Use of indol-3-carbinol in the preparation of polymyxin antibacterial potentiators and antibacterial compositions against gram-negative bacteria
By combining indole-3-methanol with polymyxin, the problems of pharmacokinetic difficulties in achieving target values and bacterial resistance in polymyxin monotherapy have been solved, achieving highly efficient bactericidal activity and delayed resistance development against Gram-negative bacteria.
Patent Information
- Application Number
- CN202311339544.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-10-17
- Publication Date
- 2026-08-25
- Estimated Expiration
- 2043-10-17
AI Technical Summary
When polymyxin is used as a monotherapy, it is difficult to achieve the target pharmacokinetic values, and it is easy to induce bacterial heterogeneous drug resistance, increasing the risk of treatment failure.
Indole-3-methanol and polymyxin are used in combination at a concentration range of 0.01-2 (μg/mL): 0.01-10 mM, preferably 0.125 (μg/mL): 1 mM, to prepare a polymyxin antibacterial synergist, forming an anti-Gram-negative bacterial drug composition.
It significantly improves the bactericidal activity of polymyxin against Gram-negative bacteria, reduces the dosage, prolongs the antibacterial effect, and delays the development of drug resistance.
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Figure CN117338770B_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of biomedical technology, and more specifically, to the application of indole-3-methanol in the preparation of polymyxin antibacterial synergists and anti-Gram-negative bacterial drug compositions. Background Technology
[0002] Polymyxins are polypeptide antibiotics obtained from cultures of *Bacillus polymyxa*. They were marketed and used clinically in the 1950s, but due to toxic side effects such as nephrotoxicity, they were gradually replaced by other drugs. In recent years, with the increasing irrational use of antibiotics, infections caused by multidrug-resistant and extensively drug-resistant Gram-negative bacilli have also increased, becoming one of the main threats to human health. Therefore, polymyxin antibiotics, as effective drugs for treating multidrug-resistant and extensively drug-resistant Gram-negative bacilli infections, are once again playing an important role in the clinical treatment of Gram-negative bacterial infections.
[0003] However, due to the toxic side effects of polymyxins, it is difficult to achieve the maximum clinically permissible dose when using polymyxin monotherapy, and it is prone to bacterial heterogeneous resistance, increasing the risk of treatment failure. Therefore, the search for polymyxin antibacterial potentiators to achieve combination therapy with other antibacterial drugs has become a research hotspot. Based on the above, this application provides the application of indole-3-methanol in the preparation of polymyxin antibacterial potentiators and anti-Gram-negative bacterial drug compositions. Summary of the Invention
[0004] To address the shortcomings of polymyxin monotherapy, such as the difficulty in achieving the maximum clinically permissible dose to the target pharmacokinetic value and the tendency for bacterial heterogeneous resistance to increase the risk of treatment failure, this application provides the application of indole-3-methanol in the preparation of polymyxin antibacterial potentiators and a drug composition against Gram-negative bacteria.
[0005] In the first aspect, this application provides the application of indole-3-methanol in the preparation of polymyxin antibacterial synergists, using the following technical solution:
[0006] Application of indole-3-methanol in the preparation of polymyxin antibacterial synergists.
[0007] Preferably, the concentrations of polymyxin and indole-3-methanol are 0.01-2 (μg / mL): 0.01-10 mM.
[0008] Preferably, the concentration of polymyxin and indole-3-methanol is 0.125 (μg / mL): 1 mM.
[0009] Preferably, the polymyxin is at least one of polymyxin B, polymyxin E, a pharmaceutically acceptable salt of polymyxin B, and a pharmaceutically acceptable salt of polymyxin E.
[0010] Preferably, the polymyxin is at least one of polymyxin B, polymyxin E, a pharmaceutically acceptable salt of polymyxin B, and a pharmaceutically acceptable salt of polymyxin E.
[0011] Secondly, this application provides an anti-Gram-negative bacterial drug composition, which adopts the following technical solution:
[0012] An anti-Gram-negative bacterial drug composition comprising polymyxin and indole-3-methanol in the above concentration ratio.
[0013] Preferably, the anti-Gram-negative bacterial drug composition further includes pharmaceutically acceptable excipients and / or carriers.
[0014] Preferably, the dosage form of the composition includes, but is not limited to, injections, ointments, or aerosols.
[0015] Preferably, the Gram-negative bacteria include, but are not limited to, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, or Pseudomonas aeruginosa.
[0016] In summary, this application has the following beneficial effects:
[0017] This application provides a novel use of indole-3-methanol in the preparation of polymyxin antibacterial synergists, disclosing that indole-3-methanol can synergistically enhance the bactericidal activity of polymyxin against Gram-negative bacteria, reduce the dosage of polymyxin, improve antibacterial efficacy, prolong antibacterial duration, and delay the development of drug resistance.
[0018] The bacteria used in this study mainly included Gram-negative opportunistic pathogens such as Klebsiella pneumoniae ATCC43816, Acinetobacter baumannii ATCC17978, Escherichia coli BW25113, and Pseudomonas aeruginosa PA14. Through studies such as minimum inhibitory concentration (MIC) determination, time-bacterial kinetics experiments, checkerboard microdilution method, and crystal violet detection of biofilm inhibition, it was found that indole-3-methanol can significantly improve the sensitivity of Gram-negative bacteria to polymyxins and can synergistically enhance the antibacterial effect of polymyxins. Attached Figure Description
[0019] Figure 1 This is a time-knockout kinetics experiment of Example 2 of this application, showing the bactericidal curves of polymyxin B and indole-3-methanol used in combination against Gram-negative bacteria.
[0020] Figure 2This is a representative heatmap of the synergistic effect of polymyxin B and indole-3-methanol in Gram-negative bacteria in the micro-checkerboard dilution assay of Example 3 of this application.
[0021] Figure 3 This diagram illustrates the inhibitory effects of polymyxin B and indole-3-methanol on biofilm formation in the crystal violet detection biofilm inhibition experiment of Example 4 of this application. Detailed Implementation
[0022] The present application will be further described in detail below with reference to the embodiments.
[0023] 1. Source of strain
[0024] This study selected four standard bacterial strains (Klebsiella pneumoniae ATCC 43816, Acinetobacter baumannii ATCC 17978, Escherichia coli BW25113, and Pseudomonas aeruginosa PA14). All strains were preserved by the Anhui Provincial Center for Antimicrobial Resistance Monitoring. The use of these clinical strains was approved by the Ethics Committee of the First Affiliated Hospital of Anhui Medical University.
[0025] 2. Reagents
[0026]
[0027] 3. Instruments
[0028]
[0029] Example 1: Determination of Minimum Inhibitory Concentration (MIC)
[0030] The minimum inhibitory concentrations (MICs) of polymyxin B and indole-3-methanol against Gram-negative bacteria, specifically Klebsiella pneumoniae ATCC 43816, Acinetobacter baumannii ATCC 17978, Escherichia coli BW25113, and Pseudomonas aeruginosa PA14, were determined.
[0031] Activated strains
[0032] Wearing masks, hats, gloves, and white coats, the entire process is carried out in a sterile environment in a biosafety cabinet. Take a sterilized 50mL centrifuge tube, add 5mL of MHB medium, take out the corresponding bacterial strain stored in a -80℃ refrigerator, use a sterile inoculation loop to pick up the bacterial solution and inoculate it into MHB medium, place it in a constant temperature shaker at 220rpm and 37℃, and incubate overnight to activate the bacterial strain.
[0033] Determination of minimum inhibitory concentration (MIC)
[0034] The activated overnight strain was diluted 100-fold with sterilized MHB medium and cultured in a constant temperature shaker at 220 rpm and 37°C until OD. 600Between 0.25 and 0.3 (OD in the embodiments of this application) 600 To induce the bacteria to enter the logarithmic growth phase (2.7%), the bacterial culture was diluted to 5 × 10⁻⁷ using MHB medium. 5 CFU / mL, for later use.
[0035] Add 2 mL of the diluted bacterial suspension to 10 mL sterile test tubes, and then add the final concentration of polymyxin B or indole-3-methanol respectively. Dilute using a serial dilution method, and incubate under the same conditions (220 rpm, 37℃) in a constant temperature shaker. After 12 h, observe the turbidity of each bacterial suspension. The concentration of the drug corresponding to the tube with clear liquid is taken as the minimum inhibitory concentration (MIC). All experiments were repeated three times. The results are shown in Table 1 below:
[0036] Table 1. Minimum inhibitory concentrations (MICs) of polymyxin B and indole-3-methanol against four Gram-negative bacteria.
[0037]
[0038] Note: PMNB represents polymyxin B; I3C represents indole-3-carbinol.
[0039] Example 2: Time-Kill Kinetics Experiment
[0040] The overnight strain was diluted 100-fold with sterilized MHB medium and cultured in a constant temperature shaker at 220 rpm and 37°C until OD. 600 Between 0.25 and 0.3 (OD in the embodiments of this application) 600 To induce the bacteria to enter the logarithmic growth phase (2.7%), the bacterial culture was diluted to 1×10⁻⁶ using MHB medium. 8 CFU / mL, for later use.
[0041] 5 mL of diluted bacterial solution was added to each of eight sterile test tubes, which were divided into groups treated with polymyxin B (1 / 4×MIC), different concentrations (0.25 mM, 0.5 mM, 1 mM) of indole-3-methanol, a combination of both, and an untreated bacterial solution (control group). The test tubes were incubated in a constant temperature shaker (37℃, 220 rpm). At different time points (2h, 4h, 8h, 12h, and 24h), 50 μL of bacterial solution was taken from each test tube and added to a sterile EP tube containing 450 μL of physiological saline. Then, 50 μL of the solution was taken from the previous EP tube and added to a second sterile EP tube containing 450 μL of physiological saline, and so on, for at least five consecutive dilutions. The bacterial solution was diluted 10 (1), 10 (2), 10 (3), 10 (4), 10 (5), 10 (6), 10 (7), 10 (8), 10 (9), 10 (10 ... 2 (2) 10 3 (3), ..., 10 n(n) times. At different time points (2h, 4h, 8h, 12h, 24h), 10μL of bacterial solution of the corresponding dilution concentration was taken from the EP tube of each gradient three times and dropped onto the fixed area of the marked MHA broth agar plate. After the liquid was absorbed, the plate was inverted and placed in a constant temperature incubator at 37℃ for overnight incubation.
[0042] Colony counts were performed at each time point, and the average value of the three points was taken to obtain the bactericidal curve of polymyxin B and indole-3-methanol combined against Gram-negative bacteria, such as... Figure 1 As shown.
[0043] Formula for calculating bacterial concentration: Bacterial concentration = Average number of colonies at the nth gradient × 10 n CFU / mL.
[0044] Figure 1 In this context, PMNB represents polymyxin B; I3C represents indole-3-carbinol. Data are from at least three independent replicate experiments; error bars represent the standard error of the mean.
[0045] (A) Klebsiella pneumoniae ATCC 43816, (B) Acinetobacter baumannii ATCC 17978, (C) Escherichia coli BW25113, (D) Pseudomonas aeruginosa PA14 were treated with PMNB (1 / 4×MIC) and I3C (0.25mM, 0.5mM, 1mM) alone or in combination, and their growth was observed at different time points.
[0046] Depend on Figure 1 The results show that: when polymyxin B is at a concentration of 1 / 4 × MIC, and 1 mM I3C is used, it effectively kills Klebsiella pneumoniae ATCC 43816 within 24 hours, with a kill rate of 5 log10 CFU / mL; in Acinetobacter baumannii ATCC 17978, the kill rate also reaches 5 log10 CFU / mL within 24 hours; in Escherichia coli BW25113, the combined use of the two drugs significantly enhances the lethality rate within 24 hours, and the bacterial count decreases by 4 log10 CFU / mL; in Pseudomonas aeruginosa PA14, the addition of I3C to polymyxin B at 1 / 4 MIC reduces the bacterial count by 3.7 log10 CFU / mL within 24 hours.
[0047] Example 3: Chessboard Micro-dilution Method
[0048] The overnight strain was diluted 100-fold with sterilized MHB medium and cultured in a constant temperature shaker at 220 rpm and 37°C until OD. 600 Between 0.25 and 0.3 (OD in the embodiments of this application) 600To induce the bacteria to enter the logarithmic growth phase (2.7%), the bacterial culture was diluted to 5 × 10⁻⁷ using MHB medium. 5 CFU / mL, for later use.
[0049] Add 200 μL of the diluted bacterial solution to each of the 96-well microplates, and follow the instructions. Figure 2 As shown, different concentrations of polymyxin B (PMNB) and indole-3-carbinol (I3C) were added to the bacterial culture in pairs, creating a final concentration gradient starting from the MIC concentration and proceeding downwards in a vertical or parallel direction with at least five serial dilutions, maintaining a consistent drug concentration in each vertical and parallel well. The culture was then incubated at 37°C for 12 hours. The lowest concentration in the culture plate that did not become turbid was taken as the effective minimum inhibitory concentration (MIC) for the combined use of the two drugs.
[0050] The formula FIC index = MIC A药联合 / MIC A药单药 +MIC B药联合 / MIC B药单药 The fractional inhibitory concentration (FIC) index was calculated, and the results are as follows: Figure 2 As shown.
[0051] The FIC index is defined as follows: synergistic effect ≤ 0.5; additive effect 0.5-1.0; irrelevant effect 1.0-2.0; antagonistic effect > 2.0.
[0052] Figure 2 In this context, PMNB represents polymyxin B; I3C represents indole-3-methanol; FIC represents the partial inhibitory concentration index (also known as FICI); all results were repeated three times.
[0053] The effects of combined PMNB and I3C use on (A) Klebsiella pneumoniae ATCC 43816, (B) Acinetobacter baumannii ATCC 17978, (C) Escherichia coli BW25113, and (D) Pseudomonas aeruginosa PA14. (E) Equivalent line plots of combined PMNB and I3C use in different Gram-negative bacteria. Black lines represent equivalent lines, and points within the intersection of equivalent lines and coordinate axes represent synergistic effects.
[0054] Depend on Figure 2 The results show that, using the checkerboard dilution method to study the synergistic effect of PMNB and I3C in combination, in the study of Klebsiella pneumoniae ATCC 43816, when the two drugs were used in combination, the MIC of PMNB was reduced by 8-fold in the presence of I3C (1 mM). Figure 2 A), FICI is about 0.188 (FICI<0.5, Figure 2E). The FICI values for Acinetobacter baumannii ATCC 17978, Escherichia coli BW25113, and Pseudomonas aeruginosa PA14 were 0.258, 0.313, and 0.281, respectively. Figure 2 E); When I3C was present, the MIC of PMNB in all three strains was reduced by 4-fold. Figure 2 (B, C, D).
[0055] Example 4: Crystal Violet Detection Experiment for Biomembrane Inhibition
[0056] The overnight strain was diluted 100-fold with sterilized MHB medium and cultured in a constant temperature shaker at 220 rpm and 37°C until OD. 600 Between 0.25 and 0.3 (OD in the embodiments of this application) 600 To induce the bacteria to enter the logarithmic growth phase (2.7%), the bacterial culture was diluted to 5 × 10⁻⁷ using MHB medium. 5 CFU / mL, for later use.
[0057] The diluted 200 μL bacterial solutions were transferred into 96-well plates and divided into five groups for treatment: blank control group, 1 / 4×MIC polymyxin B group, 1 / 2×MIC polymyxin B group, 1 mM indole-3-methanol + 1 / 4×MIC polymyxin B group, and 1 mM indole-3-methanol + 1 / 2×MIC polymyxin B group.
[0058] After incubation at 37°C for 24 hours, the culture medium was discarded using a pipette. The cells were washed three times with 1× phosphate buffer, with the last wash removing as much liquid as possible. The bacterial biofilm was then fixed with 100 μL of 95% formalin for 15 minutes. After removing the 95% formalin, the cells were allowed to air dry at room temperature. Then, 100 μL of 0.1% crystal violet solution was added for staining for 20 minutes. The cells were washed twice with 1× phosphate buffer to remove excess staining. Finally, the cells were dried at 37°C for 1 hour and dissolved in 30% acetic acid solution (30 mL acetic acid mixed with 70 mL deionized water). The absorbance of the remaining crystal violet at 590 nm was measured using a microplate reader (Tecan, Mannedorf, Switzerland). The results are as follows: Figure 3 As shown.
[0059] Figure 3 In this context, PMNB represents polymyxin B; I3C represents indole-3-carbinol; data are from at least three independent replicate experiments; error bars represent the standard error of the mean.
[0060] Depend on Figure 3The results show that in *Klebsiella pneumoniae* ATCC 43816, compared with the untreated group, the biofilm clearance rate was 25.8% after treatment with 1 / 2×MIC polymyxin B, and 86.4% after combined treatment with I3C. In *Acinetobacter baumannii* ATCC 17978, compared with the untreated group, the biofilm clearance rate was 16.7% after treatment with 1 / 4×MIC polymyxin B, and 30.6% after combined treatment with I3C; the biofilm clearance rate was 49.8% after treatment with 1 / 2×MIC polymyxin B, and 83.2% after combined treatment with I3C. In *Escherichia coli* BW25113, compared with the untreated group, the biofilm clearance rate was 49.8% after treatment with 1 / 2×MIC polymyxin B, and 83.2% after combined treatment with I3C. In Pseudomonas aeruginosa PA14, compared with the untreated group, the biofilm clearance rate was 14.1% when treated with 1 / 4×MIC polymyxin B and 58.9% when treated with I3C. The biofilm clearance rate was 25.3% when treated with 1 / 2×MIC polymyxin B and 63.7% when treated with I3C.
[0061] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.
Claims
1. The use of indole-3-methanol in the preparation of drugs for enhancing the antibacterial activity of polymyxin B against Gram-negative bacteria, characterized in that, The Gram-negative bacteria are selected from one or more of Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, and Pseudomonas aeruginosa; The concentration of indole-3-methanol is 0.25-1mM, and the dosage of polymyxin B is 1 / 4×MIC or 1 / 2×MIC. The drug can reduce the minimum inhibitory concentration of polymyxin B by more than 4 times, and the partial inhibitory concentration index is less than 0.5, and can inhibit the formation of biofilms of the Gram-negative bacteria.
2. A pharmaceutical composition for enhancing the antibacterial activity of polymyxin B against Gram-negative bacteria, characterized in that, The pharmaceutical composition comprises polymyxin B and indole-3-methanol; The Gram-negative bacteria are selected from one or more of Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, and Pseudomonas aeruginosa; The pharmaceutical composition can reduce the minimum inhibitory concentration of polymyxin B by more than 4 times, the partial inhibitory concentration index is less than 0.5, and inhibit the formation of biofilms of the Gram-negative bacteria.
3. The pharmaceutical composition for enhancing the antibacterial activity of polymyxin B against Gram-negative bacteria according to claim 2, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.
4. The pharmaceutical composition for enhancing the antibacterial activity of polymyxin B against Gram-negative bacteria according to claim 2, characterized in that, The dosage forms of the pharmaceutical composition include, but are not limited to, injections, ointments, or aerosols.