[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

WO2023065526A1 - 一种bms-833923及其衍生物的应用及药物 - Google Patents

一种bms-833923及其衍生物的应用及药物 Download PDF

Info

Publication number
WO2023065526A1
WO2023065526A1 PCT/CN2021/140984 CN2021140984W WO2023065526A1 WO 2023065526 A1 WO2023065526 A1 WO 2023065526A1 CN 2021140984 W CN2021140984 W CN 2021140984W WO 2023065526 A1 WO2023065526 A1 WO 2023065526A1
Authority
WO
WIPO (PCT)
Prior art keywords
bms
colistin
derivatives
gram
drug
Prior art date
Application number
PCT/CN2021/140984
Other languages
English (en)
French (fr)
Inventor
郑军
张年
Original Assignee
澳门大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 澳门大学 filed Critical 澳门大学
Publication of WO2023065526A1 publication Critical patent/WO2023065526A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure relates to the technical field of antibiotics, and specifically relates to an application and medicine of BMS-833923 and its derivatives.
  • ESKAPE Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli
  • Polymyxins are polycationic peptide antibiotics developed in the 1940s that kill bacteria by disrupting their cell membranes.
  • the cationic charge in polymyxin binds electrostatically to the negatively charged lipopolysaccharide of the outer membrane of Gram-negative bacteria, and then displaces the calcium and magnesium ions that stabilize the cell membrane, ultimately disrupting the integrity of the bacterial membrane.
  • polymyxins (such as colistin) can effectively kill bacteria, they have been abandoned in clinical practice because of their significant nephrotoxicity and neurotoxicity.
  • the present disclosure provides BMS-833923 and its derivatives as colistin adjuvants.
  • the present disclosure provides the use of BMS-833923 and its derivatives for the combined treatment of bacteria with colistin.
  • the bacteria are Gram-negative bacteria.
  • the Gram-negative bacteria are in logarithmic growth phase.
  • 10 ⁇ g/mL of BMS-833923 and its derivatives are added to 1 ⁇ MIC 50 colistin-treated Gram-negative bacteria.
  • BMS-833923 and its derivatives and colistin are used for joint inhibition or joint elimination of Escherichia coli.
  • BMS-833923 and its derivatives and colistin are used for joint inhibition or joint elimination of Klebsiella pneumoniae.
  • BMS-833923 and its derivatives are used with colistin for joint inhibition or joint elimination of Acinetobacter baumannii.
  • BMS-833923 and its derivatives and colistin are used for joint inhibition or joint elimination of Pseudomonas aeruginosa.
  • BMS-833923 and its derivatives and colistin are used for joint inhibition or joint clearance of at least one of pan-drug-resistant strains BAA-1800, BAA-1794 and BAA-1792.
  • the present disclosure provides the use of BMS-833923 and derivatives thereof for independent inhibition or elimination of Gram-positive bacteria.
  • the Gram-positive bacteria include at least one of Staphylococcus aureus and Bacillus subtilis.
  • the present disclosure provides an antibacterial drug, and the active ingredients of the antibacterial drug include BMS-833923 and its derivatives and colistin.
  • the present disclosure provides an antibacterial drug, the active ingredients of which include a combination of BMS-833923 or one of its derivatives and colistin.
  • the antibacterial drug is against Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, pan-drug resistant bacterial strain BAA-1800 A drug that inhibits or clears at least one of BAA-1794 and BAA-1792.
  • the present disclosure provides an antibacterial drug, the active ingredients of which include BMS-833923, its derivatives or a combination of BMS-833923 and its derivatives.
  • the antibacterial drug is a drug that inhibits or eliminates at least one of Staphylococcus aureus and Bacillus subtilis.
  • the present disclosure provides a method of treating diseases associated with bacterial infections, comprising:
  • a therapeutically effective amount of the antibacterial drug is administered to a subject in need thereof.
  • the present disclosure provides a method of treating diseases associated with Gram-positive bacterial infections, comprising:
  • a therapeutically effective amount of the antibacterial drug is administered to a subject in need thereof.
  • the Gram-positive bacteria include at least one of Staphylococcus aureus and Bacillus subtilis.
  • the present disclosure provides the use of the antibacterial drug for treating diseases related to bacterial infection.
  • the diseases related to bacterial infection include: skin infection, soft tissue infection, pneumonia, osteomyelitis, meningitis, sepsis, endocarditis, infective endocarditis.
  • the present disclosure provides the use of the antibacterial drug for treating diseases related to Gram-positive bacterial infection.
  • Fig. 1 is that in embodiment 1, identify the compound that strengthens colistin to inhibit bacterial growth and in embodiment 2, measure BMS-833923 (abbreviation BMS, the same below) and colistin combined use to large intestine by checkerboard (checkerboard) experiment The relevant test results of the synergistic effect of growth inhibition of bacillus (Escherichia coli);
  • Fig. 2 is the relevant test result that BMS-833923 in embodiment 3 promotes the bactericidal activity of colistin and BMS-833923 in embodiment 4 directly kills Gram-positive bacteria and does not produce drug resistance;
  • Figure 3 is the corresponding cell apoptosis test results when colistin and BMS-833923 are jointly treated with bacteria Escherichia coli in Example 5, wherein in Figures B to E, Ctrl represents the control group, CT represents the colistin group, and BMS represents the colistin group.
  • Ctrl represents the control group
  • CT represents the colistin group
  • BMS represents the colistin group.
  • BMS-833923 group, CT+BMS means the combination group of colistin and BMS-833923;
  • Fig. 4 is the corresponding cell apoptosis test result diagram when BMS-833923 alone treats Staphylococcus aureus (Staphylococcus aureus) in embodiment 5;
  • Ctrl represents negative control group
  • BMS represents BMS-833923 group
  • Van means the positive control group;
  • pan-drug resistance refers to the phenomenon of resistance to all empirical drugs currently recommended for corresponding bacterial infections (except colistin and tigecycline).
  • pan-drug-resistant strains refers to strains that are resistant to all empirical drugs currently recommended for corresponding bacterial infections (except colistin and tigecycline).
  • BMS-833923 and its derivatives are used as colistin adjuvants.
  • BMS-833923 is an existing well-known substance, which is usually used as a Hedgehog pathway inhibitor.
  • the BMS-833923 derivatives claimed in this disclosure can refer to the prior art, such as including but not limited to fluorinated derivatives, chlorinated derivatives etc., and the structure of its derivatives will not be described in detail here.
  • the registration number of BMS-833923 is CAS No.1059734-66-5, and its chemical name is benzamide, N-[2-methyl-5-[(methylamino)methyl]phenyl]-4-[ (4-phenyl-2-quinazolinyl)amino]-(N-[2-methyl-5-[(methylamino)methyl]phenyl]-4-[(4-phenyl-2-quinazolinyl)amino]- Benzamide).
  • BMS-833923 derivatives include but are not limited to any one of the compounds shown in the following structural formulas:
  • BMS-833923 derivatives can also be used as colistin adjuvants to jointly inhibit or eliminate Gram-negative bacteria, or independently inhibit or eliminate Gram-positive bacteria.
  • the present disclosure can sensitize pathogens to antibiotics (colistin) by using BMS-833923 and its derivatives as colistin adjuvants, expanding the therapeutic index of colistin, allowing lower, non-toxic doses of slime mold
  • BMS-833923 and its derivatives as colistin adjuvants
  • antibiotic adjuvant refers to a class of compounds that do not have antibacterial function themselves, but can act synergistically with antibiotics to promote the bactericidal activity of antibiotics against bacteria, especially resistant bacteria.
  • BMS-833923 and its derivatives provided in the present disclosure can be used to treat bacteria in combination with colistin.
  • the bacteria are mainly Gram-negative bacteria, optionally Gram-negative bacteria in logarithmic growth phase.
  • 5-15 ⁇ g/mL of BMS-833923 and its derivatives are added to 1 ⁇ MIC 50 colistin-treated Gram-negative bacteria, optionally, 1 ⁇ MIC 50 colistin-treated Add 10 ⁇ g/mL of BMS-833923 and its derivatives to Gram-negative bacteria.
  • BMS-833923 and its derivatives and colistin can be used for joint inhibition or joint clearance of E. coli, and can also be used for joint inhibition or joint clearance of Klebsiella pneumoniae (Klebsiella Pneumoniae). It can also be used for joint inhibition or joint clearance of Acinetobacter baumannii, and can also be used for joint inhibition or joint clearance of Pseudomonas aeruginosa, and it can also be used for pan-drug-resistant strains BAA-1800, At least one of BAA-1794 and BAA-1792 is co-inhibited or co-cleared.
  • BMS-833923 and its derivatives and colistin has a synergistic effect on the growth inhibition of Escherichia coli. In contrast, no such effect was observed between BMS-833923 and its derivatives and the ⁇ -lactam antibiotic ampicillin, the quinolone antibiotic norfloxacin, or the aminoglycoside antibiotic kanamycin.
  • BMS-833923 and its derivatives can enhance the effect on colistin by up to 8-512 times through synergistic effect. This shows that BMS-833923 and its derivatives can enhance the bacteriostatic activity of colistin.
  • present disclosure also provides the use of BMS-833923 and its derivatives for independently inhibiting or eliminating Gram-positive bacteria.
  • Gram-positive bacteria may include at least one of Staphylococcus aureus and Bacillus subtilis, and other Gram-positive bacteria may also be included.
  • BMS-833923 and its derivatives alone can directly inhibit the growth of Gram-positive bacteria without the assistance of other antibiotics. That is, BMS-833923 and its derivatives can directly kill Gram-positive bacteria without producing drug resistance.
  • the compound BMS-833923 provided by the present disclosure can enhance the activity of colistin against different Gram-negative bacteria by up to 8-512 times. Combined treatment of log-phase bacteria with low concentrations of colistin and BMS-833923 and its derivatives resulted in rapid and efficient bacterial clearance. It is worth noting that BMS-833923 and its derivatives can directly kill Gram-positive bacteria without any other antibiotics. Moreover, the bacteria did not produce obvious drug-resistant mutants within 30 days of continuous passage in the presence of BMS-833923 and its derivatives.
  • BMS phosphatidylserine
  • DNA fragmentation DNA fragmentation
  • chromosome condensation DNA fragmentation
  • membrane depolarization membrane depolarization
  • caspase-like Enzyme protein binding activity was significantly increased. That is, BMS is a potent adjuvant that can expand the therapeutic index of colistin to allow lower, non-toxic doses of colistin to be used clinically for effective treatment of drug-resistant bacterial infections.
  • One embodiment of the present disclosure provides BMS-833923 or its derivatives as colistin adjuvants.
  • BMS-833923 or its derivatives are used for combined treatment of bacteria with colistin.
  • the bacteria are Gram-negative bacteria. In some embodiments, the Gram-negative bacteria are in logarithmic growth phase. In some embodiments, 5-15 ⁇ g/mL of BMS-833923 is added to 1 ⁇ MIC 50 colistin-treated Gram-negative bacteria. In some embodiments, 1 ⁇ MIC 50 colistin-treated Gram-negative bacteria are supplemented with 10 ⁇ g/mL of BMS-833923 and derivatives thereof.
  • BMS-833923 or a derivative thereof and the colistin are used for joint inhibition or joint elimination of Escherichia coli.
  • BMS-833923 or a derivative thereof and the colistin are used for joint inhibition or joint elimination of Klebsiella pneumoniae.
  • BMS-833923 or a derivative thereof and the colistin are used for joint inhibition or joint elimination of Acinetobacter baumannii.
  • BMS-833923 or a derivative thereof and the colistin are used for joint inhibition or joint elimination of Pseudomonas aeruginosa.
  • BMS-833923 or a derivative thereof and the colistin are used for joint inhibition or joint elimination of at least one of pan-drug-resistant strains BAA-1800, BAA-1794 and BAA-1792.
  • One embodiment of the present disclosure also provides an application of BMS-833923 or its derivatives, where BMS-833923 or its derivatives are used to independently inhibit or eliminate Gram-positive bacteria.
  • the Gram-positive bacteria include at least one of Staphylococcus aureus and Bacillus subtilis.
  • the present disclosure also provides an antibacterial drug, the active ingredients of which include BMS-833923 and its derivatives and colistin.
  • the present disclosure also provides an antibacterial drug, the active ingredients of which include a combination of BMS-833923 or one of its derivatives and colistin.
  • antibacterial drugs for example, can be against Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, pan-drug-resistant bacterial strains BAA-1800, BAA-1794 and At least one drug that inhibits or clears BAA-1792.
  • Some embodiments of the present disclosure provide an antibacterial drug, the active ingredients of which include BMS-833923, its derivatives or a combination of BMS-833923 and its derivatives.
  • the antibacterial drug is a drug that inhibits or eliminates at least one of Staphylococcus aureus and Bacillus subtilis.
  • Some embodiments of the present disclosure provide a method of treating diseases associated with bacterial infections, comprising:
  • a therapeutically effective amount of the antibacterial drug is administered to a subject in need thereof.
  • Some embodiments of the present disclosure provide a method of treating diseases associated with Gram-positive bacterial infections, comprising:
  • a therapeutically effective amount of the antibacterial drug is administered to a subject in need thereof.
  • the Gram-positive bacteria include at least one of Staphylococcus aureus and Bacillus subtilis.
  • Some embodiments of the present disclosure provide the use of the antibacterial drug for treating diseases related to bacterial infection.
  • diseases associated with bacterial infection include: skin infection, soft tissue infection, pneumonia, osteomyelitis, meningitis, sepsis, endocarditis, infective endocarditis.
  • the present disclosure provides the use of the antibacterial drug for treating diseases related to Gram-positive bacterial infection.
  • BMS-833923 its derivatives, or the combination of the two provided by some embodiments of the present disclosure as a colistin adjuvant, for example, for joint treatment with colistin on drug-resistant bacteria, can effectively expand the efficacy of colistin Therapeutic index, allowing lower, nontoxic doses of colistin to be used clinically for effective treatment of drug-resistant bacterial infections.
  • the antibacterial drugs provided by some embodiments of the present disclosure its functional components include BMS-833923, its derivatives or a combination of the two and colistin, which can reduce the toxic and side effects by enhancing the bactericidal activity of antibiotics (colistin) The concentration of antibiotics used clinically to avoid side effects on patients.
  • BMS-833923 and its derivatives as colistin adjuvants, combined treatment with colistin against drug-resistant bacteria can effectively expand the therapeutic index of colistin, allowing lower, non-toxic doses of colistin Bacteroids are clinically used for the effective treatment of drug-resistant bacterial infections.
  • the functional ingredients contain BMS-833923 and its derivatives and colistin antibacterial drugs at the same time, which can enhance the bactericidal activity of antibiotics (colistin) so as to reduce the clinical use concentration of antibiotics with toxic side effects and avoid their adverse effects on Patients experience side effects.
  • Bacterial strains and reagents used in the examples are listed in Table 1. All antibiotics were purchased from Sigma. Apoptotic compound library (product number L3300) was purchased from SelleckChem. MHB and LB media were purchased from Becton Dickinson (BD). Compound BMS-833923 was purchased from Shanghai DC Chemicals Company. Apoptosis detection kits FITC Annexin V Apoptosis Detection Kit (Cat. No. 556547) and APO-DIRECTTM Kit (Cat. No. 556381) were purchased from BD Company. CaspACE TM FITC-VAD-FMK In Situ Marker (product number G7401) was purchased from Promega. Membrane potential detection reagent DiBAC4 (product number B438) was purchased from Invitrogen. Hoechst 33342 (product number H1339) was purchased from Thermo Fisher.
  • BMS-833923 (XL139, BMS) is a human SMO receptor inhibitor of Hedgehog signaling pathway (chemical formula shown in Figure 1B), which has potential activity in treating advanced or metastatic cancer. Its clinical trials show that the subjects have good tolerance to the drug, which proves that BMS has good safety. Therefore, BMS has the possibility of potential clinical use.
  • CFU colony forming units
  • Colistin adjuvant high-throughput screening method An WF, Tolliday NJ: Introduction: cell-based assays for high-throughput screening. Methods Mol Biol 2009, 486: 1-12.
  • the 96-well plate was incubated at 37°C, and the OD 600 was read again after 24 hours. It is best to shake the plate for 5 seconds before reading to get more accurate data.
  • the effectiveness of the high-throughput screening method was assessed by calculating the Z' value. Data were normalized by a method based on quartile means. If the OD value of the well where the compound is located is lower than 0.2, the compound is selected as the "primary hit" and subsequent verification is performed.
  • BMS can enhance the antibacterial activity of colistin.
  • test methods are as follows:
  • MIC minimum inhibitory concentration
  • Figure 2B is the bactericidal result of the following grouping treatment in the logarithmic phase (about 1 ⁇ 10 8 CFU/mL) of large intestine citrus MG1655: divided into control: 1 ⁇ PBS (Ctrl group), 1 ⁇ MIC 50 slime mold Colistin (CT group), 10 ⁇ g/ml BMS (BMS group), or a combination of 1 ⁇ MIC 50 colistin + 10 ⁇ g/ml BMS (CT+BMS group).
  • pan-drug-resistant strains BAA-1800, BAA-1794 and BAA-1792 (as shown in Figure 2C, wherein the value of CT+BMS is 0), wherein in Figure 2C, it is the logarithm of Acinetobacter baumannii Period (approximately 1 ⁇ 10 8 CFU/mL CFU/mL), treated with the following groups: CT is 1 ⁇ MIC 50 colistin, CT+BMS: 1 ⁇ MIC 50 colistin+10 ⁇ g/ml BMS .
  • BMS alone can directly inhibit the growth of Gram-positive bacteria without the assistance of other antibiotics.
  • the MIC 50 of BMS alone against Gram-negative bacteria is higher than 100 ⁇ g/mL.
  • the MIC50 of BMS against Staphylococcus aureus and Bacillus subtilis were 5 and 2.5 ⁇ g/mL, respectively (as shown in Table 2).
  • log phase Staphylococcus aureus ATCC 25904 (about 5 ⁇ 10 7 CFU/mL) or Staphylococcus aureus ATCC 43300 (MRSA) (about 5 ⁇ 10 7 CFU/mL) was treated with 10 ⁇ g/mL BMS for 8 hours All bacteria in the post-culture were cleared (as shown in Figure 2D and 2E), wherein, in Figure 2D and 2E, Ctrl was the control group (1 ⁇ PBS), BMS_5 was 5 ⁇ g/ml of BMS, and BMS_10 was 10 ⁇ g/ml BMS, Norf is 1 ⁇ MIC 50 of colistin + 10 ⁇ g/ml of BMS.
  • test methods are as follows:
  • Staphylococcus aureus ATCC 25904 in the exponential growth phase was diluted 1:100 (v/v) into 1mL MHB medium containing different concentrations of BMS, and at the same time was treated with different concentrations of norfloxa star as a positive control.
  • Bacteria were incubated at 37° C., 200 rpm, and passaged every 24 h in different concentrations (0.25, 0.5, 1, 2, 4 ⁇ MIC) of BMS or norfloxacin.
  • the culture at the second highest concentration (OD 600 ⁇ 1) that allowed growth was washed once with 1 ml of PBS, and then diluted 1:100 (v/v) to contain different concentrations (0.25, 0.5, 1, 2, 4 ⁇ MIC) in the corresponding antibacterial agents to continue passage. If changes in MIC values were observed, adjust the concentration of the corresponding antimicrobial for subsequent passages. This serial passage was repeated for 30 days and three independent replicates were performed. MIC change was calculated by dividing the respective daily MIC by the initial MIC value on day 1.
  • BMS-triggered bacterial death exhibits apoptosis-like features
  • Escherichia coli was treated with colistin alone or in combination with BMS, and four apoptotic markers of bacteria were detected: using Annex V to detect phosphatidylserine (PS) exposure; using TUNNEL to detect DNA fragmentation; using BiDAC 4 was used to detect depolarization of bacterial cell membrane; FITC-Z-VD-FMK was used to detect caspase-like substrate binding ability.
  • PS phosphatidylserine
  • TUNNEL to detect DNA fragmentation
  • BiDAC 4 was used to detect depolarization of bacterial cell membrane
  • FITC-Z-VD-FMK was used to detect caspase-like substrate binding ability.
  • test methods are as follows:
  • PI phosphatidylserine
  • Fluorescence analysis was performed on a CytoFLEX flow cytometer (Beckman Coulter LifeSciences) equipped with a 488 nm argon laser for excitation, and 525 ⁇ 40 nm (FITC channel for FITC-mediated fluorescence) and 585 ⁇ 42 nm (PE channel for PI-mediated Fluorescence) Fluorescent filters are used to detect fluorescent signals.
  • the following PMT voltages were used for detection: 77 (FSC), 72 (SSC), 162 (FITC) and 192 (PI).
  • FSC 77
  • SSC 72
  • FITC 162
  • PI PI
  • FITC-dUTP FITC-conjugated deoxyuridine triphosphate
  • samples were centrifuged at 8K for 5 mins in a 4°C centrifuge and ethanol was removed by aspiration.
  • Cells were washed twice in 1 mL of wash buffer (kit component), then resuspended in 50 ⁇ L of DNA labeling solution (prepared as described in the kit). The cells were cultured in a temperature-controlled shaking incubator at 37°C, 300rmp and protected from light for 60mins. At the end of the incubation, 1 mL of washing buffer (kit component) was added to each sample, and the samples were centrifuged at 8K for 5 mins.
  • the cells were washed again with 1 mL of washing buffer and resuspended in 500 ⁇ L of PI/RNaseA solution (if the cell density is low, adjust the amount of PI/RNaseStainingBuffer to 0.3 mL). The samples were incubated for 30 mins at room temperature in the dark. Finally 500 ⁇ L of 1 ⁇ PBS diluted samples were added and analyzed by flow cytometry.
  • Fluorescent analysis was performed by a CytoFLEX flow cytometer (Beckman Coulter Life Sciences). The parameter settings are the same as those related to PS exposure detection. Experiments were repeated three times independently, and GraphPadPrism8 was used to process data and generate pictures.
  • chromosomal aggregation observations bacteria were cultured as described above and treated with the indicated concentrations of drugs. Chromosomes were fluorescently stained using the DNA-specific dye Hoechst33342 (Invitrogen, Cat. No. H1339), which was dissolved in deionized water and a 10 mg/mL (16.23 mM) stock solution was prepared. PI was used as a counterstain and dissolved in deionized water to prepare a 1 mg/mL (1.5 mM) stock solution.
  • Hoechst33342 Invitrogen, Cat. No. H1339
  • DiBAC4 (3) is a membrane potential ( ⁇ ) sensitive dye that can be used to monitor membrane depolarization in drug-treated cells.
  • DiBAC4 (3) can cross depolarized but still intact cell membranes and bind intracellular lipid components exhibiting an enhanced fluorescent signal.
  • DiBAC4 (3) staining at each time point after drug treatment, approximately 105 cells were collected by centrifugation at 8K for 5 mins at 4°C and washed twice with 1 mL of cold 1 ⁇ PBS. Resuspend the cells in 1 mL of filtered 1 ⁇ PBS and add 1 ⁇ L of 1 mg/mL DiBAC4 (3) to each sample and incubate for 10 min at room temperature in the dark. After incubation, wash the cells 3 times with 1 mL of filtered 1x PBS. Samples were resuspended in 1 mL of filtered 1 ⁇ PBS and analyzed by CytoFLEX flow cytometer (Beckman Coulter Life Sciences). The parameter settings are the same as those related to PS exposure detection. Experiments were repeated three times independently, and GraphPadPrism8 was used to process data and generate pictures.
  • Caspases are considered to be central components of the mechanisms responsible for apoptosis in eukaryotes.
  • CaspACE TM FITC-VAD-FMK in situ labeling dye Promega, Cat. No. G7462, which is a fluorescent analog of the pan-caspase inhibitor Z-VAD-FMK, was used. thing.
  • BMS has the potential as a colistin adjuvant.
  • BMS-833923 and its derivatives provided by the present disclosure as colistin adjuvants can effectively expand the therapeutic index of colistin, allowing a relatively Low, non-toxic doses of colistin are clinically used for the effective treatment of drug-resistant bacterial infections, which can reduce the clinical use concentration of antibiotics with toxic side effects by enhancing the bactericidal activity of antibiotics (colistin) , to avoid its side effects on patients, and has broad application prospects and high market value.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Epidemiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

本公开提供了一种BMS-833923及其衍生物的应用及药物，属于抗生素技术领域。BMS-833923及其衍生物可用作粘菌素佐剂对革兰氏阴性菌进行联合抑制或清除，也可独立地对革兰氏阳性菌进行抑制或清除。其中，革兰氏阴性菌可包括大肠杆菌、肺炎克雷伯氏菌、鲍曼不动杆菌、铜绿假单胞菌、泛耐药菌株BAA-1800、BAA-1794和BAA-1792中，革兰氏阳性菌可包括金黄色葡萄球菌和枯草芽孢杆菌中的至少一种。通过将BMS-833923及其衍生物作为粘菌素的佐剂，可有效扩大粘菌素的治疗指数，允许较低的、无毒剂量的粘菌素在临床上用于抗药性细菌感染的有效治疗。

Description

一种BMS-833923及其衍生物的应用及药物
相关申请的交叉引用
本公开要求于2021年10月21日提交中国专利局的申请号为“202111226018.X”名称为“一种BMS-833923及其衍生物的应用及药物”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及抗生素技术领域，具体而言，涉及一种BMS-833923及其衍生物的应用及药物。
背景技术
许多常见细菌病原体对抗生素的耐药性日益增强，已成为威胁人类生命的全球性危机。“ESKAPE”病原体，即屎肠球菌、金黄色葡萄球菌、肺炎克雷伯菌、鲍曼不动杆菌、铜绿假单胞菌和大肠杆菌，代表了这些多重耐药病原体中的主要挑战。
目前，细菌已经对临床上几乎所有可用的抗生素都产生了耐药性，很多时候，缺乏能够有效控制和治疗细菌感染的有效手段。因此，迫切需要开发新的抗生素来缓解这种危机。然而，近年来新型抗生素的发现变得越来越困难，处在开发阶段的新型抗生素越来越少。一些因具有毒副作用而被抛弃的旧抗生素不得不被重新引入临床而作为治疗感染的手段。
多粘菌素是20世纪40年代开发的聚阳离子肽抗生素，这类抗生素通过破坏细菌细胞膜来杀死细菌。多粘菌素中的阳离子电荷与革兰氏阴性细菌外膜的带负电荷的脂多糖静电结合，然后置换稳定细胞膜的钙离子和镁离子，最终破坏细菌膜的完整性。尽管多粘菌素(如粘菌素)能够有效地杀死细菌，但由于其具有显著的肾毒性和神经毒性，此前在临床已经被放弃使用。最近，由于缺乏对多重耐药菌(例如鲍曼不动杆菌、铜绿假单胞菌和肺炎克雷伯菌)的有效治疗手段，被这些细菌感染的患者的发病率和死亡率高起。作为有效的治疗手段，粘菌素近年来被重新引入临床来治疗超级细菌的感染，尤其是革兰氏阴性超级菌的感染，并成为治疗的最后手段。然而，有研究显示，平均30％接受粘菌素治疗方案的患者出现至少轻度肾毒性。
因此，急需一种方法能扩大粘菌素的治疗指数以使其能够以较低的、无毒的剂量在临床上使用从而降低其毒副作用。
发明内容
本公开提供一种BMS-833923及其衍生物用作粘菌素佐剂。
本公开提供BMS-833923及其衍生物用于与粘菌素对细菌进行联合处理的应用。
在可选的实施方式中，细菌为革兰氏阴性菌。
在可选的实施方式中，革兰氏阴性菌处于对数生长期。
在可选的实施方式中，1×MIC 50粘菌素处理的革兰氏阴性菌中添加5-15μg/mL的BMS-833923及其衍生物。
在可选的实施方式中，1×MIC 50粘菌素处理的革兰氏阴性菌中添加10μg/mL的BMS-833923及其衍生物。
在可选的实施方式中，BMS-833923及其衍生物与粘菌素用于对大肠杆菌进行联合抑制或联合清除。
在可选的实施方式中，BMS-833923及其衍生物与粘菌素用于对肺炎克雷伯氏菌进行联合抑制或联合清除。
在可选的实施方式中，BMS-833923及其衍生物与粘菌素用于对鲍曼不动杆菌进行联合抑制或联合清除。
在可选的实施方式中，BMS-833923及其衍生物与粘菌素用于对铜绿假单胞菌进行联合抑制或联合清除。
在可选的实施方式中，BMS-833923及其衍生物与粘菌素用于对泛耐药菌株BAA-1800、BAA-1794和BAA-1792中的至少一种进行联合抑制或联合清除。
本公开提供BMS-833923及其衍生物用于独立地对革兰氏阳性菌进行抑制或清除的应用。
在可选的实施方式中，革兰氏阳性菌包括金黄色葡萄球菌和枯草芽孢杆菌中的至少一种。
本公开提供一种抗菌药物，所述抗菌药物的功效成分包括BMS-833923及其衍生物与粘菌素。
本公开提供一种抗菌药物，所述抗菌药物的功效成分包括BMS-833923或其衍生物中的一种与粘菌素的组合。
在一些实施方式中，所述抗菌药物为对大肠杆菌、肺炎克雷伯氏菌、鲍曼不动杆菌、铜绿假单胞菌、金黄色葡萄球菌、枯草芽孢杆菌、泛耐药菌株BAA-1800、BAA-1794和BAA-1792中的至少一种进行抑制或清除的药物。
本公开提供一种抗菌药物，所述抗菌药物的功效成分包括BMS-833923、其衍生物或者BMS-833923及其衍生物的组合。
在一些实施方式中，所述抗菌药物为对金黄色葡萄球菌和枯草芽孢杆菌中的至少一种进行抑制或清除的药物。
本公开提供了上文任一项所述BMS-833923的衍生物，包括以下结构式所示化合物中的任意一种：
Figure PCTCN2021140984-appb-000001
本公开提供一种治疗与细菌感染相关疾病的方法，包括：
向有此需要的受试者给药治疗有效量的所述抗菌药物。
本公开提供一种治疗与革兰氏阳性细菌感染相关疾病的方法，包括：
向有此需要的受试者给药治疗有效量的所述的抗菌药物。
在一些实施方式中，所述革兰氏阳性细菌包括金黄色葡萄球菌和枯草芽孢杆菌中的至少一种。
本公开提供所述抗菌药物，用于治疗与细菌感染相关的疾病的用途。
在一些实施方式中，所述与细菌感染相关的疾病包括：皮肤感染、软组织感染、肺炎、骨髓炎、脑膜炎、败血症、心内膜炎、感染性心内膜炎。
本公开提供所述抗菌药物，用于治疗与革兰氏阳性细菌感染相关的疾病的用途。
附图说明
为了更清楚地说明本公开实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本公开的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为实施例1中鉴定增强粘菌素抑制细菌生长的化合物以及实施例2中通过棋盘(checkerboard)实验来测量BMS-833923(简称BMS，下同)和粘菌素的联合使用时对大肠杆菌(Escherichia coli)生长抑制的协同作用的相关测试结果；
图2为实施例3中BMS-833923促进粘菌素的杀菌活性以及实施例4中BMS-833923直接杀死革兰氏阳性菌且不产生耐药性的相关测试结果；
图3为实施例5中粘菌素和BMS-833923联合处理细菌大肠杆菌时相应的细胞凋亡测试结果图，其中图B至E中，Ctrl表示对照组，CT表示粘菌素组，BMS表示BMS-833923组，CT+BMS表示粘菌素与BMS-833923的组合组；
图4为实施例5中BMS-833923单独处理金黄色葡萄球菌(Staphylococcus aureus)时相应的细胞凋亡测试结果图；其中图B至E中，Ctrl表示阴性对照组，BMS表示BMS-833923组，Van表示阳性对照组；。
术语定义
如本文所用，术语“泛耐药”是指对目前推荐用于相应细菌感染的经验用药(除多黏菌素、替加环素外)都耐药的现象。术语“泛耐药菌株”则是指对目前推荐用于相应细菌感染的经验用药(除多黏菌素、替加环素外)都耐药的菌株。
具体实施方式
为使本公开实施例的目的、技术方案和优点更加清楚，下面将对本公开实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
下面对本公开提供的BMS-833923及其衍生物的应用及药物进行具体说明。
本公开提出一种BMS-833923及其衍生物的应用，为BMS-833923及其衍生物用作粘菌素佐剂。其中，BMS-833923为现有的公知物质，通常用作Hedgehog通路抑制剂，本公开所声称的BMS-833923衍生物可参照现有技术，诸如包括但不限于氟代衍生物、氯代衍生物等，在此不对其衍生物的结构过多赘述。
其中，BMS-833923的登记号为CAS No.1059734-66-5，化学名称为苯甲酰胺,N-[2-甲基-5-[(甲氨基)甲基]苯基]-4-[(4-苯基-2-喹唑啉基)氨基]-(N-[2-methyl-5-[(methylamino)methyl]phenyl]-4-[(4-phenyl-2-quinazolinyl)amino]-Benzamide)。
在一些实施方式中，BMS-833923衍生物包括但不限于以下结构式所示化合物中的任意一种：
Figure PCTCN2021140984-appb-000002
上述BMS-833923衍生物也可以用作粘菌素佐剂对革兰氏阴性菌进行联合抑制或清除，也可独立地对革兰氏阳性菌进行抑制或清除。
本公开通过将BMS-833923及其衍生物用作粘菌素佐剂，可以使病原体对抗生素(粘菌素)敏感，扩大粘菌素的治疗指数，允许较低的、无毒剂量的粘菌素获得有效杀死细菌病原体的能力，不仅可减缓抗生素耐药性危机，而且还能有效降低对患者产生的副作用。
如本文所述，术语“抗生素佐剂”是指一类本身并不具有抗菌功能，但可与抗生素协同作用，促进抗生素对于细菌尤其是抗性细菌的杀菌活性的化合物。
可参考地，本公开提供的BMS-833923及其衍生物可用于与粘菌素对细菌进行联合处理。
其中，细菌主要为革兰氏阴性菌，可选地为处于对数生长期的革兰氏阴性菌。
在可选的实施方式中，1×MIC 50粘菌素处理的革兰氏阴性菌中添加5-15μg/mL的BMS-833923及其衍生物，可选地，1×MIC 50粘菌素处理的革兰氏阴性菌中添加10μg/mL的BMS-833923及其衍生物。
可参考地，BMS-833923及其衍生物与粘菌素可用于对大肠杆菌进行联合抑制或联合清除，也可用于对肺炎克雷伯氏菌(Klebsiella Pneumoniae)进行联合抑制或联合清除，也可用于对鲍曼不动杆菌(Acinetobacter baumannii)进行联合抑制或联合清除，也可用于对铜绿假单胞菌(Pseudomonas aeruginosa)进行联合抑制或联合清除，还可用于对泛耐药菌株BAA-1800、BAA-1794和BAA-1792中的至少一种进行联合抑制或联 合清除。
需要说明的是，BMS-833923及其衍生物和粘菌素的联合使用时对大肠杆菌生长抑制的协同作用。相比之下，在BMS-833923及其衍生物与β-内酰胺抗生素氨苄青霉素、喹诺酮类抗生素诺氟沙星或氨基糖苷类抗生素卡那霉素之间未观察到这种作用。在其它病原菌中也观察到BMS-833923及其衍生物对粘菌素抑制细菌的增强作用，包括肺炎克雷伯氏菌、鲍曼不动杆菌和铜绿假单胞菌，BMS-833923及其衍生物通过协同作用能够对粘菌素作用增强高达8-512倍。由此表明BMS-833923及其衍生物可以增强粘菌素的细菌抑制活性。
此外，本公开还提供了BMS-833923及其衍生物用于独立地对革兰氏阳性菌进行抑制或清除的应用。
其中，革兰氏阳性菌可包括金黄色葡萄球菌和枯草芽孢杆菌(Bacillus subtilis)中的至少一种，此外，也可包括其它革兰氏阳性菌。
需要说明的是，与对革兰氏阴性菌的作用不同，BMS-833923及其衍生物单独能够直接抑制革兰氏阳性菌的生长，而不需要其他抗生素的协助。也即，BMS-833923及其衍生物可直接杀死革兰氏阳性菌且不产生耐药性。
承上，本公开提供的化合物BMS-833923能够把粘菌素对不同革兰氏阴性细菌的活性增强高达8-512倍。用低浓度粘菌素和BMS-833923及其衍生物联合处理对数生长期细菌可快速有效地清除细菌。值得注意的是，BMS-833923及其衍生物本身能够直接杀死革兰氏阳性菌，无需与其它任何抗生素连用。而且，细菌在BMS-833923及其衍生物存在下连续传代30天内未产生明显的耐药突变体。初步机理研究发现，被BMS-833923及其衍生物杀死的细菌表现出细菌凋亡的特征，其中磷脂酰丝氨酸(PS)暴露、DNA断裂、染色体浓缩、膜去极化和类半胱天冬酶蛋白结合活性显着增加。也即，BMS是一种有效的佐剂，可以扩大粘菌素的治疗指数，以允许较低的、无毒剂量粘菌素在临床上用于抗药性细菌感染的有效治疗。
本公开一实施方式提供BMS-833923或其衍生物用作粘菌素佐剂。
本公开一实施方式提供一种BMS-833923或其衍生物的应用，BMS-833923或其衍生物用于与粘菌素对细菌进行联合处理。
在一些实施方式中，该细菌为革兰氏阴性菌。在一些实施方式中，革兰氏阴性菌处于对数生长期。在一些实施方式中，1×MIC 50粘菌素处理的革兰氏阴性菌中添加5-15μg/mL的BMS-833923。在一些实施方式中，1×MIC 50粘菌素处理的革兰氏阴性菌中添加10μg/mL的BMS-833923及其衍生物。
在一些实施方式中，BMS-833923或其衍生物与所述粘菌素用于对大肠杆菌进行联合抑制或联合清除。
在一些实施方式中，BMS-833923或其衍生物与所述粘菌素用于对肺炎克雷伯氏菌进行联合抑制或联合清除。
在一些实施方式中，BMS-833923或其衍生物与所述粘菌素用于对鲍曼不动杆菌进 行联合抑制或联合清除。
在一些实施方式中，BMS-833923或其衍生物与所述粘菌素用于对铜绿假单胞菌进行联合抑制或联合清除。
在一些实施方式中，BMS-833923或其衍生物与所述粘菌素用于对泛耐药菌株BAA-1800、BAA-1794和BAA-1792中的至少一种进行联合抑制或联合清除。
本公开一实施方式还提供一种BMS-833923或其衍生物的应用，BMS-833923或其衍生物用于独立地对革兰氏阳性菌进行抑制或清除。
在一些实施方式中，所述革兰氏阳性菌包括金黄色葡萄球菌和枯草芽孢杆菌中的至少一种。
可选地，本公开还提供了一种抗菌药物，其功效成分包括BMS-833923及其衍生物与粘菌素。
在一些实施方式中，本公开还提供了一种抗菌药物，其功效成分包括BMS-833923或其衍生物中的一种与粘菌素的组合。
上述抗菌药物例如可以为对大肠杆菌、肺炎克雷伯氏菌、鲍曼不动杆菌、铜绿假单胞菌、金黄色葡萄球菌、枯草芽孢杆菌、泛耐药菌株BAA-1800、BAA-1794和BAA-1792中的至少一种进行抑制或清除的药物。
本公开一些实施方式提供一种抗菌药物，所述抗菌药物的功效成分包括BMS-833923、其衍生物或者BMS-833923及其衍生物的组合。
在一些实施方式中，抗菌药物为对金黄色葡萄球菌和枯草芽孢杆菌中的至少一种进行抑制或清除的药物。
本公开一些实施方式提供一种治疗与细菌感染相关疾病的方法，包括：
向有此需要的受试者给药治疗有效量的所述抗菌药物。
本公开一些实施方式提供一种治疗与革兰氏阳性细菌感染相关疾病的方法，包括：
向有此需要的受试者给药治疗有效量的所述的抗菌药物。
在一些实施方式中，革兰氏阳性细菌包括金黄色葡萄球菌和枯草芽孢杆菌中的至少一种。
本公开一些实施方式提供所述抗菌药物，用于治疗与细菌感染相关的疾病的用途。
在一些实施方式中，与细菌感染相关的疾病包括：皮肤感染、软组织感染、肺炎、骨髓炎、脑膜炎、败血症、心内膜炎、感染性心内膜炎。
本公开提供所述抗菌药物，用于治疗与革兰氏阳性细菌感染相关的疾病的用途。
本公开一些实施方式提供的BMS-833923、其衍生物或者两者的组合用作粘菌素佐剂的应用，如用于与粘菌素对抗药性细菌进行联合处理，可有效扩大粘菌素的治疗指数，允许较低的、无毒剂量的粘菌素在临床上用于抗药性细菌感染的有效治疗。
本公开一些实施方式提供的抗菌药物，其功效成分包括BMS-833923、其衍生物或者两者的组合与粘菌素，可起到通过增强抗生素(粘菌素)的杀菌活性从而降低具有毒副作用的抗生素在临床上的使用浓度，避免其对患者产生副作用。
通过将BMS-833923及其衍生物作为粘菌素的佐剂，使其与粘菌素对抗药性细菌进行联合处理，可有效扩大粘菌素的治疗指数，允许较低的、无毒剂量的粘菌素在临床上用于抗药性细菌感染的有效治疗。功效成分同时含有BMS-833923及其衍生物与粘菌素的抗菌药物，可起到通过增强抗生素(粘菌素)的杀菌活性从而降低具有毒副作用的抗生素在临床上的使用浓度，避免其对患者产生副作用。
以下结合实施例对本公开的特征和性能作可选的详细描述。
实施例中使用的菌株与试剂：以下实施例中所用菌株均列于表1中。所有抗生素均购于Sigma公司。细胞凋亡化合物库(货号L3300)购自SelleckChem公司。MHB和LB培养基购自Becton Dickinson(BD)公司。化合物BMS-833923购自上海DC Chemicals公司。细胞凋亡检测试剂盒FITC Annexin V Apoptosis Detection Kit(货号556547)及APO-DIRECTTM Kit(货号556381)购自BD公司。CaspACE TM FITC-VAD-FMK In Situ Marker(货号G7401)购自Promega公司。膜电位检测试剂DiBAC4(货号B438)购自Invitrogen公司。Hoechst 33342(货号H1339)购自Thermo Fisher公司。
表1细菌菌株
Figure PCTCN2021140984-appb-000003
Figure PCTCN2021140984-appb-000004
实施例1
鉴定增强粘菌素抑制细菌生长的化合物
通过测量96孔板中的细菌生长来测试化合物与粘菌素联合使用对细菌的作用，得到可以增强低浓度粘菌素抑制细菌生长的化合物。
待测化合物共计689种。
在细菌培养基中加入1/4最小抑制浓度(MIC 50)的粘菌素和40μM的单个化合物，并在细菌培养16小时后检查生长情况。设定OD 600＝0.2为临界值，找出导致细菌生长OD 600<0.2的化合物可选的研究(如图1A所示)，共有17种化合物在初步筛选中被鉴定出来(如表1所示)。
当可选的降低化合物的最终浓度到20μM时，17种化合物中的15种在1/4MIC的粘菌素存在时仍然完全抑制细菌生长(OD 600<0.2)。
此外，可选的结果显示，这些化合物中的5种可以在与1/8MIC 50粘菌素的组合时完全有效抑制细菌生长。其中，BMS-833923(XL139，BMS)为Hedgehog信号通路的人SMO受体抑制剂(化学式如图1B所示)，具有潜在的治疗晚期或转移性癌症活性。其临床试验表明，受试者对该药物具有出良好的耐受性，证明BMS的具有良好的安全性。因此，BMS具有潜在临床使用的可能。
其中，相关培养和检测方法如下：
①细菌生长培养：
对于大多数实验，挑取单克隆菌落置于2mLMHB培养基中，37℃，220rpm过夜培养。然后以1:100(v/v)比例稀释到25mL新鲜MHB中继续培养。处理前培养细菌至生长对数中期(OD 600＝0.35～0.45)(约2h)。使用SpectraMaxM5酶标仪(MolecularDevices)进行OD 600测量。对于菌落形成单位(CFU)/mL测量方法如下，在每个时间点收集200μL菌液，用无菌的1×PBS，pH7.2(SantaCruzBiotechnology)洗涤两次，然后在1×PBS中连续稀释。取每个稀释度中5μL的稀释液滴于LB-琼脂平板上，并在37℃温箱中过夜培养。仅对产生20-100个菌落的稀释液进行计数，并使用以下公式计算CFU/mL值：CFU/mL值＝[菌落数×稀释因子]/(以mL为单位的体积)。每个实验独立重复三次并计算三次实验所得数据的平均值及标准差。
②粘菌素佐剂高通量筛选方法：
粘菌素佐剂高通量筛选方法参考文献：An WF,Tolliday NJ:Introduction:cell-based assays for high-throughput screening.Methods Mol Biol 2009,486:1-12。简而言之，大肠杆菌MG1655通过上述方法在LB培养基中培养至OD 600＝0.4。然后，将2μL的化合物(浓度为2mM)分装到含有48μL的LB培养基的96孔板中。接下来，用LB培养基将细菌培养物稀释至OD 600＝0.04并添加粘菌素至1μg/mL的浓度。将含有1μg/mL粘菌素的50μL稀释后细菌加入已含有化合物的96孔板中。100μL混合物中各组分的终浓度如下：稀释后细菌OD 600＝0.02、0.5μg/mL粘菌素、40μM化合物、2％DMSO。通过添加2μL纯DMSO和相同数量的细菌，将4个阴性对照设置到A12、B12、C12、D12孔中。通过将诺氟沙星添加到最终浓度为1μg/mL和相同数量的细菌，将4个阳性对照设置到E12、F12、G12、H12孔中。添加每种化合物后立即读取OD 600。将96孔板在37℃下孵育，24h后再次读取OD 600。最好在读数前摇动平板5秒，以获得更准确的数据。高通量筛选方法的有效性通过计算Z′值进行评估。数据通过基于四分位数平均值的方法进行标准化。若化合物所在孔OD值低于0.2则选取化合物作为“主要命中(primaryhit)”并进行后续验证。
实施例2
通过棋盘实验来测量BMS和粘菌素的联合使用时对大肠杆菌生长抑制的协同作用
结果显示：BMS与粘菌素的联合使用时展现出显著的协同效应(如图1C所示，该图为BMS和粘菌素与大肠杆菌MG1655协同抗菌作用的棋盘分析结果图)。相比之下，在BMS与β-内酰胺抗生素氨苄青霉素、喹诺酮类抗生素诺氟沙星或氨基糖苷类抗生素卡那霉素之间未观察到这种作用(数据未示)。
在其它病原菌中也观察到BMS对粘菌素抑制细菌的增强作用，包括肺炎克雷伯氏菌、鲍曼不动杆菌和铜绿假单胞菌，BMS通过协同作用能够对粘菌素作用增强高达8至512倍(如图1D所示)。结果表明：BMS可以增强粘菌素的细菌抑制活性。
其中，相关测试方法如下：
最小抑菌浓度(MIC)测定和棋盘实验参照美国临床实验室标准协会(CLSI)指南进行。简而言之，通过前面描述的方法在MHB培养基中将细菌菌株培养至OD 600＝0.4。然后将BMS和抗生素在MHB中进行两倍稀释，并与MHB中含有约1.5×10 6CFU/mL的细菌悬液混合在96孔微量滴定板中。在37℃下孵育24h后，MIC被定义为BMS或抗生素抑制细菌生长的最低浓度。无法确定准确MIC值时，例如BMS单独作用于革兰氏阴性菌时，则将测试最高浓度的两倍视为其MIC值。分数抑制浓度指数(FICI)由以下公式计算：FICI＝A/MIC A+B/MIC B，其中A和B分别是两种药物联合抑制细菌生长的MIC值，MIC A和MIC B是药物单独抑菌时的MIC值。协同作用定义为FICI≤0.5。
以下实施例中涉及的MIC等相关测试参照实施例2，后续不做赘述。
实施例3
BMS促进粘菌素的杀菌活性
为了证明BMS对粘菌素的协同抑制作用可以转化为对粘菌素的杀菌的增强作用， 检测了在BMS存在下粘菌素的杀菌动力学。如图2A所示，对指数期大肠杆菌(约2×10 8CFU/mL)用对照：1×PBS(Ctrl组)、1×MIC 50的粘菌素(CT组)、10μg/ml的BMS(BMS组)、或1×MIC 50的粘菌素+10μg/ml的BMS二者联合使用(CT+BMS组)对细菌进行处理来研究其对大肠杆菌的杀菌情况。
单独使用粘菌素、BMS处理对数期细菌对其存活几乎没有影响。然而，BMS有效地增强了粘菌素的杀菌活性。二者联合使用时，细菌在处理后1小时，几乎没有能够从处理过的培养物中检测到存活的细菌。此外，BMS还能够使对粘菌素具有抗性的细菌重新获得药物的敏感性。用粘菌素和BMS一起处理含有mcr-1基因的MG1655能在2小时内有效地清除对数生长的耐药细菌，而单独使用粘菌素对细菌存活几乎没有影响(如图2B所示)，其中，图2B为在大肠柑橘MG1655对数期(约1×10 8CFU/mL)用以下分组处理的杀菌结果：分为对照：1×PBS(Ctrl组)、1×MIC 50的粘菌素(CT组)、10μg/ml的BMS(BMS组)、或1×MIC 50的粘菌素+10μg/ml的BMS二者联合使用(CT+BMS组)。
同样，在鲍曼不动杆菌中也检测了BMS对粘菌素杀菌的增强作用，结果表明：在1×MIC 50粘菌素处理的细菌中添加10μg/ml BMS导致各种细菌菌株的快速被清除，包括泛耐药菌株BAA-1800、BAA-1794和BAA-1792(如图2C所示，其中CT+BMS的值为0)，其中图2C中，为在鲍曼不动杆菌的对数期期(约1×10 8CFU/mLCFU/mL)，用以下分组处理的：CT为1×MIC 50粘菌素，CT+BMS：为1×MIC 50的粘菌素+10μg/ml的BMS。
总之，这些结果表明BMS可以增强粘菌素的杀菌活性，并且在BMS存在的情况下，较低的粘菌素浓度足以清除细菌，从而达到与高浓度粘菌素的杀菌相一致的效果。
实施例4
BMS直接杀死革兰氏阳性菌且不产生耐药性
与对革兰氏阴性菌的作用不同，BMS单独能够直接抑制革兰氏阳性菌的生长，而不需要其他抗生素的协助。BMS单独对革兰氏阴性菌(如大肠杆菌或鲍曼不动杆菌)的MIC 50高于100μg/mL。然而，BMS对金黄色葡萄球菌和枯草芽孢杆菌的MIC 50分别为5和2.5μg/mL(如表2所示)。
表2 BMS对不同菌株的MIC 50
Figure PCTCN2021140984-appb-000005
Figure PCTCN2021140984-appb-000006
此外，用10μg/mL BMS处理对数期金黄色葡萄球菌ATCC 25904(约5×10 7CFU/mL)或金黄色葡萄球菌ATCC 43300(MRSA)(约5×10 7CFU/mL)，8小时后培养物中的所用细菌被清除(如图2D和2E所示)，其中，图2D和2E中，Ctrl为对照组(1×PBS)、BMS_5为5μg/ml的BMS、BMS_10为10μg/ml的BMS、Norf为1×MIC 50的粘菌素+10μg/ml的BMS。为了可选的研究BMS对细菌的杀伤作用，尝试分离出一个对BMS具有抗性的突变体。按照之前的方案，金黄色葡萄球菌ATCC 25904在不同浓度的BMS(0.25～4倍MIC 50)中传代30天，以确定金黄色葡萄球菌进化出对BMS具有表型抗性的潜力。
数据显示：金黄色葡萄球菌在连续培养30天后没有出现BMS抗性的突变体(如图2F所示)。相比之下，细菌对诺氟沙星的抗性产生迅速，培养细菌的MIC 50在这段时间内提高了400倍以上(如图2F所示)，其中，图2F中，为在金黄色葡萄球菌
用以下分组处理：BMS为10μg/ml的BMS、Norf为1×MIC 50的粘菌素+10μg/ml的BMS。。
通过上述数据表明：细菌无法在不失去生理适应性的情况下进化为对BMS产生抗性。
其中，相关测试方法如下：
耐药性形成测定：
为了通过连续传代产生抗性，将在指数生长期的金黄色葡萄球菌ATCC 25904以1:100(v/v)比例稀释到含有不同浓度BMS的1mLMHB培养基中，同时以不同浓度的诺氟沙星作为阳性对照。细菌在37℃、200rpm下孵育，并在不同浓度(0.25、0.5、1、2、4×MIC)的BMS或诺氟沙星中每隔24h进行传代。将允许生长的第二高浓度(OD 600≥1)的培养物用1ml的PBS洗涤一次，然后以1:100(v/v)的比例稀释到含有不同浓度(0.25、0.5、1、2、4×MIC)的相应抗菌剂中继续传代。如果观察到MIC值的变化，则调整相应抗菌剂的浓度以用于随后的传代。这种连续传代重复了30天，并进行三次独立重复。MIC变化的计算方法是将各自的每日MIC除以第1天的初始MIC值。
实施例5
BMS引发的细菌死亡表现出类似细胞凋亡的特征
通过显微镜研究比较在BMS存在和不存在情况下粘菌素处理后的细菌的变化。用低浓度粘菌素(1×MIC 50)单独，或与BMS联合处理大肠杆菌，然后使用Hochest 33342对处理的细菌进行染色，研究细菌染色体的形态变化。
结果发现：当使用粘菌素和BMS联合处理细菌时，大部分细菌细胞中间显示了强烈的荧光(如图3A所示)，表明细菌的染色体发生了聚集。观察到的这种形态变化类似 于先前报道的细菌凋亡。因此，推测BMS可能通过促进细菌细胞凋亡来增强细菌杀伤。
通过流式细胞术检测处理的细菌是否具有细菌凋亡的其它典型特征
单独使用粘菌素或者使用粘菌素与BMS联合处理大肠杆菌，并检测了细菌的四种凋亡标志物：使用Annex V检测磷脂酰丝氨酸(PS)的暴露；用TUNNEL来检测DNA断裂；使用BiDAC 4来检测细菌细胞膜的去极化；使用FITC-Z-VD-FMK检测类半胱天冬酶底物结合能力。
结果显示：单独使用粘菌素或BMS处理大肠杆菌时，很少能够检测到这四种标志物为阳性的细菌。然而，当粘菌素和BMS联合处理大肠杆菌时，细菌中四种标志物为阳性的细菌细胞在群体中的比例大大升高，分别达到了30％到90％(如图3B到3E所示，纵坐标分别代表：利用Ctrl：1×PBS、CT：1×MIC 50粘菌素(1μg/ml)、BMS：10μg/ml BMS、CT+BMS：10μg/ml BMS和1×MIC 50粘菌素(1μg/ml)联合，将上述四组分别同时处理4.5h的大肠杆菌MG1655中Annexin V、FITC-VAD-F-FMK、DiBAC4 (3)和TUNNEL阳性细菌细胞百分比)。
与BMS单独能够杀死革兰氏阳性细菌相一致，单独使用BMS处理的金黄色葡萄球菌也发现了明显的细菌染色体浓缩聚集(如图4A所示)。流式细胞术分析表明：BMS处理的金黄色葡萄球菌中Annex V、TUNNEL、BiDAC4及FITC-Z-VD-FMK阳性的细菌细胞与对照组比较具有显著的增加(如图4B至图4E所示，纵坐标分别代表分别利用Ctrl：1×PBS、BMS：10μg/ml BMS以及Van：10μg/ml BMS和1×MIC 50粘菌素(1μg/ml)联合，将上述四组分别同时处理4.5h的金黄色葡萄球菌ATCC25904中Annexin V、FITC-VAD-F-FMK、DiBAC4 (3)和TUNNEL阳性细菌细胞百分比)。
该结果表明：BMS通过促进细菌凋亡来增强细菌杀伤力。
其中，相关测试方法如下：
①流式细胞术检测磷脂酰丝氨酸暴露：
对于磷脂酰丝氨酸(PS)暴露的检测，细菌如上所述培养并用指定浓度的药物处理。使用FITCAnnexinV细胞凋亡检测试剂盒(BDPharmingen TM，货号556547)进行AnnexinV染色。为了识别死细胞，使用PI作为复染剂。
在药物处理后的每个时间点，通过4℃离心机以8K离心5mins收集大约10 5个细胞，并用1mL冷1×PBS洗涤两次。然后将样品重新悬浮在100μL1×结合缓冲液(试剂盒成分)中。接下来，每个样品用5μLFITC偶联的AnnexinV和5μLPI(两种试剂盒成分)处理，并在室温(25℃)下避光孵育15分钟。孵育后在每个样品中加入400μL1×结合缓冲液，并在1小时内通过流式细胞术分析样品。
荧光分析由CytoFLEX流式细胞仪(BeckmanCoulterLifeSciences)进行，该仪器配备了488nm氩激光器用于激发，以及525±40nm(FITC介导荧光的FITC通道)和585±42nm(PE通道用于PI介导的荧光)荧光滤片用于检测荧光信号。检测采用以下PMT电压：77(FSC)、72(SSC)、162(FITC)和192(PI)。为消除细胞碎片的干扰，一级阈值设为FSC-H>100，二级阈值设为SSC-H>1000。每个样本至少采集10,000个细胞，使用FITC 通道测定AnnexinV-阳性细胞，用PE通道以确定AnnexinV阳性细胞是否仍然存活(PI阴性)或已经死亡(PI阳性)。实验独立重复三次，并使用GraphPadPrism8处理数据和生成图片。
②TUNEL法和流式细胞术检测DNA碎片：
对于DNA碎片化的检测，细菌如上所述培养并用指定浓度的药物处理。使用APO-DIRECTTM试剂盒(BDBiosciences，货号556381)进行TUNEL检测。该试剂盒使用FITC偶联的脱氧尿苷三磷酸(FITC-dUTP)对DNA碎片进行染色，并使用PI作为复染剂。
在药物处理后的每个时间点，通过4℃离心机以8K离心5mins收集大约10 5个细胞，并用1mL冷1×PBS洗涤两次。然后将样品重新悬浮在1mL4％多聚甲醛(SantaCruzBiotechnology)中，并将细胞悬液置于冰上30-60mins。然后将细胞在4℃离心机中以8K离心5分钟，洗涤一次并重悬于300μL冷1×PBS中。向每个样品中加入1mL70％(v/v，在dH 2O中)冰冷的乙醇，并将样品在-20℃下储存过夜。
对于染色，样品在4℃离心机中以8K离心5mins，并通过抽吸去除乙醇。细胞在1mL洗涤缓冲液(试剂盒成分)中洗涤两次，然后重悬于50μL的DNA标记溶液(按试剂盒中所述制备)中。将细胞在温控振荡培养箱中在37℃、300rmp避光培养60mins。孵育结束时，向每个样品中加入1mL漂洗缓冲液(试剂盒成分)，样品在8K下离心5mins。细胞再次用1mL漂洗缓冲液洗涤并重悬于500μLPI/RNaseA溶液中(如果细胞密度低，将PI/RNaseStainingBuffer的用量调整为0.3mL)。样品在室温下避光孵育30mins。最后添加500μL1×PBS稀释样品并用流式细胞术进行分析。
通过CytoFLEX流式细胞仪(BeckmanCoulterLifeSciences)进行荧光分析。参数设置与PS暴露检测的相关参数设置相同。实验独立重复三次，并使用GraphPadPrism8处理数据和生成图片。
③荧光显微镜观察染色体聚集：
对于染色体聚集观察，细菌如上所述培养并用指定浓度的药物处理。使用DNA特异性染料Hoechst33342(Invitrogen，货号H1339)对染色体进行荧光染色，将其溶解在去离子水中并制备10mg/mL(16.23mM)储备溶液。PI用作复染剂溶解在去离子水中制备1mg/mL(1.5mM)储备溶液。
在药物处理后的每个时间点，通过4℃离心机以8K离心5mins收集大约10 5个细胞，并用1mL冷1×PBS洗涤两次。将细胞重新悬浮在1mL过滤的1×PBS中，并将1μL的10mg/mLHoechst33342和1mg/mL的PI添加到每个样品中，在室温下避光孵育30分钟。孵育后，将细胞点在载玻片上并使用带有APOTOME荧光显微镜的CarlZeissAxioObserver进行分析，该显微镜配备有CoolSnapHQCCD相机(RoperScientific)，使用IPLab软件(Scanalytics)进行操作。对于荧光图像，使用UV/488nm双激发分析细胞，使用标准Hoechst和PI过滤器测量荧光信号。
④流式细胞术检测膜去极化：
对于膜去极化的检测，细菌如上所述培养并用指定浓度的药物处理。使用DiBAC4 (3)(Invitrogen，货号B438)进行细胞染色，将其溶解在70％乙醇(v/v)中并制备1mg/mL储备溶液。DiBAC4 (3)是一种膜电位(ΔΨ)敏感染料，可用于监测药物处理细胞的膜去极化。DiBAC4 (3)可以穿过去极化但仍然完整的细胞膜，并与细胞内脂质成分结合，表现出增强的荧光信号。
对于DiBAC4 (3)染色，在药物处理后的每个时间点，通过4℃离心机以8K离心5mins收集大约10 5个细胞，并用1mL冷1×PBS洗涤两次。将细胞重新悬浮在1mL过滤的1×PBS中，并向每个样品中加入1μL1mg/mL DiBAC4 (3)，在室温避光孵育10分钟。孵育后，用1mL过滤的1×PBS洗涤细胞3次。将样品重新悬浮在1mL过滤的1×PBS中，并通过CytoFLEX流式细胞仪(BeckmanCoulterLifeSciences)进行分析。参数设置与PS暴露检测相关的参数设置相同。实验独立重复三次，并使用GraphPadPrism8处理数据和生成图片。
⑤流式细胞术检测细菌caspase样蛋白：
半胱天冬酶(caspase)被认为是负责真核生物细胞凋亡的机制的核心成分。为了检测药物处理过的细菌中有无caspase样蛋白的表达，采用了CaspACE TMFITC-VAD-FMK原位标记染料(Promega，货号G7462)，它是泛caspase抑制剂Z-VAD-FMK的荧光类似物。
对于caspase样蛋白检测实验，细菌如上所述培养。当细菌在MHB中生长至对数中期(OD 600＝0.4)时，用指定浓度的药物处理进行处理，同时以1:1000的比例加入CaspACE TMFITC-VAD-FMKInSituMarker(终浓度为5μM)。样品避光并在温箱中37℃、220rmp进行培养。在药物处理后的每个时间点，通过4℃离心机以8K离心5mins收集大约10 5个细胞，并用1mL冷1×PBS洗涤两次。将样品重新悬浮在1mL过滤的1×PBS中，并通过CytoFLEX流式细胞仪(BeckmanCoulterLifeSciences)进行分析。参数设置与PS暴露检测相关的参数设置相同。实验独立重复三次，并使用GraphPadPrism8处理数据和生成图片。
综上所述，本研究中鉴定出的BMS可以有效增强粘菌素的活性并降低其杀死细菌病原体的有效浓度。这种促进杀菌活性很可能是通过激活细菌凋亡来实现的。考虑到临床试验已证明BMS使用的安全性，BMS具有作为粘菌素佐剂的潜力。
以上仅为本公开可选的实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。
工业实用性
本公开提供的BMS-833923及其衍生物用作粘菌素佐剂的应用，如单独使用或者用于与粘菌素联合对抗药性细菌进行处理，可有效扩大粘菌素的治疗指数，允许较低的、无毒剂量的粘菌素在临床上用于抗药性细菌感染的有效治疗，可起到通过增强抗生素 (粘菌素)的杀菌活性从而降低具有毒副作用的抗生素在临床上的使用浓度，避免其对患者产生副作用，具有广泛的应用前景和较高的市场价值。

Claims (18)

  1. BMS-833923及其衍生物用作粘菌素佐剂。
  2. 一种BMS-833923及其衍生物的应用，其特征在于，所述BMS-833923及其衍生物用于与粘菌素对细菌进行联合处理；
    优选地，所述细菌为革兰氏阴性菌；
    优选地，所述革兰氏阴性菌处于对数生长期；
    优选地，1×MIC 50MIC 50粘菌素处理的革兰氏阴性菌中添加5-15μg/mL的BMS-833923；
    更优地，1×MIC 50MIC 50粘菌素处理的革兰氏阴性菌中添加10μg/mL的BMS-833923及其衍生物。
  3. 根据权利要求2所述的应用，其特征在于，所述BMS-833923及其衍生物与所述粘菌素用于对大肠杆菌进行联合抑制或联合清除。
  4. 根据权利要求2所述的应用，其特征在于，所述BMS-833923及其衍生物与所述粘菌素用于对肺炎克雷伯氏菌进行联合抑制或联合清除。
  5. 根据权利要求2所述的应用，其特征在于，所述BMS-833923及其衍生物与所述粘菌素用于对鲍曼不动杆菌进行联合抑制或联合清除。
  6. 根据权利要求2所述的应用，其特征在于，所述BMS-833923及其衍生物与所述粘菌素用于对铜绿假单胞菌进行联合抑制或联合清除。
  7. 根据权利要求2所述的应用，其特征在于，所述BMS-833923及其衍生物与所述粘菌素用于对泛耐药菌株BAA-1800、BAA-1794和BAA-1792中的至少一种进行联合抑制或联合清除。
  8. 一种BMS-833923及其衍生物的应用，其特征在于，所述BMS-833923及其衍生物用于独立地对革兰氏阳性菌进行抑制或清除。
  9. 根据权利要求8所述的应用，其特征在于，所述革兰氏阳性菌包括金黄色葡萄球菌和枯草芽孢杆菌中的至少一种。
  10. 一种抗菌药物，其特征在于，所述抗菌药物的功效成分包括BMS-833923及其衍生物和粘菌素；
    优选地，所述抗菌药物为对大肠杆菌、肺炎克雷伯氏菌、鲍曼不动杆菌、铜绿假单胞菌、金黄色葡萄球菌、枯草芽孢杆菌、泛耐药菌株BAA-1800、BAA-1794和BAA-1792中的至少一种进行抑制或清除的药物。
  11. 一种抗菌药物，其特征在于，所述抗菌药物的功效成分包括BMS-833923或其衍生物中的一种与粘菌素的组合；
    优选地，所述抗菌药物为对大肠杆菌、肺炎克雷伯氏菌、鲍曼不动杆菌、铜绿假单胞菌、金黄色葡萄球菌、枯草芽孢杆菌、泛耐药菌株BAA-1800、BAA-1794和BAA-1792中的至少一种进行抑制或清除的药物。
  12. 一种抗菌药物，其特征在于，所述抗菌药物的功效成分包括BMS-833923、其衍生物或者BMS-833923及其衍生物的组合；
    优选地，所述抗菌药物为对金黄色葡萄球菌和枯草芽孢杆菌中的至少一种进行抑制或清除的药物。
  13. 根据权利要求1所述的BMS-833923及其衍生物用作粘菌素佐剂，或权利要求2-7中任一项所述的应用，或权利要求8-9中任一项所述的应用，或权利要求10、权利要求11或权利要求12所述的抗菌药物，所述BMS-833923的衍生物包括以下结构式所示化合物中的任意一种：
    Figure PCTCN2021140984-appb-100001
  14. 一种治疗与细菌感染相关疾病的方法，包括：
    向有此需要的受试者给药治疗有效量的权利要求10-12中任一项所述的抗菌药物。
  15. 一种治疗与革兰氏阳性细菌感染相关疾病的方法，包括：
    向有此需要的受试者给药治疗有效量的权利要求12所述的抗菌药物；
    优选地，所述革兰氏阳性细菌包括金黄色葡萄球菌和枯草芽孢杆菌中的至少一种。
  16. 如权利要10-12中任一项所述的抗菌药物，用于治疗与细菌感染相关的疾病的用途。
  17. 根据权利要求16所述的用途，其特征在于，所述与细菌感染相关的疾病包括：皮肤感染、软组织感染、肺炎、骨髓炎、脑膜炎、败血症、心内膜炎、感染性心内膜炎。
  18. 如权利要12所述的抗菌药物，用于治疗与革兰氏阳性细菌感染相关的疾病的用途。
PCT/CN2021/140984 2021-10-21 2021-12-23 一种bms-833923及其衍生物的应用及药物 WO2023065526A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111226018.X 2021-10-21
CN202111226018.XA CN115998742A (zh) 2021-10-21 2021-10-21 一种bms-833923及其衍生物的应用及药物

Publications (1)

Publication Number Publication Date
WO2023065526A1 true WO2023065526A1 (zh) 2023-04-27

Family

ID=86019678

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/140984 WO2023065526A1 (zh) 2021-10-21 2021-12-23 一种bms-833923及其衍生物的应用及药物

Country Status (2)

Country Link
CN (1) CN115998742A (zh)
WO (1) WO2023065526A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116459240B (zh) * 2023-05-06 2024-03-19 华中科技大学协和深圳医院 Kl-2及其衍生物用于制备抗细菌感染药物中的应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101679308A (zh) * 2007-03-14 2010-03-24 埃克塞里艾克西斯公司 Hedgehog途径抑制剂
US20160168140A1 (en) * 2014-12-12 2016-06-16 Synereca Pharmaceuticals Heterocyclic compounds as antibiotic potentiators
US20190231747A1 (en) * 2015-11-12 2019-08-01 The Trustees Of Columbia University In The City Of New York Rational drug design targeting resistant gram-negative bacterial infections to polymyxin-class antibiotics
WO2021086567A1 (en) * 2019-11-01 2021-05-06 University Of Notre Dame Du Lac Compounds and methods for potentiating colistin activity

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101679308A (zh) * 2007-03-14 2010-03-24 埃克塞里艾克西斯公司 Hedgehog途径抑制剂
US20160168140A1 (en) * 2014-12-12 2016-06-16 Synereca Pharmaceuticals Heterocyclic compounds as antibiotic potentiators
WO2016094730A1 (en) * 2014-12-12 2016-06-16 Synereca Pharmaceuticals, Inc. Heterocyclic compounds as antibiotic potentiators
US20190231747A1 (en) * 2015-11-12 2019-08-01 The Trustees Of Columbia University In The City Of New York Rational drug design targeting resistant gram-negative bacterial infections to polymyxin-class antibiotics
WO2021086567A1 (en) * 2019-11-01 2021-05-06 University Of Notre Dame Du Lac Compounds and methods for potentiating colistin activity

Also Published As

Publication number Publication date
CN115998742A (zh) 2023-04-25

Similar Documents

Publication Publication Date Title
Hay et al. Drug resistance and Acanthamoeba keratitis: the quest for alternative antiprotozoal chemotherapy
Dai et al. Colistin-induced apoptosis of neuroblastoma-2a cells involves the generation of reactive oxygen species, mitochondrial dysfunction, and autophagy
Shi et al. The antimicrobial peptide LI14 combats multidrug-resistant bacterial infections
Koh et al. N-lipidated peptide dimers: effective antibacterial agents against gram-negative pathogens through lipopolysaccharide permeabilization
Kuriyama et al. Antimicrobial chemotherapy: significance to healthcare
WO2023065526A1 (zh) 一种bms-833923及其衍生物的应用及药物
US20230381157A1 (en) Rifabutin for the treatment of acinetobacter baumannii
Amiss et al. Modified horseshoe crab peptides target and kill bacteria inside host cells
Li et al. The combined antibacterial effects of sodium new houttuyfonate and berberine chloride against growing and persistent methicillin-resistant and vancomycin-intermediate Staphylococcus aureus
Celebi et al. Evaluation of antimicrobial and antibiofilm efficacy of bee venom and exosome against Escherichia coli K99 strain
Snyder et al. Antifungal activity of synthetic scorpion venom-derived peptide analogues against Candida albicans
Pengfei et al. L007-0069 kills Staphylococcus aureus in high resistant phenotypes
JP6223444B2 (ja) 蛋白質‐脂質複合体を用いた抗生物質治療の増強
WO2024187962A1 (zh) Su3327在制备降低多黏菌素细胞毒性和肾毒性的药物中的用途
US8372869B2 (en) Process for the treatment of bacterial infections using 2-phenyl-1,2-benzisoselenazol-3(2H)-one 1-oxide
US8796323B2 (en) Defensin-like molecules as novel antimicrobial agents
Rios et al. Repurposing streptomycin and chloramphenicol against bacterial pathogens by combination with diminazene aceturate
Tsutsumi-Arai et al. Fungicidal activity of grapefruit seed extract against the pathogenic Candida species causing oral candidiasis
Zhen et al. Otilonium Bromide Exhibits Potent Antifungal Effects by Blocking Ergosterol Plasma Membrane Localization and Triggering Cytotoxic Autophagy in Candida Albicans
JP6590924B2 (ja) 微生物感染を治療するための相乗的組成物
FR3057463B1 (fr) Association de composes antibiotiques efficace contre des enterobacteries
Anes et al. Reversing antimicrobial resistance in multidrug-resistant Klebsiella pneumoniae of clinical origin using 1-(1-Naphthylmethyl)-Piperazine
KR101649675B1 (ko) 황색포도상구균에 대한 항균 조성물
Chen et al. Synergistic activity of colistin combined with PFK-158 against colistin-resistant and colistin-susceptible Gram-negative bacteria
Chimezie et al. The Synergistic Potentials of Platostoma africanum and Psidium guajavaagainst Some Multi-drug Resistant Bacteria

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21961264

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21961264

Country of ref document: EP

Kind code of ref document: A1