Use of ebselen in combination with polymyxin for the preparation of a medicament for the treatment of salmonella infection

Abstract

The application belongs to the technical field of biological medicine, and discloses application of Ebselen as a synergist of polymyxin in inhibition of salmonella. It is found that Ebselen combined with polymyxin has good synergistic effect on salmonella in vitro and in vivo experiments. In a mouse salmonella infection model, the bacterial load in the liver of animals treated with EBS combined with polymyxin is significantly lower than that treated with single drug. The survival rate of animals treated with EBS combined with polymyxin is significantly higher than that treated with EBS and polymyxin alone. Compared with traditional antibiotics and antibiotic combination to kill salmonella, the synergistic bactericidal effect of Ebselen and polymyxin is less likely to induce bacterial drug resistance, and Ebselen has the characteristics of wide source, multiple effects and good treatment effect, which has good research and application significance for exploring antibiotic synergistic replacement and solving bacterial drug resistance.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and discloses the application of selenium epoxetine as a polymyxin synergist in inhibiting Salmonella. Background Technology

[0002] Salmonella is a Gram-negative bacterium that can cause foodborne illnesses in humans or farmed animals after consuming food contaminated with Salmonella. These illnesses can range from localized gastroenteritis to systemic disease. The drug resistance of Salmonella has become a threat to global public health. With rising rates of Salmonella resistance in clinical testing and limited availability of new antibiotics, polymyxins have become a last-line treatment for multidrug-resistant Salmonella.

[0003] Since the 1950s, polymyxins have been used as antibiotics to combat clinical infections caused by Gram-negative bacteria. However, due to the potential clinical toxicity of polymyxins, their use declined significantly in the 1980s. But with the increasing threat of drug-resistant bacteria, polymyxins have been reintroduced as a last resort for treating multidrug-resistant bacteria.

[0004] Extracting compounds from a compound library that have synergistic effects with polymyxins can effectively restore the sensitivity of polymyxin-resistant Enterobacteriaceae to polymyxins. This is also one of the strategies for effectively exploring antibiotic alternatives and solving the problem of bacterial resistance. Given the significant and prominent issue of polymyxin resistance in Enterobacteriaceae, screening effective polymyxin synergists to treat polymyxin-resistant Enterobacteriaceae infections has become an urgent task and deserves further research and development.

[0005] Currently, there are no reports, either domestically or internationally, of using selenium ethylstilbestrol as an synergist to enhance the sensitivity of Salmonella to polymyxin. Summary of the Invention

[0006] One objective of this invention is the application of ebuselenium (EBS) as a polymyxin synergist in the inhibition of Salmonella. Furthermore, this study found that, in vitro, combined with a specific culture medium, it achieves a better inhibitory effect. This invention uses Salmonella survival rate as a target for screening polymyxin synergists and has found that ebuselenium (EBS) can significantly reduce Salmonella survival rate. In in vivo experiments, by constructing a mouse Salmonella intestinal infection model, intraperitoneal injection of EBS combined with polymyxin can reduce the amount of Salmonella in feces, liver, and spleen and significantly improve animal survival rate.

[0007] The above technical objectives are achieved through the following solutions:

[0008] Application of selenium ether in combination with polymyxin (CS) in the preparation of drugs against Salmonella infection.

[0009] Ebselenosyl selenide (EBS) is an organoselenium compound that can cross the blood-brain barrier and possesses a wide range of biological activities. EBS has been reported to show great potential in the prevention and treatment of various human diseases, such as stroke, neurodegenerative diseases, bipolar disorder, sensorineural hearing loss, cardiovascular diseases, immune system enhancement, detoxification, and antimicrobial activity.

[0010] Preferably, in in vitro experiments, Salmonella is first cultured and incubated to the logarithmic growth phase, then incubated with LPM broth, diluted after incubation, and then combined with selenium bismuth subtilis and colistin.

[0011] Preferably, the Salmonella is the standard Salmonella strain ATCC14028 and polymyxin-resistant Salmonella.

[0012] Preferably, the bacterial concentration after incubation is 1×10⁻⁶. 6 ~5×10 6 CFU / mL.

[0013] Preferably, the pH value of LPM broth is 5.5 to 6.0.

[0014] Preferably, the concentration of selenium is 0.0625~4 mg / L, and the concentration of polymyxin is 0.5~32 mg / L.

[0015] A co-culture method includes first culturing Salmonella to the logarithmic growth phase, then incubating it in LPM broth, diluting it after incubation, and then adding ebuselenium and colistin for co-treatment. Preferably, the Salmonella is the standard Salmonella strain ATCC14028 and polymyxin-resistant Salmonella.

[0016] Preferably, the bacterial concentration after incubation is 1×10⁻⁶. 6 ~5×10 6 CFU / mL.

[0017] Preferably, the pH value of LPM broth is 5.5 to 6.0.

[0018] Preferably, the concentration of selenium is 0.0625~4 mg / L, and the concentration of polymyxin is 0.5~32 mg / L.

[0019] In in vivo experiments, by establishing a mouse model of Salmonella intestinal infection, intraperitoneal injection of ebuselenium combined with polymyxin reduced the amount of Salmonella in feces, intestines, and spleen, and significantly improved the survival rate of animals. The preferred dosage forms for this drug are injections, tablets, pills, capsules, suspensions, or emulsions.

[0020] Preferably, the dosage of polymyxin in the drug is 5 mg / kg / day, and the dosage of ibuprofen is 10 mg / kg / day.

[0021] Regarding the definition of terms used in this invention: Unless otherwise stated, the initial definitions provided herein apply to the group or term throughout the specification; for terms not specifically defined herein, the meanings that a person skilled in the art would give them should be given based on the disclosure and context.

[0022] The term "pharmaceutically acceptable" means that a carrier, delivery substance, diluent, excipient, and / or the salt formed therefrom is generally chemically or physically compatible with other components constituting a drug dosage form and physiologically compatible with receptors.

[0023] The terms "salt" and "pharmaceutical salt" refer to acidic and / or basic salts formed by the above-described compounds or their stereoisomers with inorganic and / or organic acids and bases, including zwitterionic salts (internal salts) and quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly during the final separation and purification of the compounds. Alternatively, they can be obtained by mixing the above-described compounds or their stereoisomers with an appropriate amount (e.g., equimolar amounts) of an acid or base. These salts may be obtained by precipitating in solution and collecting by filtration, by recovery after solvent evaporation, or by freeze-drying after reaction in an aqueous medium. The salts described in this invention can be hydrochlorides, sulfates, hydrobroms, hydrofluoric acids, phosphates, acetates, propionates, succinates, oxalates, malates, succinates, fumarates, maleates, tartrates, or trifluoroacetates of the compounds.

[0024] In some embodiments, one or more compounds of the present invention may be used in combination with each other. Alternatively, the compounds of the present invention may be used in combination with any other active agent to prepare a medicament or pharmaceutical composition for regulating cell function or treating diseases caused by Salmonella infection. If a group of compounds is used, these compounds may be administered to the test subject simultaneously, separately, or sequentially.

[0025] Compared with the prior art, the beneficial effects of this application are as follows:

[0026] This invention provides a treatment using ebuselenium in combination with polymyxin, which exhibits excellent synergistic effects against Salmonella in both in vitro and in vivo experiments. Compared to the traditional combination of antibiotics to kill Salmonella, the synergistic bactericidal effect of ebuselenium and polymyxin is less likely to induce bacterial resistance. Moreover, ebuselenium is widely available, has multiple therapeutic effects, and is highly effective, making it significant for research and application in exploring synergistic antibiotic alternatives and addressing bacterial resistance. Attached Figure Description

[0027] Figure 1The checkerboard thermogram and in vitro bactericidal curve of EBS combined with polymyxin against Salmonella are shown (a and b represent checkerboard thermograms of Salmonella standard strain ATCC14028 and polymyxin-resistant Salmonella (17ES), respectively; c and d represent in vitro bactericidal curves of Salmonella standard strain ATCC14028 and polymyxin-resistant Salmonella (17ES), respectively).

[0028] Figure 2 The combined use of EBS and polymyxin significantly reduced the bacterial load in different tissues and organs of Salmonella-infected mice and significantly improved the survival rate of mice. Detailed Implementation

[0029] The technical solution of the present invention will be clearly and completely described below with reference to embodiments and comparative examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0031] The polymyxin used in the following examples is polymyxin E. The ibuprofen used in the following examples has CAS Registry Number 60940-34-3 and molecular formula C. 13 H9NOSe, molecular weight 274.18.

[0032] The structure is as follows:

[0033]

[0034] Example 1: Ibuselenium enhances the in vitro susceptibility of Salmonella to polymyxins.

[0035] 1. Cool the autoclaved LPM broth (pH 5.8) for later use. The Salmonella standard strain ATCC14028 and polymyxin-resistant Salmonella (17ES) were both laboratory-preserved and commercially available.

[0036] 2. Preparations before the experiment:

[0037] (1) Prepare an appropriate amount of ibuprofen using dimethyl sulfoxide as the solvent to achieve a final drug concentration of 5120 mg / L. Mix well and filter through a filter membrane for later use. Prepare a stock solution of polymyxin with a concentration of 5120 mg / L according to CLSI for later use.

[0038] (2) Inoculate Salmonella standard strain ATCC14028 and polymyxin-resistant Salmonella (17ES) onto LB agar plates and culture until they reach the appropriate size.

[0039] 3. MIC and FICI of ebuselenium and polymyxin against Salmonella

[0040] (1) The test Salmonella standard strain ATCC14028 and polymyxin-resistant Salmonella (17ES) were inoculated into a test tube containing 4 mL MH broth and incubated in a shaker at 37°C at 180 rpm until the logarithmic growth phase.

[0041] (2) Dilute the incubated bacteria to 100 times using LPM broth with a pH of 5.8, approximately 10 6 CFU / mL, for later use;

[0042] (3) Take a sterile 96-well plate, add 180 μL LPM of broth medium to well 1, and add 100 μL LPM of broth medium to wells 2-11 respectively;

[0043] (4) Add 20 μL of medicine to the first column, blow it evenly, and then take 100 μL into the second well, and so on. Take 100 μL from the tenth well and discard it.

[0044] (5) Add 100 μL of diluted bacterial solution to wells 1 to 11, and add 200 μL of LPM broth to well 12;

[0045] (6) Repeat steps (3) to (5) three times in parallel;

[0046] (7) After incubating the inoculated 96-well plate in a 37°C incubator for 16-18 hours, read the results;

[0047] (8) Based on the MIC results, the checkerboard method was then performed. The MIC results of ebuprofen are shown in Table 1. In LPM medium, the MIC values ​​of EBS against Salmonella standard strains and polymyxin-resistant Salmonella were 4 / 16 mg / L, respectively. When EBS was used in combination with polymyxin, it significantly reduced the sensitivity of both drug-resistant and standard strains to polymyxin. The checkerboard method results are shown in Table 1. Figure 1 As shown, the checkerboard method results showed that the FICI values ​​were all less than 0.5, indicating that EBS and polymyxin have a good synergistic effect in the treatment of Salmonella.

[0048] Table 1

[0049]

[0050] 4. In vitro bactericidal curves of ebuselenium and polymyxin against Salmonella.

[0051] (1) The test Salmonella standard strain ATCC14028 and polymyxin-resistant Salmonella (17ES) were inoculated into a test tube containing 4 mL MH broth and incubated in a shaker at 37°C at 180 rpm until the logarithmic growth phase.

[0052] (2) Dilute the incubated bacteria to a 10-fold ratio using LPM broth with a pH of 5.8, approximately 10 7 CFU / mL, for later use;

[0053] (3) In an LPM broth centrifuge tube with a pH of 5.8, add 1% EBS or polymyxin with an inhibitory concentration, alone or in combination, and then add 0.4 mL of the diluted bacterial solution obtained in step (2). Vortex for 5 seconds to mix. A blank control group is set up as a quality control; the control group is identical to the experimental group except that no drug is added. The bacterial concentration at this time is approximately 5 × 10⁻⁶. 6 Approximately CFU / mL, incubated at 37℃ in a shaker at 180 rpm, with a bacterial culture volume of 4 mL;

[0054] (4) Take 100 μL of bacterial culture at 0, 3, 6, 9, and 24 h of culture time and add it to a 2 mL centrifuge tube containing 900 μL of 0.85% physiological saline for a 10-fold serial dilution. After dilution, take 25 μL of each diluted culture and drop it onto MH agar medium. Incubate at 37℃ for 18 h and count the colonies. The experimental results are statistically analyzed after three biological replicates. Count the colony counts at each time point and plot the sterilization curve.

[0055] result: Figure 1 In vitro bactericidal curves showed that after 24 hours of combined use of EBS and polymyxin, the bacterial load decreased by more than 100 times compared to the single drug, demonstrating a significant synergistic effect and showcasing the excellent in vitro bactericidal effect of the combination of EBS and polymyxin against Salmonella.

[0056] Example 2: Ibuselenium enhances the in vivo killing effect of polymyxin against Salmonella.

[0057] 1. Experimental materials: Eighty 7-week-old female C57BL / 6J mice were purchased from Southern Medical University in Guangdong Province. Mice were administered via gavage using a sterile syringe.

[0058] 2. Preparations before the experiment: Prepare stock solutions of 5 mg / kg polymyxin, 10 mg / kg EBS, and 100,000 mg / L streptomycin. Inoculate Salmonella ATCC14028 strain onto LB agar plates and incubate until appropriate size. Nine mice are used in each group, for a total of four groups (control group, polymyxin treatment group, EBS treatment group, and combined treatment group).

[0059] 3. Establishment of a mouse model of Salmonella infection

[0060] (1) Each mouse was given 20 mg of streptomycin orally to treat the intestinal flora, and water and food were withheld 4 hours in advance before treatment.

[0061] (2) One day after streptomycin treatment, approximately 10% of patients were treated with 100 μL of streptomycin via gavage. 8 The ATCC14028 bacterial culture was inoculated into a test tube containing 4 mL LB broth and incubated at 37°C and 180 rpm on a shaker until the logarithmic growth phase. The centrifuge tube was then removed (approximately 10 minutes later). 8 Bacterial count).

[0062] 4. Detection of bacterial colony count in target organs after mouse infection

[0063] (1) One day after infection, mice were given EBS (10 mg / kg, intraperitoneal injection, once a day) and polymyxin (5 mg / kg, intraperitoneal injection, once a day) as a single drug and in combination.

[0064] (2) After three days of continuous treatment with the above drug dosage, mice in the treatment group and the quality control group were sacrificed by cervical joint dislocation, and various organs were collected and colony counts were performed.

[0065] Results: Compared with the best single-drug treatment group, EBS combined with polymyxin significantly reduced target organs (spleen, liver, feces) in mice, with bacterial counts decreasing by more than 100-fold compared with single-drug treatment (see [link to study]). Figure 2 ).

[0066] 5. Survival rate test after infection in mice

[0067] (1) One day after infection, mice were given EBS (10 mg / kg, intraperitoneal injection, once a day) and polymyxin (5 mg / kg, intraperitoneal injection, once a day) as a single drug and in combination.

[0068] (2) After 7 days of continuous treatment with the drug dose of (1), the survival curves of mice in the treatment group and the quality control group were recorded and plotted.

[0069] in, Figure 2 The results in a show that in a mouse Salmonella infection model, the bacterial load in the liver of animals treated with the combination of EBS and polymyxin was significantly reduced compared with the bacterial load after treatment with EBS and polymyxin alone. Figure 2 b shows that the bacterial load in the spleen of animals treated with the combination of EBS and polymyxin was significantly lower than that treated with EBS and polymyxin alone. Figure 2 c shows that the bacterial load in the feces of animals treated with the combination of EBS and polymyxin was significantly lower than that treated with EBS and polymyxin alone. Figure 2 The results in d show that the survival rate of animals treated with the combination of EBS and polymyxin was significantly higher than that of animals treated with EBS or polymyxin alone.

[0070] Conclusion: After 7 days of treatment, the survival rate was 0% in the control group, 0% in the EBS treatment group, and 10% in the polymyxin treatment group. The survival rate of the EBS combined with polymyxin combination group was 80%. The protection rate of the EBS combined with polymyxin combination group was significantly higher than that of the polymyxin treatment group (see...). Figure 2 This indicates that the combined use of EBS and polymyxin in the treatment of Salmonella infection in vivo also has a good synergistic effect.

[0071] In summary, this invention targets the survival rate of Salmonella and screens compounds such as EBS and polymyxin to identify a potent in vitro Salmonella-killing effect when combined. Finally, a Salmonella infection mouse model was successfully established via oral administration, further demonstrating that the combination of EBS and polymyxin can reduce colony colonization in different tissues and organs and decrease the mortality rate of Salmonella-infected mice. These studies will provide insights and a foundation for the development of new drugs using polymyxin synergists.

[0072] Obviously, the above embodiments of the present invention are merely examples to clearly illustrate the technical solution of the present invention, and are not intended to limit the specific implementation of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention should be included within the protection scope of the claims of the present invention.

Claims

1. The use of a composition in the preparation of a medicament against Salmonella infection, characterized in that, The composition consists of ebuselenium and polymyxin E; the Salmonella is the standard Salmonella strain ATCC14028 and polymyxin-resistant Salmonella; the concentration of ebuselenium is 1~4 mg / L, and the concentration of polymyxin E is 0.5~32 mg / L.

2. The application according to claim 1, characterized in that, The bacterial concentration is 1×10 6 ~5×10 6 CFU / mL.

3. The application according to claim 1, characterized in that, The dosage form of the drug is injection, tablet, pill, capsule, suspension or emulsion.

4. The application according to claim 1, characterized in that, The drug may also include pharmaceutically acceptable carriers or salts.

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