CN113117048A

CN113117048A - Application of anti-multiple sclerosis drug Fty720 and polymyxin system in resisting Klebsiella pneumoniae

Info

Publication number: CN113117048A
Application number: CN202110378011.3A
Authority: CN
Inventors: 麻彤辉; 陈秀丽; 马骁驰
Original assignee: Nanjing University of Chinese Medicine
Current assignee: Nanjing University of Chinese Medicine
Priority date: 2021-04-08
Filing date: 2021-04-08
Publication date: 2021-07-16
Anticipated expiration: 2041-04-08
Also published as: CN113117048B

Abstract

An application of a multiple sclerosis resistant drug Fty720 and a polymyxin system in resisting Klebsiella pneumoniae belongs to the technical field of drug preparation. Fty720 is obtained by modifying ISP-I with immunosuppressive effect in Chinese medicinal Cordyceps extract, and can be used for treating multiple sclerosis with good tolerance and low side effect. The invention discovers that Fty720 has the effect of remarkably reversing the drug resistance of Klebsiella pneumoniae and the low-dose Fty720 can reduce the MIC of the Klebsiella pneumoniae to more than 16 mu g/mL to MIC =0.0625 mu g/mL, so that the sensitivity of the Klebsiella pneumoniae to the polymyxin is increased by 256 times, the invention also shows good synergistic drug resistance effect in the level of an animal body, can remarkably prolong the life cycle of a mouse under bacterial infection and has important reagent application value.

Description

Application of anti-multiple sclerosis drug Fty720 and polymyxin system in resisting Klebsiella pneumoniae

Technical Field

The invention relates to an application of a multiple sclerosis resistant drug Fty720 and a polymyxin system in resisting Klebsiella pneumoniae, belonging to the technical field of drug preparation.

Background

Klebsiella pneumoniae is the most common gram-negative bacillus in Klebsiella pneumoniae of Enterobacteriaceae, is planted in multiple organ parts of human respiratory tracts, intestinal tracts and the like, can cause local or even systemic infection of human bodies, and causes diseases accounting for more than 95 percent of the Klebsiella pneumoniae infection of the Enterobacteriaceae. The world health organization in 2017 publishes a list of 12 pathogenic and drug-resistant pathogens, the carbapenem-resistant Klebsiella pneumoniae is listed as one of three major bacteria with high threat to human health, and in recent years, the carbapenem-resistant Klebsiella pneumoniae shows an annual prevalence trend in clinic, so that the polymyxin with broad-spectrum resistance to gram-negative bacteria is considered as the last line of defense against pan-resistant gram-negative bacteria.

Polymyxin is a cationic polypeptide antibiotic that acts initially targeted to the outer membrane of gram-negative bacilli, with positively charged polymyxin residues binding to the phosphate group of negatively charged lipid a, resulting in divalent cations (Ca)²⁺，Mg²⁺) Transfer from negatively charged membrane lipophosphate groups, resulting in destruction of lipopolysaccharide, increased cell membrane permeability, and extravasation of cytoplasmic material. However, with the increasing clinical use of polymyxin, polymyxin-resistant pathogenic bacteria have been developed in recent years. Although polymyxin has a high antibacterial effect, high-dose polymyxin has severe nephrotoxicity and neurotoxicity to human body, which is also a dose-use limiting factor for clinical treatment of polymyxin. Therefore, the discovery of an economical, effective and small-side-effect drug combination therapy becomes a new idea for overcoming the drug resistance and clinical dose use limitation of polymyxin.

Fty720 (Fingolimod), which is modified from ISP-I which is a component with immunosuppressive action in Chinese caterpillar fungus extract, is used for treating multiple sclerosis and has good tolerance and low side effect. The subject first finds that the polymyxin-resistant drug has the effect of reversing polymyxin resistance. No report on the reversal of drug resistance of Klebsiella pneumoniae polymyxin by Fty720 (Fingolimod) is available.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a new application of a multiple sclerosis resistant drug (Fty 720) in reversing polymyxin resistance of Klebsiella pneumoniae, and finds that the Fty720 has the effect of remarkably reversing the polymyxin resistance of the Klebsiella pneumoniae, the MIC (minimum integrated circuit) of the polymyxin in resistance to the Klebsiella pneumoniae is reduced to MIC =0.0625 μ g/mL by using a low in vitro dose (8 μ g/mL), the combination of the Fty720 and the polymyxin B at an animal level can remarkably treat abdominal cavity infection caused by the polymyxin resistance to the Klebsiella pneumoniae, and the pathological life of a mouse under severe infection is prolonged.

The technical scheme adopted by the invention is as follows: the use of the anti-multiple sclerosis drug Fty720 in combination with polymyxin in antibacterial medicaments.

The antibacterial drug is a drug for resisting polymyxin-resistant Klebsiella pneumoniae.

A pharmaceutical composition for use against klebsiella pneumoniae comprising a polymyxin and Fty720 in combination with the polymyxin.

In the pharmaceutical composition, the dosage ratio of polymyxin to Fty720 is 1: 4-10.

The low-dose Fty720 and polymyxin B have obvious synergistic effect of resisting polymyxin-resistant Klebsiella pneumoniae, have good antibacterial activity in vivo and in vitro, and can obviously prolong the survival period of mice after bacterial infection.

The invention has the beneficial effects that: the Fty720 is modified as a clinically common component ISP-I with an immunosuppressive effect, is used for treating multiple sclerosis, has good tolerance and low side effect, the invention finds a new application in reversing drug resistance of Klebsiella pneumoniae and polymyxin, the Fty720 has no obvious inhibitory effect on the polymyxin-resistant Klebsiella pneumoniae after being singly used, and the low dose (8 mu g/mL) can reduce the MIC (minimum integrated concentration) of the polymyxin against the Klebsiella pneumoniae from more than 16 mu g/mL to MIC =0.0625 mu g/mL, so that the sensitivity of the Klebsiella pneumoniae to the polymyxin is increased by 256 times. And at the in vivo level in animals, the combination of Fty720 (8. mu.g/mL) and polymyxin B (2. mu.g/mL) significantly improved survival and increased survival time in mice under severe infection. The compound also shows good synergistic drug-resistant bacterium resistance in the level of animal bodies, can obviously prolong the survival period of mice under bacterial infection, and has important reagent application value.

Drawings

FIG. 1 shows the in vitro inhibitory concentration and dynamic detection of bacterial growth of Fty720 in combination with polymyxin B.

FIG. 2 is a graph of the effect of Fty720 in combination with polymyxin B on survival in mice treated for celiac infection within 0-72 hr.

Detailed Description

The present invention will be described in detail with reference to the following embodiments. It should be understood that the summary of the detailed description section is illustrative and not restrictive, i.e., does not set forth any limitations on the summary of the invention.

Defining: IC (integrated circuit)₅₀The concentration corresponding to the 50% inhibitory concentration, i.e., B/B0=50%, the lower the half inhibition, which is used to measure the sensitivity of the antibody, indicates the higher the sensitivity of the antibody.

MIC is the minimum inhibitory concentration, is an index for measuring the antibacterial activity of the antibacterial drug, and is the minimum drug concentration capable of inhibiting the growth of pathogenic bacteria in a culture medium after bacteria are cultured in vitro for 18 to 24 hours.

MBC is the lowest bactericidal concentration, which is the lowest concentration capable of killing bacteria in a culture medium, namely 99.9 percent of test microorganisms are killed, and the lowest drug concentration required by the test microorganisms with 3 orders of magnitude is reduced.

Fty720 (Fingolimod) is an immunosuppressant, is modified from ISP-I which is a component with immunosuppressive effect in Chinese caterpillar fungus extract, is used for treating multiple sclerosis, and has good tolerance and low side effect.

EXAMPLE 1 Compound Fty720 reverses the in vitro antibacterial Activity of Klebsiella pneumoniae polymyxin resistance

Drugs and reagents:

polymyxin-resistant Klebsiella pneumoniae (BAA-1705) was purchased from ATCC platforms. The nutrient broth, the beef powder and the peptone are all products of Beijing Ooboxin Biotechnology Limited liability company. Yeast extract, tryptone, are all products of OXOID, England.

Fty720 reverse Klebsiella pneumoniae polymyxin drug resistance activity assay

Reference is made to CLSI (American society for clinical standards) M07-A9 broth microdilution.The bacterial liquid with 600 nm OD value of 1 is diluted to 0.5 McLeod turbidity by a McLeod turbidimetric tube, and is continuously diluted by 1000 times to obtain experimental bacterial liquid, 200 mul of LB culture solution is added into the wells of 1, 2 rows, 11 and 12 rows of a 96-well plate by a microsyringe for later use as blank control, and 200 mul of experimental bacterial liquid is added into the other wells. Fty720 was added to the column at final concentrations of 0. mu.g/mL, 0.25. mu.g/mL, 0.5. mu.g/mL, 1. mu.g/mL, 2. mu.g/mL, 4. mu.g/mL, 8. mu.g/mL and 16. mu.g/mL, respectively, using a microsyrin in the order of 64 checkerboard from low to high, and polymyxin B was added to the column at final concentrations of 0. mu.g/mL, 0.0625. mu.g/mL, 0.125. mu.g/mL, 0.25. mu.g/mL, 0.5. mu.g/mL, 1. mu.g/mL, 2. mu.g/mL and 4. mu.g/mL, respectively, in the order of low to high concentration. And (3) placing the 96-well plate in an incubator at 37 ℃ for incubation for 24 hours, measuring the bacterial growth condition of each well by using an enzyme-labeling instrument, and recording data. Measuring OD_600nmThe value is obtained. As shown in FIG. 1A, compound Fty720 (8. mu.g/mL) and polymyxin B (0.0625. mu.g/mL) completely inhibited bacterial growth.

Dynamic in vitro monitoring of bacterial growth

OD_600nmApproximately equal to 0.4-0.6, taking bacteria liquid to shake bacteria tubes, dividing each tube into a normal control group, an Fty720 group, a polymyxin B group and a combined culture group by 10 mL, respectively adding a medicine Fty720 (8 mu g/mL), polymyxin B (2 mu g/mL) and a combined culture group (the medicine Fty720 is 8 mu g/mL and the polymyxin B is 2 mu g/mL), recording the growth condition of 0-24 hr, and recording the OD once every 1hr_600nm. As can be seen from B in fig. 1, the drug Fty720 and polymyxin B alone did not change significantly from the control group; OD in Co-culture group_600nmA significant drop occurs.

Example 2: effect of Compound Fty720 in combination with polymyxin B on survival of Abdominal infected mice

The minimal antimicrobial concentration of the co-culture was determined in vitro and it was found that compound Fty720 (8. mu.g/mL) and polymyxin B (0.0625. mu.g/mL) completely inhibited bacterial growth. Polymyxin as the last line of defense against pan-resistant gram-negative bacterial infection, patients often have lower survival rates once infected, therefore, we evaluated the effect of compound Fty720 in combination with polymyxin on survival time of mice after polymyxin-resistant klebsiella pneumoniae infection.

22-25g Balb/c mice are adaptively fed for 1 week, 100mg/kg cyclophosphamide immunodeficient mice are intravenously injected into rat tail 3 days before abdominal infection, and 2 x 10 is injected into abdominal cavity of mice 3 days later⁷200 mu L of CFU/ml bacterial liquid, and establishing an abdominal infection model, wherein the abdominal infection model is randomly divided into 4 groups (a control group, an Fty720 group, a polymyxin B group, and an Fty720+ polymyxin B group). After injecting the bacterial suspension for 1h, the control group, Fty720 group, polymyxin B group, Fty720+ polymyxin B group were given intraperitoneal injections of physiological saline, Fty720 of 8. mu.g/mL, polymyxin B of 2. mu.g/mL, Fty720 of 8. mu.g/mL, and polymyxin B of 2. mu.g/mL, respectively. The survival was observed for 72 h. As can be seen from FIG. 2, the survival time of the Fty720+ polymyxin B-treated mice is significantly longer than that of the control group and the single-drug group, and the treatment advantage is achieved.

Fty720, as a common drug with tolerance and low side effects, shows good synergistic antibacterial action in combination with low-dose polymyxin at in vivo and in vitro levels; the Fty720 low dose in vitro (8. mu.g/mL) reduced the MIC of polymyxin against Klebsiella pneumoniae to > 16. mu.g/mL to MIC = 0.0625. mu.g/mL, and the in vivo combination significantly extended the survival time of the mice after infection.

Claims

1. The use of the anti-multiple sclerosis drug Fty720 in combination with polymyxin in antibacterial medicaments.

2. Use according to claim 1, characterized in that: the antibacterial drug is a drug for resisting polymyxin-resistant Klebsiella pneumoniae.

3. A pharmaceutical composition against klebsiella pneumoniae comprising polymyxin and Fty720 in combination with polymyxin.

4. The pharmaceutical composition of claim 3, wherein the ratio of polymyxin to Fty720 is 1: 4-10.