Application of 10-undecylenic acid synergistic polymyxin in preparation of antibacterial drugs

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of 10-undecylenic acid and polymyxin in preparation of an antibacterial drug, and the antibacterial drug aims at one or more of escherichia coli, salmonella and klebsiella pneumoniae. The antibacterial agent aims at bacteria which are resistant to polymyxin. The bacteria directed by the antibacterial agent are multi-drug-resistant gram-negative bacteria. Through a chessboard method minimum inhibitory concentration test, an in-vitro bacterial growth curve proves that 10-undecylenic acid synergistically enhances the antibacterial activity of polymyxin, and meanwhile, a mouse drug-resistant bacterial infection model test proves that 10-undecylenic acid can effectively enhance the in-vivo effectiveness of polymyxin at the animal level. The invention provides a new application of 10-undecylenic acid in enhancing the antibacterial activity of polymyxin antibiotics, and can solve the technical problems of low clinical drug resistance and therapeutic index of polymyxin and the like.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and specifically relates to the application of 10-undecenoic acid combined with polymyxin in the preparation of antibacterial drugs. Background Art

[0002] Gram-negative bacteria are common pathogenic bacteria in clinical practice, seriously affecting human health and the healthy breeding of livestock and poultry. Polymyxin is one of the important antibacterial drugs for the treatment of multi-drug resistant Gram-negative bacterial infections. However, in recent years, due to the irregular use of drugs in clinical treatment, the emergence of drug-resistant bacteria has seriously weakened the clinical treatment effect of polymyxin. In recent years, the research and development of new antibacterial drugs has stagnated, and using existing drugs to restore the effectiveness of existing antibiotics is an economical and effective means. 10-Undecenoic acid is a single-chain unsaturated fatty acid that naturally exists in human sweat and has been widely used in clinical antifungal treatment. It can effectively inhibit the reproduction of fungi and is commonly used to treat skin fungal infections such as tinea capitis, tinea cruris, tinea pedis, and vulvovaginal candidiasis, and can effectively inhibit the biofilm formation of Candida albicans (Literature: Shi D, Zhao Y, Yan H, Fu H, Shen Y, Lu G, Mei H, Qiu Y, Li D, Liu W. Antifungal effects of undecylenic acid on the biofilm formation of Candida albicans [J]. Int J Clin Pharmacol Ther, 2016, 54(5): 343-353.); In addition, studies have shown that 10-undecenoic acid combined with L-arginine can induce concentration-dependent tumor cell death (Literature: Day ZI, Mayfosh AJ, Giel MC, Hong Y, Williams SA, Santavanond JP, Rau TF, Poon IK, Hulett MD. Novel Formulation of Undecylenic Acid induces Tumor Cell Apoptosis [J]. Int J Mol Sci, 2022, 23(22).). So far, the antibacterial activity of 10-undecenoic acid as an antibacterial adjuvant to enhance the antibacterial effect of antibacterial drugs has not been reported. Summary of the Invention

[0003] To solve the technical problem of treating multi-drug resistant Gram-negative bacteria, an embodiment of the present invention provides a method for enhancing the antibacterial activity of polymyxin, specifically using 10-undecenoic acid in combination with polymyxin. Polymyxin is preferably polymyxin E sulfate. The two not only simply add functions, but achieve a synergistic antibacterial effect. The embodiment of the present invention also discloses the antibacterial combination ratio of 10-undecenoic acid and polymyxin E sulfate, providing a new treatment strategy for clinical treatment of bacterial infections, especially multi-drug resistant Gram-negative bacterial infections, such as bacterial infections with polymyxin resistance gene (MCR).

[0004] One aspect of the present invention provides the use of 10-undecenoic acid combined with polymyxin in the preparation of antibacterial drugs.

[0005] Further, the bacteria targeted by the antibacterial drug are one or more of Escherichia coli, Salmonella, and Klebsiella pneumoniae.

[0006] Further, the bacteria targeted by the antibacterial drug are polymyxin-resistant bacteria.

[0007] Further, the bacteria targeted by the antibacterial drug are multi-drug resistant Gram-negative bacteria.

[0008] Another aspect of the present invention provides an antibacterial drug, which includes 10-undecenoic acid and polymyxin.

[0009] Further, the drug further includes a pharmaceutically acceptable carrier.

[0010] Further, the polymyxin is polymyxin E sulfate or polymyxin B.

[0011] Further, when the polymyxin is polymyxin E sulfate, the mass ratio of 10-undecenoic acid to polymyxin E sulfate is 4.8 - 5000:1 - 8.

[0012] Further, when the polymyxin is polymyxin E sulfate, the treatment doses of 10-undecenoic acid and polymyxin E sulfate are 313 mg / kg body weight and 2 mg / kg body weight respectively.

[0013] Further, the dosage form of the drug is one of premix, tablet, oral liquid, capsule, sustained-release tablet, injection dosage form, cream, freeze-dried powder injection dosage form. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is the result of the checkerboard method of the synergistic antibacterial effect of 10-undecenoic acid combined with polymyxin E sulfate provided by the embodiment of the present invention against multiple Escherichia coli.

[0015] Figure 2Results of checkerboard method for the synergistic antibacterial effect of 10-undecenoic acid combined with polymyxin E sulfate against multiple Salmonella strains provided by the embodiments of the present invention.

[0016] Figure 3 Results of checkerboard method for the synergistic antibacterial effect of 10-undecenoic acid combined with polymyxin E sulfate against multiple Klebsiella pneumoniae strains provided by the embodiments of the present invention.

[0017] Figure 4 Results of the synergistic bactericidal effect of 10-undecenoic acid combined with polymyxin E sulfate against Escherichia coli B2 provided by the embodiments of the present invention.

[0018] Figure 5 Results of live / dead cell staining tests for the control group, polymyxin E sulfate group, 10-undecenoic acid group, and polymyxin E sulfate combined with 10-undecenoic acid group provided by the embodiments of the present invention.

[0019] Figure 6 Western blot results of the inhibition of the expression of the resistance enzyme MCR by 10-undecenoic acid provided by the embodiments of the present invention.

[0020] Figure 7 Evaluation of the therapeutic effects of the control group, model group, polymyxin E sulfate treatment group, 10-undecenoic acid treatment group, and polymyxin E sulfate combined with 10-undecenoic acid treatment group on Escherichia coli B2-infected skin wounds in mice provided by the embodiments of the present invention. Detailed implementation manners

[0021] The present invention will be further described in detail below in combination with the specific implementation manners. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0022] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0023] 10-Undecenoic acid used in the embodiments of the present invention, CAS No.: 112-38-9, molecular formula: C 11 H 20 O2, molecular weight: 184.28, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., with a purity of 95%; a certain amount of 10-undecenoic acid was weighed and prepared into a 10-undecenoic acid stock solution with a concentration of 100 mg / mL using DMSO, and stored at -20 °C;

[0024] Polymyxin E Sulfate (Colistin), CAS No.: 1264-72-8, molecular formula: C 52 H 100 N 16 O 17 S, molecular weight: 1253.53, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., purity ≥ 19,000 U / mg; Weigh a certain amount of Polymyxin E Sulfate and prepare a mother liquor of Polymyxin E Sulfate with a concentration of 2 mg / mL, and store it at -20 °C;

[0025] The pathogenic bacteria in the examples of the present invention:

[0026] Escherichia coli B2, Escherichia coli GZP11-5, Escherichia coli EPEC M3, Escherichia coli SC11-30, Escherichia coli 21677, Escherichia coli BW25113-pACYC184-mcr-1, Escherichia coli GZP80-8, Escherichia coli GZP08-9, Escherichia coli BL21-pet-n-gst-mcr-1, Escherichia coli MG1655-pACYC184-mcr-1-flag, Salmonella-albus 2, Salmonella-albus 3, Salmonella-albus 4, Klebsiella pneumoniae 1202+45-5, Klebsiella pneumoniae K57-KP3(CM), Klebsiella pneumoniae K57-KP6(CM) are all preserved by the National Veterinary Drug Safety Evaluation Center of China Agricultural University; among them, Escherichia coli GZP11-5, Escherichia coli SC11-30, Escherichia coli 21677, Escherichia coli BW25113-pACYC184-mcr-1, Escherichia coli GZP80-8, Escherichia coli GZP08-9, Escherichia coli BL21-pet-n-gst-mcr-1, Escherichia coli MG1655-pACYC184-mcr-1-flag, Salmonella-albus 2, Salmonella-albus 3, Salmonella-albus 4, Klebsiella pneumoniae 1202+45-5 are polymyxin-resistant strains containing MCR-1; Escherichia coli B2 is a multi-drug resistant strain containing MCR-1 and blaNDM-5; Escherichia coli EPEC-M3 is a polymyxin-resistant strain containing MCR-3; Klebsiella pneumoniae K57-KP3(CM) and Klebsiella pneumoniae K57-KP6(CM) are polymyxin-resistant mutant strains without MCR-1; among them, Escherichia coli BW25113-pACYC184-mcr-1, BL21-pet-n-gst-mcr-1, MG1655-pACYC184-mcr-1-flag are polymyxin-resistant strains containing MCR-1 obtained by plasmid transfection of BW25113, BL21, and MG1655, and are constructed and stored by this laboratory.

[0027] The bacterial culture media used in the examples of the present invention:

[0028] The CAMHB broth medium was purchased from BD Becton Dickinson Medical Devices (Shanghai) Co., Ltd., and the preparation method is as follows: Weigh 22.0 g and dissolve it in 1 L of distilled water, heat it to boiling until completely dissolved, sterilize it at 121 °C under high pressure for 15 min, and set aside;

[0029] The MH agar medium was purchased from Beijing Land Bridge Technology Co., Ltd., and the preparation method is as follows: Weigh 38.0 g and dissolve it in 1 L of distilled water, heat it to boiling until completely dissolved, sterilize it at 121 °C under high pressure for 15 min, cool it to 50 °C and pour it into plates for standby;

[0030] The brain heart infusion medium (BHI broth medium) was purchased from Beijing Land Bridge Technology Co., Ltd., and the preparation method is as follows: Weigh 24.5 g of this product, heat and stir to dissolve it in 1 L of distilled water, sterilize it at 121 °C under high pressure for 15 min, and set aside.

[0031] The following describes the specific implementation of the present invention in detail in combination with specific embodiments.

[0032] Example 1. Evaluation of the antibacterial effect of the combined use of 10-undecenoic acid and polymyxin E sulfate:

[0033] Determination of the fractional inhibitory concentration index (FICI) of the combined use of 10-undecenoic acid and polymyxin E sulfate against pathogenic bacteria: The checkerboard method was used to determine the combined antibacterial effect of 10-undecenoic acid and polymyxin E sulfate against Escherichia coli B2, Escherichia coli GZP11-5, Escherichia coli EPEC M3, Escherichia coli SC11-30, Escherichia coli 21677, Escherichia coli BW25113-pACYC184-mcr-1, Escherichia coli GZP80-8, Escherichia coli GZP08-9, Escherichia coli BL21-pet-n-gst-mcr-1, Salmonella alb 2, Salmonella alb 3, Salmonella alb 4, Klebsiella pneumoniae 1202 + 45-5, Klebsiella pneumoniae K57-KP3(CM), Klebsiella pneumoniae K57-KP6(CM);

[0034] Specifically, Escherichia coli B2, Escherichia coli GZP11-5, Escherichia coli EPEC M3, Escherichia coli SC11-30, Escherichia coli BW25113-pACYC184-mcr-1, Escherichia coli GZP80-8, Escherichia coli GZP08-9, Escherichia coli BL21-pet-n-gst-mcr-1, Salmonella-albus 2, Salmonella-albus 3, Salmonella-albus 4, Klebsiella pneumoniae 1202+45-5 were cultured normally using CAMHB broth medium. The specific operation of the checkerboard method is as follows: Add 100 μL of CAMHB broth medium to a 96-well plate. 10-undecenoic acid (as drug A) and polymyxin E sulfate (as drug B) were serially diluted with CAMHB broth medium with the highest concentrations of 5 mg / mL and 8 μg / mL respectively. Then, 100 μL of CAMHB broth medium containing different concentrations of the two drugs were added along the horizontal and vertical axes of the 96-well microplate respectively. Subsequently, 100 μL of various pathogenic bacterial suspensions were added respectively, so that the final number of bacteria per well was 2×10 5 CFU, and the plate was incubated at 37 °C for 16-18 h to observe the results. Record the minimum inhibitory concentration (MIC) of each drug alone and in combination, and calculate the FICI value (fractional inhibitory concentration index) according to the following formula;

[0035] Specifically, Escherichia coli 21677, Klebsiella pneumoniae K57-KP3 (CM), Klebsiella pneumoniae K57-KP6 (CM) were cultured normally using CAMHB broth medium. The specific operation of the checkerboard method is as follows: Add 100 μL of CAMHB broth medium to a 96-well plate. 10-undecenoic acid (as drug A) and polymyxin E sulfate (as drug B) were serially diluted with CAMHB broth medium with the highest concentrations of 10 mg / mL and 8 μg / mL respectively. Then, 100 μL of CAMHB broth medium containing different concentrations of the two drugs were added along the horizontal and vertical axes of the 96-well microplate respectively. Subsequently, 100 μL of various pathogenic bacterial suspensions were added respectively, so that the final number of bacteria per well was 2×10 5 CFU, and the plate was incubated at 37 °C for 16-18 h to observe the results. Record the minimum inhibitory concentration (MIC) of each drug alone and in combination, and calculate the FICI value (fractional inhibitory concentration index) according to the following formula;

[0036] FICI = MIC of drug A in combination / MIC of drug A alone + MIC of drug B in combination / MIC of drug B alone. The judgment criteria are as follows: FICI ≤ 0.5, synergistic effect; 0.5 < FICI ≤ 1, additive effect; 1 < FICI ≤ 2, no effect; FICI > 2, antagonistic effect. That is: FICI of 10-undecenoic acid and polymyxin E sulfate = MIC (10-undecenoic acid in combination) / MIC (10-undecenoic acid alone) + MIC (polymyxin E sulfate in combination) / MIC (polymyxin E sulfate alone).

[0037] The results of the combination of 10-undecenoic acid and polymyxin E sulfate against Escherichia coli are as Figure 1 shown:

[0038] As Figure 1 A, for Escherichia coli E.coli B2 strain, the MICs of 10-undecenoic acid and polymyxin E sulfate used alone are >5 mg / mL and 8 μg / mL respectively; when 10-undecenoic acid is in the concentration range of 0.039 - 5 mg / mL and polymyxin E sulfate is in the concentration range of 1 - 4 μg / mL, the two have obvious synergistic or additive effects. Preferably, when 10-undecenoic acid is at 0.625 mg / mL and polymyxin E sulfate is at 1 μg / mL, the two have obvious synergistic effects, and the synergy index FICI is 0.1875;

[0039] As Figure 1 B, for Escherichia coli GZP11-5 strain, the MICs of 10-undecenoic acid and polymyxin E sulfate used alone are >5 mg / mL and 8 μg / mL respectively; when 10-undecenoic acid is in the concentration range of 0.1562 - 5 mg / mL and polymyxin E sulfate is in the concentration range of 1 - 4 μg / mL, the two have obvious synergistic or additive effects. Preferably, when 10-undecenoic acid is at 1.25 mg / mL and polymyxin E sulfate is at 2 μg / mL, the two have obvious synergistic effects, and the synergy index FICI is 0.375;

[0040] As Figure 1 C, for Escherichia coli EPEC M3 strain, the MICs of 10-undecenoic acid and polymyxin E sulfate used alone are >5 mg / mL and 4 μg / mL respectively. When 10-undecenoic acid is in the concentration range of 0.0781 - 2.5 mg / mL and polymyxin E sulfate is in the concentration range of 1 - 2 μg / mL, the two have obvious synergistic or additive effects. Preferably, when 10-undecenoic acid is at 1.25 mg / mL and polymyxin E sulfate is at 1 μg / mL, the two have obvious synergistic effects, and the synergy index FICI is 0.375;

[0041] As Figure 1 D, for Escherichia coli SC11-30 strain, the MICs of 10-undecenoic acid and polymyxin E sulfate used alone are 5 mg / mL and 8 μg / mL respectively. When 10-undecenoic acid is in the concentration range of 0.1562 - 2.5 mg / mL and polymyxin E sulfate is in the concentration range of 1 - 4 μg / mL, the two have obvious synergistic or additive effects. Preferably, when 10-undecenoic acid is at 0.625 mg / mL and polymyxin E sulfate is at 1 μg / mL, the synergy index FICI is 0.25;

[0042] As Figure 1 shown in Figure E, for Escherichia coli strain 21677, the MICs of 10-undecenoic acid and polymyxin E sulfate used alone are 10 mg / mL and 8 μg / mL, respectively. When 10-undecenoic acid is in the concentration range of 0.0781 - 5 mg / mL and polymyxin E sulfate is in the concentration range of 2 - 4 μg / mL, the two have obvious synergistic or additive effects. Preferably, when 10-undecenoic acid is at 2.5 mg / mL and polymyxin E sulfate is at 2 μg / mL, the synergy index FICI is 0.5;

[0043] As Figure 1 shown in Figure F, for Escherichia coli strain BW25113-pACYC184-mcr-1, the MICs of 10-undecenoic acid and polymyxin E sulfate used alone are >5 mg / mL and 4 μg / mL, respectively. When 10-undecenoic acid is in the concentration range of 0.3125 - 5 mg / mL and polymyxin E sulfate is in the concentration range of 1 - 2 μg / mL, the two have obvious synergistic or additive effects. Preferably, when 10-undecenoic acid is at 2.5 mg / mL and polymyxin E sulfate is at 2 μg / mL, the synergy index FICI is 0.5;

[0044] As Figure 1 shown in Figure G, for Escherichia coli strain GZP08-8, the MICs of 10-undecenoic acid and polymyxin E sulfate used alone are >5 mg / mL and >8 μg / mL, respectively. When 10-undecenoic acid is in the concentration range of 0.0048 - 1.25 mg / mL and polymyxin E sulfate is in the concentration range of 2 - 8 μg / mL, the two have obvious synergistic or additive effects. Preferably, when 10-undecenoic acid is at 0.15625 mg / mL and polymyxin E sulfate is at 2 μg / mL, the synergy index FICI is 0.14;

[0045] As Figure 1 shown in Figure H, for Escherichia coli strain GZP08-9, the MICs of 10-undecenoic acid and polymyxin E sulfate used alone are >5 mg / mL and 8 μg / mL, respectively. When 10-undecenoic acid is in the concentration range of 0.0195 - 2.5 mg / mL and polymyxin E sulfate is in the concentration range of 2 - 4 μg / mL, the two have obvious synergistic or additive effects. Preferably, when 10-undecenoic acid is at 0.625 mg / mL and polymyxin E sulfate is at 2 μg / mL, the synergy index FICI is 0.3125;

[0046] As Figure 1I. For Escherichia coli BL21-pet-n-gst-mcr-1 strain, the MIC values of 10-undecenoic acid and polymyxin E sulfate used alone are 5 mg / mL and 8 μg / mL respectively. When 10-undecenoic acid is in the concentration range of 0.3125 - 2.5 mg / mL and polymyxin E sulfate is in the concentration range of 1 - 4 μg / mL, there is an obvious synergistic or additive effect between the two. Preferably, when 10-undecenoic acid is at 0.625 mg / mL and polymyxin E sulfate is at 2 μg / mL, the synergy index FICI is 0.375.

[0047] The results of the combination of 10-undecenoic acid and polymyxin E sulfate against Salmonella are as Figure 2 shown:

[0048] As Figure 2 A. For Salmonella - white 2 strain, the MIC values of 10-undecenoic acid and polymyxin E sulfate used alone are >5 mg / mL and 8 μg / mL respectively. When 10-undecenoic acid is in the concentration range of 0.039 - 2.5 mg / mL and polymyxin E sulfate is in the concentration range of 1 - 4 μg / mL, there is an obvious synergistic or additive effect between the two. Preferably, when 10-undecenoic acid is at 1.25 mg / mL and polymyxin E sulfate is at 1 μg / mL, the synergy index FICI is 0.25;

[0049] As Figure 2 B. For Salmonella - white 3 strain, the MIC values of 10-undecenoic acid and polymyxin E sulfate used alone are >5 mg / mL and 8 μg / mL respectively. When 10-undecenoic acid is in the concentration range of 0.0048 - 2.5 mg / mL and polymyxin E sulfate is in the concentration range of 1 - 4 μg / mL, there is an obvious synergistic or additive effect between the two. Preferably, when 10-undecenoic acid is at 1.25 mg / mL and polymyxin E sulfate is at 1 μg / mL, the synergy index FICI is 0.25;

[0050] As Figure 2 C. For Salmonella - white 4 strain, the MIC values of 10-undecenoic acid and polymyxin E sulfate used alone are >5 mg / mL and 8 μg / mL respectively. When 10-undecenoic acid is in the concentration range of 0.0781 - 2.5 mg / mL and polymyxin E sulfate is in the concentration range of 2 - 4 μg / mL, there is an obvious synergistic or additive effect between the two. Preferably, when 10-undecenoic acid is at 0.3125 mg / mL and polymyxin E sulfate is at 2 μg / mL, the synergy index FICI is 0.28125.

[0051] The results of the combination of 10-undecenoic acid and polymyxin E sulfate against Klebsiella pneumoniae are as Figure 3 shown:

[0052] AsFigure 3 A. Against Klebsiella pneumoniae strain 1202+45-5, the MICs of 10-undecenoic acid and polymyxin E sulfate used alone are >5 mg / mL and >8 μg / mL respectively. When 10-undecenoic acid is in the concentration range of 0.0195 - 2.5 mg / mL and polymyxin E sulfate is in the concentration range of 2 - 8 μg / mL, the two have obvious synergistic or additive effects. Preferably, when 10-undecenoic acid is at 0.625 mg / mL and polymyxin E sulfate is at 2 μg / mL, the synergy index FICI is 0.1875;

[0053] Such as Figure 3 B. Against Klebsiella pneumoniae strain K57-KP3(CM), the MICs of 10-undecenoic acid and polymyxin E sulfate used alone are 10 mg / mL and >8 μg / mL respectively. When 10-undecenoic acid is in the concentration range of 2.5 - 5 mg / mL and polymyxin E sulfate is in the concentration range of 2 - 8 μg / mL, the two have obvious synergistic or additive effects. Preferably, when 10-undecenoic acid is at 5 mg / mL and polymyxin E sulfate is at 2 μg / mL, the synergy index FICI is 0.625;

[0054] Such as Figure 3 C. Against Klebsiella pneumoniae strain K57-KP6(CM), the MICs of 10-undecenoic acid and polymyxin E sulfate used alone are 10 mg / mL and >8 μg / mL respectively. When 10-undecenoic acid is in the concentration range of 2.5 - 5 mg / mL and polymyxin E sulfate is in the concentration range of 2 - 8 μg / mL, the two have obvious synergistic or additive effects. Preferably, when 10-undecenoic acid is at 5 g / mL and polymyxin E sulfate is at 4 μg / mL, the synergy index FICI is 0.75.

[0055] Example 2. Synergistic bactericidal analysis of 10-undecenoic acid combined with polymyxin E sulfate:

[0056] 2.1 Time-kill curve experiment:

[0057] Pick a single colony of Escherichia coli B2 and culture it in BHI broth medium until the logarithmic growth phase, then reserve it. Add 10-undecenoic acid, polymyxin E sulfate, and the mixture of 10-undecenoic acid and polymyxin E sulfate to CAMHB broth medium, and add the bacterial solution to make the initial bacterial count 1×10 6 CFU / mL;

[0058] Grouping and dosage are as follows:

[0059] Control group (control): containing only the bacterial solution, with the final concentrations of 10-undecenoic acid and polymyxin E sulfate being 0;

[0060] 10-Undecenoic acid: The final concentration of 10-Undecenoic acid was 2.5 mg / mL;

[0061] Colistin sulfate group (Colistin): the final concentration of colistin sulfate was 4 μg / mL;

[0062] 10-Undecenoic acid and polymyxin E sulfate combination group: the final concentration was 2.5 mg / mL 10-Undecenoic acid + 4 μg / mL polymyxin E sulfate.

[0063] After the culture solution is fully mixed, it is placed in a constant temperature incubator and cultured at 37°C. At 1h, 3h, 6h, 12h and 24h, 100 μL of the bacterial solution is spread on a 10 cm diameter MH agar culture plate, and the colonies are counted after overnight culture.

[0064] 2.2 Test results:

[0065] like Figure 4 As shown in the figure, for Escherichia coli B2 strain, compared with the control group, polymyxin E sulfate treatment alone had no significant effect on the bacterial colony count, and 10-undecenoic acid alone reduced the number of viable bacteria, but the degree of reduction was weaker than that of the control group; when 10-undecenoic acid and polymyxin E sulfate were treated together, the number of viable bacterial colonies was significantly reduced compared with the control group and the group treated with 10-undecenoic acid or polymyxin E sulfate alone. At 1 hour, the number of viable colonies could be reduced to 0Log 10 CFU / mL, which means that the combination concentration can kill bacteria quickly and effectively;

[0066] These data indicate that the combined treatment of 10-undecenoic acid and polymyxin E sulfate has a significant synergistic bactericidal effect on Escherichia coli.

[0067] Example 3, Live / dead cell staining of 10-undecenoic acid combined with polymyxin E sulfate:

[0068] 3.1. Live / dead cell staining test of 10-undecenoic acid combined with polymyxin E sulfate:

[0069] The bacterial culture was the same as in Experimental Example 2. After culturing at 37°C for 2 h, 1 μg / mL PI dye was added to label dead cells, and 1 μM SYT09 dye was added to label live cells. The cells were incubated at 37°C in the dark for 30 min, and the bacterial activity was measured at 488 nm and 561 nm using a laser confocal microscope.

[0070] 3.2 Test results:

[0071] like Figure 5As shown, for Escherichia coli strain B2, compared with the control group, there was no change in red fluorescence after treatment with polymyxin E sulfate alone, while red fluorescence increased significantly after treatment with 10-undecenoic acid alone. When 10-undecenoic acid and polymyxin E sulfate were used in combination, the increase in red fluorescence was even more significant, indicating that the combination of 10-undecenoic acid and polymyxin E sulfate increased the number of dead bacteria.

[0072] Example 4. Inhibition of the expression of the drug-resistant enzyme MCR-1 by 10-undecenoic acid:

[0073] 4.1 Determination of the translation level of Escherichia coli Mcr-1-flag protein:

[0074] Pick a single colony of Escherichia coli MG1655-pACYC184-mcr-1-flag and inoculate it into BHI broth medium. Incubate it at 37 °C with shaking to make the bacteria in the logarithmic growth phase for standby; add polymyxin E sulfate and 10-undecenoic acid to the fresh BHI broth medium to final concentrations of 0.5 μg / mL / 1 μg / mL polymyxin E sulfate, 0.5 mM / 1 mM / 2 mM 10-undecenoic acid, 0.5 μg / mL polymyxin E sulfate + 0.5 mM / 1 mM / 2 mM 10-undecenoic acid respectively. After culturing at 37 °C for 3 h, centrifuge at 8000 rpm at 4 °C for 10 min, discard the supernatant. Add 200 μL of RIPA lysis buffer to each tube of bacterial precipitate, vortex and mix well, transfer to a new 1.5 mL centrifuge tube, incubate at room temperature with shaking for 15 min, ultrasonically disrupt, centrifuge at 12000 rpm at 4 °C for 20 min, collect the supernatant. Take 2 μL and use the BCA protein concentration assay kit to detect the protein concentration. Adjust the supernatant according to the protein concentration with RIPA lysis buffer to make the sample concentrations consistent. Then, mix according to 1 μL of 5× SDS-PAGE protein loading buffer added to every 4 μL of protein sample, and treat at 90 - 100 °C for 5 - 10 min to fully denature the protein for subsequent SDS-PAGE gel electrophoresis experiment; prepare a 10% protein gel according to the PAGE gel rapid preparation kit, perform polyacrylamide gel electrophoresis on the prepared protein samples. After electrophoresis, place them in the electrotransfer cassette in the order of filter paper, PVDF, protein gel, filter paper, and use a semi-dry transfer electrotransfer instrument for electrotransfer for 10 min. After electrotransfer, use the FLAG tag antibody to characterize the MCR protein, incubate with the primary antibody using GAPDH as the internal reference protein. After the primary antibody incubation is completed, wash it 3 times with 1× TBST solution by shaking for 10 min each time. Prepare the secondary antibody, put the target protein band into the secondary antibody solution and incubate at room temperature with slow shaking for 1 - 2 h. After incubation, wash it 3 times with 1× TBST solution by shaking for 10 min each time, and use the hypersensitive ECL chemiluminescence kit for development.

[0075] 4.2 Test results:

[0076] As Figure 6 shown, for the Escherichia coli MG1655-pACYC184-mcr-1-flag strain, the Western blot results preliminarily indicated that the natural product 10-undecenoic acid could significantly inhibit the expression of the polymyxin resistance enzyme MCR, and showed a dose-dependent manner.

[0077] Example 5, Therapeutic effect of 10-undecenoic acid combined with polymyxin E sulfate on the clearance rate of bacteria in mouse wound infections:

[0078] 5.1, Test materials:

[0079] SPF-grade BALB / c mice, female, weighing 18 - 20 g, were purchased from Guangdong Vital River Laboratory Animal Technology Co., Ltd. and raised in the animal house of Hainan Food Safety Monitoring and Testing Center. The temperature in the animal house was (25 ± 2) °C, the relative humidity was (44 ± 5)%, and there was a 12-hour light-dark cycle. The mice were fed a normal diet and had free access to water. After 1 week of adaptive feeding, the experiment was carried out; all animal experiments were approved by the Animal Ethics and Morality Committee of China Agricultural University.

[0080] 5.2, Test methods:

[0081] After 1 week of adaptive feeding in the laboratory, a skin wound model was established for all mice. The main steps were as follows: The back hair of the mice was removed with an electric hair remover, and the mice were anesthetized by intraperitoneal injection of 20% urethane at a dose of 1 mg / g per mouse; the back hair removal area was disinfected with alcohol, and a circular mark was made in advance with a marker pen. The back skin was cut with a high-pressure sterilized ophthalmic scissors to make a circular wound with a diameter of 1 cm, and the skin was cut off; Subsequently, 4 groups of mice were infected with Escherichia coli B2, and 100 μL (1×10 10 CFU / mL) of Escherichia coli B2 bacterial solution was added dropwise to the wound of each mouse, and the amount of bacteria infected in each mouse was 1×10 9CFU. In the control group, PBS was dropped onto the wound surface, and the mice were waited for the skin to absorb the dropped liquid, and the status of the mice was observed until they woke up. The mice infected with Escherichia coli B2 were divided into 4 groups, namely the model group, the polymyxin E sulfate group, the 10-undecenoic acid group, and the polymyxin E sulfate + 10-undecenoic acid group, with 6 mice in each group. On the first day of Escherichia coli B2 infection, 100 μL of normal saline, 100 μL of polymyxin E sulfate (the dose of polymyxin E sulfate given to each mouse was 2 mg / kg body weight), 100 μL of 10-undecenoic acid (the dose of 10-undecenoic acid given to each mouse was 313 mg / kg body weight), and 100 μL of a mixture of polymyxin E sulfate + 10-undecenoic acid (equivalent to giving each mouse a dose of 2 mg / kg body weight of polymyxin E sulfate and a dose of 313 mg / kg body weight of 10-undecenoic acid) were directly dropped onto the skin infection site. The drug was administered every other day. After 15 days of treatment, the mice were anesthetized and sacrificed, and the wound and the surrounding skin tissue were cut off with high-pressure sterilized surgical instruments, and the subcutaneous adhesions were dissected; the skin tissue was put into a 2 mL grinding centrifuge tube, 1 mL of PBS and grinding beads were added, and it was ground in a low-temperature tissue grinder. The skin tissue grinding program was selected and ground thoroughly at 4 °C; the tissue homogenate was diluted to an appropriate multiple, 100 μL of the diluted bacterial solution was taken, dropped onto the MH agar medium, and the bacterial solution was spread evenly with a spreader, and cultured in an incubator at 37 °C for 14-16 h, and the colony growth was observed and counted.

[0082] 5.3. Test results:

[0083] As Figure 7 shown in A, the control group only made skin wounds without bacterial infection. As time went by, the wound area gradually shrank, and the healing process was natural, and it was nearly completely healed on the 15th day; the model group was infected with Escherichia coli but not treated. The wound area was larger than that of other groups, and there was still a scab on the 15th day; compared with the model group, the use of polymyxin E sulfate or 10-undecenoic acid alone had no significant effect on Escherichia coli infection. However, after the combined use of polymyxin E sulfate and 10-undecenoic acid, the healing time of the skin infection wound in mice was accelerated, and the healing effect continued to be better than that of other groups; as Figure 7 shown in B, the average value of the bacterial load in the skin wound after the combined use of polymyxin E sulfate and 10-undecenoic acid was 4.12 Log 10 CFU. The average values of the bacterial load in the skin wounds of the polymyxin E sulfate and 10-undecenoic acid treatment groups were 5.09 Log 10 CFU and 5.89 Log 10The CFU and the bacterial load in the skin wound after the combined use of polymyxin E sulfate and 10-undecenoic acid were significantly lower than those in the treatment groups of polymyxin E sulfate and 10-undecenoic acid (****, P < 0.0001), indicating that the combination of polymyxin E sulfate and 10-undecenoic acid significantly improved the bacterial clearance rate of skin wounds compared with the treatment with polymyxin E sulfate or 10-undecenoic acid alone.

[0084] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses or improvements of the present invention, including changes made by using conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. Use of 10-undecenoic acid in combination with polymyxin in the preparation of antibacterial drugs.

2. The use according to claim 1, wherein the bacteria targeted by the antibacterial drug are one or more of Escherichia coli, Salmonella, and Klebsiella pneumoniae.

3. The application according to claim 1 or 2, characterized in that, The bacteria targeted by the antibacterial drug are polymyxin-resistant bacteria.

4. The application according to claim 1 or 2, characterized in that, The bacteria targeted by the antibacterial drug are multi-drug resistant Gram-negative bacteria.

5. An antibacterial drug, characterized in that, The drug comprises 10-undecenoic acid and polymyxin.

6. The antibacterial agent according to claim 5, characterized in that, The polymyxin is polymyxin E sulfate or polymyxin B.

7. The antibacterial drug according to claim 6, characterized in that, When the polymyxin is polymyxin E sulfate, the mass ratio of 10-undecenoic acid to polymyxin E sulfate is 4.8 - 5000:1 - 8.

8. The antibacterial agent according to claim 6, wherein When the polymyxin is polymyxin E sulfate, the therapeutic doses of 10-undecenoic acid and polymyxin E sulfate are 313 mg / kg body weight and 2 mg / kg body weight, respectively.

9. The antibacterial agent according to claim 5, wherein The dosage form of the drug is one of premix, tablet, oral liquid, capsule, sustained-release tablet, injection dosage form, cream, and freeze-dried powder injection dosage form.

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