Use of SU3327 in the manufacture of a drug that enhances the antibacterial effect of polymyxin

Abstract

The present invention provides the use of C-JUN N-terminal kinase inhibitor SU3327 in the preparation of a drug for enhancing the antibacterial infection effect of polymyxin, and the use of a composition of SU3327 and polymyxin in the preparation of a drug for enhancing the antibacterial infection effect, the polymyxin being preferably polymyxin E. The present invention not only proves that SU3327 synergistically enhances the antibacterial activity of polymyxin through the Punnett Square minimum inhibitory concentration test and in vitro bacterial growth curve, but also demonstrates that SU3327 can effectively enhance polymyxin and its in vivo efficacy at the animal level through mouse drug-resistant bacteria infection model experiments. The present invention provides a new use of SU3327 in enhancing the antibacterial activity of polymyxin antibiotics, and can solve technical problems such as clinical drug resistance and low therapeutic index of polymyxin.

Description

[Technical field]

[0001] The present invention relates to the pharmaceutical field, specifically to the antibacterial field, and in particular to a method for enhancing the antibacterial activity of polymyxin using the C-JUN N-terminal kinase inhibitor SU3327. [Background technology]

[0002] Polymyxin is an antibacterial peptide found in the culture medium of Bacillus polymyxa, and there are five types, including A, B, C, D, and E. The antibacterial spectrum is similar and broad, and it is particularly effective against gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Polymyxin E (also called colistin, Polymyxin E), CAS number: 1066-17-7, English name: Colistin, trade name: Canguzis, Colistina, Colistin. In clinical applications, it is mainly in the form of sulfate or mesylate, i.e., polymyxin E sulfate and polymyxin E mesylate. Polymyxin E is used to treat enteritis caused by Escherichia coli, pneumonia caused by Klebsiella pneumoniae, and other drug-resistant strains, and is also used externally for local infections of Pseudomonas aeruginosa caused by burns and trauma, and for sensitive bacterial infections in areas such as the ears and eyes.

[0003] In the past decade, infections caused by multidrug-resistant Gram-negative bacteria have seriously threatened the health of animals and humans. Currently, polymyxin antibiotics are considered to be the last line of treatment for the clinical treatment of multidrug-resistant Gram-negative bacterial (mainly including carbapenem-resistant Escherichia coli, multidrug-resistant Klebsiella pneumoniae, etc.) infections. However, with the emergence of polymyxin resistance genes (MCR), the utilization rate of polymyxins in clinical treatment has obviously decreased. This has greatly affected the useful life of this important antibacterial drug. Therefore, the development of effective colistin synergists based on the combination of colistin has become an important therapeutic strategy for the clinical treatment of these life-threatening multidrug-resistant Gram-negative bacteria.

[0004] In response to the clinical "bacterial drug resistance crisis", combination has become the most important dosing strategy for the clinical application of polymyxin in the treatment of MDR-GNR. A literature report has disclosed multiple combinations of polymyxin. The minimum inhibitory concentrations of 135 strains of carbapenem-resistant Klebaiella pneumoniae (CRKP) with 17 different antibiotics were measured to evaluate the drug susceptibility of CRKP strains. The synergistic antibacterial activity of two combination plans based on Colistin, biapenem, and ceftazidime-avibactam against CRKP was evaluated, providing a theoretical basis for clinical dosing selection (see "Study on the in vitro antibacterial activity of Colistin in combination with other antibiotics against carbapenem-resistant Klebaiella pneumoniae", Bai Yan et al., 2016 Beijing Pharmaceutical Annual Conference, 2016). A pharmacodynamic model was constructed to describe the in vitro bactericidal effect of polymyxin E (colistin E) in combination with other antibiotics against multidrug-resistant Acinetobacter baumannii (XDR-AB). The constructed pharmacodynamic model effectively described the characteristics of the bactericidal effect of polymyxin E against XDR-AB and provided an example of modeling pharmacokinetic / pharmacodynamic data of in vitro combination of antibiotics (see "Study on Pharmacodynamic Model of Treatment of Acinetobacter baumannii Infection by Combination of Polymyxin E with Other Antibacterial Agents", Fu Wen Ting et al., Anhui Medicine, December 2020).

[0005] SU3327 (also called halicin, CAS No.: 40045-50-9) is a type of N-terminal kinase inhibitor of c-JUN, the structure of which is shown in Figure 1. SU3327 is a potent, selective and substrate-competitive JNK inhibitor with IC 50 The IC 50 It inhibits the protein-protein interaction of JNK and JIP with a potency of 239 nM. SU3327 has low activity against p38α and Akt kinases.

[0006] Currently, existing technologies disclose the combination of polymyxin with other antibacterial drugs, but most of them have very poor druggability. The present invention not only provides a synergistic effect experiment at the cell level by the combination of SU3327 and polymyxin, but also provides an animal level experiment to demonstrate its good joint bactericidal effect. Up to now, there has been no report on the application of SU3327 as a polymyxin synergist to enhance the antibacterial activity of polymyxin. Summary of the Invention [Problem to be solved by the invention]

[0007] In order to solve the technical problems of multidrug-resistant Gram-negative bacteria treatment, the present invention provides a method for enhancing the antibacterial activity of polymyxin, specifically adopting the combined use of C-JUN N-terminal kinase inhibitor SU3327 and polymyxin, the polymyxin being preferably polymyxin E, and the two achieving synergistic antibacterial action rather than simply adding functions. The present invention further discloses the combination ratio of the antibacterial components SU3327 and polymyxin E, and provides a new therapeutic strategy for the clinical treatment of bacterial infections, particularly multidrug-resistant Gram-negative bacterial infections, particularly bacterial infections with polymyxin resistance genes (MCR). [Means for solving the problem]

[0008] According to one aspect of the present invention, there is provided the use of SU3327, an N-terminal kinase inhibitor of C-JUN, in the manufacture of a medicament for enhancing the antibacterial infection effect of polymyxin.

[0009] According to another aspect of the present invention there is provided the use of a composition of the C-JUN N-terminal kinase inhibitor SU3327 and polymyxin in the manufacture of a medicament for enhancing anti-bacterial infection effect.

[0010] Further, the polymyxin is polymyxin E (ie colistin) or polymyxin B.

[0011] Furthermore, the bacterium is one in which a polymyxin resistance gene is present.

[0012] Furthermore, the bacterium is a gram-negative bacterium.

[0013] Furthermore, the Gram-negative bacterium is a multidrug-resistant Gram-negative bacterium.

[0014] Furthermore, the bacteria is one or more of Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, Klebsiella shiga, and Staphylococcus aureus, and preferably, one or more of Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, Klebsiella shiga, and Staphylococcus aureus have a polymyxin resistance gene or multidrug resistance.

[0015] Furthermore, the bacterium is Klebsiella pneumoniae, and preferably, the bacterium is Klebsiella pneumoniae in which a polymyxin resistance gene or multidrug resistance is present.

[0016] Furthermore, the mass ratio of SU3327 to polymyxin in the composition of SU3327 and polymyxin is (2.5-10:1).

[0017] Furthermore, the dosage form of the composition of SU3327 and polymyxin is one of tablets, capsules, sustained release tablets, controlled release tablets, oral liquids, syrups, injection liquid dosage forms, drop pills, and freeze-dried powder injection dosage forms.

[0018] Furthermore, the final therapeutic dose of the polymyxin E and the SU3327 is 10 mg / kg body weight. Effect of the Invention

[0019] The novel use of the C-JUN N-terminal kinase inhibitor SU3327 according to the present invention, and the method for enhancing the antibacterial activity of polymyxin, have the following excellent technical effects: (1) The present invention demonstrates that SU3327 synergistically enhances the antibacterial activity of polymyxin through the minimum inhibitory concentration test using the Punnett square method and in vitro bacterial growth curves. (2) Unlike the existing combination of polymyxin and antibiotics, the present invention provides a mouse drug-resistant bacterial infection model experiment, which demonstrates that SU3327 can effectively enhance polymyxin and its in vivo efficacy at the animal level, which is more evidence-based for subsequent clinical applications. (3) The present invention reveals that SU3327 can restore the sensitivity of polymyxin-resistant bacteria, and is further evaluating the in vivo and in vitro efficacy of the combination of the two drugs, which will be useful for the development of new antibiotic synergists to alleviate the increasingly serious problem of bacterial drug resistance. (4) The present invention provides a new application of SU3327 in enhancing the antibacterial activity of polymyxin antibiotics, and can solve the technical problems such as clinical drug resistance and low therapeutic index of polymyxins. [Brief description of the drawings]

[0020] [Figure 1] 1 is the chemical formula of SU3327 of the present invention. [Diagram 2] 1 shows the results of a joint bacteriostatic activity test using the Punnett Square method for bacterial strains when SU3327 of the present invention and polymyxin E were administered alone and in combination. [Diagram 3] FIG. 3A is a graph showing the in vitro bacterial growth curve results for K. Pneumoniae 1202 strain when SU3327 and polymyxin E of the present invention are administered alone and in combination, and FIG. 3B is a graph showing the in vitro bacterial growth curve results for K. Pneumoniae 1202-45-5 strain when SU3327 and polymyxin E are administered alone and in combination. [Figure 4] FIG. 13 is a graph showing the results of viable cell counts in the thighs of mice treated with SU3327 of the present invention and polymyxin E alone and in combination with each other for K. Pneumoniae 1202-45-5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The drug SU3327 used in the present embodiment is purchased from the US reagent company MCE, with a purity of ≧99%. Polymyxin E sulfate is purchased from Hebei Shengxue Dacheng Tangshan Pharmaceutical Co., Ltd., with a titer of ≧23000U / mg polymyxin E. A certain amount of polymyxin E sulfate is weighed and prepared into an aqueous solution with a mother liquor concentration of 16mg / mL. SU3327 is prepared into a mother liquor with a mother liquor concentration of 40mg / mL using DMSO. After all the mother liquors are prepared, they are stored in a refrigerator at -20 degrees. EXAMPLES

[0022] Example 1 Evaluation of synergistic antibacterial activity by combined use of SU3327 and polymyxin 1.1 Test strains The bacterial strains used in the study, Escherichia coli ATCC 25922 (E. coli 25922) and Staphylococcus aureus ATCC 29213 (S. aureus 29213), were purchased from the Bacterial Species Collection Center of the China Veterinary Inspection Service, and Methicillin-resistant Staphylococcus aureus USA300 (ATCC BAA-1717) was purchased from the American Type Culture Collection. The bacterial strains used in other experiments, E. coli B2, E. coli GZP08-8, K. Pneumoniae 1202, K. Pneumoniae 1202-45-5, Salmonella 1-5, and Shigella Y, were all preserved from the National Veterinary Drug Safety Evaluation Center, China Agricultural University. Among them, E. coli B2, E. coli GZP08-8, K. Pneumoniae 1202-45-5, and Salmonella 1-5 are strains with MCR-1 polymyxin resistance, while K. Pneumoniae 1202 and Shigella Y are clinically colistin-susceptible strains.

[0023] 1.2 Test Method The MIC of SU3327 administered alone is detected using the multiple dilution method. Subsequently, in a 96-well sterile microplate, the combined bacteriostatic activity test of the above strains by the checkerboard method is performed for the administration of SU3327 and polymyxin E (Colistin) alone and in combination, and the fractional inhibitory concentration index (FICI) is calculated. The FICI of SU3327 and polymyxin E = MIC (polymyxin E in combination) / MIC (polymyxin E) + MIC (SU3327 in combination) / MIC (SU3327 administered alone). When FICI ≤ 0.5, it is determined as a synergistic effect; when 0.5 < FICI ≤ 1, it is determined as an additive effect; when 0.5 < FICI < 4, it is determined as an irrelevant effect.

[0024] 1.3 Test Results The results of the combined bacteriostatic activity test of the strains by the checkerboard method for the administration of SU3327 and polymyxin E alone and in combination are as shown in Figure 2, and the specific result analysis is as follows.

[0025] For Escherichia coli B2 (E. coli B2) containing MCR-1, the MICs of polymyxin E and SU3327 are 8 μg / mL and 20 μg / mL respectively. After combination, the MICs of polymyxin E and SU3327 decreased to 0.5 μg / mL and 2.5 μg / mL respectively, and the FICI was 0.1875, which was determined as a synergistic effect.

[0026] For Klebsiella pneumoniae 1202 (K. Pneumoniae 1202) strain, which is a clinically colistin-sensitive bacterium, the MICs of polymyxin E and SU3327 are 1 μg / mL and 20 μg / mL respectively. After combination, the MICs of polymyxin E and SU3327 decreased to 0.0625 μg / mL and 2.5 μg / mL respectively, and the FICI was 0.1875, which was determined as a synergistic effect.

[0027] For the colistin-susceptible K. Pneumoniae 1202-45-5 strain, the MICs of polymyxin E and SU3327 were 16 μg / mL and 20 μg / mL, respectively. After coadministration, the MICs of polymyxin E and SU3327 decreased to 0.25 μg / mL and 5 μg / mL, respectively, with an FICI of 0.266, indicating synergistic activity.

[0028] For the colistin-susceptible E. coli GZP08-8 strain, the MICs of polymyxin E and SU3327 were 16 μg / mL and 10 μg / Ml, respectively. After coadministration, the MICs of polymyxin E and SU3327 decreased to 0.25 μg / mL and 2.5 μg / mL, respectively, with an FICI of 0.266, indicating synergistic activity.

[0029] For the Escherichia coli standard strain ATCC 25922 (E. coli 25922), the MICs of polymyxin E and SU3327 were 0.125 μg / mL and 10 μg / mL, respectively. After combined use, the MICs of polymyxin E and SU3327 decreased to 0.0625 μg / mL and 5 μg / mL, respectively, with an FICI of 1, which was determined to be an additive effect.

[0030] For the standard strain of Staphylococcus aureus ATCC 29213 (S. aureus 29213), the MICs of polymyxin E and SU3327 were greater than 128 μg / mL and 5 μg / mL, respectively, and after combined use, the MICs of polymyxin E and SU3327 decreased to 64 μg / mL and 2.5 μg / mL, respectively, with an FICI of 0.75, which was determined to be an additive effect.

[0031] For the colistin-resistant strain Salmonella 1-5, the MICs of polymyxin E and SU3327 were greater than 128 and 20 μg / mL, respectively. After coadministration, the MICs of polymyxin E and SU3327 were reduced to 1 and 2.5 μg / mL, respectively, with an FICI of 0.25, which was determined to be synergistic.

[0032] For the methicillin-resistant Staphylococcus aureus USA300 (S. aureus USA300) strain, the MICs of polymyxin E and SU3327 were greater than 128 μg / mL and 5 μg / mL, respectively, and after combined use, the MICs of polymyxin E and SU3327 decreased to 32 μg / mL and 1.25 μg / mL, respectively, with an FICI of 0.25, which was determined to be synergistic.

[0033] For the colistin-susceptible strain Shigella Y, the MICs of polymyxin E and SU3327 were 0.125 μg / mL and 5 μg / mL, respectively. After coadministration, the MICs of polymyxin E and SU3327 decreased to 0.032 μg / mL and 2.5 μg / mL, respectively, with an FICI of 0.75, which was determined to be an additive effect. EXAMPLES

[0034] Example 2: Co-kill curves of polymyxin E and SU3327 in combination 2.1 Preparation of MHA medium Take 6.0g of beef powder, 1.5g of soluble starch, 17.5g of casein hydrolysate, and 17.0g of agar, add 900mL of distilled water, adjust the pH to 7.3, and make up to 1000mL. Sterilize at 121℃ for 15 minutes under high pressure, and when cooled to 50℃, pour into a sterile plate, dry, and prepare for use.

[0035] 2.2 Test Method K. Pneumoniae 1202 and K. Pneumoniae 1202-45-5 were cultured in BHI broth for 6 to 8 hours, and then the bacterial concentration was diluted to 10 colony count using a turbidimeter. For Klebsiella pneumoniae 1202, the drug treatment concentrations were set to polymyxin E (0.25 μg / mL), SU3327 (10 μg / mL), and SU3327 (10 μg / mL) + polymyxin E (0.25 μg / mL). 0.1% DMSO solvent was given to the control bacteria. For Klebsiella pneumoniae 1202-45-5, the drug treatment concentrations were set to polymyxin E (1 μg / mL), SU3327 (20 μg / mL), and SU3327 (20 μg / mL) + polymyxin E (1 μg / mL). 100 μL of the bacterial liquid was taken 1 h, 3 h, 6 h, 12 h, and 24 h after drug treatment and spread onto an agar plate. After culturing for at least 16 h, colony counts were performed.

[0036] 2.3 Test Results The in vitro bacterial growth curve results of SU3327 and polymyxin E alone and in combination for K.Pneumoniae 1202 and K.Pneumoniae 1202-45-5 are shown in Figures 3A-3B, where Figure 3A shows the in vitro bacterial growth curve results of SU3327 and polymyxin E alone and in combination for K.Pneumoniae 1202 strain, and Figure 3B shows the in vitro bacterial growth curve results of SU3327 and polymyxin E alone and in combination for K.Pneumoniae 1202-45-5 strain. The specific results are analyzed as follows:

[0037] In the results against Klebsiella pneumoniae 1202, the bactericidal effect of the combined treatment of SU3327 (10μg / mL) and polymyxin E (0.25μg / mL) was significantly higher than that of either SU3327 or polymyxin E alone. After 24 hours of continuous treatment, the bacterial colony counts were 9.4 Log10 CFU / mL in the control treatment group, 8.9 Log10 CFU / mL in the polymyxin E treatment group, 8.35 Log10 CFU / mL in the SU3327 treatment group, and 2.05 Log10 CFU / mL in the combined treatment of polymyxin E and SU3327, and the bactericidal effect was significantly higher after the combined treatment of polymyxin E and SU3327.

[0038] In the results against Klebsiella pneumoniae 1202-45-5, the bactericidal effect of the combined treatment of SU3327 (20μg / mL) and polymyxin E (1μg / mL) was significantly higher than that of either SU3327 or polymyxin E alone. After 24 hours of continuous treatment, the bacterial colony counts were 9.35 Log10 CFU / mL in the control treatment group, 9.3 Log10 CFU / mL in the polymyxin E treatment group, 6.65 Log10 CFU / mL in the SU3327 treatment group, and 2.7 Log10 CFU / mL in the combined treatment of polymyxin E and SU3327, and the bactericidal effect was significantly higher after the combined treatment of polymyxin E and SU3327.

[0039] Test Example 3: Therapeutic effects of polymyxin E and SU3327 administered alone or in combination in a BALB / c mouse model infected with drug-resistant bacteria 3.1 Animal grouping and treatment To avoid the influence of the mouse's own immune system on the experiment, all mice were administered cyclophosphamide twice, 4 days and 1 day before infection, respectively, with 100 mg / kg of cyclophosphamide administered intraperitoneally each time to induce neutropenia and immunodeficiency in the mice. Twenty-four 6-8 week-old BALB / c female mice (body weight approximately 20 g) were used to prepare a suspension by heavy-suspending logarithmic phase Klebsiella pneumoniae 1202-45-5 bacteria in PBS buffer at a concentration of 1x10. 7 CFU / mL, and the total amount of bacteria finally injected into the thigh of each mouse was 1x10 6The mice were then randomly divided into a model control group (i.e., a vehicle-treated group), a polymyxin E-treated group (10 mg / kg body weight), a SU3327-treated group (10 mg / kg body weight), and a combination SU3327 (10 mg / kg body weight) + polymyxin E (10 mg / kg body weight) treatment group (6 mice per group). The specific treatments were as follows: Vehicle control treatment group: 0.1 mL of Klebsiella pneumoniae 1202-45-5 suspension was injected into the right thigh muscle of the abdominal cavity of the mice, and 1 hour later, 200 μL of PBS buffer was injected intraperitoneally into the mice. Polymyxin E treatment group: 0.1 mL of Klebsiella pneumoniae 1202-45-5 suspension was injected into the right thigh muscle of the abdominal cavity of the mice, and 1 hour later, 200 μL (1 mg / mL) of polymyxin E solution was intraperitoneally injected into the mice, with the final dose being 10 mg / kg body weight. SU3327 treatment group: 0.1 mL of Klebsiella pneumoniae 1202-45-5 suspension was injected into the right thigh muscle of the abdominal cavity of the mice, and 1 hour later, 200 μL of SU3327 solution (1 mg / mL) was intraperitoneally injected into the mice, the final dose was 10 mg / kg body weight. SU3327 (10mg / kg) + polymyxin E (10mg / kg) combination treatment group: 0.1mL of E. coli suspension was injected into the right thigh muscle of the mouse abdominal cavity, and 1 hour later, the mouse was intraperitoneally injected with a mixture of SU3327 (1mg / mL) and polymyxin E 200μL (1mg / mL). The final dose of SU3327 and polymyxin E was 10mg / kg body weight. The treatment was performed twice, every 8 hours. 24 hours after infection, all mice were euthanized, and then the right thigh muscle of the mouse was collected, placed in 3mL of PBS solution, and crushed using a low-temperature tissue homogenizer. Finally, 100μL of the homogenate was collected and applied to a plate for colony counting.

[0040] 3.2 Test results For Klebsiella pneumoniae 1202-45-5, the viable cell count in the thighs of mice treated with SU3327 and polymyxin E alone and in combination is shown in FIG. 4, and the results are as follows:

[0041] Compared with the solvent control group, the viable bacterial count in the mouse thighs of the polymyxin E monotherapy group was 6.82 Log10 CFU / mL, which was not significantly different from the control group; compared with the solvent control group, the viable bacterial count in the mouse thighs of the SU3327 monotherapy group was 6.55 Log10 CFU / mL, which was significantly different from the control group; compared with the solvent control group, the viable bacterial count in the mouse thighs of the polymyxin E and SU3327 co-treatment group was 5.28 Log10 CFU / mL, which was significantly different from the control group, and both were significantly different from the SU3327 monotherapy group or the polymyxin E monotherapy group. These results indicate that SU3327 can effectively enhance the in vivo efficacy of polymyxin E, providing a new therapeutic approach for the treatment of infectious diseases caused by drug-resistant Gram-negative bacteria.

[0042] The above implementation of the present disclosure is merely an example for clearly explaining the present disclosure, and does not limit the embodiments of the present disclosure. Those skilled in the art can make other different forms of changes or modifications based on the above description. It is not necessary or possible to comprehensively cover all the embodiments here. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present disclosure should be included in the scope of protection of the claims of the present disclosure.

Claims

1. Use of SU3327, an N-terminal kinase inhibitor of C-JUN, in the manufacture of a medicament which enhances the antibacterial infection effect of polymyxin.

2. Use of a composition of SU3327, an N-terminal kinase inhibitor of C-JUN, and polymyxin in the manufacture of a medicament having enhanced antibacterial infection effects.

3. 3. The use according to claim 1 or 2, characterized in that the polymyxin is polymyxin E or polymyxin B, preferably the polymyxin is polymyxin E.

4. The use according to any one of claims 1 to 3, characterized in that the bacterium is a bacterium in which a polymyxin resistance gene is present.

5. 4. The use according to any one of claims 1 to 3, characterized in that the bacterium is a gram-negative bacterium.

6. 6. The use according to claim 5, characterized in that the Gram-negative bacterium is a multidrug-resistant Gram-negative bacterium.

7. The use according to any one of claims 1 to 3, characterized in that the bacteria is one or more of Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, Klebsiella shigae, and Staphylococcus aureus, and preferably, the one or more of Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, Klebsiella shigae, and Staphylococcus aureus have a polymyxin resistance gene or multidrug resistance.

8. The use according to any one of claims 1 to 3, characterized in that the bacterium is Klebsiella pneumoniae, preferably the bacterium is Klebsiella pneumoniae in which a polymyxin resistance gene or multidrug resistance is present.

9. The use according to any one of claims 2 to 8, characterized in that the mass ratio of SU3327 to polymyxin in the composition of SU3327 and polymyxin is (2.5-10):

1.

10. The use according to any one of claims 2 to 8, characterized in that the dosage form of the composition of SU3327 and polymyxin is one of tablets, capsules, sustained release tablets, controlled release tablets, oral liquids, syrups, injection liquid dosage forms, drop pills, and freeze-dried powder injection dosage forms.

11. The use according to any one of claims 2 to 8, characterized in that the final therapeutic dose of said polymyxin E and said SU3327 is 10 mg / kg body weight.

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