Inhibitor of bacterial invasion into gingival epithelial cells and infection inhibitor

Abstract

To provide an agent that can inhibits the invasion of specific bacteria into gingival epithelial cells and effectively prevent infection with periodontal disease, as well as deal with various symptoms caused by periodontal disease.SOLUTION: Provided is an agent for suppressing the invasion of Porphyromonas gingivalis and / or Fusobacterium nucleatum bacteria into gingival epithelial cells, which comprises ε-aminocaproic acid and / or tranexamic acid as an active ingredient.SELECTED DRAWING: None

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. The gingiva, periodontal ligament, cementum, and alveolar bone are collectively referred to as the periodontal tissue. The gingival epithelium, in particular, is an important oral tissue that plays a defensive role in protecting the body from bacteria. Therefore, agents have been developed from various perspectives to prevent gingival epithelial problems or to prevent their progression.

[0003] For example, Patent Document 1 discloses an oral composition containing ε-aminoaproic acid and one or more selected from lysine, arginine, etc., which enhances the effects of preventing bleeding during chronic inflammation and suppressing edema, etc. Patent Document 2 discloses an oral composition containing tranexamic acid, known as a hemostatic agent, and tea extract, etc. [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 Fusobacterium nucleatum ( Fusobacterium nucleatum) are known to be bacteria associated with periodontal disease. These bacteria, Porphyromonas gingivalis and Fusobacterium nucleatum, first adhere to and invade gingival epithelial cells, and then suppress apoptosis of the invaded epithelial cells. As a result, cell turnover becomes abnormal, further progressing the invasion of periodontal tissues.

[0006] However, the technologies described in Patent Documents 1 and 2 above relate to the use of ε-aminocaproic acid and / or tranexamic acid to soothe gingival symptoms, and are different from the use related to bacterial invasion of gingival epithelial cells and subsequent cell damage.

[0007] Therefore, the present invention relates to an agent that can inhibit the invasion of Porphyromonas gingivalis bacteria and Fusobacterium nucleatum bacteria into gingival epithelial cells and can also treat various symptoms caused by such invasion. [Means for solving the problem]

[0008] Therefore, the present inventors conducted extensive research to solve the above problems and discovered an agent that contains ε-aminocaproic acid and / or tranexamic acid as active ingredients and can effectively inhibit the invasion of Porphyromonas gingivalis and / or Fusobacterium nucleatum bacteria into gingival epithelial cells.

[0009] That is, the present invention provides an agent for inhibiting the invasion of Porphyromonas gingivalis and / or Fusobacterium nucleatum bacteria into gingival epithelial cells, which contains ε-aminocaproic acid and / or tranexamic acid as active ingredients. The present invention also provides an agent for suppressing infection of gingival epithelial cells by Porphyromonas gingivalis and / or Fusobacterium nucleatum bacteria, which contains ε-aminocaproic acid and / or tranexamic acid as active ingredients. Furthermore, the present invention provides an agent for attenuating the pathogenicity of Porphyromonas gingivalis and / or Fusobacterium nucleatum bacteria, which comprises ε-aminocaproic acid and / or tranexamic acid as active ingredients. [Effects of the Invention]

[0010] According to the present invention, by using ε-aminocaproic acid and tranexamic acid, which are only known as antiplasmin agents, as active ingredients, it is possible to effectively prevent Porphyromonas gingivalis and Fusobacterium 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 is also highly useful as an agent for inhibiting bacterial infection of gingival epithelial cells, and can also be used as an agent for inhibiting the adhesion of these bacteria to gingival epithelial cells. Furthermore, when ε-aminocaproic acid or tranexamic acid is used as an active ingredient, it can exert excellent effects as an agent for inhibiting osteoclast activity in the oral cavity or as an agent for normalizing the turnover of gingival epithelial cells. [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 The agent of the present invention is an agent for inhibiting the invasion of bacteria (Fn) into gingival epithelial cells, and contains ε-aminocaproic acid and / or tranexamic acid as active ingredients. That is, the agent of the present invention can effectively inhibit the invasion of Porphyromonas gingivalis and Fusobacterium nucleatum into gingival epithelial cells.

[0013] Therefore, whether Porphyromonas gingivalis or Fusobacterium nucleatum is present alone in the oral cavity, or whether both Porphyromonas gingivalis and Fusobacterium nucleatum are present, the agent of the present invention can exert the effect of suppressing the invasion of these bacteria into gingival epithelial cells.

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

[0015] Tranexamic acid may also be used in the form of tranexamic acid salts, such as metal salts (e.g., sodium salt, potassium salt, magnesium salt), hydrochloride, phosphate, and sulfate.

[0016] The active ingredient of these agents of the present invention may contain ε-aminocaproic acid alone, tranexamic acid alone, or both ε-aminocaproic acid and tranexamic acid.

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

[0018] From the viewpoint of effectively inhibiting the invasion of the above bacteria into gingival epithelial cells, the content of tranexamic acid in the agent of the present invention is preferably 0.03% by mass or more, more preferably 0.05% by mass or more, even more preferably 0.09% by mass or more, and even more preferably 0.15% by mass or more. Furthermore, from the viewpoint of reducing bitterness, the content of tranexamic acid in the agent of the present invention is preferably 2% by mass or less, more preferably 1.5% by mass or less, even more preferably 1% by mass or less, and even more preferably 0.8% by mass or less. Furthermore, the content of tranexamic acid in the agent of the present invention is preferably 0.03% by mass or more and 2% by mass or less, more preferably 0.05 to 1.5% by mass, even more preferably 0.09 to 1% by mass, and even more preferably 0.15 to 0.8% by mass. When tranexamic acid is a tranexamic salt, the amount of tranexamic acid salt converted into tranexamic acid may be within the above-mentioned range.

[0019] Thus, the agent of the present invention inhibits the invasion of the above bacteria into gingival epithelial cells and also inhibits the infection of these bacteria into gingival epithelial cells, and therefore is also useful as an agent for reducing the pathogenicity of these bacteria.

[0020] Furthermore, since the agent of the present invention inhibits the invasion and infection of gingival epithelial cells by the above-mentioned bacteria, the bacteria are unable to invade deeper into periodontal tissues, and therefore it is possible to inhibit the activation of osteoclasts and prevent the elution of alveolar bone. Thus, the agent of the present invention can be used both as an inhibitor of osteoclast activity in the oral cavity and as a periodontal tissue protective agent.

[0021] The agent of the present invention is also useful as a turnover normalizer for gingival epithelial cells. That is, gingival epithelium maintains a fast metabolic mechanism in which cells are constantly replaced with new ones, known as turnover, and thereby maintains a good defense function to protect the body from bacteria. However, when the above bacteria invade the cells, the cell turnover becomes abnormal. By inhibiting bacterial invasion with the agent of the present invention, gingival epithelial cells can be rapidly replaced with new cells through turnover, thereby effectively preventing the onset of periodontal disease.

[0022] Therefore, the agent of the present invention effectively inhibits the invasion or infection of gingival epithelial cells by Porphyromonas gingivalis and / or Fusobacterium nucleatum bacteria, or effectively weakens the pathogenicity of these bacteria, thereby inhibiting bacterial adhesion to gingival epithelial cells, protecting periodontal tissues, suppressing osteoclast activity in periodontal tissues, and enabling normal maintenance of gingival epithelial cell turnover.

[0023] Thus, the agent of the present invention is suitable for application to the oral cavity because it exhibits the effects of inhibiting the invasion of the above-mentioned bacteria into gingival epithelial cells and the effects of inhibiting infection, etc. Examples of forms of such agents for application to the oral cavity include dentifrice compositions such as toothpaste and powder toothpaste, or liquid oral compositions such as mouthwash, liquid or gel dentifrice, and mouth spray.

[0024] Therefore, in addition to the above-mentioned active ingredients, the agent of the present invention can contain ingredients that are commonly used in agents for application to the oral cavity, such as dentifrice compositions and liquid oral compositions, etc. Such ingredients include, for example, foaming agents, foaming assistants, surfactants, abrasives, bulking agents, sweeteners, preservatives, medicinal ingredients, pH adjusters, adhesives, pigments, coloring agents, and fragrances. [Example]

[0025] [Evaluation Test 1 in Gingival Epithelial Cells: Example 1] The effects of ε-aminocaproic acid and tranexamic acid as active ingredients on gingival epithelial cells were evaluated according to the following procedures (1) to (6). The results of the evaluation of the effect of aminocaproic acid are shown in Table 1, and the results of the evaluation of the effect of tranexamic acid are shown in Table 2.

[0026] (1) Preparation of gingival epithelial cells Human gingival epithelial cells (Ca9-22) were obtained from the JCRB Cell Bank (JCRB0625). The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% v / v Fetal Bovine Serum (Gibco) at 37°C and a CO2 concentration of 5%.

[0027] (2) Bacterial culture Porphyromonas gingivalis ATCC 33277 (Pg) and Fusobacterium nucleatum ATCC 25586 (Fn) were obtained from ATCC. Colony cultures were performed using AnaeroColumbia RS blood agar medium (Becton Dickinson Japan), and liquid cultures were performed using GAM broth medium (Nissui Pharmaceutical) supplemented with 5.0 μg / mL hemin, 17.4 μg / mL K2HPO4, and 1.0 μg / mL vitamin K. Cultures were performed at 37°C under anaerobic conditions using Anaeropack Kenki (Mitsubishi Gas Chemical). For P. gingivalis (Pg), the bacterial solution was cultured in liquid for 24 hours, then diluted 100-fold and cultured in liquid for another 24 hours. For F. nucleatum (Fn), the bacterial solution was cultured in liquid for 24 hours, then diluted 1000-fold and cultured in liquid for another 24 hours.

[0028] (3) Application of ε-aminocaproic acid and tranexamic acid The bacterial culture obtained in (2) above was centrifuged at 3,500 g for 10 minutes at 4°C, and the precipitated bacterial cells were collected. The collected bacterial cells were washed with PBS and then DMEM by centrifugation at 3,500 g for 5 minutes at 4°C, and then adjusted to an OD600 of 1.0. Next, ε-aminocaproic acid (reagent, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the 15 mL tube containing the obtained bacterial solution to give concentrations of 0 mM, 1 mM, 10 mM, and 100 mM, and tranexamic acid (reagent, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was also added to give concentrations of 0 mM, 1 mM, 10 mM, and 100 mM, and the mixture was left to stand together with the bacterial cells at 37°C under anaerobic conditions for 1 hour.

[0029] (4) Fluorescent labeling of bacterial cells The labeling reagent, Carboxyfluorescein diacetate succinimidyl ester (CFSE, Dojindo Laboratories), was dissolved in DMSO. The bacterial suspension obtained in (3) above was centrifuged at 3,500 g at 4°C for 10 minutes, and the precipitated bacterial cells were collected. The collected bacterial cells were washed with DMEM by centrifugation at 3,500 g at 4°C for 5 minutes, and then adjusted to an OD600 of 1.0. Next, CFSE solution (dissolving DMEM) was added to each bacterial suspension at a final concentration of 10 μM, and the suspension was allowed to stand at 37°C in the dark for 30 minutes to fluorescently label the cells. The fluorescently labeled cells were washed twice with DMEM by centrifugation at 3,500 g for 5 minutes at 4°C.

[0030] (5) Cultivation of fluorescently labeled bacteria After removing the medium from a culture plate containing Ca9-22 cells (cell seeding concentration: 0.3 × 10 cells / well in a 12-well plate) that had been cultured for 24 hours, the plate was washed with PBS, and the bacterial solution prepared in (4) above was added at a multiplicity of infection of 500. The plate was then 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 cells obtained in (5) above, and the cells were washed twice with PBS. Afterwards, cells invaded by fluorescently labeled bacteria (fluorescently labeled P. gingivalis (Pg) and F. nucleatum (Fn)) were detached using 0.25% Trypsin-EDTA (Gibco) and collected in a 1.5 mL tube. The collected cells were fixed with 4% paraformaldehyde-phosphate buffer (Fujifilm Wako Pure Chemical Industries) by pipetting at 4°C for 30 minutes. PBS containing 2% FBS was added and pipetted, followed by centrifugation at 600 g for 3 minutes at 4°C. The cells were then suspended in PBS (containing 2% FBS) and used as the analysis sample.

[0032] Flow cytometry (BD, FACSVerse) was used for analysis. 500,000 cells were gated based on forward scatter (FSC) and side scatter (SSC) and the mean fluorescence intensity (MFI) was calculated from the fluorescence intensity in the FITC channel. Cells invaded by fluorescently labeled P. gingivalis (Pg) and F. nucleatum (Fn) were detected, and the cell mass was calculated. Next, this amount was converted into a value (%), with the cell amount when 0 mM of each drug was applied (invasion of Pg and Fn was not inhibited) being taken as 100%, and used as an index of evaluation.

[0033] [Table 1]

[0034] [Table 2]

[0035] [Evaluation Test 2 in 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 performed to prepare each bacterial cell treated with various agents and then fluorescently stained. The resulting bacterial cells were then used to infect gingival epithelial cells to obtain the following analysis samples. Each sample was observed using a fluorescence microscope (BZ-X810 (Keyence Corporation), Excitation: 470 nm / Extension: 495 nm, Magnification: 60x). The following figures show the treated agents, the bacterial cells, and the results of observation under a fluorescent microscope. 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 with Fn treated with 100 mM ε-aminocaproic acid (Figures 3(A-3) and (a-3)) Example 2-4: Infection with Fn treated with 100 mM tranexamic acid (Figures 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] The effect of tranexamic acid as an active ingredient on gingival epithelial cells was evaluated according to the following procedures (1) to (4). The results of the effect evaluation are shown in Table 3.

[0037] (1) Preparation of gingival epithelial cells Human oral keratinocyte cells were seeded onto a 24-well plate and cultured in Oral Keratinocyte Medium until they reached semi-confluence.

[0038] (2) Bacterial culture Using the above Porphyromonas gingivalis ATCC 33277 (Pg), the same procedure as in (2) of Evaluation Test 1 on Gingival Epithelial Cells was carried out to obtain a bacterial culture medium.

[0039] (3) Application of tranexamic acid and cetylpyridinium chloride The bacterial culture obtained in (2) above was centrifuged at 3,500 g for 10 minutes at 4°C, and the precipitated bacterial cells were collected. The collected bacterial cells were washed using PBS by centrifugation at 3,500 g for 5 minutes at 4°C, and then adjusted to an OD600 of 1.0. Next, 500 ppm of tranexamic acid and, as Comparative Example 3, 500 ppm of cetylpyridinium chloride (CPC) were added to the 15 mL tube containing the obtained bacterial liquid, and the mixture was allowed to stand for 1 hour at 37°C under anaerobic conditions. 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 added to the cells obtained in (1) above at a multiplicity of infection of 100, and cultured for 3 hours. After removing the culture supernatant, RNA was extracted using the RNeasy mini kit (QIAGEN). Reverse transcription was then performed using High Capacity RNA to cDNA (Thermo Fisher Scientific). Interleukin-6 (IL-6) gene expression was quantified using a TaqMan® probe. The values ​​obtained were corrected for the gene expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

[0041] [Table 3]

Claims

1. Contains ε-aminocaproic acid as an active ingredient, An inhibitor of the invasion of Porphyromonas gingivalis and / or Fusobacterium nucleatum bacteria into gingival epithelial cells, wherein the content of the active ingredient is 0.05% by mass or more and 1.5% by mass or less.

2. A drug containing tranexamic acid (excluding tranexamic acid salts) as an active ingredient, An inhibitor of the invasion of Porphyromonas gingivalis and / or Fusobacterium nucleatum bacteria into gingival epithelial cells, wherein the content of the active ingredient is 0.05% by mass or more and 2% by mass or less.

3. Contains ε-aminocaproic acid as an active ingredient, An agent for inhibiting infection of gingival epithelial cells by Porphyromonas gingivalis and / or Fusobacterium nucleatum bacteria, the content of the active ingredient being 0.05% by mass or more and 1.5% by mass or less.

4. A drug containing tranexamic acid (excluding tranexamic acid salts) as an active ingredient, An agent for inhibiting infection of gingival epithelial cells by Porphyromonas gingivalis and / or Fusobacterium nucleatum bacteria, the content of the active ingredient being 0.05% by mass or more and 2% by mass or less.

5. Contains ε-aminocaproic acid as an active ingredient, A pathogenicity reducer for Porphyromonas gingivalis and / or Fusobacterium nucleatum bacteria, wherein the content of the active ingredient is 0.05% by mass or more and 1.5% by mass or less.

6. A drug containing tranexamic acid (excluding tranexamic acid salts) as an active ingredient, A pathogenicity reducer for Porphyromonas gingivalis and / or Fusobacterium nucleatum bacteria, wherein the content of the active ingredient is 0.05% by mass or more and 2% by mass or less.

7. Contains ε-aminocaproic acid as an active ingredient, An agent for inhibiting adhesion of Porphyromonas gingivalis and / or Fusobacterium nucleatum bacteria to gingival epithelial cells, wherein the content of the active ingredient is 0.05% by mass or more and 1.5% by mass or less.

8. A drug containing tranexamic acid (excluding tranexamic acid salts) as an active ingredient, An agent for inhibiting adhesion of Porphyromonas gingivalis and / or Fusobacterium nucleatum bacteria to gingival epithelial cells, wherein the content of the active ingredient is 0.05% by mass or more and 2% by mass or less.

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