Therapeutic agent for regenerating periodontal tissues

Abstract

Provided is a therapeutic agent for regenerating periodontal tissues also capable of restoring periodontal tissues lost by severe periodontal disease. A therapeutic agent for regenerating periodontal tissues contains a MAP4K selective inhibitor. Alternatively, the MAP4K selective inhibitor is GNE-495 or PF06260933. Alternatively, the therapeutic agent for regenerating periodontal tissues is administered with a growth factor FGF-2. Alternatively, the therapeutic agent for regenerating periodontal tissues is characterized in that the FGF-2 is a recombinant human FGF-2. Alternatively, an agent for enhancing proliferation of periodontal ligament cels contains a MAP4K selective inhibitor. Alternatively, the therapeutic agent for regenerating periodontal tissues is in a liquid form.

Description

Periodontal tissue regeneration therapeutic agent

[0001] The present invention relates to a periodontal tissue regeneration therapeutic agent. This application claims priority based on Japanese Patent Application No. 2024-168706 filed on September 27, 2024, the content of which is incorporated herein by reference.

[0002] Periodontal disease is a disease of the periodontal tissue caused by bacterial infection. When periodontal pathogenic bacteria contained in dental plaque accumulated between teeth cause inflammation in the gums, a groove called a periodontal pocket is formed between the swollen gums and teeth due to the inflammation. Since the periodontal pocket is an environment suitable for the growth of periodontal pathogenic bacteria, it causes further progression of periodontal disease. When periodontal tissues such as gums and alveolar bone are destroyed due to the progression of periodontal disease, tooth extraction becomes necessary.

[0003] The treatment of periodontal disease consists of sterilizing periodontal pathogenic bacteria and treating the inflammation of periodontal tissues, and can subside the inflammation of periodontal tissues. However, with conventional treatment methods, it has been difficult to regenerate the periodontal tissues destroyed by periodontal disease. Therefore, even after periodontal disease is cured, there are problems such as the instability of tooth support due to the defect of periodontal tissues, the increased risk of recurrence of periodontal disease, the remaining exposed tooth roots, and the lifelong continuation of sequelae such as poor aesthetics and dentin hypersensitivity.

[0004] In response to such problems, drugs that promote the regeneration of the remaining periodontal tissues have been developed. As an example of such a drug, there is one described in Patent Document 1. The periodontal disease therapeutic agent described in Patent Document 1 contains basic fibroblast growth factor (FGF) and can regenerate periodontal tissues lost due to the progression of periodontal disease.

[0005] Japanese Patent Laid-Open No. 07-017876

[0006] However, the applicable cases of the drug described in Patent Document 1 were mainly limited to vertical bone defects in which the alveolar bone around the teeth was deficient along the tooth roots, and the regeneration effect itself was also limited. Therefore, it was still difficult to recover the periodontal tissues lost due to severe periodontal disease.

[0007] In view of the above circumstances, an object of the present invention is to provide a periodontal tissue regeneration therapeutic agent that can also recover periodontal tissues lost due to severe periodontal disease.

[0008] To solve the above problems, one aspect of the present invention includes the following:

[0009] [1] A periodontal tissue regeneration agent containing a MAP4K selective inhibitor.

[0010] [2] The periodontal tissue regeneration agent according to [1], wherein the MAP4K selective inhibitor is GNE-495, PF06260933, PF-06279789, Pro-101, or DMX-5804.

[0011] [3] A periodontal tissue regeneration agent according to [1] or [2], to be administered together with FGF-2.

[0012] [4] The periodontal tissue regeneration agent according to [3], characterized in that the FGF-2 is recombinant human FGF-2.

[0013] [5] A periodontal ligament cell proliferation enhancer containing a MAP4K selective inhibitor.

[0014] [6] A periodontal tissue regeneration agent relating to any one of [1] to [4], which is in liquid form.

[0015] A bone graft material containing a periodontal tissue regeneration agent relating to any one of [7] [1] to [4].

[0016] A periodontal tissue regeneration agent according to [8] [1] or [2], and a periodontal tissue regeneration agent kit containing at least growth factor FGF-2.

[0017] A method for administering a periodontal tissue regeneration agent according to [9] [1] or [2] and the growth factor FGF-2 for periodontal tissue regeneration treatment.

[0018] According to the present invention, it is possible to provide a periodontal tissue regeneration therapy agent that can restore periodontal tissue lost due to severe periodontal disease.

[0019] Figure 1 is a graph showing the results of cell proliferation enhancement by GNE-495 and FGF-2. Figure 2 is a graph showing the therapeutic effect of GNE-495 on alveolar bone regeneration in a periodontitis model mouse. Figure 3 is a graph showing the number of cells stimulated by FGF-2 and GNE-495 on PDLF on day 9 of culture as shown in Figure 1. Figure 4 is a graph showing the number of cells stimulated by FGF-2 and GNE-495 on PDLF on day 3 of culture.

[0020] One embodiment of the present invention will be described below. The embodiments shown below illustrate configurations for realizing the technical idea of ​​the present invention, and the present invention is not limited to these embodiments. Various modifications can be made to the technical idea of ​​the present invention within the technical scope defined by the claims described in the claims.

[0021] (Periodontal tissue regeneration agent) The periodontal tissue regeneration agent of this embodiment contains a MAP4K selective inhibitor. The term "periodontal tissue regeneration agent containing a MAP4K selective inhibitor" includes not only cases where the MAP4K selective inhibitor is contained within the formulation, but also cases where the MAP4K selective inhibitor and other components are individually packaged and provided in combination. The periodontal tissue regeneration agent of this embodiment may be in liquid form.

[0022] (MAP4K Selective Inhibitors) A MAP4K selective inhibitor refers to a molecule that selectively inhibits MAP4K. Among these, those that selectively inhibit MAP4K4 are preferred. Examples of the aforementioned MAP4K selective inhibitors include GNE-495 (CAS number: 1449277-10-4), PF06260933 (CAS number: 1811510-56-1), PF-06279789 (MAP4K4-IN-3, CAS number: 1811510-58-3), Pro-101 (Prosectin / Famlacertib, CAS number: 2375591-69-6), and DMX-5804 (CAS number: 2306178-56-1). Among these, GNE-495 is preferred.

[0023] MAP4K selective inhibitors stabilize YAP and its closely related paralog TAZ by suppressing their phosphorylation, thereby inhibiting the transcription of YAP and TAZ, which are transcription factors in the Hippo signaling pathway. Stabilized YAP and TAZ then translocate into the cell nucleus, enhancing cell proliferation.

[0024] Prior art had shown that MAP4K selective inhibitors enhance cell proliferation in cell lines in vitro. However, it had not been reported whether MAP4K selective inhibitors exhibit tissue regeneration effects in vivo, which tissues they regenerate, or preferred usage conditions for tissue regeneration.

[0025] The inventors discovered that administering a MAP4K selective inhibitor to areas where periodontal tissue is lost due to periodontal disease enhances the proliferation of periodontal ligament cells, leading to in vivo regeneration of periodontal tissue. Furthermore, the inventors discovered favorable usage conditions for periodontal tissue regeneration. As a result, they succeeded in developing a useful agent for periodontal tissue regeneration therapy, thus completing the present invention.

[0026] (Periodontal ligament cells) Periodontal ligament cells are cells derived from the periodontal ligament and include fibroblasts, osteoblasts, cementoblasts, osteoclasts, and undifferentiated mesenchymal cells. Periodontal ligament cells are known to differentiate into fibroblasts, osteoblasts, and cementoblasts depending on the situation, producing collagen to form the periodontal ligament, or differentiating into hard tissue to form the alveolar bone.

[0027] In the embodiments of the present invention, it is believed that the proliferation of periodontal ligament cells was enhanced by a MAP4K selective inhibitor, and that the proliferated periodontal ligament cells then differentiated into fibroblasts, osteoblasts, cementoblasts, etc., thereby regenerating periodontal tissue, including alveolar bone.

[0028] (FGF-2) The periodontal tissue regeneration agent of this embodiment is preferably administered together with FGF-2 (bFGF). FGF-2 (bFGF) from various species and families can be used as appropriate, but recombinant human FGF-2 (RefSeq: NP_001997) is preferred. Growth factor FGF-2 (also called basic fibroblast growth factor) is known to enhance the proliferation of a wide range of cells and is used, for example, in the periodontal tissue regeneration agent described in Patent Document 1.

[0029] However, the periodontal tissue treatment agent described in Patent Document 1 had the problem of having a limited effect in enhancing cell proliferation. Furthermore, the inventors of the present invention discovered that although FGF-2 enhances cell proliferation, its effect in enhancing cell proliferation is diminished when administered at high concentrations.

[0030] The inventors discovered that when a MAP4K selective inhibitor, which has not been reported as a periodontal tissue regeneration agent, is used in combination with FGF-2, the effect of enhancing cell proliferation is synergistically strengthened compared to FGF-2 alone.

[0031] Furthermore, the inventors of this invention discovered that by using a MAP4K selective inhibitor in combination with FGF-2, the effect of enhancing cell proliferation does not diminish even when FGF-2 is administered at high concentrations, and the effect of enhancing cell proliferation increases in a concentration-dependent manner for both.

[0032] (Bone graft material) The periodontal tissue regeneration agent of this embodiment can be used by being incorporated into a bone graft material. A bone graft material is a graft material that is embedded in the area of ​​alveolar bone defect in order to promote the regeneration of alveolar bone, etc. By incorporating the periodontal tissue regeneration agent of this embodiment into a bone graft material, bone tissue regeneration can be promoted more effectively than with conventional technology.

[0033] (Periodontal tissue regeneration agent kit) The periodontal tissue regeneration agent of this embodiment can be used as a periodontal tissue regeneration agent kit containing a MAP4K selective inhibitor and FGF-2. A periodontal tissue regeneration agent kit refers to a unit in which the MAP4K selective inhibitor and FGF-2 are provided in separate compartments. In a periodontal tissue regeneration agent kit, it is preferable that the MAP4K selective inhibitor and FGF-2 can be mixed immediately before administration.

[0034] (Method of administering the periodontal tissue regeneration agent) The method of administering the periodontal tissue regeneration agent of this embodiment includes administration to the surface of the affected area, administration into the affected tissue, etc. As an example of the administration method, it may be injected into the affected area using a microsyringe. The periodontal tissue regeneration agent of this embodiment is preferably mixed with FGF-2 immediately before administration.

[0035] The effects of the present invention will be made clearer by the following examples. However, the present invention is not limited to the following examples and can be implemented with appropriate modifications without altering its essence.

[0036] <Test Example 1: Effects of GNE-495 in vitro> The inventors first verified the effect of GNE-495 and FGF-2 on enhancing periodontal ligament cell proliferation in vitro using the following method.

[0037] (Proliferation-enhancing effect on periodontal ligament fibroblasts) Periodontal ligament fibroblasts (hereinafter referred to as PDLF) purchased from Lonza were seeded in 96-well plates and cultured in high serum levels (10% fetal bovine serum) and low serum levels (1% fetal bovine serum). PDLF cells were stimulated under the following conditions: FGF-2 alone (0, 10, 20, 50 ng / ml), GNE-495 alone (0, 0.8, 3.2 μM), and a combination of FGF-2 (0, 10, 20, 50 ng / ml) and GNE-495 (0, 0.8, 3.2 μM).

[0038] The number of PDLF cells cultured under each condition was relatively quantified on days 0, 3, 6, and 9, with day 0 being the start of culture, using WST-08 (a method using water-soluble tetrazolium salt, WST method). Figure 1 shows the absorbance at 450 nm measured with WST-08 for each culture condition and day.

[0039] With FGF-2 alone, a proliferation-enhancing effect was observed until day 3, but proliferation was suppressed thereafter. With GNE-495 alone, a proliferation-enhancing effect was observed throughout the measurement period, and this effect increased in a concentration-dependent manner with GNE-495. In combined stimulation with FGF-2 and GNE-495, the proliferation suppression observed from day 3 onwards with FGF-2 alone was lost with the addition of GNE-495, and a synergistic proliferation-enhancing effect, dependent on the respective concentrations of FGF-2 and GNE-495, was observed until day 9.

[0040] <Test Example 2: Effects of GNE-495 in vivo> (Creation of periodontitis model mice) In the jawbones of 12-week-old mice, the upper right side was used as the control group and the upper left side as the experimental group. Periodontitis model mice were created by ligating 5-0 (silk thread) to the root of the upper second molar for 14 days in the experimental group.

[0041] (Confirmation of periodontitis) After 14 days of ligation, the 5-0 (silk sutures) were removed, and the jawbones of the periodontitis model mice were observed. The distance between the cementoenamel junction (hereinafter referred to as CEJ) and the alveolar bone crest was calculated from μCT images of the jawbones in the control group and the experimental group. The results are shown in Figure 2. Compared with the control group (No Ligature), the distance between the CEJ and the alveolar bone crest was significantly longer in the experimental group (14 Days Ligature), confirming alveolar bone loss due to periodontitis.

[0042] (Periodontal tissue regeneration test) Mice of a periodontitis model after suture removal were divided into three groups: one group that received local administration of a solvent containing GNE-495 (MAP4K selective inhibitor) to the periodontitis area via microsyringe at every other day intervals; one group that received local administration of a solvent without GNE-495 to the periodontitis area via microsyringe; and one group that received no administration. The distance between the CEJ and the alveolar bone crest was calculated from μCT images 14 days after suture removal.

[0043] (Results of GNE-495 Administration) The results of the distance between the CEJ and the alveolar bone crest for each administration condition in the periodontitis model mice on the 14th day after wire removal are shown in Fig. 2. Compared with the group administered with a solution not containing GNE-495 (PBS Injection), the group administered with a solution containing GNE-495 (GNE-495 Injection) had a shorter distance between the CEJ and the alveolar bone crest, and regeneration of the alveolar bone was confirmed.

[0044] <Test Example 3: Synergistic Effect of FGF-2 and GNE-495>The synergistic growth enhancement effect of FGF-2 and GNE-495 on periodontal ligament fibroblasts was examined.

[0045] (Effect on the 9th Day of Culture) Periodontal ligament fibroblasts (hereinafter referred to as PDLF) purchased from Lonza were seeded in a 96-well plate and cultured in the presence of low serum (1% fetal bovine serum). PDLF was stimulated under each condition of FGF-2 alone (0, 10, 20, 50 ng / ml), GNE-495 alone (0, 0.8, 3.2 μM), and the combined use of FGF-2 (0, 10, 20, 50 ng / ml) and GNE-495 (0, 0.8, 3.2 μM). For the PDLF cultured under each condition, the cell count on the 9th day from the start of culture was relatively quantified using WST-08. The quantification of the cell count was performed using the absorbance at 450 nm measured by WST-08.

[0046] Fig. 3 is a graph showing the cell count on the 9th day from the start of culture by the stimulation of FGF-2 and GNE-495 on PDLF. In the figure, the vertical axis represents the cell count from the absorbance at 450 nm, and the horizontal axis represents the stimulation conditions of FGF-2 and GNE-495. The cell count before the start of stimulation is also shown in the figure. As shown in the figure, by stimulating both FGF-2 and GNE-495, the cell count is higher than that under the condition of stimulating only FGF-2, and the cell count is even higher under the condition of high concentration of GNE-495. This result shows a synergistic growth enhancement effect on periodontal ligament fibroblasts on the 9th day of culture by the stimulation of both FGF-2 and GNE-495. Note that Fig. 3 shows the cell count on the 9th day of culture in Fig. 1.

[0047] (Effect on the third day of culture) Periodontal ligament fibroblasts purchased from Lonza (hereinafter referred to as PDLF) were seeded in a 96-well plate and cultured in the presence of low serum (1% fetal bovine serum). PDLF was stimulated under each condition of FGF-2 alone (50 ng / ml), GNE-495 alone (3.2 μM), and the combination of FGF-2 (50 ng / ml) and GNE-495 (3.2 μM). For the PDLF cultured under each condition, the cell count on the third day of the start of culture was relatively quantified using WST-08. The quantification of the cell count was performed based on the absorbance at 450 nm measured by WST-08.

[0048] Figure 4 is a graph showing the cell count on the third day of the start of culture by the stimulation of FGF-2 and GNE-495 on PDLF. In the figure, the vertical axis represents the cell count from the absorbance at 450 nm, and the horizontal axis represents the conditions of the stimulation of FGF-2 and GNE-495. The cell count before the start of stimulation is also shown in the figure. As shown in the figure, the cell count increased significantly under each condition of FGF-2 stimulation, GNE-495 stimulation, and FGF-2 and GNE-495 stimulation compared to the non-stimulated control. The cell count increased significantly under the condition of GNE-495 stimulation compared to FGF-2 stimulation. The cell count increased significantly under the conditions of FGF-2 and GNE-495 stimulation compared to GNE-495 stimulation. This result shows a synergistic growth-enhancing effect on periodontal ligament fibroblasts on the third day of culture by the stimulation of both FGF-2 and GNE-495.

[0049] As described above, several embodiments of the present invention have been explained. These embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, replacements, and changes can be made without departing from the gist of the invention. These embodiments and their modifications are included in the scope and gist of the invention, and are also included in the invention described in the claims and the equivalent scope thereof.

[0050] According to the present invention, it is possible to provide a periodontal tissue regeneration therapeutic agent that enables the recovery of periodontal tissues lost due to severe periodontal disease.

Claims

1. A periodontal tissue regeneration agent containing a MAP4K selective inhibitor.

2. The periodontal tissue regeneration agent according to claim 1, wherein the MAP4K selective inhibitor is GNE-495, PF06260933, PF-06279789, Pro-101, or DMX-5804.

3. A periodontal tissue regeneration agent according to claim 1 or 2, administered together with FGF-2.

4. The periodontal tissue regeneration agent according to claim 3, characterized in that the FGF-2 is recombinant human FGF-2.

5. A periodontal ligament cell proliferation enhancer containing a MAP4K selective inhibitor.

6. A periodontal tissue regeneration agent according to any one of claims 1 to 4, which is in liquid form.

7. A bone graft material containing the periodontal tissue regeneration agent according to any one of claims 1 to 4.

8. A periodontal tissue regeneration agent kit comprising at least one of claims 1 to 4 and the growth factor FGF-2.

9. A method for administering a periodontal tissue regeneration agent according to any one of claims 1 to 4, and growth factor FGF-2 for periodontal tissue regeneration treatment.

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