Applications of SU3327 in the manufacture of drugs that enhance the antibacterial effect of polymyxins.

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 particularly relates to a method for enhancing the antibacterial activity of polymyxin by using the C-JUN N-terminal kinase inhibitor SU3327.

Background Art

[0002] Polymyxin is an antibacterial peptide discovered in the culture medium of *Bacillus polymyxa*, and there are five types including A, B, C, D and E. Their antibacterial spectra are similar to each other and have a wide range, and they act particularly strongly on Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Polymyxin E, also called colistin, has CAS No.: 1066-17-7, English name: Colistin, and trade names: Kangusisu, Colistisina, Colistin. In clinical application, it is mainly used in the form of sulfate or mesylate, that is, polymyxin E sulfate and polymyxin E mesylate. Polymyxin E is used for the treatment of enteritis caused by Escherichia coli and pneumonia caused by Klebsiella pneumoniae, and for the treatment of other drug-resistant strains, and is also used topically for local Pseudomonas aeruginosa infection caused by burns and traumas and for susceptible bacterial infections at parts such as ears and eyes.

[0003] In the past 10 years, infections caused by multidrug-resistant Gram-negative bacteria have seriously threatened the health of animals and humans. Currently, polymyxin antibacterial drugs are considered the last resort of treatment for the clinical treatment of multidrug-resistant Gram-negative bacterial infections (mainly including carbapenem-resistant Escherichia coli, multidrug-resistant Klebsiella pneumoniae, etc.). However, the emergence of polymyxin resistance gene (MCR) has significantly reduced the availability of polymyxin in clinical treatment, which greatly affects the service life of this important antibacterial drug. Therefore, the development of effective colistin synergists based on the combination of colistin has become an important treatment strategy for the clinical treatment of these life-threatening multidrug-resistant Gram-negative bacteria.

[0004] To address the clinical "bacterial drug resistance crisis," combination therapy has become the most important administration strategy for the clinical application of polymyxin in the treatment of MDR-GNR. One study disclosed various combinations of polymyxin. The minimum inhibitory concentrations for growth of 135 strains of carbapenem-resistant Klebaiella pneumoniae (CRKP) were measured using 17 different antibiotics to evaluate the drug susceptibility of CRKP strains. The synergistic antibacterial activity against CRKP of two combination therapies based on colistin, with biapenem and ceftazidime avibactam, was evaluated, providing a rationale for the selection of clinical administration (see "Study on in vitro antibacterial activity against carbapenem-resistant Klebaiella pneumoniae by combination therapy of colistin and other antibiotics," Bai Yan et al., 2016 Beijing Pharmaceutical Society Annual Meeting, disclosed 2016). A pharmacodynamic model was constructed to describe the in vitro bactericidal effect of polymyxin E (colistin E) in combination with other antibacterial agents against multidrug-resistant Acinetobacter baumannii (XDR-AB). The constructed pharmacodynamic model effectively describes the characteristics of polymyxin E's bactericidal effect against XDR-AB and provides an example of modeling pharmacokinetic / pharmacodynamic data for in vitro combination antibacterial agents (see "Study on a pharmacodynamic model of the treatment of Acinetobacter baumannii infection with polymyxin E in combination with other antibacterial agents," Fu Wen-Nuting et al., Anhui Medical Journal, 2020, No. 12).

[0005] SU3327 (also known as halicin, CAS No.: 40045-50-9) is a type of N-terminal kinase inhibitor of C-JUN, and its structure is shown in Figure 1. SU3327 is a potent, selective, and substrate-competitive JNK inhibitor, exhibiting substrate competitiveness. 50 It is 0.7 μM. SU3327 is also an IC 50 At a value of 239 nM, it inhibits the protein-protein interaction between JNK and JIP. SU3327 has low activity against p38α and Akt kinase.

[0006] Currently, existing technologies disclose the combined use of polymyxins with other antimicrobial agents, but most of these have very poor drug viability. This invention not only provides cellular-level synergistic effect experiments using SU3327 in combination with polymyxins, but also provides animal-level experiments demonstrating their good co-bactericidal effect. To date, no studies have been reported on the application of SU3327 as a polymyxin synergist to enhance the antimicrobial activity of polymyxins. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] To address the technical challenges in treating multidrug-resistant Gram-negative bacteria, the present invention provides a method for enhancing the antibacterial activity of polymyxin, specifically employing a combination of the C-JUN N-terminal kinase inhibitor SU3327 and polymyxin, preferably polymyxin E, where both achieve a synergistic antibacterial effect rather than simply adding functionality. The present invention further discloses the mixing ratio of the antibacterial components SU3327 and polymyxin E, providing a novel therapeutic strategy for the clinical treatment of bacterial infections, particularly multidrug-resistant Gram-negative bacterial infections, specifically bacterial infections in which polymyxin resistance genes (MCRs) are present. [Means for solving the problem]

[0008] According to one aspect of the present invention, the use of SU3327, an N-terminal kinase inhibitor of C-JUN, in the production of a drug that enhances the antibacterial effect of polymyxin is provided.

[0009] According to another aspect of the present invention, the use of a composition of C-JUN N-terminal kinase inhibitor SU3327 and polymyxin in the manufacture of a drug that enhances antibacterial infection effects is provided.

[0010] Furthermore, the polymyxin is polymyxin E (i.e., colistin) or polymyxin B.

[0011] Furthermore, the aforementioned bacteria are those that possess polymyxin resistance genes.

[0012] Furthermore, the aforementioned bacteria are Gram-negative bacteria.

[0013] Furthermore, the Gram-negative bacteria are multidrug-resistant Gram-negative bacteria.

[0014] Furthermore, the bacteria are one or more of Escherichia coli, Klebsiella pneumoniae, Salmonella, Bacillus subtilis, and Staphylococcus aureus, and preferably one or more of Escherichia coli, Klebsiella pneumoniae, Salmonella, Bacillus subtilis, and Staphylococcus aureus have polymyxin resistance genes or multidrug resistance.

[0015] Furthermore, the bacterium is Klebsiella pneumoniae, and preferably, the bacterium is Klebsiella pneumoniae that possesses a polymyxin resistance gene or multidrug resistance.

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

[0017] Furthermore, the dosage form of the SU3327 and polymyxin composition is one of the following: tablets, capsules, sustained-release tablets, controlled-release tablets, oral solutions, syrups, injectable solutions, dropper pills, or lyophilized powder injections.

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

[0019] The novel application of the C-JUN N-terminal kinase inhibitor SU3327 according to the present invention, and the method for enhancing the antibacterial activity of polymyxins, have the following excellent technical effects. (1) The present invention demonstrates that SU3327 synergistically enhances the antimicrobial activity of polymyxin through a minimum inhibitory concentration test using the Pannett square method and an in vitro bacterial growth curve. (2) Different from the existing combination of polymyxin and antibiotics, the present invention provides an experiment on a mouse model of drug-resistant bacterial infection, and demonstrates that SU3327 can effectively enhance polymyxin and its in vivo efficacy at the animal level, which is more convincing for subsequent clinical applications. (3) The present invention clarifies that SU3327 can restore the susceptibility of polymyxin-resistant bacteria, further evaluates the in vivo and in vitro efficacy of the combination of the two drugs, and contributes to the development of novel antibiotic synergists to alleviate the increasingly serious problem of bacterial antibiotic resistance. (4) The present invention provides a novel use of SU3327 in enhancing the antibacterial activity of polymyxin antibiotics, and can solve technical problems such as clinical drug resistance of polymyxin and low therapeutic index. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] [Figure 1] It is the chemical formula of SU3327 of the present invention. [Figure 2] It is the result of the combined bacteriostatic activity test of the strain by the checkerboard method for single administration of SU3327 and polymyxin E of the present invention and combined administration of the two drugs. [Figure 3] It is a graph showing the results of in vitro bacterial growth curves of single administration of SU3327 and polymyxin E of the present invention and combined administration of the two drugs for K. pneumoniae 1202 and K. pneumoniae 1202-45-5, wherein Figure 3A is a graph showing the results of in vitro bacterial growth curves of single administration of SU3327 and polymyxin E and combined administration of the two drugs for K. pneumoniae 1202 strain, and Figure 3B is a graph showing the results of in vitro bacterial growth curves of single administration of SU3327 and polymyxin E and combined administration of the two drugs for K. pneumoniae 1202-45-5 strain. [Figure 4] It is a result graph of the number of viable bacteria in the thigh of mice treated with single administration of SU3327 and polymyxin E of the present invention and combined administration of the two drugs for K. pneumoniae 1202-45-5. DESCRIPTION OF EMBODIMENTS

[0021] The drug SU3327 used in the examples of the present invention was purchased from MCE, a reagent company in the United States, with a purity of ≥99%. Polymyxin E sulfate was purchased from Hebei Shengxue Dacheng Tangshan Pharmaceutical Co., Ltd., and is polymyxin E with a titer of ≥23000 U / mg. A certain amount of polymyxin E sulfate is weighed and prepared into an aqueous solution with a stock solution concentration of 16 mg / mL. SU3327 is prepared into a stock solution with a concentration of 40 mg / mL using DMSO. After all stock solutions are prepared, they are stored in a refrigerator at -20°C.

Examples

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

[0023] 1.2 Test method The MIC of SU3327 when administered alone is detected using the multiple dilution method. Thereafter, according to the checkerboard method, a combined bacteriostatic activity test of the above-mentioned strain was carried out in a 96-well sterile microplate for the single administration of SU3327 and polymyxin E (Colistin) respectively and the combination of the two drugs, and the fractional inhibitory concentration index (FICI) of the combination was further calculated. For SU3327 and polymyxin E, FICI = MIC (in combination with polymyxin E) / MIC (polymyxin E alone) + MIC (in combination with SU3327) / MIC (SU3327 alone). When FICI ≤ 0.5, it is determined as synergistic effect; when 0.5 < FICI ≤ 1, it is determined as additive effect; when 1 < FICI < 4, it is determined as irrelevant effect.

[0024] 1.3 Test Results The results of the combined bacteriostatic activity test on the strain by the checkerboard method for single administration of SU3327 and polymyxin E and combination of the two drugs 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, the FICI was 0.1875, and it was determined to be a synergistic effect.

[0026] For Klebsiella pneumoniae 1202 (K. Pneumoniae 1202) strain, which is a clinical 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, the FICI was 0.1875, and it was determined to be a synergistic effect.

[0027] For the colistin-sensitive strain K. pneumoniae 1202-45-5, the MICs of polymyxin E and SU3327 were 16 μg / mL and 20 μg / mL, respectively. After co-administration, the MICs of polymyxin E and SU3327 decreased to 0.25 μg / mL and 5 μg / mL, respectively, and the FICI was 0.266, indicating a synergistic effect.

[0028] For the colistin-sensitive Escherichia coli GZP08-8 (E. coli GZP08-8) strain, the MICs of polymyxin E and SU3327 were 16 μg / mL and 10 μg / mL, respectively. After co-administration, the MICs of polymyxin E and SU3327 decreased to 0.25 μg / mL and 2.5 μg / mL, respectively, and the FICI was 0.266, indicating a synergistic effect.

[0029] For the standard Escherichia coli strain ATCC 25922 (E. coli 25922), the MICs of polymyxin E and SU3327 were 0.125 μg / mL and 10 μg / mL, respectively. After co-administration, the MICs of polymyxin E and SU3327 decreased to 0.0625 μg / mL and 5 μg / mL, respectively, and the FICI was 1, indicating an additive effect.

[0030] For the standard Staphylococcus aureus strain ATCC 29213 (S. aureus 29213), the MICs of polymyxin E and SU3327 were greater than 128 μg / mL and 5 μg / mL, respectively. After co-administration, the MICs of polymyxin E and SU3327 decreased to 64 μg / mL and 2.5 μg / mL, respectively, and the FICI was 0.75, indicating an additive effect.

[0031] For Salmonella 1-5, a colistin-resistant strain, the MICs of polymyxin E and SU3327 were greater than 128 μg / mL and 20 μg / mL, respectively. After co-administration, the MICs of polymyxin E and SU3327 decreased to 1 μg / mL and 2.5 μg / mL, respectively, and the FICI was 0.25, indicating a synergistic effect.

[0032] For methicillin-resistant Staphylococcus aureus USA300 (S. aureus USA300) strains, the MICs of polymyxin E and SU3327 were greater than 128 μg / mL and 5 μg / mL, respectively. After co-administration, the MICs of polymyxin E and SU3327 decreased to 32 μg / mL and 1.25 μg / mL, respectively, and the FICI was 0.25, indicating a synergistic effect.

[0033] For Shigella Y, a colistin-sensitive strain, the MICs of polymyxin E and SU3327 were 0.125 μg / mL and 5 μg / mL, respectively. After co-administration, the MICs of polymyxin E and SU3327 decreased to 0.032 μg / mL and 2.5 μg / mL, respectively, and the FICI was 0.75, indicating an additive effect. [Examples]

[0034] Example 2: Combined sterilization curve using polymyxin E and SU3327 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 then dilute to 1000mL. Autoclave at 121°C for 15 minutes, and when cooled to 50°C, pour into a sterile plate and dry to prepare for use.

[0035] 2.2 Test Method K. pneumoniae 1202 and K. pneumoniae 1202-45-5 were cultured in BHI broth for 6-8 hours, and then the bacterial concentration was diluted to 10^6 colonies 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). A control bacterial group was given 0.1% DMSO solvent. 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 bacterial suspension was taken at 1, 3, 6, 12, and 24 hours after drug treatment and spread onto agar plates. After incubation for at least 16 hours, colony counting was performed.

[0036] 2.3 Test Results The in vitro bacterial growth curves for K. pneumoniae 1202 and K. pneumoniae 1202-45-5, administered individually and in combination with SU3327 and polymyxin E, are shown in Figures 3A and 3B. Figure 3A shows the in vitro bacterial growth curve results for K. pneumoniae 1202 strain administered individually and in combination with SU3327 and polymyxin E, while Figure 3B shows the in vitro bacterial growth curve results for K. pneumoniae 1202-45-5 strain administered individually and in combination with SU3327 and polymyxin E. The specific results analysis is as follows.

[0037] In the results against Klebsiella pneumoniae 1202, the combined treatment groups with SU3327 (10 μg / mL) and polymyxin E (0.25 μg / mL) showed significantly higher bactericidal efficacy than treatment with SU3327 or polymyxin E alone. After continuous treatment for 24 hours, the bacterial colony count was 9.4 Log10 CFU / mL in the control group, 8.9 Log10 CFU / mL in the polymyxin E group, 8.35 Log10 CFU / mL in the SU3327 group, and 2.05 Log10 CFU / mL in the combined treatment group of polymyxin E and SU3327. The combined treatment of polymyxin E and SU3327 showed a remarkably higher bactericidal efficacy.

[0038] In the results against Klebsiella pneumoniae 1202-45-5, the combined treatment group with SU3327 (20 μg / mL) and polymyxin E (1 μg / mL) showed significantly higher bactericidal efficacy than treatment with SU3327 or polymyxin E alone. After continuous treatment for 24 hours, the bacterial colony count was 9.35 Log10 CFU / mL in the control group, 9.3 Log10 CFU / mL in the polymyxin E group, 6.65 Log10 CFU / mL in the SU3327 group, and 2.7 Log10 CFU / mL in the combined treatment group of polymyxin E and SU3327. The combined treatment of polymyxin E and SU3327 showed a significantly higher bactericidal efficacy.

[0039] Test Example 3: Therapeutic efficacy of polymyxin E and SU3327 in combination with monotherapy in a BALB / c mouse model of drug-resistant bacterial infection. 3.1 Animal sorting and processing To avoid the influence of the mice's own immune system on the experiment, all mice were administered cyclophosphamide twice, 4 days and 1 day before infection, each time by intraperitoneal injection at a dose of 100 mg / kg to induce neutropenia and immunodeficiency. A suspension was prepared by deuterating 24 6-8 week old BALB / c female mice (approximately 20g body weight) with logarithmic-phase Klebsiella pneumoniae 1202-45-5 in PBS buffer, with a concentration of 1 x 10⁻¹⁶. 7 The value is CFU / mL, and in mice, the total amount of bacteria injected last into the thigh of each mouse was 1 x 10⁻⁶. 6These are CFUs. The animals are then divided into a control group (i.e., a solvent-treated group), a polymyxin E-treated group (10 mg / kg body weight), a SU3327-treated group (10 mg / kg body weight), and a combined SU3327 (10 mg / kg body weight) + polymyxin E (10 mg / kg body weight) treatment group (6 animals in each group). The specific treatment is as follows. Solvent-controlled treatment group: 0.1 mL of Klebsiella pneumoniae 1202-45-5 suspension was injected into the right thigh muscle of the abdominal cavity of 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 mice. One hour later, 200 μL (1 mg / mL) of polymyxin E solution was injected intraperitoneally into the mice, with a final dose of 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 mice. One hour later, 200 μL of SU3327 solution (1 mg / mL) was injected intraperitoneally into the mice, with a final dose of 10 mg / kg body weight. Combination therapy with SU3327 (10 mg / kg) + polymyxin E (10 mg / kg): 0.1 mL of E. coli suspension is injected into the right thigh muscle of the mouse peritoneum. One hour later, a mixture of SU3327 (1 mg / mL) and polymyxin E (200 μL, 1 mg / mL) is intraperitoneally injected into the mouse. The final dose is 10 mg / kg body weight for both SU3327 and polymyxin E. Treatment is performed twice, every 8 hours. 24 hours after infection, all mice are euthanized, and the right thigh muscle is collected. It is placed in 3 mL of PBS solution, disrupted using a cryolyzator, and finally 100 μL of the disrupted tissue is collected, spread on a plate, and colony counting is performed.

[0040] 3.2 Test Results Figure 4 shows the results of viable bacterial counts in the thighs of mice treated with SU3327 and polymyxin E alone, and in combination with both drugs, for Klebsiella pneumoniae 1202-45-5. The results are as follows:

[0041] Compared to the solvent control group, the viable bacterial count in the thigh of mice treated with polymyxin E alone was 6.82 Log10 CFU / mL, which was not significantly different from the control group. Compared to the solvent control group, the viable bacterial count in the thigh of mice treated with SU3327 alone was 6.55 Log10 CFU / mL, which was significantly different from the control group. Compared to the solvent control group, the viable bacterial count in the thigh of mice treated with polymyxin E and SU3327 in combination was 5.28 Log10 CFU / mL, which was significantly different from the control group, and was also significantly different from the SU3327 monotherapy group or the polymyxin E monotherapy group, respectively. These results indicate that SU3327 can effectively enhance the in vivo efficacy of polymyxin E and provide a new treatment option for infectious diseases caused by drug-resistant Gram-negative bacteria.

[0042] The above-described implementations of this disclosure are merely illustrative to illustrate the disclosure and do not limit the embodiments of this disclosure. Those skilled in the art can make other different forms of variations or modifications based on the above description. It is neither necessary nor possible to cover all embodiments comprehensively. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this disclosure should be included within the scope of protection of the claims of this disclosure.

Claims

1. A composition comprising SU3327 and polymyxin E for use in synergistically treating bacterial infections, The SU3327 has the following structure 【Chemistry 1】 A composition having the following characteristics.

2. The composition according to claim 1, characterized in that the bacteria are bacteria that possess a polymyxin resistance gene.

3. The composition according to claim 1, characterized in that the bacteria are Gram-negative bacteria.

4. The composition according to claim 1, characterized in that the bacteria are one or more of Escherichia coli, Klebsiella pneumoniae, Salmonella, Bacillus subtilis, and Staphylococcus aureus.

5. The composition according to claim 1, characterized in that the bacteria possess polymyxin resistance genes or multidrug resistance.

6. The composition according to any one of claims 1 to 5, characterized in that the mass ratio of SU3327 to polymyxin E in the composition is (2.5 to 10):

1.

7. The composition according to any one of claims 1 to 5, characterized in that the dosage form of the composition comprising SU3327 and polymyxin E is one of the following: a tablet, a capsule, a sustained-release tablet, a controlled-release tablet, an oral solution, a syrup, an injectable solution, a dropper pill, or a lyophilized powder injectable dosage form.

8. The composition according to any one of claims 1 to 5, characterized in that the final therapeutic dose of polymyxin E and SU3327 is 10 mg / kg body weight.

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