Application of flufenamic acid in preparation of polymyxin E antibacterial synergist

Abstract

The invention relates to the technical field of biological medicine, in particular to application of flufenamic acid in preparation of a polymyxin E antibacterial synergist. The invention discloses an application of flufenamic acid in preparation of a polymyxin E antibacterial synergist, the flufenamic acid has no obvious antibacterial effect on salmonella when being independently used, but the flufenamic acid and polymyxin E can enhance the antibacterial activity of the polymyxin E on the salmonella and reduce the use dosage of the polymyxin E when being jointly used in and out of an animal body, so that the antibacterial effect of the polymyxin E on the salmonella can be enhanced. The antibacterial activity of the polymyxin E on salmonella is enhanced, and the drug resistance of the polymyxin E is inhibited. Flurfenamic acid is approved to be used for other purposes as FDA, and when the flurfenamic acid is combined with polymyxin E, the flurfenamic acid has the characteristic of high safety, and meanwhile, the generation of the drug resistance of the polymyxin E drugs can be effectively avoided. The invention provides a basis for mining a new antibacterial prevention and control strategy for the polymyxin E drug-resistant bacteria.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of flufenamic acid in the preparation of polymyxin E antibacterial synergists. Background Technology

[0002] Salmonella is a significant zoonotic Gram-negative bacterium that poses a serious threat to both humans and the livestock industry. In recent years, the problem of Salmonella resistance has become increasingly serious due to the improper use or abuse of antibiotics. However, the development of new antibiotics has stagnated, especially drugs for treating Gram-negative bacteria; the development of antimicrobial drugs lags far behind the rate of bacterial resistance development. Given the continuous emergence of resistance to traditional antibiotics and the lack of novel antimicrobial agents, there is an urgent need to adopt new alternative strategies to alleviate this crisis.

[0003] Combination therapy has emerged as a promising alternative, offering new treatment strategies for drug-resistant bacteria. Compared to developing new drugs, combining polymyxins with existing antibiotics not only extends the lifespan of existing antibiotics but also saves time and reduces costs. Since the discovery of the plasmid-mediated colistin resistance gene mcr-1 in 2016, polymyxins have seriously threatened their status as the "last line of defense" against Gram-negative bacteria. Therefore, to prolong the clinical antibacterial effect of polymyxins, screening polymyxin synergists to restore polymyxin sensitivity is urgently needed. Currently, there are no reports, either domestically or internationally, of flufenamic acid as a polymyxin E synergist to enhance the sensitivity of polymyxins to Salmonella. Summary of the Invention

[0004] The purpose of this invention is to provide the application of flufenamic acid in the preparation of polymyxin E antibacterial synergists, thereby addressing the problems existing in the prior art. This invention first provides the application of flufenamic acid as an antibacterial synergist for polymyxin E. The combined use of flufenamic acid and polymyxin E achieves good antibacterial effects against Salmonella in vitro. This invention targets Salmonella and screens polymyxin E antibacterial synergists based on bacterial survival rate, finding that the combined use of flufenamic acid and polymyxin E significantly enhances the antibacterial activity of polymyxin E against Salmonella. Further in vivo animal efficacy evaluation shows that the combined use of flufenamic acid and polymyxin E to treat mice infected with Salmonella significantly improves animal survival rate and reduces bacterial load in the liver, spleen, and kidneys.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides the application of flufenamic acid in the preparation of polymyxin E antibacterial synergists, wherein the bacteria include Salmonella.

[0006] Optionally, the concentration of flufenamic acid in the polymyxin E antibacterial synergist is 32 µg / mL.

[0007] This invention provides a polymyxin E antibacterial synergist, wherein the polymyxin E antibacterial synergist includes flufenamic acid; and the bacteria include Salmonella.

[0008] Optionally, the concentration of flufenamic acid in the polymyxin E antibacterial synergist is 32 µg / mL.

[0009] Optionally, the polymyxin E antibacterial synergist also includes excipients.

[0010] Optionally, the dosage form of the polymyxin E antibacterial synergist includes capsules, powders, or granules.

[0011] The present invention provides an anti-Salmonella composition comprising flufenamic acid and polymyxin E.

[0012] This invention provides the use of the above-described composition in the preparation of an anti-Salmonella medicament.

[0013] The present invention provides an anti-Salmonella drug, the drug comprising the above-described composition.

[0014] Optionally, the drug may include excipients.

[0015] The present invention discloses the following technical effects: This invention discloses the application of flufenamic acid in the preparation of polymyxin E antibacterial synergists. Flufenamic acid alone has no significant inhibitory effect on Salmonella, but the combined use of flufenamic acid and polymyxin E enhances the antibacterial activity of polymyxin E against Salmonella both in vivo and in vitro in animals, reduces the dosage of polymyxin E, and inhibits the development of polymyxin E resistance. Flufenamic acid, an FDA-approved drug for other uses, exhibits high safety when used in combination with polymyxin E, while effectively preventing the development of polymyxin E resistance. This invention provides a basis for exploring new antibacterial control strategies against polymyxin E-resistant bacteria. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1The image shows a checkerboard heatmap of the combined use of flufenamic acid (FFA) and polymyxin E against Salmonella. In the image, a and b represent the checkerboard heatmap results of the combined use of flufenamic acid and polymyxin E against polymyxin E-resistant clinical Salmonella 26FS14 and the standard Salmonella strain ATCC14028, respectively. The X-axis represents the concentration of polymyxin E, and the Y-axis represents the concentration of flufenamic acid. Figure 2 The in vitro bactericidal curves of flufenamic acid (FFA) and polymyxin E alone or in combination against Salmonella are shown; where a and b represent the in vitro bactericidal curves of polymyxin E-resistant clinical Salmonella 26FS14 and Salmonella standard strain ATCC14028, respectively. Figure 3 The effect of long-term induction of polymyxin E alone or in combination with flufenamic acid on the antibacterial activity of polymyxin E was investigated; where a and b represent the results of long-term induction of polymyxin E-resistant clinical Salmonella 26FS14 and Salmonella standard strain ATCC14028, respectively. Figure 4 This study aimed to evaluate the survival rate and bacterial load in different tissues and organs of Salmonella-infected mice when polymyxin E was used alone or in combination with flufenamic acid; where a represents survival rate; b represents bacterial load in the spleen; c represents bacterial load in the liver; and d represents bacterial load in the lungs. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] The polymyxin used in the following examples is polymyxin E. The flufenamic acid described in this invention has a CAS registry number of 530-78-9 and a molecular formula of C0.05. 14 H 10 F3NO2 has a molecular weight of 281.23. The chemical structural formula of flufenamic acid (FFA) is shown below: .

[0024] Example 1: Flufenamic acid enhances the antibacterial activity of polymyxin E in vitro. 1. Cool the autoclaved MH broth for later use. Salmonella standard strain ATCC 14028 ( Salmonella ATCC 14028), polymyxin E-resistant clinical Salmonella 26FS14 (strain 26FS14, Salmonella All strains (26FS14) were preserved in the laboratory. Strain 26FS14 is disclosed in the literature "Mechanism of Action of Isopropoxy Benzene Guanidine against Multidrug-Resistant Pathogens". The applicant has committed to distributing it to the public for 20 years from the date of application.

[0025] 2. Preparations before the experiment: Flufenamic acid with a concentration of 25600 μg / mL was prepared using dimethyl sulfoxide as a solvent, and then filtered and sterilized for later use.

[0026] Polymyxin E was prepared into a stock solution with a concentration of 1600 μg / mL for later use.

[0027] 3. Detection of the MIC of flufenamic acid and polymyxin E against Salmonella Single colonies of Salmonella were picked and added to MH broth, and incubated at 37°C until the logarithmic growth phase. The bacterial concentration was then adjusted to 10⁻⁶ using MH broth. 6Prepare CFU / mL solution. In a clear 96-well plate, add 190 μL of MH broth to column 1, and 100 μL of MH broth to columns 2 through 12. Add 10 μL of the prepared test drug to column 1, mix thoroughly using a multichannel pipette, and then transfer 100 μL to column 2. Repeat this process to serially dilute the drug up to column 10. Add 100 μL of the diluted bacterial culture to columns 1 through 11. Column 11 serves as a positive control with only bacterial culture, and column 12 contains only fresh MH broth as a negative control. Incubate the 96-well plate at 37°C for 16–18 h. Record the results when the negative control is clear. The minimum concentration at which bacterial growth is observed to be inhibited by the naked eye is defined as the MIC (Minimum Inhibitory Value) of the drug.

[0028] Based on the MIC results, the appropriate drug concentration range for the checkerboard experiment was determined, and then a checkerboard experiment was conducted using both drugs in combination. In the MIC experiment, the concentration range of flufenamic acid was 0.25–128 μg / mL, and the concentration range of polymyxin E was 0.015–8 μg / mL.

[0029] The MIC results are shown in Table 1. Flufenamic acid alone had no activity against Salmonella, with a MIC > 128 μg / mL. The MIC of ATCC 14028 strain against polymyxin E was 0.5 μg / mL, and the MIC of strain 26FS14 against polymyxin E was 4 μg / mL.

[0030] Table 1. MICs of Salmonella against different drugs 4. Flufenamic acid and polymyxin E for FICI detection of Salmonella The antibacterial effect of flufenamic acid and polymyxin E combined was tested using an 8×8 checkerboard assay. Single colonies were picked and placed in MH broth, then incubated at 37°C with shaking at 180 rpm until the logarithmic growth phase. The bacteria were then diluted with fresh MH broth to a concentration of 10⁻⁶. 6Reserve CFU / mL. In a transparent 96-well plate, add 198 μL of the diluted bacterial solution to the first column, and sequentially add 100 μL of the diluted bacterial solution to the second to eighth columns. Then add 2 μL of drug A (polymyxin E) to the first column, mix well, and pipette 100 μL and add it to the second column, and serially dilute it to the seventh column in sequence. Discard 100 μL at the seventh column. The eighth column is the single-drug group of drug B (flufenamic acid). Dilute drug B with 7 required concentration gradients in advance in a petri dish with MH broth, and pipette 100 μL and add it to the first 8 wells of the first to seventh rows respectively. The eighth row is the single-drug group of drug A. Add blank broth to the eighth row and eighth column to make up 200 μL. At the same time, add blank MH broth and diluted bacterial solution to the eleventh and twelfth columns respectively as negative and positive controls. Place the 96-well plate in an incubator at 37 °C for 18 h. When reading the results, the minimum concentration that can visibly inhibit bacterial growth with the naked eye is the MIC of this drug. The specific calculation formula for FICI is as follows: ; Among them, FICI < 0.5 is synergistic; 0.5 ≤ FICI ≤ 1 is additive; 1 < FICI ≤ 4 is irrelevant; FICI > 4 is antagonistic.

[0031] The concentration range of flufenamic acid used is 0 - 128 μg / mL, and the concentration range of polymyxin E used is 0 - 8 μg / mL. The results are as Figure 1 shown. The results show that the combined use of flufenamic acid and polymyxin E has good synergistic antibacterial effects. The combined use of the two can significantly enhance the antibacterial activity of polymyxin E by 4 - 8 times, and its FICI < 0.5. At the same time, for strain 26FS14, polymyxin E has a synergistic effect when the concentration is 0.5 - 1 μg / mL and flufenamic acid ≥ 4 - 8 μg / mL; for strain ATCC14028, polymyxin E has a synergistic effect when the concentration is 0.12 μg / mL and flufenamic acid ≥ 8 μg / mL ( Figure 1 ).

[0032] 5. In vitro bactericidal curves of flufenamic acid and polymyxin E against Salmonella Single colonies of Salmonella were picked and cultured overnight in MH medium. The next day, the bacterial cells were washed twice with PBS and then resuspended in fresh broth for later use. Different concentrations of drugs (0.25×MIC polymyxin E (CL), where the MIC of polymyxin E for strain ATCC14028 was 0.5 μg / mL and that for strain 26FS14 was 4 μg / mL), 32 µg / mL flufenamic acid (FFA), or a combination of 32 µg / mL flufenamic acid and 0.25×MIC polymyxin E (CL+FFA)) were added to the bacterial culture, with PBS added as a control group. The cultures were incubated at 37°C and 180 rpm in a shaker. Starting from the time of drug addition, 100 μL of the solution was taken at time points 0, 1, 2, 4, 8, 12, 16, 20, and 24 h, and serially diluted 10-fold with 900 μL of sterile PBS. Then, 20 μL of each diluted solution was dropwise onto MH agar medium and incubated overnight at 37°C. The bacterial count was then determined using the most effective dilution gradient, and time-kill curves were plotted. A bacterial count between 30 and 300 was considered the effective counting range.

[0033] Figure 2 In vitro bactericidal curves showed that 0.25×MIC polymyxin E alone had a certain inhibitory effect on the growth of strain 26FS14 (mcr-1 positive Salmonella) and strain ATCC 14028 (mcr-1 negative Salmonella). However, the combined use of 0.25×MIC polymyxin E and 32 µg / mL flufenamic acid completely inhibited the growth of both mcr-1 positive and mcr-1 negative Salmonella within 4–8 h, and no recovery of growth was observed within the 24 h test period.

[0034] Example 2: Flufenamic acid can inhibit the development of polymyxin E resistance. The effect of flufenamic acid combined with polymyxin E or polymyxin E alone on the minimum inhibitory concentration (MIC) of polymyxin E after several consecutive days of induction was determined. The specific method is as follows: Single colonies of the test strains (ATCC 14028 and strain 26FS14) were picked and cultured in MH broth until the logarithmic growth phase. 32 µg / mL of FFA was added in combination with different concentrations of polymyxin E (0.5×MIC, 0.25×MIC, 0.12×MIC, and 0.06×MIC, where the MIC of polymyxin E for ATCC 14028 was 0.5 μg / mL and for strain 26FS14 it was 4 μg / mL) (CL + 32 µg / mL FFA). A single-drug group containing only different concentrations of polymyxin E served as the control (CL). After adding the drug, the bacteria were incubated at 37°C with shaking at 180 rpm for 24 h. The highest polymyxin E concentration that caused bacterial growth was selected in both the single-drug and combination groups for polymyxin E MIC detection, and this bacterial culture was used as the next generation for subculturing. Based on the MIC results, different concentrations of polymyxin E (0.5×MIC, 0.25×MIC, 0.12×MIC, and 0.06×MIC) were repeatedly added to the bacterial culture as single-drug groups, or different concentrations of polymyxin E (0.5×MIC, 0.25×MIC, 0.12×MIC, and 0.06×MIC) were combined with 32 µg / mL flufenamic acid. The above steps were repeated for 29 consecutive subculturing days. Changes in the polymyxin E MIC were recorded daily, and an in vitro polymyxin E MIC change curve was plotted.

[0035] Figure 3 The results showed that after 29 days of continuous induction, the MIC of polymyxin E alone against Salmonella increased by 64 times or even higher. However, when used in combination with 32 µg / mL flufenamic acid, flufenamic acid significantly inhibited the development of polymyxin E resistance to Salmonella.

[0036] Example 3: FFA enhances the in vivo therapeutic effect of polymyxin E on Salmonella-infected mice. 1. Experimental materials: 24 six-week-old SPF-grade BALB / C mice (20 g), mouse gavage needles, and sterile syringes.

[0037] 2. Preparations before the experiment: Prepare stock solutions of 2 mg / kg polymyxin E and 32 mg / kg flufenamic acid (FFA). Inoculate strain 26FS14 onto LB agar plates and incubate until the appropriate size is reached. There are 4 groups (control group (200 μL PBS, PBS), polymyxin E treatment group (200 μL 2 mg / kg polymyxin E, CL), FFA treatment group (200 μL 32 mg / kg flufenamic acid stock solution, FFA), and combined treatment group (100 μL 2 mg / kg polymyxin E + 100 μL 32 mg / kg flufenamic acid stock solution, FFA-CL)). Single colonies of strain 26FS14 are picked and incubated in LB broth at 37°C and 180 rpm until the logarithmic growth phase, then the bacterial concentration is adjusted to 10. 8 CFU / mL, 100 μL of diluted bacterial solution was injected into the left abdomen of mice. One hour after infection, 200 μL of different drug groups were administered intraperitoneally to the right abdomen. To ensure consistent drug volume, the total volume of the two drugs in combined treatments was 200 μL. All treatments were administered once. The mice's mental state and mortality were observed daily for 5 consecutive days after treatment. On day 5, all surviving mice were euthanized by cervical dislocation. If a mouse died during treatment, its liver, lungs, and spleen were aseptically dissected. A portion of each organ was weighed, ground, and serially diluted with sterile physiological saline. 20 μL of the diluted organ solution was dropped onto LB agar containing 2 μg / mL polymyxin E and incubated at 37°C for 16 hours. Bacterial counts were then performed at appropriate dilutions, and the results were statistically analyzed. A bacterial count between 30 and 300 is generally considered valid.

[0038] The results are as follows Figure 4 As shown, compared with the polymyxin E monotherapy group, the combination therapy of flufenamic acid and polymyxin E can improve the survival rate of mice and significantly reduce the bacterial load in the target organs (spleen, liver and lungs) of mice (P<0.01).

[0039] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of flufenamic acid in the preparation of polymyxin E antibacterial synergists, characterized in that, The bacteria include Salmonella.

2. The application according to claim 1, characterized in that, The concentration of flufenamic acid in the polymyxin E antibacterial synergist is 32 µg / mL.

3. A polymyxin E antibacterial synergist, characterized in that, The polymyxin E antibacterial synergist includes flufenamic acid; the bacteria include Salmonella.

4. The polymyxin E antibacterial synergist according to claim 3, characterized in that, The concentration of flufenamic acid in the polymyxin E antibacterial synergist is 32 µg / mL.

5. The polymyxin E antibacterial synergist according to claim 3, characterized in that, The polymyxin E antibacterial synergist also includes excipients.

6. The polymyxin E antibacterial synergist according to claim 5, characterized in that, The dosage forms of the polymyxin E antibacterial synergist include capsules, powders, or granules.

7. A composition for combating Salmonella, characterized in that, The composition includes flufenamic acid and polymyxin E.

8. Use of the composition of claim 7 in the preparation of an anti-Salmonella medicament.

9. A drug for treating Salmonella, characterized in that, The drug comprises the composition of claim 7.

10. The medicament according to claim 9, characterized in that, The drug includes excipients.

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