Biofilm formation inhibitors

Abstract

To provide a novel biofilm formation inhibitor, pharmaceuticals comprising the formation inhibitor, and tools and materials surface-treated with the formation inhibitor.SOLUTION: A biofilm formation inhibitor comprises a compound illustrated by the following structural formula (JBD1) or a pharmacologically acceptable salt thereof as an active ingredient.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an agent for inhibiting biofilm formation by bacteria, which is a problem in the medical field and the like. [Background technology]

[0002] Staphylococcus aureus is a major cause of hospital-acquired infections, causing serious infections such as sepsis and wound infections. In particular, the spread of methicillin-resistant Staphylococcus aureus (MRSA), which has acquired multidrug resistance, has become a global problem in recent years. According to the Centers for Disease Control and Prevention (CDC), the estimated number of cases of MRSA infection in the United States in 2017 was 100. The number of cases was 323,700, with an estimated 10,600 deaths. The estimated medical costs for this disease are $1.7 billion (Antibiotic Resistance Threats in the United States, 2019 - CDC). One of the factors that makes infections caused by this bacterium difficult to treat is the formation of drug-resistant bacterial-matrix aggregates called biofilms. Biofilm-forming bacteria produce a matrix, an extracellular polymer complex composed of diverse components, which serves as a scaffold for adhesion to surfaces and cell-cell connections. Infections caused by biofilm formation are called biofilm infections and are a problem in a wide range of medical fields, including surgery, internal medicine, orthopedics, and urology. When biofilms form on the surfaces of medical devices such as catheters, pacemakers, and artificial joints, they not only cause local bacterial infections but also increase the risk of systemic infection due to persistent bacterial shedding. Furthermore, biofilm-forming bacteria maintain low metabolic activity, making them 10 to 1,000 times more drug-resistant than normal, making antibiotic treatment difficult. There is no effective treatment other than removing the device that is the source of the infection. Biofilm infections not only place a great burden on patients, but also lead to prolonged treatment periods, reduced quality of life, and increased medical costs. There is an urgent need to develop preventive measures.

[0003] Patent Document 1 discloses a biofilm formation inhibitor using phospholipase. However, since enzyme preparations have stability problems, there has been a demand for biofilm formation inhibitors that are low molecular weight compounds. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 2019-195281 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide an agent for inhibiting the formation of a biofilm by bacteria such as Staphylococcus aureus. [Means for solving the problem]

[0006] The present inventors have conducted extensive research to solve the above problems. As a result, they have found that The present inventors have found that the compound exhibits excellent effects in inhibiting biofilm formation, and have completed the present invention.

[0007] That is, one aspect of the present invention is a compound represented by general formula (I) or a pharmacologically acceptable derivative thereof. The present invention relates to a biofilm formation inhibitor containing an acceptable salt as an active ingredient. Another aspect of the present invention relates to a method for anti-biofilm treatment of a device, comprising the step of treating the device with a biofilm formation inhibitor. Another aspect of the present invention relates to an anti-biofilm treated device with a biofilm formation inhibitor. [Effects of the Invention]

[0008] According to the present invention, biofilms of Staphylococcus aureus and the like can be efficiently inhibited, thereby contributing to the fields of medicine, etc. Furthermore, when used in combination, the effects of existing antibacterial drugs can be improved. [Brief explanation of the drawings]

[0009] [Figure 1] Graph showing the effect of compounds on the respiratory activity of Staphylococcus aureus. Cells cultured for 4 hours in the presence of control, 20 μM JBD1, or 20 μM ANG1 were analyzed by flow cytometry. DETAILED DESCRIPTION OF THE INVENTION

[0010] The biofilm formation inhibitor of the present invention contains a compound of general formula (I) as an active ingredient. include.

[0011] [ka]

[0012] Here, R1 represents a hydrocarbon group having 1 to 10 carbon atoms which may have a substituent selected from halogen (e.g., Cl, F, or Br), a haloalkyl group, a hydroxy group, a carboxy group, an amino group, an alkylamino group, an aryl group (e.g., a phenyl group which may have a substituent), an alkoxy group, an acyl group, and a mercapto group, and the hydrocarbon group may be an aliphatic hydrocarbon group or an aromatic hydrocarbon group (e.g., a phenyl group which may have one or more of the above-mentioned substituents). The aliphatic hydrocarbon group may be any of the above, but is preferably an aliphatic hydrocarbon group. The number of carbon atoms in the aliphatic hydrocarbon group is preferably 2 to 8, more preferably 3 to 6. The aliphatic hydrocarbon group may be linear, branched, or may have a cyclic moiety. The aliphatic hydrocarbon group may have an unsaturated bond. Particularly preferred examples of R1 include a cyclohexyl group and an aliphatic hydrocarbon group having 4 carbon atoms.

[0013] R2 represents a hydrocarbon group having 2 to 10 carbon atoms which may have a substituent selected from halogen (e.g., Cl, F, or Br), a haloalkyl group, a hydroxy group, a carboxy group, an amino group, an alkylamino group, an aryl group (e.g., a phenyl group which may have a substituent), an alkoxy group, an acyl group, and a mercapto group, and the hydrocarbon group may be an aliphatic hydrocarbon group or an aromatic hydrocarbon group (e.g., a phenyl group which may have one or more of the above-mentioned substituents). An aliphatic hydrocarbon group is preferred. The number of carbon atoms in the aliphatic hydrocarbon group is preferably 2 to 6, and particularly preferably 2. The aliphatic hydrocarbon group may be linear or branched. The aliphatic hydrocarbon group may have an unsaturated bond, or may have a cyclic portion. Particularly preferred as R2 is an ethyl group.

[0014] R3 represents a hydrocarbon group having 1 to 10 carbon atoms which may have a substituent selected from a hydroxyl group, halogen (e.g., Cl, F, or Br), a haloalkyl group, a hydroxy group, a carboxy group, an amino group, an alkylamino group, an aryl group (e.g., a phenyl group which may have a substituent), an alkoxy group, an acyl group, and a mercapto group, and the hydrocarbon group may be an aliphatic hydrocarbon group or an aromatic hydrocarbon group (e.g., a phenyl group which may have one or more of the above-mentioned substituents). The aliphatic hydrocarbon group may be, but is preferably an aliphatic hydrocarbon group. The number of carbon atoms in the aliphatic hydrocarbon group is preferably 1 to 8, more preferably 1 to 6. The aliphatic hydrocarbon group may be linear, branched, or may have a cyclic moiety. The aliphatic hydrocarbon group may have an unsaturated bond. Particularly preferred examples of R3 include an aliphatic hydrocarbon group having 6 carbon atoms, a 1-phenylethyl group, and a benzyl group.

[0015] An example of a compound of general formula (I) is the following compound (JBD1): [ka]

[0016] Other examples of compounds of general formula (I) include the compounds shown below. [ka]

[0017] [ka]

[0018] The salt of the compound of general formula (I) is not particularly limited as long as it has a biofilm inhibitory effect. Examples of the acid addition salts include, but are not limited to, salts with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, and phosphoric acid; salts with acetic acid, malic acid, succinic acid, tartaric acid, citric acid, and the like; Examples of base addition salts include salts with organic acids such as carboxylic acid, oxalic acid, and tosylic acid, and salts with chlorine. Examples of base addition salts include salts with alkali metals such as sodium and potassium, salts with alkaline earth metals such as calcium and magnesium, and salts with amines such as ammonium and triethylamine.

[0019] The biofilms on which the biofilm formation inhibitor of the present invention is effective are not particularly limited as long as they are effective, but are preferably biofilms formed by bacteria belonging to the genus Staphylococcus, such as Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis), or Staphylococcus saprophyticus (S. saprophyticus). It is more preferable that the biofilm is a biofilm formed by multiple types of bacteria. The biofilm may be a biofilm formed by multiple types of bacteria. Note that the bacteria belonging to the genus Staphylococcus may be drug-resistant bacteria, and examples of drug-resistant Staphylococcus aureus include methicillin-resistant Staphylococcus aureus and vancomycin-resistant Staphylococcus aureus.

[0020] The biofilm formation inhibitor of the present invention is a compound represented by the above general formula (I) or a salt thereof. The effective amount for inhibiting biofilms is adjusted as appropriate depending on the type of compound and the type of bacteria that form the biofilm, but is, for example, 5 to 100 μM, more preferably 5 to 20 μM, as the concentration of the compound represented by general formula (I) or a salt thereof at the time of use. The compound may be adjusted to this concentration range in advance, or may be diluted to this concentration range at the time of use.

[0021] The effective concentration range can be determined, for example, as follows: First, target bacteria such as Staphylococcus aureus are cultured for a certain period of time in a liquid in which an object to be attached is immersed, and a biofilm of the bacteria is formed on the surface of the object to be attached. When a compound or a salt thereof is added and culture is performed, and a biofilm is not formed on the surface of the target object, or the amount of biofilm formed is 1 / 10 or less, preferably 1 / 50 or less, and more preferably 1 / 100 or less of that when the compound is not added, the effective concentration can be determined. The effective concentration range can also be determined based on the minimum growth concentration determined by the plate dilution method.

[0022] The biofilm formation inhibitor of the present invention may contain other antibacterial components and enzyme components. Other antibacterial components include aminoglycoside antibacterial agents such as gentamicin, kanamycin, and neomycin, and β-lactam antibacterial agents such as ampicillin and cefazolin. Other enzyme components include phospholipase A, C, or D and DNA disclosed in Patent Document 1. It may also contain a degrading enzyme (DNase I) or a polysaccharide-degrading enzyme (Dispersin B).

[0023] The biofilm formation inhibitor of the present invention can also be used in combination with disinfectants that are effective against bacteria that can form biofilms, such as chlorhexidine gluconate, benzalkonium chloride, benzethonium chloride, amphoteric surfactants, alcohol, sodium hypochlorite, povidone-iodine, and glutaral.

[0024] The above-mentioned other antibacterial components and disinfectants may be contained in the biofilm formation inhibitor of the present invention together with the compound of general formula (I) or its salt, or may be used in combination with the compound of general formula (I) or its salt when in use.

[0025] The dosage form of the biofilm formation inhibitor of the present invention can be appropriately selected depending on the application. Examples of such a preparation include liquid preparations dissolved in a solvent such as the above, aerosol preparations, and powder or solid preparations that are dissolved in a solvent when used.

[0026] The biofilm formation inhibitor of the present invention comprises an active ingredient represented by the general formula (I) or The salt may also be combined with a solvent, surfactant, pH adjuster, fragrance, colorant, antioxidant, preservative, excipient, suspending agent, dispersing agent, thickener, powdering agent, granulating agent, coating agent, or base material for topical medication to form a composition.

[0027] In the present invention, the biofilm formation inhibitor is used to prevent the attachment and / or deposition of biofilms on the surface of equipment such as medical instruments.

[0028] As described above, the amount of the biofilm formation inhibitor of the present invention to be used can be appropriately determined based on the type of target bacteria, the type and size of the target equipment, etc., and the effective concentration of the compound of general formula (I) can be determined. The compound or a salt thereof can be used as a solution or an aerosol formulation.

[0029] To use the biofilm formation inhibitor of the present invention to prevent (inhibit) the formation of a biofilm, the compound of general formula (I) or a salt thereof is applied to the surface of a target object at an effective concentration. The effect can be achieved by allowing the agent to act for a certain period of time (for example, 1 minute to 1 hour). The object is immersed in a solution of the compound of general formula (I) or a salt thereof or a composition containing the compound. immersing, washing with a solution of the compound of general formula (I) or a salt thereof or a composition containing the same; or by applying a solution of the compound of general formula (I) or a salt thereof or a composition thereof. That's fine.

[0030] The equipment on which the biofilm formation inhibitor can be used is not particularly limited as long as it is equipment on which biofilms are formed, and examples include plumbing equipment such as in kitchens, cooking areas, and bathrooms, toilet bowls and drains, drainage pipes, food manufacturing or beverage manufacturing plants, cooling water systems such as industrial cooling towers, desalination equipment, circulating water systems such as pools and artificial ponds, medical devices, and medical equipment such as medical dressings and medical tapes.

[0031] Among these, it is preferable to use it as a biofilm formation inhibitor for medical devices. Medical devices include, for example, endoscopes, dialysis machines, and implant devices. Examples of implant devices include, but are not limited to, catheters, artificial teeth, bolts for fixing bones in the treatment of fractures, rheumatism, etc., pacemakers, artificial heart valves, artificial joints, voice prostheses, and contact lenses. Examples of catheters include urinary catheters, peritoneal catheters, and central venous catheters.

[0032] The biofilm formation inhibitor can also be used as a medicine for patients with biofilm infections. The pharmaceuticals include, but are not limited to, liquids, tablets, capsules, emulsions, injections, and external preparations. For example, external preparations to be applied to the infected area, oral preparations, and injections are exemplified. When formulated as a pharmaceutical, the compound of general formula (I) or a salt thereof is used in a pharmaceutical acceptable form. The compound of general formula (I) or its salt, which is an active ingredient in the pharmaceutical composition, can be combined with other ingredients (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, surfactants, lubricants, diluents, coating agents, sugar coating agents, flavoring agents, emulsifying / solubilizing / dispersing agents, pH adjusters, isotonic agents, solubilizing agents, flavorings, coloring agents, solubilizing aids, physiological saline, etc.) to form a pharmaceutical composition. The amount of the compound of general formula (I) or its salt, which is an active ingredient in the pharmaceutical composition, is an amount effective for treating or preventing biofilm infections. The amount of the active ingredient is not particularly limited as long as it is present, and generally varies depending on the dosage form, but can be set within the range of, for example, about 0.01% by mass to about 99.9% by mass so as to achieve a desired dosage. The pharmaceutical of the present invention may contain other drugs such as antibacterial agents in addition to the compound of general formula (I). Good too. [Example]

[0033] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the embodiments of the following examples.

[0034] Biofilm formation inhibition test Staphylococcus aureus or Staphylococcus epidermidis was inoculated into Brain Heart Infusion (BHI) medium (manufactured by Becton Dickinson) and cultured overnight at 37°C to prepare a preculture medium. The cells were inoculated at 1 / 500 volume into 1% glucose-containing BHI medium (BHIG medium) containing the test compound and 5% DMSO or 5% DMSO only (control), and then plated in a 96-well plate (Corning). 200 μL of the medium was dispensed into each well and cultured at 37°C for 24 hours. The supernatant containing the planktonic cells was then removed using an aspirator, and the biofilm formed on the bottom was washed twice with 200 μL of phosphate buffer (pH 7.4). 100 μL of 0.05% crystal violet was added to the air-dried biofilm. After adding 100 ml of ... The plates were washed twice with 200 μL of phosphate buffer (pH 7.4) to remove any residual biofilm. Biofilm was quantified by measuring the absorbance at 595 nm (ABS) using an Infinite 200 PRO plate reader (Tecan). 595 When calculating the 50% inhibitory concentration (IC50) of a compound, the test was carried out at final compound concentrations of 0, 5, 10, 20, 40, 60, 80, and 100 μM, and the ABS of the control (0 μM) was measured. 595 The IC value was set at 100% and the inhibition rate at each concentration was calculated using the following formula: 50 was calculated. I C 50 = 10[log (A / B) × (50 - C) / (D - C) + log (B)] A: The minimum compound concentration showing more than 50% inhibitory activity, B: The maximum compound concentration showing less than 50% inhibitory activity, C: Inhibition rate (%) at compound concentration A, D: Inhibition rate (%) at compound concentration B

[0035] Antimicrobial susceptibility testing Staphylococcus aureus was inoculated into Brain Heart Infusion (BHI) medium (Becton Dickinson). The preculture solution was inoculated into 1% glucose-containing BHI medium (BHIG medium) containing the test compound and 5% DMSO or 5% DMSO only (control) at a volume of 1 / 500, and then 5 μL of the antibiotic dilution series was added to a 96-well plate (Corning 195 μL of the medium was dispensed into wells of a plate (manufactured by Tecan) and incubated at 37°C for 24 hours. The absorbance (ABS) at 595 nm was then measured using a plate reader (Infinite 200 PRO; manufactured by Tecan). 595 The bacterial growth was evaluated by measuring the ABS 595 The minimum inhibitory concentration (MIC) was defined as the lowest concentration of antibiotic that suppressed the increase in the value to less than 20%.

[0036] Measurement of respiratory activity Staphylococcus aureus was inoculated into Brain Heart Infusion (BHI) medium (Becton Dickinson). The overnight culture of the Staphylococcus aureus strain was inoculated into 1% glucose-containing BHI medium (BHIG medium) containing the test compound and 5% DMSO or 5% DMSO only (control) at a volume of 1 / 500, and then 2 mL of the pre-culture solution was dispensed into 12-well plates (manufactured by Corning) and cultured statically at 37°C for 4 hours. After suspending the cells, 1 mL of the culture solution was added. The cells were collected by centrifugation at 5,000 × g for 10 minutes and then resuspended in 1 mL of phosphate buffer (pH 7.4). The cells were then resuspended in the BacLight RedoxSensor Green Vitality Kit (Invitrogen). Add 1 μL of Component A (previously diluted 10-fold with DMSO) to the cell suspension. The cells were added to the PBS buffer and incubated at room temperature for 10 minutes. After collecting the cells by centrifugation at 5,000 × g for 10 minutes, they were fixed by treatment with 4% paraformaldehyde for 5 minutes. The fixed cells were resuspended in 1 mL of phosphate buffer (pH 7.4) and further diluted 10-fold with water. The results were analyzed using a flow cytometer (CytoFLEX; manufactured by Beckman Coulter). The excitation wavelength was set at 488 nm, the emission wavelength at 525 nm, and 10,000 cells were measured at a flow rate of 10 μL / min.

[0037] result We screened compounds that inhibit biofilm formation in Staphylococcus aureus and identified JBD1. Structure-activity relationship studies revealed that ANG1, which lacks one methyl group present in the structure of JBD1, does not exhibit biofilm-inhibitory activity, while ANG2 and ANG20, which are structural analogs of JBD1, exhibit biofilm-inhibitory activity (Table 1).

[0038] [ka] [ka]

[0039] [Table 1]

[0040] JBD1 was also examined for its biofilm formation inhibitory activity against various methicillin-susceptible or methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis strains. JBD1 exhibited biofilm formation inhibitory activity against methicillin-susceptible Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Staphylococcus epidermidis.

[0041] [Table 2]

[0042] In addition, when we investigated the effect on antibiotic susceptibility, it was shown that JBD1 increases the susceptibility of Staphylococcus aureus to aminoglycoside antibiotics and β-lactam antibiotics (Table 3).

[0043] [Table 3]

[0044] Since aminoglycoside antibiotics are known to exert their antibacterial effects dependent on bacterial respiratory activity, we evaluated the effects of JBD1 or ANG1 on respiratory activity using a flow cytometer and confirmed that respiratory activity was significantly enhanced in the presence of JBD1 (Figure 1). 1).

Claims

1. Contains a compound represented by general formula (I) or a pharmacologically acceptable salt thereof as an active ingredient , an inhibitor of biofilm formation by Staphylococcus aureus. 【Chemistry 1】 In general formula (I), R １ represents a hydrocarbon group having 1 to 10 carbon atoms which may have a substituent selected from halogen, a haloalkyl group, a hydroxy group, a carboxy group, an amino group, an alkylamino group, an aryl group, an alkoxy group, an acyl group, and a mercapto group; R ２ represents a hydrocarbon group having 2 to 10 carbon atoms which may have a substituent selected from halogen, a haloalkyl group, a hydroxy group, a carboxy group, an amino group, an alkylamino group, an aryl group, an alkoxy group, an acyl group, and a mercapto group; R ３ represents a hydrocarbon group having 1 to 10 carbon atoms which may have a substituent selected from halogen, a haloalkyl group, a hydroxy group, a carboxy group, an amino group, an alkylamino group, an aryl group, an alkoxy group, an acyl group, and a mercapto group.

2. The biofilm according to claim 1, wherein the compound represented by general formula (I) is the following compound: Inhibitor of formation. 【Chemistry 2】

3. The biofilm formation inhibitor according to claim 1 or 2, further comprising an antibacterial agent.

4. A pharmaceutical for inhibiting the formation of a biofilm caused by Staphylococcus aureus, comprising the biofilm formation inhibitor according to any one of claims 1 to 3.

5. A method for anti-biofilm treatment of equipment, comprising a step of treating the equipment with the biofilm formation inhibitor according to any one of claims 1 to 3.

6. The method of claim 5, wherein the device is a medical device.

7. A device whose surface has been treated with the biofilm formation inhibitor according to any one of claims 1 to 3.

8. 8. An equipment surface-treated with the biofilm formation inhibitor according to claim 7, wherein the equipment is a medical instrument.

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