Pharmaceutical composition for inhibiting gram-negative bacteria and application thereof

Abstract

The invention discloses a pharmaceutical composition. The active pharmaceutical ingredients of the pharmaceutical composition comprise polymyxin E and reduced glutathione. The composition can synergistically and efficiently inhibit gram-negative bacteria such as escherichia coli with the preservation number of ATCC 25922, pseudomonas aeruginosa with the preservation number of ATCC 27853, klebsiella pneumoniae with the preservation number of ATCC 700603, acinetobacter baumannii with the preservation number of ATCC 19606 and the like, and has an application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and relates to a pharmaceutical composition for inhibiting Gram-negative bacteria and its application. In particular, it relates to a composition with glutathione and polymyxin as active materials and its application in the preparation of a preparation for inhibiting several Gram-negative bacteria. Background Art

[0002] With the widespread use of antibiotics, the problem of bacterial drug resistance has become one of the major challenges facing global public health. Especially among Gram-negative bacteria, the emergence of multi-drug resistant and extensively drug-resistant strains has made traditional antibiotic treatments gradually ineffective.

[0003] Polymyxin belongs to polycationic antimicrobial peptides and has a strong and rapid bactericidal effect on most Gram-negative bacteria. Polymyxin disrupts the bacterial envelope in two stages: (1) It undergoes electrostatic interaction with the negative charge of lipid A, replacing Ca 2+ and Mg 2+ ; (2) The acylated chain inserts into the outer membrane to form pores, facilitating the entry of molecules into the periplasm. Then, the antibiotic enters the inner membrane through the hydrophilic group in the fatty acid chain. In addition, Sabnis et al. demonstrated in 2019 that the modified LPS precursor transported from the inner membrane to the outer membrane is also a target of polymyxin, resulting in cell lysis. In recent years, with the increasingly severe form of bacterial drug resistance, polymyxin has become one of the "last lines of defense" for clinical treatment of multi-drug resistant Gram-negative bacilli infections. However, due to the emergence of the polymyxin resistance gene (mcr-1), the therapeutic output of clinical polymyxin has been significantly reduced, seriously threatening the service life of this important antibacterial drug, and posing a great threat to both (veterinary) medical clinics and the field of food safety, which has attracted global attention. Developing innovative antibiotics to deal with drug-resistant bacteria is difficult, time-consuming, and costly. Therefore, the new drug development strategy of screening antibacterial adjuvants to restore the activity of existing antibacterial drugs is more economical and effective.

[0004] Glutathione (GSH) is an antioxidant synthesized in most Gram-negative bacteria and a few Gram-positive bacteria. In addition to maintaining a specific reduction potential, GSH is also related to antibacterial activity and biofilm clearance. It has been reported that under the action of 30 mM exogenous GSH, the growth and biofilm activity of all bacteria decreased by more than 50%, but the effects on different bacteria are different. Summary of the Invention

[0005] To solve the problems existing in the prior art, the first aspect of the present invention provides a pharmaceutical composition, and the pharmaceutical active ingredients of the pharmaceutical composition include a first active substance and a second active substance;

[0006] The first active substance is any one or a combination of two of polymyxin E and the medicinal salts of polymyxin E;

[0007] The second active substance is any one or a combination of two of reduced glutathione and the medicinal salts of reduced glutathione.

[0008] In some embodiments, by weight, in the pharmaceutical composition, the dosage ratio of the first active substance to the second active substance is 1:X1 to X2;

[0009] X1 is less than X2;

[0010] X1 is selected from 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 12000, 14000, 16000, 18000, 20000, 25000, 30000, 35000, 40000, 45000;

[0011] X2 is selected from 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 12000, 14000, 16000, 18000, 20000, 25000, 30000, 35000, 40000, 45000, 50000.

[0012] In some embodiments, the medicinal salts of polymyxin E include toluenesulfonate, methanesulfonate, malate, acetate, citrate, malonate, tartrate, succinate, lactate, benzoate, ascorbate, α-ketoglutarate, α-glycerophosphate, hydrochloride, sulfate, nitrate, bicarbonate, carbonate, phosphate, hydrobromide, and hydroiodide of polymyxin E.

[0013] In some embodiments, the medicinal salts of reduced glutathione include toluenesulfonate, methanesulfonate, malate, acetate, citrate, malonate, tartrate, succinate, lactate, benzoate, ascorbate, α-ketoglutarate, α-glycerophosphate, hydrochloride, sulfate, nitrate, bicarbonate, carbonate, phosphate, hydrobromide, and hydroiodide of reduced glutathione.

[0014] In some embodiments, the medicinal salts of reduced glutathione include sodium salt, potassium salt, calcium salt, iron salt, magnesium salt, zinc salt, aluminum salt, barium salt, and ammonium salt of reduced glutathione.

[0015] In some embodiments, the pharmaceutical composition further contains pharmaceutical excipients.

[0016] The second aspect of the present invention provides the use of the pharmaceutical composition described in the first aspect of the present invention in the preparation of a preparation for preventing a disease caused by bacteria, alleviating a disease caused by bacteria, treating a disease caused by bacteria, controlling a disease caused by bacteria, controlling a pollution caused by bacteria, and / or inhibiting the proliferation of bacteria, wherein the bacteria are selected from Escherichia coli with the preservation number of ATCC 25922, Pseudomonas aeruginosa with the preservation number of ATCC 27853, Klebsiella pneumoniae with the preservation number of ATCC 700603, and Acinetobacter baumannii with the preservation number of ATCC 19606.

[0017] In some embodiments, the preparation is selected from drugs, food additives, feed additives, and bactericides for environmental disinfection.

[0018] The present invention discloses for the first time the synergistic effect of reduced glutathione and polymyxin E. It is confirmed through an in vitro bacterial growth curve test that glutathione can significantly enhance the antibacterial efficacy of polymyxin, especially showing a significant effect against mcr-1 positive polymyxin-resistant Escherichia coli. The present invention reveals that glutathione can restore the antibacterial effect of polymyxin on drug-resistant strains, and while reducing the dosage of polymyxin, reverse the drug resistance of bacteria to polymyxin. The present invention helps to alleviate the increasingly serious problem of bacterial drug resistance and provides a new treatment strategy for the clinical treatment of bacterial infectious diseases, having important application value. Description of the Drawings

[0019] Figure 1 It is the time-kill curve of glutathione combined with polymyxin against Escherichia coli ATCC 25922.

[0020] Figure 2 It is the time-kill curve of glutathione combined with polymyxin against mcr-1 positive Escherichia coli B5.

[0021] Figure 3 It is a statistical chart of the effect of glutathione alone or combined with polymyxin on the growth of Gram-negative bacteria (standard Pseudomonas aeruginosa ATCC 27853, standard Klebsiella pneumoniae ATCC 700603, standard Acinetobacter baumannii ATCC 19606, standard Salmonella ATCC SL1344, standard Shigella ATCC 12022) after 6 hours of treatment. Detailed Embodiments

[0022] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be further described in detail below in conjunction with the drawings.

[0023] The experimental methods in the following examples 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 examples can be obtained from commercial sources unless otherwise specified.

[0024] Polymyxin (in each example of the present invention, polymyxin E is used, also known as colistin, Polymyxin E, also known as Colistin, CAS No.: 1066-17-7, code COL) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0025] Glutathione (in each example of the present invention, reduced glutathione is used, Glutathione, CAS No.: 70-18-8, code GSH) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0026] The preparation methods of the culture media involved in the following examples are as follows:

[0027] MH broth medium: Purchased from Beijing Land Bridge Technology Co., Ltd., and the preparation method is as follows: Weigh 25.0 g of the culture medium powder, dissolve it in 1 L of distilled water, adjust the pH to 7.0 ± 0.1, heat to boiling until completely dissolved, sterilize at 121 °C under high pressure for 15 min, and store it for later use after cooling.

[0028] MH solid medium: Purchased from Beijing Land Bridge Technology Co., Ltd., and the preparation method is as follows: Weigh 38.0 g of the culture medium powder, dissolve it in 1 L of distilled water, adjust the pH to 7.0 ± 0.1, heat to boiling until completely dissolved, sterilize at 121 °C under high pressure for 15 min, and pour the plate when cooled to 55 °C for later use.

[0029] Escherichia coli strain B5 is a wild Escherichia coli strain isolated from the applicant's laboratory. The bacterial species was identified as Escherichia coli by 16S rDNA, and through genomic sequencing analysis, the mcr-1 gene was positive.

[0030] Standard Escherichia coli ATCC 25922, standard Pseudomonas aeruginosa ATCC 27853, standard Klebsiella pneumoniae ATCC 700603, standard Acinetobacter baumannii ATCC 19606, standard Salmonella ATCC SL1344, and standard Shigella ATCC 12022 were purchased from the American Type Culture Collection (ATCC).

[0031] Example 1: Determination of the MIC of Polymyxin against Gram-negative Bacteria

[0032] According to the method of the CLSI standard 2024 version, the minimum inhibitory concentration (MIC) of polymyxin against various Gram-negative bacteria was determined by the broth microdilution method. The specific steps are as follows:

[0033] 1. Adjust the broth cultures of various Gram-negative bacteria in the logarithmic growth phase to 0.5 McFarland turbidity using a McFarland nephelometer, and then dilute them 100-fold with MH broth medium to obtain bacterial dilution solutions with a concentration of approximately 1×10 6 CFU / mL. The specific types of bacteria are shown in Table 1.

[0034] 2. Weigh an appropriate amount of polymyxin powder, dissolve it in deionized water to obtain a polymyxin solution with a concentration of 10240 μg / mL, and then filter it through a sterile filter membrane (pore size 0.22 μm) to obtain a stock solution.

[0035] 3. Take a 96-well microplate, add the polymyxin stock solution to the first well, and perform a 2-fold volume gradient dilution with MH broth medium to obtain 100 μL of drug solutions with different dilution degrees per well. Then inoculate 100 μL of the bacterial dilution solution into each well. The concentration range of polymyxin used is 0 - 512 μg / mL. Among them, the positive control is the bacterial dilution solution (without drug), and the blank control is MH broth medium.

[0036] 4. Incubate the 96-well microplate completed in step 3 at 37 °C for 16 - 18 h.

[0037] Experimental results: The MIC values of polymyxin are shown in Table 1. For Escherichia coli ATCC 25922, it is 2 μg / mL; for Escherichia coli E.coli B5, it is 16 μg / mL; for Pseudomonas aeruginosa ATCC 27853, it is 2 μg / mL; for Klebsiella pneumoniae ATCC700603, it is 1 μg / mL; for Acinetobacter baumannii ATCC 19606, it is 1 μg / mL; for Salmonella enterica subsp. enterica serovar Typhimurium ATCC SL1344, it is 2 μg / mL; for Shigella flexneri ATCC 12022, it is 1 μg / mL.

[0038] Example 2: Determination of the MIC of glutathione against Gram-negative bacteria

[0039] Set different concentration gradients of glutathione, and determine the minimum inhibitory concentration (MIC) of glutathione against various Gram-negative bacteria through an in vitro bactericidal experiment. The specific steps are as follows:

[0040] 1. Adjust the broth cultures of various Gram-negative bacteria in the logarithmic growth phase to 0.5 McFarland turbidity using a McFarland nephelometer. The specific types of bacteria are shown in Table 1.

[0041] 2. Weigh an appropriate amount of glutathione powder, dissolve it in MH broth medium to obtain a glutathione solution with a concentration of 60 mg / mL, and then filter it through a sterile filter membrane (pore size 0.22 μm) to obtain a glutathione stock solution.

[0042] 3. Add 1 mL of MH broth medium into a sterile 1.5 mL EP tube. By adding glutathione stock solution with different volumes, the set concentration range is MH broth medium containing glutathione at a concentration of 2 - 8 mg / mL. Then inoculate 10 μL of bacterial suspension with a turbidity of 0.5 McFarland into each tube, so that the bacterial concentration in each tube is 1×10 6 CFU / mL.

[0043] 4. Incubate the 96-well microplate completed in step 3 at 37 °C for 16 - 18 h.

[0044] Experimental results: The MIC values of glutathione are shown in Table 1. For Escherichia coli ATCC 25922, it is 8 mg / mL; for Escherichia coli E.coli B5, it is 8 mg / mL; for Pseudomonas aeruginosa ATCC 27853, it is 6 mg / mL; for Klebsiella pneumoniae ATCC700603, it is 8 mg / mL; for Acinetobacter baumannii ATCC 19606, it is 6 mg / mL; for Salmonella enterica ATCC SL1344, it is 8 mg / mL; for Shigella flexneri ATCC 12022, it is 6 mg / mL.

[0045] Table 1. MIC values of polymyxin and glutathione

[0046] Strain name Polymyxin (μg / mL) Glutathione (mg / mL) Escherichia coli ATCC 25922 2 8 Escherichia coli E.coli B5 16 8 Pseudomonas aeruginosa ATCC 27853 2 6 Klebsiella pneumoniae ATCC 700603 1 8 Acinetobacter baumannii ATCC 19606 1 6 Salmonella ATCC SL1344 2 8 Shigella ATCC 12022 1 6

[0047] Example 3: Time-kill curve of glutathione combined with polymyxin against polymyxin-resistant bacteria and standard bacteria

[0048] This example studied the colony counts of mcr-1 positive polymyxin-resistant bacteria and polymyxin-sensitive bacteria after combined use of glutathione. The polymyxin-resistant bacteria is mcr-1 positive Escherichia coli B5. The polymyxin-sensitive standard bacteria is Escherichia coli ATCC 25922.

[0049] The specific steps are as follows:

[0050] 1. Take the overnight culture bacterial suspension and dilute it 100 times with MH broth medium to obtain a bacterial dilution, and the concentration of the bacteria is about 1×10 8 CFU / mL.

[0051] 2. Take 24 test tubes. Add 2970 μL of MH broth medium and 30 μL of mcr-1 positive Escherichia coli B5 bacterial dilution into 12 test tubes and mix well; add 2970 μL of MH broth medium and 30 μL of Escherichia coli ATCC 25922 bacterial dilution into 12 test tubes and mix well; then randomly divide the 12 test tubes corresponding to each strain into 4 groups: glutathione group, polymyxin group, combined group and blank group, with 3 test tubes in each group, and perform the following treatments:

[0052] Glutathione group: Add 300 μL of glutathione solution with a concentration of 60 mg / mL to each test tube, so that the concentration of glutathione in the system is about 6 mg / mL. This is the 0 h; then culture at 37 °C and 200 rpm for 24 h.

[0053] Polymyxin group: For Escherichia coli ATCC 25922, add 4.69 μL of polymyxin solution with a concentration of 640 μg / mL to 3 test tubes respectively, so that the concentration of polymyxin in the system is about 1 μg / mL; for mcr-1 positive Escherichia coli B5, add 37.5 μL of polymyxin solution with a concentration of 640 μg / mL to 3 test tubes, so that the concentration of polymyxin in the system is about 8 μg / mL. This is the 0 h; then culture at 37 °C and 200 rpm for 24 h.

[0054] Combined group: For Escherichia coli ATCC 25922, add 300 μL of glutathione solution with a concentration of 60 mg / mL and 4.69 μL of polymyxin solution with a concentration of 640 μg / mL to 3 test tubes respectively, so that the concentration of glutathione in the system is about 6 mg / mL and the concentration of polymyxin in the system is about 1 μg / mL; for mcr-1 positive Escherichia coli B5, add 300 μL of glutathione solution with a concentration of 60 mg / mL and 37.5 μL of polymyxin solution with a concentration of 640 μg / mL to 3 test tubes respectively, so that the concentration of glutathione in the system is about 6 mg / mL and the concentration of polymyxin in the system is about 8 μg / mL. This is the 0 h; then culture at 37 °C and 200 rpm for 24 h;

[0055] Blank group: Do not add any drugs to the test tubes. This is the 0 h; then culture at 37 °C and 200 rpm for 24 h.

[0056] During the culture period, take 100 μL of the culture solution at 0 h, 4 h, 8 h, 12 h and 24 h respectively and spread it on the MH solid medium, and culture at 37 °C and 200 rpm for 24 h for colony counting.

[0057] 3. After completing step 2, use the culture time as the abscissa and the log10 value of the colony number as the ordinate to draw a time-killing curve.

[0058] The time-killing curve of Escherichia coli ATCC 25922 is shown in Figure 1 and the time-killing curve of mcr-1 positive Escherichia coli B5 is shown in Figure 2 (CFU is colony forming unit).

[0059] The results showed that at the drug concentration of this example, the glutathione group, polymyxin group, and blank group could not inhibit polymyxin-resistant bacteria and sensitive bacteria, and there was no significant difference among the three groups; the combined group could significantly inhibit polymyxin-resistant bacteria and sensitive bacteria. Compared with the glutathione group, polymyxin group, and blank group, the inhibitory effect of the combined group on polymyxin-resistant bacteria and sensitive bacteria was significantly improved and the inhibitory effect was stable.

[0060] Example 4: Effects of glutathione alone or in combination with polymyxin on the growth of standard Pseudomonas aeruginosa ATCC 27853 after 6 hours of treatment

[0061] In this example, the effects of glutathione alone or in combination with drugs on the growth of standard Pseudomonas aeruginosa ATCC 27853 were studied by colony counting method.

[0062] The specific steps are as follows:

[0063] 1. Take the overnight culture of standard Pseudomonas aeruginosa ATCC 27853 bacterial solution, dilute it 100-fold with MH broth medium to obtain a bacterial dilution, and the concentration of the bacteria is about 1×10 8 CFU / mL.

[0064] 2. Take 12 test tubes, add 2970 μL of MH broth medium and 30 μL of bacterial dilution to each tube, and mix well; then randomly divide the 12 test tubes into 4 groups: glutathione group, polymyxin group, combined group, and blank group, with 3 test tubes in each group, and perform the following treatments:

[0065] Glutathione group: Add 200 μL of glutathione solution with a concentration of 60 mg / mL (the solvent is water, the same in Examples 4-8) to each test tube, so that the concentration of glutathione in the system is about 4 mg / mL, and this is the 0 h; then culture at 37 °C and 200 rpm for 6 h;

[0066] Polymyxin group: Add 4.69 μL of polymyxin solution with a concentration of 640 μg / mL (the solvent is water, the same in Examples 4-8) to each test tube, so that the concentration of polymyxin in the system is about 1 μg / mL, and this is the 0 h; then culture at 37 °C and 200 rpm for 6 h;

[0067] Combined group: Add 200 μL of glutathione solution with a concentration of 60 mg / mL and 4.69 μL of polymyxin solution with a concentration of 640 μg / mL to each test tube, so that the concentration of glutathione in the system is about 4 mg / mL and the concentration of polymyxin in the system is about 1 μg / mL, and this is the 0 h; then culture at 37 °C and 200 rpm for 6 h;

[0068] Blank group: No drug is added to the test tube, which is the 0 h at this time; then culture at 37 °C and 200 rpm for 6 h.

[0069] During the culture period, take 100 μL of the culture solution at the 6th h and spread it on the MH solid medium, and culture at 37 °C and 200 rpm for 24 h for colony counting.

[0070] 3. After completing step 2, use the culture time as the abscissa and the log10 value of the number of colonies per mL of the bacterial solution as the ordinate to draw a graph of the bacterial growth situation. The results are shown in Figure 3 (CFU is the colony forming unit).

[0071] The results show that at the drug concentration in this example, the glutathione group, the polymyxin group and the blank group cannot inhibit the standard Pseudomonas aeruginosa ATCC 27853, and there is no significant difference among the three groups; the combined group can significantly inhibit the standard Pseudomonas aeruginosa ATCC 27853. Compared with the glutathione group, the polymyxin group and the blank group, the effect of the combined group in inhibiting the standard Pseudomonas aeruginosa ATCC 27853 is significantly improved.

[0072] Example 5: Effect of glutathione alone or in combination with polymyxin on the growth of standard Klebsiella pneumoniae ATCC 700603 after 6 h of treatment

[0073] In this example, the effect of glutathione alone or in combination with drugs on the growth of standard Klebsiella pneumoniae ATCC 700603 was studied by the colony counting method.

[0074] The specific steps are as follows:

[0075] 1. Take the overnight cultured standard Klebsiella pneumoniae ATCC 700603 bacterial solution and dilute it 100-fold with MH broth medium to obtain a bacterial dilution, and the concentration of the bacteria is about 1×10 8 CFU / mL.

[0076] 2. Take 12 test tubes, add 2970 μL of MH broth medium and 30 μL of the bacterial dilution to each tube, and mix well; then randomly divide the 12 test tubes into 4 groups: the glutathione group, the polymyxin group, the combined group and the blank group, with 3 test tubes in each group, and perform the following treatments:

[0077] Glutathione group: Add 300 μL of glutathione solution with a concentration of 60 mg / mL to each test tube, so that the concentration of glutathione in the system is about 6 mg / mL, which is the 0 h at this time; then culture at 37 °C and 200 rpm for 6 h;

[0078] Polymyxin group: Add 2.34 μL of polymyxin solution with a concentration of 640 μg / mL to each test tube, so that the concentration of polymyxin in the system is about 0.5 μg / mL. This is the 0 h; then incubate at 37 °C and 200 rpm for 6 h.

[0079] Combined group: Add 300 μL of glutathione solution with a concentration of 60 mg / mL and 2.34 μL of polymyxin solution with a concentration of 640 μg / mL to each test tube, so that the concentration of glutathione in the system is about 6 mg / mL and the concentration of polymyxin in the system is about 0.5 μg / mL. This is the 0 h; then incubate at 37 °C and 200 rpm for 6 h.

[0080] Blank group: Do not add any drugs to the test tubes. This is the 0 h; then incubate at 37 °C and 200 rpm for 6 h.

[0081] During the incubation period, take 100 μL of the culture solution at the 6th h and spread it on the MH solid medium, and incubate at 37 °C and 200 rpm for 24 h for colony counting.

[0082] 3. After completing step 2, use the incubation time as the abscissa and the log10 value of the number of colonies per mL of the bacterial solution as the ordinate to plot the bacterial growth situation graph. The results are shown in Figure 3 (CFU is colony-forming unit).

[0083] The results show that at the drug concentration in this example, the glutathione group, the polymyxin group, and the blank group could not inhibit Klebsiella pneumoniae ATCC 700603, and there was no significant difference among the three groups; the combined group could significantly inhibit Klebsiella pneumoniae ATCC 700603. Compared with the glutathione group, the polymyxin group, and the blank group, the effect of the combined group in inhibiting Klebsiella pneumoniae ATCC 700603 was significantly improved.

[0084] Example 6: Effect of glutathione alone or combined with polymyxin treatment for 6 h on the growth of Acinetobacter baumannii ATCC 19606

[0085] In this example, the effect of glutathione alone or in combination on the growth of Acinetobacter baumannii ATCC 19606 was studied by the colony counting method.

[0086] The specific steps are as follows:

[0087] 1. Take the overnight culture of Acinetobacter baumannii ATCC 19606 bacterial solution and dilute it 100-fold with MH broth medium to obtain a bacterial dilution solution, and the concentration of the bacteria is about 1×10 8 CFU / mL.

[0088] 2. Take 12 test tubes, add 2970 μL of MH broth medium and 30 μL of bacterial dilution to each tube, and mix well. Then randomly divide the 12 test tubes into 4 groups: glutathione group, polymyxin group, combination group, and blank group, with 3 test tubes in each group, and perform the following treatments:

[0089] Glutathione group: Add 200 μL of glutathione solution with a concentration of 60 mg / mL to each test tube, so that the concentration of glutathione in the system is 4 mg / mL. This is the 0 h. Then incubate at 37 °C and 200 rpm for 6 h.

[0090] Polymyxin group: Add 2.34 μL of polymyxin solution with a concentration of 640 μg / mL to each test tube, so that the concentration of polymyxin in the system is 0.5 μg / mL. This is the 0 h. Then incubate at 37 °C and 200 rpm for 6 h.

[0091] Combination group: Add 200 μL of glutathione solution with a concentration of 60 mg / mL and 2.34 μL of polymyxin solution with a concentration of 640 μg / mL to each test tube, so that the concentration of glutathione in the system is approximately 4 mg / mL and the concentration of polymyxin in the system is approximately 0.5 μg / mL. This is the 0 h. Then incubate at 37 °C and 200 rpm for 6 h.

[0092] Blank group: Do not add any drugs to the test tubes. This is the 0 h. Then incubate at 37 °C and 200 rpm for 6 h.

[0093] During the incubation period, take 100 μL of the culture solution at 6 h and spread it on the MH solid medium, and incubate at 37 °C and 200 rpm for 24 h for colony counting. <x

[0094] 3. After completing step 2, use the incubation time as the abscissa and the log10 value of the number of colonies per mL of the bacterial solution as the ordinate to plot the bacterial growth curve. The results are shown in Figure 3 (CFU is the colony forming unit).

[0095] The results show that at the drug concentrations in this example, the glutathione group, polymyxin group, and blank group could not inhibit Acinetobacter baumannii ATCC 19606, and there was no significant difference among the three groups; the combination group could significantly inhibit Acinetobacter baumannii ATCC 19606. Compared with the glutathione group, polymyxin group, and blank group, the inhibitory effect of the combination group on Acinetobacter baumannii ATCC 19606 was significantly improved.

[0096] Example 7: Effect of glutathione alone or in combination with polymyxin on the growth of Salmonella enterica subsp. enterica serovar Typhimurium ATCC SL1344 after 6 h of treatment

[0097] In this example, the effects of glutathione alone or in combination with other drugs on the growth of standard Salmonella ATCC SL1344 were studied by colony counting method.

[0098] The specific steps are as follows:

[0099] 1. Take the overnight cultured standard Salmonella ATCC SL1344 bacterial solution, dilute it 100 times with MH broth medium to obtain a bacterial dilution, and the concentration of the bacteria is about 1×10 8 CFU / mL.

[0100] 2. Take 12 test tubes, add 2970 μL of MH broth medium and 30 μL of bacterial dilution to each test tube, and mix well; then randomly divide the 12 test tubes into 4 groups: glutathione group, polymyxin group, combination group and blank group, with 3 test tubes in each group, and perform the following treatments:

[0101] Glutathione group: Add 300 μL of glutathione solution with a concentration of 60 mg / mL to each test tube, so that the concentration of glutathione in the system is about 6 mg / mL. This is the 0 h; then culture at 37 °C and 200 rpm for 6 h;

[0102] Polymyxin group: Add 4.69 μL of polymyxin solution with a concentration of 640 μg / mL to each test tube, so that the concentration of polymyxin in the system is about 1 μg / mL. This is the 0 h; then culture at 37 °C and 200 rpm for 6 h;

[0103] Combination group: Add 300 μL of glutathione solution with a concentration of 60 mg / mL and 4.69 μL of polymyxin solution with a concentration of 640 μg / mL to each test tube, so that the concentration of glutathione in the system is about 6 mg / mL and the concentration of polymyxin in the system is about 1 μg / mL. This is the 0 h; then culture at 37 °C and 200 rpm for 6 h;

[0104] Blank group: Do not add any drugs to the test tubes. This is the 0 h; then culture at 37 °C and 200 rpm for 6 h.

[0105] During the culture period, take 100 μL of the culture solution at the 6th h and spread it on the MH solid medium, and culture at 37 °C and 200 rpm for 24 h for colony counting.

[0106] 3. After completing step 2, use the culture time as the abscissa and the log10 value of the number of colonies in each mL of the bacterial solution as the ordinate to draw a graph of the bacterial growth situation. The results are shown in Figure 3 (CFU is the colony forming unit).

[0107] The results showed that at the drug concentration of this example, the glutathione group, polymyxin group, combination group, and blank group could not inhibit the standard Salmonella ATCC SL1344, and there was no significant difference among the four groups.

[0108] Example 8: Effect of glutathione alone or in combination with polymyxin on the growth of standard Shigella ATCC 12022 after 6 hours of treatment

[0109] In this example, the effect of glutathione alone or in combination with drugs on the growth of standard Shigella ATCC 12022 was studied by the colony counting method.

[0110] The specific steps are as follows:

[0111] 1. Take the overnight culture of standard Shigella ATCC 12022 bacterial solution, dilute it 100-fold with MH broth medium to obtain a bacterial dilution, and the concentration of the bacteria is about 1×10 8 CFU / mL.

[0112] 2. Take 12 test tubes, add 2970 μL of MH broth medium and 30 μL of bacterial dilution to each test tube, and mix well; then randomly divide the 12 test tubes into 4 groups: glutathione group, polymyxin group, combination group, and blank group, with 3 test tubes in each group, and perform the following treatments:

[0113] Glutathione group: Add 200 μL of glutathione solution with a concentration of 60 mg / mL to each test tube, so that the concentration of glutathione in the system is about 4 mg / mL. This is the 0 h; then culture at 37 °C and 200 rpm for 6 h;

[0114] Polymyxin group: Add 2.34 μL of polymyxin solution with a concentration of 640 μg / mL to each test tube, so that the concentration of polymyxin in the system is about 0.5 μg / mL. This is the 0 h; then culture at 37 °C and 200 rpm for 6 h;

[0115] Combination group: Add 200 μL of glutathione solution with a concentration of 60 mg / mL and 2.34 μL of polymyxin solution with a concentration of 640 μg / mL to each test tube, so that the concentration of glutathione in the system is about 4 mg / mL and the concentration of polymyxin in the system is about 0.5 μg / mL. This is the 0 h; then culture at 37 °C and 200 rpm for 6 h;

[0116] Blank group: Do not add any drugs to the test tubes. This is the 0 h; then culture at 37 °C and 200 rpm for 6 h.

[0117] During the culture period, take 100 μL of the culture solution at the 6th hour and spread it on the MH solid medium, and culture at 37 °C and 200 rpm for 24 h for colony counting.

[0118] 3. After completing Step 2, plot the bacterial growth curve with the culture time as the abscissa and the log10 value of the number of colonies per mL of bacterial suspension as the ordinate. The results are shown in Figure 3 (CFU stands for colony-forming unit).

[0119] The results showed that at the drug concentration in this example, the glutathione group, the polymyxin group, the combination group and the blank group could not inhibit Shigella flexneri ATCC 12022, and there was no significant difference among the four groups.

[0120] Therefore, it can be seen that glutathione combined with polymyxin can restore the inhibitory effect of polymyxin on polymyxin-resistant Escherichia coli and reduce the amount of polymyxin used against standard strains (standard Escherichia coli ATCC 25922, standard Pseudomonas aeruginosa ATCC 27853, standard Klebsiella pneumoniae ATCC 700603, standard Acinetobacter baumannii ATCC 19606), that is, glutathione combined with polymyxin can inhibit polymyxin-resistant bacteria and standard bacteria.

[0121] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit 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 general, according to the principle of the present invention, this application intends to cover any modification, use or improvement of the present invention, including those that depart from the scope disclosed in this application but are made by conventional techniques known in the art.

Claims

1. A pharmaceutical composition, wherein the pharmaceutically active ingredients of the pharmaceutical composition include a first active substance and a second active substance; The first active substance is any one or a combination of two of polymyxin E and pharmaceutically acceptable salts of polymyxin E; The second active substance is any one or a combination of two of reduced glutathione and pharmaceutically acceptable salts of reduced glutathione.

2. The pharmaceutical composition according to claim 1, wherein By weight, in the pharmaceutical composition, the dosage ratio of the first active substance to the second active substance is 1:X1 to X2; X1 is less than X2; X1 is selected from 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 12000, 14000, 16000, 18000, 20000, 25000, 30000, 35000, 40000, 45000; X2 is selected from 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 12000, 14000, 16000, 18000, 20000, 25000, 30000, 35000, 40000, 45000, 50000.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that, The pharmaceutically acceptable salts of polymyxin E include toluenesulfonate, methanesulfonate, malate, acetate, citrate, malonate, tartrate, succinate, lactate, benzoate, ascorbate, α-ketoglutarate, α-glycerophosphate, hydrochloride, sulfate, nitrate, bicarbonate, carbonate, phosphate, hydrobromide and hydroiodide of polymyxin E.

4. The pharmaceutical composition according to claim 1 or 2, characterized in that, The pharmaceutically acceptable salts of reduced glutathione include toluenesulfonate, methanesulfonate, malate, acetate, citrate, malonate, tartrate, succinate, lactate, benzoate, ascorbate, α-ketoglutarate, α-glycerophosphate, hydrochloride, sulfate, nitrate, bicarbonate, carbonate, phosphate, hydrobromide and hydroiodide of reduced glutathione.

5. The pharmaceutical composition according to claim 1 or 2, characterized in that, The pharmaceutically acceptable salts of reduced glutathione include sodium salt, potassium salt, calcium salt, iron salt, magnesium salt, zinc salt, aluminum salt, barium salt and ammonium salt of reduced glutathione.

6. The pharmaceutical composition according to any one of claims 1-5, characterized in that, The pharmaceutical composition further contains pharmaceutical excipients.

7. Use of the pharmaceutical composition according to any one of claims 1-6 in the preparation of a preparation for preventing a disease caused by bacteria, alleviating a disease caused by bacteria, treating a disease caused by bacteria, controlling a disease caused by bacteria, controlling pollution caused by bacteria and / or inhibiting the proliferation of bacteria, wherein the bacteria are selected from Escherichia coli with the preservation number of ATCC 25922, Pseudomonas aeruginosa with the preservation number of ATCC 27853, Klebsiella pneumoniae with the preservation number of ATCC 700603 and Acinetobacter baumannii with the preservation number of ATCC19606.

8. The application according to claim 6, characterized in that The preparation is selected from drugs, food additives, feed additives, bactericides for environmental disinfection.

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