Application of composition containing polymyxin in resisting klebsiella pneumoniae

Abstract

The invention discloses an application of a composition containing polymyxin in resisting klebsiella pneumoniae. The pharmaceutical composition provided by the invention comprises an active ingredient and an antibiotic synergist, the active ingredient is polymyxin, and the antibiotic synergist is HM43239 or a pharmaceutically acceptable salt thereof. The pharmaceutical composition not only is effective to polymyxin-sensitive klebsiella pneumoniae, but also has an obvious inhibition effect on polymyxin-resistant clinical strain klebsiella pneumoniae, and meanwhile, has a protection effect on a klebsiella pneumoniae mouse infection model; meanwhile, the antibacterial dosage of the polymyxin is low, and the safety is improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a composition containing the antibiotic polymyxin and its adjuvant HM43239 and the application of the composition in the treatment of Klebsiella pneumoniae infection. Background Technology

[0002] Infections caused by Klebsiella pneumoniae have become a serious threat to public health. The World Health Organization (WHO) has listed carbapenem-resistant Enterobacteriaceae, including Klebsiella pneumoniae, as the most dangerous pathogens in urgent need of new treatments. Due to the lack of new antibiotics, the Infectious Diseases Society of America (IDSA) and the Centers for Disease Control and Prevention (CDC) have identified multidrug-resistant Klebsiella pneumoniae as the most important pathogen. Since the discovery of penicillin in 1929, more than 20 different classes of antibiotics have been discovered in the following 30 years. However, despite technological and economic advancements, only six new classes of antibiotics have been approved in the past 20 years, none of which are specifically targeting Klebsiella pneumoniae.

[0003] The slowdown in the discovery of new antibiotics, coupled with resistance mechanisms such as the evolution and horizontal transfer of multidrug-resistant bacteria, has resulted in a limited number of antibiotics available clinically. Antibiotic potentiators, substances that possess little or no antibacterial activity but can significantly enhance the activity of existing antibiotics, have attracted widespread attention in recent years. Because antibiotic potentiators can restore the activity of antibiotics that have developed resistance, they are of great significance in alleviating the resistance problem. Furthermore, the development of antibiotic potentiators can serve as an effective complementary strategy to the research and development of new antibiotics, compensating for their low success rate.

[0004] Polymyxins are cationic peptide antibiotics that electrostatically bind to negatively charged lipids A on the bacterial outer membrane (OM), thereby disrupting the membrane. In recent years, due to the increasing prevalence of multidrug-resistant Klebsiella pneumoniae, polymyxins have gradually become a "last line of defense" against multidrug-resistant Gram-negative bacteria. Therefore, polymyxins are considered a promising target drug for developing new antibiotic adjuvants.

[0005] HM43239 is an orally active selective FLT3 inhibitor. As a reversible type I inhibitor, HM43239 directly inhibits FLT3 kinase activity and regulates p-STAT5, p-ERK SYK, JAK1 / 2, and TAK1. HM43239 inhibits the proliferation of leukemia cells and induces apoptosis. However, there are no reports of direct inhibition or combined bactericidal effects against bacteria. Summary of the Invention

[0006] The purpose of this invention is to provide a pharmaceutical composition comprising polymyxin and HM43239, which is effective not only against polymyxin-sensitive Klebsiella pneumoniae but also has a significant inhibitory effect on polymyxin-resistant Klebsiella pneumoniae. Furthermore, it reduces the dosage of polymyxin, greatly reducing the occurrence of toxic side effects such as nephrotoxicity, and has significant clinical significance.

[0007] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a pharmaceutical composition comprising an active ingredient and an antibiotic potentiator.

[0008] The active ingredient is polymyxin.

[0009] The antibiotic potentiator is HM43239 or a pharmaceutically acceptable salt thereof.

[0010] The chemical formula of HM43239 is C 29 H 33 ClN6, PubChem CID number 135390910, has the following structural formula:

[0011] In this invention, the active ingredient and the antibiotic synergist can be used in combination or formulated into a mixed preparation.

[0012] The polymyxin is selected from polymyxin B and / or polymyxin E.

[0013] The mass ratio of the polymyxin to the HM43239 is 1:(0.05-1613), preferably 1:(5-50), and more preferably 1:50.

[0014] In a second aspect, the present invention provides a pharmaceutical preparation comprising the aforementioned pharmaceutical composition.

[0015] Preferably, the pharmaceutical composition described in this invention can be prepared into injectable formulations, oral formulations, tablets, suspensions, or capsules using conventional techniques in the art. This invention preferably prepares the pharmaceutical composition described in this invention into injectable formulations.

[0016] Depending on the formulation, the content of the pharmaceutical composition in the formulation, expressed as a mass fraction, can be 1-99%, preferably 5-90%; the excipients used in the formulation can be conventional excipients in the art, provided that they do not affect the efficacy of the drug of the present invention or do not react with the pharmaceutical composition of the present invention; the preparation method of the formulation can be a conventional preparation method in the art.

[0017] According to an embodiment of the present invention, the concentration of polymyxin in the pharmaceutical preparation is 0.031–8 µg / mL; According to an embodiment of the present invention, the concentration of HM43239 in the pharmaceutical preparation is 0.391 to 50 µg / mL.

[0018] Thirdly, the present invention provides the use of the pharmaceutical composition or pharmaceutical preparation described herein in the preparation of a product for treating diseases caused by Klebsiella pneumoniae in subjects.

[0019] The Klebsiella pneumoniae strain is selected from one or more of the following strains: polymyxin-sensitive Klebsiella pneumoniae and polymyxin-resistant Klebsiella pneumoniae.

[0020] Preferably, the polymyxin-sensitive Klebsiella pneumoniae is selected from ATCC13883.

[0021] Preferably, the polymyxin-resistant Klebsiella pneumoniae is selected from one or more of the following strains: KP38218, AH12, and KP38206.

[0022] The disease is selected from one or more of the following groups: Klebsiella pneumoniae pneumonia, Klebsiella pneumoniae bloodstream infection, Klebsiella pneumoniae intraperitoneal infection, and Klebsiella pneumoniae urinary tract infection.

[0023] The subjects were mammals; The dosage of the pharmaceutical composition in this invention can be appropriately varied depending on the target population, route of administration, or formulation of the drug, but the premise is to ensure that the pharmaceutical composition can achieve an effective blood drug concentration in mammals.

[0024] Compared with the prior art, the beneficial effects achieved by the present invention are: 1. The pharmaceutical composition of the present invention has a significant positive effect in treating Klebsiella pneumoniae infection. It is effective not only against polymyxin-sensitive Klebsiella pneumoniae, but also has a significant inhibitory effect on polymyxin-resistant clinical strains of Klebsiella pneumoniae. In particular, the synergistic therapeutic effect is best when the molar concentration ratio of polymyxin to HM43239 is 1:50.

[0025] 2. Compared with existing drugs for treating Klebsiella pneumoniae infection, the drug composition provided by this invention can significantly reduce the dosage of polymyxin and improve safety.

[0026] 3. In addition, the pharmaceutical composition provided by the present invention also has a protective effect against a mouse model of Klebsiella pneumoniae infection. Attached Figure Description

[0027] Figure 1 This is a bacterial pyrogram of the Klebsiella pneumoniae standard strain ATCC13883, which corresponds to polymyxin B.

[0028] Figure 2 This is a bacterial thermogram of KP38218, a clinically resistant strain of Klebsiella pneumoniae corresponding to polymyxin B.

[0029] Figure 3 This is a bacterial thermogram of AH12, a clinically resistant strain of Klebsiella pneumoniae corresponding to polymyxin B.

[0030] Figure 4 This is a bacterial thermogram of KP38206, a clinically resistant strain of Klebsiella pneumoniae corresponding to polymyxin B.

[0031] Figure 5 This is a bacterial pyrogram of the Klebsiella pneumoniae standard strain ATCC13883 corresponding to polymyxin E.

[0032] Figure 6 This is a bacterial thermogram of Kp38218, a clinically resistant strain of Klebsiella pneumoniae corresponding to polymyxin E.

[0033] Figure 7 This is a bacterial thermogram of AH12, a clinically resistant strain of Klebsiella pneumoniae corresponding to polymyxin E.

[0034] Figure 8 This is a bacterial thermogram of KP38206, a clinically resistant strain of Klebsiella pneumoniae corresponding to polymyxin E.

[0035] Figure 9 This is a graph showing the thermal stability of histones for a single drug.

[0036] Figure 10 This is a comparison of the survival rates of female mice treated with a combination of HM43239 and polymyxin B.

[0037] In the above diagram, Polymyxin B and PB refer to polymyxin B, and Colistin refers to polymyxin E. Detailed Implementation

[0038] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

[0039] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0040] Unless otherwise specified, all reagents, materials, instruments, etc. used in the following examples are commercially available.

[0041] In the following embodiments, the standard strains involved were obtained from the American Type Culture Collection Center (ATCC).

[0042] In the following embodiments, the clinically resistant strains involved were obtained from the Microbiology Laboratory of the Department of Laboratory Medicine, Shenzhen People's Hospital.

[0043] The public can obtain the above-mentioned standard strains and drug-resistant strains through public channels.

[0044] In the following examples, LB broth culture medium was prepared as follows: 8g of Tryptone Powder, 8g of NaCl, and 4g of YEAST EXTRACT were weighed and dissolved in 800 mL of distilled water, autoclaved at 121°C for 15 minutes, and stored at 4°C.

[0045] In the following examples, the PBS buffer was prepared as follows: 8g of NaCl, 0.2g of KCl, 3.58g of Na2HPO4·12H2O and 0.24g of KH2PO4 were weighed and dissolved in 1000mL of distilled water, autoclaved at 121°C for 15 minutes, and stored at room temperature.

[0046] In the following embodiments, the HM43239 and polymyxin involved were purchased from Shanghai Taosu Biochemical Technology Co., Ltd.

[0047] Example 1: Preparation of the pharmaceutical composition Formula 1: Polymyxin B and HM43239 are mixed at a mass ratio of 1:5.

[0048] Formula 2: Polymyxin B and HM43239 are prepared at a mass ratio of 1:25.

[0049] Formula 3: Polymyxin B and HM43239 are prepared at a mass ratio of 1:50.

[0050] The aforementioned polymyxin B can be replaced with polymyxin E.

[0051] Effect test: 1. The combined antibacterial effect of HM43239 and polymyxin In this experiment, the standard strain of Klebsiella pneumoniae ATCC13883, the clinically resistant strain of Klebsiella pneumoniae AH12, the clinically resistant strain of Klebsiella pneumoniae KP38218, and the clinically resistant strain of Klebsiella pneumoniae KP38206 were cultured at 37°C and 220 rpm with shaking until OD600 = 0.6-0.8. The combined effect of HM43239 with polymyxin B or polymyxin E was determined by checkerboard experiment to further illustrate the present invention.

[0052] The specific steps of the chessboard experiment are as follows: A) Adjust the density of bacterial cultures with OD600 = 0.6 to 0.8 to OD600 = 0.001 respectively; B) Serially dilute polymyxin by 2 times along the horizontal axis and serially dilute HM43239 by 2 times along the vertical axis; C) Set up a blank group (with LB broth medium) and a control group (with LB broth medium and bacterial culture). D) Control the final volume of each well in the 96-well plate to 100 µL. After incubation for 18 h, add 10 µL MTT to each well and incubate at 37°C in the dark for 30 min. Measure the absorbance at 600 nm using a microplate reader. Calculate the bacterial inhibition rate (%) using the formula: 1 - (OD experimental group - OD blank) / (OD control group - OD blank) × 100%. Plot a bacterial calorimeter (see results). Figure 1-8 (As shown); the MIC values ​​of HM43239 and polymyxin used alone and the graded inhibitory concentrations (FIC index) of the combined use of the two were calculated based on the bacterial inhibition rate.

[0053] The MIC value is defined as the minimum concentration of a compound that completely inhibits bacterial growth. FIC = MICab / MICa + MICba / MICb = FICa + FICb, where MICab is the MIC value of HM43239 bound to polymyxin, MICba is the MIC value of polymyxin bound to HM43239, MICa or MICb is the MIC value of HM43239 or polymyxin, and FICa or FICb is the FIC value of HM43239 or polymyxin.

[0054] The results showed that FIC < 0.5, indicating that the combination of polymyxin and compound HM43239 had a good synergistic effect in inhibiting bacterial growth. Furthermore, heatmap data showed that the presence of HM43239 reduced the MIC of polymyxin to 1 / 256-1 / 8 of its original value.

[0055] 2. Mechanism study of HM43239 enhancing polymyxin B activity in Klebsiella pneumoniae In this experiment, the mechanism by which HM43239 enhances polymyxin B activity in Klebsiella pneumoniae was explored through thermal proteomics analysis, in order to further illustrate the present invention.

[0056] The specific steps are as follows: 1) Inoculate the standard strain of Klebsiella pneumoniae (ATCC13883) at a volume ratio of 1:100 into 80 ml of fresh LB broth and incubate at 37°C with shaking at 220 rpm until OD (outlet volume) is reached. 600 =0.5.

[0057] 2) Preparation of the control group: Take 40 ml of OD 600Add an appropriate amount of DMSO to the bacterial culture at a concentration of 0.5, incubate at 37°C for 10 min; centrifuge at 4000g for 5 min, then wash with 20 ml of PBS (containing the same concentration of DMSO); resuspend in PBS solution containing the drug (HM43239), allowing OD to... 600 It is 10.

[0058] 3) Preparation of single-drug groups: Take 40 ml of OD 600 Add an appropriate amount of HM43239 to a bacterial culture with a concentration of 0.5, incubate at 37°C for 10 min; centrifuge at 4000g for 5 min, then wash with 20 ml of PBS (containing the same concentration of HM43239); resuspend in PBS solution containing the drug (HM43239), and allow OD to... 600 It is 10.

[0059] 4) Gradient temperature heating treatment: Add 100ul of bacterial solution of the above density to each well of the PCR plate, centrifuge the plate at 4000g for 5min, discard 80ul of the supernatant; then start the heating experiment, heat for 3min, and immediately return to room temperature for 3min.

[0060] The specific steps of gradient temperature heating treatment are as follows: A) Heating PCR tubes at 16 temperature points (37°C, 40°C, 43°C, 46°C, 49°C, 52°C, 55°C, 58°C, 61°C, 64°C, 67°C, 70°C, 73°C, 76°C, 80°C, 83°C) for 3 minutes each. Immediately after heating, remove the tubes and incubate at room temperature for 3 minutes. After 3 minutes, proceed with the sample quick-freezing step.

[0061] B) Lysis: The lysis buffer was lysed with 30 μL of lysis solution (final concentration: 50 μg / ml lysozyme, 0.8% NP-40, 1× protease inhibitor (Roche), 250 U / ml benzonase, and 1 mM MgCl2 in PBS) at room temperature for 20 min with shaking. The sample was then rapidly frozen in liquid nitrogen. Three freeze-thaw cycles were then performed (liquid nitrogen, followed by 1 min vortexing on a PCR instrument at 25°C). Finally, the plate was centrifuged at 2000g for 5 min to remove debris. The supernatant was filtered through a 0.45 μm 96-well filter plate (Millipore, catalog number: MSHVN4550) and centrifuged at 500g for 5 min to remove protein polymers. The filtered solution was mixed 1:1 with the 2× sample solution (180 mM Tris, pH 6.8, 4% SDS, 20% glycerol) until enzyme digestion.

[0062] C) Enzymatic desalting: Add an appropriate amount of protein to a final concentration of 5 mM DTT, incubate at 37°C for 1 h, then return to room temperature; add 10 mM iodoacetamide, incubate at room temperature in the dark for 45 min. Dilute the sample 4-fold with 25 mM ammonium bicarbonate, add trypsin at a protein-to-trypsin ratio of 50:1, and incubate at 37°C overnight; the next day, add formic acid to adjust the pH to less than 3 to terminate the digestion.

[0063] D) Labeled samples: Based on the peptide quantification results, take 5 μL of each sample for TMT labeling for 1 h; add ammonia to terminate the reaction; mix the labeled samples, vortex, centrifuge to the bottom of the tube; freeze-dry under vacuum.

[0064] E) Fractionation: Add 1 μL of 5% ammonia to each sample to terminate the fractionation; after merging the samples, heat dry the samples and perform RP fractionation; elute with 10 gradient sequences: 6% (940 μL ammonia, 60 μL acetonitrile), 9%, 12%, 15%, 18%, 21%, 25%, 30%, 35%, 50%; elute with 90 μL for the six gradients: 6%, 9%, 12%, 15%, 18%, 21%; elute with 85 μL for the four gradients: 25% (merged into 6% tubes), 30% (9%), 35% (12%), 50% (21%); finally, heat dry the samples and store them at -20°C until they are used in the instrument.

[0065] F) Mass Spectrometry Setup: Prepare mobile phases A (100% water, 0.1% formic acid) and B (80% acetonitrile, 0.1% formic acid); dissolve the lyophilized powder in 10 µL of mobile phase A, centrifuge at 14000 g for 20 min at 4°C, and inject 1 µg of the supernatant as sample for LC-MS analysis; LC elution conditions are shown in Table 1; use an ORBITRAP ECLIPSE mass spectrometer with the FAIMS Pro™ Interface selected, the compensation voltage CV switching between -45 and -65 every 1 second, a Nanospray Flex™ (NSI) ion source, setting the ion spray voltage to 2.0 kV, the ion transfer tube temperature to 320°C, and data-dependent acquisition mode for mass spectrometry, with a full scan range of m / z 350-1500 and a first-order mass spectrometry resolution of 120000 (200 The mass spectrometry (MS / z) resolution was set to 30000 (200 m / z), the AGC was 4×10⁵, and the maximum C-trap injection time was 50 ms. The secondary mass spectrometry detection was performed in "Top Speed" mode, with the secondary mass spectrometry resolution set to 30000 (200 m / z), the AGC to 5×10⁴, the maximum injection time to 54 ms, and the peptide fragmentation collision energy to 36%. Raw mass spectrometry detection data (.raw) was generated.

[0066] Table 1 Separating Gradients

[0067] Through data processing and bioinformatics analysis, the overall protein expression changes in the control group and the single-drug group were studied.

[0068] The results are as follows Figure 9 As shown, red indicates proteins with increased thermal stability, green indicates proteins with decreased thermal stability, and blue indicates proteins with no change in thermal stability. Compared with the control group, 793 proteins in the single-drug group showed increased thermal stability, and 428 proteins showed decreased thermal stability (thermal melting point shift greater than 1°C). Among them, LpxL_1 and NagA are likely associated with bacterial resistance to polymyxins. Therefore, HM43239 may enhance the activity of polymyxin B in Klebsiella pneumoniae through LpxL_1 and NagA.

[0069] 3. The combined medication has a protective effect against Klebsiella pneumoniae infection in a mouse model. In this embodiment, HM43239 and polymyxin B are combined to form a pharmaceutical composition and administered in combination to a mouse infection model to further illustrate the present invention.

[0070] The specific steps are as follows: 1) Establishment of a pulmonary infection model: Sixty 6-8 week old female BALB / c mice (approximately 20 g in weight) were injected into their lungs with a lethal dose of clinical polymyxin B-resistant Klebsiella pneumoniae strain SZ206 to establish an infection model. The concentration of clinical polymyxin B-resistant Klebsiella pneumoniae strain KP38206 was 1.0 × 10⁻⁶. 8 CFUs / each; 2) Group treatment: Female mice were randomly divided into six groups (n = 8 mice / group) and treated with intraperitoneal injection; the survival rate of female mice was observed for 7 consecutive days. The study included six groups: a control group (injected with PBS), treatment group I (injected with 0.2 mg / kg polymyxin B alone), treatment group II (injected with 10 mg / kg HM43239 alone), treatment group III (injected with a mixture of 0.2 mg / kg polymyxin B and 1 mg / kg HM43239, in a mass ratio of 1:5), treatment group IV (injected with a mixture of 0.2 mg / kg polymyxin B and 5 mg / kg HM43239, in a mass ratio of 1:25), and treatment group V (injected with a mixture of 0.2 mg / kg polymyxin B and 10 mg / kg HM43239, in a mass ratio of 1:50).

[0071] The results are as follows Figure 10As shown, female mice (survival rate 87.5%) had a better survival rate within 7 days after treatment with polymyxin B (0.2 mg / kg) in combination with HM43239 (10 mg / kg) than the control group treated with polymyxin B alone (survival rate 25%) or HM43239 alone (survival rate 25%), indicating that the combination of HM43239 and polymyxin B significantly improved the survival rate of mice.

[0072] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A pharmaceutical composition comprising an active ingredient and an antibiotic potentiator; The active ingredient is polymyxin; The antibiotic potentiator is HM43239 or a pharmaceutically acceptable salt thereof; The chemical formula of HM43239 is C 29 H 33 ClN6, PubChem CID number 135390910, has the following structural formula: 。 2. The pharmaceutical composition according to claim 1, characterized in that, The polymyxin is selected from polymyxin B and / or polymyxin E.

3. The pharmaceutical composition according to any one of claims 1 or 2, characterized in that, The mass ratio of the polymyxin to the HM43239 is 1:(0.05-1613).

4. A pharmaceutical preparation comprising the pharmaceutical composition according to any one of claims 1-3.

5. The pharmaceutical preparation according to claim 4, characterized in that, The pharmaceutical preparations include injectable preparations, oral preparations, tablets, suspensions, and capsules.

6. The pharmaceutical preparation according to any one of claims 4 or 5, characterized in that, The mass fraction of the pharmaceutical composition in the pharmaceutical preparation is 1-99%; The concentration of polymyxin in the pharmaceutical preparation is 0.031–8 µg / mL; The concentration of HM43239 in the pharmaceutical preparation is 0.391–50 µg / mL.

7. Use of the pharmaceutical composition of any one of claims 1-3 or the pharmaceutical preparation of any one of claims 4-6 in the preparation of a product for treating a disease caused by Klebsiella pneumoniae in a subject.

8. The use according to claim 7, characterized in that, The Klebsiella pneumoniae strain is selected from one or more of the following strains: polymyxin-sensitive Klebsiella pneumoniae and polymyxin-resistant Klebsiella pneumoniae.

9. The use according to any one of claims 7 or 8, characterized in that, The disease is selected from one or more of the following groups: Klebsiella pneumoniae pneumonia, Klebsiella pneumoniae bloodstream infection, Klebsiella pneumoniae intraperitoneal infection, and Klebsiella pneumoniae urinary tract infection.

10. The use according to any one of claims 7-9, characterized in that, The subjects were mammals.

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