Inhibitor of bacterial vaginosis, inhibitor of autoinducer-2 of bacterial vaginosis-associated bacteria, inhibitor of biofilm formation of bacterial vaginosis-associated bacteria, enhancer of antibacterial power of antibiotic against bacterial vaginosis-associated bacteria, and method for enhancing the antibacterial power

Abstract

To provide an inhibitor of bacterial vaginosis.SOLUTION: An inhibitor of bacterial vaginosis comprises lactic acid and / or a salt thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] One embodiment of the present disclosure relates to an inhibitor of bacterial vaginosis. Another embodiment of the present disclosure relates to an autoinducer-2 inhibitor of bacteria associated with bacterial vaginosis. Yet another embodiment of the present disclosure relates to an inhibitor of biofilm formation of bacteria associated with bacterial vaginosis. Another embodiment of the present disclosure relates to an agent for enhancing the antibacterial activity of an antibacterial agent against bacteria associated with bacterial vaginosis in a biofilm, and a method for enhancing the antibacterial activity of an antibacterial agent against bacteria associated with bacterial vaginosis in a biofilm. [Background technology]

[0002] Quorum sensing is a microbial signaling mechanism that senses the density of bacteria by sensing the extracellular concentration of autoinducers, signal substances produced by the same bacteria, and controls specific gene expression and phenotype accordingly. Quorum sensing is known to be involved in bacterial infection, pathogenicity, biofilm formation, toxin production, etc.

[0003] Gardnerella vaginalis, the main causative bacterium of bacterial vaginosis, possesses a quorum-sensing mechanism that uses autoinducer-2 (AI-2) as a signaling molecule. AI-2 is known to be involved in biofilm formation and colonization in the vagina by Gardnerella vaginalis, contributing to bacterial vaginosis. Therefore, the use of AI-2 inhibitors can inhibit quorum sensing in Gardnerella vaginalis, potentially preventing bacterial vaginosis.

[0004] Furthermore, because biofilms have the effect of inhibiting the penetration of antibiotics and other drugs and protecting the bacteria present within, Gardnerella vaginalis within biofilms is more likely to exhibit drug resistance than bacteria in a free-floating state. Therefore, to improve the efficacy of drugs for the prevention or treatment of bacterial vaginosis, it would be effective to inhibit the quorum sensing of Gardnerella vaginalis with an AI-2 inhibitor and suppress the formation of biofilms by Gardnerella vaginalis.

[0005] Previously, subtilosin and benzoyl peroxide have been reported to reduce the amount of AI-2 produced by Gardnerella vaginalis and inhibit biofilm formation (Non-Patent Documents 1 and 2). However, the components found in Non-Patent Documents 1 and 2 are difficult to obtain and expensive, and their safety when administered intravaginally has not been fully investigated. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Ammar Algburi et al., Subtilosin Prevents Biofilm Formation by Inhibiting Bacterial Quorum Sensing, Probiotics & Antimicro. Prot. (2017) 9:81-90, DOI 10.1007 / s12602-016-9242-x. [Non-patent document 2] Ammar Algburi et al., Benzoyl Peroxide Inhibits Quorum Sensing and Biofilm Formation by Gardnerella vaginalis 14018, Infectious Diseases in Obstetrics and Gynecology. Vol. 2018, Article ID 1426109, https: / / doi.org / 10.1155 / 2018 / 1426109. Summary of the Invention [Problem to be solved by the invention]

[0007] One object of the present disclosure is to provide an agent for inhibiting bacterial vaginosis. Another object of the present disclosure is to provide an AI-2 inhibitor for bacteria associated with bacterial vaginosis. Yet another object of the present disclosure is to provide an agent for inhibiting biofilm formation by bacteria associated with bacterial vaginosis. Yet another object of the present disclosure is to provide an agent for enhancing the antibacterial activity of an antibacterial agent against bacteria associated with bacterial vaginosis in a biofilm, or a method for enhancing the antibacterial activity of an antibacterial agent against bacteria associated with bacterial vaginosis in a biofilm. [Means for solving the problem]

[0008] The present inventors conducted extensive research to solve the above-mentioned problems and found that lactic acid and / or its salts exhibit excellent inhibitory activity against AI-2 when used in a concentration range in which antibacterial activity is not exerted (i.e., a concentration range in which bacterial growth and / or development is not inhibited). They also found that lactic acid and / or its salts can be used as active ingredients in AI-2 inhibitors for bacterial vaginosis-associated bacteria. Furthermore, the present inventors found that the use of lactic acid and / or its salts in a concentration range in which antibacterial activity is not exerted can inhibit the formation of biofilms by bacterial vaginosis-associated bacteria. They also found that lactic acid and / or its salts can be used as active ingredients in inhibitors of bacterial vaginosis or biofilm formation by bacterial vaginosis-associated bacteria. Furthermore, the present inventors found that the use of lactic acid and / or its salts in a concentration range in which antibacterial activity is not exerted enhances the antibacterial activity of antibacterial agents against bacterial vaginosis-associated bacteria in biofilms. The present disclosure was completed based on these findings and further research.

[0009] As one embodiment of the present disclosure, there is provided the invention having the following aspects relating to the use for inhibiting bacterial vaginosis. Item 1-1. An agent for inhibiting bacterial vaginosis, containing lactic acid and / or a salt thereof. Item 1-2. The agent for suppressing bacterial vaginosis according to Item 1-1, which is administered to achieve a concentration of lactic acid and / or a salt thereof in the vagina of 11 to 22 mM. Item 1-3: An inhibitor of bacterial vaginosis according to Item 1-1 or 1-2, which is used for bacterial vaginosis caused by Gardnerella vaginalis. Item 1-4. A vaginal preparation for suppressing bacterial vaginosis, comprising the agent for suppressing bacterial vaginosis according to any one of Items 1-1 to 1-3. Item 1-5. A vaginal preparation for suppressing bacterial vaginosis according to Item 1-4, further comprising an antibacterial agent. Item 1-6. Use of lactic acid and / or a salt thereof for producing an agent for inhibiting bacterial vaginosis. Item 1-7. Lactic acid and / or its salts used in the treatment of inhibiting bacterial vaginosis. Item 1-8. A method for suppressing bacterial vaginosis, which comprises administering lactic acid and / or a salt thereof to the vagina of a person in need of suppression of bacterial vaginosis.

[0010] Another embodiment of the present disclosure provides the following invention relating to the use of inhibiting AI-2 in bacteria associated with bacterial vaginosis. Item 2-1. An AI-2 inhibitor of bacterial vaginosis-associated bacteria, containing lactic acid and / or a salt thereof. Item 2-2. The AI-2 inhibitor according to Item 2-1, which is applied to a body site where inhibition of AI-2 of bacterial vaginosis-associated bacteria is desired, so that lactic acid and / or a salt thereof reaches a concentration of 11 to 22 mM. Item 2-3: The AI-2 inhibitor according to Item 2-1 or 2-2, wherein the bacterial vaginosis-associated bacterium is a bacterium of the genus Gardnerella. Item 2-4. A preparation for inhibiting AI-2 in bacteria associated with bacterial vaginosis, comprising the AI-2 inhibitor according to any one of Items 2-1 to 2-3. Item 2-5. A formulation for inhibiting AI-2 according to Item 2-4, further comprising an antibacterial agent. Item 2-6. Use of lactic acid and / or a salt thereof for producing an AI-2 inhibitor for bacteria associated with bacterial vaginosis. Item 2-7. Lactic acid and / or a salt thereof for use in a treatment for inhibiting AI-2 of bacteria associated with bacterial vaginosis. Item 2-8. A method for inhibiting AI-2 in bacteria associated with bacterial vaginosis, comprising administering an effective amount of lactic acid and / or a salt thereof to a body site where inhibition of AI-2 in bacteria associated with bacterial vaginosis is desired.

[0011] Another embodiment of the present disclosure provides the following invention relating to the use of a composition for inhibiting biofilm formation by bacteria associated with bacterial vaginosis. Item 3-1. An agent for inhibiting biofilm formation by bacteria associated with bacterial vaginosis, comprising lactic acid and / or a salt thereof. Item 3-2. The biofilm formation inhibitor according to Item 3-1, which is applied to a body site where inhibition of biofilm formation by bacterial vaginosis-associated bacteria is desired, so that lactic acid and / or a salt thereof reaches a concentration of 11 to 22 mM. Item 3-3. The biofilm formation inhibitor according to Item 3-1 or 3-2, wherein the bacterial vaginosis-associated bacterium is a bacterium of the genus Gardnerella. Item 3-4. A preparation for inhibiting biofilm formation by bacteria associated with bacterial vaginosis, comprising the biofilm formation inhibitor according to any one of Items 3-1 to 3-3. Item 3-5. A preparation for inhibiting biofilm formation according to Item 3-4, further comprising an antibacterial agent. Item 3-6. Use of lactic acid and / or a salt thereof for producing an agent for inhibiting the formation of a biofilm by bacteria associated with bacterial vaginosis. Item 3-7. Lactic acid and / or a salt thereof for use in a treatment to inhibit the formation of a biofilm of bacteria associated with bacterial vaginosis. Item 3-8. A method for inhibiting biofilm formation by bacteria associated with bacterial vaginosis, comprising administering an effective amount of lactic acid and / or a salt thereof to a body site where inhibition of biofilm formation by bacteria associated with bacterial vaginosis is desired.

[0012] Another embodiment of the present disclosure provides the following invention relating to the use of enhancing the antibacterial activity of an antibacterial agent against bacterial vaginosis-associated bacteria in a biofilm. Item 4-1. An agent for enhancing the antibacterial activity of an antibacterial agent against bacterial vaginosis-associated bacteria in a biofilm, the agent comprising lactic acid and / or a salt thereof. Item 4-2. The antibacterial activity enhancer according to Item 4-1, which is applied to a body site where enhanced antibacterial activity of an antibacterial agent against bacterial vaginosis-associated bacteria in a biofilm is required, so that lactic acid and / or a salt thereof is at a concentration of 11 to 22 mM. Item 4-3. The antibacterial activity enhancer according to Item 4-1 or 4-2, wherein the bacterial vaginosis-associated bacterium is a bacterium of the genus Gardnerella. Item 4-4. A preparation for enhancing the antibacterial activity of an antibacterial agent against bacteria associated with bacterial vaginosis, comprising the antibacterial activity enhancer according to any one of Items 4-1 to 4-3. Item 4-5. A preparation for enhancing the antibacterial activity of an antibacterial agent according to Item 4-4, further comprising an antibacterial agent against bacteria associated with bacterial vaginosis. Item 4-6. Use of lactic acid and / or a salt thereof for producing an agent for enhancing the antibacterial activity of an antibacterial agent against bacterial vaginosis-associated bacteria in a biofilm. Items 4-7. A method for enhancing the antibacterial activity of an antibacterial agent against bacterial vaginosis-associated bacteria in a biofilm in a preparation containing the antibacterial agent, the method comprising blending lactic acid and / or a salt thereof into the preparation containing the antibacterial agent. Item 4-8. The method according to Item 4-7, wherein the amount of lactic acid and / or a salt thereof in the preparation is set so that the concentration of lactic acid and / or a salt thereof is 11 to 22 mM at the body site to which the preparation is administered. Item 4-9. The method according to Item 4-7 or 4-8, wherein the preparation is a vaginal preparation. Item 4-10. The method according to any one of Items 4-7 to 4-9, wherein the bacterial vaginosis-associated bacterium is a bacterium of the genus Gardnerella. Item 4-11. The method according to any one of Items 4-7 to 4-10, wherein the preparation is a vaginal preparation. [Effects of the Invention]

[0013] According to one embodiment of the present disclosure, bacterial vaginosis can be suppressed by inhibiting quorum sensing of bacterial vaginosis-associated bacteria. According to another embodiment of the present disclosure, AI-2 of bacterial vaginosis-associated bacteria can be inhibited without exerting antibacterial activity, thereby inhibiting quorum sensing of bacterial vaginosis-associated bacteria occurring in the vagina. According to yet another embodiment of the present disclosure, biofilm formation by bacterial vaginosis-associated bacteria can be effectively suppressed without exerting antibacterial activity. Furthermore, the antibacterial agent enhancer of the present disclosure can enhance the antibacterial activity of an antibacterial agent against bacterial vaginosis-associated bacteria in a biofilm. Therefore, by applying the enhancer and an antibacterial agent of the present disclosure to the vagina where a biofilm of bacterial vaginosis-associated bacteria has formed, bacterial vaginosis-associated bacteria in the biofilm can be effectively antibacterialized. [Brief explanation of the drawings]

[0014] [Figure 1]This shows the results of evaluating the AI-2 inhibitory activity of lactic acid. The bar graph shows the measurement results for relative luminescence unit (RLU), and the dots show the measurement results for viable cell count (CFU / mL). [Figure 2] These are the results of evaluating the inhibitory effect of lactic acid on biofilm formation by Gardnerella vaginalis. The bar graph shows the absorbance (OD595) measurement results, and the dots show the viable cell count (CFU / mL). DETAILED DESCRIPTION OF THE INVENTION

[0015] 1.Definition In this disclosure, the notation "X to Y" regarding a numerical range refers to a range of X or more and Y or less.

[0016] In the present disclosure, the term "bacterial vaginosis inhibitor" refers to a component used for inhibiting bacterial vaginosis. In addition, in the present disclosure, the term "inhibition of bacterial vaginosis" refers to the inhibition of the onset of bacterial vaginosis, the prolongation of the period until the onset of bacterial vaginosis, or the inhibition of the progression of bacterial vaginosis, and includes the prevention of bacterial vaginosis and the inhibition of the worsening of the symptoms of bacterial vaginosis.

[0017] In this disclosure, bacterial vaginosis-associated bacteria refers to bacteria that overgrow in bacterial vaginosis.

[0018] In the present disclosure, an AI-2 inhibitor of bacteria associated with bacterial vaginosis refers to a component used to inhibit AI-2 produced by bacteria associated with bacterial vaginosis.

[0019] In the present disclosure, an inhibitor of biofilm formation by bacteria associated with bacterial vaginosis refers to a component used to inhibit biofilm formation by bacteria associated with bacterial vaginosis.

[0020] In the present disclosure, an agent for enhancing the antibacterial activity of an antibacterial agent against bacterial vaginosis-associated bacteria in a biofilm refers to a component used to enhance the antibacterial activity of an antibacterial agent against bacterial vaginosis-associated bacteria in a biofilm. Also, in the present disclosure, a method for enhancing the antibacterial activity of an antibacterial agent against bacterial vaginosis-associated bacteria in a biofilm refers to a method performed to enhance the antibacterial activity of an antibacterial agent against bacterial vaginosis-associated bacteria in a biofilm.

[0021] 2. Inhibitor of bacterial vaginosis In one embodiment of the present disclosure, there is provided an inhibitor of bacterial vaginosis (hereinafter, also referred to as "inhibitor") comprising lactic acid and / or a salt thereof. The inhibitor of the present disclosure will be described in detail below.

[0022] [Lactic acid and / or its salts] The inhibitor of the present disclosure contains lactic acid and / or its salt as an active ingredient for inhibiting bacterial vaginosis. Lactic acid and / or its salt have AI-2 inhibitory activity and can inhibit quorum sensing by bacteria associated with bacterial vaginosis. Based on this mechanism, lactic acid and / or its salt can exert an effect of inhibiting the formation of biofilms by bacteria associated with bacterial vaginosis, thereby enabling the inhibition of bacterial vaginosis.

[0023] The salt of lactic acid used in the present disclosure is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, ammonium salt, etc. These salts of lactic acid may be used alone or in combination of two or more.

[0024] In the inhibitor of the present disclosure, one kind of lactic acid and its salts may be selected and used alone, or two or more kinds may be used in combination. Among lactic acid and its salts, lactic acid is a preferred example.

[0025] [Concentration used] Lactic acid and / or its salts can inhibit AI-2, a bacterial vaginosis-associated bacterium, even at low concentrations where the antibacterial activity of lactic acid and / or its salts is not exerted. Therefore, in one embodiment of the inhibitor of the present disclosure, lactic acid and / or its salts are administered at a concentration that does not exert the antibacterial activity of lactic acid and / or its salts in the vagina. The intravaginal concentration of lactic acid and / or its salts, within which the antibacterial activity of lactic acid and / or its salts is not exerted and the inhibitory activity against AI-2, a bacterial vaginosis-associated bacterium, can be, for example, 11 to 22 mM, preferably 16 to 22 mM, 16 to 19 mM, or 19 to 22 mM. Here, the "concentration of lactic acid and / or its salts in the vagina" refers to the intravaginal concentration of lactic acid and / or its salts contained in the inhibitor of the present disclosure that is reached after administration into the vagina. For example, when the inhibitor of the present invention is provided in the form of a vaginal tablet, the concentration of lactic acid and / or its salts contained in the vaginal tablet is the concentration reached after the vaginal tablet is dissolved in the vagina. By using lactic acid and / or its salts at such a concentration that does not exert antibacterial activity, bacterial vaginosis can be suppressed while avoiding the adverse effects of lactic acid and / or its salts on normal vaginal bacteria that are not involved in bacterial vaginosis.

[0026] [Indications, dosage and administration] The inhibitor of the present disclosure is used by vaginal administration to suppress bacterial vaginosis. The inhibitor of the present disclosure can suppress the formation of a biofilm by inhibiting AI-2 of bacterial vaginosis-related bacteria, and can therefore be used for the purpose of preventing bacterial vaginosis or suppressing the worsening of bacterial vaginosis symptoms.

[0027] The type of bacterial vaginosis to which the inhibitor of the present disclosure is applicable is not particularly limited, and the inhibitor can be applied to bacterial vaginosis caused by any bacterial vaginosis-associated bacteria, such as Gardnerella bacteria (e.g., Gardnerella vaginalis), Prevotella bacteria, Peptostreptococcus bacteria, Mobiluncus bacteria, or Mycoplasmas bacteria (e.g., Mycoplasma hominis). In one embodiment of the inhibitor of the present disclosure, the inhibitor is preferably applied to bacterial vaginosis caused by at least one of Gardnerella bacteria, Prevotella bacteria, and Mobiluncus bacteria.

[0028] Furthermore, since the inhibitors disclosed herein can enhance the antibacterial activity of antibacterial drugs against bacterial vaginosis-associated bacteria in biofilms, they can also be used in combination with antibacterial drugs for the prevention or treatment of bacterial vaginosis.

[0029] The dosage of the inhibitor of the present disclosure may be an effective amount that exhibits the effect of inhibiting bacterial vaginosis. A preferred example of the dosage of the inhibitor of the present disclosure is set to a concentration in the vagina that does not exhibit antibacterial activity of lactic acid and / or its salts, but exhibits inhibitory activity against AI-2, a bacterial vaginosis-associated bacterium. The number of doses of the inhibitor of the present disclosure may be appropriately set within a range that exhibits the effect of inhibiting bacterial vaginosis, and may be, for example, about 1 to 3 times per day, preferably about once per day.

[0030] [Preparations containing inhibitors] The inhibitor of the present disclosure is used by incorporating it into a vaginal preparation (a preparation for vaginal administration). The dosage form of the vaginal preparation incorporating the inhibitor of the present disclosure is not particularly limited, and may be any of liquid, semi-solid, solid, etc., as long as it can be administered into the vagina. Specific examples of vaginal preparations incorporating the inhibitor of the present disclosure include ointments, creams, gels, vaginal tablets (vaginal suppositories), liquids, etc. Among these preparations, ointments are preferred. These preparations can be prepared by formulating them using additives appropriate for the dosage form according to known methods described in the General Provisions for Preparations in the 18th Edition of the Japanese Pharmacopoeia, etc.

[0031] The content of lactic acid and / or a salt thereof in the vaginal preparation containing the inhibitor of the present disclosure may be appropriately set depending on the type of vaginal preparation, etc., so that the lactic acid and / or a salt thereof reaches the concentration described in the above-mentioned "Usage concentration" column in the vagina after administration, and may be, for example, 0.1 to 3 wt %, preferably 0.15 to 3 wt %, and more preferably 0.2 to 3 wt %.

[0032] The vaginal preparation containing the inhibitor of the present disclosure may contain an antibacterial agent, if necessary. Lactic acid and / or a salt thereof has the effect of enhancing the antibacterial activity of the antibacterial agent against bacterial vaginosis-associated bacteria in biofilms. Therefore, when the vaginal preparation containing the inhibitor of the present disclosure contains an antibacterial agent, not only the AI-2 inhibitory activity of lactic acid and / or a salt thereof but also the antibacterial activity of the antibacterial agent against the causative bacteria of bacterial vaginosis can be enhanced and exerted, thereby making it possible to more effectively suppress bacterial vaginosis.

[0033] The type of antibiotic used may be any that has an antibacterial effect against bacteria associated with bacterial vaginosis, with suitable examples including antibiotics that have an antibacterial effect against Gardnerella vaginalis. Specific examples of such antibiotics include metronidazole, clindamycin, chloramphenicol, and tetracycline antibiotics (minocycline, doxycycline, etc.). These antibiotics may be used alone or in combination.

[0034] When an antibacterial agent is contained in a vaginal preparation containing the inhibitor of the present disclosure, the content thereof may be appropriately set depending on the type of antibacterial agent used, the dosage form and administration amount of the inhibitor of the present disclosure, etc., but may be, for example, 0.1 to 2% by weight, preferably 0.5 to 2% by weight, and more preferably 1 to 2% by weight.

[0035] The vaginal preparation containing the inhibitor of the present disclosure may contain other pharmacological ingredients in addition to the above-mentioned ingredients, as needed. Furthermore, the vaginal preparation containing the inhibitor of the present disclosure may contain base materials or additives other than the above-mentioned ingredients, as needed, to form a desired formulation.

[0036] 3. AI-2 inhibitors In another embodiment of the present disclosure, there is provided an AI-2 inhibitor for bacterial vaginosis-associated bacteria, comprising lactic acid and / or a salt thereof. The AI-2 inhibitor of the present disclosure is described in detail below.

[0037] [Lactic acid and / or its salts] The AI-2 inhibitor of the present disclosure contains lactic acid and / or a salt thereof as an active ingredient for inhibiting AI-2 of bacterial vaginosis-associated bacteria. The types of lactic acid salts used in the AI-2 inhibitor of the present disclosure are as described in the section "2. Bacterial vaginosis inhibitors" above.

[0038] The AI-2 inhibitor of the present disclosure may be selected from lactic acid and its salts and used alone or in combination of two or more thereof. Among lactic acid and its salts, lactic acid is a preferred example.

[0039] [Concentration used] Lactic acid and / or its salts can inhibit AI-2 in bacterial vaginosis-associated bacteria even at low concentrations where the antibacterial activity of lactic acid and / or its salts is not exerted. Therefore, in one embodiment of the AI-2 inhibitor of the present disclosure, the AI-2 inhibitor is applied and used at a concentration at which the antibacterial activity of lactic acid and / or its salts is not exerted at the application site. The concentration of lactic acid and / or its salts at the application site, which does not exert the antibacterial activity of lactic acid and / or its salts but can exert AI-2 inhibitory activity, can be, for example, 11 to 22 mM, preferably 16 to 22 mM, 16 to 19 mM, or 19 to 22 mM. Here, the "concentration of lactic acid and / or its salts at the application site" refers to the concentration at which lactic acid and / or its salts contained in the AI-2 inhibitor of the present disclosure act at the site where inhibition of AI-2 in bacterial vaginosis-associated bacteria is desired, i.e., the concentration reached by lactic acid and / or its salts contained in the AI-2 inhibitor of the present disclosure at the application site. By using lactic acid and / or its salts at a concentration that does not exert antibacterial activity in this way, it is possible to prevent or treat infections caused by bacterial vaginosis-related bacteria that control quorum sensing using AI-2 as a signal substance, while avoiding the adverse effects of lactic acid and / or its salts on normal bacteria that are not involved in the infection.

[0040] [Indications, dosage and administration] The AI-2 inhibitors of the present disclosure are used to inhibit AI-2 in bacterial vaginosis-associated bacteria, which regulate quorum sensing using AI-2 as a signaling substance. The AI-2 inhibitors of the present disclosure can inhibit quorum sensing by inhibiting AI-2 in bacterial vaginosis-associated bacteria. As a result, the AI-2 inhibitors of the present disclosure can suppress pathogenicity, biofilm formation, toxin production, etc., caused by bacterial vaginosis-associated bacteria, thereby preventing or suppressing the progression of infections caused by bacterial vaginosis-associated bacteria. Therefore, the AI-2 inhibitors of the present disclosure can be used as agents for preventing or suppressing the progression of infections caused by bacterial vaginosis-associated bacteria by applying them to body sites where the bacteria are present (e.g., the oral cavity, skin, vagina, etc.). Furthermore, since the AI-2 inhibitors of the present disclosure inhibit quorum sensing in bacterial vaginosis-associated bacteria, they can effectively exert the antibacterial activity of antibacterial agents against bacterial vaginosis-associated bacteria in biofilms, and can therefore be used in combination with antibacterial agents to combat bacteria in biofilms.

[0041] Examples of bacteria associated with bacterial vaginosis include bacteria of the genus Gardnerella, such as Gardnerella vaginalis; bacteria of the genus Prevotella; bacteria of the genus Peptostreptococcus; bacteria of the genus Mobiluncus; and bacteria of the genus Mycoplasma, such as Mycoplasma hominis. Among these, preferred examples of bacteria to which the AI-2 inhibitors of the present disclosure are applied include bacteria of the genus Gardnerella, bacteria of the genus Prevotella, and bacteria of the genus Mobiluncus, and more preferred examples include bacteria of the genus Gardnerella.

[0042] Inhibiting AI-2 in bacterial vaginosis-associated bacteria can inhibit their quorum sensing, thereby suppressing the formation of biofilms that cause bacterial vaginosis. Therefore, in one embodiment, the AI-2 inhibitors disclosed herein can be used as inhibitors of bacterial vaginosis. Furthermore, because the AI-2 inhibitors disclosed herein can effectively exert the antibacterial activity of antibacterial agents against bacterial vaginosis-associated bacteria in biofilms, they can also be used in combination with antibacterial agents to inhibit Gardnerella vaginalis in biofilms.

[0043] The dosage of the AI-2 inhibitor of the present disclosure may be an effective amount capable of exerting inhibitory activity against AI-2 of bacteria associated with bacterial vaginosis. A preferred example of the dosage of the AI-2 inhibitor of the present disclosure per administration is set so that lactic acid and / or a salt thereof does not exert antibacterial activity at the administration site but exerts AI-2 inhibitory activity. The frequency of administration of the AI-2 inhibitor of the present disclosure may be appropriately set within a range in which the inhibitory activity against AI-2 of bacteria associated with bacterial vaginosis is effectively exerted, and may be, for example, about 1 to 3 times per day, preferably about once per day.

[0044] [Preparations containing AI-2 inhibitors] The AI-2 inhibitor of the present disclosure is formulated into a desired dosage form and used as a preparation for inhibiting AI-2 in bacteria associated with bacterial vaginosis.

[0045] The dosage form of the preparation containing the AI-2 inhibitor of the present disclosure is not particularly limited, and may be any of solid, semi-solid, or liquid.

[0046] A formulation incorporating the AI-2 inhibitor of the present disclosure may also contain an antibacterial agent. A formulation comprising the AI-2 inhibitor of the present disclosure and an antibacterial agent can enhance the antibacterial activity of the antibacterial agent against bacterial vaginosis-associated bacteria in biofilms. The types of antibacterial agents are as described above in the section "2. Bacterial vaginosis inhibitors." When an antibacterial agent is contained in a formulation incorporating the AI-2 inhibitor of the present disclosure, the content of the antibacterial agent in the transmucosal formulation may be appropriately determined depending on the type of formulation, etc., but may be, for example, 0.1 to 2 wt %, preferably 0.5 to 2 wt %, and more preferably 1 to 2 wt %.

[0047] Specific examples of formulations incorporating the AI-2 inhibitor of the present disclosure include transmucosal formulations, topical skin preparations, oral care products, foods and beverages, etc. Among these, transmucosal formulations, topical skin preparations, and oral care products are preferred.

[0048] When the AI-2 inhibitor of the present disclosure is incorporated into a transmucosal preparation (i.e., when provided as a transmucosal preparation for inhibiting AI-2 in bacteria associated with bacterial vaginosis), lactic acid and / or a salt thereof may be combined with other additives, medicinal ingredients, and the like to prepare a desired form. Examples of transmucosal preparations include vaginal preparations, and specific examples of vaginal preparations include ointments, creams, gels, vaginal tablets (vaginal suppositories), and liquid preparations. When the AI-2 inhibitor of the present disclosure is incorporated into a transmucosal preparation, the content of lactic acid and / or a salt thereof in the transmucosal preparation may be appropriately determined depending on the type of transmucosal preparation, etc., so that the concentration of lactic acid and / or a salt thereof at the application site is as described in the "Use Concentration" column above; for example, 0.1 to 3 wt %, preferably 0.15 to 3 wt %, and more preferably 0.2 to 3 wt %.

[0049] When the AI-2 inhibitor of the present disclosure is incorporated into a topical skin preparation (i.e., when provided as a topical skin preparation for inhibiting AI-2 in bacteria associated with bacterial vaginosis), lactic acid and / or a salt thereof may be combined with other additives, medicinal ingredients, etc. to prepare a desired form. Examples of topical skin preparations include topical skin pharmaceuticals and cosmetics, specifically creams, lotions, gels, emulsions, liquids, poultices, patches, liniments, aerosols, aqueous ointments, and packs; and cosmetics such as aqueous ointments, creams, emulsions, lotions, packs, and gels. Among these, topical skin preparations are preferred. When the AI-2 inhibitor of the present disclosure is incorporated into a topical skin preparation, the content of lactic acid and / or its salts in the topical skin preparation may be appropriately set depending on the type of topical skin preparation, etc., so that the concentration of lactic acid and / or its salts at the applied site is as described in the "Usage concentration" column above, and may be, for example, 0.1 to 3 wt %, preferably 0.15 to 3 wt %, and more preferably 0.2 to 3 wt %.

[0050] When the AI-2 inhibitor of the present disclosure is incorporated into an oral care product (i.e., when provided as an oral care product for inhibiting AI-2 in bacteria associated with bacterial vaginosis), lactic acid and / or a salt thereof may be combined with other additives, medicinal ingredients, etc. to prepare a desired form. Oral care products may be any product that can be applied to the oral cavity and remain there for a certain period of time, and specific examples include oral hygiene products such as liquid dentifrices, toothpastes, mouthwashes, mouth fresheners (e.g., mouth sprays), oral pastes, and gum massage creams. When the AI-2 inhibitor of the present disclosure is incorporated into an oral care product, the content of lactic acid and / or a salt thereof in the oral care product may be appropriately determined depending on the type of oral care product, etc., so that the concentration of lactic acid and / or a salt thereof at the application site is as described in the "Use Concentration" column above. Examples include, for example, 0.1 to 3 wt %, preferably 0.15 to 3 wt %, and more preferably 0.2 to 3 wt %.

[0051] When the AI-2 inhibitor of the present disclosure is incorporated into a food or beverage (i.e., when provided as a food or beverage for inhibiting AI-2 in bacteria associated with bacterial vaginosis), lactic acid and / or a salt thereof can be prepared into the desired form, either directly or in combination with other food ingredients or additives. Examples of such foods and beverages include general foods and beverages, as well as foods with health claims (including foods for specified health uses, foods with nutrient functions, and foods with functional claims), and foods for patients. The form of these foods and beverages is not particularly limited, but specific examples include beverages such as tea drinks, energy drinks, fruit juice drinks, carbonated drinks, and lactic acid drinks; supplements such as capsules (soft capsules and hard capsules), tablets, granules, powders, jellies, and liposome preparations; and luxury items such as gummies, candies, and jellies. Among these foods and beverages, supplements are preferred. When the AI-2 inhibitor of the present disclosure is incorporated into a food or beverage, the content of lactic acid and / or its salts in the food or beverage may be appropriately set depending on the type of food or beverage, etc., so that the lactic acid and / or its salts reach the concentration described in the "Usage Concentration" column at the site of application (in the oral cavity, in the intestinal tract, etc.), and may be, for example, 0.1 to 3 wt %, preferably 0.15 to 3 wt %, and more preferably 0.2 to 3 wt %.

[0052] 4. Biofilm formation inhibitors In another embodiment of the present disclosure, there is provided an agent for inhibiting the formation of a biofilm of bacterial vaginosis-associated bacteria, comprising lactic acid and / or a salt thereof. The biofilm formation inhibitor of the present disclosure is described in detail below.

[0053] [Lactic acid and / or its salts] The biofilm formation inhibitor of the present disclosure contains lactic acid and / or a salt thereof as an active ingredient for inhibiting biofilm formation by bacterial vaginosis-associated bacteria. The types of lactic acid salts used in the biofilm formation inhibitor of the present disclosure are as described in the section "2. Bacterial vaginosis inhibitor" above.

[0054] In the biofilm formation inhibitor of the present disclosure, one kind of lactic acid and its salts may be selected and used alone, or two or more kinds may be used in combination. Among lactic acid and its salts, lactic acid is a preferred example.

[0055] [Concentration used] The inhibitory effect of lactic acid and / or its salts on the formation of biofilms by bacteria associated with bacterial vaginosis can be exerted even at low concentrations at which the antibacterial activity of lactic acid and / or its salts is not exerted, and therefore, in one embodiment of the biofilm formation inhibitor of the present disclosure, the agent is applied and used at a concentration at which the antibacterial activity of lactic acid and / or its salts is not exerted at the application site. The concentration of lactic acid and / or its salts at the application site can be in a range of 11 to 22 mM, preferably 16 to 22 mM, 16 to 19 mM, or 19 to 22 mM, for example. Here, the "concentration of lactic acid and / or a salt thereof at the application site" refers to the concentration at which lactic acid and / or a salt thereof contained in the biofilm formation inhibitor of the present disclosure acts at the site where it is desired to inhibit biofilm formation by bacterial vaginosis-associated bacteria, i.e., the concentration reached by lactic acid and / or a salt thereof contained in the biofilm formation inhibitor of the present disclosure at the site to which the biofilm formation inhibitor is applied. By using lactic acid and / or a salt thereof at a concentration at which antibacterial activity is not exerted, it is possible to inhibit biofilm formation by bacterial vaginosis-associated bacteria while avoiding the adverse effects of lactic acid and / or a salt thereof on normal bacteria not involved in infection.

[0056] [Indications, dosage and administration] The biofilm formation inhibitor of the present disclosure is used to inhibit biofilm formation by bacterial vaginosis-associated bacteria that regulate quorum sensing using AI-2 as a signaling substance. Biofilms formed by bacterial vaginosis-associated bacteria on skin or mucous membranes are involved in protecting pathogenic bacteria, expressing bacterial pathogenicity, and producing bacterial toxins. Therefore, by applying the biofilm formation inhibitor of the present disclosure to body parts where bacterial vaginosis-associated bacteria biofilms are formed (in the oral cavity, skin, vagina, etc.) or body parts where such biofilms are likely to form (in the oral cavity, skin, vagina, etc.), it can prevent or inhibit the progression of infections caused by bacterial vaginosis-associated bacteria and restore healthy skin and mucous membranes. Furthermore, because the biofilm formation inhibitor of the present disclosure can effectively exert the antibacterial activity of antibacterial agents against bacterial vaginosis-associated bacteria in biofilms, it can also be used in combination with antibacterial agents to inhibit the antibacterial activity of bacterial vaginosis-associated bacteria in biofilms.

[0057] In the biofilm formation inhibitor of the present disclosure, the biofilm to be inhibited may be one formed by bacteria associated with bacterial vaginosis, and examples include biofilms formed by bacteria of the genus Gardnerella, such as Gardnerella vaginalis; Prevotella; Peptostreptococcus; Mobiluncus; and Mycoplasma, such as Mycoplasma hominis. In the biofilm formation inhibitor of the present disclosure, a suitable example of a biofilm to be inhibited is a biofilm formed by at least one of bacteria of the genus Gardnerella, Prevotella, and Mobiluncus.

[0058] For example, bacterial vaginosis can be prevented or treated by inhibiting the formation of a biofilm of bacterial vaginosis-associated bacteria in the vagina. Furthermore, the biofilm formation inhibitor of the present disclosure can effectively exert the antibacterial activity of an antibacterial agent against bacterial vaginosis-associated bacteria in a biofilm, and therefore can be used in combination with an antibacterial agent to inhibit the bacterial vaginosis-associated bacteria in a biofilm.

[0059] The amount of the biofilm formation inhibitor of the present disclosure to be applied may be an effective amount that can exert its biofilm formation inhibitory effect. A suitable example of the amount of the biofilm formation inhibitor of the present disclosure to be applied per administration is set to a concentration at the administration site where lactic acid and / or a salt thereof does not exert its antibacterial activity but is able to exert its biofilm formation inhibitory effect on bacterial vaginosis-associated bacteria. The number of administrations of the biofilm formation inhibitor of the present disclosure may be appropriately set within a range in which the biofilm formation inhibitory effect is effectively exerted, and may be, for example, about 1 to 3 times per day, preferably about once per day.

[0060] [Preparations containing biofilm formation inhibitors] The biofilm formation inhibitor of the present disclosure is formulated into a desired dosage form and used as a preparation for inhibiting biofilm formation by bacteria associated with bacterial vaginosis.

[0061] The dosage form of the preparation containing the biofilm formation inhibitor of the present disclosure is not particularly limited, and may be any of solid, semi-solid, or liquid.

[0062] A formulation incorporating the biofilm formation inhibitor of the present disclosure may also contain an antibacterial agent. A formulation comprising the biofilm formation inhibitor of the present disclosure and an antibacterial agent can enhance the antibacterial activity of the antibacterial agent against bacteria in biofilms. The types of antibacterial agents are as described above in "2. Bacterial vaginosis inhibitors." When an antibacterial agent is contained in a formulation incorporating the biofilm formation inhibitor of the present disclosure, the content of the antibacterial agent in the formulation is the same as that described above in "3. AI-2 inhibitors."

[0063] Specific examples of preparations incorporating the biofilm formation inhibitor of the present disclosure include transmucosal preparations, topical skin preparations, oral care products, foods and beverages, etc. Specific examples of preparations incorporating the biofilm formation inhibitor of the present disclosure are the same as those described above for "3. AI-2 inhibitor."

[0064] 5. Antibacterial enhancer In another embodiment of the present disclosure, there is provided an agent for enhancing the antibacterial activity of an antibacterial agent against bacteria in a biofilm (hereinafter, also referred to as an "enhancing agent"), which comprises lactic acid and / or a salt thereof. The enhancing agent of the present disclosure will be described in detail below.

[0065] [Lactic acid and / or its salts] The enhancer of the present disclosure contains lactic acid and / or a salt thereof as an active ingredient for enhancing the antibacterial activity of an antibacterial agent against bacterial vaginosis-associated bacteria in a biofilm. The types of lactic acid salts used in the enhancer of the present disclosure are as described in the section "2. Bacterial vaginosis inhibitors" above.

[0066] In the enhancer of the present disclosure, one kind of lactic acid and its salts may be selected and used alone, or two or more kinds may be used in combination. Among lactic acid and its salts, lactic acid is a suitable example.

[0067] [Concentration used] Lactic acid and / or a salt thereof can enhance the antibacterial activity of an antibacterial agent even at low concentrations where the antibacterial activity of lactic acid and / or a salt thereof is not exerted. Therefore, in one embodiment of the enhancer of the present disclosure, the enhancer is applied and used at a concentration at which the antibacterial activity of lactic acid and / or a salt thereof is not exerted at the application site. The concentration of lactic acid and / or a salt thereof at the application site, which does not exert the antibacterial activity of lactic acid and / or a salt thereof and can enhance the antibacterial activity of an antibacterial agent, is, for example, 11 to 22 mM, preferably 16 to 22 mM, 16 to 19 mM, or 19 to 22 mM. Here, the "concentration of lactic acid and / or a salt thereof at the application site" refers to the concentration at which lactic acid and / or a salt thereof contained in the enhancer of the present disclosure acts at the site where the antibacterial activity of an antibacterial agent is desired to be enhanced, i.e., the concentration of lactic acid and / or a salt thereof contained in the enhancer of the present disclosure at the application site.

[0068] [Indications, dosage and administration] The enhancer of the present disclosure is used as an additive component to enhance the antibacterial activity of an antibacterial agent against bacterial vaginosis-associated bacteria in a biofilm. When a biofilm is formed on the skin, in the oral cavity, in the vagina, or the like, the biofilm prevents the antibacterial agent from penetrating and weakens the antibacterial activity of the antibacterial agent. However, by applying the enhancer of the present disclosure in combination with an antibacterial agent to a biofilm, an effective antibacterial effect can be exerted against bacterial vaginosis-associated bacteria in the biofilm.

[0069] The type of antibacterial agent whose antibacterial activity is to be enhanced by the enhancer of the present disclosure is not particularly limited, and examples include the antibacterial agents exemplified above in "2. Agents for inhibiting bacterial vaginosis."

[0070] In the enhancer of the present disclosure, the type of bacterial vaginosis-associated bacteria targeted by the antibacterial agent whose antibacterial activity is enhanced is not particularly limited, and examples include those exemplified in "3. AI-2 inhibitors" above. In the enhancer of the present disclosure, suitable examples of bacterial vaginosis-associated bacteria targeted by the antibacterial agent whose antibacterial activity is enhanced include bacteria of the genus Gardnerella, bacteria of the genus Prevotella, and bacteria of the genus Mobiluncus.

[0071] For example, bacterial vaginosis can be prevented or treated by effectively inhibiting bacterial vaginosis-related bacteria in biofilms within the vagina, and therefore the enhancer of the present disclosure can be suitably used for the purpose of enhancing the antibacterial activity of antibacterial agents used to prevent or treat bacterial vaginosis.

[0072] [Preparations containing enhancers] The potentiator of the present disclosure is formulated into a desired dosage form and used as a preparation for potentiating the antibacterial activity of an antibacterial agent.

[0073] The dosage form of the preparation into which the enhancer of the present disclosure is blended is not particularly limited, and may be any of solid, semi-solid, or liquid.

[0074] A formulation incorporating the enhancer of the present disclosure preferably also contains an antibacterial agent. A formulation containing the enhancer of the present disclosure and an antibacterial agent may be applied to body sites (skin, oral cavity, vagina, etc.) where a biofilm of bacterial vaginosis-associated bacteria has formed. The types of antibacterial agents are as described in the section "2. Bacterial vaginosis inhibitors" above.

[0075] Furthermore, when the formulation containing the enhancer of the present disclosure does not contain an antibacterial agent, the formulation containing the enhancer of the present disclosure and the formulation containing the antibacterial agent may be applied simultaneously or in any order to a body part (skin, oral cavity, vagina, etc.) where a biofilm of bacterial vaginosis-associated bacteria has formed.

[0076] Specific examples of preparations incorporating the enhancer of the present disclosure include transmucosal preparations, topical skin preparations, oral care products, foods and beverages, etc. Specific examples of preparations incorporating the enhancer of the present disclosure, the content of lactic acid and / or a salt thereof in the preparation, and the type and content of the antibacterial agent when the preparation contains an antibacterial agent are the same as those described above in "3. AI-2 inhibitor."

[0077] 6. Methods for enhancing antibacterial activity Another embodiment of the present disclosure provides a method for enhancing the antibacterial activity of an antibacterial agent against bacterial vaginosis-associated bacteria in a biofilm in a formulation containing the antibacterial agent, comprising incorporating lactic acid and / or a salt thereof into the formulation containing the antibacterial agent (hereinafter sometimes referred to as a "method for enhancing antibacterial activity").

[0078] The amount of lactic acid and / or its salt used in the method for enhancing antibacterial activity disclosed herein, the type of antibacterial agent, the type of bacterial vaginosis-associated bacteria targeted by the antibacterial agent, the content of the antibacterial agent in the formulation, the type of formulation, etc. are as described in the section "5. Antibacterial activity enhancer" above. [Example]

[0079] The present disclosure will be specifically described below using examples, but the present invention is not limited to these examples.

[0080] Test Example 1: Verification of AI-2 inhibitory activity 1. Test Method The AI-2 inhibitory activity was measured using reporter bacteria that emit light upon recognition of AI-2. The test materials and measurement conditions used are as follows.

[0081] (1) Test materials Reporter bacteria The reporter bacterium used was Vibrio harveyi MM32 strain (ATCC BAA-1121, luxN::Cm, luxS::Tn5Ka).

[0082] ·Km-LB medium Km-LB medium was prepared by sterilizing LB medium containing 10 g / L sodium chloride, 10 g / L bactotryptone, and 5 g / L yeast extract under high pressure (121°C, 15 minutes) and then aseptically adding kanamycin sulfate to a concentration of 50 mg / L.

[0083] AB medium First, an aqueous solution containing 0.3 M sodium chloride, 0.05 M magnesium sulfate, and 2 g / L casamino acids (vitamin-free, Difco) was adjusted to pH 7.5 with potassium hydroxide and autoclaved (121 °C, 15 min). 100 mL of this solution was mixed with 1 mL of 1 M potassium phosphate buffer (a buffer adjusted to pH 7.0 by mixing 1 M potassium dihydrogen phosphate aqueous solution and 1 M dipotassium hydrogen phosphate aqueous solution), 1 mL of an aqueous solution containing 0.1 M L-arginine, 2 mL of an aqueous solution containing 50 wt% glycerol, 0.01 mL of an aqueous solution containing 0.1 mg / mL riboflavin, and 0.01 mL of an aqueous solution containing 10 mg / mL thiamine hydrochloride. The mixture was then sterilized by filtration using a 0.22 μm filter to obtain AB medium.

[0084] ·HMF-containing liquid 2.35 mg of 4-hydroxy-5-methyl-3(2H)-furanone (HMF) (CAS: 19322-27-1), 1 mL of ethanol, and 9 mL of sterile distilled water were mixed in a glass container to prepare an HMF-containing solution (containing 2,000 μM HMF). HMF is known to have AI-2 activity, and in this study, HMF was used as an AI-2 surrogate.

[0085] Specimen Lactic acid was added to an aqueous solution containing 10% by volume of ethanol so that the concentration was 20 times the concentration shown in Table 1, to prepare various samples. [Table 1]

[0086] (2) Measurement conditions The reporter bacteria were pre-cultured in Km-LB medium. The absorbance at 600 nm (OD ) of the resulting pre-culture solution was measured. 600 ) and add an appropriate amount of Km-LB medium to obtain the OD 600 The reporter bacteria solution was adjusted to 0.50, and then diluted 2,500 times with AB medium to obtain a reporter bacteria diluted solution.

[0087] In a sterile glass test tube, 0.9 mL of the reporter bacteria dilution and 0.05 mL of the sample were mixed. The pH was adjusted using 1 N NaOH to a value that would prevent lactic acid from exerting antibacterial activity against the reporter bacteria. The mixture of the reporter bacteria dilution and the sample was incubated for 10 minutes at 30°C with stirring (180 rpm) in the dark. Next, 0.05 mL of HMF-containing solution was added, and the mixture was incubated for 4 hours at 30°C with stirring (180 rpm) in the dark. At this point, the sample was diluted 20-fold. The final concentration listed in Table 1 is the lactic acid concentration at this point. After incubation, 0.1 mL of the solution was transferred to a luminescence measurement plate, and the luminescence intensity at 578 nm (the luminescence intensity of the sample) was measured using a microplate reader (SpextraMax iD5, measurement mode: LUM endpoint, Molecular Devices). The number of viable reporter bacteria in the solution after incubation was also measured using the colony counting method.

[0088] As a blank, an aqueous solution containing 10% by volume of ethanol was used instead of the specimen, and an aqueous solution containing 10% by volume of ethanol was used instead of the HMF-containing solution, and the luminescence intensity (blank luminescence intensity) was measured under the same conditions as above. The relative luminescence intensity (RLU) of the specimen was calculated by subtracting the measured luminescence intensity of the blank from the measured luminescence intensity of the specimen.

[0089] 2. Test Results The results are shown in Figure 1. When the lactic acid concentration was 11 to 22 mM, the viable cell count was equivalent to that without lactic acid, confirming that lactic acid at concentrations of 11 to 22 mM did not exhibit antibacterial activity. Furthermore, when the lactic acid concentration was 11 to 22 mM, a concentration-dependent decrease in relative luminescence intensity (RLU) was observed, confirming that lactic acid has AI-2 inhibitory activity. In other words, the results of this test revealed that lactic acid exhibits AI-2 inhibitory activity at low concentrations that do not exhibit antibacterial activity.

[0090] Test Example 2: Verification of the biofilm formation inhibitory effect of Gardnerella vaginalis 1. Test Method The inhibitory effect on biofilm formation was evaluated using Gardnerella vaginalis. The test materials and measurement conditions used were as follows.

[0091] (1) Test materials Gardnerella vaginalis Gardnerella vaginalis (JCM 11026T) was used.

[0092] ·sBHI liquid medium 37 g of brain heart infusion (BHI) broth (Becton Dickinson Japan) was mixed with 1000 mL of distilled water, sterilized by high-pressure steam (121°C, 15 minutes), and then 30 mL of horse serum (heat-inactivated horse serum from New Zealand, ThermoFisher) was added to obtain sBHI liquid medium.

[0093] sBHI agar medium 52 g of brain heart infusion (BHI) agar (Becton Dickinson Japan Co., Ltd.) was mixed with 1000 mL of distilled water, sterilized by high-pressure steam (121°C, 15 minutes), and then 30 mL of horse serum (heat-inactivated horse serum from New Zealand, ThermoFisher) was added and the mixture was placed in a petri dish to prepare sBHI agar medium.

[0094] BHIG liquid medium 37 g of Brain Heart Infusion (BHI) broth (Becton Dickinson Japan Co., Ltd.) and 10 g of glucose were mixed with 1000 mL of distilled water, and the mixture was sterilized by high-pressure steam (121°C, 15 minutes) to obtain a BHI liquid medium.

[0095] Lactic acid-containing BHIG liquid medium Lactic acid was added to the BHI liquid medium to give concentrations of 0 mM, 11 mM, 13 mM, 16 mM, 19 mM, or 22 mM to obtain lactic acid-containing BHIG liquid medium.

[0096] Specimen Lactic acid was used.

[0097] (2) Measurement conditions Biofilm formation Gardnerella vaginalis was smeared on sBHI agar medium, placed in an Anaerobox containing Anaeropacks, and cultured at 37°C for 4 days. The cultured Gardnerella vaginalis was then inoculated into sBHI liquid medium, placed in an Anaerobox containing Anaeropacks, and cultured at 37°C for 1 day in a 20% CO2 incubator. The resulting culture was diluted 100-fold with 10 mL of BHIG liquid medium or 10 mL of lactic acid-containing BHIG liquid medium. 0.2 mL was dispensed into each well of a 96-well plate, capped, and placed in an Anaerobox containing Anaeropacks and cultured at 37°C for 24 hours. The viable cell count and biofilm volume were then measured as described below.

[0098] Measurement of viable bacteria count The wells were pipetted until the biofilm on the bottom was completely detached and suspended, and the number of viable bacteria was measured by colony counting (using sBHI agar medium, an Anaerobox containing an Anaeropack, at 37°C for 2 days).

[0099] Measurement of biofilm volume After gently removing the culture medium without damaging the biofilm in the wells, 0.1 mL of PBS was added and pipetted once to wash the biofilm on the bottom of the wells and remove planktonic cells. The 96-well plate was then inverted and left to dry at 60°C for 60 minutes to allow the biofilm to adhere to the bottom of the wells. 0.2 mL of 0.01% crystal violet staining solution was then added to each well and left to stand at room temperature for 15–20 minutes. 0.2 mL of sterile water was then added to each well and pipetted once to wash the biofilm on the bottom of the wells. 0.2 L of 95% ethanol was then added and the staining solution was extracted by pipetting. The resulting extract was diluted appropriately and the absorbance at 595 nm (sample absorbance) was measured. As a blank, BHIG liquid medium was used instead of the Gardnerella vaginalis culture medium, and the absorbance at 595 nm (blank absorbance) was measured under the same conditions as above. The absorbance (OD595) of the sample was calculated by subtracting the measured absorbance of the blank from the measured absorbance of the sample.

[0100] 2. Test Results The results are shown in Figure 2. The viable cell counts at lactic acid concentrations of 11 to 22 mM were equivalent to those without lactic acid, confirming that lactic acid at concentrations of 11 to 22 mM did not exhibit antibacterial activity. Furthermore, a concentration-dependent decrease in absorbance (OD595) was observed at lactic acid concentrations of 11 to 22 mM, confirming that lactic acid has an inhibitory effect on Gardnerella vaginalis biofilm formation. These test results demonstrate that lactic acid can inhibit Gardnerella vaginalis biofilm formation at low concentrations that do not exhibit antibacterial activity. Given that lactic acid was confirmed to have AI-2 inhibitory activity in Test Example 1, it is believed that lactic acid inhibits quorum sensing in Gardnerella vaginalis through AI-2 inhibition, resulting in the suppression of biofilm formation at low concentrations that do not exhibit antibacterial activity. Furthermore, since Gardnerella vaginalis is the causative bacterium of bacterial vaginosis, the results of this study also revealed that lactic acid has an inhibitory effect on bacterial vaginosis.

[0101] Test Example 3: Verification of the effect on the antibacterial activity of antibiotics 1. Test Method The antibacterial activity of antibacterial drugs against free-floating Gardnerella vaginalis and Gardnerella vaginalis in biofilms was evaluated. The test materials and measurement conditions used were as follows.

[0102] (1) Test materials Gardnerella vaginalis Gardnerella vaginalis (JCM 11026T) was used.

[0103] sBHI liquid medium, sBHI agar medium, BHIG liquid medium, and BHIG liquid medium containing lactate According to the method described in Test Example 2 above, sBHI liquid medium, sBHI agar medium, BHIG liquid medium, and lactic acid-containing BHIG liquid medium were prepared.

[0104] (2) Test conditions (2-1) Verification of antibacterial activity against airborne Gardnerella vaginalis Preparation of suspended bacterial suspension Gardnerella vaginalis was smeared on sBHI agar medium, placed in an Anaerobox containing Anaeropacks, and cultured at 37°C for 4 days. The cultured Gardnerella vaginalis was then inoculated into sBHI liquid medium, placed in an Anaerobox containing Anaeropacks, and cultured at 37°C for 1 day in a 20% CO2 incubator. The resulting culture was diluted 100-fold with BHIG liquid medium, and 5 mL of the diluted solution was dispensed into glass test tubes, placed in an Anaerobox containing Anaeropacks, and cultured at 37°C for 1 day. The resulting culture was transferred to a centrifuge tube and centrifuged, after which the supernatant was removed. An additional 5 mL of PBS was added, pipetted, and centrifuged again. After removing the supernatant, 5 mL of sterile water was added to obtain a suspended bacterial solution (10 7-8 CFU / mL) was obtained.

[0105] Measurement of minimum inhibitory concentration and minimum bactericidal concentration A two-fold serial dilution of metronidazole or clindamycin was prepared using sBHI liquid medium. 0.2 mL of the diluted antibiotic solution was dispensed into a 96-well plate, and 1 / 50 of the volume of the suspended bacterial solution was added. The plate was then placed in an Anaerobox containing Anaeropacks and incubated at 37°C for 24 hours.

[0106] After incubation, the presence or absence of growth of Gardnerella vaginalis in each well was confirmed, and the minimum inhibitory concentrations (MIC) of metronidazole and clindamycin were determined.

[0107] After the incubation, 0.02 mL of the culture medium was dropped onto sBHI agar medium, placed in an Anaerobox containing Anaeropack, and incubated at 37°C for 24 hours. After incubation, the presence or absence of colony formation was confirmed, and the minimum bactericidal concentrations (MBC) of metronidazole and clindamycin were determined.

[0108] (2-2) Verification of antibacterial activity against Gardnerella vaginalis in biofilm Biofilm preparation Gardnerella vaginalis was smeared on sBHI agar medium and placed in an Anaerobox containing Anaeropacks and cultured at 37°C for 4 days. The cultured Gardnerella vaginalis was then inoculated into sBHI liquid medium, placed in an Anaerobox containing Anaeropacks, and cultured at 37°C for 1 day in a 20% CO2 incubator. The resulting culture medium was diluted 100-fold with 10 mL of BHIG liquid medium or lactate-containing BHIG liquid medium. 0.2 mL of the diluted solution was dispensed into each well of a 96-well plate, capped, and placed in an Anaerobox containing Anaeropacks and cultured at 37°C for 24 hours. The culture medium was then gently removed without damaging the biofilm in the well. Then, 0.2 mL of PBS was added and pipetted once to wash the biofilm on the bottom of the well and remove any planktonic cells.

[0109] Measurement of minimum inhibitory concentration and minimum bactericidal concentration A two-fold serial dilution of metronidazole or clindamycin was prepared in sBHI liquid medium. 0.2 mL of the antibiotic dilution was added to the well containing the biofilm, which was then placed in an Anaerobox containing an Anaeropack and incubated at 37°C for 24 hours.

[0110] After incubation, the presence or absence of growth of Gardnerella vaginalis in each well was confirmed, and the minimum inhibitory concentrations (MIC) of metronidazole and clindamycin were determined.

[0111] After the incubation, the biofilm on the bottom of the well was completely detached and suspended by pipetting to prepare a biofilm-derived suspension. 0.02 mL of this biofilm-derived suspension was added dropwise to sBHI agar medium, placed in an Anaerobox containing Anaeropack, and incubated at 37°C for 24 hours. After incubation, the presence or absence of colony formation was confirmed, and the minimum bactericidal concentrations (MBC) of metronidazole and clindamycin were calculated.

[0112] 2. Test Results The results are shown in Table 2. Metronidazole and clindamycin demonstrated excellent antibacterial activity against free-floating Gardnerella vaginalis, but little antibacterial activity was observed against Gardnerella vaginalis in biofilms formed in the absence of lactic acid. In contrast, metronidazole and clindamycin demonstrated excellent antibacterial activity against Gardnerella vaginalis in biofilms formed in the presence of lactic acid. In other words, these test results confirmed that lactic acid, when used in a low concentration range where it does not exert antibacterial activity, can enhance the antibacterial activity of antibiotics against bacteria present in biofilms.

[0113] [Table 2]

Claims

1. An agent for inhibiting bacterial vaginosis, comprising lactic acid and / or a salt thereof.

2. The agent for suppressing bacterial vaginosis according to claim 1, which is administered so that the concentration of lactic acid and / or a salt thereof in the vagina becomes 11 to 22 mM.

3. A vaginal preparation for suppressing bacterial vaginosis, comprising the agent for suppressing bacterial vaginosis according to claim 1 or 2.

4. The vaginal preparation for suppressing bacterial vaginosis according to claim 3, further comprising an antibacterial agent.

5. An autoinducer-2 inhibitor for bacterial vaginosis-associated bacteria, comprising lactic acid and / or a salt thereof.

6. The autoinducer-2 inhibitor according to claim 5, which is applied to a body site where inhibition of autoinducer-2 of bacterial vaginosis-associated bacteria is desired, so that lactic acid and / or a salt thereof reaches a concentration of 11 to 22 mM.

7. The autoinducer-2 inhibitor according to claim 5 or 6, wherein the bacterial vaginosis-associated bacterium is a Gardnerella bacterium.

8. An agent for inhibiting the formation of biofilms by bacteria associated with bacterial vaginosis, comprising lactic acid and / or a salt thereof.

9. The biofilm formation inhibitor according to claim 8, which is applied to a body site where inhibition of biofilm formation by bacterial vaginosis-associated bacteria is desired, so that lactic acid and / or a salt thereof reaches a concentration of 11 to 22 mM.

10. The biofilm formation inhibitor according to claim 8 or 9, wherein the bacterial vaginosis-associated bacterium is a Gardnerella bacterium.

11. An agent for enhancing the antibacterial activity of an antibacterial agent against bacteria associated with bacterial vaginosis in a biofilm, the agent comprising lactic acid and / or a salt thereof.

12. The antibacterial activity enhancer according to claim 11, which is applied to a body site where enhanced antibacterial activity of an antibacterial agent against bacterial vaginosis-associated bacteria in a biofilm is desired, so that lactic acid and / or a salt thereof reaches a concentration of 11 to 22 mM.

13. The antibacterial activity enhancer according to claim 11 or 12, wherein the bacterial vaginosis-associated bacterium is a Gardnerella bacterium.

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