An anti-Acinetobacter baumannii composition and its application

Abstract

The present invention relates to the field of anti-infective drug technology, and more particularly to an anti-Acinetobacter baumannii composition and its use. The anti-Acinetobacter baumannii composition comprises compound D44 and an antibiotic in a mass ratio of 1:(0.01-16). The chemical structure of compound D44 is shown in Formula I. The present invention screened and developed a composition with anti-infective properties. Combining compound D44 with an antibiotic effectively inhibits the growth of Acinetobacter baumannii. In particular, the combination of compound D44 and daptomycin induces structural disintegration of the lipid bilayer through a double membrane targeting mechanism, leading to the lysis and death of Acinetobacter baumannii. This invention provides a new treatment strategy for severe infections caused by drug-resistant Acinetobacter baumannii and has significant application value.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-infective drugs, and in particular to an anti-Acinetobacter baumannii composition and application thereof. Background Art

[0002] Antibiotics are a crucial pillar of modern medicine. In many parts of the world, the emergence of multidrug-resistant and even pandrug-resistant bacterial pathogens is becoming increasingly frequent, posing a serious challenge to public health and healthcare safety. To address this threat, the development of new antibiotics targeting highly prevalent pathogens is urgently needed. Enterobacteriaceae (such as Escherichia coli and Klebsiella pneumoniae) have become widespread representatives of extended-spectrum β-lactamase (ESBL)-producing and carbapenem-resistant Gram-negative bacteria. Furthermore, non-fermenting bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannii are exhibiting similar resistance trends. These multidrug-resistant and pandrug-resistant Gram-negative bacteria have become a significant health threat.

[0003] Currently, single antimicrobial therapy is still the dominant treatment option for infections caused by Gram-positive or Gram-negative bacteria, with combination therapy being the only treatment option for severe infections caused by multidrug-resistant bacteria. However, bacteria will eventually develop varying degrees of resistance to new or approved antimicrobial drugs, necessitating an urgent need to explore new therapies through more basic research, translational medicine, and clinical practice. Against this backdrop, the value of drug combination strategies is becoming increasingly prominent. These strategies can shorten treatment, improve efficacy, and reduce toxicity through synergistic effects, thereby reducing the probability of bacteria developing resistance to new antibiotics and effectively treating infections caused by resistant bacteria. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the present invention provides an anti-Acinetobacter baumannii composition and application thereof.

[0005] In a first aspect, the present invention provides an anti-Acinetobacter baumannii composition, comprising:

[0006] Compound D44 and antibiotics; the mass ratio of compound D44 to antibiotics is 1:(0.01-16);

[0007] The chemical structure of the compound D44 is shown in Formula I:

[0008] .

[0009] Furthermore, the antibiotic is a cyclic lipopeptide antibiotic or a glycopeptide antibiotic, and the mass ratio of the compound D44 to the antibiotic is 1:(0.1~4).

[0010] Furthermore, the antibiotic is a cyclic lipopeptide antibiotic; the mass ratio of the compound D44 to the cyclic lipopeptide antibiotic is 1:(0.25~4).

[0011] Furthermore, the cyclic lipopeptide antibiotic is daptomycin, and the mass ratio of the compound D44 to daptomycin is 1:(0.5~2).

[0012] Furthermore, the concentration of the compound D44 is 4-32 μg / mL; the concentration of daptomycin is 4-32 μg / mL.

[0013] During the research process, the present invention discovered that compound D44 and daptomycin themselves do not have the effect of inhibiting Acinetobacter baumannii, but after combining the two, compound D44 and daptomycin can produce significantly better synergistic antibacterial activity against Acinetobacter baumannii. Compound D44 destroys the permeability barrier of the bacterial outer membrane, promotes the penetration of daptomycin and targets the inner membrane, triggering the structural collapse of the membrane lipid bilayer, and ultimately triggering cell dehydration shrinkage, leading to bacterial lysis and death.

[0014] In addition, the present invention compares various combinations of compound D44 and antibiotics. Although the FICI index of compound D44 and daptomycin is not optimal, it exhibits the best antibacterial activity and the best therapeutic effect on mouse infection.

[0015] In a second aspect, the present invention provides a product comprising the aforementioned composition; the product is a medicine, an antibacterial agent, a disinfectant or a preservative.

[0016] Furthermore, the product is one or more of a solid dosage form, a semisolid dosage form, a liquid dosage form or a gaseous dosage form.

[0017] The solid dosage forms of the present invention include one or more of tablets, capsules, granules, powders, pills, or suppositories. The semisolid dosage forms of the present invention include one or more of ointments, creams, or gels. The liquid dosage forms of the present invention include one or more of solutions, syrups, injections, suspensions, or emulsions. The gaseous dosage forms of the present invention include aerosols.

[0018] In a third aspect, the present invention provides use of the aforementioned composition in the preparation of anti-infective drugs.

[0019] Furthermore, the anti-infective drug is a drug for resisting Acinetobacter baumannii infection.

[0020] The present invention has the following beneficial effects:

[0021] The present invention combines compound D44 with the cyclic lipopeptide antibiotic daptomycin to obtain an anti-infective composition. Each component of the anti-infective composition provided by the present invention cannot inhibit Acinetobacter baumannii on its own, but the combination of the two exhibits a synergistic effect, resulting in a significant inhibitory effect on Acinetobacter baumannii. The anti-infective composition provided by the present invention can be prepared into a pharmaceutical or antibacterial agent and can be used to prevent and treat various infectious diseases caused by Acinetobacter baumannii, thus having important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction will be given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Picture 1 This is the bactericidal curve of D44 provided in Example 3 of the present invention used in combination with daptomycin against Acinetobacter baumannii.

[0024] Picture 2 This is the bactericidal curve of D44 provided in Example 3 of the present invention in combination with vancomycin against Acinetobacter baumannii.

[0025] Picture 3 This is the bactericidal curve of D44 provided in Example 3 of the present invention in combination with meropenem against Acinetobacter baumannii.

[0026] Picture 4 This is the sterilization curve of D44 provided in Example 3 of the present invention in combination with ceftazidime against Acinetobacter baumannii.

[0027] Picture 5 This is the sterilization curve of D44 provided in Example 3 of the present invention in combination with aztreonam against Acinetobacter baumannii.

[0028] Picture 6 This is the bactericidal curve of D44 provided in Example 3 of the present invention in combination with linezolid against Acinetobacter baumannii.

[0029] Picture 7 This is the sterilization curve of D44 provided in Example 4 of the present invention and daptomycin in combination with different ratios against Acinetobacter baumannii.

[0030] Picture 8 The NPN method provided in Example 4 of the present invention is used to determine the effect of the combined use of D44 and daptomycin on the permeability of the bacterial outer membrane; wherein, p <0.01; p<0.001; p <0.0001.

[0031] Picture 9 The PI method provided in Example 4 of the present invention was used to determine the effect of the combined use of D44 and daptomycin on cell membrane integrity; wherein, the D44+daptomycin group was compared with the D44, daptomycin and control groups. p All <0.01.

[0032] Picture 10 The OD provided in Example 4 of the present invention 600nm The effect of combined use of D44 and daptomycin on the growth curve of Acinetobacter baumannii was dynamically monitored by the method; among them, the D44+daptomycin group was compared with the D44, daptomycin and control groups at the same time point. p All <0.0001, D44 compared with the control group at the same time point p <0.0001.

[0033] Picture 11 This is the effect of the combined use of D44 and daptomycin provided in Example 5 of the present invention on the bacterial morphology of Acinetobacter baumannii. DETAILED DESCRIPTION

[0034] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0035] Unless otherwise specified, the experimental methods involved in the following examples are all conventional methods in the art. For example, reference can be made to experimental manuals in the art, or the conditions recommended by the manufacturer's instructions.

[0036] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources.

[0037] The experimental instruments involved in the following examples are:

[0038] A shaking incubator (ZWY-100H, Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.), a round-bottom 96-well cell culture plate (3799, Corning), a McFadden turbidimeter (DENSICHEK, Bio-Merieux), a vortexer (MS 3digital, IKA), an autoclave (SQL510C, YAMATO), a biological safety cabinet (Class II, Nuaire), and a high-precision electronic balance (XS105, Mettler, USA) were used.

[0039] Example 1

[0040] The present invention verifies the inhibitory effects of compound D44 in combination with daptomycin, vancomycin, meropenem, ceftazidime, and aztreonam on Acinetobacter baumannii, specifically including the following:

[0041] 1. The measurement methods include:

[0042] Test strains include A. baumannii ATCC 19606, A. baumannii ATCC 17978, A. baumannii 24-13, A. baumannii 24-14, A. baumannii 24-18, A. baumannii 24-22, A. baumannii 24-24, A. baumannii 24-25, A. baumannii 24-32, A. baumannii 24-33.

[0043] Two days before the experiment, remove each strain from the -80°C freezer, inoculate it onto an MHA plate using the ice chip streak method, and incubate it overnight at 37°C. Pick 3-5 well-isolated, morphologically consistent colonies from the agar plate and inoculate them into 3 mL of CAMH medium. Incubate it at 37°C overnight.

[0044] The minimum inhibitory concentration (MIC) of each drug combination against Acinetobacter baumannii was determined using the chessboard method, and the fractional inhibitory concentration index (FICI) was calculated. The specific method is as follows: the McFarland concentration of the bacterial solution was measured using a McFarland turbidimeter, and the bacterial solution was adjusted to 0.5 McFarland (approximately 1×10 8CFU / mL), and dilute it 100 times with CAMH medium for standby. The bacterial solution should be used within 15 minutes after preparation; refer to the CLSI standard, and serially dilute the stock solutions of D44 and each antibiotic drug with CAMH broth culture medium for standby; add 100 μL of each antibiotic drug solution to the twelfth column of the round-bottom 96-well plate, and add 50 μL of CAMH broth culture medium to the remaining wells. Perform serial dilution to the second column by the two-fold dilution method, so that the concentration of each antibacterial drug in the first column is 0; add 50 μL of D44 with different concentrations after two-fold dilution to each row from the first row to the seventh row, and add 50 μL of CAMH broth culture medium to the eighth row; the diluted drug concentrations are all 4 times the final experimental concentration, and the solution in each well is 100 μL at this time; take 100 μL of the prepared bacterial solution and add it to the round-bottom 96-well plate, and the inoculation concentration is about 5×10 5 CFU / mL; wrap the 96-well plate with plastic wrap and place it in a static culture at 37 °C for 16 - 20 h; take out the 96-well plate and observe with the naked eye whether there is colony deposition at the bottom of the wells. The minimum concentration of the drug contained in the wells without bacterial growth is the MIC value (refer to the CLSI standard). Calculate the fractional inhibitory concentration index FICI, FICI = MIC 甲药联用 / MIC 甲药单用 +MIC 乙药联用 / MIC 乙药单用 . The criteria for judging synergy are as follows: FICI ≤ 0.5, synergy; 0.5 < FICI ≤ 1, additive effect; 1 < FICI ≤ 2, no effect; FICI > 2, antagonistic effect. The test strains include both ATCC standard strains and clinical isolates.

[0045] The test results are shown in Table 1 - Table 6: The combination of D44 with daptomycin, vancomycin, meropenem, ceftazidime, aztreonam, and linezolid has a broad synergistic antibacterial effect against Acinetobacter baumannii. After the combination of D44 with each of the above antibiotics, the MIC values are significantly reduced. Among them, the MIC of daptomycin is significantly reduced by 8 - 64 times, the MIC of vancomycin is significantly reduced by 64 - 256 times, the MIC of meropenem is significantly reduced by 4 - 64 times, the MIC of ceftazidime is significantly reduced by 4 - 64 times, the MIC of aztreonam is significantly reduced by 4 - 32 times, and the MIC of linezolid is significantly reduced by 4 - 8 times; through the calculation of the fractional inhibitory concentration index (FICI), the synergy is obvious, and the FICI values are all less than 0.5. The average FICI values are < 0.108; < 0.076; < 0.133; < 0.155; < 0.147; < 0.413, indicating that the combination of the two produces a strong synergistic antibacterial effect. The antibacterial activities of the combination of D44 with daptomycin and vancomycin against Acinetobacter baumannii are better than those of the combination of D44 with meropenem, ceftazidime, aztreonam, and linezolid against Acinetobacter baumannii.

[0046] Table 1 FICI values ​​of D44 combined with daptomycin

[0047]

[0048] Table 2 FICI values ​​of D44 combined with vancomycin

[0049]

[0050] Table 3 FICI values ​​of D44 combined with meropenem

[0051]

[0052] Table 4 FICI values ​​of D44 combined with ceftazidime

[0053]

[0054] Table 5 FICI values ​​of D44 combined with aztreonam

[0055]

[0056] Table 6 FICI values ​​of D44 combined with linezolid

[0057]

[0058] Example 2

[0059] The present invention uses D44 in combination with daptomycin, vancomycin, meropenem, ceftazidime, aztreonam, and linezolid to determine the time-kill curve against Acinetobacter baumannii, including the following:

[0060] Measurement methods include:

[0061] Store in -80℃ freezer A. baumannii ATCC19606 strain was removed and quickly streaked onto MHA plates, and then incubated in a 37°C incubator overnight. 3-5 single colonies were picked from the plate and inoculated into 3 mL of CAMH medium. The culture was shaken at 37°C and 220 rpm for 2-3 hours. The bacterial solution cultured to the logarithmic growth phase was inoculated into 5 mL of fresh CAMH medium at a ratio of 1:100 and incubated at 37°C overnight. The next day, the bacterial solution was diluted to approximately 2 × 10 6 CFU / mL, and dispensed into sterile small test tubes, 5 mL each.

[0062] Referring to the MIC values ​​of D44 and antibiotics alone, the antibiotic alone group, D44 alone group, and antibiotic and D44 combination group were added to the tubes with a final concentration of 1 / 4×MIC, respectively. A bacterial growth control group without antibiotics and D44 was set up, with 5 mL in each tube. In addition, to explore the optimal concentration ratio of D44 to daptomycin, the concentrations of the daptomycin and D44 combination group were set as follows: D44 (32µg / mL): daptomycin (32µg / mL) = 1:1, D44 (32µg / mL): daptomycin (16µg / mL) = 2:1, D44 (32µg / mL): daptomycin (8µg / mL) = 4:1, D44 (16µg / mL): daptomycin (32µg / mL) = 1:2, and D44 (8µg / mL): daptomycin (32µg / mL) = 1:4. Samples were taken at 0, 2, 4, 6, 8 and 24 hours for colony count using the drip method.

[0063] Drip flow colony count method: Add 100µL of bacterial solution to 900µL of 0.85% NaCl, and continue diluting ten-fold. Place 10µL of the appropriate dilution onto an MHA plate. Tilt the plate to allow the solution to flow down in a linear pattern. Set up three biological replicates for each dilution. After absorbing the solution, incubate the plate upside down in a 37°C incubator overnight. Adjust the test concentration based on the results.

[0064] With Time (h) as the horizontal axis, Log 10 CFU / mL was used as the vertical axis, and the bactericidal curve was drawn using GraphPad Prism 5. The criterion for judging drug synergy was that the colony count of the combination group decreased by ≥2 Log compared with the group with the strongest drug effect when using only one drug during the observation period. 10 CFU / mL, and the colony count of the combination group decreased by ≥2Log compared with the initial inoculation volume 10 CFU / mL, it was judged to be a synergistic effect.

[0065] The test results are as follows Pictures 1~6 As shown: D44 combined with various antibiotics A. baumannii The bactericidal curve of ATCC19606 showed that the colony count of the group treated with D44 in combination with various antibiotics (daptomycin, vancomycin, meropenem, ceftazidime, aztreonam, linezolid) decreased by ≥2Log compared with the group treated with the most effective drug alone. 10 CFU / mL, and the colony count of the combination group decreased by ≥2Log compared with the initial inoculation volume 10 CFU / mL, so the synergistic effect of D44 in combination with various antibiotics (daptomycin, vancomycin, meropenem, ceftazidime, aztreonam, linezolid) was verified; Picture 7As shown: Taking the time point 8h when the colony count of the combination group decreased the most compared with the group with the strongest efficacy of each drug as an example, the antibacterial effects of D44 and daptomycin at different concentration ratios were compared, and the synergistic antibacterial effects at different concentration ratios were obtained: 1:1>1:2>2:1>1:4>4:1.

[0066] Example 3

[0067] The present invention uses D44 in combination with daptomycin, vancomycin and meropenem to determine its effect on A. baumannii Therapeutic effects of ATCC19606 systemically infected mice.

[0068] Measurement methods include:

[0069] take out A. baumannii Inoculate 5 mL of BHI medium with ice chips from a frozen stock of ATCC19606 strain 19606 at 37°C with shaking at 220 rpm for 6 hours. Then, transfer the culture to fresh BHI medium at a ratio of 1:50 and incubate at 37°C for 18 hours. Dilute the culture with 5% highly active yeast solution to prepare the infection medium. Simultaneously, dilute the solution several times with 0.85% NaCl and drip 10 µL onto MHA plates. Allow to dry, invert, and count colonies. Female ICR mice weighing 18-22 g were randomly divided into groups of 5 per group. After the abdomen was disinfected with iodine, 0.5 mL of a 100% minimal lethal dose (MLD) bacterial solution was intraperitoneally injected. The D44 and daptomycin combination groups and their respective control groups (D44 alone and daptomycin alone) were administered intravenously once, 1 hour after infection. The other combination groups and their respective control groups were administered intravenously twice, 1 hour and 6 hours after infection. Each drug was prepared in normal saline at a volume of 0.2 mL / 20 g. The infected control group received an equal volume of normal saline. The number of animals surviving for 7 days was observed, and the survival rate of each group was calculated.

[0070] The results are shown in Tables 7 to 9: Intraperitoneal infection of mice in each group A. baumanniiATCC19606 was administered intravenously and observed for 7 consecutive days. The results showed that the survival rate of the infected control group animals was 0%; the survival rate of the animals in the 40 mg / kg D44 single dose group was 0%; the survival rate of the animals in the daptomycin 40 mg / kg, 80 mg / kg, and 120 mg / kg single dose groups was 0%. After the combination with 40 mg / kg D44, the survival rate of the animals in each combination group increased to 100%, which was significantly improved compared with the survival rate of the single dose group. This shows that the antibacterial activity in vivo is enhanced after the combination of daptomycin and D44, and the D44 and daptomycin dose ratios of 1:1, 1:2, and 1:3 all have good synergistic antibacterial activity in vivo. In addition, after daptomycin 5mg / kg, 10mg / kg, and 20mg / kg were combined with 40mg / kg of D44, the survival rates of animals in each combination group were 0%, 60%, and 100%, respectively, indicating that the in vivo synergistic antibacterial activity of D44 to daptomycin at a dose ratio of 2:1 was better than that of 4:1, and no in vivo synergistic antibacterial activity was observed at a dose ratio of 1:8. The survival rates of animals in the meropenem 20mg / kg and 40mg / kg single-dose groups were 0% and 20%, respectively. After combined with 40mg / kg of D44, the survival rates of animals in the combination-dose groups increased to 60% and 80%, respectively, which were significantly higher than the survival rates of animals in the single-dose groups, indicating that the in vivo antibacterial activity of meropenem and D44 was enhanced after combination. The survival rate of animals in the single-dose groups of vancomycin 80mg / kg and 160mg / kg was 0%. After the combination of 40mg / kg dose of D44, the survival rate of animals in each combination dose group increased to 100%, which was significantly higher than the survival rate of the single-dose group. This shows that the antibacterial activity of vancomycin and D44 in vivo is enhanced after the combination of vancomycin and D44. The above results show that the combination of D44 and meropenem, and the combination of 1901 and vancomycin can A. baumannii D44 has a protective effect against systemic infection and mortality in mice caused by ATCC19606. D44, when combined with daptomycin, meropenem, and vancomycin, exhibits strong in vivo anti-Acinetobacter baumannii activity and is effective in treating Acinetobacter baumannii infections. The combination of D44 and daptomycin is more effective than the combination of D44 and meropenem or D44 and vancomycin.

[0071] Table 7 Daptomycin combined with D44 antibiotic A. baumannii Death rate of mice in each group infected with ATCC19606

[0072]

[0073] Infectious dose 1×10 7 CFU / mouse.

[0074] Table 8 Meropenem combined with D44 A. baumanniiDeath rate of mice in each group infected with ATCC19606

[0075]

[0076] Infectious dose 1×10 7 CFU / mouse.

[0077] Table 9 Vancomycin combined with D44 antibiotics A. baumannii Death rate of mice in each group infected with ATCC19606

[0078]

[0079] Infectious dose 1×10 7 CFU / mouse.

[0080] Example 4

[0081] The present invention investigates the level of damage to the inner and outer membranes of Acinetobacter baumannii by combining D44 with daptomycin, specifically including the following:

[0082] Measurement methods include:

[0083] Store in -80℃ freezer A. baumannii Remove the ATCC19606 strain and quickly streak it onto an MHA plate. Incubate it in a 37°C incubator overnight. Pick 3-5 single colonies from the plate and inoculate them into 3 mL of CAMH medium. Incubate the culture at 37°C with shaking at 220 rpm for 2-3 hours. Dilute the bacterial suspension to a McFarland concentration of 0.5 (approximately 1 × 10 8 CFU / mL) and aliquoted into sterile glass tubes. Add D44, DAP, and D44+DAP to each tube, respectively, and set up a control group of bacteria without drug. Incubate with shaking at 180 rpm and 37°C.

[0084] After 2 hours of incubation, 1 mL of bacterial suspension was collected from each well and centrifuged at 12,300 g for 3 minutes. The cells were washed once with 5 mM HEPES (pH 7.2) and resuspended in the same volume of 5 mM HEPES. N-Phenyl-1-naphthylamine (NPN) was added to a final concentration of 10 µM. Each resuspension was added to a 96-well plate with a black bottom, 100 µL per well. The fluorescence of each well was measured using a microplate reader with an excitation wavelength of 355 nm and an emission wavelength of 405 nm. The NPN uptake factor was calculated as (fluorescence value) = 样品 +NPN - Fluorescence value 样品不含NPN ) / (fluorescence value HEPES+NPN -Fluorescence value HEPES 不含 NPN).

[0085] At 0 h, 2 h, and 4 h, 1 mL of bacterial suspension was collected and centrifuged at 12,300 g for 3 min. The cells were washed once with sterile 1× PBS and thoroughly resuspended in the same volume of 1× PBS. Propidium Iodide (PI) fluorescent reagent was added to a final concentration of 2.5 µM. Each resuspension was added to a 96-well plate with a black bottom, 100 µL per well. Fluorescence values ​​were measured in each well using a microplate reader. Excitation wavelength was 535 nm, and emission wavelength was 617 nm. Samples were taken at each time point to measure the OD value of the bacterial suspension. 600 nm value.

[0086] The test results are as follows Picture 8-10 As shown: NPN is a hydrophobic fluorescent probe that is soluble in the lipid bilayer. When the cell outer membrane is damaged, NPN can penetrate the outer membrane and enter the phospholipid layer, generating fluorescence. The amount of NPN entering the cell is monitored by detecting the increase in the solution's fluorescence intensity, thus reflecting the permeability of the bacterial outer membrane. In this experiment, samples were taken at various time points and centrifuged to remove the compound and resuspend them in HEPES. NPN was then added and fluorescence values ​​were measured. This method eliminates the influence of compound fluorescence values. Measurements were performed 2 hours after drug treatment, and the results showed ( Picture 8 ), compared with the control group, there was no significant change in the NPN fluorescence intensity in the daptomycin alone group ( p >0.05); the NPN fluorescence intensity in the D44 alone group increased significantly ( p <0.0001); the NPN fluorescence intensity in the D44 combined with daptomycin group increased significantly ( p <0.01).

[0087] The results showed that: daptomycin alone could not destroy the cell outer membrane and could not increase the outer membrane permeability; D44 alone and D44 combined with daptomycin both destroyed the cell outer membrane and increased the cell outer membrane permeability. It is worth noting that the fluorescence intensity of the D44 alone group was significantly higher than that of the combined group ( p <0.0001). This may be due to the fact that daptomycin, a cyclic lipopeptide antibiotic, targets cell membrane phospholipids. Its lipophilic tail inserts into the bacterial cell membrane, enabling it to bind to negatively charged phospholipids (such as phosphatidylglycerol). D44, on the other hand, disrupts bacterial outer membrane permeability by inhibiting lipopolysaccharide synthesis. It is hypothesized that the combined use of D44 and daptomycin allows daptomycin to penetrate the outer membrane and target the inner or outer membrane phospholipid bilayer. During this process, daptomycin may occupy the hydrophobic binding domains of outer membrane phospholipids, resulting in competitive inhibition with NPN. Furthermore, bacterial death induced by the combined use may lead to membrane collapse, further reducing the number of NPN binding sites. Ultimately, although the NPN fluorescence intensity in the D44 combination group was higher than in the control group, the magnitude of the increase was significantly lower than in the D44 alone group.

[0088] PI is a nuclear staining reagent. The intact bacterial cell membrane (inner membrane) has a barrier function. PI cannot penetrate the intact lipid bilayer structure and therefore cannot enter the cell. When the bacterial cell inner membrane becomes permeable or pores are formed due to toxins, environmental stress, or drugs (such as antibiotics), PI can penetrate the damaged membrane and enter the cell. PI is embedded in double-stranded DNA and releases red fluorescence. The measurement results show ( Picture 9 ), 4 hours after drug treatment, compared with the control group, there was no significant change in the PI fluorescence values ​​of the D44 alone group and the daptomycin alone group ( p >0.05, while the D44 combined with daptomycin group showed a significant increase in PI fluorescence value ( p <0.01). Mechanism analysis showed that although D44 can destroy the permeability of the bacterial outer membrane, it has no significant effect on the inner cell membrane when used alone, and PI cannot penetrate the intact lipid bilayer to enter the cell. Daptomycin is a membrane lipid-targeting antibiotic. When used alone, it is difficult for it to access the inner membrane phospholipid target because it cannot penetrate the intact outer membrane. When the two drugs are used in combination, D44-mediated outer membrane destruction significantly improves the transmembrane permeation efficiency of daptomycin, allowing it to fully access the inner membrane phospholipids and cause membrane damage. This dual outer membrane-inner membrane targeting ultimately leads to the loss of membrane integrity, allowing PI to enter the cell and bind to DNA. 600nm Dynamic detection results of values ​​( Picture 10 ) further confirmed that the absorbance of the bacterial solution decreased significantly and the turbidity of the bacterial solution decreased 4 hours after D44 was combined with daptomycin, indicating that membrane damage caused the lysis and death of a large number of bacteria.

[0089] This study showed that the combination of D44 and daptomycin has a synergistically enhanced double-membrane targeting effect on Acinetobacter baumannii. After D44 destroys the outer membrane of the cell, it promotes daptomycin to penetrate the outer membrane and act on the inner membrane, thereby destroying the integrity of the cell membrane and ultimately leading to bacterial lysis and death.

[0090] Example 5

[0091] The present invention investigates the effects of the combined use of D44 and daptomycin on the bacterial morphology of Acinetobacter baumannii, specifically including the following:

[0092] Measurement methods include:

[0093] Store in -80℃ freezer A. baumannii Remove the ATCC19606 strain and quickly streak it onto an MHA plate. Incubate it in a 37°C incubator overnight. Pick 3-5 single colonies from the plate and inoculate them into 3 mL of CAMH medium. Incubate the culture at 37°C with shaking at 220 rpm for 4-5 hours. Dilute the bacterial suspension to a McFarland concentration of 0.5 (approximately 1 × 10 8CFU / mL) and aliquoted into sterile glass tubes. Add D44 (32 µg / mL), DAP (32 µg / mL), and D44 (32 µg / mL) + DAP (32 µg / mL) to each tube, respectively. A control group containing no drug was also established. Culture was shaken at 180 rpm and 37°C for 6 h.

[0094] Transfer 1.5 mL of bacterial solution to each 1.5 mL EP tube and centrifuge at 3000 × g for 10 min (a total of 3 mL of bacterial solution can be centrifuged multiple times). Discard the supernatant and wash the cells one to two times with 1× PBS. Discard the supernatant. Fill each EP tube with 2.5% glutaraldehyde fixative along the length of the tube, taking care not to disperse the cells. Seal the tube with parafilm and fix overnight at 4°C. The next day, send the sample to Zhongke Baice Co., Ltd. for scanning electron microscopy (SEM).

[0095] The test results are as follows Picture 11 As shown: In the D44 alone group, the bacteria changed from long rods to shorter, oval-shaped, and the outer layer of the bacteria became smooth. In the daptomycin alone group, no obvious changes were seen in the bacteria. In the D44 and daptomycin combination group, due to the changes in the permeability of the bacterial cell outer membrane and the destruction of the inner membrane, the double membrane damage caused cell dehydration and shrinkage, the bacterial surface collapsed and shrank, and the morphology was distorted.

[0096] Analysis of the antibacterial mechanism of the combined use of D44 and daptomycin: Studies have confirmed that daptomycin, a cyclic lipopeptide antibiotic primarily targeting Gram-positive bacteria, has a lipophilic tail that inserts into the bacterial cell membrane and specifically binds to negatively charged phospholipids (such as phosphatidylglycerol). By inhibiting bacterial protein synthesis and preventing bacterial cell wall synthesis, it inhibits bacterial growth. It is worth noting that neither daptomycin nor D44 has individual antibacterial activity against Acinetobacter baumannii. This study found that the combination of D44 and daptomycin can produce synergistic antibacterial activity against Acinetobacter baumannii. Its dual-membrane targeting mechanism is manifested as follows: D44 destroys the permeability barrier of the bacterial outer membrane, promoting daptomycin penetration and targeting the inner membrane, triggering the structural collapse of the membrane lipid bilayer, ultimately triggering cell dehydration and shrinkage, leading to bacterial lysis and death.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. An anti-Acinetobacter baumannii composition, characterized in that: include: Compound D44 and antibiotics; The mass ratio of the compound D44 to the antibiotic is 1:(0.01~16); The antibiotics include: one or more of daptomycin, vancomycin, meropenem, ceftazidime, aztreonam or linezolid; the chemical structure of the compound D44 is shown in Formula I: 。 2. The composition according to claim 1, characterized in that The antibiotic is daptomycin or vancomycin, and the mass ratio of the compound D44 to the antibiotic is 1:(0.1~4).

3. The composition according to claim 2, characterized in that The antibiotic is daptomycin or vancomycin, and the mass ratio of the compound D44 to the antibiotic is 1:(0.25~4).

4. The composition according to claim 3, characterized in that The antibiotic is daptomycin, and the mass ratio of the compound D44 to daptomycin is 1:(0.5~2).

5. The composition according to claim 4, characterized in that The concentration of the compound D44 is 4-32 μg / mL; the concentration of daptomycin is 4-32 μg / mL.

6. A product, characterized in that The product comprises the composition according to any one of claims 1 to 5; the product is a medicine, an antibacterial agent, a disinfectant or an antiseptic.

7. The product according to claim 6, characterized in that The product is in the form of one or more of a solid dosage form, a semisolid dosage form, a liquid dosage form or a gaseous dosage form.

8. Use of the composition according to any one of claims 1 to 5 in the preparation of an anti-infective drug; the anti-infective drug is a drug for treating Acinetobacter baumannii infection.

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