Use of phenolic quinolines or pharmaceutically acceptable salts in the preparation of a medicament for the treatment of drug resistant bacteria and in the preparation of a medicament for the treatment of pneumonia

Abstract

The present application relates to a kind of naphtholquinone or pharmaceutically acceptable salt in the application of drug-resistant bacteria-resistant drug and the preparation of drug for treating pneumonia, naphtholquinone is the antibacterial drug of inhibiting drug-resistant bacteria reproduction or killing drug-resistant bacteria, by the proposal of phenol quinoline or pharmaceutically acceptable salt to solve the technical problems existing in the prior art, to solve the technical problems existing in the prior art, bauman acinetobacter LAC-4 mainly produces drug resistance to sulfonamides, in clinic, the scheme for treating bauman acinetobacter LAC-4 infection is very limited.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the use of a phenolquinone or a pharmaceutically acceptable salt in the preparation of drugs for treating drug-resistant bacteria and drugs for treating pneumonia. Background Technology

[0002] Antimicrobial resistance has become a major challenge in global public health. The emergence and prevalence of multidrug-resistant (MDR), extensively drug-resistant (XDR), and even pan-drug-resistant (PDR) bacteria pose a significant threat to human health. Currently, drug-resistant bacteria, represented by ESKAPE pathogens, pose a major threat to global human health. It is estimated that as of 2019, AMR (antimicrobial resistance) bacterial pathogens have directly caused approximately 1.3 million deaths worldwide. ESKAPE pathogens include Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii LAC-4, Pseudomonas aeruginosa, and Enterobacteriaceae.

[0003] Acinetobacter baumannii LAC-4 is a well-known Gram-negative opportunistic pathogen that can cause serious infections, particularly in immunocompromised populations such as the elderly, newborns, postoperative patients, and other critically ill patients, making it difficult to control and treat. Furthermore, it can cause a range of infections, including bacteremia, urinary tract infections, skin and soft tissue infections, and surgical site infections. In recent years, the annual prevalence of Acinetobacter baumannii LAC-4 has been steadily increasing and is recognized as a major contributor to nosocomial infections worldwide. Initially, Acinetobacter baumannii LAC-4 was primarily resistant to sulfonamides. However, with the escalation of antibiotic use, its resistance spectrum has expanded to include a variety of key antimicrobial agents, including cephalosporins, aminoglycosides, quinolones, and carbapenems. Clinically, treatment options for Acinetobacter baumannii LAC-4 infections are very limited, primarily including tigecycline, polymyxin, and linezolid. Unfortunately, strains resistant to these antibiotics have emerged in the past decade. Globally, approximately 45% of Acinetobacter baumannii LAC-4 isolates exhibit multidrug resistance (MDR), and the prevalence of Acinetobacter baumannii LAC-4 MDR is four times higher than that of other Gram-negative pathogens, including Klebsiella pneumoniae and Pseudomonas aeruginosa.

[0004] The global demand for novel antimicrobial therapies is urgent, necessitating increased efforts in drug discovery and development. However, the lengthy and challenging drug discovery and development process exacerbates the difficulty of delivering new therapies in a timely manner. Therefore, there is an urgent need to develop strategies to improve drug development efficiency and accelerate the introduction of effective drugs into clinical practice. Drug reuse (also known as drug repositioning) and drug combination therapy can significantly reduce development time and costs, and are key strategies for novel drug development.

[0005] Naphtholquin is an original antimalarial drug invented in my country in the last century. Pharmacological studies have shown that naphtholquin has a killing effect on various Plasmodium schizonts and gametophytes and tissue-stage protozoa of certain strains of Plasmodium, and is suitable for the treatment of falciparum malaria, vivax malaria, and drug-resistant malaria. Currently, there are no research reports on the use of naphtholquin alone or in combination with other drugs in the fight against drug-resistant bacteria.

[0006] Therefore, in view of the above problems, the present invention urgently needs to provide the application of phenolquinone or pharmaceutically acceptable salts in the preparation of drugs against drug-resistant bacteria and drugs for treating pneumonia. Summary of the Invention

[0007] The purpose of this invention is to provide the application of phenolquinone or pharmaceutically acceptable salts in the preparation of drugs against drug-resistant bacteria and drugs for treating pneumonia. By proposing the use of phenolquinone or pharmaceutically acceptable salts in the preparation of drugs against drug-resistant bacteria, this invention addresses the technical problem in the prior art that Acinetobacter baumannii LAC-4 is mainly resistant to sulfonamides, and that clinical treatment options for Acinetobacter baumannii LAC-4 infection are very limited.

[0008] This invention provides the use of naphtholquinone or a pharmaceutically acceptable salt in the preparation of drugs against drug-resistant bacteria, where naphtholquinone is an antibacterial drug that inhibits the growth of drug-resistant bacteria or kills drug-resistant bacteria.

[0009] Preferably, the drug-resistant bacteria include one of Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus, or Enterococcus faecalis.

[0010] Preferably, the naphtholquin or pharmaceutically acceptable salt is at least one selected from naphtholquin phosphate, hydrochloride, sulfate or maleate.

[0011] Preferably, the pharmaceutically acceptable salt of naphtholquin is selected from at least one of naphtholquin phosphate, naphtholquin hydrochloride, naphtholquin sulfate, or naphtholquin maleate;

[0012]

[0013] The above are the molecular structural formulas of naphtholquinone (Formula 1), naphtholquinone phosphate (Formula 2), naphtholquinone hydrochloride (Formula 3), naphtholquinone sulfate (Formula 4), and naphtholquinone maleate.

[0014] Preferably, the naphtholquin is used in combination with other antibacterial drugs.

[0015] Preferably, other antibacterial agents include pentamidine.

[0016] Preferably, the antibacterial drug is prepared as an oral dosage form, tablet, or capsule.

[0017] Preferably, the daily dose of the antibacterial drug for Acinetobacter baumannii LAC-4 is 10-20 mg / kg.

[0018] The present invention also provides the use of naphtholquinone or a pharmaceutically acceptable salt or solvate in the preparation of a medicament for treating pneumonia.

[0019] Preferably, the pneumonia is caused by drug-resistant bacteria; the drug-resistant bacteria include one of Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus, or Enterococcus faecalis.

[0020] The application of a phenolquinone or pharmaceutically acceptable salt provided by this invention in the preparation of drugs against drug-resistant bacteria and drugs for treating pneumonia has the following advantages compared with the prior art:

[0021] 1. Naphtholquin is an antibacterial drug that inhibits the growth of drug-resistant bacteria or kills drug-resistant bacteria.

[0022] 2. Naphthol quinone or pharmaceutically acceptable salts or solvates can be used to prepare drugs for treating pneumonia. Attached Figure Description

[0023] Figure 1 The results of antibacterial experiments on Acinetobacter baumannii using naphthol phosphate alone and in combination with pentamidine are shown; (a) is the time-bactericidal curve of naphthol phosphate, and (b) is the time-bactericidal curve of pentamidine combined with naphthol phosphate.

[0024] Figure 2 The results of toxicity tests on naphthol phosphate quinolone phosphate in A549 cells, Huh7 cells, and Vero cells;

[0025] Figure 3 Results of hemolysis of erythrocytes in A549, Huh7, and Vero cells by naphthol phosphate;

[0026] Figure 4 The antibacterial activity of naphthol phosphate against A549 cells infected with Acinetobacter baumannii;

[0027] Figure 5 The images show scanning electron microscope (SEM) and transmission electron microscope (TEM) images of Acinetobacter baumannii before and after treatment with naphthol phosphate. In the image, (a) is the SEM image, with the left image being the blank control sample and the right image being the test sample; and (b) is the TEM image, with the left image being the blank sample and the right image being the test sample.

[0028] Figure 6 The results of the assessment of damage to the cell membrane of Acinetobacter baumannii after treatment with naphthol phosphate include: (a) bacterial membrane potential test results; (b) extracellular ATP test results; (c) β-galactosidase test results; and (d) PI staining results.

[0029] Figure 7 The results show the oxidative stress damage to Acinetobacter baumannii after treatment with naphthol phosphate; (a) NADH test results; (b) H2O2 test results; (c) intracellular ROS level test results; (d) SOD test results; (e) MDA test results. Detailed Implementation

[0030] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] This embodiment provides the application of naphtholquin or a pharmaceutically acceptable salt in the preparation of drugs against drug-resistant bacteria. Naphtholquin is an antibacterial drug that inhibits the growth of drug-resistant bacteria or kills drug-resistant bacteria.

[0032] Specifically, drug-resistant bacteria include one of Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus, or Enterococcus faecalis.

[0033] Specifically, the naphtholquinone or pharmaceutically acceptable salt is at least one selected from naphtholquinone phosphate, hydrochloride, sulfate or maleate.

[0034] Specifically, the pharmaceutically acceptable salt of naphtholquin is selected from at least one of naphtholquin phosphate, naphtholquin hydrochloride, naphtholquin sulfate, or naphtholquin maleate;

[0035]

[0036] The above are the molecular structural formulas of naphtholquinone (Formula 1), naphtholquinone phosphate (Formula 2), naphtholquinone hydrochloride (Formula 3), naphtholquinone sulfate (Formula 4), and naphtholquinone maleate.

[0037] Specifically, the naphtholquin is used in combination with other antibacterial drugs.

[0038] Specifically, other antibacterial drugs include pentamidine.

[0039] Specifically, the antibacterial drug is prepared as an oral dosage form, tablet, or capsule.

[0040] Specifically, the daily dose of the antibacterial drug for Acinetobacter baumannii LAC-4 is 10-20 mg / kg.

[0041] The present invention also provides the use of naphtholquinone or a pharmaceutically acceptable salt or solvate in the preparation of a medicament for treating pneumonia.

[0042] Specifically, pneumonia caused by drug-resistant bacteria; drug-resistant bacteria include one of Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus, or Enterococcus faecalis.

[0043] Example 1

[0044] (1) Acinetobacter baumannii LAC-4, methicillin-resistant Staphylococcus aureus USA-300-R and Enterococcus faecalis HJP554 were selected as targets, and the antibacterial activity of naphthol phosphate was evaluated by micro-broth dilution method.

[0045] Acinetobacter baumannii LAC-4, methicillin-resistant Staphylococcus aureus USA-300-R, and Enterococcus faecalis HJP554 were inoculated into cationic broth (Ca-MHB) and cultured to the logarithmic phase.

[0046] Naphthol phosphate at a concentration of 2000 μg / mL was prepared using sterile deionized water, filtered through a 0.22 μm filter membrane, and then serially diluted in 96-well microplates using Ca-MHB medium.

[0047] The experiment requires four replicates. 100 μL of the corresponding concentration of naphthol phosphate is added to each replicate. Then, bacterial suspension is added to three of the replicates as the experimental group, and 100 μL of Ca-MHB medium is added to one replicate as the solvent control group. 100 μL of bacterial suspension is added to each well, resulting in a final inoculum size of 5 × 10⁻⁶. 5 CFU / mL. The concentrations of naphthol phosphate were set successively at 1000 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, 15.625 μg / mL, 7.81 μg / mL, 3.90 μg / mL and 1.95 μg / mL.

[0048] The experimental group consisted of 100 μL Ca-MHB and 100 μL bacterial suspension.

[0049] A control group was prepared by adding 100 μL of water to 100 μL of Ca-MHB as solvent.

[0050] The positive control group and the solvent control group were incubated at 37°C for 16-18 hours. The minimum concentration at which no bacterial growth was observed to the naked eye was taken as the MIC value of the drug. The test was considered valid if the liquid in the wells of the blank control group and the solvent control group was clear and transparent with no bacterial growth, and the wells of the positive control group showed obvious precipitation and bacterial growth. Otherwise, the test was repeated.

[0051] The minimum inhibitory concentration (MIC) of naphthol phosphate against Acinetobacter baumannii LAC-4, methicillin-resistant Staphylococcus aureus, and Enterococcus faecalis was determined using the micro-broth dilution method, as shown in Table 1.

[0052] As shown in Table 1, the MIC value of naphthol phosphate against Acinetobacter baumannii LAC-4 was 62.5 μg / mL, and the MIC value against methicillin-resistant Staphylococcus aureus and Enterococcus faecalis was 31.25 μg / mL. The results indicate that naphthol phosphate has broad-spectrum antibacterial activity.

[0053] Table 1. MIC determination results of naphthol phosphate and naphthol phosphate.

[0054]

[0055] (2) The drug susceptibility index of naphthol phosphate and naphthol phosphate combined with pentamidine against Acinetobacter baumannii LAC-4 was quantitatively detected by in vitro combined drug susceptibility test using the checkerboard method.

[0056] Acinetobacter baumannii LAC-4 was inoculated into cationic broth (Ca-MHB) and cultured to the logarithmic growth phase, then diluted to 1×10⁻⁶. 6 CFU / mL.

[0057] The two compounds in the combined drug therapy were diluted 2-fold laterally (Drug A) and 2-fold vertically (Drug B) on two U-shaped 96-well plates, resulting in a final experimental concentration that was diluted 4-fold.

[0058] The two diluted drugs were completely combined at different concentrations and added to a 96-well plate at a 1:1 volume ratio (50 μL each). Then, 100 μL of bacterial suspension was added and the plate was incubated at 37°C for 16-18 hours.

[0059] A positive control group was set up with Acinetobacter baumannii that grew only in the culture medium.

[0060] The control group, consisting only of culture medium and drug, was used as a solvent.

[0061] When reading the results, place the 96-well plate in a well-lit area, observe the turbidity of the broth in the wells and whether there is colony deposition at the bottom of the wells, record the MIC values ​​of the two drugs used alone and the MIC values ​​of sterile growth when used in combination, and obtain the combination drug index.

[0062] Calculation and interpretation criteria of the combination drug index (FICI): FICI = MIC A联用 / MIC A单用 +MIC B联用 / MIC B单用 FICI ≤ 0.5, synergistic effect; FICI > 0.5-1, additive effect; FICI > 1-2, no effect; FICI > 2, antagonistic effect.

[0063] Table 2 shows that the MIC value of pentamidine alone against Acinetobacter baumannii was 250 μg / mL, the MIC value of pentamidine combined with naphthol phosphate was 62.5 μg / mL, and the MIC values ​​of naphthol phosphate and pentamidine before and after combination were 62.5 μg / mL and 15.625 μg / mL, respectively, with FICI ≤ 0.5, indicating that the combination of pentamidine and naphthol phosphate has a potential synergistic effect.

[0064] Table 2. Results of the checkerboard method combined with drug sensitivity testing

[0065]

[0066] (3) Antimicrobial kinetics were studied by monitoring the in vitro time-killing curves of naphthol phosphate alone and in combination with pentamidine.

[0067] Acinetobacter baumannii LAC-4 was inoculated into cationic broth (Ca-MHB) and cultured to the logarithmic growth phase, then diluted to 1×10⁻⁶. 6 CFU / mL was added at a 1:1 ratio to a sterile Erlenmeyer flask containing naphthol phosphate, resulting in final concentrations of 0.5MIC, MIC, 2MIC, 4MIC, and 8MIC, where the MIC values ​​are the minimum inhibitory concentrations (MICs) for Acinetobacter baumannii LAC-4 as shown in Table 1. A solvent control group was also prepared.

[0068] Based on the results of the combined antibacterial experiment, the concentration of pentamiprid (PT) and naphthol phosphate (NQP) in combination was determined to be 1 / 4 MIC. PT 1 / 4MIC NQP 1 / 2MIC PT +1 / 2MIC NQP Simultaneously, a solvent control group was prepared. After shaking, the samples were incubated at 37°C. Samples were taken at 0, 0.5, 1, 2, 4, 8, 23, and 24 hours, diluted with physiological saline (containing 0.05% poloxamer), and spotted onto solid culture medium. The samples were incubated at 37°C for 24 hours, and observation and counting were performed using plate culture. A time-kill curve was plotted. (See figure). Figure 1 (a) and Figure 1 (b)

[0069] Figure 1 In (a), naphthol phosphate exhibits a strong bactericidal effect at the MIC concentration, rapidly killing all drug-resistant bacteria and reducing the viable count to 0 after 1 hour of treatment. Treatment with naphthol phosphate at concentrations of 2, 4, and 8 MIC completely kills all bacteria within 0.5 hours.

[0070] Figure 1In (b), when naphtholquinoline phosphate and pentamidine were used in combination, the bacteria in the control group (without any added drug) grew normally. The bacterial growth trend when naphtholquinoline phosphate was used alone was the same as that in the control group, but the bacterial concentration was still lower than that in the control group. When pentamidine was used alone, it showed a decreasing trend within 1 hour after administration, followed by a slow increase. However, the combination of pentamidine and naphtholquinoline phosphate was able to completely kill the bacteria within 6 hours, demonstrating excellent synergistic bactericidal effect.

[0071] (4) The cytotoxicity of naphthol phosphate to A549 cells, Huh7 cells and Vero cells was detected by the CCK8 method. The hemolytic ability of naphthol phosphate to erythrocytes was also investigated.

[0072] The cytotoxicity of naphthol phosphate was detected using the CCK-8 assay kit.

[0073] A549, HuH7, and Vero cells were grown in DMEM medium supplemented with a mixture of 10% FBS and 1% penicillin-streptomycin. Then, 200 μL of cells were added to 96-well plates at a density of 3 × 10⁻⁶ cells per well. 4 Cells were seeded at a density of [number] cells per well in a 96-well microtiter plate. After incubation overnight at 37°C and 5% CO2, the cells were washed twice with PBS. 200 μL of serially diluted DMEM solution (using DMEM as a negative control) was added to each well. After incubation for 24 hours, the buffer was removed, and then 110 μL of CCK8 was added to each well. After 4 hours of incubation, the absorbance was measured at 450 nm using a microplate reader. Figure 2 As shown.

[0074] from Figure 2 As can be seen, at a concentration of 125 μg / mL, naphthol phosphate reduced cell viability to 20% and 10% in Vero and Huh7 cells, respectively. However, within the range of 31.25–62.5 μg / mL, it had no significant effect on cell viability. Naphthol phosphate at a concentration of 500 μg / mL had no effect on the viability of A549 cells.

[0075] (5) Red blood cell hemolysis test

[0076] Whole blood was extracted from Balb / c mice. 1.5 mL of mouse whole blood was centrifuged at 3000 rpm for 10 minutes at 4°C to separate red blood cells from the plasma. The red blood cells were washed three times with saline (0.9% NaCl) and resuspended in saline. A 2.5% v / v red blood cell suspension was prepared by resuspending the red blood cells in physiological saline. 700 μL of the red blood cell suspension was added to a 1.5 mL centrifuge tube, centrifuged at 3000 rpm for 10 minutes, and the supernatant was discarded. The test drug was dissolved in physiological saline (0.9% NaCl), and 700 μL of different concentrations of naphthol phosphate were added to the red blood cell pellet. An equal volume of physiological saline (C0.9% NaCl) was added to the negative control. - The positive control, when treated with an equal volume of distilled water, exhibited osmotic hemolysis (C). + After incubation at 37°C for 1 hour, the cells were centrifuged again to remove intact red blood cells. The absorbance of the supernatant was measured at 540 nm. Hemolysis was described as the percentage of hemolyzed blood cells, calculated as hemolysis rate (%) = (absorbance - absorbance C). - ) / (Absorbance C) + - Absorbance C - The result was calculated as () × 100%. The experiment was repeated three times for statistical analysis.

[0077] like Figure 3 As shown in the mouse erythrocyte hemolysis assay, naphthol phosphate at a concentration of 125 μg / mL caused almost no hemolysis, a concentration higher than the MIC of Acinetobacter baumannii LAC-4. Even at concentrations as high as 500 μg / mL, naphthol phosphate only caused mild hemolysis. These results indicate that the use of naphthol phosphate within a reasonable concentration range is feasible.

[0078] (6) An infection model was established using the A549 cell line to study the antibacterial activity against infected cells.

[0079] A549 cells were seeded into 96-well plates (2 × 10⁶ cells per well). 4 Cells were cultured at 37°C for 24 hours in a 5% CO2 incubator (cells / well). Acinetobacter baumannii LAC-4 suspension was added to the cells at a multiplicity of infection (MOI) of 200. After infecting the cells with LAC-4 at 37°C for 2 hours, A549 cells were washed three times with PBS to remove free bacteria, and then treated with 1 / 2 MIC, 1 MIC, and 2 MIC of naphthol phosphate for another 2 hours at 37°C. Cells were washed three times with PBS and lysed in 100 μL of cold PBS containing 0.1% Triton X-100. The count of surviving bacteria was determined by inoculating serial dilutions of cell lysates onto MHA agar.

[0080] like Figure 4As shown, approximately 27,833 ± 4,611 cells adhered to the surface of A549 cells 2 hours after infection. Treatment with 31.25 μg / mL (0.5 MIC) naphthol phosphate significantly reduced the number of surviving adherent bacteria compared to the control group (p < 0.0001). Naphthol phosphate at concentrations of 2 MIC, 4 MIC, and 8 MIC killed all adhering bacteria.

[0081] (7) Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to observe changes in bacterial cells.

[0082] The bacteria were cultured to the logarithmic phase and diluted with culture medium to OD0. 600 ≈1.0, add MIC concentration of naphthol phosphate for 1 h, centrifuge at 3000g for 10 min to collect the precipitate, with no drug treatment as a negative control. Fix the precipitate with 2.5% glutaraldehyde at 4° for 12 h, wash twice with PBS, fix with 1% osmium tetroxide, rinse with buffer and dehydrate with ethanol gradient, etc., then dry, embed, section and stain, and observe with scanning electron microscope (SEM) and transmission electron microscope (TEM).

[0083] like Figure 5 (a) SEM images show that the untreated control group bacteria exhibit normal morphology, characterized by regular shape, uniform size, and clearly distinguishable surface features. In contrast, SEM images of the naphthol phosphate-treated group show wrinkles and various deformations on the bacterial surface, strongly demonstrating the ability of naphthol phosphate to disrupt bacterial membrane integrity.

[0084] like Figure 5 (b) TEM images show that the bacteria in the control group have regular morphology, smooth edges and clear outlines, and uniform distribution of intracellular contents, while the bacteria treated with naphthol phosphate exhibit vacuolization, plasma membrane shedding, irregular edges and cell membrane deformation.

[0085] (8) Cell membrane permeability was assessed by measuring changes in bacterial membrane potential, β-galactosidase, and extracellular ATP before and after treatment with naphthol phosphate, as well as the fluorescence intensity under a fluorescence microscope after PI staining.

[0086] Membrane potential: Bacteria were cultured to the logarithmic phase and diluted with culture medium to OD. 600 ≈1.0, prepare 3 equal parts of bacterial suspension, add disc3(5) probe, incubate in a 37℃ biochemical incubator in the dark for 1.5h, then add sterile aqueous solution of naphthol quinolone phosphate, adjust the concentration to 1 / 2MIC, MIC, add sterile water to the negative control group, continue incubation at 37℃ for 15min, the final concentration of disc3(5) is 50nM, add the sample to the 96-well plate in the dark, set the excitation wavelength of the microplate reader to 622nm and the emission wavelength to 670nm, measure the change in fluorescence intensity of the sample, see Figure 6 (a).

[0087] ONPG experiment: Bacteria were cultured to the logarithmic phase and diluted with culture medium to OD0.05. 600 ≈1.0, prepare three equal portions of bacterial suspension, add n-nitrobenzene-β-D-galactopyranoside (ONPG), then add sterile aqueous solution of naphthol quinolone phosphate, adjusting the concentration to 1 / 2 MIC and MIC. Add sterile water to the negative control group. Incubate at 37℃ for 1 h. The final concentration of ONPG is 3 mM. Add the sample to a 96-well plate in the dark and measure the absorbance of the sample solution at 420 nm. See [reference needed]. Figure 6 (c)

[0088] Extracellular ATP detection experiment: The same number of cells were collected, and sterile aqueous solution of naphthol quinoline phosphate was added and incubated at 37°C for 1 h. The supernatant was collected by centrifugation, and the ATP level in the supernatant was measured using an ATP assay kit (Beyotime, A22066). In short, luciferase catalyzes the conversion of ATP and D-luciferin into light, and the light was measured by a multi-functional microplate reader (SpectraMax i3x; MolecularDevices, Shanghai, China). See [link to relevant documentation]. Figure 6 (b)

[0089] PI staining experiment: Bacteria were cultured to the logarithmic phase and diluted with culture medium to OD. 600 The concentration was approximately 1.0, and the solution was added to the shaken tube containing the sample at a 1:1 ratio. The final concentration of naphthol phosphate was 0.5xMIC and MIC, with a solvent control group set up. After bacterial administration, the bacterial cells were collected by centrifugation and washed three times with physiological saline (containing 0.05% poloxamer). The bacterial cells were resuspended in physiological saline, and the probe PI was added and incubated in the dark for 15 min. Finally, the sample was dropped onto a glass slide, observed and photographed under a fluorescence microscope, and quantified by ImageJ analysis. Figure 6 (d) and Figure 6 (e).

[0090] like Figure 6 As shown in (a), the fluorescence intensity observed in the naphthol phosphate-treated group was significantly higher than that in the control group, indicating membrane depolarization.

[0091] like Figure 6 As shown in (c), the degree of membrane potential depolarization increases with increasing concentration of naphthol phosphate; naphthol phosphate increases the release of extracellular ATP in a dose-dependent manner.

[0092] like Figure 6 As shown in (b), the absorbance measured at 420 nm showed that the absorbance in the naphthol phosphate treatment group was significantly higher than that in the control group.

[0093] like Figure 6(d) and Figure 6 As shown in (e), when Acinetobacter baumannii LAC-4 was treated with 1 / 2MIC and MIC naphthol phosphate and stained with propidium iodide (PI), an increase in the number of dead (red) bacteria was observed, indicating that the permeability of the Acinetobacter baumannii LAC-4 cell membrane was increased after treatment with naphthol phosphate.

[0094] (9) The relevant biochemical indicators of bacterial oxidative damage before and after treatment with naphthol phosphate were determined: ROS, H2O2, NADH, MDA, and SOD.

[0095] NADH: Sample collection was performed using the same method as the ROS method. Indicated cells were collected, and intracellular NADH levels were determined using the NAD+ / NADH assay kit (Beyotime, S0175) and WST-8 (an upgraded alternative to MTT) according to the manufacturer's instructions. Cell lysates were incubated at 60°C for 30 minutes to remove NAD+, and then WST-8 was reduced to formazan by NADH and 1-mPMS (1-methoxy-5-methylazine methyl sulfate). To analyze total NADH levels, absorbance was measured at 450 nm and analyzed on a plate reader (SpectraMax I3; Molecular Devices, Shanghai, China). Figure 7 (a).

[0096] H2O2: Sample collection was performed using the same method as ROS. After organism lysis, the supernatant was collected, and intracellular H2O2 levels were measured using a hydrogen peroxide detection kit (Shanghai Bio-Tech Research Institute, S0038, China) according to the manufacturer's instructions. Figure 7 (b)

[0097] Intracellular ROS level detection: Bacteria were cultured to the logarithmic growth phase and diluted with culture medium to OD0.05. 600 ≈1.0, add naphthol phosphate and incubate at 37℃ for 1 hour. Adjust the concentration of naphthol phosphate to 0, 1 / 2 MIC, MIC, 2 MIC, 4 MIC, and 8 MIC. Centrifuge to collect bacterial pellet, resuspend the pellet in DCFH-DA (10 μM) diluted in culture medium, and continue incubation at 37℃ for 20 minutes to allow the probe to enter the cells. After the reaction, wash the cells twice with physiological saline containing 0.05% poloxamer to thoroughly remove any probes that have not entered the cells; transfer the sample to 200 μL per well of a 96-well plate, and measure the fluorescence intensity (excitation wavelength 488 nm; emission wavelength 525 nm) using a multi-functional microplate reader (SpectraMax i3x, Shanghai). See [link to microplate reader]. Figure 7 (c)

[0098] SOD and MDA: Intracellular SOD levels were measured using the WST-8 Total Superoxide Dismutase Assay Kit (Beyotime, S0101) according to the manufacturer's operating procedures. Intracellular MDA levels were measured using the Lipid Peroxidation Assay Kit (Icoin, KTB9050), see [link to kit]. Figure 7 (d) and Figure 7 (e).

[0099] like Figure 7 As shown in (a), the NADH level in the naphthol phosphate quinoline treatment group was higher than that in the control group. As a product of the TCA cycle, the increase in NADH level stimulates the oxidative phosphorylation process.

[0100] like Figure 7 (b) and Figure 7 As shown in (c), hydrogen peroxide levels increased significantly, and superoxide ROS levels also rose markedly. Excessive ROS can damage intracellular DNA, iron-sulfur protein clusters, lipids, and other important cellular components in bacterial cells.

[0101] like Figure 7 (d) and Figure 7 (e) The level of SOD secreted by the bacteria did not increase significantly, and therefore was insufficient to offset the damage caused by excessive ROS. Therefore, naphthol phosphate greatly increased the level of ROS in bacterial cells, accelerating bacterial death; as the accumulation of ROS induced lipid peroxidation in the cell membrane, the content of malondialdehyde (MDA) in Acinetobacter baumannii LAC-4 gradually increased, which in turn damaged the integrity of the cell membrane and enhanced its permeability.

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of naphthoquine or a pharmaceutically acceptable salt thereof and pentamidine in the preparation of a drug against drug-resistant bacteria, characterized in that: The drug-resistant bacteria are selected from one of Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus or Enterococcus faecium.

2. Use according to claim 1, characterized in that: The pharmaceutically acceptable salt of naphthoquine is at least one selected from a phosphate salt, a hydrochloride salt, a sulfate salt or a maleate salt of naphthoquine.

3. Use according to claim 1, characterized in that: The preparation of the antibacterial drug is one of a tablet or a capsule.

4. Use according to claim 1, characterized in that: The daily dose of the antibacterial drug for Acinetobacter baumannii LAC-4 is 10-20 mg / kg.

5. The use of naphthoquine or a pharmaceutically acceptable salt thereof and pentamidine in the preparation of a medicament for treating pneumonia, characterized in that: The pneumonia is pneumonia caused by drug-resistant bacteria; the drug-resistant bacteria are selected from one of Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus or Enterococcus faecium.

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