Use of INE963 in the preparation of drugs against drug-resistant bacteria

Abstract

This invention provides an application of INE963 in the preparation of drugs against drug-resistant bacteria. This invention reveals for the first time that INE963 can specifically target bacterial membrane phosphatidylglycerol and lipopolysaccharides unique to the outer membrane of Gram-negative bacteria. This dual-targeting strategy endows INE963 with good antibacterial specificity and low toxicity. INE963 targets PG and inhibits the Agr system, thus combating MRSA and its biofilm; it increases outer membrane permeability by targeting LPS, synergistically combating drug-resistant Acinetobacter baumannii with colistin, exhibiting advantages such as high antibacterial efficiency and good safety.

Description

Technical Field

[0001] This invention belongs to the field of novel antibacterial drug technology, and in particular relates to the application of INE963 in the preparation of drugs against drug-resistant bacteria. Background Technology

[0002] Antibiotic resistance has become a global public health challenge, directly causing approximately 1.27 million deaths annually. Methicillin-resistant Staphylococcus aureus (MRSA) and carbapenem-resistant Acinetobacter baumannii (CRAB) are listed as priority bacterial pathogens by the World Health Organization. Due to their high virulence, ease of biofilm formation, and prominent multidrug resistance, clinical treatment is particularly challenging. The widespread use of colistin as a last-line treatment for CRAB infections has led to the rapid emergence of drug-resistant strains, resulting in treatment failure and accelerating the spread of resistance.

[0003] Currently, treatment options for the aforementioned pathogens are significantly inadequate. Traditional antibiotics often operate on a single-target mechanism, allowing bacteria to rapidly develop resistance through target mutations, enzyme inactivation, or efflux pump activation. While cell membrane-targeting antibacterial agents have emerged in recent years and can circumvent some traditional resistance mechanisms, they struggle to overcome biomembrane barriers, resulting in limited efficacy against complex, multidrug-resistant strains. Therefore, there is an urgent need to develop anti-drug-resistant bacterial strategies that can enhance resistance barriers and employ novel mechanisms of action.

[0004] INE963, molecular formula: C 19 H 26 N6O2S, CAS Registry No.: 2640567-43-5, IUPAC System Nomenclature: 4-(aminomethyl)-1-{5-[2-methoxy-6-(isopropyl)pyridin-3-yl]imidazo[2,1-b][1,3,4]thiadiazol-2-yl}piperidin-4-ol, is an imidazothiadiazole derivative and an antiparasitic compound that has completed Phase I clinical trials, with its safety preliminarily verified. A search revealed no published patents or literature reporting its antibacterial or antibiofilm activity, nor any technical solutions for its use in inhibiting MRSA and its biofilm, or in combination with colistin for treating drug-resistant Acinetobacter baumannii. This technological direction currently represents an unexplored and uncharted area. Summary of the Invention

[0005] The technical problem to be solved by this invention is to overcome the shortcomings of the existing technology and provide an application of INE963 in the preparation of drugs against drug-resistant bacteria. It breaks through the limitation of the existing technology in which INE963 is only used as an antiparasitic drug, and for the first time uses it against MRSA and its biofilm, and in combination with colistin against drug-resistant Acinetobacter baumannii.

[0006] To address the aforementioned technical problems, this invention provides an application of INE963 in the preparation of drugs against drug-resistant bacteria.

[0007] Furthermore, in the above-described applications, the anti-drug-resistant bacterial drug is an anti-Gram-negative bacterial drug.

[0008] In the above application, the drug for treating drug-resistant bacteria is a drug for treating methicillin-resistant Staphylococcus aureus.

[0009] Furthermore, the aforementioned application refers to the use of anti-drug-resistant bacteria drugs for treating skin wound infections, peritonitis, and biofilm-associated implant infections.

[0010] In the above-mentioned application, the anti-drug-resistant bacteria drug is used to inhibit bacterial biofilm formation and remove mature biofilms, wherein the bacteria are methicillin-resistant Staphylococcus aureus.

[0011] Based on a general technical concept, the present invention provides the use of a pharmaceutical composition in the preparation of an anti-drug resistant bacterial drug, the pharmaceutical composition comprising INE963 and colistin.

[0012] Furthermore, in the above-described applications, the anti-drug-resistant bacterial drug is an anti-Gram-negative bacterial drug.

[0013] In the above-described application, the drug for treating drug-resistant bacteria is specifically designed to treat colistin-resistant Acinetobacter baumannii.

[0014] Further, as described above, the anti-drug drug is used to treat peritonitis caused by drug-resistant Acinetobacter baumannii.

[0015] Application of INE963 in the preparation of an inhibitor that suppresses the bacterial quorum sensing Agr system.

[0016] Compared with the prior art, the advantages of the present invention are as follows: This invention provides an application in the preparation of drugs against drug-resistant bacteria. This invention reveals for the first time that INE963 can specifically target bacterial membrane phosphatidylglycerol (PG) and lipopolysaccharide (LPS) specific to the outer membrane of Gram-negative bacteria. The former is present in extremely low amounts in mammalian cell membranes, while the latter is absent in mammalian cells. This dual-targeting strategy endows INE963 with excellent antibacterial specificity and low toxicity. Based on this innovative discovery, this invention has the following significant technical effects: (1) Anti-MRSA and its biofilm by targeting PG and inhibiting the Agr system: The MIC of INE963 against MRSA is 1~4 μg / mL, and it can clear intracellular MRSA at a concentration of 4×MIC, while vancomycin is ineffective against intracellular bacteria. At a concentration of 1 / 4×MIC, INE963 significantly inhibits the synthesis of virulence factors, reducing EPS, eDNA and extracellular protein to 39.73%, 19.56% and 22.14% of the control group, respectively. In terms of anti-biofilm, 1 μg / mL can destroy pre-formed biofilm, and 4 μg / mL can clear mature biofilm on the surface of titanium sheets. Drug resistance evaluation showed that INE963 induced no drug resistance for 18 consecutive days, and the MIC induced by 35 days only increased slightly, while the MIC of the ciprofloxacin control group increased 32 times. In terms of in vivo efficacy, mouse wound infection, peritonitis and prosthetic joint infection models all confirmed that it has significant in vivo antibacterial activity, and no obvious cytotoxicity and organ damage were observed at therapeutic concentrations.

[0017] (2) INE963 increases outer membrane permeability by targeting LPS, synergistically combating drug-resistant Acinetobacter baumannii with colistin: INE963 alone exhibits weak antibacterial activity against Acinetobacter baumannii (MIC ≥ 32 μg / mL). However, it shows a significant synergistic effect when used in combination with colistin, with a partial inhibitory concentration index (FIC) ≤ 0.5, and is also effective against colistin-resistant clinical isolates. Time-bactericidal curves show that the bacterial load in the INE963-colistin combination group was reduced by more than three orders of magnitude compared to the single-drug group. Resistance evaluation shows that INE963 can effectively delay the development of colistin resistance. In terms of in vivo efficacy, in a colistin-resistant Acinetobacter baumannii-induced peritonitis model, the bacterial load in the liver and lungs of the combination group was significantly lower than that of the single-drug group. Furthermore, safety evaluation shows that the combination of INE963 and colistin did not increase hepatotoxicity or nephrotoxicity, nor did it cause pathological damage to major organs. Attached Figure Description

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0019] Figure 1 This is a quantitative analysis diagram of the number of viable bacteria in HaCaT cells after INE963 treatment in Experiment 2 of this invention.

[0020] Figure 2 This is the case of INE963 continuously inducing bacterial resistance in Experiment 3 of this invention.

[0021] Figure 3 This shows the microscopic changes of bacteria after treatment with INE963 in Experiment 4 of this invention.

[0022] Figure 4The fluorescence intensities of SYTOX Green (A in the figure), DiSC3(5) (B in the figure), and BCECF-AM (C in the figure) after INE963 treatment in Experiment 5 of this invention are shown.

[0023] Figure 5 This is an enrichment analysis diagram of differentially expressed proteins obtained by proteomics screening after INE963 treatment in Experiment 6 of this invention.

[0024] Figure 6 This is a representative molecular dynamics image of the interaction between INE963 and the bacterial membrane in Experiment 6 of this invention.

[0025] Figure 7 The results of the anti-biofilm activity detection of INE963 in Experiment 7 of this invention are shown.

[0026] Figure 8 The figures show the expression levels of virulence factors such as EPS (A) in the figure, extracellular DNA (B) in the figure, and protein (C) in the figure after INE963 treatment in Experiment 8 of this invention.

[0027] Figure 9 This is a volcano diagram of differentially expressed biomembrane-related genes in MRSA after INE963 treatment in Experiment 9 of this invention.

[0028] Figure 10 This is a representative image of the molecular docking between INE963 and AgrB, a key protein in the quorum sensing system, in Experiment Nine of this invention.

[0029] Figure 11 This is a representative image of molecular docking between INE963 and LPS in Experiment Nine of this invention.

[0030] Figure 12 This invention demonstrates the synergistic antibacterial effect of the combination of INE963 and colistin in common Gram-negative clinical isolates in Experiment 10 of this invention.

[0031] Figure 13 The bacterial viable counts are those obtained from Experiment 10 of this invention after 16 hours of combined administration of 1 / 16× and 1 / 8× MIC colistin with INE963.

[0032] Figure 14 This invention demonstrates the effect of INE963 in delaying the development of colistin resistance in Experiment 10.

[0033] Figure 15 In Experiment 10 of this invention, after the combined use of INE963 and colistin, the change in bacterial outer membrane permeability over time was determined using an NPN probe.

[0034] Figure 16The figure shows the results of the cell membrane permeability investigation in Experiment 11 of this invention. Figure A shows the change of membrane potential over time detected by the DiSC3 (5) probe; Figure B shows the change of intracellular pH over time detected by the BCECF-AM fluorescent probe.

[0035] Figure 17 In Experiment Twelve of this invention, the isothermal titration calorimetry method was used to verify the binding affinity between INE963 and LPS.

[0036] Figure 18 This is a wound infection model caused by MRSA in Experiment Thirteen of this invention. In the figure, A is a representative image of the wound healing process within 7 days. In the figure, B is the viable bacterial load in the wound on days 1, 3, and 5.

[0037] Figure 19 The above figures represent the viable bacterial counts in the liver (A) of mice, kidneys (B) and lungs (C) of mice after treatment with INE963 in Experiment Thirteen of this invention.

[0038] Figure 20 These are representative scanning electron microscope images of the biofilm on the surface of the prosthesis implant in Experiment Thirteen of this invention.

[0039] Figure 21 This shows the congestion and edema of various organs in the abdominal cavity of mice in Experiment Thirteen of this invention.

[0040] Figure 22 This is a count of viable bacteria in the liver (A) and lungs (B) of mice in Experiment Thirteen of this invention. Detailed Implementation

[0041] The present invention will be further described below with reference to specific preferred embodiments, but this does not limit the scope of protection of the present invention.

[0042] The materials, reagents, and instruments used in the following examples are all commercially available. Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art.

[0043] Example 1 An application of INE963 in the preparation of anti-drug-resistant bacteria drugs, which are drugs against Gram-positive bacteria.

[0044] Experiment 1: Determination of the antibacterial activity of INE963.

[0045] Referring to the CLSI 2023 M100 operating procedure, the logarithmic phase bacteria were adjusted to 0.5 McFarland turbidity, and INE963 was added in a serially diluted MH broth at a volume ratio of 1:100. The MIC was determined after incubating the 96-well plate at 37 °C for 16-18 h. The MIC was then transferred to the highest concentration of bacterial solution and spread onto blood agar plates. After incubation for 24 h, the MBC was determined.

[0046] Table 1 shows the results of the antibacterial activity assay for INE963.

[0047] Table 1: Results of antibacterial activity assay.

[0048]

[0049] The results in Table 1 show that INE963 exhibits significant antibacterial activity against Gram-positive bacteria, including MRSA, with MIC values ​​ranging from 1 μg / mL to 8 μg / mL, and MBC values ​​ranging from 4 μg / mL to 16 μg / mL. However, when used alone, INE963 shows weak antibacterial activity against Gram-negative pathogens such as Acinetobacter baumannii, with MIC values ​​>32 μg / mL.

[0050] Example 2 The application of INE963 in the preparation of anti-resistant bacterial drugs. These anti-resistant drugs are for treating MRSA infections. The anti-MRSA drugs can be used for intracellular MRSA bacterial infections.

[0051] Experiment 2: Determination of intracellular bactericidal effect.

[0052] HaCaT cells in logarithmic growth phase were adjusted to approximately 3 × 10⁻⁶. 6 Cells were inoculated at a density of 1 / mL into 6-well plates for adherent culture to obtain a cell suspension. *Staphylococcus aureus* USA300 was washed with PBS and adjusted to 0.5 McFarland turbidity, then mixed with an equal volume of the cell suspension and incubated at 37°C for 1 h. 100 μg / mL gentamicin was added for 90 min to eliminate extracellular bacteria. After discarding the supernatant, cells were treated with INE963 for 3 h, followed by lysis with 2% Triton X-100 for 20 min. The lysate was plated on MH agar plates for cell counting, and Gram staining was used to verify the extracellular bacterial elimination effect. Vancomycin (VAN) and linezolid (LZD) were used as conventional antibiotic controls, and the Ctrl group served as a blank control.

[0053] Figure 1 The figure shows the quantitative analysis of intracellular viable bacteria count in HaCaT cells after INE963 treatment. As shown in the figure, after 3 h of treatment with INE963 at a concentration of 4×MIC, the number of intracellular viable bacteria decreased significantly; while vancomycin and linezolid, even at a concentration of 200 μg / mL, did not show intracellular antibacterial activity.

[0054] Experiment 3: Induction of drug resistance assay.

[0055] On day 1, the MIC of the drug against the MRSA standard strain USA300 was determined. After incubation for 16-18 hours, the bacterial suspension from wells at a subinhibitory concentration (0.5×MIC) was diluted 1000-fold with MH broth. Subsequently, the diluted bacterial suspension and different concentrations of the drug were added to 96-well plates, and the MIC was determined again. This process was repeated for 35 days. Ciprofloxacin treated in the same way was used as a positive control.

[0056] Figure 2 This illustrates the situation where INE963 continuously induces bacterial resistance. As shown in the figure, although sub-inhibitory concentrations of ciprofloxacin (… ciprofloxacin CIP can significantly induce drug resistance mutations in MRSA, causing its MIC to increase significantly to 32 times; however, even after 18 consecutive days of treatment with subinhibitory concentrations of INE963, no significant increase in drug resistance was observed in MRSA, and its MIC value remained unchanged. Even after 35 consecutive days of treatment, its MIC value only increased to 4 times.

[0057] Experiment 4: Detection of microscopic changes in bacteria.

[0058] The standard MRSA strain USA300 in the logarithmic growth phase was diluted with TSB broth containing INE963 to a final bacterial concentration of 1×10⁻⁶. 8 CFU / ml, while the control group was treated with fresh TSB broth containing 1% DMSO. The bacteria after treatment were then observed using transmission electron microscopy (TEM).

[0059] Figure 3 Microscopic changes in bacteria after treatment with INE963. As shown in the figure, after 1 hour of treatment with INE963 at a concentration 5 times the MIC, transmission electron microscopy images revealed swollen and ruptured bacterial cell membranes, and edematous and disordered intracellular structures in the INE963-treated group. Based on these ultrastructural changes, we preliminarily hypothesize that INE963 exerts its antibacterial effect by acting on the cell membrane.

[0060] Experiment 5: Membrane permeability detection.

[0061] The USA300 bacteria in logarithmic growth phase were washed with HEPES solution, and the OD630 nm of the bacterial suspension was adjusted to 0.05. Next, 2 μmol / L SYTOX Green, DiSC3(5), and 20 μmol / L BCECF-AM staining solutions were prepared. Then, the bacterial suspensions were incubated with SYTOX Green for 15 min in the dark, DiSC3(5) for 1 h in the dark, and BCECF-AM for 30 min in the dark, respectively. Then, 10 µL of different concentrations of the test drug and 90 µL of the bacterial-stain mixture were added to each well of a 96-well black plate. Finally, the fluorescence intensity changes of SYTOX Green and BCECF-AM staining solutions every 5 min within 30 min and the fluorescence intensity changes of DiSC3(5) staining solution every 30 s within 5 min were detected using a multi-mode microplate reader.

[0062] Figure 4 The figures show the fluorescence intensities of SYTOX Green (A), DiSC3(5) (B), and BCECF-AM (C) after INE963 treatment. The results of these fluorescent probe detections all indicate that INE963 treatment significantly increased the levels of SYTOX Green, DiSC3(5), and BCECF-AM in a concentration-dependent manner, suggesting that INE963 exerts its antibacterial effect by acting on the cell membrane and increasing the membrane potential, and that this antibacterial effect is PMF-dependent.

[0063] Experiment 6: Proteomics Analysis.

[0064] 6.1 Enrichment analysis of differentially expressed proteins: Staphylococcus aureus USA300 was cultured to the logarithmic growth phase, and the OD630nm was adjusted to 0.2. The cells were then treated with INE963 at 5×MIC or 1% DMSO for 1 h. After low-temperature collection, washing with PBS, and liquid nitrogen freezing, the cells were lysed with SDT solution, and protein quantification was performed. Following enzymatic digestion, desalting, concentration, and formic acid reconstitution, differentially expressed proteins were screened using TMT labeling and mass spectrometry analysis, based on a fold change ≥1.5 and P<0.05.

[0065] Figure 5 This figure shows the enrichment analysis of differentially expressed proteins obtained through proteomics screening after INE963 treatment. As shown in the figure, the differentially expressed proteins after INE963 treatment were mainly enriched in bacterial cell membrane proteins.

[0066] 6.2 All-Atom Molecular Dynamics (MD) Simulation: The initial molecular structure of INE963 was constructed using Chem3D software, and the CHARMM simulation was performed. A GUI platform was used to construct a phospholipid bilayer model simulating the MRSA cell membrane, consisting of DOPC and DOPG in a 7:3 ratio. Gromacs 2018.4 was used to perform system equilibrium and 500 ns all-atom molecular dynamics simulations. The simulation results were visualized and analyzed using Gromacs tools and VMD software.

[0067] Figure 6 This is a representative molecular dynamics image of the interaction between INE963 and the bacterial membrane. Molecular dynamics simulations show that INE963 rapidly adsorbs onto the bacterial cell membrane surface, forming hydrogen bonds with phospholipid phosphate groups to achieve a stable binding. Adsorption and membrane insertion are completed in 100 ns, and it is stably embedded on the outer side of the phospholipid bilayer after several hundred nanoseconds.

[0068] Example 3 Application of INE963 in the preparation of drugs that inhibit bacterial biofilm formation.

[0069] Experiment 7: Determination of anti-biofilm ability.

[0070] Bacterial biofilms were constructed under different conditions using microplates and titanium sheets, respectively. The biofilms were washed three times with sterile physiological saline and stained with methyltetrazoate (XTT) and phenazine dimethyl sulfate (PMS) solutions at a working concentration of 0.2 μg / mL at a ratio of 1:1 for 2 h to 4 h. The absorbance was measured at 450 nm to evaluate the ability of INE963 to inhibit biofilm growth or disperse existing biofilms.

[0071] Figure 7 The results show the anti-biofilm activity of INE963. Figure A represents the inhibitory effect of INE963 on MRSA biofilm in microplates; as shown, INE963 significantly inhibits biofilm formation at a concentration of 0.125 μg / mL, exhibiting a clear dose-dependent effect.

[0072] Figure B shows the scavenging effect of INE963 on mature MRSA biofilms in microplates. The figure demonstrates that INE963 significantly disperses existing biofilms at a concentration of 1 μg / mL. Since biofilms commonly colonize in clinical settings primarily using artificial titanium grafts, the anti-biofilm effect of INE963 on titanium sheets was also investigated.

[0073] In the figure, C represents the removal effect of INE963 on the mature MRSA biofilm on the titanium sheet. As shown in the figure, INE963 has a significant dispersing effect on the biofilm formed on the titanium sheet at a concentration of 4 μg / mL, and this dispersing effect is dose-dependent.

[0074] Experiment 8: Determination of extracellular EPS, DNA and extracellular protein content.

[0075] The overnight cultured Staphylococcus aureus USA300 was adjusted to 0.5 McFarland turbidity, treated with INE963, and cultured for 24 h. The biofilm was collected, ultrasonically dispersed, and centrifuged. The supernatant was precipitated with ethanol, dried, and reconstituted. The absorbance at 260 nm and 280 nm was measured using a NanoDrop 2000.

[0076] Extracellular polysaccharides (EPS) were determined using the phenol-sulfuric acid method: Staphylococcus aureus USA300 cultured overnight was adjusted to 0.5 McFarland turbidity, and the bacterial suspension was co-cultured with INE963 for 12 h. The supernatant was collected by centrifugation, precipitated with ethanol, redissolved, and reacted with phenol-sulfuric acid. The absorbance at 490 nm was measured, and the relative EPS secretion was calculated as (experimental group / control group) × 100%.

[0077] Figure 8 The expression levels of virulence factors such as EPS (A) secreted by MRSA, extracellular DNA (B) and protein (C) were measured after treatment with INE963. As shown in the figure, after treatment with 1 / 4×MIC of INE963, the levels of extracellular DNA, extracellular protein and EPS were significantly reduced to 39.73%, 19.56% and 22.14% of those in the control group, respectively.

[0078] Experiment 9: Transcriptomics Sequencing.

[0079] Logarithmic growth phase Staphylococcus aureus USA300 was collected and treated in tryptone soybean (TSB) broth with 5×MIC INE963 and 1% (vol / vol) DMSO at 37°C and 180 rpm for 1 hour. After centrifugation to collect the bacterial cells, total RNA was extracted using the EZNA bacterial RNA extraction kit, and ribosomal RNA was removed using the Ribo-Zero magnetic bead kit. Subsequently, the RNA was analyzed using TruSeq. TM A cDNA library was constructed using a strand-specific RNA library preparation kit, and transcriptome sequencing was performed on the Illumina sequencing platform. After quality control, the raw data were used to screen and analyze differentially expressed genes using the edgeR software package in the R language.

[0080] Figure 9 This is a volcano plot showing the differentially expressed biofilm-related genes in MRSA after INE963 treatment. The figure shows that, compared to the control group, the INE963-treated strain exhibited 1491 differentially expressed genes; among them, genes related to the Agr quorum sensing system were significantly downregulated.

[0081] Analysis of the binding energies of INE963 with Agr B and LPS using molecular docking technology: First, the three-dimensional structures of Agr B and LPS were constructed using AlphaFold3 and AutoDock Tools 1.5.7. Energy optimization was then performed using Rosetta Relax, and pretreatment with PyMOL was used to remove water molecules and add hydrogen atoms. The INE963 structure was obtained from the PubChem database. The prepared acceptor and ligand were molecularly docked using the CB-DOCK2 online platform. The optimal conformation was screened based on the binding energy, and its binding site and interaction mode were evaluated. Furthermore, the non-covalent interactions between the ligand and acceptor were analyzed using the PLIP online tool. Finally, PyMOL was used to visualize the three-dimensional conformation of the docked complex.

[0082] Figure 10 This image shows a representative molecular docking of INE963 with AgrB, a key protein in the quorum sensing system. As shown, the INE963 molecule is embedded within the hydrophobic binding cavity of the AgrB protein. INE963 and AgrB form a number of hydrogen bonds and hydrophobic interactions. Based on Vina's binding energy prediction using the AMBER force field, the binding energy between INE963 and AgrB is -7.1 kcal / mol, indicating a good affinity between the two.

[0083] Figure 11 This image shows a representative molecular docking image of the interaction between INE963 and LPS. As shown, the INE963 molecule is embedded in the hydrophobic binding cavity of the LPS protein. INE963 and LPS form a certain number of hydrogen bonds and hydrophobic interactions, and their docking binding energy is -5.589 kcal / mol, indicating that they have good affinity.

[0084] Example 4 The application of INE963 and colistin in the preparation of an antibacterial drug. The antibacterial drug is an anti-Gram-negative bacterial infection agent.

[0085] Example 5 The use of a pharmaceutical composition in the preparation of an antimicrobial drug, wherein the pharmaceutical composition is a combination of INE963 and colistin, and the antimicrobial drug is an antimicrobial drug against Gram-negative bacteria or against colistin-resistant Acinetobacter baumannii infection.

[0086] Experiment 10: Chessboard Dilution Test.

[0087] 10.1 Evaluation of the antibacterial effect of combined drug use: Bacteria in the logarithmic growth phase were diluted with MH broth to a concentration of 1×10⁻⁶. 6CFU / mL, and added to a 96-well plate together with the serially diluted drugs of INE963 and colistin. After incubation at 37°C for 16 h to 18 h, the MIC concentrations of the drugs in combination and when used alone were determined, and the fractional inhibitory concentration index (FICI) was calculated. The calculation formula of FICI value: FICI=MIC_(A(in combination)) / MIC_(A(used alone)) +MIC_(B(in combination)) / MIC_(B(used alone)) FICI is defined as: FICI≤0.5, synergistic effect; 0.5<FICI<1, partial synergistic effect; 1≤FICI≤4, indifferent effect; FICI>4, antagonistic effect.

[0088] Figure 12 shows the synergistic antibacterial effect produced by the combination of INE963 and colistin on common Gram-negative clinical isolates. As shown in the figure, taking FIC index≤0.5 as the criterion for synergy, the FIC indices of multiple clinical strains such as Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii and Escherichia coli are all no more than 0.5, which confirms that this drug combination produces a synergistic antibacterial effect on common Gram-negative bacteria, and the synergistic effect on Acinetobacter baumannii is particularly significant.

[0089] 10.2 Bacterial viable count of sub-inhibitory concentration of colistin combined with INE963: Take colistin-resistant Acinetobacter baumannii in the logarithmic growth phase and adjust the bacterial suspension concentration to 0.5 McFarland standard, set up a growth control group, colistin monotherapy groups (1 / 8×MIC and 1 / 16×MIC), INE963 monotherapy group and combination groups (1 / 8×MIC or 1 / 16×MIC colistin + 4 μg / mL INE963). After the drugs and the bacterial suspension are incubated at 37°C for 16 h, the bacterial solution of each group is subjected to gradient dilution, then spread on blood agar plates, and colony counting is carried out after incubation to evaluate the bactericidal effect of the combined medication.

[0090] Figure 13 shows the bacterial viable count after 16 h of combined medication of 1 / 16× and 1 / 8×MIC colistin with INE963. As shown in the figure, compared with monotherapy, the combined medication can reduce the bacterial load by at least 3 orders of magnitude, exhibiting significant synergistic bactericidal activity.

[0091] 10.3 Detect the effect of combined medication on the development of bacterial resistance through continuous resistance induction: Sub-inhibitory concentration of drugs are used for continuous induction of drug resistance. Colistin-resistant Acinetobacter baumannii is co-incubated with colistin monotherapy group and the colistin and INE963 combined medication group respectively, the change of MIC of colistin is recorded, the development of drug resistance in the two groups is compared, and the delaying effect of INE963 on the development of colistin resistance is evaluated.

[0092] Figure 14 This study demonstrates the effect of INE963 in delaying the development of colistin resistance. As shown in the figure, the MIC of the colistin monotherapy group increased significantly with each passage, reaching 8 times the initial value; while the colistin MIC of the group treated with INE963 showed only slight fluctuations, with an increase of no more than 2 times. These results confirm that INE963 can significantly delay the development of colistin resistance in the strains.

[0093] Experiment 11: Bacterial permeability.

[0094] 11.1 Extracellular membrane permeability: Bacteria in the logarithmic growth phase were washed and resuspended in 5 mM HEPES buffer and adjusted to OD630nm=0.1. They were then treated with colistin alone or in combination with INE963, with 1% DMSO as a negative control. N-phenyl-1-naphthylamine (NPN) probe was added to a final concentration of 10 μM, and the mixture was incubated at room temperature in the dark for 10 min. Fluorescence intensity was then detected at an excitation wavelength of 350 nm and an emission wavelength of 420 nm.

[0095] Figure 15 After combined administration of INE963 and colistin, the changes in bacterial outer membrane permeability over time were measured using an NPN probe. As shown in the figure, after treatment with INE963 or colistin alone, there was no significant change in NPN fluorescence intensity. However, the NPN fluorescence intensity in the combined treatment group increased significantly in a concentration-dependent manner, indicating that INE963 exerts its sensitizing effect on colistin by acting on the bacterial outer membrane and disrupting its permeability.

[0096] 11.2 Intracellular membrane permeability: After washing, the logarithmic growth phase bacteria were resuspended in 5 mM HEPES buffer and adjusted to OD630nm=0.05. Colistin was administered alone or in combination with INE963, with 1% DMSO as a negative control. After incubation in DiSC3(5) staining solution for 1 h in the dark and BCECF-AM staining solution for 30 min in the dark, 10 µL of the test drug at different concentrations and 90 µL of the bacterial and staining mixture were added to each 96-well black plate. Finally, the fluorescence intensity changes of BCECF-AM staining solution every 5 min within 30 min and the fluorescence intensity changes of DiSC3(5) staining solution every 30 s within 5 min were detected using a multi-mode microplate reader.

[0097] Figure 16The results of the study on intracellular membrane permeability are shown in Figure A, where the membrane potential changes over time as detected by the DiSC3 (5) probe; and Figure B, where the intracellular pH changes over time as detected by the BCECF-AM fluorescent probe. Both results indicate that after treatment of bacteria with INE963 and colistin, both DiSC3 (5) and BCECF-AM increased significantly in a concentration-dependent manner. This suggests that the combined use of INE963 and colistin can lead to depolarization of the bacterial cell membrane potential and cause changes in intracellular pH, indicating that this combination can exert a synergistic bactericidal effect by disrupting the integrity and function of the bacterial cell membrane.

[0098] Experiment 12: Isothermal titration calorimetry (ITC) determination.

[0099] A 10–20 μM LPS and 200 μM INE963 solution was prepared using buffer solution. The sample cell contained LPS solution and the reference cell contained ultrapure water. The titration needle was loaded with INE963 solution. 2.5 μL solutions were injected in 20 batches at 25 °C and 300 rpm. Detection was performed using a Nano-ITC instrument, and the data were analyzed using NanoAnalyze 3.12.5 software.

[0100] Figure 17 The binding affinity between INE963 and LPS was verified using isothermal titration calorimetry. As shown in the figure, the ITC results further confirm that INE963 can directly bind to LPS, with a dissociation constant of 1.2 × 10⁻⁶. -7 M indicates that there is a specific, high-affinity interaction between the two.

[0101] Example 6 The use of INE963 in the preparation of a medicament for treating skin wound infections, peritonitis, or biofilm-associated implant infections caused by methicillin-resistant Staphylococcus aureus.

[0102] Example 7 The use of INE963 in the preparation of a drug for treating peritonitis caused by drug-resistant Acinetobacter baumannii.

[0103] Experiment Thirteen: Detection of in vivo antibacterial effect.

[0104] 13.1 MRSA-induced mouse wound infection model: Six- to eight-week-old female ICR mice were selected, anesthetized, and had their backs shaved. A wound approximately 5 mm in diameter was created on the back and inoculated with 50 μL of 1×10⁻⁶ MRSA-induced lesion. 7 CFU / mL MRSA bacterial suspension. One hour after infection, 2% (w / w) of the test drug was applied to the back. Bacterial load was measured on the wound on days 1, 3, and 5 post-infection, and the wound healing status was observed by taking photos every other day for a total observation period of 7 days.

[0105] Figure 18 This is a wound infection model caused by MRSA. Figure A shows a representative image of the wound healing process over 7 days. Figure B shows the viable bacterial load in the wound on days 1, 3, and 5. As shown in Figure A, on day 7 after administration, the solvent group showed significant edema and suppuration, indicating poor wound healing ability. The INE963 group and the fusidic acid positive control group showed significantly reduced wound exudation, rapid drying and shrinkage, and significantly better wound healing than the solvent group. Figure B shows the wound bacterial load test results, indicating that on days 3 and 5 after administration, the number of bacteria in the wounds of the INE963 group and the fusidic acid group was significantly lower than that of the solvent group, proving that INE963 has a stable and long-lasting antibacterial effect in vivo, and its anti-infective efficacy is comparable to that of the clinical antibacterial drug fusidic acid.

[0106] 13.2. Construction of an MRSA-induced mouse peritonitis-septicemia model: Six- to eight-week-old female ICR mice were selected, and 500 μL of 0.5 McFarland standard logarithmic growth phase USA300 bacterial solution containing 5% (wt / vol) mucin was injected intraperitoneally. One hour after infection, mice were injected intraperitoneally once with either a solvent (5% polyoxyethylene castor oil + ethanol, prepared with physiological saline at a 1:1 ratio) or 8 mg / kg INE963. Mice were sacrificed 24 hours after drug treatment, and liver, lung, and kidney tissues were collected for viable bacterial counts. Figure 19 The figures show the viable bacterial counts in the liver (A), kidney (B), and lung (C) of mice after treatment with INE963. As shown, a single administration of INE963 at a dose of 8 mg / kg significantly reduced the bacterial load in the liver, kidney, and lungs of infected mice.

[0107] 13.3. Construction of an MRSA-induced mouse model of prosthetic arthritis: Six- to eight-week-old female ICR mice were randomly divided into three groups: a sham-operated control group, a solvent control group (5% polyoxyethylene castor oil + ethanol mixture diluted 1:1 with physiological saline), and an INE963 administration group (30 mg / kg), administered via local injection once daily for 7 days. Overnight cultured Staphylococcus aureus USA300 bacterial suspension was washed twice with phosphate-buffered saline and resuspended in physiological saline for later use. After anesthesia, the hair around the knee joint was removed and the area was disinfected with iodine. The skin was incised to expose the knee joint, and a 0.3 mm × 0.8 mm needle was implanted as a femoral prosthesis. After suturing the wound, approximately 1 × 10⁻⁶ needles were inoculated locally. 7CFU bacterial suspension. Mice were sacrificed on day 21 after model establishment. The implant was removed, washed twice with phosphate-buffered saline, fixed overnight in 2.5% (wt / vol) glutaraldehyde, and then dehydrated sequentially with gradient concentrations of ethanol. The samples were then coated with gold-palladium and observed under a scanning electron microscope.

[0108] Figure 20 These are representative scanning electron micrographs of biofilms on the surface of prosthetic implants. As shown in the figure, the surface of the implant in the solvent control group was covered with a dense biofilm and a large number of bacteria, while only scattered bacteria were visible in the INE963 treatment group, with no complete biofilm formation, indicating that INE963 has a good in vivo anti-biofilm effect.

[0109] 13.4. Construction of a mouse peritonitis-septicemia model induced by colistin-resistant Acinetobacter baumannii: Logarithmically growing Acinetobacter baumannii was adjusted to 0.5 McFarland turbidity with 5% mucin solution. Then, 500 μL of the solution was injected intraperitoneally into 6-8 week old female ICR mice. One hour post-infection, mice were given a single intraperitoneal injection of the following: solvent (5% polyoxyethylene castor oil + ethanol mixture, diluted 1:1 with physiological saline), 4 mg / kg INE963, 5 mg / kg colistin, or a combination of INE963 and colistin (4 mg / kg + 5 mg / kg). Mice were sacrificed 24 hours post-infection, and liver and lungs were harvested for colony-forming unit counting.

[0110] Figure 21 This shows the congestion and edema of various organs in the abdominal cavity of mice. Figure 22 The figures show viable bacterial counts in the liver (A) and lungs (B) of mice. As shown, the bacterial counts in the liver and lungs of mice treated with the combination of INE963 and colistin were significantly reduced, showing a statistically significant difference compared to the solvent group and the single-drug group. This result indicates that the combination therapy of INE963 and colistin exhibits significant antibacterial efficacy against drug-resistant Acinetobacter baumannii in vivo.

[0111] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention.

Claims

1. The application of INE963 in the preparation of drugs against drug-resistant bacteria.

2. The application according to claim 1, characterized in that, The drug used to combat drug-resistant bacteria is a drug that is effective against Gram-negative bacteria.

3. The application according to claim 1, characterized in that, The drug used to combat drug-resistant bacteria is a drug that is effective against methicillin-resistant Staphylococcus aureus.

4. The application according to claim 2, characterized in that, The anti-drug-resistant bacteria drug is used to treat skin wound infections, peritonitis, and biofilm-associated implant infections.

5. The use of a pharmaceutical composition in the preparation of an anti-drug resistant bacterial drug, characterized in that, The pharmaceutical composition includes INE963 and colistin.

6. The application according to claim 5, characterized in that, The drug used to combat drug-resistant bacteria is a drug that is effective against Gram-negative bacteria.

7. The application according to claim 5, characterized in that, The drug used to combat drug-resistant bacteria is a drug for Acinetobacter baumannii resistant to colistin.

8. The application according to claim 5, characterized in that, The drug is used to treat peritonitis caused by drug-resistant Acinetobacter baumannii.

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