Use of varenicline in the preparation of an antibiotic potentiator

Abstract

The application discloses application of varenicline in preparation of an antibiotic enhancer, and belongs to the technical field of medicinal chemistry.The application proves that varenicline can improve the bacteriostatic effect of an antibiotic when the antibiotic is used in combination with varenicline.The effect is achieved by inhibiting cystathionine gamma-lyase activity, thereby inhibiting the generation of hydrogen sulfide and reducing antibiotic resistance.The inhibiting capacity of varenicline on bacterial cystathionine gamma-lyase activity is obviously superior to that on human cystathionine gamma-lyase, and varenicline has the potential to develop into an antibiotic enhancer.

Description

Application of varenicline in the preparation of antibiotic enhancers Technical Field

[0001] This invention relates to the field of medicinal chemistry, and more particularly to the use of varenicline in the preparation of antibiotic enhancers. Background Technology

[0002] The gradual loss of effectiveness of antimicrobial drugs has attracted widespread attention worldwide. Due to the overuse and misuse of antibiotics, many have become ineffective, leading to a growing problem of bacterial resistance. The adverse effects of bacterial resistance on human society, including increased biosecurity threats, exacerbated environmental pollution, and constraints on economic development, necessitate not only strengthened control over antibiotic abuse but also the urgent development of novel treatment strategies for drug-resistant bacteria.

[0003] Developing antibiotic enhancers is a relatively new antibacterial strategy targeting hydrogen sulfide (H2S)-mediated bacterial tolerance and resistance. Bacterial tolerance develops before resistance; various environmental stresses can induce tolerance, including starvation, hypoxia, heat shock, oxidative stress, and DNA damage. Studies have shown that tolerance develops rapidly under intermittent antibiotic exposure. If the starting strain carries a tolerance mutation, resistance develops even faster, highlighting the importance of finding compounds that can eliminate resistant and persistent bacteria. In 2021, Evgeny Nudler, Professor of Biochemistry and Molecular Pharmacology at NYU Grossman School of Medicine, first pointed out that bacterial tolerance can be suppressed and persistent bacteria (a small subset of resistant bacteria) eliminated by disrupting the bacterial H2S-mediated defense system. Since most antibiotics, such as gentamicin, oxacillin, and nalidixic acid, exert their bactericidal effects primarily through oxidative stress (Fenton's reaction), endogenously produced hydrogen sulfide (H2S) can counteract oxidative stress, protecting bacteria from its harmful effects. Inhibiting bacterial H2S production can make resistant or persistent bacteria susceptible to available clinical antibiotics. Therefore, finding compounds that inhibit bacterial H2S production is a beneficial strategy to curb bacterial resistance and drug development, and this method holds promise as a broad-spectrum antibacterial approach. Cystathion-gamma lyase is an important enzyme in the H2S generation process, and studies have found that bacterial cystathion-gamma lyase and human cystathion-gamma lyase are isoenzymes. If specific inhibitors (as antibiotic enhancers) are used to inhibit bacterial cystathion-gamma lyase but do not inhibit or only slightly inhibit human cystathion-gamma lyase, this targeted inhibition of bacterial resistance and drug development will have little or no impact on human H2S production, resulting in minimal impact on the human body. Summary of the Invention

[0004] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides the application of varenicline in the preparation of antibiotic enhancers, which solves the technical problem of antibiotic resistance and proves that varenicline can inhibit bacterial H2S production, that is, varenicline is a specific inhibitor of bacterial cystathionine γ-lyase.

[0005] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0006] In a first aspect, the present invention provides the use of varenicline in the preparation of antibiotic enhancers, wherein the varenicline has the structure shown in formula (I):

[0007]

[0008] Optionally, the antibiotic includes gentamicin. The dosage of the antibiotic and enhancer is 0.004-0.008 μg of gentamicin and greater than 500 μmol of varenicline per liter of system.

[0009] Optionally, the antibiotic enhancer is a reagent that inhibits bacterial tolerance and / or resistance.

[0010] Optionally, the varenicline combined with antibiotics enhances the antibacterial ability against Gram-negative bacilli and / or Gram-positive cocci and reduces the minimum inhibitory concentration (MIC) value of the antibiotics to inhibit bacterial growth.

[0011] Preferably, the Gram-negative bacilli include *Pseudomonas aeruginosa*, wherein the antibiotic and enhancer used are 0.008 μg gentamicin and greater than 500 μmol varenicline per liter of system; the Gram-positive cocci include *Staphylococcus aureus*, wherein the antibiotic and enhancer used are 0.004 μg gentamicin and greater than 500 μmol varenicline per liter of system.

[0012] Secondly, the present invention provides the use of varenicline in the preparation of cystathionine γ-lyase inhibitors.

[0013] Optionally, the cystathionine γ-lyase is a bacterial cystathionine γ-lyase.

[0014] Thirdly, the present invention provides the use of vareniclan in the preparation of reagents that inhibit the production of hydrogen sulfide by bacteria.

[0015] Optionally, the bacteria include Pseudomonas aeruginosa and Staphylococcus aureus.

[0016] The beneficial effects of this invention are as follows: Using the three-dimensional structure of bacterial cystathionine γ-lyase as a model, this invention establishes a theoretical screening method for inhibitors. Thousands of compounds, including those from natural product libraries, clinical compound libraries, and antiviral drug libraries, are theoretically screened to obtain compounds that may have inhibitory activity against bacterial cystathionine γ-lyase. Then, using the previously constructed bacterial cystathionine γ-lyase inhibitor screening method, these compounds are experimentally screened through enzyme activity testing, and their activity is compared with that of human cystathionine γ-lyase inhibitors.

[0017] Through drug screening according to the above scheme, this invention demonstrates that varenicline has a preferred inhibitory effect on bacterial cystathionine γ-lyase. The compound, when combined with antibiotics, enhances the antibacterial effect against various bacterial species. This invention demonstrates that varenicline can inhibit bacterial cystathionine γ-lyase while having minimal inhibitory effect on human cystathionine γ-lyase. Therefore, varenicline can be combined with various antibiotics to treat bacterial infectious diseases in humans with minimal harm.

[0018] This invention demonstrates that when varenicline is used in combination with the antibiotic gentamicin, it can significantly enhance the antibacterial effect of gentamicin and reduce the MIC value of gentamicin in inhibiting bacterial growth. Varenicline and gentamicin have a synergistic effect and can be used as antibiotic enhancers to inhibit bacterial tolerance and / or resistance.

[0019] Specific inhibition of bacterial cystathionine-gamma lyase and targeting the hydrogen sulfide-mediated defense system promises to be a broad-spectrum antibacterial strategy, potentially revolutionary. Once discovered, specific inhibitors are expected to be studied in preclinical trials, becoming enhancers for a range of antibiotics. This strategy would benefit the control of infectious diseases, alleviating patient suffering, reducing mortality, lowering healthcare costs, and generating significant social benefits. Attached Figure Description

[0020] Figure 1 shows the inhibition curves of different concentrations of varenicline on human cystathionine γ-lyase.

[0021] Figure 2 shows the inhibition curves of different concentrations of varenicline on bacterial cystathionine γ-lyase.

[0022] Figure 3 shows the KB disc method for detecting the efficacy of gentamicin combined with varenicline against gentamicin-resistant Pseudomonas aeruginosa. In the figure, A represents the use of 1 μL of DMSO alone, with no antibacterial effect; B represents the use of 5 μg of gentamicin alone, with an inhibition zone of 1.46 cm; C represents the use of 5 μg of gentamicin + 1 μL of DMSO, with an inhibition zone of 1.46 cm; D represents the use of 5 μg of gentamicin + 5 μg of varenicline, with an inhibition zone of 1.67 cm; E represents the use of 5 μg of varenicline alone, with no antibacterial effect (because varenicline is dissolved in DMSO, an equal amount of DMSO was used for separate verification).

[0023] Figure 4 shows the detection of the minimum inhibitory concentration (MIC) of gentamicin against gentamicin-resistant Pseudomonas aeruginosa.

[0024] Figure 5 shows the growth curve of gentamicin combined with varenicline against gentamicin-resistant Pseudomonas aeruginosa.

[0025] Figure 6 shows the measurement of gentamicin-resistant Pseudomonas aeruginosa biofilms using the crystal violet staining method.

[0026] Figure 7 shows the KB disk method for detecting the efficacy of gentamicin combined with varenicline against Staphylococcus aureus. In Figure 7, A shows no antibacterial effect when 1 μL of DMSO was used alone; B shows an inhibition zone of 1.61 cm when 5 μg of gentamicin was used alone; C shows an inhibition zone of 1.58 cm when 5 μg of gentamicin was combined with 1 μL of DMSO; D shows an inhibition zone of 1.81 cm when 5 μg of gentamicin was combined with 5 μg of varenicline; E shows no antibacterial effect when 5 μg of varenicline was used alone (because varenicline was dissolved in DMSO, an equal amount of DMSO was used for separate verification).

[0027] Figure 8 shows the minimum inhibitory concentration (MIC) of gentamicin against Staphylococcus aureus.

[0028] Figure 9 shows the growth curve of Staphylococcus aureus against Staphylococcus aureus using gentamicin combined with varenicline.

[0029] Figure 10 shows the measurement of Staphylococcus aureus biofilm using the crystal violet staining method. Detailed Implementation

[0030] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0031] To better understand the above technical solutions, exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that the present invention can be understood more clearly and thoroughly, and that the scope of the present invention can be fully conveyed to those skilled in the art.

[0032] Throughout the experiment, all operating methods, procedures, and reaction conditions of the substrate were designed and implemented according to methods familiar to those skilled in the art.

[0033] The inhibitor molecules used in the following examples were partly purchased from MCE (MedChemExpress) or other common commercial sources.

[0034] In the following examples, the antibiotics were dissolved in physiological saline using commercially available gentamicin aqueous solution to the concentration required for each example.

[0035] The human cystathionine γ-lyase inhibitor screening kit used in this invention, according to application number 202110381567.8, comprises the following components:

[0036] Fluorescent probe NI-CO-HCYS, cystathionine γ-lyase, pyridoxal 5-phosphate, and borate-borax buffer (pH=8). The fluorescent probe NI-CO-HCYS was synthesized using method 202110381567.8. The cystathionine γ-lyase was purchased from Beijing Aibisin, and the pyridoxal 5-phosphate and borate-borax buffer were purchased from a general reagent company.

[0037] The concentration of the fluorescent probe substrate NI-CO-HCYS was 2 mmol / L; the concentration of the human cystathionine γ-lyase was 1.3 mg / mL; the concentration of pyridoxal 5-phosphate was 1 mmol / L; and the concentration of borate-borax buffer (pH=8) was 50 mmol / L.

[0038] The screening method for bacterial cystathionine γ-lyase inhibitors used in this invention refers to the method reported in the literature (Yan JiaY, Wang Y, Zhang Y, etc. Profiling cystathionine β / γ-lyase in complex biosamples using novel activatable fluorogens. Analytical Chemistry. 2022, 94(2):1203-1210).

[0039] doi:10.1021 / acs.analchem.1c04393), including the following components:

[0040] Fluorescent probe Azmc, bacterial cystathionine γ-lyase, pyridoxal 5-phosphate, and borate-borax buffer (pH=8). Fluorescent probe Azmc was synthesized using a method reported in the literature (Thorson MK, Majtan T, Kraus JP, Barrios AM. Identification of cystathionine β-synthase inhibitors using a hydrogensulfide selective probe. Angew Chem Int Edit, 2013, 52(17):4641-4644).

[0041] doi:10.1002 / anie.201300841), bacterial cystathionine γ-lyase was purchased from Crystal Biopharma, pyridoxal 5-phosphate and borate-borax buffer were purchased from a general reagent company.

[0042] The concentration of the fluorescent probe substrate Azmc is 0.5 mmol / L; the concentration of the bacterial cystathionine γ-lyase is 10 mmol / L; the concentration of pyridoxal 5-phosphate is 0.1 mmol / L; and the concentration of borate-borax buffer (pH=8) is 50 mmol / L.

[0043] The experimental process of this invention uses a fluorescence microplate reader for fluorescence detection.

[0044] Following the drug screening procedure described above, this invention demonstrated that the following compound inhibits thioether γ-lyase. Quantitative analysis using the KB disc method, broth dilution method, bacterial growth curves, and bacterial biofilm analysis confirmed that this compound significantly enhances the antibacterial effect of the antibiotic gentamicin. The following compound, varenicline (CAS No. 249296-44-4), is in powder form. It is dissolved in DMSO (dimethyl sulfoxide) to obtain a stock solution for preservation. In the examples described below, it is dissolved in physiological saline to a suitable concentration for each use, ensuring that the final DMSO concentration is less than 0.1%. 0.1% is considered to be harmless to cells.

[0045]

[0046] Example 1

[0047] Determination of the inhibitory effect of varenicline on human cystathionine γ-lyase

[0048] The specific implementation process is as follows:

[0049] 1) Human cystathionine γ-lyase and fluorescent probe NI-CO-HCYS stock solution were both stored in a -80℃ freezer;

[0050] 2) Thaw 1.3 mg / mL human cystathionine γ-lyase at room temperature in a cryopreservation plate (-4 to 4℃), take 5 μL and dilute it in 89.5 μL of borate-borax buffer (pH=8), and add it to the detection plate;

[0051] 3) Take 1 μL of the inhibitor varenicline at different concentrations (0 mM, 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, 2.5 mM) and add it to the solution obtained in step 2).

[0052] 4) Add 2.5 μL of 1 mM PLP (pyridoxal phosphate) coenzyme to the solution obtained in step 3) above;

[0053] 5) Add 2 μL of 2 mM NI-CO-HCYS to the solution obtained in step 4) above, incubate at 37°C using a fluorescence microplate reader, and monitor the fluorescence value emitted at 540 nm after excitation at 450 nm using a fluorescence microplate reader. Detect the fluorescence value while incubating, and incubate for 22 h.

[0054] 6) Statistical analysis of fluorescence emission values ​​at 450 nm excitation and 540 nm before and after incubation for each group. The fluorescence change value of the control group (inhibitor concentration of 0) before and after incubation was set as 100. The residual activity value was obtained by comparing the fluorescence change values ​​of different inhibition groups before and after incubation with this control group. Using GraphPad Prism8 software, a graph was plotted with the logarithm of the inhibitor concentration (logC(vareniclan)) on the x-axis and the corresponding residual activity value on the y-axis. The results are shown in Figure 1. Figure 1 shows the effect of different inhibitor concentrations on the proportion of enzyme activity inhibited, indicating the inhibitory ability of the compound, expressed as the inhibitor concentration at which half the enzyme activity is inhibited. IC50 50 The formula for calculation is Y = 100 / (1 + 10^((X - LogIC)). 50 ))), where Y represents the remaining activity fraction, X represents the common logarithm of the inhibitor compound concentration, and ∧ refers to the exponentiation algorithm.

[0055] It can be seen that the above compounds have an inhibitory effect on human cystathionine γ-lyase, IC50. 50 The value was 7.545 μM, indicating that only a high dose of varenicline could inhibit the activity of human cystathionine γ-lyase.

[0056] Example 2

[0057] Determination of the inhibitory effect of varenicline on bacterial cystathionine γ-lyase

[0058] The specific implementation process is as follows:

[0059] 1) Both bacterial cystathionine γ-lyase and fluorescent probe Azmc stock solutions were stored in a -80℃ freezer;

[0060] 2) Thaw 10 mM bacterial cystathionine γ-lyase at room temperature in a cryopreservation plate (-4 to 4°C), take 5 μL and dilute it in 70 μL borate-borax buffer (pH=8), and add it to the detection plate;

[0061] 3) Take 1 μL of the inhibitor varenicline at different concentrations (0 mM, 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, 2.5 mM) and add it to the solution obtained in step 2).

[0062] 4) Add 10 μL of 0.1 mM L-cysteine ​​to the solution obtained in step 3) above;

[0063] 5) Add 10 μL of 0.1 mM PLP to the solution obtained in step 4) above;

[0064] 6) Add 4 μL of 0.5 mM Azmc to the solution obtained in step 5) above, incubate at 37°C using a fluorescence microplate reader, and monitor the fluorescence value emitted at 450 nm after excitation at 380 nm using a fluorescence microplate reader. Detect the fluorescence value while incubating, and incubate for 1 h.

[0065] 7) Statistically analyze the fluorescence values ​​emitted at 380 nm excitation and 450 nm for each group before and after incubation. Using the fluorescence change value of the control group (inhibitor concentration of 0) before and after incubation as 100, compare the fluorescence change values ​​of different inhibition groups before and after incubation to obtain the residual activity value. Use GraphPad Pris8 software to plot the logarithm of the inhibitor concentration (logC(vareniclan)) on the x-axis and the corresponding residual activity value on the y-axis. The results are shown in Figure 2. Figure 2 shows the ratio of different inhibitor concentrations to enzyme activity inhibition, indicating the inhibitory ability of the compound, expressed as the inhibitor concentration at which half the enzyme activity is inhibited. IC50 50 The formula for calculation is Y = 100 / (1 + 10^((X - LogIC)). 50 ))), where Y represents the remaining activity fraction, X represents the common logarithm of the inhibitor compound concentration, and ∧ refers to the exponentiation algorithm.

[0066] It can be seen that the above compounds have a significant inhibitory effect on bacterial cystathionine γ-lyase, IC50. 50 The value was 2.958 μM, indicating that varenicline can effectively inhibit the activity of bacterial cystathionine γ-lyase.

[0067] Examples 1 and 2 above demonstrate that varenicline mainly inhibits bacterial cystathionine γ-lyase, while having minimal inhibitory effect on human cystathionine γ-lyase, indicating that varenicline is specific for both bacterial and human cystathionine γ-lyase.

[0068] Example 3

[0069] KB disk assay for the inhibitory effect of varenicline combined with gentamicin on Pseudomonas aeruginosa.

[0070] The specific implementation process is as follows:

[0071] Gentamicin-resistant Pseudomonas aeruginosa in the logarithmic growth phase was evenly spread on a pre-prepared MH plate with a turbidity of 0.7 (0.2 for blank saline). Sterile filter paper was placed on the plate, and drugs were added to the filter paper in the following ways: 1 μL DMSO, 5 μg gentamicin, 5 μg gentamicin + 1 μL DMSO, 5 μg gentamicin + 5 μg varenicline, and 5 μg varenicline. After incubation at 35°C and 5% CO2 for 24 h, the inhibition zones produced by the drugs were observed.

[0072] As shown in Figure 3, the inhibition zone of gentamicin combined with varenicline was significantly larger than that of gentamicin alone.

[0073] Example 4

[0074] Determination of the inhibitory effect of gentamicin combined with varenicline on Pseudomonas aeruginosa using the broth dilution method

[0075] To determine the minimum inhibitory concentration (MIC) of gentamicin-resistant Pseudomonas aeruginosa against gentamicin: Add 100 μL of MH liquid medium to wells F1-F10 of a 96-well plate. Add 100 μL of 516 μg / mL gentamicin to well F1, mix well, and then add 100 μL to well F2. Repeat this process until well F10. After mixing, discard 100 μL. The gentamicin concentrations from F1 to F10 are 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, and 0.5 μg / mL, respectively. Adjust the bacterial suspension to a turbidity of 0.7 using a turbidimeter (blank saline is 0.2). At this point, the bacterial count is 1-2 × 10⁻⁶. 8 CFU / mL, diluted with MH liquid medium to 1×10⁻⁶. 6 CFU / mL, 100 μL of gentamicin was added to wells F1 to F11 respectively. Well F11 was supplemented with 100 μL of MH liquid medium as a positive control. Well F12 was supplemented with 200 μL of MH liquid medium as a blank control. At this point, the gentamicin concentrations in wells F1-F10 were 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL, respectively. Wells G1-G12 were replicates of wells F1-F12. After incubation at 35℃ and 5% CO2 for 24 h, the clear, transparent wells showing no bacterial growth were observed as the MIC of this strain. The MIC of this gentamicin-resistant Pseudomonas aeruginosa was 16 μg / mL.

[0076] Add 100 μL of LB liquid medium to wells H1-H10 of a 96-well plate and dilute to 1×10⁻⁶. 6CFU / mL bacterial culture was incubated with gentamicin and varenicline to achieve final gentamicin concentrations of 32 μg / mL (H1 / H2), 16 μg / mL (H3 / H4), 8 μg / mL (H5 / H6), and 4 μg / mL (H7 / H8), and a final varenicline concentration of 128 μg / mL. 100 μL of varenicline at a concentration of 256 μg / mL was added to well C9, 100 μL of LB broth was added to well H10 as a positive control, and 200 μL of LB broth was added to well H11 as a blank control. After incubation at 35℃ and 5% CO2 for 24 h, the addition of varenicline reduced the original MIC to half, i.e., 8 μg / mL.

[0077] The results are shown in Figure 4. In the figure, the solutions in wells F1-F4 and G1-G4 were all clear, indicating no bacterial growth. Therefore, the MIC of gentamicin was 16 μg / mL. The solutions in wells H1-H6 were also clear, indicating no bacterial growth. H1 and H2 were gentamicin 32 μg / mL combined with varenicline; H3 and H4 were gentamicin 16 μg / mL combined with varenicline; H5 and H6 were gentamicin 8 μg / mL combined with varenicline; and H7 and H8 were gentamicin 4 μg / mL combined with varenicline. This indicates that the combination of gentamicin and varenicline reduced the MIC of gentamicin from 16 μg / mL to 8 μg / mL. Well H9 was treated with the same concentration of varenicline as wells H1-H8, and the solution was turbid, indicating bacterial growth. Varenicline alone did not inhibit bacterial growth. Well H10 was a positive control, and well H11 was a blank control. The combined use of gentamicin and varenicline reduced the minimum inhibitory concentration (MIC) of gentamicin to half of its original value, namely 8 μg / mL.

[0078] Example 5

[0079] Growth curve of gentamicin combined with varenicline against gentamicin-resistant Pseudomonas aeruginosa

[0080] The bacteria were divided into a blank control group (culture medium only), a positive control group (culture medium and bacteria), a gentamicin 8 μg / mL group, a gentamicin 8 μg / mL + varenicline 500 μM group, and a gentamicin 8 μg / mL + varenicline 200 μM group. The bacteria were incubated in a shaker at 37℃ for 48 h. Samples of 200 μL were taken at 0 h, 6 h, 12 h, 24 h, and 48 h, and absorbance was measured at 600 nm, and a line graph was plotted. The results are shown in Figure 5. Gentamicin combined with varenicline inhibited the growth of gentamicin-resistant Pseudomonas aeruginosa. Specifically, gentamicin at 8 μg / mL combined with varenicline 500 μM significantly inhibited the growth of gentamicin-resistant Pseudomonas aeruginosa. Varenicline concentrations above 500 μmol / L showed a significant antibacterial effect, which was statistically significant.

[0081] Example 6

[0082] Crystal violet staining method for measuring gentamicin-resistant Pseudomonas aeruginosa biofilm

[0083] The cells were divided into three groups: a blank control group (culture medium only), a positive control group (culture medium and bacteria), a gentamicin 2 μg / mL group, and a gentamicin 2 μg / mL + varenicline 500 μM group. The bacterial culture and drug-culture medium mixture was added to 96-well PVC plates and incubated at 37℃ for 24 h. The culture medium was then discarded, and the cells were washed three times with sterile water, stained with 0.1% crystal violet for 15 min, washed three times with sterile water, and the absorbance was measured at 570 nm after dissolving the crystal violet in 95% ethanol. A bar chart was plotted, and statistical analysis was performed using an unpaired t-test with GraphPad Pris8 software. The results are shown in Figure 6. Gentamicin at 2 μg / mL combined with varenicline 500 μM significantly inhibited the formation of gentamicin-resistant Pseudomonas aeruginosa biofilms (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; no statistically significant difference in ns).

[0084] Example 7

[0085] The specific procedure for detecting the inhibitory effect of gentamicin combined with varenicline on Staphylococcus aureus using the KB disk method is as follows:

[0086] Staphylococcus aureus in the logarithmic growth phase was evenly spread on a pre-prepared MH plate with a turbidity of 0.7 (0.2 for blank saline). Sterile filter paper was placed on the plate, and drugs were added to the filter paper as follows: 1 μL DMSO, 5 μg gentamicin, 5 μg gentamicin + 1 μL DMSO, 5 μg gentamicin + 5 μg varenicline, and 5 μg varenicline. After incubation at 35°C in a 5% CO2 incubator for 24 h, the inhibition zones produced by the drugs were observed.

[0087] As shown in Figure 7, the inhibition zone of gentamicin combined with varenicline was significantly larger than that of gentamicin alone.

[0088] Example 8

[0089] Determination of the inhibitory effect of gentamicin combined with varenicline on Staphylococcus aureus using the broth dilution method

[0090] To determine the minimum inhibitory concentration (MIC) of gentamicin against Staphylococcus aureus: Add 100 μL of MH liquid medium to wells A1-A11 of a 96-well plate. Add 100 μL of gentamicin (516 μg / mL) to well A1, mix well, and then add 100 μL to well A2. Repeat this process until well A10. After mixing, discard 100 μL. The gentamicin concentrations from A1 to A10 are 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, and 0.5 μg / mL, respectively. Adjust the bacterial suspension to a turbidity of 0.7 using a turbidimeter (blank saline is 0.2). At this point, the bacterial count is 1-2 × 10⁻⁶. 8 CFU / mL, diluted with MH liquid medium to 1×10⁻⁶. 6 CFU / mL, 100 μL of gentamicin was added to wells A1 to A11 respectively. 100 μL of MH liquid medium was added to well A11 as a positive control, and 200 μL of MH liquid medium was added to well A12 as a blank control. At this point, the gentamicin concentrations in wells A1-A10 were 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL, respectively. Wells B1-B12 were replicates of wells A1-A12. After incubation at 35℃ and 5% CO2 for 24 hours, the clear, transparent wells showing no bacterial growth were observed as the MIC of this strain of Staphylococcus aureus, which was 16 μg / mL.

[0091] Add 100 μL of LB liquid medium to wells C1-C10 of a 96-well plate and dilute to 1×10⁻⁶. 6 CFU / mL bacterial culture was incubated with gentamicin and varenicline to achieve final gentamicin concentrations of 32 μg / mL (C1 / C2), 16 μg / mL (C3 / C4), 8 μg / mL (C5 / C6), and 4 μg / mL (C7 / C8), and a final varenicline concentration of 128 μg / mL. 100 μL of varenicline at a concentration of 256 μg / mL was added to well C9, 100 μL of LB broth was added to well C10 as a positive control, and 200 μL of LB broth was added to well C11 as a blank control. After incubation at 35℃ and 5% CO2 for 24 h, the addition of varenicline reduced the original MIC to half, i.e., 8 μg / mL.

[0092] The results are shown in Figure 8. In Figure 8, the solutions in wells A1-A4 and B1-B4 were all clear, indicating no bacterial growth. Therefore, the MIC of gentamicin against Staphylococcus aureus was 16 μg / mL. The solutions in wells C1-C6 were also clear, indicating no bacterial growth. Wells C1 and C2 contained gentamicin at 32 μg / mL combined with varenicline; wells C3 and C4 contained gentamicin at 16 μg / mL combined with varenicline; wells C5 and C6 contained gentamicin at 8 μg / mL combined with varenicline; and wells C7 and C8 contained gentamicin at 4 μg / mL combined with varenicline. This indicates that the combination of gentamicin and varenicline reduced the MIC of gentamicin from 16 μg / mL to 8 μg / mL. Well C9 contained varenicline at the same concentration as in wells C1-C8, but the solution was turbid, indicating bacterial growth. Varenicline alone did not inhibit bacterial growth. Well C10 was a positive control, and well C11 was a blank control. Gentamicin combined with varenicline reduced the minimum inhibitory concentration (MIC) of gentamicin to half of its original value, i.e., 8 μg / mL.

[0093] Example 9

[0094] Growth curve of gentamicin combined with varenicline against Staphylococcus aureus

[0095] The study included a blank control group (culture medium only), a positive control group (culture medium and bacteria), a gentamicin 4 μg / mL group, a gentamicin 4 μg / mL + varenicline 500 μM group, and a gentamicin 4 μg / mL + varenicline 200 μM group. All samples were incubated at 37℃ in a shaker for 48 h. Samples of 200 μL were taken at 0 h, 6 h, 12 h, 24 h, and 48 h, and absorbance was measured at 600 nm. Line graphs were plotted. The results are shown in Figure 9. Gentamicin combined with varenicline inhibited the growth of Staphylococcus aureus. Specifically, gentamicin at 4 μg / mL combined with varenicline 500 μM significantly inhibited the growth of Staphylococcus aureus.

[0096] Example 10

[0097] Crystal violet staining method for measuring Staphylococcus aureus biofilm

[0098] The cells were divided into three groups: a blank control group (culture medium only), a positive control group (culture medium and bacteria), a gentamicin 1 μg / mL group, and a gentamicin 1 μg / mL + varenicline 500 μM group. The bacterial culture and drug-culture medium mixture was added to 96-well PVC plates and incubated at 37℃ for 24 h. The culture medium was then discarded, and the cells were washed three times with sterile water, stained with 0.1% crystal violet for 15 min, washed three times with sterile water, and the absorbance was measured at 570 nm after dissolving the crystal violet in 95% ethanol. A bar chart was plotted, and statistical analysis was performed using an unpaired t-test with GraphPad Pris8 software. The results are shown in Figure 10. Gentamicin at 1 μg / mL combined with varenicline 20 μM significantly inhibited the formation of standard Staphylococcus aureus biofilm (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; no statistically significant difference in ns).

[0099] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of varenicline in the preparation of antibiotic enhancers, characterized in that, Varenicline, in combination with antibiotics, acts on Gram-negative bacilli and Gram-positive cocci, and varenicline has the structure shown in formula (I): (I); the antibiotic is gentamicin; the Gram-negative bacillus is Pseudomonas aeruginosa, and the Gram-positive coccus is Staphylococcus aureus.

2. The application according to claim 1, characterized in that, The antibiotic enhancer is a reagent that inhibits bacterial tolerance and / or drug resistance.

3. The application according to claim 1, characterized in that, The combination of varenicline and antibiotics enhances the antibacterial ability against Gram-negative bacilli and Gram-positive cocci, and lowers the minimum inhibitory concentration (MIC) value of antibiotics to inhibit bacterial growth.

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