Application of pyridoxal phosphate in preparation of drugs or antibiotic synergists for treating bacterial infectious diseases

Abstract

The invention discloses an application of pyridoxal phosphate in preparation of drugs or antibiotic synergists for treating bacterial infectious diseases. According to the invention, the pyridoxal phosphate (the active form of vitamin B6) has a relatively strong drug-resistant pathogenic bacterium resisting effect in vivo and in vitro when being combined with the antibiotic polymyxin for use for the first time, and the curative effect of the polymyxin can be effectively improved; when the pyridoxal phosphate and the polymyxin are used for treating gram-negative bacterium infectious diseases, the survival rate of greater wax moths and mice can be remarkably improved and the inflammation level of the organism can be reduced through combined medication of the pyridoxal phosphate and the polymyxin. The invention provides a new thought for the control of drug-resistant pathogenic bacteria, and also provides a new strategy for the prevention and treatment of increasingly serious gram-negative bacterial infection.

Description

Technical Field

[0001] The invention relates to application of pyridoxal phosphate in preparing medicines or antibiotic synergists for treating bacterial infectious diseases, and belongs to the technical field of medicines. Background Art

[0002] The successful application of antibiotics has laid a milestone in modern medicine. The discovery and widespread use of antibiotics have not only saved countless lives, but also played a vital role in the prevention and control of bacterial diseases in livestock and poultry farming, ensuring a stable supply of animal-derived food. However, the emergence of antibiotic-resistant strains has seriously weakened the effectiveness of this traditional treatment strategy. The problem of antibiotic resistance has become one of the biggest challenges facing global public health. Polymyxin E is a cationic polypeptide antibiotic that is widely considered to be the ultimate choice for treating multidrug-resistant bacterial infections, especially infections caused by carbapenem-resistant Enterobacteriaceae. Mechanistically, polymyxin is a bactericidal antibiotic that interacts with the lipopolysaccharide (LPS) of the outer membrane of Gram-negative bacteria, destroys the structure of the membrane and changes its permeability, leading to the leakage of cell contents, thereby causing bacterial death. It is currently believed that there are four main mechanisms of polymyxin resistance: modification of the outer membrane lipopolysaccharide structure; activation of the broad-spectrum efflux pump system; degradation of drug proteins; and heterogeneous resistance of bacteria. Among them, the mcr plasmid-mediated resistance mechanism has become a key factor in the spread of polymyxin E resistance. The mcr gene encodes a phosphoethanolamine transferase protein, which enables bacteria to reduce the binding of polymyxin to the cell membrane by changing the structure of LPS, thereby significantly reducing its antibacterial effect. Although polymyxin is widely used as a last line of defense to treat severe infections, especially against drug-resistant Gram-negative bacteria, the widespread use of this antibiotic, especially in animal husbandry, has led to the accumulation and spread of mcr-related resistance genes. In 2015, the mcr-1 gene was first reported to appear in clinical bacteria, marking the beginning of the cross-species spread of mcr-mediated resistance mechanisms. The mcr-1 gene is rapidly transferred horizontally in bacterial populations through plasmids, which greatly reduces the clinical efficacy of polymyxin, especially in Enterobacteriaceae. In 2020, mcr-1 and mcr-3 genes have been found to appear at high frequencies in animal and food-derived bacteria, and even in some areas, the detection rate of pig-derived bacteria is as high as more than 50%. Due to the rapid spread of the mcr gene and its ability to spread rapidly through horizontal gene transfer, it has brought severe challenges to global antibiotic treatment. Although the research and development of new antibiotics has been advancing, only more than 20 new antibiotics have entered clinical use worldwide since the 21st century, and bacterial resistance to new drugs has rapidly developed. Although new antibiotics can temporarily control the spread of drug-resistant bacteria, after long-term use, bacteria will soon develop resistance to these drugs, leading to the emergence of more multidrug-resistant bacteria with a wide spectrum of resistance and even super-resistant bacteria. Therefore, relying solely on the research and development of new drugs is no longer the only way to solve the problem of drug resistance. In this case, it becomes increasingly important to improve the efficacy of existing antibiotics, especially through sensitization strategies.

[0003] Pyridoxal phosphate (PLP), as the biologically active form of vitamin B6, is an essential cofactor for 238 different enzyme reactions, involved in multiple biological processes such as amino acid, sugar and lipid metabolism. The recommended daily intake for adults is 1.4 mg, which can be supplemented in the form of tablets or injections. Studies have found that about 1.5% of the genes in prokaryotes are responsible for encoding PLP-dependent enzymes, and these enzymes are associated with a reduced risk of cardiovascular disease, venous thrombosis, diabetes and inflammatory bowel disease (IBD). Therefore, PLP-dependent enzymes not only show potential value in disease treatment, but are also important targets for antibiotics.

[0004] At the same time, the study also showed that changes in the bacterial growth environment have a significant impact on their growth and survival, and excessive metabolic activity can even directly cause death. This suggests the great potential of enhancing the bactericidal efficacy of antibiotics through metabolic reprogramming. This strategy, combined with the mcr plasmid-mediated drug resistance mechanism, may provide an effective way to develop new treatments, inhibit the spread of polymyxin-resistant bacteria, and improve the success rate of clinical treatment. Summary of the invention

[0005] Purpose of the invention: The first purpose of the present invention is to provide the use of pyridoxal phosphate in the preparation of a drug or antibiotic synergist for treating bacterial infectious diseases. The second purpose of the present invention is to provide a pharmaceutical composition for treating bacterial infectious diseases.

[0006] Technical solution: The present invention provides an application of a biologically active form of vitamin B6 in the preparation of a drug or antibiotic enhancer for treating bacterial infectious diseases, wherein the biologically active form of vitamin B6 is pyridoxal phosphate.

[0007] Furthermore, the molecular formula of the pyridoxal phosphate is C 8 H 10 NO 6 P.

[0008] Furthermore, the bacteria are Gram-negative pathogens.

[0009] Furthermore, the Gram-negative pathogen is a drug-resistant bacterium carrying the mcr-1, mcr-3 or mcr-8 gene.

[0010] Furthermore, the bacteria are one or more of E. coli DH5α (pUC19-mcr-1), E. coli G92 mcr-1, S. enterica SC2016090mcr-3, K. pneumoniae D120 mcr-8, etc.

[0011] Furthermore, the antibiotic is a polypeptide antibacterial drug.

[0012] Furthermore, the polypeptide antibacterial drug is polymyxin.

[0013] Furthermore, the drug or antibiotic synergist for bacterial infectious diseases is in the form of freeze-dried powder injection or injection. The pyridoxal phosphate supplement is in the form of capsule, tablet or injection.

[0014] The present invention also provides a pharmaceutical composition for treating bacterial infectious diseases, wherein the pharmaceutical composition comprises pyridoxal phosphate in the active form of vitamin B6 and polymyxin.

[0015] Furthermore, the final concentration of the pyridoxal phosphate is 5 to 20 mM.

[0016] Furthermore, the final concentration of the polymyxin is 0.125-8 μg / mL.

[0017] Furthermore, the mass ratio of pyridoxal phosphate to polymyxin is 0.1 to 40:1.

[0018] 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 when used in combination with the antibiotic polymyxin, it has a strong anti-Gram-negative pathogen effect both in vivo and in vitro, which is specifically manifested in that the amount of bacteria can be effectively reduced; when used to treat diseases infected with E. coli G92 mcr-1, the combined use of drugs can significantly increase the survival rate of greater wax moth and mice, and reduce the body's inflammation level. Current studies have found that the use of pyridoxal phosphate in combination with polymyxin can increase the sensitivity of drug-resistant bacteria to antibiotics. This discovery provides a new perspective for the development of new resistance prevention and control strategies, and also provides a new method for the prevention and control of increasingly serious Gram-negative bacterial infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is an in vitro evaluation of the effectiveness of pyridoxal phosphate in enhancing antibiotics, where CON means blank control.

[0020] Figure 2 This is an in vitro evaluation of the effect of pyridoxal phosphate on enhancing the effectiveness of antibiotics in various mcr-positive strains. In Figure b, CON means blank control, (a) is Escherichia coli DH5α (pUC19-mcr-1), (b) is Escherichia coli G92 (mcr-1), (c) is Salmonella SC2016090 (mcr-3), and (d) is Klebsiella pneumoniae D120 (mcr-8).

[0021] Figure 3 These are the pattern diagrams and result diagrams of the combined treatment of pyridoxal phosphate and polymyxin for the infection of Glechoma wax moth and the abdominal infection of mice. Among them, PBS means blank control. DETAILED DESCRIPTION

[0022] 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.

[0023] The LB broth medium used in the examples is an aqueous solution containing 10 g / L tryptone, 5 g / L yeast extract powder and 10 g / L NaCl; the MHB broth medium is an aqueous solution containing 2 g / L beef powder, 1.5 g / L soluble starch and 17.5 g / L acid hydrolyzed casein; the M9CA medium is an aqueous solution containing 2.0 g / L casamino acids, 1.5 g / L NaCl and 10 g / L broth. 2 HPO 4 6.8 g / L, KH 2 PO 4 3.0 g / L, NH 4 Cl 1.0g / L, NaCl 0.5g.

[0024] The specific information of the strains used in the examples and their sources or literature is shown in Table 1.

[0025] Table 1

[0026] Test strains Source or literature Escherichia coli DH5α (pUC19-mcr-1) Reference 1 Escherichia coli G92 (mcr-1) Reference 1 Salmonella SC2016090 (mcr-3) Reference 2 Klebsiella pneumoniae D120 (mcr-8) Reference 3

[0027] Note:

[0028] Document 1 is T.Xu et al.A Dietary Source ofHigh Level ofFluoroquinoloneTolerance in mcr-Carrying Gram-Negative Bacteria.Research.Research, (2023).

[0029] Document 2 is R.Li et al., The genomic epidemiology ofmcr-positive Salmonellaenterica in clinical patients from 2014to 2017in Sichuan,China and global epidemiological features.JInfect, (2022).

[0030] Document 3 is X.Yang et al., Emergence ofmcr-8.2-bearingKlebsiellaquasipneumoniae of animal origin.J Antimicrob Chemother, (2019).

[0031] Example 1 In vitro evaluation of pyridoxal phosphate to enhance the effectiveness of antibiotics

[0032] The metabolites were combined with the antibiotic polymyxin to replenish mcr-positive resistant bacteria, and substances that can effectively enhance the effectiveness of antibiotics were screened. The survival rate of bacteria under different treatments was determined by the drop plate counting method. A single colony of Escherichia coli G92 (mcr-1) was picked and cultured overnight in a shaker at 37°C, with the culture condition of 200r / min, and then diluted 1:100 into fresh MH broth, expanded for 4h to the logarithmic phase, and then washed and resuspended with M9CA basal culture medium. The blank group was set as the bacterial solution resuspended with M9CA alone, and the experimental group included the addition of polymyxin (COL, 4μg / mL) as a single drug group, and polymyxin (COL, 4μg / mL) was mixed with different metabolites (Fructose-1,6-diphosphate (fructose-1,6-diphosphate, 10mM), Uric acid(uric acid, 10mM), L-ornithine(L-ornithine, 10mM), Citrulline(citrulline, 10mM), N-acetyl-5-hydroxytryptamine(N-acetyl-5-hydroxytryptamine, 10mM), L-tryptophan(L-tryptophan, 10mM), Pyridoxal5'-phosphate(pyridoxal 5'-phosphate, 10mM), Tyramine(tyramine, 10mM), Cytosine(cytosine, 10mM), L-homoserine(L-homoserine, 10mM), N,N-dimethylglycine(N,N-dimethylglycine, 10mM), Aspartic Acid (aspartic acid, 10mM) was added at the same time as the combined use group, and the action time was 4h. Then, 50μL of the bacterial solution of the single drug group or the combined use group was diluted tenfold, and 50μL of the bacterial solution was spread on the LB plate and cultured in a 37℃ incubator overnight. After 18h, the number of colonies was counted, the bacterial count of each group was calculated, and three biological replicates were set. The results showed that the bactericidal effect was most significant when polymyxin was used in combination with pyridoxal phosphate ( Figure 1b). The MIC of pyridoxal phosphate against Escherichia coli G92 (mcr-1) was 62.5 μg / mL. In order to further evaluate the scope of application of the sensitization effect of pyridoxal phosphate, polymyxin (COL, 4 μg / mL) and pyridoxal phosphate (PLP, 5, 10, 20 mM) were added simultaneously as a combined use group. The results showed ( Figure 1 c) When used in combination with polymyxin, 20 mM is the optimal concentration for pyridoxal phosphate supplementation.

[0033] Escherichia coli G92 (mcr-1) was cultured overnight and diluted with MHB broth at 1:1000. The absorbance of the bacterial solution at 600 nm was adjusted to 0.1. Polymyxin (1 / 2 times MIC) with or without pyridoxal phosphate (10 mM) was added and cultured at 37°C with shaking at 200 rpm for 12 h. The absorbance of the bacterial solution at 600 nm was measured every 1 h. The bacterial growth curve results showed that the combination of pyridoxal phosphate and polymyxin limited bacterial proliferation ( Figure 1 d). To determine the effect of PLP on the evolution of polymyxin resistance, E. coli G92 (mcr-1) was serially subcultured in medium containing polymyxin alone (1 / 2 times MIC) or in combination with pyridoxal phosphate (10 mM) for 20 days, for a total of 40 generations, and the MIC was measured every 5 generations. The results show ( Figure 1 e), in the presence of colistin, the MIC value increased 8-fold. In contrast, a 2-fold decrease in colistin MIC was observed in the colistin and pyridoxal phosphate combination group, indicating that the addition of PLP effectively prevented the evolution of colistin resistance.

[0034] Example 2 In vitro evaluation of pyridoxal phosphate for enhancing the effectiveness of antibiotics in various multidrug-resistant bacteria

[0035] To further evaluate the scope of application of the sensitization effect of pyridoxal phosphate, we selected the engineered bacteria E. coli DH5α (pUC19-mcr-1) and clinical E. coli G92 (mcr-1), S. enterica SC2016090 (mcr-3), and K. pneumoniae D120 (mcr-8) for the same survival rate determination experiment. At this time, the selected polymyxin concentration was 2 times the MIC, and the blank control group was set as the bacterial solution with only polymyxin added after resuspending with M9CA. The concentrations of pyridoxal phosphate were 5, 10, and 20 mM. The results showed that compared with the use of colistin alone, pyridoxal phosphate supplementation increased the sensitivity of multidrug-resistant bacteria carrying the mcr resistance gene to antibiotics, and the effect was particularly significant when the concentration of pyridoxal phosphate was 20 mM ( Figure 2 a).

[0036] The bacteria were stained with a live / dead fluorescent staining probe.

[0037] (pUC19-mcr-1), E. coli G92 (mcr-1), S. enterica SC2016090 (mcr-3) and K. pneumoniae D120 (mcr-8) were diluted in MHB medium for 4 h and then resuspended in M9CA medium. Then, the bacteria were co-cultured with polymyxin (2 times MIC) and PLP (10 mM) alone or in combination. Fluorescent probes SYTO 9 (2.5 μM) and PI (5 μM) were added to the bacterial solution and incubated in the dark for 30 min at room temperature. CytExpert flow cytometer was used for measurement. Flow cytometry results showed that pyridoxal phosphate supplementation increased the mortality of mcr-positive bacteria compared with colistin alone ( Figure 2 b). Laser scanning confocal microscopy was used to distinguish live bacteria (green) from dead bacteria (red). The results were consistent with those of flow cytometry ( Figure 2 c). In conclusion, pyridoxal phosphate can reverse the resistance of mcr-positive resistant bacteria to polymyxin.

[0038] Example 3 Evaluation of the effect of combined treatment with pyridoxal phosphate and polymyxin in animal infection models

[0039] G. mellonella infection model: Prepare enough G. mellonella larvae (Hui Yu De Biotechnology Company, body length about 2 cm) and divide them into 3 groups, 8 in each group. Inject 1.0×10 6 CFU of E.coli G92 (mcr-1) (10 μL per larva). 1 h after infection, 10 μL of PBS was injected into the left caudal pedicle of the larvae in the "PBS" group, 10 μL of polymyxin (5 mg / kg) was injected into the right second-to-last caudal pedicle of the larvae in the "COL" group, and 10 μL of a mixture of polymyxin (5 mg / kg) and pyridoxal phosphate (0.25 mg / kg) was injected into the right second-to-last caudal pedicle of the larvae in the "COL+PLP" group. The survival rate of the greater wax moth larvae was continuously observed and recorded for 5 days.

[0040] Mouse intraperitoneal infection model: Prepare enough female BALB / c mice (Comparative Medicine Center of Yangzhou University, 6-8 weeks old), and inject the mice intraperitoneally with PBS (n=12, 300 μL) or 10 mg / kg PLP (n=6) for 5 consecutive days. On the 5th day, inject 5.0×10 8CFU of E.coli G92 (100 μL per mouse). One hour after infection, the mice in the PBS group were randomly divided into a blank control group (n=6) and an antibiotic group (n=6, COL group). The blank control group received a single intraperitoneal injection of 300 μL of PBS, and the antibiotic treatment group and the PLP group were given a dose of 2 mg / kg of polymyxin. The survival rate of the mice was monitored for 5 days. To determine the bacterial load in mouse organs, mice were treated in the same manner and all mice were euthanized 48 hours after infection. The liver and kidney were removed aseptically and divided into two parts for CFU estimation, HE staining and ELISA detection.

[0041] ELISA method was used to determine the level of inflammatory factors in serum: an ELISA detection kit for inflammatory factors was used, including pro-inflammatory factors IL-1β, IL-6, TNF-α, IFN-γ and anti-inflammatory factors IL-4 and IL-10 (Enzyme-linked Biologicals, ml098416, ml098430, ml002095, ml002277, ml064310, ml037873). First, prepare the standard and serum samples to be tested. Each sample was repeated three times, and 50 μL of sample was added to each well. 50 μL of antibodies (HRP-IL-1β, HRP-IL-6, HRP-TNF-α, HRP-IFN-γ, HRP-IL-4, HRP-IL-10) that had been pre-labeled with biotin were added to each well containing the sample. The experimental operation required gently shaking the well plate to ensure that the reaction was evenly mixed, and then the well plate was placed in a constant temperature environment of 37°C for incubation for 1 hour. After the incubation is complete, the liquid in the well needs to be completely poured out and each well is washed with sufficient washing solution. After each wash, gently shake for 30 seconds to remove unbound substances, and then tap the bottom of the well with absorbent paper to ensure cleanliness. This washing process needs to be repeated three times to ensure that all non-specific binding is removed as much as possible. After the washing step, 80 μL of HRP (horseradish peroxidase) labeled with affinity streptavidin is added to each well, and after gently shaking again to mix, it is incubated again at 37°C for 30 minutes. After incubation, the previous washing steps are repeated to ensure that all unreacted enzyme markers are removed. Next, 50 μL of substrate A and substrate B are added to each well at the same time under dark conditions. These substrates react with enzyme-labeled antibodies to produce color changes. After incubation at 37°C in the dark for 10 minutes, the reaction is stopped by adding 50 μL of stop solution. Finally, the absorbance of each well is measured at a wavelength of 450 nm using an enzyme reader. By comparing the absorbance of the sample with the pre-prepared standard curve, the concentration of specific inflammatory factors in the sample is calculated.

[0042] The experimental results are shown in Figure 3The results of the G. mellonella infection model showed that the survival rate of infected larvae treated with PBS (PBS group) was 0% within 5 days after infection, and the survival rate of infected larvae treated with polymyxin (COL group) was 25% within 5 days after infection. In contrast, the combined treatment (COL+PLP group) achieved a survival rate of 75%, which was significantly higher than that of single drug treatment ( Figure 3 c).

[0043] The mouse intraperitoneal infection model showed that the survival rate of mice treated with polymyxin alone (COL group) was 0% within 5 days after infection. In contrast, the combined treatment (COL+PLP group) achieved a survival rate of 66%, which was significantly higher than that of the single drug treatment ( Figure 3 d). Supplementation of pyridoxal phosphate can also reduce the pathogen load in the liver and kidney of mice ( Figure 3 e), reducing the level of inflammation. At the same time, compared with the single drug treatment group, the pro-inflammatory factors IL-1β, IL-6, TNF-α, and IFN-γ in the combined use group all decreased, and the anti-inflammatory factor IL-4 was upregulated, indicating that the immune system played an active role in the early stage of infection ( Figure 3 f). In line with this, hematoxylin and eosin (HE) staining was performed to evaluate liver and kidney damage. In the liver or kidney, pathological lesions such as inflammatory cell infiltration were the most severe in the PBS group, and polymyxin treatment alleviated some of the lesions, while the lesions in the polymyxin and pyridoxal phosphate combined treatment group basically disappeared.

[0044] In summary, the combined use of pyridoxal phosphate and polymyxin can exert potential antibacterial effects against MCR-positive bacteria both in vivo and in vitro.

Claims

1. Use of a biologically active form of vitamin B6 in the preparation of a drug for treating bacterial infectious diseases or an antibiotic synergist, characterized in that: The biologically active form of vitamin B6 is pyridoxal phosphate.

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

3. The use according to claim 2, characterized in that: The Gram-negative pathogen is a drug-resistant bacterium carrying the mcr-1, mcr-3 or mcr-8 gene.

4. The use according to claim 1, characterized in that: The bacteria are one or more of E. coli DH5α (pUC19-mcr-1), E. coli G92 mcr-1, S. enterica SC2016090mcr-3, K. pneumoniae D120 mcr-8, etc.

5. The use according to claim 1, characterized in that: The antibiotic is a polypeptide antibacterial drug.

6. The use according to claim 5, characterized in that: The polypeptide antibacterial drug is polymyxin.

7. A pharmaceutical composition for treating bacterial infectious diseases, characterized in that: The pharmaceutical composition comprises pyridoxal phosphate in the active form of vitamin B6 and polymyxin.

8. The pharmaceutical composition according to claim 7, characterized in that The final concentration of the pyridoxal phosphate is 5-20 mM.

9. The pharmaceutical composition according to claim 7, characterized in that The final concentration of the polymyxin is 0.125-8 μg / mL.

10. The pharmaceutical composition according to claim 7, characterized in that The mass ratio of pyridoxal phosphate to polymyxin is 0.1 to 40:1.

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