JP5484648B2 - Bone mineralization promoting factor - Google Patents

Description

  This application claims the priority from Japanese Patent Application No. 2003-059130, which is incorporated herein by reference.

  The present invention relates to a means for promoting bone mineralization at a desired site in a living body. More specifically, the present invention relates to a method for promoting bone mineralization and a factor for promoting mineralization used therein. In particular, it relates to a novel use of DMP1 (Dentin matrix protein 1).

As pharmacologically active agents having osteogenic potential, factors that cause osteoinduction by differentiating osteogenic cells such as rhBMP-2, rhBMP-7, and TGF-β, bFGF, PTH and the like are known. However, the existence of substances that increase the rate of calcification of the bone matrix and form mature bone that has been calcified earlier has not been known so far.
Bone, dentin, and cementum are calcified with calcium phosphate deposited in the extracellular matrix. The extracellular matrix is mainly collagen, but also includes a non-collagenous matrix, and most of the non-collagenous matrix is an acidic phosphorylated protein. It has been believed that the collagen fibers function as a scaffold on which hydroxyapatite crystals are deposited, and that acidic phosphorylated proteins are involved in calcification of the extracellular matrix. The acidic phosphorylated protein identified by recombinant DNA technology was first named AG1 (J. BiolChem 1993; 268: 12624-12630: Non-Patent Document 1) and later changed to DMP1 (J. Histchem Cytochem 1994; 42: 1527-1531: Non-patent document 2). A cDNA clone of DMP1 was identified from a rat tooth cDNA library and has been considered to be specific for dental dentin (J. Histchem Cytochem 1994; 42: 1527-1531). Later, it was shown that its expression is also present in other calcified tissues.
Recently, the present inventor has found that DMP1 gene is present in the genome even in birds, which are animal species lacking the ability to form teeth, and DMP1 is expressed in bone tissue. It was shown that there is an important function in the organization (J. Mol Evol 2000; 48: 160-166: Non-Patent Document 4). Further, the present inventor has found that in bone tissue of birds and mammals, the DMP1 gene is not expressed in osteoblasts that secrete bone matrix, and is not expressed in calcified bone matrix regardless of animal species. It was specifically expressed in the existing bone cells, and the protein was found to be distributed only in the bone matrix surrounding the bone cells (J. Bone Miner Res2001; 16: 2017-2026: Non-patent document 5). A characteristic aspect of DMP1 is that it has a very high content of acidic amino acids and is highly phosphorylated in the tissue, so that it is highly negatively charged in the tissue.
J. et al. BiolChem 1993; 268: 12624-12630 J. et al. Histchem Cytochem 1994; 42: 1527-1531 J. et al. Histchem Cytochem 1994; 42: 1527-1531 J. et al. Mol Evol 2000; 48: 160-166 J. et al. Bone Miner Res 2001; 16: 2017-2026

An object of the present invention is to provide a novel means for promoting bone mineralization, and particularly to find a new use of DMP1. Since DMP1 is highly negatively charged in tissues, it is presumed that it binds calcium and thereby promotes calcification of the extracellular matrix. To date, several non-collagenous proteins have been identified in calcified tissue, but none have promoted extracellular matrix calcification in vivo. In order to obtain possible adaptations of DMP1 in hard tissue engineering, the characteristics of DMP1 molecules and their possible functions in the mineralization process were investigated.
In order to solve the above-mentioned problems, the present inventor has established means for specifically expressing DMP1 in bone tissue. From the examination of its function, the production of an appropriate amount of DMP1 does not cause side effects on the living body, and the bone density can be reduced. The present invention was completed by finding out that it promotes the rise, and consequently, calcification of bone.
The present invention consists of the following.
1. A method for promoting bone mineralization, wherein a target bone formation site is in a negatively charged state.
2. The method of item 1, wherein the negative ion charge state is achieved by the presence of the compound.
3. 3. The method according to item 2 above, wherein the compound is DMP1 or a DMP1 derivative.
4). 4. The method of item 2 or 3, wherein the presence of the compound is due to filling.
5. 4. The method according to item 2 or 3 above, wherein the presence of the compound is due to an increase in production by a gene transfer method in selective bone tissue.
6). 5. The method according to item 4 above, wherein the presence of the compound is binding or coexistence with the matrix substance.
7). 7. The method according to item 6 above, wherein the matrix substance to be bound is collagen.
8). A bone mineralization promoting factor comprising DMP1 or a DMP1 gene-carrying vector used in the method according to any one of 2 to 7 above.
9. 9. The calcification promoting factor according to 8 above, which is a derivative of DMP1 or the gene-carrying vector.
10. 10. The calcification promoting factor according to 8 or 9 above, wherein the gene-carrying vector carries a promoter suitable for specific expression regulation in bone.
11. A bone or tooth formation method using the bone mineralization promoting method according to any one of 1 to 7 above.
12 A method for treating a bone or dental disease and / or disorder using the method for promoting bone mineralization according to any one of 1 to 7 above.
13. A therapeutic agent for a bone or dental disease and / or disorder comprising the calcification promoting factor according to any one of the above items 8 to 10 as an active ingredient.
14 A bone or tooth forming agent comprising the calcification promoting factor according to any one of 8 to 10 as an active ingredient.
15. A bone or dental pharmaceutical composition comprising the calcification promoting factor according to any one of 8 to 10 as an active ingredient.

FIG. 1 shows the measurement of bone density of cortical bone on the distal side of the femur of a mouse.
FIG. 2 is a diagram in which the bone density of cortical bone at the diaphysis of the femur of a mouse was measured.

Explanation of symbols

The horizontal axis in FIG. [15 slices were prepared distally from the distal side to the diaphysis / femur. Normally, calcification starts from the distal side and the degree of calcification (bone density) increases toward the diaphysis / femur], the vertical axis indicates bone density (mg / cm ³ ), and Tg2M is 5 times the force of DMP1 Expression, Tg3H means 30-fold forced expression of DMP1, and WT means wild type. ● represents Tg2M, ▲ represents Tg3H, and ◯ represents a wild type. Each value is an average value. In FIG. 2, the horizontal axis represents Tg2M, Tg3H, and WT, and the vertical axis represents bone density (mg / cm ³ ).

DMP1 is an acidic protein that is an extracellular matrix protein having 400 to 550 amino acids. The amino acid sequence and gene sequence are disclosed by Toyosawa et al. (J Mol Evol 1999; 48: 160-166, Gene 1999; 234: 307-314, J Mol Evol 2000; 50: 31-38, etc.). The DMP1 gene is a known gene, and the sequence of DMP1 has been reported from 10 kinds of animals including humans. From amino acid sequence comparisons, all species of DMP1 are known to begin with a hydrophobic leader sequence containing 16-21 amino acids. The cell-adhesive peptide RGD motif is not specific to the DMP1 molecule but has been found to be present in many other acidic phosphorylated proteins such as osteopontin, bone sialoprotein, and dentin sialophosphoprotein. The Arg-Gly-Asp sequence is strictly conserved with the mammalian DMP1 sequence and is thought to have its important biological functions. In addition, DMP1 contains a very large amount of acidic amino acids, and a large number of motif sequences including Ser undergo phosphorylation. Therefore, it is judged that DMP1 is highly negatively charged in tissues. This suggests that DMP1 has a strong binding ability with calcium ions and is necessary for calcification of the extracellular matrix.
One feature of the present invention is that a relationship between the negative ion state at the bone formation site and the bone mineralization rate has been found. The inventor selectively expressed the DMP1 gene in bone tissue and accumulated the protein in the bone matrix. And the expression level was adjusted by changing various conditions, and it was confirmed that the optimal concentration exists in the production amount of DMP1 and the bone mineralization rate. It was confirmed by the present invention that DMP1 is a highly negatively charged protein in the tissue, and this is strongly related to the rate of bone mineralization. Calcification is the deposition of calcium phosphate in the extracellular matrix, but DMP1 attracts calcium ions and collagen attracts phosphoric acid, so that calcium ions and phosphate accumulate at the same site to form calcium phosphate crystals. It is considered that calcification is promoted.
In the present invention, it was confirmed that DMP1 and its derivatives function as calcification promoting factors. It has already been established that DMP1 can be produced by genetic engineering, and it is possible to use recombinant DMP1 produced in large quantities in a system using a known and widely used E. coli host promoter system. In particular, post-translational modifications such as sugar chain structure are not necessary, and DMP1 produced by E. coli is sufficient. In the present invention, the DMP1 derivative is a sequence mutation such as one to several substitutions / deletions / additions of amino acids that maintain the calcification-promoting function of DMP1, particularly a conserved sequence confirmed in mammals, and DMP1 A synthetic polypeptide consisting of several tens of amino acids partially mimicking the amino acid sequence is also targeted. Examples of the derivative include a protein having a scaffold for bone formation or a biodegradable synthetic resin. As proteins, collagen, particularly type I collagen, and polylactic acid derivatives can be suitably used as biodegradable synthetic resins. The bond may be any physical bond or chemical bond, but is preferably a disulfide bond. Further, even if DMP1 is not derivatized, it may be a mixture with a protein having a matrix structure or a biodegradable synthetic resin.
In the calcification promoting means of the present invention, the amount of DMP1 used to promote calcification per 1 ml of material may be any concentration as long as it promotes bone mineralization. For example, it is usually 0.1 μg or more, preferably several μg, more preferably 5 to 20 μg.
For example, in order to fill DMP1 into the target bone forming part, it is preferably prepared in a paste form by general-purpose means by blending with a known composition. For example, a blended composition with collagen, a polylactic acid derivative, or the like can be used.
A vector carrying the DMP1 gene or a derivative gene thereof can exhibit the bone mineralization promoting function of the present invention by means of specific gene introduction into bone tissue. The object of the present invention can also be achieved by means for carrying a gene in a self-propagating vector. That is, a vector carrying the DMP1 gene can also be included in the calcification promoting factor of the present invention. Desirably, the gene introduction means is based on an integration method, for example, a liposome method, a calcium phosphate method, an electroporation method, or the like. However, a transient expression method such as a liposome method, a calcium phosphate method, an electroporation method, a viral vector method, an atelocollagen method, or the like may be used. The vector to be used is not particularly limited, and it is applicable to all recombinant vectors capable of introducing an inserted DNA fragment by a usual recombination experiment, such as a known plasmid, phage, cosmid, BAC, YAC, recombinant virus, transposon, etc. be able to. The vector can be constructed together with a promoter and an enhancer which are naturally suitable for a combination known per se. For example, a commercially available protein expression vector in which a promoter suitable for a normal host is inserted can be used.
Specific examples include ZAP Express (manufactured by Stratagene), pSVK3 (manufactured by Amersham Pharmacia Biotech), pEGFP-C1 (manufactured by Clontech), and atelocollagen. In the examples of the present invention, the mouse pro-α1 (I) collagen promoter was used as the promoter, but other promoters such as osteocalcin promoter may be used. Since this promoter is a promoter of a protein that is secreted specifically by bone-forming cells, it provides a powerful means for specifically expressing DMP1 in bone tissue.
The DMP1 polynucleotide is inserted into the vector by ligating the polynucleotide or a DNA fragment containing the polynucleotide so as to be placed under the control of the promoter downstream of the promoter in the vector. In addition, a structure in which a Kozak sequence (Kozak, M., Gene, 234, 187, (1999)) is inserted between the promoter and DMP1, or a DNA encoding a polypeptide serving as a tag is inserted downstream of DMP1. A vector having the same is also preferably used. The polypeptide to be a tag is not particularly limited, and examples thereof include a FLAG tag (BioTechniques, 7, 580, (1989)).
A homologous recombination technique (AA Vertes et al., Biosci. Biotechnol. Biochem., 57, 2036 (1993)), or a transposon or insertion that directly inserts a promoter-linked DMP1 polynucleotide into the chromosome of the target cell. It can be expressed using a sequence (AA Vertes et al., Molecular Microbiol., 11, 739 (1994)) and the like.
DMP1 or a derivative thereof thus prepared can be directly loaded into the bone formation site where bone mineralization is desired. Further, a vector carrying the DMP1 gene or a derivative gene thereof can be administered by direct injection, gene gun, injection or the like.
According to the present invention, DMP1 or a derivative thereof is used for filling a joint part of a fracture, filling a bone after removal of bone due to infection, filling a joint such as ceramic after extensive resection by tumor or osteomyelitis and existing bone, alveoli It can be effectively used for filling the alveolar bone after removal of the alveolar bone due to pus leakage, filling for alveolar ridge formation of dentures, filling for alveolar bone formation for implant implantation, and the like. With the introduction of the present invention, after osteogenic cells grow and extracellular matrix is produced, the level of calcification rises early, and early function can be demonstrated as mature bone.
Moreover, the pharmaceutical composition containing the calcification promoting factor of the present invention as an active ingredient can contain a pharmaceutically acceptable salt. Examples of pharmacologically acceptable salts include conventional non-toxic salts, that is, acid addition salts and salts with various bases. More specifically, inorganic acid salts such as hydrochloric acid, nitric acid and sulfuric acid, organic acid salts such as acetic acid, citric acid, fumaric acid and tartaric acid, sulfonic acid salts such as methanesulfonic acid and p-toluenesulfonic acid, and alanine and leucine Amino acid salts such as glutamic acid, and inorganic base salts such as alkali metal salts (for example, sodium salts, potassium salts), alkaline earth metal salts (for example, magnesium salts, calcium salts), and triethylamine salts, pyridine salts, picoline salts, ethanol Organic amine salts such as amine salts, triethanolamine salts, dicyclohexylamine salts, N, N′-dibenzylethylenediamine salts and the like can be mentioned.
The technical scope of the present invention is not limited to the bone mineralization promoting method and the bone mineralization promoting factor, but the bone or tooth formation method using the bone mineralization promoting method, the bone or dental disease, and It extends to methods for treating disorders. Furthermore, the present invention includes a pharmaceutical composition containing the calcification promoting factor of the present invention as an active ingredient, and includes a therapeutic agent for bone or dental diseases and / or disorders, and further a bone or dental formation agent.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

PNASSβ (CLONTECH) for selective expression in the bone tissue from the start codon to the stop codon of the translation region of the rat DMP1 gene with the pro-α1 (I) collagen promoter (Reference: J Cell Biol. 1995; 129: 1421-1432). Lab., Inc.) using a restriction enzyme to construct a construct for producing a transgenic mouse. Using this construct, a gene was introduced into a C57B / L fertilized egg to produce a DMP1 transgenic mouse, and the bone density of the bone tissue in which DMP1 was forcibly expressed (4 groups per group, 3 groups) was examined. The bone density at each site of the femur at 3 months of age was measured. The expression level of DMP1 in bone tissue was examined by Northern, the expression site was confirmed again by in situ hybridization, and about 5 times normal expression of DMP1 was forcibly expressed, about 30 times normal expression. These were compared with normal mice.
FIG. 1 shows the measurement of bone density of cortical bone on the distal side of the femur of a mouse. The horizontal axis is sliced in 0.5 mm increments from the distal side. For each of up to 15 slices, each bone density (mg / cm ³ ) of the cortical bone was measured with a peripheral quantitative computed tomography (pQCT). In long shafts such as the femur, the bone density of cortical bone increases as it goes distally from the epiphyseal cartilage to the shaft. In the DMP1 transgenic mouse (Tg2M group) in which DMP1 was forcibly expressed about 5 times the normal, the bone density was significantly increased in each slice portion as compared to the normal mouse (WT group). On the other hand, the DMP1 transgenic mice (Tg3H group) in which DMP1 was forcibly expressed about 30 times normal did not change in bone density compared to normal mice. It has been confirmed that there is an adaptive amount for supplementing DMP1, and that no effect is obtained when a large amount is accumulated in bone tissue. That is, when it is forced to express about 5 times the normal, the rate of increase in bone density of cortical bone is faster than normal, which means that calcification is promoted. Here, the effect of DMP1 was confirmed by the gene introduction method as an experimental system, but this effect proved that the same effect can be induced even if DMP1 itself is filled directly or together with a matrix substance such as collagen in the target site. Think of it as being.
FIG. 2 shows the bone density of cortical bone at the diaphysis of the femur of a mouse. The horizontal axis represents each Tg2M (DMP1 transgenic mouse in which DMP1 is forcibly expressed about 5 times normal), Tg3H (DMP1 transgenic mouse in which DMP1 is forcibly expressed about 30 times normal), WT (normal) from the left. Each bone density (mg / cm ³ ) was measured in the same manner as above. As a result, there was no difference in bone density between the Tg2M, Tg3H group and the WT group. This indicates that DMP1 no longer induces excessive bone mineralization without pharmacological action under conditions of plateaus with increased bone mineral density and bone density.

  The means of the present invention provides an effective means for facilitating early bone formation by increasing the speed of calcification by facilitating calcification in the extracellular matrix portion that becomes a scaffold for calcification during bone formation. . As a result, it provides new clinical significance in bone treatment. In particular, the means of the present invention promotes bone mineralization during the bone formation period, but when the level of mineralization is increased and mature bone formation is completed, the function of promoting mineralization is no longer exhibited. Therefore, it can be a therapeutic means for bone diseases with excellent safety without causing side effects such as hypercalcification.

Claims (6)

Partially mimics the mammalian DMP1, or one or several substitutions, deletions or additions of the mammalian DMP1 amino acid sequence that maintains the calcification promoting function of DMP1, or the mammalian DMP1 amino acid sequence. A synthetic polypeptide consisting of several tens of amino acids (hereinafter referred to as DMP1 derivative) and type I collagen are administered in combination, and in combination or coexistence thereof, calcification of the target bone formation site is promoted. A bone or tooth forming agent comprising DMP1 or a DMP1 derivative. The bone or tooth forming agent according to claim 1, wherein the administration of DMP1 or DMP1 derivative is by filling the target bone formation site. The bone or tooth forming agent according to claim 1, wherein the administration of DMP1 or DMP1 derivative is due to an increase in production by a gene transfer method in selective bone tissue. The bone or tooth forming agent according to any one of claims 1 to 3 , wherein the DMP1 or the DMP1 derivative is DMP1. The DMP1 or DMP1 derivative is a sequence variant of one to several substitutions, deletions or additions of the amino acid sequence of mammalian DMP1 that maintains the calcification promoting function of DMP1. The bone or tooth forming agent described. The bone or tooth forming agent according to any one of claims 1 to 2, wherein the DMP1 or DMP1 derivative is a synthetic polypeptide consisting of several tens of amino acids partially mimicking the amino acid sequence of mammalian DMP1.

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