An antibacterial agent and preparation method thereof

Abstract

The present invention provides an antibacterial agent and a preparation method thereof. The antibacterial agent is used to fight multidrug-resistant Gram-negative bacteria. The antibacterial agent comprises: a CO-releasing molecule for releasing CO, wherein the CO-releasing molecule comprises CORM-401 powder; and an antibiotic solution. The present invention solves the technical problem that there is currently a lack of safe and effective methods to destroy the outer membrane barrier of multidrug-resistant Gram-negative bacteria, resulting in poor antibacterial effects of antibiotics, and achieves the technical effect of safely and effectively destroying the outer membrane barrier of multidrug-resistant Gram-negative bacteria, thereby improving the antibacterial effect of multidrug-resistant Gram-negative bacteria.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an antibacterial agent and a preparation method thereof. Background Art

[0002] The emergence of multidrug-resistant Gram-negative bacteria, such as multidrug-resistant Pseudomonas aeruginosa (P. aeruginosa), multidrug-resistant Escherichia coli (E. coli), and multidrug-resistant Acinetobacter baumannii (A. baumannii), has posed a serious threat to global public health. The unique outer membrane structure of these bacteria leads to their strong antibiotic resistance and makes them an increasingly important factor in the increased mortality associated with antibiotic-resistant infections. The outer membrane structure of Gram-negative bacteria is composed of two monolayers, of which the inner monolayer is composed of asymmetric glycerophospholipids, which is essential for maintaining the structural stability of the outer membrane. The outer monolayer is densely covered with lipopolysaccharide molecules, forming a strong barrier that effectively blocks the entry of antimicrobial drugs, including a variety of commonly used clinical antibiotics. Due to the limited drug accessibility and low bioavailability of multidrug-resistant Gram-negative bacteria, these bacteria are prone to cause serious and sometimes even fatal hospital-acquired infections, such as bacterial pneumonia. In this context, there is an urgent need to explore effective strategies to disrupt the outer membrane barrier and improve the antibacterial effect against multidrug-resistant Gram-negative bacteria.

[0003] At present, there are two main approaches to this problem. The first approach involves cationic polymer-mediated perturbation of the outer membrane, which is achieved through electrostatic and hydrophobic interactions between cationic polymers and LPS. Although this approach is effective, it faces serious disadvantages in practical applications, such as high systemic toxicity, the risk of inducing immune and other adverse reactions, and high development costs. In addition, given the key role of glycerophospholipids (GPL) and LPS in the outer membrane barrier and the need for them to be transported across the membrane from the synthesis site to the outer membrane through various transport proteins, the development of small molecule inhibitors that can selectively target synthesis-related enzymes and protein transporters has become the focus of current research. Recent progress has developed several small molecule drugs for this purpose. Cerastecins and the quinoline compound G907 can inhibit MsbA activity and block the turnover of LPS from the cytoplasm to the inner membrane. Similarly, Thanatin and Murepavadin can inhibit the activity of LptA-G, preventing the transport of LPS from the periplasmic space to the outer membrane and its subsequent anchoring. However, most of these drugs can only inhibit specific steps in the biosynthesis or transport process and are not sufficient to destroy the outer membrane barrier. On the one hand, these drugs must enter bacteria to work, and the outer membrane barrier firmly hinders their entry, further weakening the destructive effect. On the other hand, because the targets and mechanisms of action of these drugs make them selective for specific strains or specific types of lipopolysaccharides, this selectivity limits their broad-spectrum antibacterial activity.

[0004] The problem is that there is currently a lack of safe and effective methods to destroy the outer membrane barrier of multidrug-resistant Gram-negative bacteria, resulting in poor antibacterial effect of antibiotics. Summary of the invention

[0005] The present invention solves the technical problem that there is currently a lack of safe and effective methods to destroy the outer membrane barrier of multidrug-resistant Gram-negative bacteria, resulting in poor antibacterial effect of antibiotics, and achieves the technical effect of safely and effectively destroying the outer membrane barrier, thereby improving the antibacterial treatment effect of multidrug-resistant Gram-negative bacteria.

[0006] To solve the above problems, the present invention provides an antibacterial agent, which is used to fight multi-drug resistant Gram-negative bacteria. The antibacterial agent comprises: CO releasing molecules for releasing CO, wherein the CO releasing molecules comprise CORM-401 powder; and an antibiotic solution.

[0007] Compared with the prior art, the technical effect achieved by adopting this technical solution is: CO is an important endogenous signal molecule, which is biologically inert and has good biocompatibility at low concentrations, and is non-selective to bacteria. Due to the ultra-low molecular weight and high diffusivity of CO, it can quickly penetrate bacteria and inhibit ATP synthesis by accelerating mitochondrial metabolic exhaustion. The synthesis and transport of glycerophospholipids and lipopolysaccharides in bacteria involve a series of complex biological processes, which are heavily dependent on ATP hydrolysis for energy supply. CORM-401 powder is a CO-releasing molecule that is sensitive to oxidants and can be used for pathological studies mediated by inflammation and oxidative stress. Therefore, by utilizing the unique properties of CO and the huge energy requirements required for the synthesis and transport of GPL and LPS in bacteria, the present invention effectively and safely destroys the outer membrane barrier of multidrug-resistant Gram-negative bacteria by targeted inhibition of ATP synthesis and the synthesis and transport of GPL and LPS, promotes the penetration of various antibacterial drugs, and significantly improves their antibacterial effects. At the same time, it can also reduce the dosage of antibiotics, improve the efficacy, and significantly reduce the risk of multidrug resistance.

[0008] In one example of the present invention, the antibiotic solution includes at least one of a levofloxacin solution, a meropenem solution and an azithromycin solution.

[0009] Compared with the existing technology, the technical effect achieved by adopting this technical solution is: levofloxacin, meropenem and azithromycin are widely used first-line antibiotics, and their permeability to the outer membrane barrier of Gram-negative bacteria is very limited, so the antibacterial results are intuitive and easy to observe.

[0010] In one example of the present invention, the antimicrobial agent further comprises an exciter, and the exciter is used to excite the CO-releasing molecules to release CO.

[0011] Compared with the existing technology, the technical effect achieved by adopting this technical solution is as follows: under the action of the stimulant, the CORM-401 molecule can release CO, which can then destroy the outer membrane of multidrug-resistant Gram-negative bacteria, allowing antibiotics to enter the interior of the bacteria and exert an antibacterial effect.

[0012] In one embodiment of the present invention, the activator is H2O2.

[0013] Compared with the existing technology, the technical effect achieved by adopting this technical solution is: although the mixture of free CORM-401 and antibiotics cannot effectively adhere to the outer membrane surface of the outer membrane of multidrug-resistant Gram-negative bacteria, it can still release CO when H2O2 is added or ROS exists in the infection site, which can then destroy the outer membrane, allowing the antibiotics to enter the bacteria and exert an antibacterial effect.

[0014] In one embodiment of the present invention, the antimicrobial agent further comprises a buffer.

[0015] In one embodiment of the present invention, the buffer comprises a phosphate buffer.

[0016] Compared with the prior art, the technical effect achieved by adopting this technical solution is that the buffer solution can dilute the antimicrobial agent, thereby avoiding the precipitation problem caused by excessively high concentration of the antimicrobial agent, thereby improving the performance and safety of the antimicrobial agent.

[0017] In one example of the present invention, the concentration of the levofloxacin solution is 4 μg / mL, the concentration of the meropenem solution is 8 μg / mL, and the concentration of the azithromycin solution is 32 μg / mL.

[0018] In one example of the present invention, the concentration of H2O2 is 100 μM.

[0019] In one example of the present invention, the concentration of the CORM-401 solution is 20 μg / mL.

[0020] Compared with the prior art, the technical effect achieved by adopting this technical solution is that the combination of antibiotic solution at this concentration and CO has a good antibacterial effect.

[0021] The present invention also provides a method for preparing the antibacterial agent in any of the above examples, the preparation method comprising the following steps: S10: obtaining a CORM-401 solution; S20: obtaining an antibiotic solution; S30: mixing the CORM-401 solution and the antibiotic solution to obtain the antibacterial agent.

[0022] Compared with the prior art, the technical effect achieved by adopting this technical solution is as follows: the above preparation method is simple to operate and easy to implement. CORM-401 solution can be obtained by dissolving CORM-401 powder with methanol. At the same time, CORM-401 solutions of different concentrations can also be prepared according to the situation.

[0023] In one embodiment of the present invention, the preparation method further comprises the following steps: S40: adding phosphate buffer to the antibacterial agent in S30 to obtain a final antibacterial agent.

[0024] Compared with the prior art, the technical effect achieved by adopting this technical solution is that the above preparation method is simple to operate and easy to implement. After adding phosphate buffer, the final antibacterial agent is not easy to precipitate. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The results of in vitro antibacterial activity test of free CORM-401 and antibiotic mixture against multidrug-resistant Pseudomonas aeruginosa provided in the embodiment of the present invention Figure 1 .

[0026] Figure 2The results of in vitro antibacterial activity test of free CORM-401 and antibiotic mixture against multidrug-resistant Pseudomonas aeruginosa provided in the embodiment of the present invention Figure 2 .

[0027] Figure 3 The results of in vitro antibacterial activity test of free CORM-401 and antibiotic mixture against multidrug-resistant Pseudomonas aeruginosa provided in the embodiment of the present invention Figure 3 .

[0028] Figure 4 The results of in vitro antibacterial activity test of free CORM-401 and antibiotic mixture against multidrug-resistant Pseudomonas aeruginosa provided in the embodiment of the present invention Figure 4 .

[0029] Figure 5 The results of in vitro antibacterial activity test of free CORM-401 and antibiotic mixture against multidrug-resistant Pseudomonas aeruginosa provided in the embodiment of the present invention Figure 5 .

[0030] Figure 6 The results of in vitro antibacterial activity test of free CORM-401 and antibiotic mixture against multidrug-resistant Pseudomonas aeruginosa provided in the embodiment of the present invention Figure 6 .

[0031] Figure 7 The results of in vitro antibacterial activity test of free CORM-401 and antibiotic mixture against multidrug-resistant Pseudomonas aeruginosa provided in the embodiment of the present invention Figure 1 .

[0032] Figure 8 The results of in vitro antibacterial activity test of free CORM-401 and antibiotic mixture against multidrug-resistant Pseudomonas aeruginosa provided in the embodiment of the present invention Figure 2 .

[0033] Fig. 9 Results of the penetration test of the antibiotics provided in the embodiments of the present invention alone and in combination with CORM-401 on multidrug-resistant Pseudomonas aeruginosa Figure 1 .

[0034] Fig.10 Results of the penetration test of the antibiotics provided in the embodiments of the present invention alone and in combination with CORM-401 on multidrug-resistant Pseudomonas aeruginosa Figure 2 .

[0035] Fig.11 Results of in vivo experimental test of CORM-401 and LEV mixed solution against Pseudomonas aeruginosa pneumonia provided in the embodiment of the present invention Figure 1 .

[0036] Fig.12 Results of in vivo experimental test of CORM-401 and LEV mixed solution against Pseudomonas aeruginosa pneumonia provided in the embodiment of the present invention Figure 2 .

[0037] Fig.13 Results of in vivo experimental test of CORM-401 and LEV mixed solution against Pseudomonas aeruginosa pneumonia provided in the embodiment of the present invention Figure 3 .

[0038] Fig.14 Immunofluorescence staining test results of lung tissue provided by the embodiment of the present invention Figure 1 .

[0039] Fig.15 Immunofluorescence staining test results of lung tissue provided by the embodiment of the present invention Figure 2 .

[0040] Fig.16 Immunofluorescence staining test results of lung tissue provided by the embodiment of the present invention Figure 3 .

[0041] Fig.17 The test results of the immunomodulatory effect of the mixed solution of CORM-401 and LEV on Pseudomonas aeruginosa pneumonia provided in the embodiment of the present invention Figure 1 .

[0042] Fig.18 The test results of the immunomodulatory effect of the mixed solution of CORM-401 and LEV on Pseudomonas aeruginosa pneumonia provided in the embodiment of the present invention Figure 2 .

[0043] Fig.19 The test results of the immunomodulatory effect of the mixed solution of CORM-401 and LEV on Pseudomonas aeruginosa pneumonia provided in the embodiment of the present invention Figure 3 .

[0044] Fig. 20 The test results of the immunomodulatory effect of the mixed solution of CORM-401 and LEV on Pseudomonas aeruginosa pneumonia provided in the embodiment of the present invention Figure 4 .

[0045] Fig.21 A graph showing HbCO concentration in mouse plasma after different treatments provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0046] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0047] Embodiment 1:

[0048] The invention provides an antibacterial agent, which is used for resisting multi-drug resistant Gram-negative bacteria. The antibacterial agent comprises: a CO releasing molecule for releasing CO, wherein the CO releasing molecule comprises CORM-401 powder; and an antibiotic solution.

[0049] Specifically, in response to these practical challenges faced by current progress, the present invention pioneered the use of CO gas to destroy the outer membrane barrier of multidrug-resistant Gram-negative bacteria. CO is an important endogenous signal molecule that is biologically inert and has good biocompatibility at low concentrations and is non-selective for bacteria. Due to the ultra-low molecular weight and high diffusivity of CO, it can quickly penetrate bacteria and inhibit ATP synthesis by accelerating mitochondrial metabolic exhaustion. The synthesis and transport of glycerophospholipids and lipopolysaccharides in bacteria involve a series of complex biological processes, which are heavily dependent on ATP hydrolysis for energy supply. CORM-401 powder is a CO-releasing molecule that is sensitive to oxidants and can be used for pathological studies mediated by inflammation and oxidative stress. Therefore, by utilizing the unique properties of CO and the huge energy requirements required for the synthesis and transport of GPL and LPS in bacteria, the present invention effectively and safely destroys the outer membrane barrier of multidrug-resistant Gram-negative bacteria by targeted inhibition of ATP synthesis and the synthesis and transport of GPL and LPS, promotes the penetration of various antibacterial drugs, and significantly improves their antibacterial effects. At the same time, it can reduce the use of antibiotics, improve efficacy, and significantly reduce the risk of multidrug resistance.

[0050] Furthermore, the antibiotic solution includes at least one of a levofloxacin solution, a meropenem solution and an azithromycin solution.

[0051] Specifically, levofloxacin, meropenem and azithromycin are widely used first-line antibiotics, which have very limited permeability to the outer membrane barrier of Gram-negative bacteria, so the antibacterial results are intuitive and easy to observe. The above antibiotic solutions can act on bacteria with CORM-401 alone, or in combination with CORM-401.

[0052] Furthermore, the antimicrobial agent also includes an exciter, which is used to excite the CO-releasing molecules to release CO.

[0053] Specifically, under the action of the stimulant, the CORM-401 molecule can release CO, which can then destroy the outer membrane of multidrug-resistant Gram-negative bacteria, allowing antibiotics to enter the interior of the bacteria and exert an antibacterial effect.

[0054] Furthermore, the activator is H2O2.

[0055] Specifically, although the mixture of free CORM-401 and antibiotics cannot effectively attach to the outer membrane surface of multidrug-resistant Gram-negative bacteria, it can still release CO in the presence of H2O2 or ROS at the infection site, which can then destroy the outer membrane, allowing the antibiotic to enter the interior of the bacteria and exert an antibacterial effect.

[0056] Furthermore, the antimicrobial agent also includes a buffer.

[0057] Specifically, the buffer includes phosphate buffer.

[0058] Specifically, the buffer solution can dilute the antimicrobial agent, thereby avoiding precipitation problems caused by excessively high concentrations of the antimicrobial agent, thereby improving the performance and safety of the antimicrobial agent.

[0059] Further, the concentration of the levofloxacin solution is 4 μg / mL, the concentration of the meropenem solution is 8 μg / mL, the concentration of the azithromycin solution is 32 μg / mL, the concentration of H2O2 is 100 μM, and the concentration of the CORM-401 solution is 20 μg / mL.

[0060] Specifically, the antibiotic solution at this concentration combined with CO has a good antibacterial effect.

[0061] Embodiment 2:

[0062] The present invention also provides a method for preparing the antibacterial agent in any of the above examples, the preparation method comprising the following steps:

[0063] S10: Obtain CORM-401 solution;

[0064] S20: Obtain antibiotic solution;

[0065] S30: mixing the CORM-401 solution and the antibiotic solution to obtain an antibacterial agent.

[0066] Specifically, the above preparation method is simple to operate and easy to implement. The CORM-401 solution can be obtained by dissolving CORM-401 powder in methanol. At the same time, CORM-401 solutions of different concentrations can also be prepared according to the situation.

[0067] Furthermore, the preparation method further comprises the following steps:

[0068] S40: adding phosphate buffer to the antibacterial agent in S30 to obtain a final antibacterial agent.

[0069] Specifically, the above preparation method is simple to operate and easy to implement. After adding phosphate buffer, the final antibacterial agent is not easy to precipitate.

[0070] Embodiment three:

[0071] The present invention conducted an in vitro antibacterial verification experiment on the ability of CO to destroy the outer membrane of multi-drug resistant Gram-negative bacteria, as follows:

[0072] Weigh 10 mg of CORM-401 powder, dissolve it in methanol and prepare CORM-401 methanol solutions of different concentrations (0.8 mg / mL or 6 mg / mL) for later use.

[0073] Weigh 10 mg of levofloxacin (LEV), meropenem (MEC), and azithromycin (AZM) respectively; dissolve levofloxacin in DMSO to a concentration of 1.28 mg / mL; dissolve meropenem and azithromycin in methanol to a concentration of 1.28 mg / mL.

[0074] According to the needs of subsequent experiments, the CORM-401 methanol solution was mixed with the antibiotic solution, and PBS was used for dilution according to the actual situation. To trigger the release of CO, different volumes of H2O2 were added to the mixed solution of CORM-401 and antibiotics.

[0075] The antibiotics were diluted along the horizontal axis to maintain the concentration of 64, 32, 16, 8, 4, 2, 1, 0 μg / mL. CORM-401 was diluted along the vertical axis to maintain the concentration of 40, 20, 10, 0) μg / mL. 10 μL H2O2 and 170 μL Pseudomonas aeruginosa suspension were added. The final concentration of H2O2 was 100 μM.

[0076] A checkerboard assay was performed, and P. aeruginosa at an OD value of 0.4 was inoculated into a 96-well plate. Then, levofloxacin solution, meropenem solution, and azithromycin solution were added vertically to the plate at concentrations ranging from 1 to 64 μg / mL. At the same time, CORM-401 was added horizontally to each row of the plate at a concentration range of 10 to 40 μg / mL. After the addition of antibiotics and CORM-401, H2O2 was also added to a final concentration of 100 μM. The minimum inhibitory concentration (MIC) was defined as the lowest antibiotic concentration at which there was no significant bacterial growth after incubation at 37°C for 24 hours. The bacterial growth curve was monitored every hour for 8 hours in the presence of each antibiotic and CORM-401 alone and in combination. The concentrations of levofloxacin, meropenem, azithromycin, and CORM-401 were set at 4, 8, 32, and 20 μg / mL, respectively, while the H2O2 concentration was kept constant at 100 μM. In addition, 100 µL of bacterial suspension without visible growth was taken for plate count test, and the minimum bactericidal concentration (MBC) was defined as the lowest antibiotic concentration at which no colonies were formed. Figure 1 , Figure 1This is the in vitro antibacterial activity test result of free CORM-401 and antibiotic mixture against multidrug-resistant Pseudomonas aeruginosa (MDR P. aeruginosa). Figures 1 to 6 Representative heatmap of CORM-401 in combination with levofloxacin (LEV), meropenem (MEC), and azithromycin (AZM) against P. aeruginosa. Growth curves of P. aeruginosa over 8 hours in the presence of 4 μg / mL LEV, 8 μg / mL MEC, and 32 μg / mL AZM alone and in combination with 20 μg / mL CORM-401. Data are presented as mean ± SD (n = 3).

[0077] It is clear that the inhibitory effect of 20 μg / mL CORM-401 combined with 4 μg / mL LEV or 8 μg / mL MEC was comparable to that of either antibiotic alone at 16 μg / mL. Similarly, the antibacterial effect of 20 μg / mL CORM-401 combined with 32 μg / mL AZM was comparable to that of 64 μg / mL AZM alone. These results indicate that CO significantly reduced the minimum inhibitory concentration (MIC) of these antibiotics by 4-fold and 2-fold for LEV and MEC and 2-fold for AZM, indicating that CO-induced outer membrane barrier disruption facilitated antibiotic penetration. Bacterial growth curves further showed that the CO-antibiotic combination rapidly and persistently inhibited bacterial proliferation, with significant inhibitory effects observed after only 1 hour of incubation and lasting for more than 8 hours. In summary, CO significantly enhanced the antibacterial effects of LEV, MEC, and AZM against multidrug-resistant P. aeruginosa, both in terms of bacterial growth inhibition and overall antibacterial activity.

[0078] See also Figures 7 and 8 , is the in vitro antibacterial activity test results of free CORM-401 and antibiotic mixture against multidrug-resistant Pseudomonas aeruginosa. Figure 7 The images of the culture plates after using 4 μg / mL LEV, 16 μg / mL MEC and 32 μg / mL AZM alone and in combination with 20 μg / mL CORM-401; Figure 8 Effects of CO-antibiotic combinations on the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of all tested antibiotics. Data are presented as mean ± SD (n = 3). Plate count results after 24 h of treatment with different formulations, Figures 7 and 8The results showed that the combination of CO and the three antibiotics effectively eliminated all bacteria, while a large number of viable bacteria still survived in the groups using antibiotics or CORM-401 alone. This indicates that CO significantly enhanced the antibacterial effect of antibiotics. Additional minimum bactericidal concentration (MBC) assays showed that CO reduced the MBC of LEV, MEC, and AZM from 32 μg / mL, 32 μg / mL, and 128 μg / mL to 4 μg / mL, 16 μg / mL, and 32 μg / mL, respectively, an 8-fold, 2-fold, and 4-fold decrease, respectively. These findings confirm the potentiation effect of CO on antibiotic activity, even in a mixed solution with CORM-401, and demonstrate the potential of the CO-induced outer membrane barrier disruption strategy to reduce antibiotic doses and mitigate the risk of developing multidrug resistance.

[0079] The relevant samples were analyzed using an Agilent 1260 Infinity high-performance liquid chromatography (HPLC) system. Levofloxacin (4 μg / mL) and meropenem (8 μg / mL) were incubated with P. aeruginosa cultures with or without CORM-401 (20 μg / mL) for 1, 2, and 4 hours, respectively. After incubation, the cultures were centrifuged to collect the supernatant for subsequent drug concentration determination. For the detection of levofloxacin, an eluent consisting of 10% acetic acid and acetonitrile (volume ratio of 80:20) was used, and the UV detection wavelength was set to 300 nm. For the detection of meropenem, a phosphate buffer containing 10 mM potassium dihydrogen phosphate (pH 3) and acetonitrile (volume ratio of 90:10) was used, and the UV detection wavelength was set to 298 nm. The parameters of HPLC were set as follows: flow rate of 1.0 mL / min, injection volume of 20 μL, and analytical reversed-phase C-18 ODS column [ZORBAX Eclipse XDB-C18, 150 mm × 4.6 mm, 5 μm]. The control group was a culture containing only levofloxacin (4 μg / mL) or meropenem (8 μg / mL) without P. aeruginosa. The results were processed by peak area integration, and the permeability was calculated using the following formula: Permeability (%) = (1 - sample peak area / control peak area) × 100. The test results are shown in Figure 3 .

[0080] See also Figure 9-10, the results of the test of the penetration of antibiotics alone and in combination with CORM-401 on multidrug-resistant Pseudomonas aeruginosa, the permeability of levofloxacin (LEV) and meropenem (MEC) to Pseudomonas aeruginosa at different incubation time points. Within the first 2 hours, the penetration efficiency of LEV increased from 2.60% to 10.96%, an increase of 4.4 times; the penetration efficiency of MEC increased from 2.83% to 9.75%, an increase of 3.5 times. Therefore, the membrane-disrupting effect of CORM-401 significantly enhanced the permeability of levofloxacin (LEV) and meropenem (MEC) to multidrug-resistant Pseudomonas aeruginosa. However, with the extension of incubation time, the CO-enhanced permeability gradually slowed down, which may be due to the increase in outer membrane permeability leading to antibiotic leakage, or bacterial lysis to release previously permeated antibiotics.

[0081] Embodiment 4:

[0082] Pseudomonas aeruginosa pneumonia is a serious inflammatory lung disease with a complex course and high mortality rate, posing a serious threat to burn patients and those with impaired immune systems. In view of the significant membrane-destructive effect of CO observed in in vitro experiments and its significant enhancement of the efficacy of antibiotics, this example selected 24 female ICR mice aged 6-8 weeks and weighing 20-25 grams to establish a pneumonia infection model induced by Pseudomonas aeruginosa to explore the feasibility of combining the membrane-destructive effect of CO with antibiotics to combat Gram-negative bacterial infections. It is worth noting that after the mice are infected, CORM-401 can release CO in the presence of ROS in the infected site.

[0083] First, each mouse was anesthetized by intraperitoneal injection of 75 mg / kg sodium pentobarbital, followed by intratracheal injection of 20 μL of Pseudomonas aeruginosa (5 × 10 7CFU / mL) to establish a pneumonia mouse model. Four hours after infection, the mice were randomly divided into four treatment groups, with 6 mice in each group, and 20 μL of different solutions were given: phosphate buffer, CORM-401 (600 μg / mL), levofloxacin (120 μg / mL), and a mixed solution of CORM-401 (600 μg / mL) and levofloxacin (120 μg / mL). Eight hours after infection, each group was given the same dose of drugs again. At the same time, 6 healthy mice were selected as controls. Twenty-four hours later, orbital blood was collected to measure the carboxyhemoglobin (HbCO) concentration, and then all mice were euthanized, and lung and brain tissues were collected to calculate the lung-brain ratio. Lung tissues were ground under ice bath conditions to measure the number of colony-forming units (CFU) in the supernatant and evaluate the expression levels of inflammatory cytokines. In addition, lung tissues were fixed, embedded, sectioned, and then subjected to hematoxylin-eosin (H&E) staining and immunohistochemical staining using antibodies against CD86, CD206, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-10 (IL-10).

[0084] See also Figure 11-13 , which is the result of in vivo experimental detection of CORM-401 and LEV mixed solution on Pseudomonas aeruginosa pneumonia, shows a schematic diagram of the method of establishing a multidrug-resistant Pseudomonas aeruginosa pneumonia model in mice, and details the steps from infection to treatment; quantification of residual bacteria by colony forming unit counts in lung tissue after different treatments; lung to brain weight ratio of mice receiving different treatments; data are expressed as mean ± standard deviation (n=3). p<0.05, p≤0.01, p≤0.001 indicate statistical significance, and ns indicates that there is no significant difference between the LEV+CORM-401 treatment group and other groups.

[0085] See also Fig.12 In vivo, both CORM-401 and levofloxacin showed limited antibacterial effects, reducing the levels of multidrug-resistant Pseudomonas aeruginosa by 0.83 and 2.05 log units, respectively. However, the combination of levofloxacin and CORM-401 resulted in a significant reduction in bacterial counts, from 6.49 to 3.49 log units. This suggests that the membrane-disrupting effects of CO significantly enhance the ability of levofloxacin to penetrate multidrug-resistant Pseudomonas aeruginosa in vivo, resulting in a significant antibacterial effect. Changes in lung weight are a key biomarker for lung histopathology; lung weight typically increases significantly following bacterial infection. See Fig.13, the lung weights of mice with pneumonia treated with PBS were significantly higher than those of healthy mice. Neither CORM-401 nor levofloxacin alone significantly reduced lung weights, suggesting that their therapeutic effects in vivo are limited. Notably, the combination of levofloxacin and CORM-401 significantly promoted the recovery of lung tissue physiology, with the lung weights of the treated groups almost matching those of healthy mice.

[0086] The lung tissue was sectioned and stained with H&E. The test results are shown in Figure 14-16 , is the result of immunofluorescence staining of lung tissue, Hematoxylin-eosin (H&E) staining analysis of lung tissue after 24 hours of different treatments; Immunofluorescence staining shows different phenotypes of macrophages in lung sections after different treatments. Further analysis found that alveolar fusion was significant and inflammatory cell infiltration was extensive in the PBS, CORM-401 and levofloxacin groups. In contrast, the combination treatment group maintained a complete alveolar structure and significantly reduced the infiltration of inflammatory cells. This result was mainly attributed to the combination of levofloxacin and CORM-401, which effectively eliminated the inflammatory response caused by bacteria, as well as the powerful anti-inflammatory effect of CO itself. In order to further explore the role of the combination of CORM-401 and levofloxacin in regulating inflammation, immunofluorescence staining was performed on lung tissue sections. See Fig.15 and Fig.16 , PBS, CORM-401, and levofloxacin treatment groups mainly showed pro-inflammatory M1 macrophages (CD86-positive macrophages). In contrast, the combination of levofloxacin and CORM-401 led to a decrease in pro-inflammatory M1 macrophages and a significant increase in anti-inflammatory M2 macrophages (CD206-positive macrophages).

[0087] In addition, the expression levels of pro-inflammatory cytokines TNF-α and IL-1β and anti-inflammatory cytokine IL-10 in lung tissues were analyzed by immunohistochemistry and ELISA. The test results are shown in Figures 17 to 20, is the result of the test of the immunomodulatory effect of CORM-401 and LEV mixed solution on Pseudomonas aeruginosa pneumonia. Immunohistochemistry shows the different phenotypes of macrophages in lung sections after different treatments and the expression levels of tumor necrosis factor TNF-α, interleukin IL-1β and IL-10; after 24 hours of different treatments, the expression levels of TNF-α, IL-1β and IL-10 in mouse lung tissues; data are expressed as mean ± standard deviation (n=3). p<0.05, p≤0.01, p≤0.001 indicate statistical significance, and ns indicates that there is no significant difference between the LEV+CORM-401 treatment group and other groups. Compared with the PBS group, the expression of TNF-α and IL-1β in the CORM-401 and levofloxacin treatment groups decreased to a certain extent, while the expression of IL-10 increased. However, compared with the combination treatment group, its inhibitory and enhancing effects on inflammatory cytokines were not significant. Immunohistochemical staining results and ELISA results ( Figures 18 to 20 ). These results show that the combination of CORM-401 and levofloxacin effectively eliminated multidrug-resistant Pseudomonas aeruginosa while significantly inhibiting the inflammatory response caused by infection. This accelerates the process of lung tissue returning to normal biological function and state.

[0088] In addition, the combination of CORM-401 and levofloxacin showed good biosafety, as changes in blood HbCO concentrations were monitored throughout the period and it was found that the HbCO levels in the levofloxacin and CORM-401 combination group were not significantly different from those in healthy mice. The test results are shown in Fig.21 , Fig.21 Figure 3 shows the HbCO concentration in mouse plasma after different treatments. Fig.21 Data are expressed as mean ± SD (n = 3). ns indicates no significant difference between the LEV+CORM-401 treatment group and other groups.

[0089] In summary, the embodiments provided by the present invention demonstrate the potential of CO gas as an effective strategy to disrupt the outer membrane (OM) barrier of multidrug-resistant Gram-negative bacteria. This approach significantly promoted the penetration of multiple first-line antibiotics, thereby significantly improving their efficacy against multidrug-resistant Gram-negative infections (including pneumonia caused by multidrug-resistant Pseudomonas aeruginosa). It is worth noting that the significant synergistic effect of CO on the antibacterial activity of various first-line antibiotics in mixed solutions further strengthened our belief that CO-mediated outer membrane disruption strategies play a key role in effectively combating MDR GNB. This strategy not only enhances the penetration of antibiotics into multidrug-resistant Gram-negative bacteria and reduces the required antibiotic dose, but also improves the efficacy of antibiotics, thereby significantly reducing the risk of multidrug resistance. Therefore, this method provides a very promising solution for the clinical effective treatment of multidrug-resistant Gram-negative infections and provides better prospects for the development of new antibiotics.

[0090] It is worth noting that CORM-401 in the above example was synthesized in the laboratory according to the prior art method. Three antibiotics: levofloxacin, meropenem and azithromycin were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Mouse IL-6, IL-1β and TNF-α enzyme-linked immunosorbent assay (ELISA) kits were purchased from Proteintech (USA), and mouse HbCOELISA kits were purchased from Nanjing Enzyme Immunity Biotechnology Co., Ltd. Pseudomonas aeruginosa (ATCC 27853) was derived from the American Type Culture Collection (ATCC). The animal experiments in the embodiments of the present invention have been approved by the Animal Care and Use Committee of Wenzhou Medical University, with approval number wydw2023-0431. The female ICR mice (6-8 weeks old, weighing 20-25 grams) used in the experiment were purchased from Beijing SPF Biotechnology Co., Ltd. All animals were adaptively fed for one week before the experiment.

[0091] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined by the claims.

Claims

1. An antibacterial agent, characterized in that The antibacterial agent is used to fight against multi-drug resistant Gram-negative bacteria, and the antibacterial agent includes: A CO-releasing molecule for releasing CO, wherein the CO-releasing molecule comprises CORM-401 powder; An antibiotic solution, wherein the antibiotic solution comprises at least one of a levofloxacin solution, a meropenem solution and an azithromycin solution; The antimicrobial agent further comprises an exciter, which is used to excite the CO-releasing molecules to release CO.

2. The antibacterial agent according to claim 1, characterized in that The activator is H2O2.

3. The antibacterial agent according to claim 1, characterized in that The antimicrobial agent also includes a buffer.

4. The antibacterial agent according to claim 3, characterized in that The buffer comprises a phosphate buffer.

5. The antibacterial agent according to claim 1, characterized in that The concentration of the levofloxacin solution is 4 μg / mL, the concentration of the meropenem solution is 8 μg / mL, and the concentration of the azithromycin solution is 32 μg / mL.

6. The antibacterial agent according to claim 2, characterized in that The concentration of H2O2 was 100 μM.

7. A method for preparing the antibacterial agent according to any one of claims 1 to 6, characterized in that: The preparation method comprises the following steps: S10: Obtain CORM-401 solution; S20: Obtain antibiotic solution; S30: mixing the CORM-401 solution and the antibiotic solution to obtain the antibacterial agent.

8. The preparation method according to claim 7, characterized in that: The preparation method further comprises the following steps: S40: adding phosphate buffer to the antibacterial agent in S30 to obtain a final antibacterial agent.

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