent application, wherein the polysaccharide is sulfur-56-200820980 chondroitin or hyaluronic acid. The releasable preparation according to any one of claims 1 to 9, wherein the content of the polysaccharide is 4 x 10% by weight or less. 11. The releasable preparation according to claim 10, wherein the content is 4 x 10% by weight to 1 x 10% by weight. 12. The releasable preparation according to any one of claims 1 to 11, wherein the porous particles have a diameter of 1 x 1 02 μm or less. 1 3 . For example, the Xu Fang type preparation of the patent scope of Item 12, wherein the diameter is Lu 1 from m to lxl 〇 2 / / m. 14. The releasable preparation according to any one of claims 1 to 13, wherein the porous particles have a specific surface area of from 5 x 10 m 2 /g to 2 x 10 2 m 2 /g. 15. The releasable preparation according to any one of claims 1 to 14, wherein the porous particles have a porosity of from 3 x 10% to 8 x 10%. 16. The releasable preparation according to any one of claims 1 to 15, wherein the in vivo absorption of the porous particles is within 6 months. 17. The releasable preparation according to any one of claims 1 to 16, wherein < wherein the porous particles are produced by spray drying. 18. The releasable preparation according to any one of claims 1 to 17, wherein the divalent metal ion is selected from the group consisting of zinc ions, magnesium ions, calcium ions, barium ions, barium ions, and copper ions. . 19. The releasable preparation according to claim 18, wherein the divalent metal ion is a zinc ion. 20. The releasable preparation according to any one of claims 1 to 19, which is a therapeutic agent for abnormal bone metabolism. -57-200820980 2 1. A releasable preparation as claimed in any one of claims 1 to 9 which is a fracture healing accelerator. 22. The bone defect filling material comprising the releasable preparation according to any one of claims 1 to 19. 2 3 . A method for preparing a sputum release preparation, which comprises the steps of: spray-drying a suspension containing crystals of gold phosphate and polysaccharides to obtain a porous particle containing calcium phosphate nanocrystals and polysaccharides. 'Incorporating the aforementioned porous particles into a solution containing an osteoclast formation inhibitor, the same class or variant or a modification thereof, and forming an osteoclast formation inhibitor, its congener or variant or The modified body is carried on the entire inner surface of the pores of the porous particles, and the porous plasmid containing the osteoclast formation inhibitor, the homologous substance or the variant or the modified body thereof is added to contain two A solution of a valent metal ion, whereby the osteoclast formation inhibitor, its congener or variant or a modification thereof is combined with the entire inner surface of the pore of the porous particle to intervene in the divalent metal ion. -58-