Active ingredient for inhibiting CRKP, medicine and application

Abstract

The invention belongs to the field of medicines, and particularly discloses an active ingredient for inhibiting CRKP, a medicine and application, the active ingredient is composed of a component A and a component B, wherein the component A is polymyxin B and a pharmaceutically acceptable salt of the polymyxin B; the component B is a compound with a structure shown in a formula 1 # imgabs0 # and a pharmaceutically acceptable salt of the compound. Innovative research shows that the combination of the component A and the component B with a special structure can accidentally synergistically improve the inhibition and killing effects of CRKP, especially CKP type CRKP, and can obtain a good antibacterial effect and reduce the drug resistance of CRKP under the condition that the administration dosage of polymyxin B is 0.5 times of MIC (Minimum Inhibitory Concentration).

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the field of CRKP antibacterial drugs. Background Art

[0002] Klebsiella pneumoniae is one of the main pathogenic bacteria causing hospital-acquired infections, often causing pneumonia, bacteremia, and urinary tract infections, ranking second in the clinical isolation rate. Carbapenem antibiotics (such as imipenem and meropenem) are key therapeutic drugs for Klebsiella pneumoniae infections, but their widespread use has led to an increasing detection rate of Carbapenem-resistant Klebsiella pneumoniae (CRKP).

[0003] CRKP infections are common in immunocompromised patients, those who have long-term use of broad-spectrum antibiotics, or those who receive invasive treatments (such as ventilators and catheters). Due to limited available antibacterial drugs, the fatality rate is as high as over 40%, and it significantly increases the length of hospital stay and treatment costs.

[0004] Current treatment regimens rely on antibiotics such as polymyxin B, polymyxin E, tigecycline, and ceftolozane / avibactam. However, when these drugs are used alone to treat CRKP infections, problems such as continuous medication leading to bacterial drug resistance may occur.

[0005] In addition, the most important mechanism of Klebsiella pneumoniae resistance to carbapenem antibacterial drugs is the production of carbapenemases. Currently, the commonly seen carbapenemases in clinical practice are type A (KPC type), type B (NDM, IMP, VIM type), and type D (OXA-48 type). Since there are significant differences in the drug resistance mechanisms of different subtypes of CRKP, treatment methods for a certain subtype are often difficult to directly apply to other subtypes. Currently, only a few antibiotics show partial efficacy and have significant toxicity risks, and there is a lack of an optimized drug combination for KPC-type CRKP. Summary of the Invention

[0006] Aiming at the problems of unsatisfactory antibacterial effect and easy development of drug resistance of CRKP in clinical treatment, the first object of the present invention is to provide an active ingredient for inhibiting CRKP, aiming to provide an active ingredient that can effectively inhibit CRKP, especially KPC-type CRKP.

[0007] The second object of the present invention is to provide the application of the above-mentioned active ingredient in the preparation of a drug for inhibiting CRKP.

[0008] The third object of the present invention is to provide a drug for inhibiting CRKP containing the above-mentioned active ingredient.

[0009] An active ingredient for inhibiting CRKP, which is composed of ingredient A and ingredient B;

[0010] Among them, the component A is polymyxin B and its pharmaceutically acceptable salts;

[0011] The component B is a compound having the structure of Formula 1 and its pharmaceutically acceptable salts;

[0012]

[0013] The innovative research of the present invention shows that combining component A and component B with a special structure can unexpectedly synergistically improve the inhibitory and killing effects on CRKP, especially CKP-type CRKP. A good antibacterial effect can be obtained at a dosing dose of 0.5 times the MIC of polymyxin B, and the drug resistance of CRKP can be reduced.

[0014] In the present invention, for the active ingredient for inhibiting CRKP, the weight ratio of component A to component B is 1:1 to 80; further, it can be 1:30 to 50.

[0015] The present invention also provides an application of the active ingredient in the preparation of a drug for inhibiting CRKP.

[0016] The preferred application of the present invention is the application of the active ingredient in the preparation of a drug for inhibiting KPC-type CRKP, and the KPC-type CRKP is CRKP carrying the blaKPC-2 gene.

[0017] The research of the present invention shows that innovatively combining component A and component B with a special structure can unexpectedly synergistically improve the sensitivity of CKP-type CRKP. Furthermore, a good antibacterial effect can be obtained at a relatively low dosing dose. In addition, the active ingredient of the present invention has good safety and can reduce the drug resistance of CRKP.

[0018] In the present invention, the drug is for treating any indication caused by CRKP infection. For example, as an optional scheme, the drug is a drug for treating at least one indication among respiratory-related pneumonia, sepsis, urinary tract infection, and meningitis caused by CRKP infection.

[0019] In the application of the present invention, the active ingredient is combined with pharmaceutically acceptable excipients to prepare a pharmaceutically acceptable dosage form of the drug.

[0020] The present invention also provides a drug for inhibiting CRKP, which contains a pharmaceutically effective amount of the active ingredient.

[0021] The drug for inhibiting CRKP of the present invention contains pharmaceutically acceptable excipients. The excipients include, for example, excipients, flavoring agents, disintegrants, stabilizers, lubricants, and the like.

[0022] In the present invention, the drug for inhibiting CRKP has a pharmaceutically acceptable dosage form. The dosage form may be, for example, an oral dosage form, an injection dosage form, etc.

[0023] The drug for inhibiting CRKP according to the present invention is a drug for treating KPC-type CRKP infection.

[0024] Beneficial effects

[0025] In the present invention, component A and component B with a special structure are innovatively combined, which can unexpectedly synergistically improve the inhibitory and killing effects on CRKP, especially CKP-type CRKP. Good antibacterial effects can be obtained at a lower dosage, and the long-term drug resistance of CRKP can be reduced. Description of the drawings

[0026] Figure 1 Total-time killing curves of polymyxin B (PMB) combined with formula 1 against two polymyxin B-resistant Klebsiella pneumoniae strains (CRKP5(a) and CRKP215(b)) Detailed implementation manners

[0027] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0028] Example 1: Static time-killing experiment based on a single point

[0029] I. Materials and methods

[0030] 1. Experimental instruments

[0031] AUW120D type one in a hundred thousandth electronic analytical balance (Shimadzu Corporation, Japan);

[0032] Ultra-clean workbench (Fuxia Medical Technology Co., Ltd., Zhejiang);

[0033] Vertical pressure sterilizer (Boxun Biotech Co., Ltd., Shanghai);

[0034] Smart-S ultrapure water machine (Shanghai Hantai Instruments Co., Ltd., Shanghai);

[0035] XW-80A type vortex mixer (Shanghai Huxi Analytical Instrument Factory Co., Ltd., Shanghai);

[0036] KQ3200DE type numerically controlled ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd., Kunshan);

[0037] Infinite200 multi-functional microplate reader (Tecan Group Ltd., Switzerland);

[0038] TGL16 Desktop High-Speed Refrigerated Centrifuge (Changsha Yingtai Instrument Co., Ltd., Changsha);

[0039] SPX-150B-Z Biochemical Incubator (Shanghai Heta Instrument Co., Ltd., Shanghai);

[0040] -80°C Ultra-Low Temperature Refrigerator (Haier Group, Qingdao).

[0041] 2. Experimental Reagents

[0042] Polymyxin B Sulfate, Tigecycline, Minocycline Hydrochloride 9-Amino, Imipenem (Monohydrate), Doripenem (Monohydrate), Amikacin, Fosfomycin Sodium, Dextro-Glucose-6-Phosphate Disodium Salt, Cefepime, Aztreonam were purchased from Shanghai Macklin Biochemical Co., Ltd.;

[0043] Meropenem Hydrochloride Form 1, Trihydrate was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0044] Anhydrous Sodium Carbonate was purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd.;

[0045] Cation Adjusted Minca Agar (CAMH) was purchased from Qingdao Hope Bio-Technology Co., Ltd.;

[0046] Hydrolyzed Casein Tryptone Broth (MH), Technical Agar Powder were purchased from Guangdong Huankai Microbial Sci & Tech Co., Ltd.;

[0047] Physiological Sodium Chloride Solution was purchased from Sichuan Kelun Pharmaceutical Co., Ltd.;

[0048] Ultra-pure water was prepared in the laboratory.

[0049] 3. Preparation of Experimental Drugs

[0050] Preparation of Polymyxin B (PMB) Stock Solution: Weigh 18.84 mg of Polymyxin B (purity ≥ 6000 IU / mg) precisely with an analytical balance into a 20 mL brown sample bottle. Use a pipette to accurately measure 16 mL of sterile injection water and add it to the sample bottle. Vortex until completely dissolved to prepare a Polymyxin B stock solution with a concentration of 1000 mg / L. The stock solution was filtered through a sterile filter with a pore size of 0.22 μm in a laminar flow hood, aliquoted, and stored in a -80°C ultra-low temperature freezer for later use;

[0051] Preparation of Tigecycline (TIG) Stock Solution: Weigh 16.32 mg of Tigecycline (purity 98%) precisely with an analytical balance into a 20 mL brown sample bottle. Use a pipette to accurately measure 16 mL of sterile injection water and add it to the sample bottle. Vortex until completely dissolved to prepare a Tigecycline stock solution with a concentration of 1000 mg / L. The stock solution was filtered through a sterile filter with a pore size of 0.22 μm in a laminar flow hood, aliquoted, and stored in a -80°C ultra-low temperature freezer for later use;

[0052] Preparation of minocycline (MNO) stock solution: Weigh 13.46 mg of minocycline (purity 96%) precisely with an analytical balance into a 50 mL sterile EP tube. Use a pipette to accurately measure 32 mL of sterile injection water and add it to the EP tube. Vortex until completely dissolved to prepare a minocycline stock solution with a concentration of 375 mg / L. The stock solution is filtered through a sterile filter with a pore size of 0.22 μm in a laminar flow hood, aliquoted, and stored in a -80°C ultra-low temperature freezer for later use;

[0053] Preparation of meropenem (MEM) stock solution: Weigh 11.92 mg of meropenem (purity 98%) precisely with an analytical balance into a 20 mL brown sample vial. Use a pipette to accurately measure 8 mL of sterile injection water and add it to the sample vial. Vortex until completely dissolved to prepare a meropenem stock solution with a concentration of 1280 mg / L. The stock solution is filtered through a sterile filter with a pore size of 0.22 μm in a laminar flow hood, aliquoted, and stored in a -80°C ultra-low temperature freezer for later use;

[0054] Preparation of imipenem (IPM) stock solution: Weigh 11.08 mg of imipenem standard (purity 98%) precisely with an analytical balance into a 20 mL brown sample vial. Use a pipette to accurately measure 8 mL of sterile injection water and add it to the sample vial. Vortex until completely dissolved to prepare an imipenem stock solution with a concentration of 1280 mg / L. The stock solution is filtered through a sterile filter with a pore size of 0.22 μm in a laminar flow hood, aliquoted, and stored in a -80°C ultra-low temperature freezer for later use;

[0055] Preparation of doripenem (DOR) stock solution: Weigh 10.95 mg of doripenem (purity 99%) precisely with an analytical balance into a 20 mL brown sample vial. Use a pipette to accurately measure 8 mL of sterile injection water and add it to the sample vial. Vortex until completely dissolved to prepare a doripenem stock solution with a concentration of 1300 mg / L. The stock solution is filtered through a sterile filter with a pore size of 0.22 μm in a laminar flow hood, aliquoted, and stored in a -80°C ultra-low temperature freezer for later use;

[0056] Preparation of amikacin (AMK) stock solution: Weigh 13.06 mg of amikacin (purity 98%) precisely with an analytical balance into a 20 mL brown sample vial. Use a pipette to accurately measure 8 mL of sterile injection water and add it to the sample vial. Vortex until completely dissolved to prepare an amikacin stock solution with a concentration of 1600 mg / L. The stock solution is filtered through a sterile filter with a pore size of 0.22 μm in a laminar flow hood, aliquoted, and stored in a -80°C ultra-low temperature freezer for later use;

[0057] Preparation of fosfomycin (FOS) stock solution: Weigh 76.31 mg of fosfomycin (purity 97.5%) precisely with an analytical balance into a 20 mL brown sample bottle. Use a pipette to accurately measure 8 mL of sterile injection water and add it to the sample bottle. Vortex until completely dissolved to prepare a fosfomycin stock solution with a concentration of 9300 mg / L. The stock solution is filtered through a sterile filter with a pore size of 0.22 μm in a laminar flow hood, aliquoted, and stored in a -80°C ultra-low temperature freezer for later use;

[0058] Preparation of aztreonam (ATM) stock solution: Weigh 19.60 mg of aztreonam (purity 98%) precisely with an analytical balance into a 20 mL brown sample bottle. Use a pipette to accurately measure 8 mL of sterile injection water and add it to the sample bottle. Vortex until completely dissolved to prepare an aztreonam stock solution with a concentration of 2400 mg / L. The stock solution is filtered through a sterile filter with a pore size of 0.22 μm in a laminar flow hood, aliquoted, and stored in a -80°C ultra-low temperature freezer for later use;

[0059] Preparation of stock solution of Formula 1: Weigh 19.09 mg of Formula 1 (purity 95%) precisely with an analytical balance into a 20 mL brown sample bottle. Add an appropriate amount of sodium carbonate solution. Use a pipette to accurately measure 10 mL of sterile injection water and add it to the sample bottle. Vortex until completely dissolved to prepare a stock solution of Formula 1 with a concentration of 2000 mg / L. The stock solution is filtered through a sterile filter with a pore size of 0.22 μm in a laminar flow hood, aliquoted, and stored in a -80°C ultra-low temperature freezer for later use;

[0060] Preparation of cefepime (FEP) stock solution: Weigh 16.33 mg of cefepime (purity 98%) precisely with an analytical balance into a 20 mL brown sample bottle. Use a pipette to accurately measure 8 mL of sterile injection water and add it to the sample bottle. Vortex until completely dissolved to prepare a cefepime stock solution with a concentration of 2000 mg / L. The stock solution is filtered through a sterile filter with a pore size of 0.22 μm in a laminar flow hood, aliquoted, and stored in a -80°C ultra-low temperature freezer for later use;

[0061] Preparation of glucose-6-phosphate disodium (G-6-P) stock solution: Weigh 20.41 mg of glucose-6-phosphate disodium (purity 98%) precisely with an analytical balance into a 20 mL brown sample bottle. Use a pipette to accurately measure 8 mL of sterile injection water and add it to the sample bottle. Vortex until completely dissolved to prepare a glucose-6-phosphate disodium stock solution with a concentration of 2500 mg / L. The stock solution is filtered through a sterile filter with a pore size of 0.22 μm in a laminar flow hood, aliquoted, and stored in a -80°C ultra-low temperature freezer for later use.

[0062] 4. Preparation of bacterial culture medium

[0063] Prepare the cation - adjusted broth medium required for bacterial resuscitation, culture, and growth in real - time according to the proportion of adding 21 g of broth dry powder medium to 1 L of ultrapure water. The prepared medium is placed in a blue - capped bottle and then autoclaved at 115 °C for 30 min. After sterilization, place the CAMH in the laminar flow hood and wait until it has completely cooled before using it for bacterial culture.

[0064] 5. Experimental strains and their preservation and resuscitation

[0065] 5.1 Experimental strains: The CRKP strains used in this experiment were derived from clinical specimens of patients in the Second Xiangya Hospital. After genotype and phenotype identification, 10 CRKP strains carrying the blaKPC - 2 gene were screened out, showing resistance to β - lactam antibiotics. The gene detection and phenotype detection results of these 10 CRKP strains are shown in Table 1.

[0066] Table 1 Gene detection and phenotype detection results of 10 CRKP strains

[0067]

[0068]

[0069] Among these 10 Klebsiella pneumoniae strains, the distribution width of the MIC values for polymyxin B was from 1 mg / L to 16 mg / L, and the corresponding MIC values are shown in Table 2. It was thus judged that 4 strains were resistant to polymyxin B (MIC≥4 mg / L), 4 strains were intermediate - sensitive to polymyxin B (4 mg / L > MIC≥2 mg / L), and 2 strains were sensitive to polymyxin B (MIC < 2 mg / L).

[0070] Table 2 MIC value results of 10 CRKP strains

[0071]

[0072] 5.2 Preservation and resuscitation of experimental strains

[0073] The experimental method used glycerol cryopreservation to preserve the bacterial strains. After thawing the glycerol - cryopreserved bacteria in a 37 °C oven for 30 min, inoculate the bacteria onto an MH agar plate under the working conditions of a laminar flow hood using a sterile inoculation loop, and then place the plate in a 37 °C constant - temperature incubator and incubate for about 18 hours.

[0074] Preparation of MH agar: Prepare an appropriate amount of MH agar medium according to the ratio of adding 21 g of hydrolyzed casein peptone broth dry powder medium and 15 g of agar powder to 1 L of ultrapure water. After autoclaving at 115 °C for 30 min, cool it until it is not hot to the touch. After manual homogenization, pour about 15 mL of MH agar medium into a sterile petri dish, spread it evenly without bubbles, and then cover it and let it cool. Prepare three plates in parallel for each concentration gradient to ensure the reliability of the experimental results.

[0075] 6. Evaluation of bactericidal effects of different combination drug therapies based on single points

[0076] Refer to the American CLSI antimicrobial susceptibility testing performance standards and determine the static time bactericidal effects of the experimental CRKP strains at 24 h under different drug combinations according to the following steps.

[0077] 6.1 Preparation of different antimicrobial CAMH broths:

[0078] Take 10 mL of CAMH broth into a sterilized test tube respectively, add the antimicrobial stock solution according to Table 3, and add the antimicrobial drugs into the test tubes containing 10 mL of CAMH broth respectively. The prepared solutions are used to draw the time-kill curves of single drugs. In addition, prepare polymyxin B + tigecycline, polymyxin B + minocycline, polymyxin B + meropenem, polymyxin B + imipenem, polymyxin B + doripenem, polymyxin B + amikacin, polymyxin B + fosfomycin, polymyxin B + aztreonam, polymyxin B + formula 1, and polymyxin B + cefepime solutions by mixing the polymyxin B stock solution and other stock solutions, and add them into the test tubes containing 10 mL of CAMH broth respectively. The prepared solutions are used to draw the time-kill curves of combination drug therapies. For the experiments containing fosfomycin, add 25 mg / L G-6-P to the broth. The final concentration of each antimicrobial drug in the system is the peak free plasma drug concentration that can be achieved at the maximum clinical dosing dose.

[0079] Table 3 Preparation of different antimicrobial CAMH broths for time-kill curves

[0080]

[0081] 6.2 Preparation of bacterial suspensions:

[0082] Resuscitate and culture the CRKP strains according to the above operations. Pick a single colony and use 0.5 mL of sterile CAMH broth medium to dilute it to a concentration of about 5×10 8 CFU / mL. After mixing the prepared mixture evenly, place it in an incubator at 37 °C for 24 h. Take 1 tube of 10 mL of sterile CAMH broth from each group and add 10 μL of the bacterial solution as the growth group for control.

[0083] 6.3 Inoculation of bacterial suspensions and colony counting:

[0084] Mix the above-prepared mixture evenly and place it in an incubator at 37°C. Take out the test tubes at 0 h and 24 h, pipette 500 μL of the bacterial liquid into a sterile EP tube, centrifuge at 15000 rpm for 10 min, aspirate the supernatant, and then add an equal dose of normal saline for reconstitution. The treated samples are serially diluted 10-fold with normal saline, and 100 μL of the sample solution at an appropriate dilution is evenly spread onto the surface of blank agar. Three plates are prepared in parallel for each gradient. The agar plates after spreading are placed in an incubator at 37°C for 18 - 24 h and then counted.

[0085] 6.4 Evaluation of bactericidal effect and combined effect:

[0086] The bactericidal effect is that at 24 h, the bacterial liquid concentration decreases by ≥ 3 log 10 CFU / mL compared with the initial bacterial liquid concentration; the bacteriostatic effect is that at 24 h, the bacterial liquid concentration decreases by < 3 log 10 CFU / mL but ≥ 2 log 10 CFU / mL.

[0087] The synergistic effect is that at 24 h, when comparing the bacterial liquid concentration of the combined group with that of the single drug group with the best bactericidal effect, the reduction in the number of colonies is ≥ 2 log 10 CFU / mL; the irrelevant effect is that at 24 h, when comparing the bacterial liquid concentration of the combined group with that of the single drug group with the best bactericidal effect, the reduction in the number of colonies is ≤ 2 log 10 CFU / mL; the antagonistic effect is that at 24 h, when comparing the bacterial liquid concentration of the combined group with that of the single drug group with the best bactericidal effect, the increase in the number of colonies is ≥ 2 log 10 CFU / mL.

[0088] II. Evaluation results of the bactericidal effects of different combined drug regimens at a single time point

[0089] The results of the 24-hour static time bactericidal experiments of polymyxin B and the other 10 antibacterial drugs alone and in combination against 10 strains of CRKP are shown in Tables 4 to 13. CFU t refers to the average concentration of CRKP within t hours. When all antibacterial drugs are used alone, except that the bacterial liquid concentrations of two strains, CRKP2404 and CRKP5095, decrease by 2 - 3 log 10 CFU / mL under the action of tigecycline and doripenem respectively, showing a bacteriostatic effect, the bacterial liquid concentrations in other groups increase compared with the initial bacterial liquid concentration at 24 h. Therefore, when these antibacterial drugs are used alone, there is no bactericidal or bacteriostatic effect on most strains.

[0090] Table 4 Δlog 10 CFU 0-24 values of polymyxin B and tigecycline alone or in combination

[0091]

[0092]

[0093] Table 5 Δlog of polymyxin B and minocycline used alone or in combination 10 CFU 0-24 value

[0094]

[0095] Table 6 Δlog of polymyxin B and meropenem used alone or in combination 10 CFU 0-24 value

[0096]

[0097] Table 7 Δlog of polymyxin B and imipenem used alone or in combination 10 CFU 0-24 value

[0098]

[0099]

[0100] Table 8 Δlog of polymyxin B and doripenem used alone or in combination 10 CFU 0-24 value

[0101]

[0102] Table 9 Δlog of polymyxin B and amikacin used alone or in combination 10 CFU 0-24 value

[0103]

[0104] Table 10 Δlog of polymyxin B and fosfomycin used alone or in combination 10 CFU 0-24 value

[0105]

[0106]

[0107] Table 11 Δlog of polymyxin B and aztreonam used alone or in combination 10 CFU 0-24 value

[0108]

[0109] Table 12 Polymyxin B and Formula 1 Monotherapy or Combination Therapy Δlog 10 CFU 0-24 value

[0110]

[0111] Table 13 Polymyxin B and Cefepime Monotherapy or Combination Therapy Δlog 10 CFU 0-24 value

[0112]

[0113]

[0114] In summary, the combination of polymyxin B and Formula 1 can achieve a better inhibitory effect on KPC-type CRKP compared with the combination of other components.

[0115] Example 2: Time-Kill Curve Experiment Based on the Whole Time Period

[0116] I. Research Method:

[0117] Referring to the American CLSI Antimicrobial Susceptibility Testing Performance Standards, the whole-time period time-kill curve experiment of the drug combination of polymyxin B and Formula 1 against 2 drug-resistant strains (CRKP215 and CRKP5) with an MIC value of 4 mg / L was carried out according to the following steps.

[0118] 1. Preparation of CAMH broth with different drug combinations: Take 15 mL of CAMH broth in a sterilized test tube. Add 1 mL of sterile water to the blank group, 1 mL of polymyxin B solution with a concentration of 32 μg / mL to the monotherapy group, and 0.5 mL of polymyxin B solution with a concentration of 64 μg / mL and 0.5 mL of Formula 1 solution with a concentration of 3200 μg / mL to the combination therapy group. The final concentration of polymyxin B in the system is 0.5×MIC.

[0119] 2. Preparation of bacterial suspension: Resuscitate and culture strains CRKP5 and CRKP215 according to the above operation. Pick a single colony into 0.5 mL of sterile CAMH broth medium and dilute it to a concentration of about 5×10 8 CFU / mL, and then take 16 μL and inoculate it into the above 16 mL of CAMH broth containing drugs to make the final concentration about 5×10 5 CFU / mL.

[0120] 3. Inoculation of bacterial suspension and colony counting: Mix the prepared mixture well and place it in an incubator at 37°C. Take out the test tube at 0 h, 2 h, 4 h, 8 h, 10 h, and 24 h. The subsequent sample treatment is the same as that in "5.3 Inoculation of bacterial suspension and colony counting in Example 1".

[0121] 4. Plotting the time-killing curve: The concentration of the original bacterial suspension = the number of colony-forming units × dilution factor × 10 CFU / mL. Taking time as the abscissa and the logarithm of the number of colonies as the ordinate, plot the killing curve.

[0122] II. Evaluation results of the bactericidal effect of the combined drug combinations throughout the whole period

[0123] As Figure 1 shown, under the condition of 0.5-fold MIC of polymyxin B, polymyxin B alone has no bactericidal effect on 2 strains of CRKP. Within the initial 2 - 4 h, the bactericidal effect of polymyxin B on 2 strains of CRKP exceeds 3 log 10 CFU / mL. After that, CRKP develops rapid drug resistance, and the growth curve shows initial inhibition followed by a significant increase until it is similar to the growth curve of the control group. Meanwhile, the bactericidal effect of the combination of polymyxin B and Formula 1 within 0 - 4 h is similar to that of polymyxin B alone.

[0124] However, the regeneration of the drug-resistant subpopulation of all strains in the group of the combination of polymyxin B and Formula 1 is significantly inhibited. After CRKP215 is killed, there is no regeneration phenomenon. CRKP5 has slight regeneration after 10 h, but the number of bacteria does not exceed 2 log 10 CFU / mL within 24 h, showing a significant bactericidal effect. For CRKP5 and CRKP215, the Δlog 10 CFU 0-24 are -3.34 and -5.72 respectively, and the combination of polymyxin B and Formula 1 shows a significant synergistic bactericidal effect.

Claims

1. An active ingredient for inhibiting CRKP, characterized in that, Composed of component A and component B; Among them, the said component A is polymyxin B and its pharmaceutically acceptable salts; The said component B is a compound having the structure of formula 1 and its pharmaceutically acceptable salts; 2. The active ingredient for inhibiting CRKP according to claim 1, wherein The weight ratio of the said component A to component B is 1:1 to 80.

3. Use of the active ingredient according to any one of claims 1 to 2 in the preparation of a drug for inhibiting CRKP.

4. The application according to claim 3, characterized in that Use of the said active ingredient in the preparation of a drug for inhibiting KPC-type CRKP, and the said KPC-type CRKP is CRKP carrying the blaKPC-2 gene.

5. The application according to claim 3 or 4, characterized in that, The said drug is a drug for treating at least one indication among respiratory-related pneumonia, sepsis, urinary tract infection, and meningitis caused by CRKP infection.

6. The application according to any one of claims 3 to 5, characterized in that Combining the said active ingredient with pharmaceutically acceptable excipients to prepare a drug in a pharmaceutically acceptable dosage form.

7. A drug for inhibiting CRKP, characterized in that, Containing a pharmaceutically effective amount of the active ingredient according to any one of claims 1 to 2.

8. The drug for inhibiting CRKP according to claim 7, wherein Containing pharmaceutically acceptable excipients.

9. The drug for inhibiting CRKP according to claim 7, wherein, Having a pharmaceutically acceptable dosage form.

10. The drug for inhibiting CRKP according to any one of claims 7 to 9, characterized in that, It is a drug for treating KPC-type CRKP infection.

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