Furadantin or pharmaceutical composition or compound preparation containing effective components of furadantin and application of furadantin or pharmaceutical composition or compound preparation

Abstract

The invention discloses a new application of nitrofurantoin alone or in combination with tigecycline. The application refers to the application of nitrofurantoin alone or in combination with tigecycline in inhibition of tigecycline drug-resistant bacterium infection. According to the present invention, the single use of furantoin or the combination of furantoin and tigecycline provides strong antibacterial activity for lon gene mutation and tet (X4) mediated tigecycline drug-resistant bacteria, and the furantoin single use or the combination of furantoin and tigecycline can enhance the survival rate of greater wax moths, the colonization of the tet (X4)-mediated tigecycline drug-resistant bacteria in mouse organs can be effectively reduced, and the healing of skin wound infection caused by tet (X4)-mediated tigecycline drug-resistant bacteria can be remarkably promoted by the combined medication. Furantoin designed depending on interaction sensitivity and tigecycline combined medication can be used for resisting tigecycline drug-resistant pathogens and treating diseases caused by tigecycline drug-resistant bacterium infection, and meanwhile, the evolution of tigecycline drug resistance can be prevented.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and relates to nitrofurantoin or a pharmaceutical composition or a compound preparation containing nitrofurantoin as an active ingredient and its application, and particularly relates to the application of the combined use of nitrofurantoin and tigecycline in the prevention and treatment of tigecycline-resistant bacterial infections. Background Art

[0002] The emergence and spread of multi-drug resistant bacteria have posed a serious threat to human health. Tigecycline is the first glycylcycline antibiotic approved for clinical intravenous administration. It can overcome existing tetracycline resistance mechanisms and thus exert a broad-spectrum antibacterial effect, and is usually used to treat severe pulmonary infections, bloodstream infections, and abdominal infections. Nowadays, tigecycline has become the last line of defense for the treatment of multi-drug resistant bacterial infections. However, the emergence of plasmid-mediated mobile tigecycline resistance genes (such as tet(X) and tmexCD-toprJ and their variants) has severely restricted the clinical efficacy of tigecycline. Therefore, it is urgent to use innovative methods to deal with the related infections caused by tigecycline-resistant bacteria. The molecular formula of tigecycline is C 29 H 39 N 5 O 8 . The chemical structural formula of tigecycline is shown in Formula 1:

[0003]

[0004] Cross-sensitivity is a common phenotype in the evolution of bacterial drug resistance. It is a phenomenon in which bacteria change their genetic material or physiological and biochemical characteristics to adapt to the pressure of a certain class of antibacterial drugs and produce an increase in sensitivity to another class or several classes of antibacterial drugs. The cross-sensitivity antibacterial strategy can increase the sensitivity of drug-resistant pathogenic bacteria to existing antibacterial drugs by utilizing specific interactions between drugs. Therefore, by using the cross-sensitivity of bacteria, effective treatment can be achieved not only after the strain has developed drug resistance, but also the efficacy of existing antibacterial drugs can be improved. In-depth study of the cross-sensitivity mechanism of bacteria will be conducive to further revealing the generation and formation rules of drug-resistant strains and providing a theoretical basis for new antibacterial drugs targeting drug-resistant bacteria.

[0005] Based on this, treating tigecycline-resistant bacterial infections by taking advantage of cross-sensitivity is a potential antibacterial strategy. Among them, nitrofurantoin is a cyclic amide containing 5-nitrofurans, and it has been used for the treatment and prevention of urinary tract infections for more than 60 years, and the acidic pH environment in urine has a significant enhancing effect on the antibacterial activity of nitrofurantoin. The antibacterial mechanism of nitrofurantoin can generally be summarized as: non-specifically affecting the normal synthesis of proteins, DNA, and RNA in bacteria by inhibiting biochemical processes in bacterial energy metabolism. The molecular formula of nitrofurantoin is C 8 H 6 N4 O 5 The chemical structural formula of nitrofurantoin is shown in Formula 2:

[0006]

[0007] Currently, the difficulty of developing new drugs has increased. Since the 21st century, only more than 20 antibiotics have entered clinical application. Under the premise that the research and development of new antibiotics is slow and the harm of super drug-resistant bacteria is widespread, the strategy of enhancing the antibacterial effect of existing antibiotics has become an inevitable choice. As one of the "last line of defense" antibiotics for treating bacterial infections caused by multi-drug resistant bacteria, tigecycline still faces the dilemma of continuously emerging drug-resistant bacteria with mutant genes and drug-resistant genes. However, it is still unclear whether tigecycline can have a synergistic effect with nitrofurantoin to solve the problem of new drug-resistant bacteria. Summary of the Invention

[0008] Object of the Invention: The technical problem to be solved by the present invention is to provide the use of nitrofurantoin or a pharmaceutical composition or compound preparation containing an effective ingredient of nitrofurantoin.

[0009] Another technical problem to be solved by the present invention is to provide a pharmaceutical composition or compound preparation containing an effective ingredient of nitrofurantoin.

[0010] Technical Solution: To solve the above technical problems, the present invention provides the use of nitrofurantoin or a pharmaceutical composition or compound preparation containing an effective ingredient of nitrofurantoin in at least one of the following:

[0011] (a) Preparing a product or drug for inhibiting or killing the activity of tigecycline-resistant bacteria;

[0012] (b) Preparing a product or drug for treating tigecycline-resistant bacterial infections;

[0013] (c) Preparing a product or drug for preventing and / or treating infectious diseases mediated by tigecycline-resistant bacterial infections.

[0014] Wherein, the composition containing an effective ingredient of nitrofurantoin further includes tigecycline.

[0015] Wherein, the tigecycline-resistant bacteria are tigecycline-resistant bacteria mediated by lon gene mutation or tet(X4)-positive pathogenic bacteria.

[0016] Wherein, the tet(X4)-positive pathogenic bacteria are one or more of E. coli DH5α(pUC19-tet(X4)) or E. coli B3-1tet(X4).

[0017] Wherein, the product for inhibiting or killing the activity of tigecycline-resistant bacteria includes a tigecycline-resistant bacteria bacteriostatic agent or bactericide.

[0018] Among them, the products or drugs for preparing for preventing and / or treating infectious diseases mediated by tigecycline-resistant bacterial infections include products or drugs for preparing for reducing the expression levels of pro-inflammatory factors IL-1β, IL-6, TNF-α and / or INF-γ.

[0019] Among them, the products or drugs for preparing for preventing and / or treating infectious diseases mediated by tigecycline-resistant bacterial infections include products or drugs for preparing for increasing the expression levels of anti-inflammatory factors IL-4 and / or IL-10.

[0020] Among them, in the composition containing nitrofurantoin as an active ingredient, the mass ratio of nitrofurantoin to tigecycline is 0.5-4:1.

[0021] The content of the present invention further includes a pharmaceutical composition or a combined preparation containing nitrofurantoin as an active ingredient, and the pharmaceutical composition or the combined preparation includes nitrofurantoin and tigecycline.

[0022] Among them, the mass ratio of nitrofurantoin to tigecycline is 0.5-4:1.

[0023] Among them, the combined use of nitrofurantoin and tigecycline prevents the evolution of tigecycline resistance.

[0024] Among them, when the combined preparation of nitrofurantoin and tigecycline is used to treat tigecycline-resistant bacterial infections mediated by lon gene mutations in Galleria mellonella, the concentration of nitrofurantoin is 8 or 4 mg / kg of Galleria mellonella.

[0025] Preferably, the concentration of tigecycline is 2 or 1 mg / kg of Galleria mellonella.

[0026] Among them, when the combined preparation of nitrofurantoin and tigecycline is used to treat tigecycline-resistant bacterial infections mediated by lon gene mutations in mice, the concentration of nitrofurantoin is 8 or 4 mg / kg of mice.

[0027] Preferably, the concentration of tigecycline is 2 or 1 mg / kg of mice.

[0028] Among them, when the combined preparation of nitrofurantoin and tigecycline is used to treat tigecycline-resistant bacterial infections mediated by tet(X4) in rats, the concentration of nitrofurantoin is 10 mg / kg of rats.

[0029] Preferably, the concentration of tigecycline is 20 mg / kg of rats.

[0030] Among them, the combined preparation includes a combined preparation that enhances the survival rate of Galleria mellonella and reduces the inflammation of mouse organs and rat skin tissues.

[0031] The present invention shows that the compound preparation of nitrofurantoin and tigecycline can effectively reduce the amount of tigecycline-resistant bacteria in vivo and in vitro.

[0032] The method is a non-disease diagnosis and treatment method. For example, it is used as a positive control for developing sensitive drugs against tigecycline-resistant bacteria.

[0033] Beneficial effects: Compared with the prior art, the present invention has the following outstanding and significant advantages: The present invention discloses for the first time that the combined use of nitrofurantoin and tigecycline has a strong effect against tigecycline-resistant bacteria infection in vivo and in vitro. Specifically, nitrofurantoin has significant bactericidal activity against tigecycline-resistant bacteria with lon gene mutations, and the combined use of nitrofurantoin and tigecycline has a significant bactericidal effect against tigecycline-resistant bacteria mediated by tet(X4). The compound preparation of nitrofurantoin alone or in combination with tigecycline significantly reduces the pathogen load in the liver, spleen, and kidneys of mice infected with tigecycline-resistant bacteria with lon gene mutations, and reduces the body's inflammation level. The compound preparation of nitrofurantoin and tigecycline significantly reduces the pathogen load in the skin wounds of rats infected with tigecycline-resistant bacteria mediated by tet(X4), reduces the inflammation level of the wound, and improves the wound healing degree. In addition, the combined use of nitrofurantoin and tigecycline prevents the evolution of tigecycline resistance. The invention provides a new perspective for the development of new drug resistance prevention and control strategies, and also provides a new method for the prevention and control of increasingly serious tigecycline-resistant bacteria infections. Description of the Drawings

[0034] Figure 1 For the screening of antibiotics that are cross-sensitive to laboratory-evolved tigecycline-resistant bacteria, and the in vitro evaluation of the effect of cross-sensitive antibiotics in inhibiting and killing tigecycline-resistant bacteria mediated by lon gene mutations.

[0035] Figure 2 For the in vitro evaluation of the effectiveness of the combined administration of nitrofurantoin and tigecycline in inhibiting the development of tigecycline resistance.

[0036] Figure 3 For the in vivo evaluation of the effect of the combined administration of nitrofurantoin and tigecycline in controlling the infection caused by tigecycline-resistant bacteria mediated by lon gene mutations, where CON means blank control.

[0037] Figure 4 For the screening of antibiotics that are cross-sensitive to tigecycline-resistant bacteria mediated by tet(X4), and the in vivo evaluation of the effect of the combined administration of nitrofurantoin and tigecycline in controlling the infection caused by tigecycline-resistant bacteria mediated by tet(X4), where CON means blank control. Detailed Embodiments

[0038] The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several variations and improvements can also be made, which should also be regarded as belonging to the protection scope of the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all obtained from regular biochemical reagent stores unless otherwise specified. In the following examples, quantitative experiments are all set with three repeated experiments, and the results are averaged.

[0039] ICR female mice and Wistar female rats were purchased from the Comparative Medicine Center of Yangzhou University.

[0040] The specific information of the strains and their sources or references used in the examples is shown in Table 1.

[0041] Table 1

[0042]

[0043]

[0044] Note: Literature 1 is Liu Y, Yang K, Jia Y, et al. Cysteine Potentiates Bactericidal Antibiotics Activity Against Gram-Negative Bacterial Persisters. Infect. Drug Resist., 2020, 13, 2593 - 2599.

[0045] Literature 2 is Zhang H, Li Y, Jiang Y, et al. Rapid and accurate antibiotics susceptibility determination of tet(X)-positive E.coli using RNA biomarkers. Microbiology Spectrum, 2021, 9, e00648 - 21.

[0046] Literature 3 is Y. Liu, Y. Jia, K. Yang, R. Li, et al. Anti-HIV agent azidothymidine decreases Tet(X)-mediated bacterial resistance to tigecycline in Escherichia coli. Commun. Biol., 2020, 3, 162.

[0047] Screening of Antibiotics with Cross-Sensitivity to Laboratory-Evolved Tigecycline-Resistant Bacteria in Example 1, and Evaluation of the in Vitro Effects of Cross-Sensitive Antibiotics on Inhibiting and Killing Tigecycline-Resistant Bacteria Mediated by lon Gene Mutation

[0048] After laboratory evolution experiments on Escherichia coli EC25922 and Salmonella SE13076 under the stimulation of sub-inhibitory concentration of tigecycline, tigecycline-resistant strains were obtained.

[0049] The minimum inhibitory concentration (MIC) of 46 antibiotics against laboratory-evolved tigecycline-resistant bacteria was detected by the microbroth dilution method (European Committee on Antimicrobial Susceptibility Testing, EUCAST, 2024). The steps for detecting the minimum inhibitory concentration are as follows:

[0050] 1. Suspend the laboratory-evolved tigecycline-resistant bacteria with MH liquid medium (components per liter: beef extract powder 2.0 g, soluble starch 1.5 g, acid-hydrolyzed casein 17.5 g) to obtain a bacterial suspension with a bacterial concentration of 1×10 6 CFU / mL.

[0051] 2. Dissolve 46 antibiotics from 14 families to obtain solutions of Tigecycline, Vancomycin, Colistin, Amikacin, Gentamicin, Tobramycin, Streptomycin, Kanamycin, Neomycin, Spectinomycin, Ciprofloxacin, Levofloxacin, Ofloxacin, Norfloxacin, Enrofloxacin, Moxifloxacin, Ampicillin, Piperacillin, Carbenicillin, Ticarcillin, Oxacillin, Amoxicillin, Cefepime, Cefuroxime, Ceftazidime, Ceftiofur, Ceftriaxone, Aztreonam, Meropenem, Imipenem, Minocycline, Tetracycline, Oxytetracycline, Chlortetracycline, Azithromycin, Clarithromycin, Tylosin, Erythromycin, Rifampicin, Novobiocin, Nitrofurantoin, Fusidic acid, Tiamulin, Clindamycin, Chloramphenicol, Florfenicol with a concentration of 5120 μg / mL.

[0052] 3. Take a 96-well plate and add MH broth medium (190 μL per well in the first column and 100 μL per well in the remaining columns). Add the antibiotic solution prepared in step 2 (10 μL per well) to each well in the first column, and perform two-fold serial dilution to the tenth well with 100 μL. Discard 100 μL of the liquid from the tenth well, and then add 100 μL of the test bacterial suspension prepared in step 1 to each well. The final concentrations in the wells are 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0, 64.0, or 128.0 μg / mL. Incubate statically at 37 °C for 16 - 20 h. Observe the lowest concentration in the well that inhibits bacterial growth, which is the MIC. Set negative control wells in the 96-well plate, and add 100 μL of MH broth medium to each negative control well.

[0053] The analysis results are shown in Figure 1 a. The results show that nitrofurantoin exhibits cross-sensitivity to tigecycline-resistant Escherichia coli EC25922 and Salmonella SE13076 evolved in the laboratory.

[0054] After gene editing, the ACGCC at positions 1618 - 1622 of the lon gene in Escherichia coli EC25922 was mutated to A, and the G at position 97 of the lon gene in Salmonella SE13076 was mutated to A, obtaining tigecycline-resistant strains EC25922-TIG and SE13076-TIG mediated by lon gene mutations.

[0055] The minimum inhibitory concentrations (MICs) of tigecycline and nitrofurantoin against tigecycline-resistant bacteria with lon gene mutations were detected using the microbroth dilution method (European Committee on Antimicrobial Susceptibility Testing, EUCAST, 2024).

[0056] The experimental results are shown in Table 2. The results show that the MICs of tigecycline against tigecycline-resistant Escherichia coli EC25922 and Salmonella SE13076 with lon gene mutations increased from 0.0625 μg / mL and 0.5 μg / mL to 8 μg / mL and 16 μg / mL, respectively; the MICs of nitrofurantoin against tigecycline-resistant Escherichia coli EC25922 and Salmonella SE13076 with lon gene mutations decreased from 16 μg / mL and 32 μg / mL to 4 μg / mL, respectively.

[0057] Table 2

[0058]

[0059] The Kirby-Bauer disk diffusion method was used to detect the in vitro antibacterial effect of nitrofurantoin against tigecycline-resistant bacteria mediated by lon gene mutation: The tigecycline-resistant bacteria mediated by lon gene mutation were suspended in LB liquid medium (components per liter: 10.0 g of tryptone, 5.0 g of yeast extract powder, 10.0 g of sodium chloride) to obtain a bacterial suspension with a bacterial concentration of 1×10 6 CFU / mL. A 90-mm petri dish was taken, and 16 mL of LB solid medium (components per liter: 10.0 g of tryptone, 5.0 g of yeast extract powder, 10.0 g of sodium chloride, 15.0 g of agar) was added. The 100-μL bacterial suspension to be tested prepared above was evenly spread on the LB solid medium. Finally, a sterile filter paper containing 20 μg of nitrofurantoin was placed. It was statically cultured at 37°C for 24 h, and the diameter of the inhibition zone without bacterial growth was measured.

[0060] The bactericidal curve was used to detect the in vitro bactericidal effect of nitrofurantoin against tigecycline-resistant bacteria mediated by lon gene mutation: The tigecycline-resistant bacteria mediated by lon gene mutation were suspended in LB liquid medium to obtain a bacterial suspension with a bacterial concentration of 1×10 6 CFU / mL. The bacteria were diluted 1000-fold in 5 ml of broth containing 256 or 512 μg / mL of nitrofurantoin (the concentration was 16 times the MIC of the antibiotic that produced cross-sensitivity to the original strain). Every hour, 50 μL of the sample was taken and serially diluted in 450 μL of 0.9% sterile physiological saline, and then dropped evenly on the sterile LB solid medium. It was statically cultured at 37°C for 16 h, and the viable bacterial load was measured.

[0061] The analysis results are shown in Figure 1 Figure b. Nitrofurantoin had good antibacterial and bactericidal activities against tigecycline-resistant Escherichia coli EC25922 and Salmonella SE13076 mediated by lon gene mutation in vitro.

[0062] Example 2 In vitro efficacy evaluation of the combined administration of nitrofurantoin and tigecycline in inhibiting the development of tigecycline resistance

[0063] Determination of mutant prevention concentration (MPC): Tigecycline and its combination with nitrofurantoin were added at different concentrations in MH solid medium (composition per liter: beef extract powder 6.0 g, soluble starch 1.5 g, acid hydrolysate casein 17.5 g, agar 17.0 g), and the final concentrations in the solid medium were 0, 1.0, 2.0, 4.0, 8.0, 16.0 times the minimum inhibitory concentration (the minimum inhibitory concentrations of tigecycline against Escherichia coli EC25922 and Salmonella SE13076 were 0.0625 μg / mL and 0.5 μg / mL respectively, and the minimum inhibitory concentrations of nitrofurantoin against Escherichia coli EC25922 and Salmonella SE13076 were 16 μg / mL and 32 μg / mL respectively). 100 μL of EC25922 and SE13076 with a concentration of 1.0×10 10 CFU were inoculated into the corresponding resistant MH solid medium. Incubate statically at 37 °C for 72 h, and the MPC of the antibiotic was determined as the lowest concentration that could inhibit the development of drug resistance.

[0064] After continuous in vitro passage of Escherichia coli EC25922 and Salmonella SE13076 under the stimulation of sub-inhibitory concentrations of tigecycline and its combination with nitrofurantoin, the speed of drug resistance acquisition by the strains under the two treatments was observed.

[0065] The analysis results are shown in Figure 2 . The results showed that the combination of tigecycline and nitrofurantoin could slow down the evolution of drug resistance of EC25922 and SE13076 to tigecycline.

[0066] Example 3 Evaluation of the in vivo effect of nitrofurantoin and its combination with tigecycline in controlling infections caused by tigecycline-resistant bacteria mediated by lon gene mutation

[0067] Greater wax moth infection model: 128 greater wax moth larvae (Tianjin Huiyude Biotechnology Co., Ltd., 300 mg) were taken and randomly divided into Escherichia coli EC25922 infection group, Salmonella SE13076 infection group, tigecycline-resistant Escherichia coli EC25922-TIG infection group mediated by lon gene mutation, and tigecycline-resistant Salmonella SE13076-TIG infection group mediated by lon gene mutation. Each group was further divided into CON control group, tigecycline monotherapy group, nitrofurantoin monotherapy group, and nitrofurantoin and tigecycline combination therapy group, with 8 larvae in each subgroup. By adding 10 μL of bacterial suspension (1×10 6The larvae were infected by injecting 10 μL of the bacterial suspension (1×10⁷ CFU) into the second-to-last right leg of the larvae. One hour later, 10 μL of PBS (0.01 M, pH = 7.2), 2 mg / kg tigecycline, 8 mg / kg nitrofurantoin, or a combination of 1 mg / kg + 4 mg / kg tigecycline and nitrofurantoin were used to treat the second-to-last left leg of the infected larvae. During the 7-day observation, the survival status of the greater wax moth larvae was examined and recorded.

[0068] Mouse intraperitoneal infection model: 96 female ICR mice (Comparative Medicine Center of Yangzhou University, 6 - 8 weeks old, body weight 20 ± 2 g) were randomly divided into Escherichia coli EC25922 infection group, Salmonella SE13076 infection group, tigecycline-resistant Escherichia coli EC25922-TIG infection group mediated by lon gene mutation, and tigecycline-resistant Salmonella SE13076-TIG infection group with lon gene mutation. Each group was further divided into a CON control group, a tigecycline monotherapy group, a nitrofurantoin monotherapy group, and a combined treatment group of nitrofurantoin and tigecycline, with 6 mice in each subgroup. After 3 days of adaptive feeding with free access to food and water, the mice were fasted and water-deprived for 12 h before infection. A mouse infection model was established by injecting 200 μL of the bacterial suspension (5×10 8 CFU) into the left side of the mouse peritoneal cavity. One hour after infection, 200 μL of PBS (CON), 2 mg / kg tigecycline (TIG), 8 mg / kg nitrofurantoin (NIT), or a combination of 1 mg / kg + 4 mg / kg tigecycline and nitrofurantoin (T + N) were injected into the right side of the mouse peritoneal cavity. After 48 h of treatment, the mice were sacrificed by cervical dislocation, and the livers, spleens, and kidneys of the infected mice were taken. The organ colony counts were performed using LB agar plates, and the organ lesions were observed by HE staining.

[0069] The analysis results are shown in Figure 3 . The results showed that compared with the survival rate of the greater wax moth in the CON control group, the survival rates of the greater wax moths infected with EC25922 and SE13076 were significantly enhanced by tigecycline and the combined administration of tigecycline and nitrofurantoin ( Figure 3 c), while the survival rates of the greater wax moths infected with EC25922-TIG and SE13076-TIG were significantly enhanced by nitrofurantoin and the combined administration of nitrofurantoin and tigecycline ( Figure 3 d); compared with the CON control group of mice, the bacterial loads in the organs of the mice infected with EC25922 and SE13076 were significantly reduced by tigecycline and the combined administration of tigecycline and nitrofurantoin ( Figure 3 e, Figure 3 g) and the inflammatory changes in their organs were alleviated ( Figure 3 f), while the bacterial loads in the organs of the mice infected with EC25922-TIG and SE13076-TIG were significantly reduced by nitrofurantoin and the combined administration of nitrofurantoin and tigecycline ( Figure 3 e, Figure 3g) and reduce the inflammatory changes in its organs Figure 3 f), indicating that the combination therapy of nitrofurantoin and tigecycline can effectively relieve the burden of Galleria mellonella and mice infected with tigecycline-resistant bacteria mediated by lon gene mutation.

[0070] Example 4 Screening of antibiotics with cross-sensitivity produced by tet(X4)-mediated tigecycline-resistant bacteria, and in vivo evaluation of the effect of combined administration of nitrofurantoin and tigecycline on controlling infections caused by tet(X4)-mediated tigecycline-resistant bacteria

[0071] The minimum inhibitory concentration (MIC) of 46 antibiotics against tet(X4)-mediated tigecycline-resistant bacteria was detected by the microbroth dilution method (European Committee on Antimicrobial Susceptibility Testing, EUCAST, 2024).

[0072] The experimental results are shown in Table 3. The results showed that the minimum inhibitory concentration of tigecycline against Dh5α-pUC19-tet(X4) increased from 0.25 μg / mL of Dh5α-pUC19 to 16 μg / mL; the minimum inhibitory concentration of nitrofurantoin against Dh5α-pUC19-tet(X4) decreased from 1 μg / mL of Dh5α-pUC19 to 0.25 μg / mL.

[0073] Table 3

[0074]

[0075] The in vitro bactericidal effect of the combination of nitrofurantoin and tigecycline on tet(X4)-mediated tigecycline-resistant bacteria B3-1 was detected by the bactericidal curve: tet(X4)-mediated tigecycline-resistant bacteria B3-1 was suspended in LB liquid medium to obtain a bacterial suspension with a bacterial concentration of 1×10 6 CFU / mL. The bacteria were diluted 1000-fold in 5 ml of broth containing 32 μg / mL of tigecycline and nitrofurantoin. 50 μL of the sample was taken every hour and serially diluted in 450 μL of 0.9% sterile physiological saline, and then dropped evenly on the sterile LB solid medium at a uniform dose. Incubate statically at 37 °C for 16 h, and determine the viable bacterial load.

[0076] Rat skin wound infection model: Twelve female Wistar rats (Comparative Medicine Center of Yangzhou University, 6-8 weeks old, weighing 60±10 g) were randomly divided into a CON control group, a tigecycline monotherapy group, a nitrofurantoin monotherapy group, and a combination therapy group of tigecycline and nitrofurantoin, with 3 rats in each group. They were adaptively fed for 3 days, during which they had free access to food and water, and were fasted and watered for 12 h before infection. A 2-cm2 wound. A rat skin wound infection model was established by dropping 100 μL of Escherichia coli B3-1 bacterial suspension (1×10 6 CFU) onto the wound. One hour after infection, 100 μL of PBS (CON), 20 mg / kg tigecycline (TIG), 10 mg / kg nitrofurantoin (NIT), or 20 mg / kg + 10 mg / kg combination of tigecycline and nitrofurantoin (T+N) were dropped onto the wounds of the rats, respectively. The body weight of the rats was monitored daily, and the wounds were photographed every 3 days to observe the degree of healing for 12 days. On the 12th day, the rats were euthanized. The wound tissues at the infected sites of the rats were taken, and colony counting was performed using LB agar plates. The wound healing was observed by HE staining and Masson staining, and the levels of inflammatory factors in the wounds at the infected sites were measured by enzyme-linked immunosorbent assay (ELISA). An ELISA kit (Mlbio, Shanghai, China) was used to quantify the levels of cytokines (IL-1β, IL-6, TNF-α, INF-γ, IL-4, IL-10).

[0077] Determination of inflammatory factor levels in the wounds at the infected sites by ELISA: An ELISA kit for detecting inflammatory factors was used, including the pro-inflammatory factor IL-1β kit (Mlbio, ml106733), IL-6 kit (Mlbio, ml098430), TNF-α ELISA kit (Mlbio, ml002095), IFN-γ ELISA kit (Mlbio, ml002277), and anti-inflammatory factor IL-4 ELISA kit (Mlbio, ml064310) and IL-10 ELISA kit (Mlbio, ml037888). Three replicate wells were set for each group. 50 μL of the diluted standard product and 50 μL of the ground sample of the tissue to be tested were added into the reaction wells. Subsequently, 50 μL of the detection antibody labeled with horseradish peroxidase (HRP) (HRP-IL-1β, HRP-IL-6, HRP-TNF-α, HRP-IFN-γ, HRP-IL-4, HRP-IL-10) was immediately added. The membrane plate was covered and incubated at 37 °C for 1 h. The liquid in the wells was discarded, each well was filled with the diluted 1× washing buffer, the washing liquid was shaken off, and the wells were patted dry with absorbent paper. This operation was repeated 5 times. 50 μL of each of the substrate A and B solutions in the respective ELISA kits for detecting inflammatory factors were added to each well, gently shaken and mixed evenly, and incubated at 37 °C in the dark for 10 min. Subsequently, 50 μL of the stop solution was added to each well, and the absorbance at a wavelength of 450 nm was measured using an enzyme-linked immunosorbent assay reader. With the concentration of the standard as the abscissa and the OD value as the ordinate, a standard curve was plotted, and the corresponding concentration was obtained from the standard curve according to the OD value of the sample.

[0078] The analysis results are shown in Figure 4The results showed that nitrofurantoin exhibited cross-sensitivity against tet(X4)-mediated tigecycline-resistant Escherichia coli ( Figure 4 a). In the in vitro efficacy verification, the combination of tigecycline and nitrofurantoin significantly eliminated tet(X4)-mediated tigecycline-resistant Escherichia coli ( Figure 4 b). In the rat skin wound infection model, without affecting the body weight of the rats ( Figure 4 d), the combination of tigecycline and nitrofurantoin significantly promoted the healing of skin wounds ( Figure 4 e, Figure 4 f), reduced the bacterial load in the skin wounds ( Figure 4 g), simultaneously inhibited the expression of pro-inflammatory factors IL-1β, IL-6, TNF-α, and INF-γ, and promoted the expression of anti-inflammatory factors IL-4 and IL-10 ( Figure 4 h), and alleviated inflammation ( Figure 4 i). This indicates that the combination of tigecycline and nitrofurantoin can treat infections caused by tet(X4)-mediated tigecycline-resistant Escherichia coli both in vitro and in vivo.

[0079] In summary, the combination of tigecycline and nitrofurantoin can be considered as a potential treatment option for tigecycline-resistant bacterial infections, with significant bactericidal effects both in vivo and in vitro, and can significantly inhibit the inflammatory response caused by tigecycline-resistant bacterial infections.

Claims

1. Use of nitrofurantoin or a pharmaceutical composition or compound preparation containing the active ingredient of nitrofurantoin in at least one of the following: (a) Preparation of products or medicines for inhibiting or killing the activity of tigecycline-resistant bacteria; (b) preparing products or medicines for use against infections caused by tigecycline-resistant bacteria; (c) Preparation of products or medicines for the prevention and / or treatment of infectious diseases mediated by tigecycline-resistant bacteria.

2. The use according to claim 1, characterized in that: The composition containing nitrofurantoin as an effective ingredient also includes tigecycline.

3. The use according to claim 1 or 2, characterized in that: The tigecycline-resistant bacteria are lon Tigecycline resistance mediated by gene mutation or tet (X4) Positive pathogens.

4. The use according to claim 3, characterized in that: Said tet (X4) Positive pathogens are carriers tet (X4) Bacteria with drug resistance genes.

5. The use according to claim 1 or 2, characterized in that: The product for inhibiting or killing the activity of tigecycline-resistant bacteria includes tigecycline-resistant bacteria inhibitors or bactericides.

6. The use according to claim 1 or 2, characterized in that: The preparation of the product or medicine for preventing and / or treating infectious diseases mediated by tigecycline-resistant bacteria includes preparing a product or medicine for reducing the expression level of pro-inflammatory factors IL-1β, IL-6, TNF-α and / or INF-γ.

7. The use according to claim 1 or 2, characterized in that: The preparation of the product or medicine for preventing and / or treating infectious diseases mediated by tigecycline-resistant bacteria includes the preparation of the product or medicine for increasing the expression level of the anti-inflammatory factors IL-4 and / or IL-10.

8. The use according to claim 2, characterized in that: The mass ratio of nitrofurantoin to tigecycline in the composition containing nitrofurantoin as an effective ingredient is 0.5 to 4:

1.

9. A pharmaceutical composition or compound preparation containing nitrofurantoin as an active ingredient, characterized in that: The pharmaceutical composition or compound preparation comprises nitrofurantoin and tigecycline.

10. The pharmaceutical composition or compound preparation containing nitrofurantoin as an effective ingredient according to claim 8, characterized in that: The mass ratio of nitrofurantoin to tigecycline is 0.5 to 4:1.

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