JP2010095489A - Toxin production inhibitor of pathogenic bacteria - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop a toxin production inhibitor without inducing resistant bacteria and having a wide anti-bacterial spectrum by solving a problem in that since the abuse of antibiotics brought infection within a hospital, the development of an infection-treating agent without inducing the resistant bacteria is desired, and as the treating agent hardly inducing the resistant bacteria, medicines without showing bactericidal effects and only inhibiting the production of the toxin attract attentions, but they have a defect of having a narrow anti-bacterial spectrum in conventional methods. <P>SOLUTION: This ethylene diamine tetra-acetic acid is found out to bring the reduction of production of the toxin as a result of inhibiting the synthesis of an auto-inducer of bacteria through the inhibition of polyphosphoric acid synthesis in the bacteria. That means, the above medicine is the treating agent and preventing agent for reducing the toxin production. The above medicine is the treating and preventing agent having a wide anti-bacterial spectrum capable of acting both on Gram positive bacteria and Gram negative bacteria, and also considered to hardly induce the resistant bacteria. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to methods for the treatment and prevention of bacterial infections in patients. The method of the present invention is applicable to most Gram negative and Gram positive infections. The present invention suppresses the production of toxins by inhibiting the synthesis of the cell signaling substance itself that controls the production of toxins, and can be used for the suppression and treatment of bacterial infections.

  Antibiotics such as antibiotics are used to treat infections, but abuse of antibacterial agents has led to the emergence of multidrug-resistant bacteria that are not effective at all, leading to nosocomial infections. . At present, there are no effective treatments for nosocomial infections, and there remain major medical problems.

  The main pathogenic bacteria for this nosocomial infection are Pseudomonas aureus and Staphylococcus aeruginosa. Since these bacteria pose a serious threat to human health, we will refer to Pseudomonas aeruginosa as a representative example of a gram-negative bacterial infection and S. aureus as a representative example of a gram-positive bacterial infection. This is not intended to limit the scope of the invention in any way.

Pseudomonas aeruginosa and Staphylococcus aureus are both typical opportunistic pathogens in humans, infecting patients with compromised immune function and sometimes dying. Both pathogenic bacteria have high resistance to disinfectants and antibiotics, and many of them have acquired drug resistance, so once they develop, they are difficult to treat.

  Antibiotics can no longer exert their efficacy against pathogenic bacteria that have acquired multidrug resistance. That is, there is a limit to the use of antibacterial agents including antibiotics, and the development of therapeutic and preventive drugs that have completely different mechanisms of action from antibiotics is desired. Furthermore, this new therapeutic and preventive drug requires a condition that resistant bacteria are unlikely to appear.

  In recent years, it has been clarified that toxin production of many pathogenic bacteria including multidrug-resistant bacteria is promoted by a cell signaling substance called an autoinducer produced by itself. The structure of this autoinducer differs depending on the bacterial species, and in particular, the structure is completely different between Gram-negative bacteria and Gram-positive bacteria. Gram-negative bacteria are compounds called homoserine lactone, whereas gram-positive bacteria are cyclic oligopeptides.

Therefore, attempts have been made to control the pathogenicity by adjusting the autoinducer level. To suppress the action of the autoinducer, Princeton University used a substance (analog) with a structure very similar to that of the autoinducer in 2003 (Patent Document 1), and Haptogen Limited in 2006 A method for suppressing toxin production was developed using the antibody (Patent Document 2). Since these methods do not suppress the growth of pathogenic bacteria but suppress only the production of toxins, they are attracting attention as infectious disease therapeutic agents and preventive agents in which selective pressure does not occur and resistant bacteria hardly appear.
Special table 2003-532698 Special table 2006-508910

  However, these methods listed above target autoinducers with different structures depending on the individual pathogenic bacteria, so the problem is the narrow antibacterial spectrum that only affects specific pathogenic bacteria. . That is, when an infectious disease occurs, it is necessary to identify an infected pathogenic bacterium, and in addition to the complexity, it often takes time to identify the pathogenic bacterium and the symptoms often worsen. Therefore, development of a new toxin production inhibitor having a broad antibacterial spectrum is desired.

  The present invention provides a method for controlling the toxicity of pathogenic bacteria to humans, animals and plants by suppressing the synthesis of bacterial autoinducers, and provides a method with a wide antibacterial spectrum in which resistant bacteria are unlikely to appear. .

  As a result of intensive studies to solve the problems in the previous period, the present inventor has inhibited ethylene autotetraducer synthesis through inhibition of polyphosphate synthesis in bacteria through ethylenediaminetetraacetic acid (hereinafter referred to as EDTA). As a result, the present invention was completed.

  Inhibition of autoinducer synthesis of pathogenic bacteria by EDTA results in a decrease in toxin production of pathogenic bacteria. That is, the present invention is a method for regulating toxin production of pathogenic bacteria using EDTA. Since it suppresses the synthesis of the autoinducer itself, it is a method that has a broad antibacterial spectrum and makes it difficult for resistant bacteria to appear.

  Bacteria to be treated or prevented include Actinobacillus actinomycetemcomitans, Acinetobacter baumani, Bordetella sp. Bordetella sp. (Campylobacter sp.), Capnocytophaga sp., Cardiobacterium hominis, Eikenella corrodens, Ferrari genus Hellis cell illus ducreyi, Haemophilus influenzae, Helicobacter pylori, Kingella kingae, Legionella pneumopterum palaeum (Citrobacter sp.), Enterobacter sp., Escherichia coli, Klebsiella pneumoniae, Proteus sp., Salmonella enteritidis (Salmonella enteritidis) onella typhi, Serratia marcescens, Shigella sp., Yersinia enterocolitica, Yersinia pestis, Neisserhisitis, Neisserhisitis ), Moraxella catarrhalis, Veillonella sp., Bacteroides fragilis, Bacteroides sp., Prebella sp. m sp.), Spirolum minus, Aeromonas sp., Plesiomonas shigeroides, Vibrio cholerae, Vibrio chorioe V. vulnificus, Acinetobacter sp., Flavobacterium sp., Pseudomonas aeruginosa, Burkholderia-Pepperidia burdum lei), Xanthomonas maltophilia, Stenotrophomonas maltophila, Staphylococcus aureus, Bacillus sp. ), Clostridium spp., And Streptococcus spp.

  While antimicrobial treatments act directly on bacteria to cause death, the present invention targets autoinducers for the purpose of reducing toxin production. For this reason, it is considered that the possibility of emergence of a strain resistant to the treatment method is much lower.

  EDTA acts through the inhibition of polyphosphate synthesis because it chelates and removes the magnesium necessary for the activity of polyphosphate synthase. Many drug-resistant bacteria have acquired resistance by discharging drugs that have entered the cells to the outside of the cells, but even when EDTA is discharged outside the cells, extracellular magnesium can be removed and the activity of polyphosphate synthase Therefore, the effectiveness is not lost. That is, it is considered that the possibility that resistant bacteria appear is lower than that of the conventional method.

  It is contemplated that the present invention can be administered to treat bacterial infections or as a prophylactic agent when the risk of infection is high. If an infection already exists, EDTA can be administered alone or in combination with an antimicrobial antibody or antibiotic or other antimicrobial therapy.

  The dosage of this drug may vary widely depending on the disease or disorder to be treated, the tissue, age and condition of the individual to be treated, and ultimately the appropriate dosage to be used by the physician is determined. I think that.

  This drug can be formulated as a therapeutic or prophylactic agent for human, animal or plant infections. Treatments can also be used to increase the effectiveness of existing treatments.

  The composition may be provided as a drug containing only EDTA, and can generally be provided in a sealed container, and further provided as part of a kit. EDTA may be mixed with a plurality of other drugs and administered.

  The method of the present invention can be applied to short-term or long-term, acute or chronic diseases or disorders caused by bacteria.

  The therapeutic and prophylactic methods of the invention can further include administration of antibiotics. The antibiotic may be a β-lactam antibiotic such as penicillin or a penicillin derivative, kanamycin, ampicillin, chloramphenicol, tetracycline, fluoroquinolone, gentamicin, imipenem, and / or carbenicillin, or combinations thereof. Administration of EDTA may be performed at the same time as or before or after administration of antibiotics. The therapeutic and prophylactic methods of the present invention are equally applicable to humans, veterinary medicine and even plant pathology.

  The pharmaceutical composition can be administered by oral (including buccal or sublingual), rectal, intranasal, cutaneous, intravaginal, subcutaneous, intramuscular, intravenous or intradermal route.

  Pharmaceutical compositions prepared for oral administration can be provided as capsules or tablets, powders or granules, solutions, syrups or suspensions.

  Pharmaceutical compositions prepared for topical application can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. Pharmaceutical compositions prepared for topical application to the eye include eye drops wherein the active ingredient is dissolved or suspended in an aqueous solvent. Pharmaceutical compositions prepared for topical application in the mouth include buccal tablets, lozenges and mouthwashes.

  Pharmaceutical compositions prepared for rectal administration can be provided as suppositories or enemas.

  A pharmaceutical composition wherein the carrier prepared for intranasal administration is a solid, includes a crude powder administered by rapid inhalation through the nasal cavity in a manner inhaled through the nose. Compositions suitable for administration as spray nasal drops or nasal drops, where the carrier is a liquid, include aqueous or oily solutions of EDTA.

  Pharmaceutical compositions prepared for administration by inhalation include particulate matter or mist produced by various types of metered pressure pressurized spray devices, nebulizers or inhalers.

  Pharmaceutical compositions prepared for intravaginal administration can be provided as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

  Pharmaceutical compositions prepared for enteral administration include aqueous and non-aqueous sterile injection solutions and aqueous and non-aqueous sterile suspensions. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

  Next, the principle that the EDTA of the present invention exhibits a toxin production inhibitory action will be described.

(Test Example 1: Inhibitory effect on intracellular polyphosphate accumulation)
It has been shown that reduction of intracellular polyphosphate accumulation reduces autoinducer synthesis and suppresses toxin production (Raid et al., 2000). Polyphosphate is synthesized by an enzyme called polyphosphate kinase, which requires magnesium. Since EDTA has the property of depriving magnesium, it is expected to suppress the activity of polyphosphate kinase and reduce the amount of accumulated polyphosphate in cells. Therefore, Staphylococcus aureus was cultured in TSB medium (tryptic soy broth) to a low cell density (absorbance 600 nm = 0.9), and the cells were collected by centrifugation, and then TSB containing EDTA was collected. It was suspended in a medium and prepared to have a high cell density (absorbance 600 nm = 1.8). Addition of EDTA is dissolved in distilled water to 100 mmol / liter (hereinafter abbreviated as mM), adjusted to pH = 7.0 with 2 mol / liter sodium hydroxide, and the final concentration is 10 mM. The addition was performed as follows. Thereafter, the cells were cultured for 3 hours, and the amount of increase in intracellular polyphosphate accumulation was measured. The result is shown in FIG. EDTA has been shown to significantly reduce intracellular polyphosphate accumulation.

(Test Example 2; Transcriptional repression of autoinducer synthesis-related genes)
In the above experiment, we decided to confirm that the synthesis of the autoinducer was actually suppressed. The autoinducer synthesis-related gene (agrB) is read and transcribed into mRNA (messenger RNA), and the transcribed mRNA is converted into protein. It means that it is decreasing. Therefore, the amount of mRNA of the autoinducer synthesis-related gene (agrB) was examined by a quantitative PCR (real-time PCR) method. The result is shown in FIG. EDTA also clearly suppressed the transcription of mRNA for autoinducer synthesis-related genes. That is, EDTA is shown to suppress autoinducer synthesis by reducing the amount of polyphosphate accumulated in the cells, and this is the principle of EDTA that has a toxin production-suppressing effect.

(Example 1: Effect on Pseudomonas aeruginosa)
Next, the effect of the present invention to suppress toxin production will be described with reference to experimental examples.
The present invention was added at 1 mM to 10 mM, and Pseudomonas aeruginosa was cultured at 37 ° C. for 24 hours in Luria-Bertani medium (LB medium). Thereafter, pyocyanin toxin and elastase, which are pathogenic factors of this bacterium, were measured. The produced pyocyanin toxin was determined by measuring the absorbance (wavelength 570 nm) of the supernatant obtained by removing the cells by centrifugation. The elastase activity was determined by adding CBB elastin (CBB; red pigment) to the supernatant obtained in the same manner as above and culturing for 10 hours, and measuring the absorbance (wavelength wavelength: 405 nm). The amount of protein in the precipitate (bacteria) obtained by centrifugation was measured and used as a measure of the amount of bacteria. The results are shown in Table 1. The addition of EDTA caused a slight decrease in the amount of bacterial cells (the amount of bacterial protein), while the production of pyocyanin toxin was greatly reduced. Elastase, the major toxin of this pathogenic bacterium, was also found to be significantly suppressed, as was the case with pyocyanin toxin. This result shows that EDTA specifically suppressed the production of toxins while hardly inhibiting the growth of Pseudomonas aeruginosa.

(Example 2: Effect on Staphylococcus aureus)
Staphylococcus aureus is cultured to a low cell density (absorbance absorbance 600 nm = 0.9) in TSB medium, and the cells are collected by centrifugation and then suspended in TSB medium containing 10 mM inhibitor. And adjusted to a high cell density (absorbance 600 nm = 1.8). Then, after culturing for 3 hours, the mRNA amount of the pathogenicity related factor (catalase) gene (catA) was examined by a quantitative PCR method. The result is shown in FIG. Addition of EDTA decreased the gene expression level of catalase. That is, EDTA was shown to have an effect of suppressing the expression of virulence factors of Staphylococcus aureus.

  It can be used as an infectious disease therapeutic agent and prophylactic agent in place of antibiotics that are losing efficacy due to the emergence of resistant bacteria. In addition, it can be used as an effective therapeutic agent and preventive agent for nosocomial infections that currently have no effective treatment method. Furthermore, it can be used as an effective therapeutic agent and preventive agent for periodontal disease and bad breath. In addition to humans, there is a possibility that they can be used as therapeutic and preventive drugs for infectious diseases of animals and plants such as livestock and pets. Other than that, it can be used as a biofilm production inhibitor or preventive agent produced by bacteria that clog the sewer pipe.

It shows the effect of EDTA on the amount of increase or decrease of polyphosphate accumulated in the cells of S. aureus. It represents the effect of EDTA on the amount of transcription of autoinducer synthesis-related gene (agrB) into mRNA in S. aureus. It shows the effect of EDTA on the amount of transcription of the toxin catalase gene to mRNA in S. aureus.

Claims (3)

  Infectious disease therapeutic agent and preventive agent as a toxin production inhibitor of pathogenic bacteria containing ethylenediaminetetraacetic acid as a medicinal ingredient.   Infectious disease treatment and prevention as a solution, suspension, powder, granule, fine granule, tablet, capsule, syrup, and intravenous or intramuscular injection inhibitor of toxin production containing ethylenediaminetetraacetic acid medicine. Bacteria to be treated or prevented include Actinobacillus actinomycetemcomitans, Acinetobacter baumani, Bordetella sp. Bordetella sp. (Campylobacter sp.), Capnocytophaga sp., Cardiobacterium hominis, Eikenella corrodens, Ferrari genus Hellis cell illus ducreyi, Haemophilus influenzae, Helicobacter pylori, Kingella kingae, Legionella pneumopterum palaeum (Citrobacter sp.), Enterobacter sp., Escherichia coli, Klebsiella pneumoniae, Proteus sp., Salmonella enteritidis (Salmonella enteritidis) onella typhi, Serratia marcescens, Shigella sp., Yersinia enterocolitica, Yersinia pestis, Neisserhisitis, Neisserhisitis ), Moraxella catarrhalis, Veillonella sp., Bacteroides fragilis, Bacteroides sp., Prebella sp. m sp.), Spirolum minus, Aeromonas sp., Plesiomonas shigeroides, Vibrio cholerae, Vibrio chorioe V. vulnificus, Acinetobacter sp., Flavobacterium sp., Pseudomonas aeruginosa, Burkholderia-Pepperidia burdum lei), Xanthomonas maltophilia, Stenotrophomonas maltophila, Staphylococcus aureus, Bacillus sp. ), Clostridium spp., And Streptococcus spp., The therapeutic and prophylactic agents according to any one of claims 1-2.

Images