Inhibitor of bacterial invasion into gingival epithelial cells and infection inhibitor

Abstract

It inhibits the invasion of specific bacteria into gingival epithelial cells, effectively preventing periodontal infection. Not only can it prevent periodontal disease, but it can also deal with various symptoms caused by periodontal disease. This relates to agents that SOLUTION: A periodontal tissue protective agent containing ε-aminocaproic acid and / or tranexamic acid as active ingredients.

Description

[Technical Field]

[0001] The present invention relates to an agent for inhibiting bacterial invasion into gingival epithelial cells and an agent for inhibiting infection. [Background technology]

[0002] The gingiva is a soft tissue composed of gingival epithelium and adjacent connective tissue. In addition, the periodontal ligament, cementum, and alveolar bone are collectively referred to as periodontal tissues. However, gingival epithelium is an important oral tissue that plays a defensive role in protecting the body from bacteria. Therefore, in order to prevent problems with the gingival epithelium or to prevent the progression of the problems, In the past, agents have been developed from various viewpoints.

[0003] For example, Patent Document 1 discloses a method for producing a compound selected from ε-aminoaporonic acid, lysine, arginine, etc. The oral composition containing one or more of the compounds is disclosed, and is effective in preventing bleeding during chronic inflammation. Patent Document 2 also describes a drug known as a hemostatic agent, which has enhanced anti-inflammatory and edema suppressing effects. An oral composition containing nexamic acid and tea extract has been disclosed. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 4-300816 [Patent Document 2] Japanese Patent Application Publication No. 2018-203714 Summary of the Invention [Problem to be solved by the invention]

[0005] By the way, Porphyromonas gingivalis ( Porphyromonas gingivalis ) and Fusobak Therium nucleatum ( Fusobacterium nucleatum ) is known as a periodontal disease-related bacterium. These Porphyromonas gingivalis and Fusobacterium nucleatum are also First, they adhere to and invade gingival epithelial cells, and then induce apoptosis of the invaded epithelial cells. As a result, cell turnover becomes abnormal, and the cells become more susceptible to invasion of periodontal tissues. This continues to progress.

[0006] However, the techniques described in Patent Documents 1 and 2 above do not involve the use of ε-aminocaproic acid and / or The present invention relates to the use of tranexamic acid to soothe gingival symptoms, and to the prevention of bacterial infection of the gingival epithelium. This is different from the use related to cell invasion and subsequent cell damage.

[0007] Therefore, the present invention provides a method for producing Porphyromonas gingivalis bacteria and Fusobacterium nuclei. It inhibits the invasion of P. creatum bacteria into gingival epithelial cells and alleviates various symptoms caused by this invasion. It relates to agents that can be used to treat this condition. [Means for solving the problem]

[0008] Therefore, the present inventors have conducted extensive research to solve the above problems, and as a result, have found that ε-aminocaproic acid By using lactic acid and / or tranexamic acid as the active ingredient, it is possible to Prevents the invasion of gingival epithelial cells by squirrel and / or Fusobacterium nucleatum bacteria. We have discovered an agent that can suppress this.

[0009] That is, the present invention provides a method for treating rheumatoid arthritis with ε-aminocaproic acid and / or tranexamic acid as an active ingredient. Porphyromonas gingivalis and / or Fusobacterium nucleatum bacteria The present invention provides an agent that inhibits invasion into gingival epithelial cells. The present invention also provides a method for treating rheumatoid arthritis, comprising administering ε-aminocaproic acid and / or tranexamic acid as an active ingredient. Porphyromonas gingivalis and / or Fusobacterium nucleatum bacteria in the gums The present invention provides an agent for suppressing infection in epithelial cells. Furthermore, the present invention provides a method for treating a skin ulcer comprising ε-aminocaproic acid and / or tranexamic acid as an active ingredient. , Porphyromonas gingivalis and / or Fusobacterium nucleatum bacterial disease The present invention provides a potency attenuator. [Effects of the Invention]

[0010] According to the present invention, ε-aminocaproic acid and trastuzumab, which are known only as antiplasmin agents, are used. By using nexamic acid as the active ingredient, it is possible to It can effectively prevent S. nucleatum from invading gingival epithelial cells. Therefore, the present invention is not only useful as an agent for inhibiting the invasion of these bacteria into gingival epithelial cells, but also It is also highly useful as an inhibitor of bacterial infection of gingival epithelial cells, and it is effective in preventing the bacterial infection of gingival epithelial cells. It can also be used as an agent to inhibit adhesion to skin cells. In addition, if ε-aminocaproic acid or tranexamic acid is used as an active ingredient, as an inhibitor of osteoclast activity in the oral cavity, or as a turnover normalizer of gingival epithelial cells can also exert excellent effects. [Brief explanation of the drawings]

[0011] [Figure 1] Photographs showing the results of Example 2-1 in Evaluation Test 2 on gingival epithelial cells. Enlarged photographs of parts of Figures 1(A-1), (B-1), and (C-1) are shown in Figures 1(a-1), (b-1), and (c-1), respectively. [Figure 2]Photographs showing the results of Example 2-2 in Evaluation Test 2 on gingival epithelial cells. Enlarged photographs of parts of Figures 2(A-2), (B-2), and (C-2) are shown in Figures 2(a-2), (b-2), and (c-2), respectively. [Figure 3] Photographs showing the results of Examples 2-3 and -4 in Evaluation Test 2 on gingival epithelial cells. Enlarged photographs of parts of Figures 3(A-3) and (B-3) are shown in Figures 3(a-3) and (b-3), respectively. [Figure 4] Photographs showing the results of Comparative Example 2-1 in Evaluation Test 2 on gingival epithelial cells. Enlarged photographs of parts of Figures 4 (Ax-1), (Bx-1), and (Cx-1) are shown in Figures 4 (ax-1), (bx-1), and (cx-1), respectively. [Figure 5] Photographs showing the results of Comparative Example 2-2 in Evaluation Test 2 on gingival epithelial cells. Enlarged photographs of parts of Figure 5 (Ax-2), (Bx-2), and (Cx-2) are shown in Figure 4 (ax-2), (bx-2), and (cx-2), respectively. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will be described in detail below. The agent of the present invention is Porphyromonas gingivalis ( Porphyromonas gingivalis (Pg )) and / or Fusobacterium nucleatum ( Fusobacterium nucleatum (Fn) ) into gingival epithelial cells, and is an inhibitor of the invasion of bacteria into gingival epithelial cells, The agent of the present invention contains ranexamic acid as an active ingredient. Effectively inhibits the invasion of Bacillus ingivalis and Fusobacterium nucleatum into gingival epithelial cells It is possible.

[0013] Therefore, if Porphyromonas gingivalis or Fusobacterium nuclei is found in the oral cavity, Even when P. gingivalis and P. gingivalis are present alone, Even if both B. nucleatum and B. nucleatum are present, the agent of the present invention can inhibit the growth of these bacteria in gingival epithelial cells. It is possible to exert an effect of suppressing invasion into the

[0014] As the ε-aminocaproic acid, ε-aminocaproic acid salt may be used. Salts that form ε-aminocaproic acid salt include alkali salts such as sodium salts and potassium salts. alkaline earth metal salts such as calcium salts and magnesium salts; hydrochlorides, sulfates, etc. Examples include inorganic acid salts.

[0015] Tranexamic acid may also be used as tranexamic acid. Examples of the salts that can be formed include metal salts such as sodium salts, potassium salts, and magnesium salts, hydrochlorides, and lysates. Examples of the salts include phosphates and sulfates.

[0016] The agent of the present invention may contain ε-aminocaproic acid alone as an active ingredient, It may contain tranexamic acid alone or ε-aminocaproic acid and tranexamic acid. It may contain both.

[0017] The content of ε-aminocaproic acid effectively inhibits the invasion of the above bacteria into gingival epithelial cells. From this viewpoint, the amount of the compound in the agent of the present invention is preferably 0.03% by mass or more, and more preferably 0.03% by mass or more. It is preferably 0.05% by mass or more, more preferably 0.1% by mass or more, and even more preferably The content of ε-aminocaproic acid is 0.13 mass % or more. From this viewpoint, the amount of the compound in the agent of the present invention is preferably 1.5% by mass or less, more preferably 1. % by mass or less, more preferably 0.5% by mass or less, and even more preferably 0 The content of ε-aminocaproic acid in the agent of the present invention is preferably 0.2% by mass or less. The content is preferably 0.03 mass % or more and 1.5 mass % or less, and more preferably 0.05 to 1 mass %. %, more preferably 0.1 to 0.5 mass %, and even more preferably 0.1 3 to 0.2 mass% When ε-aminocaproic acid salt is used as ε-aminocaproic acid, The amount of caproate converted into ε-aminocaproic acid may be within the above content range.

[0018] The content of tranexamic acid is thought to effectively inhibit the invasion of the above bacteria into gingival epithelial cells. From this viewpoint, the content of the agent of the present invention is preferably 0.03% by mass or more, more preferably 0.0 It is 5% by mass or more, more preferably 0.09% by mass or more, and even more preferably The content of tranexamic acid is 0.15% by mass or more. Therefore, the content in the agent of the present invention is preferably 2% by mass or less, more preferably 1.5% by mass or less. and more preferably 1 mass % or less, and even more preferably 0.8 mass % or less. The content of tranexamic acid in the agent of the present invention is preferably 0.03 mass %. % or more and 2% or less by mass, more preferably 0.05 to 1.5% by mass, and even more preferably Preferably, it is 0.09 to 1 mass %, and more preferably, it is 0.15 to 0.8 mass %. When tranexamic acid is tranexamic acid salt, The amount converted to ranexamic acid may be within the above content range.

[0019] Thus, the agent of the present invention inhibits the invasion of the above bacteria into gingival epithelial cells, and Since the agent of the present invention also inhibits the infection of gingival epithelial cells by these bacteria, it is believed that the agent of the present invention can effectively inhibit the infection of gingival epithelial cells by these bacteria. It is also useful as a pathogenicity attenuator.

[0020] Furthermore, the agent of the present invention inhibits the invasion and infection of the above bacteria into gingival epithelial cells. This prevents bacteria from invading deeper into the periodontal tissue, suppressing the activation of osteoclasts. It is also possible to suppress the erosion of alveolar bone by inhibiting osteoclast activity in the oral cavity. It can be used as a sex inhibitor and a periodontal tissue protector.

[0021] The agent of the present invention is also useful as an agent for normalizing the turnover of gingival epithelial cells. In other words, the gingival epithelium has a rapid metabolic mechanism that constantly replaces cells with new ones, known as turnover. By maintaining normal function of the immune system, the body's defenses against bacteria are maintained well. However, when the above bacteria invade cells, cell turnover becomes abnormal. The agent of the present invention inhibits bacterial invasion, and the turnover of gingival epithelial cells This allows for rapid replacement with new cells, effectively preventing the onset of periodontal disease. become.

[0022] Therefore, the agent of the present invention can effectively prevent the growth of Porphyromonas gingivalis and / or Fusobacterium diffusum. Effectively inhibiting the invasion or infection of gingival epithelial cells by Bacterium nucleatum bacteria, or Or, it effectively weakens the pathogenicity of these bacteria, inhibiting their adhesion to gingival epithelial cells, Protects periodontal tissue, inhibits osteoclast activity in periodontal tissue, and promotes the turnover of gingival epithelial cells. This allows the server to be maintained normally.

[0023] Thus, the agent of the present invention has the effect of inhibiting the invasion of the above bacteria into gingival epithelial cells and the effect of inhibiting infection. Since it exhibits the above effects, it is suitable for use as an agent for application to the oral cavity. The agent may be in the form of a toothpaste, a toothpaste powder, or other toothpaste composition, or a mouthwash, liquid, or gel. Examples include liquid oral compositions such as gel-type dentifrice and mouth sprays.

[0024] Therefore, the agent of the present invention contains, in addition to the above-mentioned active ingredients, a toothpaste composition, a liquid oral composition, etc. The composition may contain ingredients that are commonly used in preparations for oral application. Examples of such additives include foaming agents, foaming assistants, surfactants, abrasives, bulking agents, sweeteners, preservatives, and pharmaceuticals. Examples include active ingredients, pH adjusters, adhesives, pigments, coloring materials, fragrances, etc. [Example]

[0025] [Evaluation Test 1 in Gingival Epithelial Cells: Example 1] According to the following procedures (1) to (6), the activity of ε- The effects of aminocaproic acid and tranexamic acid were evaluated. The results of the evaluation of the effects of aminocaproic acid are shown in Table 1, and the results of the evaluation of the effects of tranexamic acid are shown in Table 2. Shown below.

[0026] (1) Preparation of gingival epithelial cells Human gingival epithelial cells (Ca9-22) were obtained from the JCRB Cell Bank (JCRB0 625). Dulbecco's Modified Eagle Medium:DMEM(G ibco) and added 10% v / v Fetal Bovine Serum (Gibco). The cells were cultured in the medium containing the above-mentioned solution at a temperature of 37°C and a CO2 concentration of 5%.

[0027] (2) Bacterial culture Porphyromonas gingivalis ATCC 33277(Pg), and Fusobacterium nucleatum ATCC 25586 (Fn) was obtained from ATCC. Colony culture was performed on Anello Columbia RS blood agar medium (Becton Dickinson Japan). For liquid culture, GAM broth medium (Nissui Pharmaceutical Co., Ltd.) was used, containing 5.0 μg / mL He The medium was supplemented with 17.4 μg / mL K2HPO4 and 1.0 μg / mL Vitamin K. The cells were incubated under anaerobic conditions at a temperature of 37°C using Anaeropack Kenki (Mitsubishi Gas Chemical Co., Ltd.). and cultured. P. gingivalis (Pg) was cultured in liquid for 24 hours, and then diluted 100 times. In addition, the bacteria were cultured in liquid for 24 hours. F. nucleatum (Fn) was cultured in liquid for 24 hours. After that, the cells were diluted 1000 times and further cultured in liquid for 24 hours before use.

[0028] (3) Application of ε-aminocaproic acid and tranexamic acid The bacterial culture obtained in (2) above was centrifuged at 4°C and 3,500 g for 10 minutes. The precipitated cells were collected. The collected cells were incubated in PBS and then in DMEM at 4°C for 3, After washing by centrifugation at 500 g for 5 minutes, the mixture was adjusted to OD600=1.0. Next, the resulting bacterial solution was placed in a 15 mL tube and diluted with 0 mM, 1 mM, 10 mM, and 1 ε-aminocaproic acid (reagent, Fujifilm Wako Pure Chemical Industries, Ltd.) was added to make the final concentration 0.00 mM. In addition, tranexamic acid (reagent, Fuji) was used at 0 mM, 1 mM, 10 mM, and 100 mM concentrations. The mixture was then incubated at 37°C under anaerobic conditions for 1 hour. Placed it.

[0029] (4) Fluorescent labeling of bacterial cells The labeling reagent, Carboxyfluorescein diacetate succinimidyl ester (CFSE, Dojindo) (manufactured by Chemical Co., Ltd.) was dissolved in DMSO. The bacterial suspension obtained in (3) above was centrifuged at 4°C and 3,500 g for 10 minutes. The collected bacterial cells were then centrifuged at 4°C, 3,500g, and 5 min using DMEM. After washing by centrifugation for 1 minute, the OD600 was adjusted to 1.0. Then, CFSE solution (dissolving DMEM) was added at a final concentration of 10 μM. The plate was left to stand at 37°C in the dark for 30 minutes for fluorescent labeling. The fluorescently labeled cells were centrifuged at 3,500 g for 5 minutes at 4°C using DMEM. Washed twice.

[0030] (5) Cultivation of fluorescently labeled bacteria Ca9-22 cells cultured for 24 hours (cell seeding concentration: 0.3 × 106 cells / well) After removing the medium from the culture plate (1 in a 12-well plate), PBS The bacterial solution prepared in (4) above was washed using a Multiplicity of In The cells were added at an infection rate of 500 and cultured for 2 hours at 37°C and a CO2 concentration of 5%.

[0031] (6) Analysis method for cells invaded by bacteria The medium was removed from the Ca9-22 obtained in (5) above, and the cells were washed twice with PBS. Fluorescently labeled bacteria ( Detachment of cells invaded by fluorescently labeled P. gingivalis (Pg) and F. nucleatum (Fn). The collected cells were then collected in a 1.5 mL tube. Add phosphate buffer (Fujifilm Wako Pure Chemical Industries, Ltd.) and pipette. Incubate at 4°C for 30 minutes. After adding PBS containing 2% FBS and pipetting, the cells were incubated at 4°C for 1 hour. Centrifuge at 600g for 3 minutes and suspend in PBS (containing 2% FBS). The resulting mixture was used as an analysis sample.

[0032] Flow cytometry (BD, FACSVerse) was used for analysis. . forward scatter (FSC), side scatter (SSC) Based on the gating, 500,000 cells were obtained in the FITC channel. Calculate the mean fluorescence intensity (MFI) from the fluorescence intensity. The cells were then invaded by fluorescently labeled P. gingivalis (Pg) and F. nucleatum (Fn). The amount of cells was determined. Next, for this amount, when 0 mM of each drug was applied (invasion of Pg and Fn), The amount of cells in the control group (uninhibited) was converted into a value (%), with the amount of cells in the control group (uninhibited) being 100%, and used as an index of evaluation.

[0033] [Table 1]

[0034] [Table 2]

[0035] Evaluation test 2 on gingival epithelial cells: Examples 2-1 to 2-3 and Comparative Examples 2-1 to 2-2 ] The same procedures as in (1) to (6) of Evaluation Test 1 were carried out, and after treatment with various drugs, fluorescent staining was performed. Each of the bacterial cells was then infected into gingival epithelial cells, and the following results were obtained: Each sample was analyzed using a fluorescence microscope (BZ-X810 ( (manufactured by GE Healthcare, Excitation: 470 nm / Extension: 495 nm, Magnification: 60x) Observations were conducted. The figures showing the treated agents, the bacterial cells, and the observation results using a fluorescent microscope are as follows: be. Example 2-1: Infection of Pg treated with 100 mM tranexamic acid (Figure 1) Example 2-2: Infection of Pg treated with 100 mM ε-aminocaproic acid (Figure 2) Example 2-3: Infection of Fn treated with 100 mM ε-aminocaproic acid (Figure 3( A-3) and (a-3)) Example 2-4: Fn treated with 100 mM tranexamic acid was infected (Figure 3(B-3) ) and (b-3)) Comparative Example 2-1: Pg infected with 0 mM drug (Figure 4) Comparative Example 2-2: Infection with Fn at 0 mM of drug (Figure 5)

[0036] [Evaluation Test 3 in Gingival Epithelial Cells: Example 3 and Comparative Example 3] Following the procedures (1) to (4) below, the activity of trastuzumab as an active ingredient in gingival epithelial cells was investigated. The effect of nexamic acid was evaluated. The results of the effect evaluation are shown in Table 3.

[0037] (1) Preparation of gingival epithelial cells Human oral keratinocytes were cultured in a 24-well plate. Oral Kera cells were seeded and grown until semi-confluent. Cultured in tinocyte medium.

[0038] (2) Bacterial culture The above-mentioned Porphyromonas gingivalis ATCC 33277 (Pg) was used to investigate the effect of Porphyromonas gingivalis on gingival epithelial cells. The same procedure as in (2) of Evaluation Test 1 was carried out to obtain a bacterial culture solution.

[0039] (3) Application of tranexamic acid and cetylpyridinium chloride The bacterial culture obtained in (2) above was centrifuged at 4°C and 3,500 g for 10 minutes. The precipitated cells were collected. The collected cells were then centrifuged at 3,500 g for 5 minutes at 4°C using PBS. After washing by centrifugation, the OD600 was adjusted to 1.0. Next, 500 ppm of tranexamic acid was added to the 15 mL tube containing the bacterial solution. In Comparative Example 3, 500 ppm of cetylpyridinium chloride (CPC) was added. The mixture was left to stand for 1 hour under anaerobic conditions at 37°C. A control (PBS) containing no bacterial solution or drug was also prepared.

[0040] (4) Method for analyzing the presence or absence of inflammation in gingival epithelial cells The bacteria obtained in (3) above were used to calculate the Multiplicity of Infection (MPI) 100 was added to the cells obtained in (1) above, and cultured for 3 hours. After removing the culture supernatant, the R was isolated using the RNeasy mini kit (QIAGEN). The RNA was extracted. Then, High capacity RNA to cDNA (Ther The reverse transcription reaction was carried out using TaqMan ( Using a registered trademark probe, gene expression of Interleukin-6 (IL-6) was detected. The obtained values ​​were determined based on the amount of glyceraldehyde-3-phosphine. The analysis was performed by correcting for the gene expression level of GAPDH. analyzed.

[0041] Table 3

Claims

1. A periodontal tissue protecting agent containing ε-aminocaproic acid and / or tranexamic acid as active ingredients.

2. An agent for inhibiting osteoclast activity in periodontal tissue, which comprises ε-aminocaproic acid and / or tranexamic acid as active ingredients.

3. A turnover normalizing agent for gingival epithelial cells, which contains ε-aminocaproic acid and / or tranexamic acid as active ingredients.

4. The agent according to any one of claims 1 to 3, wherein the content of ε-aminocaproic acid is 0.03% by mass or more and 1.5% by mass or less.

5. The agent according to any one of claims 1 to 4, wherein the content of tranexamic acid is 0.03% by mass or more and 2% by mass or less.

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