Application of combination of ebbeselenium and polymyxin in preparation of salmonella infection resisting medicine

Abstract

The invention belongs to the technical field of biological medicine, and discloses application of ebb-selenium as a polymyxin synergist in inhibition of salmonella. According to the application, the combination of ebb selenium and polymyxin has a good synergistic effect on salmonella in vitro and in vivo experiments. In a mouse salmonella infection model, compared with the bacterium carrying amount after single-drug treatment, the bacterium carrying amount in the animal liver after EBS and polymyxin combined treatment has the effect that the bacterium carrying amount in the animal liver is obviously reduced. The survival rate of animals treated by the combination of the EBS and the polymyxin is obviously increased compared with the survival rate of the animals treated by single medicines of the EBS and the polymyxin. Compared with the traditional method for killing salmonella through combination of antibiotics, the method has the advantages that the generation of bacterial drug resistance is not easily induced through the synergistic sterilization of the ebb-selenium and the polymyxin, and the ebb-selenium has the characteristics of wide source, multiple effects and good treatment effect, and has better research and application significance for excavating the synergistic replacement of the antibiotics and solving the bacterial drug resistance.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and discloses the application of ebselen as a polymyxin synergist in inhibiting Salmonella. Background Art

[0002] Salmonella is a Gram-negative bacterium. After humans or farm animals consume food contaminated with Salmonella, it can cause foodborne diseases, ranging from local gastroenteritis to systemic diseases. The problem of Salmonella drug resistance has posed a threat to global public health. With the increasing drug resistance rate of Salmonella in clinical tests and the limited supply of new antibiotics, polymyxin has become the last line of defense for treating multi-drug resistant Salmonella.

[0004] Extracting compounds with synergistic effects with polymyxin from compound libraries can effectively restore the sensitivity of polymyxin-resistant Enterobacteriaceae bacteria to polymyxin, which is also one of the strategies for effectively exploring alternative methods for antibiotics and solving the problem of bacterial drug resistance. Based on the fact that the problem of polymyxin resistance in Enterobacteriaceae bacteria is quite important and prominent, it has become an urgent task to screen effective polymyxin synergists to treat infections of polymyxin-resistant Enterobacteriaceae, which is worthy of further research and development.

[0005] Currently, there is no report at home and abroad on using ebselen as a synergist to enhance the sensitivity of Salmonella to polymyxin. Summary of the Invention

[0006] One of the purposes of the present invention is the application of ebselen as a polymyxin synergist in inhibiting Salmonella. At the same time, this application finds that in an in vitro environment, better inhibitory effects can be achieved in combination with a specific culture medium. The present invention uses the survival rate of Salmonella as the target for screening polymyxin synergists, and screens that ebselen (EBS) can significantly reduce the survival rate of Salmonella. 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 the survival rate of animals.

[0007] The above technical purpose is achieved through the following scheme:

[0008] The application of the combination of ebselen and polymyxin (CS) in the preparation of drugs for treating Salmonella infections.

[0009] Ebselen (EBS) is an organic selenium compound that can penetrate the blood-brain barrier and has a wide range of biological activities. It is reported that EBS shows broad potential in the prevention and treatment of various human diseases, such as stroke, neurodegenerative diseases, bipolar disorder, sensorineural deafness, cardiovascular diseases, immune system enhancement, detoxification and anti-microbial effects, etc.

[0010] Preferably, in in vitro experiments, Salmonella is first cultured and incubated until the logarithmic growth phase, then incubated with LPM broth. After incubation is completed, it is diluted, and then ebselen and colistin are added for combined use.

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

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

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

[0014] Preferably, the concentration of ebselen is 0.0625 - 4 mg / L, and the concentration of colistin is 0.5 - 32 mg / L.

[0015] A co-culture method includes first culturing and incubating Salmonella until the logarithmic growth phase, then incubating with LPM broth. After incubation is completed, it is diluted, and then ebselen and colistin are added for combined use. Preferably, the Salmonella are Salmonella standard strain ATCC14028 and polymyxin-resistant Salmonella.

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

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

[0018] Preferably, the concentration of ebselen is 0.0625 - 4 mg / L, and the concentration of colistin is 0.5 - 32 mg / L.

[0019] In in vivo experiments, by constructing a mouse Salmonella intestinal infection model, intraperitoneal injection of ebselen combined with colistin can reduce the amount of Salmonella in feces, intestines, and spleens and significantly improve the survival rate of animals. The preferred dosage forms of the drug are injection, tablet, pill, capsule, suspension, or emulsion.

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

[0021] Regarding the definitions of the terms used in the present invention: Unless otherwise specified, the initial definitions provided for the terms in this article apply to the group or term throughout the specification; for terms not specifically defined in this article, their meanings should be given according to the disclosure content and context, which can be understood by those skilled in the art.

[0022] The term "pharmaceutically acceptable" means that a carrier, vehicle, diluent, excipient, and / or the salts formed are generally chemically or physically compatible with the other components constituting a pharmaceutical dosage form and are physiologically compatible with the receptor.

[0023] The terms "salt" and "pharmaceutically acceptable salt" refer to acid and / or base salts formed from the above-mentioned compounds or their stereoisomers with inorganic and / or organic acids and bases, including zwitterionic salts (inner salts), and also including quaternary ammonium salts, such as alkylammonium salts. These salts can be directly obtained during the final separation and purification of the compound. They can also be obtained by appropriately (e.g., equimolarly) mixing the above-mentioned compound or its stereoisomer with a certain amount of acid or base. These salts may form precipitates in solution and be collected by filtration, or recovered after evaporation of the solvent, or prepared by lyophilization after reaction in an aqueous medium. The salts described in the present invention can be hydrochloride, sulfate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, fumarate, maleate, tartrate, or trifluoroacetate of the compound.

[0024] In certain embodiments, one or more compounds of the present invention can be used in combination with each other. Optionally, the compounds of the present invention can also be combined with any other active agent for preparing a drug or pharmaceutical composition for regulating cell function or treating diseases caused by Salmonella infection. If a group of compounds is used, these compounds can be administered to the subject simultaneously, separately, or sequentially.

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

[0026] The present invention provides a treatment of combining ebselen with polymyxin, which has good synergistic effects in both in vitro and in vivo experiments against Salmonella. Compared with the combination of traditional antibiotics to kill Salmonella, the synergistic bactericidal effect of the compound ebselen and polymyxin is not easy to induce the generation of bacterial drug resistance. Moreover, ebselen has the characteristics of wide sources, multiple effects, and good therapeutic effects, and has good research and application significance for exploring antibiotic synergistic alternatives and solving bacterial drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Showing the checkerboard heat map and in vitro bactericidal curve of EBS combined with polymyxin against Salmonella (a and b represent the checkerboard heat maps of Salmonella standard strain ATCC14028 and polymyxin-resistant Salmonella (17ES) respectively; c and d represent the in vitro bactericidal curves of Salmonella standard strain ATCC14028 and polymyxin-resistant Salmonella (17ES) respectively);

[0028] Figure 2It is shown that the combination of EBS and polymyxin significantly reduces the bacterial load in different tissues and organs of mice infected with Salmonella and significantly improves the survival rate of mice. Detailed implementation manners

[0029] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments and comparative examples of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative work shall fall within the scope of protection of the present invention.

[0030] Unless otherwise specified, the test methods used in the following embodiments are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0031] The polymyxin used in the following embodiments is polymyxin E. Ebselen used in the following embodiments has a CAS registration number of 60940-34-3 and a molecular formula of C 13 H9NOSe with a molecular mass of 274.18.

[0032] The structural formula is as follows:

[0033]

[0034] Example 1 Ebselen enhances the sensitivity of Salmonella to polymyxin in vitro

[0035] 1. Cool the autoclaved LPM broth with a pH value of 5.8 for standby. The Salmonella standard strain ATCC 14028 and the polymyxin-resistant Salmonella (17ES) are both stored in the laboratory and can also be obtained commercially.

[0036] 2. Preparation before the experiment:

[0037] (1) Prepare an appropriate amount of ebselen with dimethyl sulfoxide as the solvent to make the final drug concentration 5120 mg / L. After mixing, filter with a filter membrane and reserve. Polymyxin is prepared into a stock solution with a concentration of 5120 mg / L according to CLSI for standby.

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

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

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

[0041] (2) The incubated bacteria were diluted 100-fold with LPM broth at a pH of 5.8 to approximately 10 6 CFU / mL and set aside;

[0042] (3) A sterile 96-well plate was taken. 180 μL of LPM broth medium was added to the first well, and 100 μL of LPM broth medium was added to each of the second to eleventh wells;

[0043] (4) 20 μL of the drug was added to the first column. After pipetting evenly, 100 μL was aspirated and transferred to the second well, and so on. 100 μL was aspirated and discarded from the tenth well;

[0044] (5) 100 μL of the diluted bacterial solution was added to wells 1 to 11, and 200 μL of LPM broth medium was added to the twelfth well;

[0045] (6) Steps (3) to (5) were repeated for three parallel replicates;

[0046] (7) The inoculated 96-well plate was placed in an incubator at 37°C and incubated for 16 - 18 h, after which the results were read;

[0047] (8) According to the MIC results, the checkerboard method was then performed. The MIC results of ebselen are shown in Table 1. In the LPM medium, the MIC values of EBS against the standard strain of Salmonella and polymyxin-resistant Salmonella were 4 / 16 mg / L respectively; when MAG was used in combination with polymyxin, it could significantly reduce the sensitivity of drug-resistant bacteria and standard bacteria to polymyxin. The results of the checkerboard method are as Figure 1 shown, and the checkerboard method results showed that all FICI values were less than 0.5, indicating that MAG and polymyxin had good synergistic effects in the treatment of Salmonella.

[0048] Table 1

[0049]

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

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

[0052] (2) The incubated bacteria were diluted 10-fold with LPM broth at a pH of 5.8 to approximately 10 7CFU / mL, for standby;

[0053] (3) In an LPM broth centrifuge tube with a pH value of 5.8, add MAG at a concentration of 1 or polymyxin at an inhibitory concentration alone or in combination, and then add 0.4 mL of each of the diluted bacterial solutions obtained by diluting in step (2). Vortex for 5 s to mix evenly. Set up a blank control group as a quality control. Except for not adding drugs, the other conditions of the control group are the same as those of the experimental group. At this time, the bacterial solution concentration is about 5×10 6 CFU / mL or so, place it in a shaker at 37°C and culture at 180 rpm. The dosage of the bacterial solution system is 4 mL;

[0054] (4) At the culture times of 0, 3, 6, 9, and 24 h, respectively, take 100 μL of the bacterial solution and add it to a 2 mL centrifuge tube containing 900 μL of 0.85% normal saline for 10-fold serial dilution. After dilution, take 25 μL and drop it on the MH agar medium, incubate in a 37°C incubator for 18 h, and perform counting. The experimental results are statistically analyzed after three biological replicates. Count the number of colonies at each time point and draw a bactericidal curve graph.

[0055] Results: Figure 1 The in vitro bactericidal curve shows that the bacterial load after 24 h of the combination of EBS and polymyxin decreased by more than 100 times compared with the single drug, and the synergy was significant, demonstrating that the combination of EBS and polymyxin has excellent effects on killing Salmonella in vitro.

[0056] Example 2 Ebselen Enhances the Killing Effect of Polymyxin on Salmonella in Vivo

[0057] 1. Test materials: Purchase 80 7-week-old C57BL / 6J female mice from Southern Medical University in Guangdong Province, mouse gavage needles, and sterile syringes.

[0058] 2. Preparation before the test: Prepare a polymyxin stock solution of 5 mg / kg, an Ebselen stock solution of 10 mg / kg, and streptomycin of 100000 mg / L. Inoculate the Salmonella ATCC14028 strain onto an LB agar culture plate and culture until it reaches an appropriate size. There are 9 mice in each group, and a total of 4 groups (control group, polymyxin treatment group, Ebselen treatment group, combination treatment group).

[0059] 3. Establishment of a Salmonella-infected mouse model

[0060] (1) Treat the intestinal flora of each mouse orally with 20 mg of streptomycin, and cut off water and food 4 h before the treatment.

[0061] (2) One day after the streptomycin treatment, gavge 100 μL of approximately 10 8ATCC 14028 bacterial solution. The tested ATCC 14028 strain was inoculated into a test tube containing 4 mL of LB broth and incubated in a shaker at 37 °C at 180 rpm until the logarithmic growth phase, and then the centrifuge tube was taken out (about 10 8 bacterial quantity).

[0062] 4. Detection of the number of bacterial colonies colonized in target organs after mouse infection

[0063] (1) One day after mouse infection, EBS (10 mg / kg, intraperitoneal injection, once a day) and polymyxin (5 mg / kg, intraperitoneal injection, once a day) were administered alone and in combination.

[0064] (2) After continuous treatment with the above drug doses for 3 days, the mice in the treatment group and the quality control group were sacrificed by cervical dislocation, various organs were collected, and colony counting was performed.

[0065] Results: Compared with the best single-drug treatment group, after combined treatment with EBS and polymyxin, the target organs (spleen, liver, feces) of mice were significantly reduced, and the bacterial quantity decreased by more than 100 times compared with single-drug treatment (see Figure 2 ).

[0066] 5. Survival rate test after mouse infection

[0067] (1) One day after mouse infection, EBS (10 mg / kg, intraperitoneal injection, once a day) and polymyxin (5 mg / kg, intraperitoneal injection, once a day) were administered alone and in combination.

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

[0069] Among them, Figure 2 a shows that: in the mouse Salmonella infection model, after combined treatment with EBS and polymyxin, the amount of bacteria carried in the liver of the animals showed a significant decrease compared with that after treatment with the single drug EBS and polymyxin; Figure 2 b shows that: after combined treatment with EBS and polymyxin, the amount of bacteria carried in the spleen of the animals showed a significant decrease compared with that after treatment with the single drug EBS and polymyxin; Figure 2 c shows that: after combined treatment with EBS and polymyxin, the amount of bacteria carried in the feces of the animals showed a significant decrease compared with that after treatment with the single drug EBS and polymyxin; Figure 2 d shows that: after combined treatment with EBS and polymyxin, the survival rate of the animals showed a significant increase compared with that after treatment with the single drug EBS and polymyxin.

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

[0071] In summary, the present invention targets the survival rate of Salmonella and screens out that the combination of compound EBS and polymyxin has a good effect on killing Salmonella in vitro. Finally, a mouse model of Salmonella infection was successfully established by oral infection with Salmonella, further confirming that the combination of EBS and polymyxin can reduce the colonization of colonies in different tissues and organs and reduce the mortality rate of mice infected with Salmonella. These studies will provide ideas and a basis for the new drug research and development of polymyxin synergists.

[0072] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the technical solutions of the present invention, and are not intended to limit the specific embodiments of the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the claims of the present invention shall be included within the protection scope of the claims of the present invention.

Claims

1. Use of ebselen in combination with polymyxin in the preparation of a medicament for treating Salmonella infection.

2. The application according to claim 1, wherein The Salmonella is Salmonella standard strain ATCC14028 and / or polymyxin-resistant Salmonella.

3. The application according to claim 1, wherein The polymyxin is polymyxin E.

4. The application according to claim 1, wherein The concentration of the bacterial solution is 1×10 6 ~5×10 6 CFU / mL.

5. The application according to claim 3 or 4, wherein the concentration of ebselen is 0.0625 - 4 mg / L, and the concentration of polymyxin is 0.5 - 32 mg / L.

6. The application according to claim 1, wherein The dosage form of the medicament is injection, tablet, pill, capsule, suspension or emulsion.

7. The application according to claim 6, characterized in that, The dosage of polymyxin in the medicament is 5 mg / kg / day, and the dosage of ebselen is 10 mg / kg / day.

8. The application according to claim 1, characterized in that, The medicament further comprises a pharmaceutically acceptable carrier or salt.

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