Application of 5-fluorouracil in preparation of medicines for treating bacterial infectious diseases or antibiotic synergists

Abstract

The invention discloses application of 5-fluorouracil in preparation of medicines for treating bacterial infectious diseases or antibiotic synergists, and belongs to the technical field of medicines. The invention discloses an anti-cancer drug 5-fluorouracil as a novel antibiotic synergist for the first time, and the anti-cancer drug 5-fluorouracil is used for treating bacterial infectious diseases. Specifically speaking, the invention finds that low-dose 5-fluorouracil and fosfomycin combined medication can effectively and synergistically inhibit the growth of gram-positive bacteria and gram-negative bacteria. The combined medication scheme shows remarkable advantages in the aspect of preventing and treating infection of multiple drug-resistant bacteria, especially for clinically important methicillin-resistant staphylococcus aureus (MRSA). Through combined use of 5-fluorouracil and fosfomycin, MRSA can be effectively eradicated, formation of a biofilm can be inhibited, and mature biofilm bacteria are removed. In conclusion, a new thought is provided for developing a novel drug-resistant pathogenic bacterium prevention and control strategy, and a new treatment scheme is also provided for coping with multi-drug-resistant bacterial infection and biological membrane related persistent infection.

Description

Technical Field

[0001] The invention relates to application of 5-fluorouracil in preparing a drug for bacterial infectious diseases or preparing an antibiotic synergist, and belongs to the technical field of medicine. Background Art

[0002] Staphylococcus aureus is a Gram-positive pathogen and one of the leading causes of hospital- and community-acquired infections, which can lead to serious consequences. More worryingly, Staphylococcus aureus is resistant to multiple antibiotics. One of the representative strains is methicillin-resistant Staphylococcus aureus (MRSA), which was clinically discovered in the early 1960s. The rapid spread of MRSA around the world poses a major threat to public health and has become one of the leading causes of serious, life-threatening infections. For many years, the traditional treatment of MRSA has been highly dependent on antibiotics, but with the widespread use of antibiotics, the strain has become resistant to a variety of existing antibiotics. Due to the rapid spread of MRSA infections and the complex resistance mechanisms, the mortality rate of related diseases has increased significantly. According to statistics, nearly 150,000 cases of MRSA infections are reported in the European Union each year, resulting in more than 7,000 deaths. In China, according to the CHINET monitoring system, the infection rate of MRSA has remained above 30% over the past five years.

[0003] Despite the ongoing efforts to develop new antimicrobial drugs in recent years, only a small number of new antibiotics have been approved in the past few decades. Therefore, new antimicrobial strategies are urgently needed. Drug combinations of antibiotics and adjuvants provide new ideas for overcoming drug development bottlenecks, bypassing the stagnant new drug development process and resensitizing resistant bacteria. The combined use of antibiotics and adjuvants not only enhances the efficacy of antibiotics, but also extends the life of existing antibiotics and delays the emergence of resistant strains. In addition, these synergistic therapies can reduce antibiotic toxicity, treatment time and dosage. In this context, older antibiotics such as polymyxin and fosfomycin (FOS) have regained the attention of researchers. Fosfomycin, a bactericidal agent discovered more than 40 years ago, has significant activity against both Gram-negative and Gram-positive bacteria, including multidrug-resistant (MDR) strains. Its unique mechanism of action is to reduce the possibility of cross-resistance with other antimicrobial agents by inhibiting the early stages of peptidoglycan synthesis. This singularity makes fosfomycin a potential candidate for the treatment of systemic infections. Although the clinical application of fosfomycin is gradually increasing, there are still few studies on its combination therapy, which deserves further exploration to fully utilize its therapeutic potential. Based on this, the sensitization strategy to improve the antibacterial effect of existing antibiotics has become an inevitable choice, and enhancing the effectiveness of older antibiotics by approved antibiotics or non-antibiotics has become a highly promising antibacterial strategy. Summary of the invention

[0004] Purpose of the invention: The first purpose of the present invention is to provide an application of 5-fluorouracil in the preparation of a drug for bacterial infectious diseases or an antibiotic synergist. The second purpose of the present invention is to provide a pharmaceutical composition and its application in the preparation of a drug for bacterial infectious diseases or an antibiotic synergist.

[0005] Technical solution: The present invention provides the use of 5-fluorouracil in the preparation of drugs for bacterial infectious diseases or the preparation of antibiotic enhancers.

[0006] Furthermore, the molecular formula of the 5-fluorouracil is C4H3FN2O2.

[0007] Furthermore, the bacteria are Gram-positive bacteria or Gram-negative bacteria.

[0008] Furthermore, the Gram-positive bacteria include Staphylococcus aureus.

[0009] Furthermore, the Gram-positive bacteria include S. aureus G16, S. aureus MRSA 1530 and S. aureus MRSAT144.

[0010] Furthermore, the Gram-negative bacteria include Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii.

[0011] Furthermore, the Gram-negative bacteria include E. coli ATCC 25922, S. Typhimurium (mcr-3), P. aeruginosa PA01, A. baumannii ATCC 19606, K. pneumoniae ATCC 700603 and K. pneumoniae D120 (mcr-8).

[0012] Furthermore, the antibiotic is fosfomycin.

[0013] Furthermore, the molecular formula of the fosfomycin is C3H7O4P.

[0014] The present invention also provides a pharmaceutical composition, which comprises 5-fluorouracil and fosfomycin.

[0015] Furthermore, the concentration of 5-fluorouracil is 2-256 μg / mL.

[0016] Furthermore, the final concentration of fosfomycin is 0.0156-16 μg / mL.

[0017] Furthermore, the mass ratio of 5-fluorouracil to fosfomycin is 0.125:1 to 16410:1.

[0018] The present invention also provides the use of the above-mentioned pharmaceutical composition in preparing drugs for bacterial infectious diseases or antibiotic enhancers.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention discloses for the first time the use of 5-fluorouracil as an antibiotic synergist in the treatment of bacterial infectious diseases. Specifically, Gram-positive bacteria or Gram-negative bacteria can be effectively eliminated by the combined use of low-dose 5-fluorouracil and fosfomycin; in particular, methicillin-resistant Staphylococcus aureus that is resistant to both β-lactam and cephalosporin antibiotics can be effectively eradicated, biofilm formation can be inhibited, and mature biofilms can be removed at the same time. The present invention provides a new perspective for the development of new resistance prevention and control strategies, and also provides a new treatment method for coping with multidrug-resistant bacterial infections and biofilm-related persistent infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is an in vitro efficacy evaluation of 5-fluorouracil-enhanced fosfomycin against MRSAT144, wherein CON means blank control.

[0021] Figure 2 In vitro evaluation of 5-fluorouracil-enhanced fosfomycin against Gram-negative bacteria.

[0022] Figure 3 The combined use of 5-fluorouracil and fosfomycin inhibits the formation of MRSAT144 biofilm and eliminates mature biofilm, wherein CON means blank control.

[0023] Figure 4 These are the pattern diagrams and result diagrams of G. mellonella infection and mouse peritonitis infection treated with the combination of 5-fluorouracil and fosfomycin, where Vehicle means the PBS negative control. DETAILED DESCRIPTION

[0024] The technical scheme of the present invention is further described below in conjunction with the accompanying drawings. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several modifications and improvements can also be made, which should also be considered to belong to the protection scope of the present invention. The experimental methods in the following embodiments are conventional methods unless otherwise specified. The experimental materials used in the following embodiments are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative experiments in the following embodiments are all repeated three times, and the results are averaged.

[0025] The LB broth medium used in the embodiment is an aqueous solution containing 10 g / L tryptone, 5 g / L yeast extract powder and 10 g / L NaCl; the LB solid medium is LB broth medium plus 15 g / L Agra; the MHB broth medium is an aqueous solution of 2 g / L beef powder, 1.5 g / L soluble starch and 17.5 g / L acid hydrolyzed casein.

[0026] The antibiotics and non-antibiotics used in the examples were purchased from Yuanye Biotechnology Co., Ltd.

[0027] C57 female mice were purchased from the Comparative Medicine Center of Yangzhou University.

[0028] The specific information of the strains used in the examples and their sources or references are shown in Table 1.

[0029] Table 1

[0030] Test strains Source or literature Escherichia coli ATCC25922 Reference 1 Salmonella Typhimurium (mcr-3) This laboratory keeps Klebsiella pneumoniae ATCC 700603 Reference 2 Klebsiella pneumoniae D120 (mcr-8) This laboratory keeps Pseudomonas aeruginosa PA01 Reference 3 Acinetobacter baumannii ATCC19606 Reference 4 <![CDATA[Staphylococcus aureus G16 (RIF R )]]> Reference 5 Staphylococcus aureus MRSA1530 Reference 5 Staphylococcus aureus MRSAT144 Reference 5

[0031] Note: Document 1 is Shi J, Chen C, Wang D, et al. The antimicrobial peptide LI14combats multidrug-resistantbacterial infections. Commun Biol. 2022Sep 7;5(1):926.

[0032] Document 2 is Rasheed JK, Anderson GJ, Yigit H, et al. Characterization of the extended-spectrum beta-lactamase reference strain, Klebsiellapneumoniae K6 (ATCC 700603), which produces the novel enzyme SHV-18. Antimicrob Agents Chemother. 2000 Sep; 44 (9): 2382-8.

[0033] Document 3 is Hancock RE, Decad GM, Nikaido H. Identification of the proteinproducing transmembrane diffusionpores in the outer membrane of Pseudomonasaeruginosa PA01. Biochim Biophys Acta. 1979 Jul 5; 554 (2): 323-31.

[0034] Reference 4 is Tsubouchi T, Suzuki M, Niki M, et al. Complete Genome Sequence of Acinetobacter baumannii ATCC 19606 T , a Model Strain of Pathogenic Bacteria Causing Nosocomial Infection. Microbiol Resour Announc. 2020 May 14;9(20):e00289-20.

[0035] Reference 5 is Liu Y, Ding S, Dietrich R, et al. A biosurfactant-inspired heptapeptide with improved specificity to kill MRSA. Angew Chem Int Ed Engl. 2017,56(6):1486-1490.

[0036] Example 15 - Evaluation of the in vitro effectiveness of 5-fluorouracil in enhancing fosfomycin against MRSA T144

[0037] 1. Use the checkerboard method to test the synergistic activity of fosfomycin and 5-fluorouracil against S. aureus G16 (RIF R ), MRSA 1530 and MRSA T144. Specifically, the checkerboard assay with two-fold serial dilution of drugs (8×8) was used to evaluate the synergistic effect between fosfomycin and antibiotics or non-antibiotics with different mechanisms. After incubation with the bacterial suspension (1.5×10 6 CFU / mL) for 18 hours, the absorbance at 600 nm was measured using a microplate reader. The fractional inhibitory concentration index (FICI) was calculated from two biological replicates of each combination using the formula: FICI = FICa + FICb = MICab / MICa + MICba / MICb. An FICI of ≤0.5 indicates synergistic activity, 0.5 < FICI ≤ 4 indicates no interaction, and FICI > 4 indicates antagonistic activity. The experiment was repeated independently 3 times.

[0038] 2. The plate count method was used to determine the effect of 5-fluorouracil (5-FU) and fosfomycin in killing MRSAT144. The overnight culture of MRSAT144 was diluted 1:1000 into fresh MHB and incubated at 37°C with continuous shaking (200 rpm) for 4 hours. The culture was then treated with fosfomycin (1 μg / mL), 5-fluorouracil (16 μg / mL) or their combination for 24 hours. The bacterial count was determined at 0, 4, 8 and 24 hours using the plate colony count method. MHB containing PBS was used as a negative control. Each experiment was repeated 3 times.

[0039] 3. Analyze bacterial viability using flow cytometry. 6 CFU / mL) were treated with fosfomycin (1 μg / mL), 5-fluorouracil (16 μg / mL) or their combination at 37°C and 220 rpm for 6 hours. Then, according to the kit instructions, LIVE / DEAD containing PI (5 mM, 3 μL) and SYTO 9 (0.835 mM, 3 μL) was used. TM BacLight TM The bacterial suspension was stained with a bacterial viability detection kit for 15 minutes. After washing twice with sterile PBS, the samples were analyzed using a flow cytometer with FITC and PI channels. The data were analyzed using CytExpert2.0 software (Beckman, USA) and the bacterial survival rates of the different treatment groups were compared.

[0040] 4. Use confocal microscopy to image MRSAT144 exposed to fosfomycin (1 μg / mL), 5-fluorouracil (16 μg / mL), or a combination thereof. The specific operation method is to inoculate the bacterial culture into a sterile 6-well plate containing the drug, place the sterile cell slide in LB liquid culture medium, and incubate at 37 ° C for 24 hours. After rinsing with PBS three times to remove planktonic cells, the bacterial viability was assessed using a bacterial viability kit. After incubation in the dark for 15 minutes, the stained cell slides were examined under a confocal microscope (Leica TCS SP2, Heidelberg, Germany).

[0041] 5. Scanning electron microscopy was used to observe the morphological changes of bacteria treated with fosfomycin (1 μg / mL), 5-fluorouracil (16 μg / mL) or the combination. 6CFU / mL) was incubated with fosfomycin and / or 5-fluorouracil at 37°C for 8 hours, and the bacterial suspension was centrifuged (5000×g, 5 minutes) and gently washed twice with PBS. The bacterial pellet was then fixed with 2.5% glutaraldehyde (Solarbio, Beijing, China) at 4°C for 24 hours. Subsequently, the bacteria were dehydrated through a series of ethanol gradients (40%, 50%, 60%, 70%, 80%, 90% and 100%; 10 minutes for each gradient). After a metal film was formed on the surface of the sample using an ion sputtering instrument, the bacterial morphology was detected using a scanning electron microscope Gemini SEM 300 (ZEISS, Germany).

[0042] 6. Use transmission electron microscopy to examine the ultrastructural changes of bacteria treated with fosfomycin (1 μg / mL), 5-fluorouracil (16 μg / mL), or their combination. The specific method is to culture the bacteria in the exponential phase (10 6 CFU / mL) were incubated with the drug at 37°C for 8 hours. The bacterial suspension was then centrifuged and resuspended in 1 mL of a fixative consisting of 2.5% glutaraldehyde and 5% formaldehyde. The fixed bacteria were washed three times with 0.1 M cacodylate buffer and post-fixed with 1% osmium tetroxide for 1 hour. After washing three times with water, the bacteria were further dehydrated using an ethanol gradient (1 minute each: 50%, 60%, 70%, 80%, 90% and 100%). The Epon TM The resin infiltrated the samples and polymerized at 75°C for 48 hours. Ultrathin sections were cut with a diamond knife, picked up on copper grids, and stained with lead citrate. Cell micrographs were examined using a JEM1011 TEM (JEOL, Tokyo, Japan).

[0043] The experimental results are shown in Figure 1 . The results showed that (A) the combination of 5-fluorouracil and fosfomycin had a synergistic effect in inhibiting the growth of S. aureus G16 and MRSA 1530, with the fractional inhibition concentration index (FICI) values ​​of 0.3125 and 0.375, respectively. It was proved that the combination had a synergistic effect on the growth inhibition of Gram-positive bacteria. (B) The combination of fosfomycin and 5-fluorouracil had a synergistic effect in inhibiting the growth of MRSAT144, with a FICI value of 0.375. (C) The synergistic time-kill curve of 5-fluorouracil and fosfomycin against MRSAT144 determined by the plate count method. (D) The live / dead bacterial ratio detected by flow cytometry. Live bacteria are stained green and dead bacteria are stained red. (E) The bacterial survival rate measured by flow cytometry. (F) Confocal microscopy analysis. (G) SEM / TEM micrographs. These results indicate that low-dose fosfomycin or 5-fluorouracil, when used alone, failed to kill MRSAT144, but when used in combination, they could effectively kill MRSAT144.

[0044] Example 25 - In vitro evaluation of fluorouracil-enhanced fosfomycin against Gram-negative bacteria

[0045] The synergistic activity of the combination of 5-fluorouracil and fosfomycin against Gram-negative pathogens was determined by the checkerboard assay. The specific method was to evaluate the synergistic effect between fosfomycin and 5-fluorouracil using a two-fold serial dilution of the drug (8×8). 5-fluorouracil + fosfomycin were mixed with bacterial suspension (1.5×10 6 CFU / mL) for 18 hours, and the absorbance at 600 nm was measured using a microplate reader. The fractional inhibitory concentration index (FICI) was calculated from two biological replicates of each combination using the formula: FICI = FICa + FICb = MICab / MICa + MICba / MICb. FICI of ≤ 0.5 indicates synergy.

[0046] The experimental results are shown in Figure 2 The results showed that the combination of 5-fluorouracil and fosfomycin had synergistic activity against a variety of Gram-negative pathogens (E. coli ATCC 25922, S. typhimurium (mcr-3), P. aeruginosa PA01, A. baumannii ATCC 19606, K. pneumoniae ATCC 700603, and K. pneumoniae D120 (mcr-8)), indicating that this combination has a broad-spectrum antibacterial activity.

[0047] Example 35 - Combination of fluorouracil and fosfomycin inhibits MRSAT144 biofilm formation and eliminates mature biofilms.

[0048] The cell crawler with a diameter of 24 mm is placed at the bottom of each well in a 6-well plate (Corning, USA). 2 mL of MRSAT144 bacterial culture treated with fosfomycin, 5-fluorouracil or a combination thereof is added to each well. After incubation at 37 ° C for 24 hours, planktonic bacteria are removed and the biofilm is stained with 1% crystal violet (Solarbio) for 15 minutes. The slides are then washed 3 times with distilled water and the biofilm is dissolved with 33% acetic acid. The absorbance at 570 nm is measured using a microtiter plate reader to quantify the formation of biofilm. 5-fluorouracil and fosfomycin are simultaneously photographed using a confocal microscope to form and remove biofilm.

[0049] Dilute the mid-exponential phase MRSAT144 culture to 10 6CFU / mL. In a 6-well plate, each well was filled with 2 mL of bacterial suspension, and the plate was incubated at 37°C for 36 hours to form a mature biofilm. After discarding the planktonic bacteria, 2 mL of MHB containing fosfomycin, 5-fluorouracil, or a combination thereof was added to the remaining biofilm cells, and the plate was incubated at 37°C for 24 hours. After incubation, the plate was sonicated for 10 minutes to detach adherent bacteria, and the number of viable bacteria was determined by microbial counting.

[0050] The experimental results are shown in Figure 3 The results showed that the use of fosfomycin or 5-fluorouracil alone had no inhibitory or clearing effect on the biofilm of MRSAT144, while the combination of 5-fluorouracil and fosfomycin could significantly inhibit the formation of MRSA T144 biofilm and clear the mature biofilm.

[0051] Example 45 - Evaluation of the in vivo effectiveness of fluorouracil and fosfomycin against MRSAT144.

[0052] G. mellonella infection model: Prepare enough G. mellonella larvae (Hui Yu De Biotechnology Company, about 2 cm in length) and divide them into 4 groups, 8 in each group. Inject 1.0×10 8 CFU of MRSAT144 (10 μL per larva). 1 h after infection, 10 μL of fosfomycin (10 mg / kg), 5-fluorouracil (40 mg / kg) and their combination were injected into the left caudal foot of the larvae. The survival rate of the greater wax moth larvae was continuously observed and recorded for 5 days. The negative control was injected with 10 μL of PBS.

[0053] Mouse peritonitis infection model: Prepare enough female ICR mice (Comparative Medicine Center of Yangzhou University, 6-8 weeks old) and divide them into 4 groups, 6 mice in each group. Inject 1.0×10 8 CFU of MRSAT144 (100 μL per mouse) was injected intraperitoneally 1 h after infection with 100 μL of fosfomycin (10 mg / kg), 5-fluorouracil (40 mg / kg) and their combination, and the mortality of mice was monitored within two days.

[0054] The ICR mice were divided into four groups, with 8 mice in each group. 1.0×10 8 CFU of MRSA T144 (100 μL per mouse) were injected intraperitoneally 1 h after infection with 100 μL of fosfomycin (10 mg / kg), 5-fluorouracil (40 mg / kg) and their combination. The survival rate of mice was monitored within 7 days after infection, and the bacterial load in the homogenate of heart, liver, spleen, lung and kidney was determined by colony counting.

[0055] The experimental results are shown in Figure 4In the wax moth infection model, larvae infected with MRSAT144 treated with fosfomycin alone died within 48 hours. The survival rate of wax moth in the control group and 5-fluorouracil alone after 5 days of treatment was only 37.5%, while the survival rate in the combined treatment group reached 100% (P<0.0001)( Figure 4 B). In addition, the in vivo efficacy of the combination therapy was also confirmed in a mouse peritonitis infection model, where the combination of fosfomycin and 5-fluorouracil provided a higher survival rate compared with the single treatment groups (P = 0.0071) ( Figure 4 C). By measuring the bacterial load in mouse organs, the combination treatment group (10+40 mg / kg) resulted in a CFU reduction of approximately 2-3 log10 ( Figure 4 D). These encouraging results suggest that 5-FU and fosfomycin exhibit synergistic effects against MRSAT144 in vivo.

[0056] In summary, the combined use of 5-fluorouracil and fosfomycin can exert potential antibacterial effects against MRSAT144 both in vivo and in vitro.

Claims

Application of 1.5-fluorouracil in the preparation of drugs for bacterial infectious diseases or the preparation of antibiotic enhancers.

2. The use according to claim 1, characterized in that: The bacteria are Gram-positive bacteria or Gram-negative bacteria.

3. The use according to claim 2, characterized in that: The Gram-positive bacteria include Staphylococcus aureus.

4. The use according to claim 2, characterized in that: The Gram-negative bacteria include Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii.

5. The use according to claim 1, characterized in that: The antibiotic is fosfomycin.

6. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises 5-fluorouracil and fosfomycin.

7. The pharmaceutical composition according to claim 5, characterized in that The concentration of 5-fluorouracil is 2-256 μg / mL.

8. The pharmaceutical composition according to claim 5, characterized in that The final concentration of fosfomycin is 0.0156-16 μg / mL.

9. The pharmaceutical composition according to claim 5, characterized in that The mass ratio of 5-fluorouracil to fosfomycin is 0.125:1 to 16410:

1.

10. Use of the pharmaceutical composition according to any one of claims 6 to 9 in the preparation of drugs for bacterial infectious diseases or antibiotic synergists.

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