JPH0717876A - Agent for treatment of periodental disease - Google Patents

Abstract

PURPOSE:To obtain the therapeutic agent excellent in regeneration of periodental tissue and dentine, useful, e.g. for the regeneration of periodental tissue lost by the progress of paradentitis and having excellent safety and stability by compounding basic fibroblast growth factor and its homolog. CONSTITUTION:This therapeutic agent is obtained by compounding basic fibroblast growth factor (bFGF) and/or its homolog (preferably human bFGF or its homology. It is preferable to use the bFGF produced by microorganism such as E. coli or cultured cell with the use of gene recombination to stably obtain the pharmaceutical agent on an industrial scale. Further, the pharmaceutical agent is preferably applied at the affected part or its neighborhood 1-3 times a day at a single dose of 1-1000mug in the case of human bFGF and/or its homolog.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a therapeutic agent for periodontal disease containing a basic fibroblast growth factor and / or its homologue. More specifically, periodontitis, cysts, oral cancer, tooth extraction, jaw deformity, and gingival diseases caused by caries, periodontal tissue diseases such as periodontal ligament tissue, alveolar bone, cementum, jaw bone and dentin deformation. , A drug used to promote healing of defects or pathological conditions.

[0002]

2. Description of the Related Art Among periodontal diseases, it is becoming clear that the cause of periodontitis is caused by bacteria that form colonies on the root surface. Emphasis is placed on removing plaque from the tooth surface and keeping it plaque-free.

Also, in periodontal surgery performed for severe periodontitis, the causative factors such as plaque and tartar adhering to the root surface in the pocket and pathological tissue are similarly removed to clean the oral cavity. The purpose is to improve in an environment that makes it more effective. Furthermore, recently, in addition to these therapies, chemotherapy with antibiotics has been tried.

However, although these therapies eliminate the lesions and prevent the progression of periodontitis, they do not actively repair and regenerate the destroyed periodontal tissue or absorbed alveolar bone, and they do The healing form depends on one's healing ability.

[0005] The healing forms include regeneration healing or new attachment healing and reattachment.
hment) divided into healing. Regenerative healing is a healing mode in which postoperative healing is accompanied by regeneration of the cementum in which the implantation of collagen fibers capable of obtaining functional recovery is recognized. on the other hand,
Reattachment refers to a long epithelial attachment observed on the root surface of the tooth or an accumulation image of collagen fiber bundles on the root surface for which functional recovery cannot be expected.

[0006] Many basic and clinical studies have been conducted so far, but most of them are histopathological studies, in which epithelial adhesion due to invasion of epithelial cells (down growth) from the dentition toward the root tip side. This indicates that it is difficult for new adhesion. Particularly in advanced periodontitis, complicated pocket formation and various types of bone destruction are involved, and bone regeneration is not possible in cases of high bone resorption. Also, Melcher (Melcher, AH: J.Periodontol., 47,256-26
(0,1976), proliferation of periodontal ligament-derived cells is necessary for recovery of periodontal tissue with new attachment to the exposed root surface,
It has been reported that when the cells that reach the root surface are epithelial cells, gingiva-derived cells, or bone-derived cells, long junctional epithelial formation, root resorption, and ankylosis occur.

From the above, for the purpose of obtaining functional recovery, (1) treatment of the root surface with citric acid (2) local administration of fibronectin (3) epithelium using a highly biocompatible filter "Tissue regeneration induction method (GTR method)" that blocks the invasion of sex cells and induces periodontal ligament cells to the root surface (4) For the resorption of alveolar bone, to the bone defect site using various transplant materials Transplantation is being studied in basic and clinical ways.

However, (1) is harmful to cells,
(2) is the stability and antigenicity of high molecular weight fibronectin, (3) is the need for re-operation for extraction, and
(4) has various problems such as collection, storage, sterilization, or absorbability of transplant material, and has periodontal tissue and alveolar bone regeneration that have safety, easiness of formulation, and effectiveness. The development of a drug that promotes

[0009]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a therapeutic agent for periodontal disease which is excellent in safety, stability and effectiveness.

[0010]

The present inventors have succeeded in developing a drug useful as a therapeutic agent for periodontal disease using basic fibroblast growth factor and / or its homologue, and the present invention Has been completed.

Basic fibroblast growth factor (also referred to as basic fibroblast growth factor; hereinafter referred to as bFGF) is
Pituitary gland, brain, retina, luteum, adrenal gland, kidney, placenta, prostate,
It is a peptidic cell growth factor whose presence has been confirmed in thoracic lines, chondrosarcoma, and macrophages ("Cell growth factor part II" edited by the Japanese Society for Tissue Culture, pages 15-20, Asakura Shoten). FGF initially shows a strong proliferative effect on fibroblasts such as BALB / c3T3 cells (D. Gospodarowi
cz: Nature 249: 123, 1974), but then promotes proliferation of most cells of mesodermal origin, especially vascular endothelial cells (D. Gospodarowicz: National Cancer).
Institute Monograph 48: 109.1978) also promotes the proliferation of satellite cells in skeletal muscle (RE Allen: E
xp. CellRes. 152: 154, 1984). In recent years, clinical application of bFGF in wound treatment and application of FGF to blood vessel repair using angiogenic action have been performed. Further, recently, a fibroblast-derived epidermal growth factor was discovered, and it is considered that the production of the fibroblast-derived epidermal growth factor is increased by activating the fibroblast with bFGF.

In the treatment of periodontal disease, these bFG
There are no reports of application of F and / or its homologues. The present inventors have found that these bFGF and / or homologues thereof have excellent periodontal tissue regeneration and alveolar bone regeneration promotion effects and are useful for the treatment of periodontal disease.

The bFGF and / or homologues thereof in the present invention are isolated or purified from those produced in microorganisms or cultured cells by natural or genetic recombination techniques, or by chemically or biologically modifying them. Is obtained by Further, as bFGF, human bFGF or a homolog thereof is particularly preferable.

Further, in the therapeutic agent for periodontal disease of the present invention,
A homologue of bFGF may be used as the active ingredient. Here, the homologue of bFGF means the following or the following polypeptide.

BFGF produced in certain mammals
A polypeptide consisting of an amino acid sequence substantially the same as the above.
A substantially identical amino acid sequence means 1 in the amino acid sequence.
~ 6 amino acids substituted with another type of amino acid and having the biological activity of bFGF.

BFGF produced in a particular mammal
A polypeptide in which an additional amino acid segment is added to the N-terminal and / or the C-terminal of, or the N-terminal and / or the C-terminal of the above-mentioned polypeptide. The additional amino acid segment consists of 1 to 12 amino acids, b
It means one that does not impair the biological activity of FGF or the biological activity of the above-mentioned polypeptide.

Human bFGF is a polypeptide of 146 amino acids. In the therapeutic agent for periodontal disease of the present invention,
As the homologue of human bFGF (the above-mentioned homologue), for example, a polypeptide of 146 amino acids described in Published Japanese Patent Application No. 2-504468 may be used. This polypeptide comprises a cysteine at position 69 (Cys) and a cysteine at position 87 (C, which constitutes the amino acid sequence of human bFGF.
ys) are each replaced by serine (Ser).

As the above-mentioned homologues, for example, the amino acid 154 described in Published Japanese Patent Application No. 63-500036.
Individual polypeptides may be used. This polypeptide is a human bFGF with an 8 amino acid segment added to the N-terminus.

Further, a polypeptide of 147 amino acids having Met- added at the N-terminal, and published patent publication Sho 63-5.
The polypeptide of 157 amino acids in which a segment of 11 amino acids has been added to the N-terminus described in 01953 may be used.

In the agent for treating periodontal disease of the present invention, bF
GF and its homologues may be used alone or in combination. Furthermore, as described above, there are a plurality of homologues of bFGF, and these homologues may be used alone or in combination.

Since the amount of bFGF present in the living body is extremely small, in order to stably supply the therapeutic agent for periodontal disease of the present invention industrially, microorganisms such as Escherichia coli or BFG produced in cultured cells
It is particularly preferred to use F or its homologues. b
When a gene for producing FGF or a homologue thereof (in this case, the above-mentioned polypeptide in general) is incorporated into a microorganism or a cultured cell, the one produced by the microorganism or the cultured cell is generally the N-terminal of bFGF and And / or C-terminus, or the N-terminus and / or C-terminus of the above-mentioned polypeptide with an additional amino acid segment added, that is, the above-mentioned polypeptide.

The therapeutic agent for periodontal diseases of the present invention has an excellent periodontal tissue regeneration-promoting action and regenerates periodontal tissues (cementum, periodontal ligament, alveolar bone) lost due to the progression of periodontitis. In addition to related diseases, for example, repair of bone tissue after tooth extraction and extraction of cyst / oral cancer, reconstruction of resorbed and atrophied alveolar bone, accelerated fixation of implant material, and loss of dentin caused by caries It is useful for regeneration and the like and used for treating various periodontal diseases.

In addition, the active ingredient of the present invention is very safe, and in human bFGF, for example, in a single-dose toxicity test, LD ₅₀ values by both subcutaneous administration and oral administration in male and female rats are both 75 mg / kg or more. LD ₅₀ values by subcutaneous and transdermal administration in dogs are 5 and 3.36 mg / kg or more, respectively,
Even at the maximum dose that can be administered to rats and dogs, no death was observed in either sex.

Thus, the agent for treating periodontal disease according to the present invention comprises bFGF and / or its homologues in a pharmaceutically acceptable carrier such as a solvent, an isotonicity agent, an emulsifying agent, a suspending agent, a stabilizing agent, etc. In addition to the agent and the filler used in the dental field, it can be used as an external preparation such as a liquid preparation, an emulsion, a gel preparation, an injection preparation, a patch preparation, an injection preparation, and a powder preparation.

The therapeutic agent for periodontal disease of the present invention is a tooth extraction,
It can be used by administering to the root surface, peeled gingival surface, alveolar bone surface or the vicinity thereof after periodontal surgery or smooth surface lubrication.

The dose may be appropriately increased or decreased depending on the symptom to be treated and the site, but in the case of human bFGF and / or its homologue, if 0.1 to 1000 μg is applied to the affected area or its vicinity 1 to 3 times a day, The desired therapeutic effect on periodontal disease is exhibited.

[0027]

[Formulation Example: bFGF aqueous solution]

 Ingredient ─────────────────────────────── Human bFGF 2.775mg Normal saline adjusted to 0.555ml ─ ──────────────────────────────

The action of human bFGF to promote periodontal tissue regeneration was tested. The test method and test results are shown below.

[Test Example] Effect on Regeneration after Artificial Periodontal Tissue Defect After Canine Artificial Human Periodontal Tissue Regeneration After Artificial Periodontal Tissue Defect After Beagle Dog Examined. Establish a healthy periodontal tissue by brushing, etc., remove the gingival mucosal flaps of the maxillary left and right anterior teeth by the conventional method to expose the bone, and penetrate the bone and periodontal membrane from the buccal surface corresponding to the tooth root to form dentin. Artificial defect reaching up to
mm, depth 2 mm). Cement was removed from the cavity bottom of the defect using a chisel, and dentin was exposed.

Then, on the right side (experimental side), 50 μg of human b
An aqueous solution containing FGF was placed on the left side (control side) of human bF.
An aqueous solution containing no GF was administered, and the gingival mucosal flap was returned to its original position and sutured. One week after the surgery, tooth extraction was performed, and four weeks later, the tooth was sacrificed, and a pathological sample was prepared by the Azan staining method, and observed with an optical microscope.

The observation results on the experimental side are shown in FIG. 1, and the observation results on the control side are shown in FIG. Both were observed at a magnification of 10 times. On the experimental side, (1) an image was observed in which the bone (a in FIG. 1) newly formed from the alveolar bone cuts on the crown side and the apex side proliferated and invaded toward the central portion of the defective cavity. Osteoblasts (b in FIG. 1) were lined up on the margin of the trabecular bone, and osteoclasts were also observed, indicating a tendency for active bone remodeling. (2) The periodontal ligament (c in FIG. 1) newly formed between the newly formed bone and the root surface has a wider width and is rich in capillaries than existing periodontal ligament, and its fibers (Fig. The running of d) in No. 1 was being obliquely or vertically arranged on the root surface, and an image of embedding in the new cementum (e in FIG. 1) was observed. Further, (3) the new cementum was formed on the root surface barely exposed from the existing cementum surface, and the new cementum was formed almost uniformly in the absorption cavities of the root surface dentin. The new periodontal ligament between the new bone and the root surface was similar to the existing periodontal ligament.

On the other hand, on the control side, as in the case of (4) the experimental side, the bone (a in FIG. 2) newly formed from the alveolar bone cut portions on the crown side and the apex side was located in the central portion of the defective cavity. Although there was an image of in-proliferation and invasion, the degree of new bone formation was lower than that of the experimental side. (5) The degree of growth of fibers of the new periodontal ligament formed between the new bone and the root surface was also low. Also, (6) new cement material (e in FIG. 2)
Although it was formed from the existing cementitious surface to the root surface, its thickness decreased toward the central part of the defect cavity, and the formation degree was lower than that of the experimental side. Further, (7) formation of long junctional epithelium due to invasion of inner marginal epithelial cells, root resorption and ankylosis were not observed in both experimental side and control side.

As a result, human bFGF showed a clear accelerating effect on the improvement of the destroyed periodontal tissue, which was important for the treatment of periodontal disease, the defect and the regeneration of the absorbed alveolar bone.

[0034]

As is apparent from the above results, human bF
GF has an excellent periodontal tissue and dentin regeneration effect,
Excellent healing against periodontitis, tooth extraction, oral cancer, jaw deformity, etc. and gum, periodontal tissues such as cementum, periodontal tissues such as cementum, jaw bone and dentin that are deformed, defective, or pathological. Has a promoting effect.

From the above, the therapeutic agent for periodontal disease of the present invention is used for regeneration of periodontal tissues (cementum, periodontal ligament, alveolar bone) lost due to the progression of periodontitis, after tooth extraction, and after cyst / oral cancer extraction. It is expected to be applied to the repair of bone defects in bones, the reconstruction of alveolar bone that has resorbed and atrophied, the promotion of the fixation of implant materials, and the regeneration of dentin that has been damaged by caries.

[Brief description of drawings]

FIG. 1 is an optical microscope observation view of a pathological specimen on an experimental side in a periodontal tissue regeneration test after surgery for artificial periodontal tissue defect in a dog.

FIG. 2 is an optical microscopic observation view of a control-side pathological specimen in a periodontal tissue regeneration test after canine artificial periodontal tissue defect surgery.

[Explanation of symbols]

 a new bone b osteoblast c periodontal ligament d filament e new cementum

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] September 28, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Brief description of the drawing

[Correction method] Change

[Correction content]

[Brief description of drawings]

FIG. 1 is an optical micrograph of an experimental pathological specimen in a periodontal tissue regeneration test after canine artificial periodontal tissue defect surgery.

FIG. 2 is an optical micrograph of a control side pathological specimen in a periodontal tissue regeneration test after canine artificial periodontal tissue defect surgery.

[Explanation of Codes] a New bone b Osteoblast c Periodontal ligament d Fiber e New cementum

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Akio Terashima 14 Shinomiya Minamikawaracho, Yamashina-ku, Kyoto City, Kyoto Prefecture Central Research Institute of Kaken Pharmaceutical Co., Ltd.

Claims (2)

[Claims] 1. A therapeutic agent for periodontal disease containing a basic fibroblast growth factor and / or a homologue thereof. 2. The therapeutic agent for periodontal disease according to claim 1, wherein the basic fibroblast growth factor and / or its homologue is human basic fibroblast growth factor and / or its homologue.