Application of carnosine and aminoglycoside antibiotics or polymyxin antibiotics in preparation of anti-infective drugs

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a pharmaceutical composition, which comprises: (a) carnosine; and (b) at least one antibiotic selected from the group consisting of aminoglycoside antibiotics and polymyxin antibiotics. Researches find that carnosine has the effect of enhancing the bactericidal effect of aminoglycoside antibiotics or polymyxin antibiotics, carnosine and the two antibiotics are jointly used for resisting bacterial infection, and the carnosine and the two antibiotics have a remarkable synergistic effect and can jointly act to achieve the remarkable bacterial inhibition effect which cannot be achieved by a single antibiotic.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of carnosine in combination with aminoglycoside antibiotics or polymyxin antibiotics in the preparation of anti-infective drugs. Background Technology

[0002] In anti-infective therapy, bacterial resistance has become a major challenge in global public health. Current methods for controlling drug-resistant bacteria mainly include: First, using newer antibiotics, such as modern novel antibiotics; second, using combination antibiotics, leveraging synergistic mechanisms to produce a synergistic effect and kill drug-resistant bacteria; third, increasing the dosage of antibiotics, appropriately increasing the dosage within a safe range to improve bactericidal efficacy; and fourth, finely adjusting the administration method of antibiotics, for example, for time-dependent antibiotics, extending the infusion time using intravenous microinfusion pumps to enhance the bactericidal effect against drug-resistant bacteria.

[0003] However, the above methods still face limitations in their effectiveness against drug-resistant bacteria, such as: the development of new antibiotics lags behind the evolution of drug resistance; combination therapy may increase the risk of adverse reactions or produce antagonistic effects; and dosage adjustment is limited by the antibiotic therapeutic window.

[0004] To address the aforementioned issues, Chinese patent application CN115192561A discloses the application of sodium formate in the preparation of anti-infective drugs. This research found that sodium formate can significantly increase bacterial sensitivity to antibiotics, making antibiotics that were previously ineffective or inefficient against pathogenic bacteria effective or highly effective, thereby killing the bacteria and achieving an anti-infective effect. However, the number of known drugs that can increase bacterial sensitivity to antibiotics is limited, and there is still an urgent need to develop more drugs that can improve bacterial sensitivity to antibiotics or reverse bacterial resistance. Summary of the Invention

[0005] To overcome the problems existing in the prior art, the primary objective of this invention is to provide a pharmaceutical composition.

[0006] A second objective of this invention is to provide a method for preparing a pharmaceutical composition.

[0007] A third object of the present invention is to provide the use of a pharmaceutical composition in the preparation of a medicament for treating bacterial infections.

[0008] A fourth objective of this invention is to provide the use of carnosine in the preparation of medicaments that enhance or reverse the sensitivity of bacteria to aminoglycoside antibiotics and polymyxin antibiotics.

[0009] A fifth object of the present invention is to provide the use of carnosine and quinolone antibiotics in the preparation of medicaments for antibacterial infection.

[0010] The above-mentioned objective of the present invention is achieved through the following technical solution: The primary objective of this invention is to provide a pharmaceutical composition comprising: (a) carnosine; and (b) at least one antibiotic selected from aminoglycoside antibiotics and polymyxin antibiotics.

[0011] Carnosine (β-alanyl-L-histidine) is an endogenous dipeptide in the human body. Under physiological conditions, carnosine can buffer pH, act as an antioxidant, and chelate heavy metals. Its pH buffering effect is mainly reflected in improving athletic ability and performance. Current research on the medicinal uses of carnosine focuses primarily on improving blood lipids, alleviating depression, and treating neurodegenerative diseases, with less attention paid to its role in combating bacterial infections.

[0012] This invention is the first to discover that carnosine can significantly enhance the bactericidal effects of aminoglycoside antibiotics or polymyxin antibiotics. When carnosine is used in combination with these two classes of antibiotics to combat bacterial infections, they exhibit a significant synergistic effect, achieving a significant bacterial inhibition effect that cannot be achieved by a single antibiotic. This invention provides technical support for the clinical treatment of bacterial infections, offering safe and effective adjuvant drugs and controlling bacterial resistance.

[0013] Preferably, the aminoglycoside antibiotic is selected from one or more of gentamicin, neomycin, spectinomycin, streptomycin, and tobramycin; and / or the polymyxin antibiotic is one or more of polymyxin B and polymyxin E.

[0014] The specific types of aminoglycoside antibiotics or polymyxin antibiotics mentioned above should not be construed as limiting the scope of protection of this invention. This is because although there are hundreds of antibiotics, they can be classified according to their chemical structure and antibacterial mechanism; similar chemical structures have the same antibacterial mechanism, thus eliminating the need for individual verification. Those skilled in the art, based on the concept of this invention, can readily deduce that the other aminoglycoside antibiotics or polymyxin antibiotics mentioned above can also be applied to the pharmaceutical compositions described in this invention, achieving the aforementioned expected technical effects.

[0015] Preferably, in the above-mentioned pharmaceutical composition, the molar ratio of (a) to (b) is 10 to 350,000:1; the molar ratio of (a) to (b) is 800 to 312,500:1. Specifically, the molar ratio of (a) to (b) can be 100:1, 500:1, 1000:1, 3000:1, 5000:1, 8000:1, 10000:1, 30000:1, 50000:1, 80000:1, 100000:1, 150000:1, 200000:1, 250000:1, 300000:1, etc., or any range formed by the above values, and the present invention is not limited thereto.

[0016] More preferably, the in vitro concentration of carnosine is 15-100 mM. The above concentration refers to the concentration used when acting on bacteria in vitro. When carnosine is applied to animals or humans, the concentration can be converted according to standards.

[0017] The second objective of this invention is to provide a method for preparing a pharmaceutical composition by uniformly mixing carnosine, aminoglycoside antibiotics, and polymyxin antibiotics.

[0018] Furthermore, the present invention provides the use of the above-described pharmaceutical composition in the preparation of a medicament for treating bacterial infections.

[0019] Preferably, the anti-infective drug, in addition to its effectiveness in treating bacterial infections in mice and humans, can also be used in veterinary medicine to treat bacterial infections in pets, livestock, and other organisms (such as mammals and rodents). Other examples of animals include horses, dogs, and cats.

[0020] Furthermore, the anti-infective drug also contains pharmaceutically acceptable excipients. Even further, the anti-infective drug is an oral formulation, an injectable formulation, or a topical formulation.

[0021] Preferably, when the antibiotic is selected from aminoglycoside antibiotics, the bacteria are Gram-positive and Gram-negative bacteria; when the antibiotic is selected from polymyxin antibiotics, the bacteria are Gram-positive bacteria.

[0022] Preferably, when the antibiotic is selected from aminoglycoside antibiotics, the bacteria are Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, agalactolyticus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, Aeromonas hydrophila, Klebsiella pneumoniae, Acinetobacter baumannii, Streptococcus pyogenes, or drug-resistant bacteria of any of the above bacteria; When the antibiotic is selected from polymyxin antibiotics, the bacteria are Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, agalactolytica streptococci, Enterococcus faecalis, Streptococcus pyogenes, or drug-resistant strains of any of the above bacteria.

[0023] It should be noted that these bacteria are common pathogens, and their drug-resistant strains are also common. Furthermore, *Escherichia coli* and methicillin-resistant *Staphylococcus aureus* are model bacteria for studying bacterial resistance. Therefore, these bacteria are good representatives of both drug-resistant and non-drug-resistant bacteria. Although the bacteria listed in the embodiments of this invention include *Streptococcus agalactiae*, *Enterococcus faecalis*, *Pseudomonas aeruginosa*, *Escherichia coli*, *Aeromonas hydrophila*, *Klebsiella pneumoniae*, *Acinetobacter baumannii*, *Streptococcus pyogenes*, and methicillin-resistant *Staphylococcus aureus*, especially since most of the verification experiments in this invention use multidrug-resistant *Escherichia coli* Y-17 and methicillin-resistant *Staphylococcus aureus* MRSA-7 as research subjects, these bacteria cannot be used as a limitation on the scope of protection of this invention because: (1) *Escherichia coli* and methicillin-resistant *Staphylococcus aureus* are model bacteria for studying drug resistance mechanisms; (2) bacteria can have drug-resistant and non-drug-resistant states, i.e., drug-resistant and non-drug-resistant strains of the same bacterium. The clinical Escherichia coli strains of this invention are in a drug-resistant state, and their sensitivity to antibiotics is enhanced after the addition of carnosine. Therefore, based on the above principles, it can be inferred that many more strains are also suitable for the concept of this invention.

[0024] Furthermore, the present invention provides the use of carnosine in the preparation of drugs that improve or reverse the sensitivity of bacteria to aminoglycoside antibiotics and polymyxin antibiotics.

[0025] The carnosine mentioned above enhances bacterial susceptibility to antibiotics in antibacterial drugs. The mechanism by which carnosine increases bacterial susceptibility to antibiotics may involve increased damage to cell membrane structures, leading to a greater amount of antibiotic entering the bacterial cell and thus increasing susceptibility. Alternatively, carnosine may upregulate the siderophore nonribosomal peptide biosynthesis pathway, thereby reversing bacterial resistance.

[0026] Preferably, the aminoglycoside antibiotic is selected from one or more of gentamicin, neomycin, spectinomycin, streptomycin, and tobramycin; and / or the polymyxin antibiotic is one or more of polymyxin B and polymyxin E.

[0027] Furthermore, the present invention provides the use of carnosine and quinolone antibiotics in the preparation of medicaments for antibacterial infection.

[0028] This invention has discovered through research that carnosine and quinolone antibiotics can produce a synergistic effect, working together to achieve a significant bacterial inhibition effect that cannot be achieved by a single antibiotic.

[0029] Preferably, the bacteria are Gram-negative bacteria. More preferably, the bacteria are Escherichia coli.

[0030] Preferably, the molar ratio of carnosine to quinolone antibiotic is 10~350000:1; and / or the quinolone antibiotic is ciprofloxacin.

[0031] Preferably, the molar ratio of carnosine to quinolone antibiotics is 800 to 312,500:1. Specifically, the molar ratio of carnosine to quinolone antibiotics can be 100:1, 500:1, 1000:1, 3000:1, 5000:1, 8000:1, 10000:1, 30000:1, 50000:1, 80000:1, 100000:1, 150000:1, 200000:1, 250000:1, 300000:1, etc., or any range formed by the above values, and the present invention is not limited thereto.

[0032] Compared with the prior art, the present invention has the following beneficial effects: (1) Through research, this invention found that carnosine can significantly improve the sensitivity of bacteria to aminoglycoside antibiotics and polymyxin antibiotics. When carnosine and these two types of antibiotics are used in combination to fight bacterial infections, there is a significant synergistic effect between the two, which can work together to achieve a significant bacterial inhibition effect that cannot be achieved by a single antibiotic.

[0033] (2) The present invention uses carnosine in combination with aminoglycoside antibiotics and polymyxin antibiotics. On the one hand, it can achieve a significant anti-infection effect under low concentration of antibiotics; on the other hand, the reduced amount of antibiotics used can also significantly reduce the possibility of bacteria developing drug resistance. Attached Figure Description

[0034] Figure 1 This is a statistical graph showing the results of adding carnosine to improve the sensitivity of multidrug-resistant Escherichia coli to multiple antibiotics in Example 2.

[0035] Figure 2 This is a statistical graph showing the results of adding carnosine in Example 3 to improve the sensitivity of multidrug-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus to aminoglycoside antibiotics.

[0036] Figure 3 This is a statistical chart showing the results of adding carnosine to improve the sensitivity of various bacterial strains to gentamicin in Example 4.

[0037] Figure 4-7 The diagram illustrates the effects of different concentrations of multidrug-resistant methicillin-resistant Staphylococcus aureus (MRSA), different carnosine concentrations, different antibiotic concentrations, and treatment durations on bacterial survival rates.

[0038] Figure 8-10 The diagram illustrates the effects of different antibiotic concentrations, different carnosine concentrations, and treatment times on the survival rate of Escherichia coli.

[0039] Figure 11 This is a statistical graph showing the effect of carnosine synergistically with gentamicin on the PI fluorescence value of Escherichia coli in Example 6.

[0040] Figure 12 A statistical graph showing the results of in vitro bactericidal experiments of carnosine-enhanced polymyxin B against drug-resistant strains.

[0041] Figure 13 A statistical graph showing the results of in vitro bactericidal experiments of carnosine-enhanced polymyxin E against drug-resistant strains. Detailed Implementation

[0042] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field, and the strains are all clinically isolated strains.

[0043] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0044] Example 1: Preparation of bacterial samples Pick a single bacterial colony and inoculate it into 5 mL of LB or BHI medium, and incubate at 37°C with shaking at 200 rpm until OD500. 600 =0.5; Dilute the bacterial culture 200 times with fresh LB or BHI medium to achieve a bacterial concentration of 5 × 10⁻⁵. 6 CFU / mL. Add 100 μL of LB or BHI medium to each well of a 96-well plate. Except for the negative control, add 10 μL of diluted bacterial suspension to each well to achieve a bacterial count of 5 × 10⁻⁶ cells / well. 4 CFU / mL.

[0045] Example 2: Combined Antimicrobial Susceptibility Testing of Carnosine with Multiple Types of Antibiotics against Bacteria (1) Prepare multidrug-resistant Escherichia coli Y-17 and Y-21 according to Example 1 (the minimum inhibitory concentration is shown in Table 1).

[0046] Following the CLSI guidelines, saturated bacteria were transferred 1:100 to fresh LB medium and cultured at 37°C with shaking at 200 rpm until OD was reached. 600 = 0.5. Dilute the bacterial culture 200 times with fresh LB medium to achieve a bacterial concentration of 5 × 10⁻⁵. 6 CFU / mL. Add 100 μL of LB medium to each well of a 96-well plate, and add twice the final concentration of the antibiotic stock solution to column 10. Serially dilute the antibiotic to obtain media containing a gradient of antibiotic concentrations. Except for the negative control, add 10 μL of the diluted bacterial solution to each well to achieve a bacterial count of 5 × 10⁻⁶ cells / well. 4CFU. Incubate at 37°C for 18 hours and read the minimum inhibitory concentration.

[0047] Table 1. Minimum inhibitory concentrations (MICs, micrograms / mL) of Y-17 and Y-21 against six antibiotics.

[0048] (2) Based on the MIC results, the micro-culture medium dilution checkerboard method was used to test two strains of Escherichia coli in a 96-well sterile microplate. The dilution factors were designed as 1, 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, and 1 / 64 MICs for the combination of carnosine and various antibiotics. 180 μL of LB medium was added to the first column, and 90 μL of medium was added to the remaining columns. 20 μL of carnosine was added to the first column, and the horizontal gradient was applied to the seventh column. Antibiotics were added to the first eight wells of the first row, and the vertical gradient was applied to the seventh row. 90 μL of medium and drug were fixed in each well. After the test bacterial suspension was mixed, 10 μL was pipetted into each well of the 96-well plate using an eight-well pipette, for a total volume of 100 μL. At the same time, MICs of carnosine alone, MICs of antibiotics alone, negative wells (without bacteria), and positive wells (without drug) were set up as controls. The concentration used when using carnosine alone was the same as the concentration used when using them in combination. The samples were incubated in a carbon dioxide incubator at 37℃ and 5% CO2 for 16 h. The results were observed and recorded. Each experiment was repeated twice.

[0049] The formula for calculating the FIC index is: FIC Index = (MIC of drug A in combination / MIC of drug A alone) + (MIC of drug B in combination / MIC of drug B alone). The interpretation criteria for the FIC index are as follows: when the FIC index is less than 0.5, the two drugs have a synergistic effect; when the FIC index is between 0.5 and 1, the two drugs have an additive effect; when the FIC index is greater than 1 and less than 2, the two drugs have no effect; when the FIC index is greater than 2, the two drugs have an antagonistic effect. The FIC index is represented by FICI (FIC Index) in the following experimental results.

[0050] See results Figure 1 As shown in the figure, carnosine can improve the sensitivity of Escherichia coli to gentamicin and ciprofloxacin. Specifically, the FIC index of the combined ciprofloxacin and gentamicin susceptibility test for both E. coli strains was ≤ 0.5, indicating that carnosine exhibits a synergistic effect with ciprofloxacin and gentamicin. Furthermore, the results show that the synergistic effect of carnosine and gentamicin on improving the bactericidal effect is optimal.

[0051] Example 3: Combined Antimicrobial Susceptibility Test of Carnosine and Aminoglycoside Antibiotics against Bacteria Following Example 1, multidrug-resistant Escherichia coli strain Y-17 and methicillin-resistant Staphylococcus aureus MRSA-7 were prepared. Then, carnosine was used to conduct combined drug susceptibility tests with several aminoglycoside antibiotics, including neomycin, spectinomycin, streptomycin, tobramycin, gentamicin, and neomycin sulfate (the concentration of carnosine used was 125 mM). Specific experimental procedures are detailed in Example 2. The changes in the MIC values ​​of various antibiotics against Y-17 and MRSA-7 are shown in Table 2.

[0052] Table 2. MIC determination results of carnosine combined with multiple aminoglycoside antibiotics against Y-17 and MRSA-7.

[0053] As shown in Table 2, the FIC index of carnosine combined with various aminoglycoside antibiotics on Escherichia coli strain Y-17 and methicillin-resistant Staphylococcus aureus MRSA-7 showed that the interaction between carnosine and aminoglycoside antibiotics was synergistic.

[0054] See results Figure 2 As shown in the figure, carnosine generally improved the sensitivity of *Escherichia coli* Y-17 and methicillin-resistant *Staphylococcus aureus* MRSA-7 to aminoglycoside antibiotics. In both strains, the FIC index in the combined susceptibility test for aminoglycoside antibiotics was ≤ 0.5, indicating a synergistic effect between carnosine and aminoglycoside antibiotics. Furthermore, the results showed that the synergistic effect of carnosine and gentamicin resulted in the best bactericidal effect.

[0055] Example 4: The universality of carnosine synergistically with gentamicin in eliminating multidrug-resistant bacteria Multiple bacterial samples were prepared according to Example 1. The bacterial strains included Gram-positive bacteria: methicillin-resistant Staphylococcus aureus (MRSA). S. aureus MRSA-1, 3, 5, 6, 8 and 9), agalactiae (Streptococcus agalactiae) S. agalactiae GBS, GBS-1535), Enterococcus faecalis ( E.faecium Fae 1, 5) and Gram-negative bacteria: drug-resistant Pseudomonas aeruginosa ( P. aeruginosa 3, 10), drug-resistant Escherichia coli ( E.Coli Y-6, Y-9 and K12), Aeromonas hydrophila ( A. hydrophila LP-2), Klebsiella pneumoniae ( K. pneumoniae 66, 69), Acinetobacter baumannii ( A.baumannii 25, 64). A combined drug susceptibility test was performed on each strain (the concentration of carnosine used was 125 mM). The specific experimental steps are shown in Example 2. The changes in MIC values ​​of each strain are shown in Table 3.

[0056] Table 3. MIC determination results of carnosine combined with gentamicin against various bacterial strains.

[0057] See results Figure 3 As shown in the figure, the sensitivity of these bacteria to gentamicin was significantly improved in general after the addition of carnosine. Analysis of the results showed that the FIC (Fixed Ingredient Criterion) in the carnosine-gentamicin susceptibility test was ≤ 0.5, indicating that carnosine and gentamicin exhibited a synergistic effect in these strains.

[0058] Example 5: In vitro bactericidal experiment of carnosine-enhanced gentamicin against drug-resistant strains. Using MRSA-7 and Escherichia coli K12 as research subjects, this study investigated the effect of carnosine on enhancing bacterial susceptibility to gentamicin by adding different concentrations of carnosine or antibiotics, and by different initial bacterial loads and treatment times. The bacterial samples were first divided into four groups: an M9 medium control group (Control), a carnosine group, a gentamicin group, and a gentamicin + carnosine combination group. For the methicillin-resistant Staphylococcus aureus (MRSA-7) strain, the gentamicin concentration was 200 μg / mL (approximately 400 μM), and the carnosine concentration was 15 mM. For the Escherichia coli K12 strain, the gentamicin concentration was 0.75 μg / mL (approximately 1.5 μM), and the carnosine concentration was 30 mM. The system volume for both groups was 5 mL. Each group had three biological replicates. After adding the corresponding drugs, each group was incubated at 37℃ and 200 rpm in a shaker for 6 hours. Then, 100 μL of each sample was serially diluted, and 5 μL was spotted onto LB agar plates. After incubation at 37℃ for 12 hours, the bacterial count was recorded. The survival rate of the treated bacteria was calculated using the formula: Survival rate (%) = (Number of bacteria in the treated sample / Number of bacteria in the control sample) × 100%, as detailed below: (1) The synergistic effect of carnosine and gentamicin on killing MRSA-7 strains is dose-dependent. To understand the synergistic effect of carnosine and gentamicin in killing MRSA at different initial bacterial counts, different initial bacterial counts (1×10⁻⁶) were treated with 15 mM carnosine and 200 μg / mL gentamicin. 6 ~5×10 6 MRSA-7 (CFU / mL) was treated with gentamicin or carnosine alone for 1×10⁻⁶ CFU / mL. 6 The initial bacterial count of CFU / mL was used as a control, and the survival rate was calculated.

[0059] The results are as follows Figure 4 As shown, the synergistic killing effect of carnosine and gentamicin on MRSA increased with decreasing initial bacterial load. In the figure, *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001.

[0060] (2) The synergistic effect of carnosine and gentamicin on killing MRSA-7 / K12 strains is antibiotic concentration-dependent. To investigate whether there is a gradient effect between gentamicin concentration and bactericidal efficiency, a concentration of 2×10⁻⁶ was used. 6 CFU / mL was the initial bacterial load. Based on the addition of 15 mM carnosine to MRSA-7 strain and 10 mM carnosine to K12 strain, different concentrations of gentamicin (MRSA-7 strain: 150~300 μg / mL, K12 strain: 0.0625~0.75 μg / mL) were added and incubated for 6 hours. Viable cell counts were then performed to compare the bacterial survival rate at the same carnosine concentration with different concentrations of gentamicin.

[0061] The results are as follows Figure 5 and Figure 8 As shown in the figure, in the MRSA-7 group, the bacterial survival rate was 19.75% when only carnosine was added. As the concentration of gentamicin increased, the bacterial survival rate decreased from 1.79% to 0.033%, while the bactericidal efficiency increased from 1.22 times to 598 times. In the K12 group, the bacterial survival rate was 95.75% when only carnosine was added. As the concentration of gentamicin increased, the bacterial survival rate decreased from 87.3% to 3.53%, while the bactericidal efficiency increased from 1.1 times to 273.5 times.

[0062] (3) The synergistic effect of carnosine and gentamicin on killing MRSA-7 / K12 strains is carnosine concentration-dependent. To investigate whether there is a gradient effect between carnosine concentration and bactericidal efficiency, a concentration of 2 × 10⁻⁶ was used. 6 CFU / mL was the initial bacterial load. Based on the addition of 200 μg / mL gentamicin to MRSA-7 strain and 0.75 μg / mL gentamicin to K12 strain, different concentrations of carnosine (2.5–15 mM and 2.5–30 mM) were added and incubated for 6 hours. Viable cell counts were then performed. It was found that compared to 10 mM carnosine, the synergistic bactericidal effect of 15 mM carnosine and gentamicin was not significant, indicating that the synergistic effect of carnosine and gentamicin was dose-dependent.

[0063] The results are as follows Figure 6 and Figure 9 As shown in the figure, in the MRSA-7 group, the bacterial survival rate was 39.66% when only gentamicin was added. As the concentration of carnosine increased, the bacterial survival rate decreased from 7.59% to 0.02%, while the bactericidal efficiency increased from 5.23 times to 1983 times. In the Escherichia coli K12 group, the bacterial survival rate was 75% when only antibiotics were added. As the concentration of carnosine increased, the bacterial survival rate decreased from 35.56% to 0.51%, while the bactericidal efficiency increased from 2.11 times to 147.1 times.

[0064] (4) The synergistic effect of carnosine and gentamicin on killing MRSA-7 / K12 strains is time-dependent. Further investigation revealed that the absorbance (OD) of bacterial cells treated for different durations was measured when 10 mM carnosine and 200 μg / mL gentamicin were added. 600 The study analyzed the bacterial colony-forming units and calculated the survival rate, observing the relationship between the sterilization efficiency and time.

[0065] The results are as follows Figure 7 and Figure 10 As shown in the figure, based on the survival rate results, it can be found that the synergistic killing effect of carnosine and gentamicin on MRSA-7 and K12 is not obvious in the first 2 hours after incubation, but increases significantly from 2 to 4 hours, and continues to increase slowly from 4 to 8 hours. According to the overall absorbance (OD)... 600 The results of the colony-forming units also showed that the synergistic killing effect of carnosine and gentamicin on MRSA-7 and K12 mainly began to take effect 2 hours after treatment.

[0066] The bactericidal efficiency of both strains treated with gentamicin and carnosine was higher at every time point than that treated with gentamicin alone, and the bactericidal multiple increased significantly with prolonged treatment time. Specifically, in the MRSA-7 group, the survival rate was 93.46% after 2 hours with gentamicin alone, but decreased to 48.46% after the addition of carnosine, with a 1.93-fold increase in sensitivity. After 4 hours of treatment, the survival rate decreased from 18.85% with gentamicin alone to 0.76% with the addition of carnosine, with a 24.80-fold increase in sensitivity. Moreover, the number of viable bacteria decreased significantly with prolonged treatment time, and sensitivity increased significantly, with an 88-100-fold increase in sensitivity after 6-8 hours. It was also found that the addition of carnosine had no effect on bacterial survival rate within 8 hours.

[0067] In the Escherichia coli K12 group, the survival rate was 87.93% after 2 hours with only gentamicin added, but the survival rate decreased to 1.78% after the addition of carnosine, and the sensitivity increased by 49.4 times. After 4 hours of treatment, the survival rate decreased from 40.16% with only gentamicin added to 0.13% with the addition of carnosine, and the sensitivity increased by 308.92 times. After 4 hours of treatment, the effect of carnosine on the sensitivity of the strain gradually decreased. It was also found that the addition of carnosine had no effect on the survival rate of the bacteria within 8 hours.

[0068] Example 6: Preliminary exploration of the bactericidal mechanism of carnosine-enhanced gentamicin To investigate the mechanism by which carnosine enhances bacterial susceptibility to gentamicin, Escherichia coli K12 was used as the research subject to study the metabolic mechanism by which carnosine enhances bacterial gentamicin susceptibility. (1) Increase bacterial sensitivity to gentamicin by disrupting membrane permeability Gentamicin, an aminoglycoside antibiotic, relies on penetrating the bacterial cell membrane to enter the cell and bind to ribosomes, interfering with protein synthesis. If carnosine increases the membrane permeability of Escherichia coli K12, it can promote the intracellular accumulation of gentamicin, thereby enhancing its antibacterial effect.

[0069] This section uses a PI staining experiment to verify the effect of carnosine on the permeability of the K12 membrane of *Escherichia coli*. The experimental procedure was as follows: bacterial samples were divided into four groups: M9 medium control group (Control), carnosine group, gentamicin group, and gentamicin + carnosine group (Combination). Each group of bacteria was treated differently according to the sterilization conditions. After washing the bacterial cells, the bacterial count was adjusted to 10⁻⁶ cells / mL using M9 medium. 6 CFU / mL, add 2 μg / mL PI dye to each 1 mL of bacterial culture, shake to mix, incubate at 37°C in the dark for 20 minutes, and then use flow cytometry to detect the cells at excitation light of 544 nm and emission light of 620 nm.

[0070] See results Figure 11 As shown in the figure, the fluorescence value of bacteria significantly increased when gentamicin was added along with carnosine, increasing by 5.36 times and 8.15 times compared to gentamicin alone and carnosine alone, respectively. This indicates that carnosine can disrupt the cell membrane permeability of Escherichia coli K12, allowing gentamicin to more easily penetrate the membrane barrier, accumulate intracellularly, and target ribosomes, accelerating protein synthesis disorders and enhancing antibacterial sensitivity. In the figure, *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001.

[0071] Example 7: The universality of carnosine synergistic with polymyxin antibiotics in eliminating multidrug-resistant bacteria. Multiple bacterial samples were prepared according to Example 1. The bacterial strains included Gram-positive bacteria: methicillin-resistant Staphylococcus aureus (MRSA). S. aureus MRSA-2, 7, 10 and SH-7, SH-12), agalactiae ( S. agalactiae WT-GBS, AR-GBS), Enterococcus faecalis ( E.Faecium Fae 36, 37, 38, 39, 40, 41), Streptococcus pyogenes ( S. Pyogenes GAS 1, β1, 3). Combined drug susceptibility testing was performed on each strain using polymyxin B and carnosine, and polymyxin E and carnosine (with carnosine used at a concentration of 125 mM). Specific experimental procedures are detailed in Example 2. Changes in MIC values ​​for each strain are shown in Tables 4 and 5.

[0072] Table 4. MIC determination results of carnosine combined with polymyxin B on various bacterial strains.

[0073] Table 5. MIC determination results of carnosine combined with polymyxin E on various bacterial strains.

[0074] As shown in Tables 4 and 5, the susceptibility of these bacteria to gentamicin was significantly improved in general after the addition of carnosine. Analysis of the results showed that the FIC index ≤ 0.5 in the carnosine-gentamicin susceptibility test was 100%, indicating that carnosine and gentamicin exhibited a synergistic effect in the vast majority of strains.

[0075] Example 8: In vitro bactericidal experiment of carnosine-enhanced polymyxin antibiotics against drug-resistant strains. Using MRSA-12 as the research subject, this study investigated the effect of carnosine on enhancing bacterial sensitivity to gentamicin by adding different concentrations of carnosine or antibiotics. The bacterial samples were first divided into four groups: LB medium control, carnosine group, polymyxin group (PM), and polymyxin + carnosine combination group. The in vitro sterilization system consisted of 5 mL of medium. Polymyxin B concentrations for treating methicillin-resistant Staphylococcus aureus MRSA-12 were 10 μg / mL and 20 μg / mL, polymyxin E concentrations were 20 μg / mL, 40 μg / mL, and 80 μg / mL, and carnosine concentrations were 50 mM and 100 mM. Each group had three biological replicates. After adding the corresponding drugs, each group was incubated at 37℃ and 200 rpm in a shaker for 6 hours. Then, 100 μL of each sample was serially diluted, and 5 μL was spotted onto LB agar plates. After incubation at 37℃ for 12 hours, the bacterial count was recorded. The survival rate of the treated bacteria was calculated using the formula: Survival rate (%) = (Number of bacteria in the treated sample / Number of bacteria in the control sample) × 100%. The results are shown below. Figure 12 , 13 In the figure, *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001.

[0076] Depend on Figure 12 and Figure 13It is evident that in methicillin-resistant Staphylococcus aureus (MRSA12), the bacterial survival rate was 99.36% when carnosine was added alone. After adding polymyxin B, the bacterial survival rate decreased to 0.0012%, and further decreased with increasing concentrations of both colistin B and carnosine. Conversely, when polymyxin E was added, the bacterial survival rate was 99.62% when carnosine was added alone. After adding polymyxin E, the bacterial survival rate decreased to 0.2152%, and further decreased with increasing concentrations of both colistin E and carnosine. Both results demonstrate that carnosine significantly enhances the bactericidal efficiency of polymyxin antibiotics.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A pharmaceutical composition, characterized in that, include: (a) Carnosine; (b) At least one antibiotic selected from aminoglycoside antibiotics and polymyxin antibiotics.

2. The pharmaceutical composition according to claim 1, characterized in that, The aminoglycoside antibiotics are selected from one or more of gentamicin, neomycin, spectinomycin, streptomycin, and tobramycin; and / or The polymyxin antibiotics are one or more of polymyxin B and polymyxin E.

3. The pharmaceutical composition according to claim 1, characterized in that, The molar ratio of (a) to (b) is 10~350000:

1.

4. Use of the pharmaceutical composition according to any one of claims 1-3 in the preparation of a medicament for antibacterial infection.

5. The application according to claim 4, characterized in that, When the antibiotic is selected from aminoglycoside antibiotics, the bacteria are Gram-positive and Gram-negative bacteria; when the antibiotic is selected from polymyxin antibiotics, the bacteria are Gram-positive bacteria.

6. The application according to claim 5, characterized in that, When the antibiotic is selected from aminoglycoside antibiotics, the bacteria are Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, agalactolytic streptococcus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, Aeromonas hydrophila, Klebsiella pneumoniae, Acinetobacter baumannii, Streptococcus pyogenes, or drug-resistant bacteria of any of the above bacteria. When the antibiotic is selected from polymyxin antibiotics, the bacteria are Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, agalactolytica streptococci, Enterococcus faecalis, Streptococcus pyogenes, or drug-resistant strains of any of the above bacteria.

7. Application of carnosine in the preparation of drugs that improve or reverse the sensitivity of bacteria to aminoglycoside antibiotics and polymyxin antibiotics.

8. The application according to claim 7, characterized in that, The aminoglycoside antibiotics are selected from one or more of gentamicin, neomycin, spectinomycin, streptomycin, and tobramycin; and / or The polymyxin antibiotics are one or more of polymyxin B and polymyxin E.

9. Application of carnosine and quinolone antibiotics in the preparation of drugs for antibacterial infection.

10. The application according to claim 9, characterized in that, The molar ratio of carnosine to quinolone antibiotics is 10 to 350,000:1; and / or the quinolone antibiotic is ciprofloxacin.

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