A drug for reducing bacterial resistance, its preparation method and application

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a drug for reducing bacterial resistance, its preparation method, and its application. This invention is the first to discover a novel use for the AMPA receptor antagonist Fanapanel (ZK200775) in inhibiting New Delhi metallo-β-lactamase-1 (NDM-1). This invention further relates to the application of Fanapanel in combination with β-lactam antibiotics in the preparation of synergistic antibacterial drugs. Experimental results show that Fanapanel can significantly enhance the sensitivity of NDM-1-expressing drug-resistant bacteria to β-lactam antibiotics, exhibiting significant synergistic antibacterial effects both in vitro and in vivo, while also possessing low cytotoxicity and low hemolytic activity, and good safety. This invention provides a new strategy for treating NDM-1-mediated drug-resistant bacterial infections.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a drug for reducing bacterial resistance, its preparation method, and its application. Background Technology

[0002] New Delhi metallo-β-lactamase-1 (NDM-1) belongs to the B1 class of metallo-β-lactamases (MBLs) and can efficiently hydrolyze almost all β-lactam antibiotics, including carbapenems. This poses a risk of ineffectiveness for core drugs used in the clinical treatment of multidrug-resistant Gram-negative bacterial infections. More seriously, the NDM-1 encoding gene can be horizontally transferred between different bacterial strains via plasmids, accelerating the spread of resistance. Therefore, developing safe and effective NDM-1 inhibitors for combination therapy with existing β-lactam antibiotics to restore their antibacterial activity is an urgent and crucial strategy for addressing the current drug resistance crisis.

[0003] Currently, the main research and development strategies for NDM-1 inhibitors include the following categories: (1) metal coordination inhibitors, such as thiol compounds, carboxylic acids, and boric acids; (2) metal chelation inhibitors, such as ethylenediaminetetraacetic acid and aspergillin A; and (3) covalent inhibitors, such as ebuselenoline and 3-bromopyruvic acid. Although the above research has made some progress, most of the NDM-1 inhibitors reported so far are still in the laboratory research stage and generally suffer from problems such as low inhibitory activity, high cytotoxicity, or poor pharmacokinetic properties.

[0004] Fanapanel (ZK200775) is a known AMPA receptor antagonist with good neuroprotective and anticonvulsant effects, and has demonstrated good safety in clinical trials. Currently, there are no reports regarding Fanapanel having NDM-1 inhibitory activity or reversing NDM-1-mediated bacterial resistance. Summary of the Invention

[0005] In view of the above-mentioned technical problems existing in the prior art, the present invention aims to provide a new NDM-1 inhibitor, Fanapanel, which can be used in combination with β-lactam antibiotics to effectively reverse NDM-1-mediated bacterial resistance and has high in vitro and in vivo safety.

[0006] In a first aspect, the present invention provides the use of Fanapanel or a pharmaceutically acceptable salt thereof in the preparation of NDM-1 type metallo-β-lactamase inhibitors.

[0007] In this invention, the molecular formula of Fanapanel (also known as ZK200775) is C 14 H 15F3N3O6P has the following chemical structural formula: .

[0008] In a second aspect, the present invention provides the use of Fanapanel or a pharmaceutically acceptable salt thereof in the preparation of a medicament for improving the antibiotic sensitivity of bacteria, wherein the bacteria are drug-resistant bacteria expressing NDM-1 type metallo-β-lactamases, and the antibiotic is a β-lactam antibiotic.

[0009] Thirdly, the present invention provides the use of Fanapanel or a pharmaceutically acceptable salt thereof in combination with an antibiotic in the preparation of a medicament for inhibiting or killing bacteria, wherein the antibiotic is a β-lactam antibiotic and the bacteria are drug-resistant bacteria expressing NDM-1 type metallo-β-lactamases.

[0010] Fourthly, the present invention provides the use of Fanapanel or a pharmaceutically acceptable salt thereof in combination with an antibiotic in the preparation of a medicament for the prevention and / or treatment of drug-resistant bacterial infections, wherein the antibiotic is a β-lactam antibiotic and the drug-resistant bacteria are drug-resistant bacteria expressing NDM-1 type metallo-β-lactamases.

[0011] Furthermore, in the above applications, the β-lactam antibiotic is a carbapenem antibiotic. Optionally, the β-lactam antibiotic may also be selected from other β-lactam antibiotics, such as penicillin antibiotics, cephalosporin antibiotics, cephamycin antibiotics, and penicillene antibiotics.

[0012] Furthermore, the β-lactam antibiotic is selected from one or more of meropenem, doripenem, imipenem, panipenem, and biapenem.

[0013] Furthermore, the drug-resistant bacteria expressing NDM-1 are Gram-negative bacteria expressing NDM-1 type metallo-β-lactamase.

[0014] Furthermore, the bacteria are Enterobacteriaceae bacteria that express NDM-1 type metallo-β-lactamase.

[0015] Optionally, the drug-resistant bacteria expressing NDM-1 may also be other Gram-negative bacteria expressing NDM-1, including but not limited to Pseudomonas aeruginosa, Acinetobacter baumannii, and Vibrio cholerae.

[0016] Fifthly, the present invention provides a pharmaceutical composition for inhibiting or killing bacteria, the composition comprising Fanapanel or a pharmaceutically acceptable salt thereof, and a β-lactam antibiotic; wherein the bacteria are drug-resistant bacteria expressing NDM-1 type metallo-β-lactamases.

[0017] Furthermore, in the pharmaceutical composition, the β-lactam antibiotic is a carbapenem antibiotic.

[0018] Optionally, the β-lactam antibiotic may also be selected from other β-lactam antibiotics, such as penicillin antibiotics, cephalosporin antibiotics, cephamycin antibiotics, and penicillene antibiotics.

[0019] Furthermore, the β-lactam antibiotic is selected from one or more of meropenem, doripenem, imipenem, panipenem, and biapenem.

[0020] Furthermore, the drug-resistant bacteria expressing NDM-1 are Gram-negative bacteria expressing NDM-1 type metallo-β-lactamase.

[0021] Furthermore, the bacteria are Enterobacteriaceae bacteria that express NDM-1 type metallo-β-lactamase.

[0022] Optionally, the drug-resistant bacteria expressing NDM-1 may also be other Gram-negative bacteria expressing NDM-1, including but not limited to Pseudomonas aeruginosa, Acinetobacter baumannii, and Vibrio cholerae.

[0023] Furthermore, the pharmaceutical composition is a fixed-dose combination preparation or an individually packaged combination preparation, the combination preparation containing a first formulation unit and a second formulation unit that are independent of each other, the first formulation unit containing Fanapanel or a pharmaceutically acceptable salt thereof, the second formulation unit containing the β-lactam antibiotic, the first formulation unit and the second formulation unit being used for simultaneous or sequential administration.

[0024] Optionally, the pharmaceutical composition may also contain a pharmaceutically acceptable carrier, excipient, or diluent.

[0025] Optionally, the dosage form of the pharmaceutical composition includes, but is not limited to, injections, tablets, capsules, or powder for injection.

[0026] In a sixth aspect, the present invention provides the use of Fanapanel or a pharmaceutically acceptable salt thereof in the in vitro inhibition of NDM-1 type metallo-β-lactamase activity for non-disease therapeutic purposes.

[0027] Furthermore, the application includes contacting Fanapanel with NDM-1 type metallo-β-lactamase or bacteria expressing NDM-1 type metallo-β-lactamase to inhibit NDM-1 enzyme activity.

[0028] Furthermore, the application includes contacting Fanapanel, together with antibiotics, with bacteria expressing NDM-1 type metallo-β-lactamases.

[0029] Furthermore, the application improves the bacteria's sensitivity to antibiotics.

[0030] Furthermore, the application has an inhibitory effect on bacteria.

[0031] Furthermore, the antibiotic is a β-lactam antibiotic.

[0032] Optionally, the in vitro application includes use in drug screening, scientific research, or diagnostic kits.

[0033] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention reveals for the first time the novel function of Fanapanel (ZK200775) as an NDM-1 inhibitor. In vitro enzyme inhibition experiments show that Fanapanel has significant inhibitory activity against NDM-1, IC50... 50 The value is 19.45 μM.

[0034] (2) This invention confirms that the combination of Fanapanel and β-lactam antibiotics has a significant synergistic antibacterial effect. In vitro antibacterial experiments showed that, in the presence of 64 μg / mL Fanapanel, the minimum inhibitory concentration (MIC) of meropenem against NDM-1-expressing Escherichia coli was reduced by 8 to 64 times, with a FICI value <0.5, demonstrating a synergistic antibacterial effect. Time-bactericidal curve experiments confirmed that the combination of 32 μg / mL Fanapanel and 16 μg / mL meropenem could completely kill recombinant NDM-1-positive Escherichia coli within 4 hours.

[0035] (3) This invention confirms the significant therapeutic effect of the combination of Fanapanel and meropenem in vivo. In a mouse model of systemic infection, the combination of meropenem and Fanapanel increased the survival rate of infected mice from 10% to 60%, significantly higher than the 40% in the meropenem monotherapy group. Simultaneously, the combined treatment significantly reduced the bacterial load in the liver, kidneys, and spleen of infected mice, and pathological sections showed that the combined treatment effectively alleviated pathological damage in these organs.

[0036] (4) This invention demonstrates that Fanapanel has good in vitro and in vivo safety. Cytotoxicity experiments showed that the survival rate of HEK-293T cells and HUVECs cells was still greater than 80% at concentrations as high as 128 μg / mL. Hemolysis experiments showed that Fanapanel did not cause significant hemolysis of erythrocytes in the concentration range of 2–128 μg / mL, demonstrating good blood compatibility. Attached Figure Description

[0037] Figure 1This is a graph showing the results of the determination of the Michaelis constant (Km) of NDM-1 enzyme kinetics.

[0038] Figure 2 This is a graph showing the half-maximal inhibitory concentration (IC50) of Fanapanel against NDM-1. The horizontal axis represents the concentration of Fanapanel, calculated using the common logarithm base 10.

[0039] Figure 3 This is an in vitro time-kill curve of Fanapanel and Meropenem, alone or in combination. Figure 3 Figure A shows the experimental results for E. coli BL21(DE3) / pET26b-NDM-1. Figure 3 Figure B shows the experimental results for E. coli 110034.

[0040] Figure 4 This is the result and quantitative analysis of the inhibition zone in the disc diffusion assay using Fanapanel or captopril in combination with meropenem, among which... Figure 4 Figure A shows the inhibition zone. Figure 4 Figure B shows the results of the quantitative analysis.

[0041] Figure 5 This is a graph showing the cytotoxicity assay results of Fanapanel on HEK-293T cells and HUVECs cells.

[0042] Figure 6 This is a graph showing the results of hemolytic activity assays for different concentrations of Fanapanel.

[0043] Figure 7 It describes the state and manifestations of infected mice, among which... Figure 7 Figure A shows that the infected model group exhibited symptoms such as curling up, lethargy, and disheveled hair. Figure 7 Figure B shows the diarrhea symptoms observed in the infection model group. Figure 7 Figure C shows the blank control group mice, which exhibited normal behavior.

[0044] Figure 8 This is a survival curve of mice infected with NDM-1 positive bacteria under different drug treatment conditions.

[0045] Figure 9 This is a graph showing the quantitative analysis of bacterial load in target organs of infected mice when Fanapanel and meropenem are used in combination.

[0046] Figure 10 These are histopathological HE staining images of mouse liver, spleen, and kidney.

[0047] Figure 11The figure shows the results of the zinc ion recovery experiment, illustrating the effect of exogenous ZnCl2 on the inhibition of NDM-1 activity by Fanapanel.

[0048] Figure 12 This is a Lineweaver-Burk double reciprocal plot of the inhibition kinetics of Fanapanel and EDTA on NDM-1.

[0049] Figure 13 This is a schematic diagram of the molecular docking between Fanapanel and NDM-1. Detailed Implementation

[0050] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods known in the art, or in accordance with the operating procedures recommended by the relevant reagent or instrument manufacturers.

[0051] To facilitate understanding of the technical solutions of this invention, the key terms involved in this invention are explained below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0052] NDM-1: New Delhi metallo-β-lactamase-1, belongs to the B1 class of metallo-β-lactamases, and can hydrolyze almost all β-lactam antibiotics, including carbapenems.

[0053] IC 50 Half-inhibition concentration (WIC) refers to the concentration of inhibitor required to inhibit 50% of enzyme activity in in vitro experiments.

[0054] MIC: Minimum inhibitory concentration, refers to the lowest concentration of antimicrobial drug required to inhibit the visible growth of bacteria in in vitro experiments.

[0055] FICI: Graded Antimicrobial Concentration Index, used to evaluate the effect of two drugs used in combination. FICI ≤ 0.5 indicates synergistic effect, 0.5 < FICI ≤ 4 indicates no effect, and FICI > 4 indicates antagonistic effect.

[0056] Example 1: In vitro NDM-1 inhibitory activity of Fanapanel 1. Construction of recombinant engineered bacteria The recombinant plasmid (NDM-1 recombinant plasmid) and E. coli BL21 (DE3) were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0057] BL21(DE3) competent cells were removed from the -80℃ cryogenic freezer and thawed on ice. In a clean bench, 1 μL of the NDM-1 recombinant plasmid was added to 100 μL of BL21(DE3) competent cells and gently mixed with a pipette. The competent cells were then incubated on ice for 30 min, followed by heat shock at 42℃ for 90 s, and then incubated on ice for another 2 min. 900 μL of LB liquid medium was added to the competent cells, and the cells were incubated in a constant temperature shaking incubator at 37℃ and 220 rpm for 1 h. The competent cell culture was then evenly spread onto LB solid medium containing 25 μg / mL kanamycin and incubated upside down at 37℃ overnight.

[0058] 2. Isolation and purification of NDM-1 (1) Preparation of solution LB medium: Weigh 12.5g of LB medium powder into a conical flask containing 500mL of ddH2O, dissolve it, autoclave and cool to room temperature before use.

[0059] Kanamycin-resistant LB agar medium: Weigh 12.5g of LB medium and 7.5g of agar powder into a conical flask containing 500mL of ddH2O and dissolve. Autoclave and cool to 55-60℃, then add 1250μL of 10mg / mL kanamycin sulfate to the solution and mix well. Pour the mixture into sterile petri dishes and cool for later use.

[0060] Lysis buffer: Weigh 2.383 g of 4-hydroxyethylpiperazine ethanesulfonic acid and 3.506 g of NaCl into an Erlenmeyer flask containing 200 mL of ddH2O, dissolve them, add 200 μL of Triton X-100 and mix well. Adjust the pH to approximately 8.0; that is, 50 mM 4-hydroxyethylpiperazine ethanesulfonic acid + 300 mM NaCl + 0.1% Triton X-100, pH ≈ 8.0.

[0061] Binding buffer: Weigh 2.383 g of 4-hydroxyethylpiperazine ethanesulfonic acid and 3.506 g of NaCl into an Erlenmeyer flask containing 200 mL of ddH2O, dissolve them, add 400 μL of Triton X-100 and mix well. Adjust the pH to approximately 8.0; that is, 50 mM 4-hydroxyethylpiperazine ethanesulfonic acid + 300 mM NaCl + 0.2% Triton X-100, pH ≈ 8.0.

[0062] Washing buffer: Weigh 2.383g of 4-hydroxyethylpiperazine ethanesulfonic acid, 3.506g of NaCl, 0.681g of imidazole and 0.003g of ZnCl2 into a 200mL Erlenmeyer flask containing ddH2O, dissolve, and adjust the pH to approximately 8.0; that is, 50mM 4-hydroxyethylpiperazine ethanesulfonic acid + 300mM NaCl + 50mM imidazole + 0.1mM ZnCl2, pH≈8.0.

[0063] Elution buffer: Weigh 2.383g of 4-hydroxyethylpiperazine ethanesulfonic acid, 3.506g of NaCl, 3.404g of imidazole and 0.003g of ZnCl2 into an Erlenmeyer flask containing 200mL of ddH2O, dissolve, and adjust the pH to approximately 8.0; that is, 50mM 4-hydroxyethylpiperazine ethanesulfonic acid + 300mM NaCl + 250mM imidazole + 0.1mM ZnCl2, pH≈8.0.

[0064] HEPES buffer: Weigh 5.95 g of 4-hydroxyethylpiperazine ethanesulfonic acid into 500 mL of ddH2O to a final concentration of 50 mM, and adjust the pH to approximately 7.0. Then add bovine serum albumin to a final concentration of 50 μg / mL, dimethyl sulfoxide to a final concentration of 1.25%, and Triton X-100 to a final concentration of 0.01%.

[0065] Cefotaxime working solution: Add 20 μL of 10 mM cefotaxime stock solution to 3.98 mL of HEPES buffer, mix well, and obtain a working solution with a final concentration of 0.05 mM.

[0066] NDM-1 enzyme working solution: Add 100 μL of 1 μM enzyme stock solution to 400 μL of HEPES buffer, mix well, and obtain an enzyme working solution with a final concentration of 0.2 μM.

[0067] Captopril stock solution: Weigh an appropriate amount of captopril powder and dissolve it in HEPES buffer to a final captopril concentration of 20 mM. Store at -20°C for later use.

[0068] Inhibitor solution: Dissolve the inhibitor in dimethyl sulfoxide to a final inhibitor concentration of 20 mM, and store at -20°C for later use.

[0069] (2) Expression and purification of NDM-1 NDM-1 Expression: E. coli BL21(DE3) strain containing the pET26b-NDM-1 plasmid was inoculated onto LB agar plates containing 25 μg / mL kanamycin. Single colonies were picked using a sterile disposable inoculating loop and incubated overnight at 37°C with shaking at 150 rpm in 10 mL of LB broth containing the same concentration of kanamycin. The next day, 5 mL of the overnight bacterial culture was inoculated into two 500 mL aliquots of LB broth containing 25 μg / mL kanamycin and incubated at 37°C with shaking at 150 rpm for approximately 5 h, until the OD600 reached 0.6. The incubation temperature was then lowered to 18°C, and IPTG was added to a final concentration of 0.1 mM. Expression was induced for 20 h at 18°C ​​with shaking at 200 rpm. After induction, the bacterial culture was aliquoted into 50 mL centrifuge tubes and centrifuged at 4°C with centrifugation at 10,000 rpm for 5 min to collect the bacterial cells. The bacterial pellet was resuspended and washed once with PBS, centrifuged again, and then stored at -80℃ for later use.

[0070] Purification of NDM-1: ① Cell lysis: The bacterial pellet stored at -80℃ was resuspended in 20 mL of lysis buffer containing 200 μL of protease inhibitor, 250 μL of lysozyme (0.25 mg / mL), and 20 μL of DNase. The suspension was then placed on ice and sonicated at 400 W for 15 min, with a 5-second interval between induction and 5-second pauses. After lysis, the lysate was centrifuged at 4℃ and 10,000 rpm for 5 min to remove cell debris. The supernatant was collected and filtered through a 0.45 μm pore size filter membrane; the resulting filtrate was the clear crude enzyme solution. ② Nickel column affinity purification: At 4℃, after draining the storage buffer from the Ni-NTA pre-packed column by gravity, equilibrate the column with twice the column volume of binding buffer. Then, pass 2 mL of crude enzyme solution onto the nickel column twice. Next, wash the column with twice the column volume of elution buffer, obtaining 2 mL of washing buffer each time, repeating 6 times. Finally, elute the target protein on the column with 2 mL of elution buffer, obtaining 2 mL of eluent each time, repeating 4 times. ③ Sodium dodecyl sulfate-polyacrylamide gel electrophoresis identification: Perform gel electrophoresis on the above crude enzyme solution, lysed precipitate, flow-through buffer, washing buffer, and eluent according to the gel electrophoresis kit instructions. Electrophoresis is performed at a constant voltage of 150V for 50 min. After electrophoresis, remove the gel and stain it in Coomassie Brilliant Blue rapid staining solution, followed by destaining and result observation.

[0071] 3. Determination of enzyme kinetic parameters (1) Experimental Methods: To evaluate the activity of the purified NDM-1 enzyme, enzyme kinetics were determined using cefotaxime as a substrate. The experiment was conducted in 96-well plates with a total reaction volume of 200 µL, containing cefotaxime substrate at a final concentration of 0-30 µM and NDM-1 enzyme at a final concentration of 10 nM. The reaction was started immediately after mixing the substrate and enzyme, and the absorbance was continuously monitored at 492 nm using a microplate reader, with data recorded every 10 s for a total of 60 s. The absorbance change data in the first 30 s of the reaction were used to calculate the initial reaction rate, and the Michaelis constant (Km value) was calculated by plotting a Michaelis kinetic curve using GraphPad Prism 8.0 software for nonlinear fitting. The Km value reflects the affinity between the enzyme and the substrate; the smaller the value, the stronger the binding ability between the enzyme and the substrate.

[0072] (2) Experimental results: The Km value of NDM-1 is as follows Figure 1 As shown, the Km value is 1.37 µM, indicating that the purified NDM-1 has good enzyme activity and can be used for subsequent inhibitor activity evaluation.

[0073] 4. IC 50 Measurement (1) Experimental method: Dissolve Fanapanel in sterile dimethyl sulfoxide and then dilute with HEPES buffer.

[0074] The concentration range of fanapanel was 0-300 μM, and the concentration of the substrate cefotaxime was 10 μM. Different concentrations of fanapanel were first co-incubated with the enzyme for 15 min, then the substrate was added to initiate the reaction. After mixing thoroughly, the absorbance change was immediately measured at 492 nm. Measurements were taken every 10 s for 1 min, and the increase in hydrolysis product in the first 30 s was used to calculate the initial reaction rate. Each experiment was performed in at least three parallel measurements.

[0075] The residual enzyme activity was calculated after adding different concentrations of inhibitor. The residual enzyme activity R(100%) = (Vi / V0) × 100% (Vi: the rate of substrate hydrolysis by the enzyme in the presence of the inhibitor; V0: the rate of substrate hydrolysis by the enzyme in the absence of the inhibitor). A nonlinear fitting plot of the inhibitor concentration versus residual enzyme activity was plotted using Graphpad Prism 8.0 to obtain the IC50. 50 value.

[0076] (2) Experimental Results Fanapanel's IC for NDM-1 50 The measurement results are as follows Figure 2 As shown, Fanapanel exhibited good inhibitory activity against NDM-1, with an IC50 value of 19.45 µM.

[0077] Example 2: In vitro antibacterial experiment of Fanapanel combined with β-lactam antibiotics 1. Checkerboard method combined with drug sensitivity test 1.1 Experimental Methods The experimental strains were the recombinant strain expressing NDM-1: E. coli BL21(DE3) / pET26b-NDM-1 and the clinical strains expressing NDM-1: E. coli 110034 and E. coli BAA-2452.

[0078] The MIC values ​​of meropenem / inhibitors (Fanapanel or captopril) alone and in combination against NDM-1-expressing strains were determined according to the microbroth dilution method established by the Clinical and Laboratory Standards Institute (CLSI). For single-drug MIC determination, under aseptic conditions, 200 μL of meropenem, Fanapanel, or captopril at a concentration of 2048 μg / mL was added to the first well of a 96-well plate. 100 μL of culture medium was added to each well in wells 2-12. A two-fold serial dilution was then performed from well 1 to well 11, with 100 μL of bacterial culture added to each well to achieve a final concentration range of 0-1024 μg / mL for meropenem, Fanapanel, or captopril. For combination MIC determination, a checkerboard method was used. Meropenem was serially diluted two-fold horizontally, and different concentrations of Fanapanel or captopril were added vertically to achieve a final concentration range of 0-128 μg / mL for both drugs. NDM-1 positive Escherichia coli culture was adjusted to a 0.5 MCF McFarland standard using LB medium, then diluted 100-fold. 100 μL of the culture was added to each well, bringing the total volume to 200 μL per well, resulting in a final bacterial concentration of 5 × 10⁻⁶. 5 CFU / mL. Incubate the 96-well plate in a 37°C incubator for 16-20 hours, observe and record bacterial growth, and use the lowest drug concentration at which no bacterial growth is visible to the naked eye as the MIC.

[0079] Calculation of the synergistic antimicrobial index (FICI): The FICI value can be calculated according to a specific equation, i.e., FICI = FIC. A + FIC B FIC A The MIC value of drug A when used in combination with other drugs / the MIC value of drug A when used alone, FIC BIt is the MIC value of drug B when used in combination / the MIC value of drug B when used alone, and is used to evaluate the interaction of two different drugs when used in combination. If FICI ≤ 0.5, it indicates that the combination of the two drugs has a synergistic effect. If 0.5 < FICI ≤ 4, there is no relevant effect when the two drugs are used in combination. If FICI > 4, it indicates that there is an antagonistic effect when the two drugs are used in combination. The smaller the FICI index, the stronger the synergistic effect of the drugs.

[0080] 1.2 Experimental results The MIC values and FICI values of meropenem when used alone and in combination with Fanapanel against drug-resistant strains expressing NDM-1 are shown in Table 1. When Fanapanel is used alone, its MIC value against Escherichia coli expressing NDM-1 > 1024 μg / mL, indicating that Fanapanel has no obvious antibacterial activity by itself. When Fanapanel is used in combination with meropenem, under the condition that the concentration of Fanapanel is 64 μg / mL, the MIC value of meropenem drops to 2 - 8 μg / mL, with a decrease of 8 to 64 times, indicating that Fanapanel can significantly restore the antibacterial activity of meropenem against drug-resistant bacteria. Compared with the positive control captopril, Fanapanel shows a better effect of reducing the MIC of meropenem in strains E. coli BL21(DE3) / pET26b-NDM-1 and E. coli 110034. Further quantitative evaluation of the combined effect through the FICI value shows that in most strains, the FICI value of the combination of Fanapanel and meropenem < 0.5, indicating that the two have a significant synergistic antibacterial effect.

[0081] Table 1 Synergistic antibacterial activity of the combination of Fanapanel and meropenem against Escherichia coli expressing NDM-1

[0082] 2. Time-kill curve / / 2.1 Experimental method The recombinant strain *E. coli* BL21(DE3) / pET26b-NDM-1 expressing NDM-1 and the clinically isolated drug-resistant strain *E. coli* 110034 were selected as experimental strains. A blank control group (containing only culture medium and bacterial suspension), a meropenem monotherapy group, a Fanapanel monotherapy group, and a combination therapy group (meropenem + Fanapanel) were set up. For the recombinant strain, the concentration of meropenem monotherapy was 16 μg / mL, the concentration of Fanapanel monotherapy was 32 μg / mL, and the concentration in the combination therapy group was 16 μg / mL meropenem + 32 μg / mL Fanapanel. For the clinical strain, the concentration of meropenem monotherapy was 16 μg / mL, the concentration of Fanapanel monotherapy was 64 μg / mL, and the concentration in the combination therapy group was 16 μg / mL meropenem + 64 μg / mL Fanapanel. Take sterile shaking tubes and add 10 mL of LB liquid medium (containing the corresponding drug concentration, 25 μg / mL kanamycin) to each tube. Then, add 100 μL of bacterial suspension at a concentration of 0.5 McFarland standard to each shaking tube and incubate at 37°C until the logarithmic growth phase (approximately 4-6 hours). Subsequently, adjust the bacterial suspension concentration in the logarithmic growth phase to 1.0 McFarland standard using LB liquid medium, adding 100 μL to each shaking tube. Perform 10-fold serial dilutions of each group of bacterial suspensions in 96-well plates using LB liquid medium. Add 10 μL of each dilution to MH plates, let the plates stand for approximately 30 minutes, then invert them and incubate overnight at 37°C for 16-20 hours. Observe and record the colony count. Plot time on the x-axis, logarithmic... 10 Plot a time-kill curve with CFU / mL as the ordinate.

[0083] 2.2 Experimental Results The results of the time-sterilization curve experiment are as follows: Figure 3As shown: For both test strains (E. coli BL21(DE3) / pET26b-NDM-1 and clinical isolate E. coli 110034), the blank control group showed normal growth, and Fanapanel alone did not show significant antibacterial activity at the tested concentrations. Although 16 μg / mL meropenem showed some inhibitory effect on both bacteria at 2 h, the bacteria could recover growth with prolonged culture time, indicating that meropenem alone could not achieve a sustained inhibitory effect. Combined administration showed a significant synergistic effect: 32 μg / mL Fanapanel combined with 16 μg / mL meropenem completely killed E. coli BL21(DE3) / pET26b-NDM-1 within 4 hours, and no bacterial recovery was observed within 10 hours. For E. coli 110034, 64 μg / mL Fanapanel combined with 16 μg / mL meropenem also significantly and persistently inhibited bacterial growth.

[0084] 3. Paper diffusion experiment 3.1 Experimental Methods The recombinant strain *E. coli* BL21(DE3) / pET26b-NDM-1 expressing NDM-1 was used as the experimental strain. First, 100 µL of the recombinant bacterial suspension (OD600 = 0.1) was spread evenly on an LB agar plate containing kanamycin using a sterile swab. Then, a paper disc containing 10 µg of meropenem was placed in the center of the agar plate, and different concentrations of Fanapanel solution (0, 8, and 16 µg / mL) were added to the center of each disc. Finally, the plate was inverted and incubated at 37°C for 16–20 h, and the diameter of the inhibition zone was measured.

[0085] 3.2 Experimental Results The results of the paper diffusion experiment are as follows Figure 4 As shown, the diameter of the inhibition zone in the combination therapy group was significantly larger than that in each single drug group, and at a concentration of 16 μg / mL, both the compound Fanapanel and the combination of captopril and meropenem produced obvious inhibition zones with a diameter of more than 20 mm.

[0086] Example 3: Cytotoxicity and Hemolysis Assays of Fanapanel 1. Cytotoxicity assay 1.1 Experimental Methods The effect of Fanapanel on the viability of HEK-293T cells and HUVECs was detected using the CCK-8 assay. Both cell lines were revived, passaged, and then collected at 5 × 10⁶ cells per well. 4Cells were seeded at a density of 100 μL of cell suspension in each well of a 96-well plate and incubated at 37°C and 5% CO2 for 24 hours. The old culture medium was discarded and replaced with complete culture medium containing different concentrations of Fanapanel (4-128 μg / mL), and incubated for another 24 hours, with 6 replicates for each concentration. After incubation, the supernatant was discarded, and 100 μL of complete culture medium containing 10% CCK-8 reagent was added to each well, and incubated for another 2 hours. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated using the following formula: Cell viability (%) = [(OD experimental group - OD blank group) / (OD control group - OD blank group)] × 100%. Wherein, the experimental group consists of wells containing different concentrations of Fanapanel, the control group consists of wells without the drug, and the blank group consists of wells without cells.

[0087] 1.2 Experimental Results Experimental results are as follows Figure 5 As shown, as the concentration of Fanapanel increased to 128 μg / mL, the survival rate of HEK-293T cells and HUVECs cells remained greater than 80%, indicating that Fanapanel had no significant cytotoxicity to mammalian cells within this concentration range and had good in vitro biosafety.

[0088] 2. Hemolysis test 2.1 Experimental Methods Rabbit whole blood samples were purchased from Beijing Solarbio Science & Technology Co., Ltd. First, the blood samples were gently mixed with an equal volume of PBS buffer and centrifuged at 3000 rpm for 10 minutes. The supernatant was discarded to remove plasma and most other blood cells. The precipitated red blood cells were collected and washed three times with PBS buffer, finally diluted with PBS to obtain a 5% (v / v) red blood cell suspension. The compound Fanapanel was pre-dissolved in dimethyl sulfoxide and then serially diluted twofold with PBS buffer to prepare a series of solutions with concentration gradients ranging from 128 μg / mL to 2 μg / mL. Two control groups were set up: a positive control containing 0.5% Triton X-100 to induce complete hemolysis; and a negative control containing only 5% red blood cell suspension and an equal volume of PBS to determine background hemolysis levels. Subsequently, in each well of a 96-well plate, 100 μL of 5% erythrocyte suspension was added, followed by 100 μL of Fanapanel solution at different concentrations. The plate was then incubated at 37°C in a 5% CO2 incubator for 1 hour. After incubation, the reaction solution from each well was transferred to a 1.5 mL EP tube and centrifuged at 4°C and 3500 rpm for 10 minutes. Finally, 100 μL of the supernatant was transferred to a new 96-well plate, and the absorbance (OD540) was measured at 540 nm using a microplate reader. The hemolysis rate was calculated using the following formula: Hemolysis rate (%) = [(OD experimental group - OD negative control) / (OD positive control - OD negative control)] × 100%.

[0089] 2.2 Experimental Results Experimental results are as follows Figure 6 As shown, the positive control group treated with 0.5% Triton X-100 completely disrupted the erythrocyte membrane, achieving a hemolysis rate of 100%. In contrast, Fanapanel exhibited good blood compatibility at all experimental concentrations. Even at the highest concentration of 128 μg / mL, Fanapanel did not induce significant erythrocyte hemolysis, and the hemolysis rate remained at a low level, showing no significant difference from the negative control group. These results indicate that Fanapanel has no significant destructive effect on the erythrocyte membrane within the experimental concentration range (from the lowest concentration to 128 μg / mL) and possesses good blood compatibility.

[0090] Example 4: In vivo pharmacodynamics of Fanapanel compared to β-lactam antibiotics 1. Establishment of a mouse model of systemic infection 1.1 Experimental Methods The experiment began with picking a single colony from an MH plate containing *E. coli* 110034, inoculating it onto LB broth, and incubating it at 37°C and 150 rpm for 4-6 hours. The colonies were then washed twice with sterile PBS and resuspended. The bacterial concentration was adjusted to 6 × 10⁻⁶ using a bacterial turbidimeter. 8 CFU / mL was prepared for use. Subsequently, mice that had undergone one week of acclimatization were randomly divided into four groups of five mice each: one group served as a blank control group, receiving an equal volume of PBS intraperitoneally; the other three groups served as infection model groups, receiving different concentrations (4.5 × 10⁻⁶ CFU / mL) intraperitoneally. 8 CFU / mL, 3×10 8 CFU / mL, 1.5×10 8 200 μL of bacterial suspension (CFU / mL) was added. Twelve hours after infection, the weight, appetite, mental state, and mortality of mice in each group were observed and recorded for two consecutive days to screen for a suitable dose for establishing a systemic infection model.

[0091] 1.2 Experimental Results As shown in Table 2, when mice were intraperitoneally injected with 200 μL of a concentration of 4.5 × 10⁻⁶... 8 When the concentration of NDM-1-positive bacterial culture at CFU / mL was high, the mortality rate in mice reached 100%. Therefore, this dose was determined as the modeling dose for the systemic infection model in this study. Twelve hours after modeling, compared with the blank control group (e.g., ... Figure 7 As shown in the figure, the infected mice exhibited clinical manifestations such as curling up, reduced activity, sluggishness, lethargy, diarrhea, weight loss, and disheveled fur. Dissection revealed a dark-colored liver and an unpleasant odor emanating from the abdominal cavity; all infected mice died within 24 hours. These results indicate that a systemic infection model in mice has been successfully established.

[0092] Table 2. Infection status of mice in each model group after bacterial injection.

[0093] 2. Survival rate experiment of mouse systemic infection model 2.1 Experimental Methods Mice that had undergone one week of acclimatization were randomly divided into 5 groups of 10 mice each: blank control, infection model group, meropenem monotherapy group, Fanapanel monotherapy group, and Fanapanel + meropenem combination group. Subsequently, except for the blank control group which received an intraperitoneal injection of an equal volume of sterile PBS, all other groups of mice received a uniform intraperitoneal injection of 200 μL of a 4.5 × 10⁻⁶ mcg solution. 8A systemic infection model was established using a bacterial suspension at CFU / mL (this dose was determined to be the lethal dose in previous experiments). Drug intervention began 2 hours after infection. Meropenem was administered at a dose of 10 mg / kg, and Fanapanel at a dose of 10 mg / kg. The blank control group and the infection model group received an equal volume of 5% dimethyl sulfoxide solution intraperitoneally, while the treatment groups received the corresponding doses intraperitoneally, administered every 12 hours for a total of 4 doses. Mice survival was observed during and after drug administration. For 96 hours from infection, the survival status of mice in each group was recorded every 12 hours, and the survival rate was calculated. Finally, at the end of the 96-hour experiment, all surviving mice were euthanized.

[0094] 2.2 Experimental Results The results are as follows Figure 8 As shown, the mortality rate of mice in the model group and the Fanapanel-only group was extremely high within 24 hours of infection, exceeding 90%, indicating that Fanapanel alone had no therapeutic effect on infected mice. In contrast, the meropenem-only group showed a certain therapeutic effect on infected mice, with a survival rate of 40%. Furthermore, after four treatments, the survival rate of infected mice in the meropenem-fanapanel combination group increased from 10% in the model group to 60%, higher than that in the meropenem-only group. These results indicate that although Fanapanel alone is ineffective in treating infected mice, the combination of this compound and meropenem can produce a synergistic effect, effectively improving the survival rate of infected mice.

[0095] 3. Target organ bacterial load and HE staining experiment 3.1 Experimental Methods First, mice that had undergone one week of acclimatization were randomly divided into 5 groups of 6 mice each: a blank control group, an infection model group, a meropenem monotherapy group, a Fanapanel monotherapy group, and a Fanapanel + meropenem combination group. After grouping, the blank control group received an intraperitoneal injection of an equal volume of sterile PBS, while all other groups received an intraperitoneal injection of 200 μL of a 4.5 × 10⁻⁶ mcg solution. 8Infection was established using bacterial suspension at CFU / mL. Two hours after modeling, drug intervention began: the blank control group and the infection model group were injected with an equal volume of sterile 5% dimethyl sulfoxide solvent; each treatment group received an intraperitoneal injection of the set dose (meropenem alone 10 mg / kg, compound Fanapanel alone 10 mg / kg, and the combination treatment group received an equal dose of both meropenem and the compound), repeated every 12 hours for a total of 4 times. Finally, the mice were euthanized, their bodies disinfected with 75% alcohol, and dissected. The liver, spleen, and kidneys of each group were removed and weighed. Then, 1 mL of sterile PBS was added, and the tissue homogenate was prepared using a cross-handled glass homogenizer. The homogenate was serially diluted tenfold with sterile PBS in 96-well plates, and 10 μL of each dilution was dropped onto MH agar plates. The plates were incubated at 37°C for 16-20 hours before the results were observed. In addition, a portion of organ samples were fixed in formalin for 24 hours and then stained with hematoxylin and eosin (HE). Histopathological observation was used to comprehensively evaluate the therapeutic effect of the combination of Fanapanel and meropenem on infected mice.

[0096] 3.2 Experimental Results Quantitative analysis of bacterial load in the liver, kidney, and spleen of mice in different treatment groups is as follows: Figure 9 As shown in the figure. Data from the blank control group are omitted to more clearly compare the differences between the treatment groups and the model group. Figure 9 As shown, compared with the model group, there was no statistically significant difference in organ bacterial load between the Fanapanel monotherapy group and the model group. However, the combination therapy of meropenem and Fanapanel significantly reduced the bacterial load in the liver, kidney, and spleen of infected mice. Furthermore, the bacterial load in each organ of the combination therapy group was significantly lower than that of the meropenem monotherapy group. These results indicate that Fanapanel monotherapy has no clearance effect on target organs in NDM-1 positive infected mice in vivo. However, the combination of Fanapanel and meropenem can enhance the in vivo antibacterial activity of meropenem and effectively reduce the bacterial load in the liver, kidney, and spleen.

[0097] Pathological sections of mouse liver, spleen, and kidney organs, as shown below Figure 10 As shown, compared with the blank control group, the infection model group showed severe damage: hepatocellular degeneration, sporadic inflammatory cell infiltration in the liver tissue; activated macrophage aggregates and numerous lymphocytes in the spleen tissue, blurred boundaries between the white and red pulp, and narrowing or irregular renal tubular lumens. After treatment with meropenem or fanapanel alone, the observed pathological features were highly similar to the model group, with no significant improvement. Hepatocellular degeneration persisted, and inflammatory responses in the spleen and kidneys remained. In contrast, the combined use of meropenem and fanapanel effectively alleviated the pathological damage in all organs, approaching the normal range.

[0098] Example 5: Study on the mechanism of action of Fanapanel in inhibiting NDM-1 1. Zinc ion recovery experiment 1.1 Experimental Methods The experiment used cefotaxime as the substrate and selected the metal chelating agent EDTA and captopril as positive controls. The specific steps are as follows: First, the purified NDM-1 enzyme solution (final concentration 10 nM) was pre-incubated with a fixed concentration (32 µg / mL) of Fanapanel at 37 °C for 15 min; then, zinc chloride (ZnCl2) solutions of different concentration gradients (0 µM, 5 µM, 50 µM, 500 µM) were added to the system, and incubation was continued for 5 min; finally, the substrate was added to carry out the reaction, and the absorbance value was immediately measured at a wavelength of 492 nm using a microplate reader. The residual enzyme activity of each group was calculated based on the initial reaction rate.

[0099] 1.2 Experimental Results The results are as follows Figure 11 As shown, the inhibitory effect of the positive control EDTA on NDM-1 exhibited a significant zinc ion dependence: with the continuous increase of the exogenous ZnCl2 concentration in the reaction system, the NDM-1 enzyme activity inhibited by EDTA showed a dose-dependent recovery, indicating that EDTA inactivates the enzyme through a chelation mechanism that deprives the active site of zinc ions. In contrast, captopril and Fanapanel (32 µg / mL) showed relatively stable inhibitory effects on NDM-1; even under conditions of excessive zinc ion supplementation, the enzyme activity of NDM-1 remained inhibited, and no activity recovery was observed.

[0100] 2. Lineweaver-Burk Double Reciprocal Curve Analysis 2.1 Experimental Methods The experiment used cefotaxime as a substrate, and the substrate was diluted to a series of concentration gradients (0-30 µM) to determine the initial reaction rate. In a 50 mM HEPES buffer system at pH 7.5, the enzyme concentration was fixed at 10 nM, and different concentration gradients of the compound Fanapanel were incubated at 37 °C for 15 min to ensure sufficient binding of the inhibitor to the enzyme's active site. The inhibitor concentration gradients were set at IC50. 50The absorbance was measured at a wavelength of 492 nm using an ELISA reader after 15 min, with each concentration gradient of cefotaximeter added immediately. The absorbance was measured every 10 s for a total of 1 min. EDTA was used as a positive control, with three replicates per group. The initial reaction rate under each condition was calculated using the first 30 s of the reaction. A Lineweaver-Burk double reciprocal curve was plotted with the reciprocal of the substrate concentration (1 / [S]) on the x-axis and the reciprocal of the reaction rate (1 / [V]) on the y-axis. By analyzing the changes in the intercepts of the curves on the coordinate axes and the trends in the Michaelis constant (Km) and the maximum reaction rate (Vmax), the type of inhibition of NDM-1 by the compound (competitive, non-competitive, or uncompetitive inhibition) was determined.

[0101] 2.2 Experimental Results The results are as follows Figure 12 As shown, the three straight lines of the positive control EDTA all intersect the negative half of the X-axis, and with increasing EDTA concentration, the maximum Vmax decreases, while the Km value remains unchanged. This characteristic indicates that EDTA exhibits a non-competitive inhibitory effect on NDM-1. However, the enzyme inhibitory mechanism of the compound Fanapanel is completely different from that of the positive control EDTA. In the LB curve of this compound, the three straight lines intersect the positive half of the Y-axis, and after the addition of Fanapanel, with increasing inhibitory concentration, Vmax remains essentially unchanged, while the Km value increases accordingly. This characteristic clearly indicates that Fanapanel is a competitive inhibitor, possibly by depriving Zn... 2+ The conclusion that NDM-1 is inhibited by other mechanisms precisely verifies the results of the aforementioned zinc ion recovery experiment.

[0102] 3. Molecular docking analysis 3.1 Experimental Methods Molecular docking software DOCK 6.1 was used for prediction. The acceptor structure was selected from the NDM-1 crystal structure (PDB ID: 7uox) from the protein database (http: / / www.rcsb.org / ). The acceptor was preprocessed using UCSF ChimeraX 1.7: all water molecules, solvent molecules, and hydrogen atoms were removed, retaining only one chain and the catalytically essential bridging water molecule to obtain the docking acceptor. The ligand Fanapanel's two-dimensional structure was transformed into a three-dimensional structure using the online tool build3d38 (TLDR website), minimizing energy to generate its three-dimensional conformation file. During docking, the grid box was set with the key binuclear zinc ion catalytic center in the NDM-1 active pocket as the positioning point to ensure sufficient coverage of the substrate binding region. Finally, from the multiple conformations generated by docking, the conformation with the most favorable binding free energy was selected for detailed intermolecular interaction analysis.

[0103] 3.2 Experimental Results Molecular docking results as follows Figure 13 As shown, Fanapanel can bind to the active pocket of NDM-1. Its phosphonate group forms a tight metal coordination with the two zinc ions in the catalytic center through multiple oxygen atoms, effectively replacing the key bridging water molecule that plays a nucleophilic attack role in the hydrolysis reaction. Furthermore, the molecule constructs a broad hydrogen bond network with amino acid residues such as Asn220, Cys208, and His250 in the active pocket through its polar atoms, combining non-covalent interactions such as van der Waals forces to stably bind the ligand to the catalytic center of NDM-1. This binding mode directly shields the substrate binding site spatially, structurally supporting the competitive inhibition conclusion determined by the aforementioned Lineweaver-Burk kinetic analysis. This reveals that Fanapanel exerts its inhibitory effect on NDM-1 by directly interfering with zinc ions and occupying the enzyme's catalytic center, rather than through simple metal chelation.

[0104] Based on the disclosure and teachings of the foregoing specification, those skilled in the art can make appropriate changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and changes to the present invention should also fall within the protection scope of the claims of the present invention. Furthermore, although some specific terms are used in this specification, these terms are only for convenience of explanation and do not constitute any limitation on the present invention.

Claims

1. The use of Fanapanel or a pharmaceutically acceptable salt thereof in the preparation of NDM-1 type metallo-β-lactamase inhibitors.

2. The use of Fanapanel or a pharmaceutically acceptable salt thereof in the preparation of drugs that enhance bacterial susceptibility to antibiotics, characterized in that, The bacteria are drug-resistant bacteria expressing NDM-1 type metallo-β-lactamases, and the antibiotic is a β-lactam antibiotic.

3. The use of Fanapanel or a pharmaceutically acceptable salt thereof in combination with an antibiotic in the preparation of a medicament for the prevention and / or treatment of drug-resistant bacterial infections, characterized in that the antibiotic is a β-lactam antibiotic and the drug-resistant bacteria are drug-resistant bacteria expressing NDM-1 type metallo-β-lactamases.

4. The application as described in any one of claims 2 or 3, characterized in that, The β-lactam antibiotics mentioned are carbapenem antibiotics.

5. The application as described in claim 4, characterized in that, The β-lactam antibiotic is selected from meropenem.

6. A pharmaceutical composition for inhibiting or killing bacteria, characterized in that, The composition comprises Fanapanel or a pharmaceutically acceptable salt thereof, and a β-lactam antibiotic; the bacteria are drug-resistant bacteria expressing NDM-1 type metallo-β-lactamases.

7. The pharmaceutical composition according to claim 6, characterized in that, The β-lactam antibiotics mentioned are carbapenem antibiotics.

8. The pharmaceutical composition according to claim 7, characterized in that, The carbapenem antibiotic is meropenem.

9. The pharmaceutical composition according to any one of claims 6-8, characterized in that, The pharmaceutical composition is a fixed-dose combination preparation or an individually packaged combination preparation, the combination preparation containing a first formulation unit and a second formulation unit that are independent of each other, the first formulation unit containing Fanapanel or a pharmaceutically acceptable salt thereof, the second formulation unit containing the β-lactam antibiotic, the first formulation unit and the second formulation unit being used for simultaneous or sequential administration.

10. Application of Fanapanel in the in vitro inhibition of NDM-1 type metallo-β-lactamase activity for non-disease therapeutic purposes.

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