Use of 5-fluorouracil in the manufacture of a medicament for a bacterial infectious disease or in the manufacture of an antibiotic potentiator

Abstract

The application discloses application of 5-fluorouracil in preparation of a drug for bacterial infectious diseases or preparation of an antibiotic synergist, and belongs to the technical field of medicines.The application discloses, for the first time, that the anticancer drug 5-fluorouracil is used as a novel antibiotic synergist for treating bacterial infectious diseases.In detail, the application finds that low-dose 5-fluorouracil combined with fosfomycin can effectively and synergistically inhibit the growth of gram-positive bacteria and gram-negative bacteria.This combined drug use scheme has a significant advantage in preventing and treating multiple drug-resistant bacteria infection, especially for clinically important methicillin-resistant Staphylococcus aureus (MRSA).The combined use of 5-fluorouracil and fosfomycin can not only effectively eradicate MRSA, but also inhibit the formation of a biofilm, and meanwhile, clear the mature biofilm bacteria.In summary, the application provides a new idea for developing a novel drug-resistant pathogenic bacteria prevention and control strategy, and also provides a new treatment scheme for coping with multiple drug-resistant bacterial infection and biofilm-related persistent infection.

Description

Technical Field

[0001] This invention relates to the application of 5-fluorouracil in the preparation of drugs for bacterial infectious diseases or in the preparation of antibiotic potentiators, belonging to the field of pharmaceutical technology. Background Technology

[0002] Staphylococcus aureus is a Gram-positive pathogen and a leading cause of hospital- and community-acquired infections, potentially leading to serious consequences. More concerningly, Staphylococcus aureus is resistant to multiple antibiotics. A representative strain is methicillin-resistant Staphylococcus aureus (MRSA), which was clinically discovered in the early 1960s. The rapid global spread of MRSA poses a significant threat to public health and is a leading cause of life-threatening infections. For many years, traditional treatment for MRSA relied heavily on antibiotics, but with the widespread use of antibiotics, this strain has developed resistance to many existing antibiotics. Due to the rapid spread of MRSA infection and its complex resistance mechanisms, the mortality rate of related diseases has increased significantly. Statistics show that the European Union reports nearly 150,000 cases of MRSA infection annually, resulting in over 7,000 deaths. In China, according to the CHINET monitoring system, the infection rate of MRSA has consistently remained above 30% over the past five years.

[0003] Despite ongoing efforts to develop new antimicrobial drugs in recent years, only a small number of novel antibiotics have been approved over the past few decades. Therefore, new antimicrobial strategies are urgently needed. Combinations of antibiotics and adjuvants offer a new approach to overcoming drug development bottlenecks, bypassing stagnant new drug development processes and resensitizing drug-resistant bacteria. The combined use of antibiotics and adjuvants not only enhances the efficacy of antibiotics but also extends the lifespan of existing antibiotics and delays the emergence of drug-resistant strains. Furthermore, these synergistic therapies can reduce antibiotic toxicity, treatment duration, and dosage. Against this backdrop, older antibiotics such as polymyxins and fosfomycin (FOS) have regained researchers' attention. Fosfomycin, a bactericidal agent discovered more than 40 years ago, exhibits significant activity against both Gram-negative and Gram-positive bacteria, including multidrug-resistant (MDR) strains. Its unique mechanism of action lies in reducing the likelihood of cross-resistance with other antimicrobial agents by inhibiting the early stages of peptidoglycan synthesis. This unique property makes fosfomycin a potential candidate drug for treating systemic infections. Although the clinical use of fosfomycin is gradually increasing, research on its application in combination therapy is still limited, and this area warrants further exploration to fully utilize its therapeutic potential. Therefore, sensitization strategies to enhance the antibacterial effects of existing antibiotics are inevitable, and enhancing the effectiveness of older antibiotics with approved or non-antibiotic formulations is a highly promising antibacterial strategy. Summary of the Invention

[0004] Objective of the Invention: The first objective of this invention is to provide the use of 5-fluorouracil in the preparation of medicaments for bacterial infectious diseases or in the preparation of antibiotic potentiators. The second objective of this invention is to provide a pharmaceutical composition and its use in the preparation of medicaments for bacterial infectious diseases or in the preparation of antibiotic potentiators.

[0005] Technical solution: This invention provides the application of 5-fluorouracil in the preparation of drugs for bacterial infectious diseases or in the preparation of antibiotic potentiators.

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

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

[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 comprising 5-fluorouracil and fosfomycin.

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

[0016] Furthermore, the final concentration of the 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-described pharmaceutical composition in the preparation of medicaments for bacterial infectious diseases or antibiotic potentiators.

[0019] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: This invention discloses for the first time the application of 5-fluorouracil as an antibiotic potentiator in the treatment of bacterial infectious diseases. Specifically, the combined use of low-dose 5-fluorouracil and fosfomycin can effectively eliminate Gram-positive or Gram-negative bacteria; in particular, it can effectively eradicate methicillin-resistant Staphylococcus aureus (MRSA), which is resistant to both β-lactam and cephalosporins, inhibiting biofilm formation and simultaneously clearing mature biofilms. This invention provides a new perspective for developing novel drug resistance control strategies and offers a new treatment method for addressing multidrug-resistant bacterial infections and biofilm-related persistent infections. Attached Figure Description

[0020] Figure 1 The in vitro efficacy of 5-fluorouracil-enhanced fosfomycin against MRSAT144 was evaluated, where CON represents the blank control.

[0021] Figure 2 To enhance the in vitro evaluation of fosfomycin's effectiveness against Gram-negative bacteria with 5-fluorouracil.

[0022] Figure 3 The combined use of 5-fluorouracil and fosfomycin was used to inhibit the formation of MRSAT144 biofilm and to remove mature biofilms. CON represents the blank control.

[0023] Figure 4 The diagram shows the pattern and results of treatment with a combination of 5-fluorouracil and fosfomycin for infection with giant wax moth and peritonitis in mice. In this diagram, "Vehicle" represents the PBS negative control. Detailed Implementation

[0024] The technical solution of the present invention will be further described below with reference to the accompanying drawings. It should be noted that those skilled in the art can make several modifications and improvements without departing from the principle of the present invention, and these should also be considered within the scope of protection of the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were all purchased from conventional biochemical reagent stores. The quantitative experiments in the following embodiments were all performed in triplicate, and the results were averaged.

[0025] The LB broth medium used in the examples was an aqueous solution containing 10 g / L tryptone, 5 g / L yeast extract and 10 g / L NaCl; the LB solid medium was the LB broth medium with 15 g / L Agra added; the MHB broth medium was an aqueous solution containing 2 g / L beef meal, 1.5 g / L soluble starch and 17.5 g / L acid-hydrolyzed casein.

[0026] All 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 on the strains used in the examples and their sources or literature is shown in Table 1.

[0029] Table 1

[0030] Test strain Source or literature Escherichia coli (Escherichiacoli) ATCC25922 Reference 1 Salmonella Typhimurium (mcr-3) This laboratory preserves Klebsiella pneumoniae (ATCC 700603) Reference 2 Klebsiella pneumoniae D120 (mcr-8) This laboratory preserves Pseudomonas aeruginosa PA01 Reference 3 Acinetobacter baumannii ATCC 19606 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 efficacy 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. A FICI of ≤0.5 indicates synergistic activity, 0.5 < FICI ≤ 4 indicates no interaction, and FICI > 4 indicates antagonism. The experiment was repeated independently 3 times.

[0038] 2. The efficacy of 5-fluorouracil (5-FU) and fosfomycin in killing MRSAT144 was determined using a plate count method. Overnight cultures of MRSAT144 were diluted 1:1000 in fresh MHB and incubated at 37°C with continuous shaking (200 rpm) for 4 hours. The cultures were then treated with fosfomycin (1 μg / mL), 5-fluorouracil (16 μg / mL), or a combination thereof for 24 hours. Bacterial counts were determined at 0, 4, 8, and 24 hours using a plate count method. MHB with PBS served as a negative control. Each experiment was repeated three times.

[0039] 3. Flow cytometry was used to analyze bacterial viability. The exponential phase bacteria MRSAT144(10) were analyzed. 6 CFU / mL) was treated with fosfomycin (1 μg / mL), 5-fluorouracil (16 μg / mL), or a combination thereof at 37°C and 220 rpm for 6 hours. Then, following the kit instructions, LIVE / DEAD was used with PI (5 mM, 3 μL) and SYTO 9 (0.835 mM, 3 μL). TM BacLight TM The bacterial viability assay kit stained the bacterial suspension for 15 minutes. After washing twice with sterile PBS, the samples were analyzed using a flow cytometer with FITC and PI channels. Data were analyzed using CytExpert 2.0 software (Beckman, USA), and bacterial survival rates were compared between different treatment groups.

[0040] 4. Image MRSAT144 exposed to fosfomycin (1 μg / mL), 5-fluorouracil (16 μg / mL), or a combination thereof using confocal microscopy. Specifically, bacterial cultures were inoculated into sterile 6-well plates containing the drug, and sterile cell smears were placed in LB broth and incubated at 37°C for 24 hours. After washing three times with PBS to remove airborne cells, bacterial viability was assessed using a bacterial viability kit. After incubation in the dark for 15 minutes, the stained cell smears were examined under a confocal microscope (Leica TCS SP2, Heidelberg, Germany).

[0041] 5. Scanning electron microscopy (SEM) is used to observe morphological changes in bacteria treated with fosfomycin (1 μg / mL), 5-fluorouracil (16 μg / mL), or a combination thereof. Exponential phase bacterial cultures (10 μg / mL) were then analyzed. 6After incubating the bacterial suspension (CFU / mL) with fosfomycin and / or 5-fluorouracil at 37°C for 8 hours, the suspension was centrifuged (5000×g, 5 min) 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 using a series of ethanol gradients (40%, 50%, 60%, 70%, 80%, 90%, and 100%; 10 min for each gradient). After forming a metallic film on the sample surface using ion sputtering, bacterial morphology was examined using a Gemini SEM 300 (ZEISS, Germany).

[0042] 6. Transmission electron microscopy was used to examine the ultrastructural changes of bacteria treated with fosfomycin (1 μg / mL), 5-fluorouracil (16 μg / mL), or a combination thereof. Specifically, exponential phase bacterial cultures (10 μg / mL) were examined. 6 The bacterial suspension (CFU / mL) was incubated with the drug at 37°C for 8 hours. The bacterial suspension was then centrifuged and resuspended in 1 mL of a fixative solution consisting of 2.5% glutaraldehyde and 5% formaldehyde. The fixed bacteria were washed three times with 0.1 M dimethylarsine 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 solution was then used with Epon. TM Samples were impregnated with resin and polymerized at 75°C for 48 hours. Ultrathin sections were cut with a diamond scalpel, picked up on a copper grid, 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 fractional inhibitory concentration indices (FICI) of 0.3125 and 0.375, respectively. This demonstrates that the combination has a synergistic inhibitory effect on the growth 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-killing curve of 5-fluorouracil and fosfomycin against MRSAT144 determined by plate counting method. (D) The live / dead bacteria ratio detected by flow cytometry. Live bacteria are stained green, and dead bacteria are stained red. (E) Bacterial viability measured by flow cytometry. (F) Confocal microscopy analysis. (G) SEM / TEM microscopic images. These results all indicate that low-dose fosfomycin or 5-fluorouracil alone failed to kill MRSAT144, but the combination of the two 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 using the checkerboard method. Specifically, the synergistic effect between fosfomycin and 5-fluorouracil was assessed using a checkerboard assay with two serial dilutions (8×8). 5-fluorouracil + fosfomycin were separately reacted with bacterial suspensions (1.5×10⁻⁶). 6 After incubation for 18 hours with CFU / mL, absorbance was measured at 600 nm using a microplate reader. The fractional index of inhibition (FICI) was calculated from two biological replicates for each combination using the formula: FICI = FICa + FICb = MICab / MICa + MICba / MICb. A FICI ≤ 0.5 indicates synergistic effect.

[0046] The experimental results are shown in Figure 2 The results showed that the combination of 5-fluorouracil and fosfomycin exhibited 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 broad-spectrum antibacterial activity.

[0047] Example 35 - The combined use of fluorouracil and fosfomycin inhibits the formation of MRSAT144 biofilm and removes mature biofilms.

[0048] Cell slides with a diameter of 24 mm were 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 was added to each well. After incubation at 37°C for 24 hours, airborne bacteria were removed, and the biofilm was stained with 1% crystal violet (Solarbio) for 15 minutes. The slides were then washed three times with distilled water, and the biofilm was dissolved in 33% acetic acid. The absorbance at 570 nm was measured using a microtiter plate reader to quantify biofilm formation. Confocal microscopy was used to image the effects of 5-fluorouracil and fosfomycin on biofilm formation and removal.

[0049] MRSAT144 culture in mid-exponential phase was diluted to 10 in MHB medium. 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 isolate the adhered 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 treatment with fosfomycin or 5-fluorouracil alone had no inhibitory or scavenging effect on the biofilm of MRSAT144, while treatment with a combination of 5-fluorouracil and fosfomycin significantly inhibited the formation of MRSA T144 biofilm and cleared mature biofilm.

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

[0052] Large wax moth infection model: Sufficient large wax moth larvae (Huiyude Biotechnology Co., Ltd., approximately 2 cm in length) were prepared and divided into 4 groups of 8 larvae each. Each larva was injected with 1.0 × 10⁻⁶ ppm of chlorpyrifos larvae into its right caudal leg. 8 CFU of MRSAT144 (10 μL per larva). One hour after infection, 10 μL of fosfomycin (10 mg / kg), 5-fluorouracil (40 mg / kg), and combinations thereof were injected into the left caudal leg of the larvae. The survival rate of the large wax moth larvae was continuously observed and recorded for 5 days. The negative control was an injection of 10 μL of PBS.

[0053] Mouse peritonitis infection model: Sufficient female ICR mice (Yangzhou University Comparative Medicine Center, 6-8 weeks old) were prepared and divided into 4 groups of 6 mice each. 1.0 × 10⁻⁶ mg / L was injected into the right side of the mouse peritoneum. 8 Mice were infected with CFU MRSAT144 (100 μL per mouse). One hour later, they were injected intraperitoneally with 100 μL of fosfomycin (10 mg / kg), 5-fluorouracil (40 mg / kg), or a combination thereof. Mice mortality was monitored over two days.

[0054] The ICR mice were then divided into four groups of eight. 1.0 × 10⁻⁶ mg / L was injected into the right side of the abdominal cavity of each mouse. 8 MRSA T144 CFU (100 μL per mouse) was used to infect mice. One hour after infection, mice were intraperitoneally injected with 100 μL of fosfomycin (10 mg / kg), 5-fluorouracil (40 mg / kg), or combinations thereof. The survival rate of mice was monitored within 7 days after infection, and the bacterial load in homogenates from the heart, liver, spleen, lung, and kidney was determined by colony counting.

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

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

Claims

1. Use of 5-fluorouracil and fosfomycin for the preparation of a medicament for the treatment of a bacterial infectious disease, characterized in that, The bacteria are Salmonella typhimurium or Acinetobacter baumannii; the Salmonella typhimurium is mcr-3 pathogenic bacteria; the concentration of 5-fluorouracil is 2-256 μg / mL, the concentration of fosfomycin is 0.0156-16 μg / mL, and the mass ratio of 5-fluorouracil to fosfomycin is 0.125:1 ~ 16410:1.

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