JP2009067700A - Bone-forming agent and bone-forming method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a bone-forming agent capable of forming a new bone even in a periodontal disease in the state wherein existing teeth is hardly left. <P>SOLUTION: The bone-forming agent comprises a peptide comprising (a) an amino acid sequence described in the sequence number 1 or (b) an amino acid sequence described in the sequence number 2 or a pharmaceutically acceptable salt thereof. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a bone forming agent containing a specific peptide and a bone forming method.

  Conventionally, recombinant human bone morphogenetic protein (rhBMP) has been reported as a substance causing bone formation (see Non-Patent Documents 1 and 2). In addition, platelet rich plasma (PRP), platelet-derived growth factor (PDGF), and the like have been reported as substances that indirectly assist bone formation (see Non-Patent Document 2). However, only rhBMP is known as a substance that brings about bone formation in soft tissue, and the development of candidate substances that bring about effective bone formation is further desired.

  In addition, periodontal tissue is in a special environment with cementum and alveolar bone across the periodontal ligament, so it is thought that not only bone formation but also regeneration of cementum and periodontal ligament is important. Yes.

  On the other hand, although Emdogain (registered trademark) (manufactured by BIORA, Sweden), a substance extracted from porcine tooth germ, has not been disclosed, its main component is an enamel matrix derivative, such as periodontal disease. Therefore, regeneration of cementum can be brought about especially among damaged periodontal tissues (gingiva, periodontal ligament, alveolar bone, cementum), and lost periodontal tissue can be recovered to some extent (Non-Patent Document 3). To 6). However, since Emdogain (registered trademark) is a biological sample, there is a risk that there may be unknown risk factors such as viruses.

Wozney JM et al. J. Cell Sci. Suppl. 1990; 13: 149-56 Tomoyasu A et al. Biochem. Biophys. Res. Commun. 2007; 361: 62-7 Hammarstroem L et al. J. Clin. Periodontol 1997; 24 (9): 669-77 Gestrelius S et al. J. Clin. Periodontol 1997; 24 (9): 678-84 Zetterstroem O et al. J. Clin. Periodontol 1997; 24 (9): 697-704 Heijl et al. J. Clin. Periodontol 1997; 24 (9): 705-14

  An object of the present invention is to provide an osteogenic agent capable of newly forming bone in periodontal tissue in which almost no existing bone remains.

  As a result of studying to solve the above-mentioned problems, a) a partial sequence of a protein induced in vivo by emdogain, a) an amino acid sequence WYQNMIR described in SEQ ID NO: 1, or b) a peptide represented by amino acid sequence WYQNMLR described in SEQ ID NO: 2 The present invention has been completed by finding that bone formation is effectively promoted in soft tissues.

  Therefore, the present invention relates to an osteogenic agent containing at least a) an amino acid sequence represented by SEQ ID NO: 1, or b) a peptide comprising the amino acid sequence represented by SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof.

  In the osteogenic agent of the present invention, the peptide content is preferably 7.5 to 30 mg / mL.

  The present invention also relates to a method for forming bone in animals other than humans by locally administering 10-60 mg / kg of an osteogenic agent in terms of an active ingredient.

  By using the osteogenic agent of the present invention, new bone can be formed even in soft tissue where there is no existing bone. Further, in rats, bone can be dramatically formed at a rate of about 2 weeks by a single administration, so that it can be used as a simpler and more effective bone formation agent.

  In addition, since the peptide of the present invention is derived from emdogain, it can regenerate cementum in addition to bone formation, and as a result, it can regenerate periodontal tissues including periodontal ligament and alveolar bone. It can be an effective periodontal tissue regenerative drug.

  The osteogenic agent of the present invention contains, as an active ingredient, at least a) a peptide containing the amino acid sequence WYQNMIR described in SEQ ID NO: 1, or b) a peptide containing the amino acid sequence WYQNMLR described in SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof. . That is, a peptide in which several amino acids to several tens of amino acids are added to both ends of the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 2, specifically, about 1 to 7 can be used in the present invention. .

  The peptide of the present invention can be produced by an ordinary synthesis method such as a liquid phase method or a solid phase method, but is generally produced by a solid phase synthesis such as a Boc method or an Fmoc method. Of course, DNA encoding the peptide of the present invention can be incorporated into a vector and produced in a host cell.

  The osteogenic agent of the present invention may contain both a peptide containing the amino acid sequence shown in SEQ ID NO: 1 and a peptide containing the amino acid sequence shown in SEQ ID NO: 2.

  The peptide of the present invention was derived in vivo from emdogain, and no toxic reaction was observed in the normal trial range.

  The osteogenic agent of the present invention is preferably applied topically and applied directly to the open wound surface.

  As the dosage form of the osteogenic agent of the present invention, any dosage form usually used for topical application can be used, but examples thereof include liquids, emulsions, gels, etc. In order to give releasability, an emulsion or gel is preferable, and it is desirable to use it in a syringe.

  The osteogenic agent of the present invention can be produced by combining a peptide as an active ingredient with a pharmaceutically acceptable base material. As a usable substrate, propylene glycol is preferable from the viewpoint of maintaining the sustained release effect of the peptide, but is not limited thereto. When propylene glycol is used, it is completely absorbed in about 14 days.

  For the osteogenic agent of the present invention, biocompatible carriers such as collagen and atelocollagen, which are usually used in this field, can also be used.

  In the osteogenic agent of the present invention, the concentration of the active ingredient peptide is preferably 7.5 to 30 mg / mL, more preferably 10 to 20 mg / mL, and most preferably about 15 mg / mL. When the peptide concentration is lower than 7.5 mg / mL, bones tend not to be formed.

  The dose of the osteogenic agent of the present invention is preferably 10 to 60 mg / kg, more preferably 20 to 40 mg / kg in terms of the active ingredient peptide. If it is less than 10 mg / kg, bones tend to be hardly formed.

  Since the osteogenic agent of the present invention can form new bone even in soft tissues where there is no existing bone, it should be applied not only to periodontal disease but also to other cartilage and bone defects in general. Can do. Such diseases include traumatic bone injury, osteoporosis, jaw cysts, jaw tumors and the like. In addition, since the peptide that is an active ingredient of the osteogenesis agent of the present invention is derived from emdogain, it causes cementum regeneration in addition to bone formation, and as a result, periodontium including periodontal ligament and alveolar bone lost. It is considered that tissue regeneration can be achieved, and can be an effective periodontal tissue regeneration agent.

  EXAMPLES Hereinafter, although this invention is demonstrated using an Example, this invention is not limited to these.

Reference example 1
Powdered Emdogain (registered trademark) (BIORA, Sweden) was adjusted to 30.0 mg / mL in propylene glycol and inoculated subcutaneously on the back of 6-week-old male Sprague-Dawley rats, 7 days later. The formed round body was excised. That is, the tissue was fixed in formalin, embedded in paraffin, pasted on a slide glass as a 7 μm-thick section, and dried in a 40 ° C. paraffin dryer. The part corresponding to the circular body was left under a stereomicroscope, and the other part was removed with a scalpel (No. 11, Keisei Medical, Tokyo). Round bodies are scraped and dissolved in the Laemmli sample buffer in the sample tube, and the lysate is electrophoresed with 12.5% SDS-polyacrylamide gel for 55 minutes, 40 mA, and then fixed with 40% methanol and 15% ethanol. The silver was stained and the protein was visualized. The visualized protein was excised from the gel and the peptide was fragmented overnight at 37 ° C. in 12.5 μg / L trypsin prepared with 50 mM ammonium bicarbonate. The sample was crystallized in 10 mg / mL 2,5 dihydroxybenzoic acid distilled water. It was analyzed by direct MALDI-Qq-TOF MS / MS QSTApulsar i (Applied Biosystems, Foster City, California, USA).

  As a result, a plurality of fragments were obtained, but a peak showing strong ionic strength at a molecular weight of 1,010 was obtained. The amino acid sequence of the longest fragment was 192 amino acids. Various fragments contained a common 7 amino acid residue sequence, ie WYQNMLR or WYQNMIR. These sequences were searched for the database NCBInr (mammalian) using the database search software MASCOT, bovine amelogenin II precursor (WYQNMLR), amelogenin leucine dominant amelogenin polypeptide (LRAP (SEQ ID NO: 3)), and It was found to be homologous to porcine amelogenin precursor (WYQNMIR) (J. Periodontol 2005; 76: 1934-1941).

Example 1
Dissolve the peptide WYQNMIR (SEQ ID NO: 1) in distilled water to 15.0 mg / mL, cool 1 mL of it in ice water, and prepare it for later excision so that the injection site can be clearly understood. 40 mg of impression material (Nissin, Alflex dust-free normal set, medical device permission number 26BZ5014, Kyoto) was added and mixed. 0.3 mL was injected subcutaneously into the back of Sprague-Dawley rats using a 1 mL Terumo syringe with a 19 G × 1 1/2 (outer diameter 1.10 mm, inner diameter 0.78 mm) needle. 14 days after the injection, the tissue was excised, fixed in formalin, made into paraffin sections, stained with hematoxylin and eosin, bone tissue 1 stained in purple, cartilage tissue 2 stained in purple, and calcium in the cartilaginous tissue. A tissue image showing cartilaginous ossification was confirmed in which calcified cartilage tissue 3 with advanced deposition was seen (FIG. 1).

It is a tissue image (magnification × 10) of hematoxylin and eosin staining of a rat subcutaneous tissue injected with the peptide of SEQ ID NO: 1. Bone tissue 1 stained in magenta, cartilage tissue 2 stained in bluish purple, and calcified cartilage tissue 3 in which calcium deposition has progressed in the cartilage tissue are observed.

Explanation of symbols

1 Bone tissue 2 Cartilage tissue 3 Calcified cartilage tissue

Claims (3)

at least,
An osteogenic agent comprising a) an amino acid sequence represented by SEQ ID NO: 1 or b) a peptide comprising the amino acid sequence represented by SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof. The osteogenic agent according to claim 1, wherein the content of the peptide is 7.5 to 30 mg / mL. The bone formation method in the animal except a human by converting 10 to 60 mg / kg locally in conversion of the osteogenic agent of Claim 1 into an active ingredient.

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