Application of dronedarone hydrochloride as polymyxin synergist

Abstract

The invention relates to application of dronedarone hydrochloride in preparation of a polymyxin synergist. A checkerboard method-minimum inhibitory concentration test, a growth curve test and a sterilization curve test prove that dronedarone hydrochloride and colistin have a good in-vitro synergistic antibacterial effect; an isothermal titration calorimetry test confirms that dronedarone hydrochloride can play a synergistic antibacterial role by combining cardiolipin and phosphatidyl glycerol. Meanwhile, a mouse ExPEC drug-resistant strain abdominal cavity infection model shows that the dronedarone hydrochloride can remarkably enhance the in-vivo treatment effect of the colistin. Compared with drug research and development of unknown active compounds, the dronedarone hydrochloride as a marketed drug can effectively accelerate the research and development process of new drugs and reduce the risk of drug research and development failure, which is of great significance in prolonging the antibacterial activity of existing antibiotics and solving the problem of bacterial drug resistance.

Description

Technical Field

[0001] The invention discloses a new use of dronedarone hydrochloride, and further relates to the use of dronedarone hydrochloride in preparing a polymyxin synergist, belonging to the field of medical biotechnology. Background Art

[0002] Polymyxins are a group of polypeptide antibiotics produced by Bacillus polymyxa, which have strong antibacterial activity against most Gram-negative bacteria. They mainly interact with lipopolysaccharide (LPS) on the outer membrane of Gram-negative bacteria, competitively replace divalent cations on the phosphate groups of membrane lipids, thereby destroying the integrity of the cell membrane phospholipid bilayer, causing osmotic imbalance and exerting bactericidal effects. Due to its potential nephrotoxicity and neurotoxicity, polymyxins were gradually replaced by other types of antibiotics (such as aminoglycosides) in the 1980s. In recent years, with the widespread spread of multidrug-resistant (MDR) Gram-negative bacteria, medical and veterinary clinics are gradually facing a situation where no drugs are available. Therefore, polymyxin antibiotics have been re-used to treat infections caused by MDR Gram-negative bacteria, especially carbapenem-resistant Enterobacteriaceae (CRE).

[0003] Extraintestinal pathogenic E. coli (ExPEC) mainly colonizes other tissues and organs outside the intestine and causes infection, which not only poses a continuous health threat to women, newborns, the elderly and people with weakened immune function, but also brings serious harm to the breeding industry, causing huge economic losses. In recent years, with the extensive use of antibiotics, multidrug-resistant E. coli has increased day by day, and resistant strains can transmit drug-resistant plasmids to other E. coli through pili, causing them to become drug-resistant, increasing the difficulty of treating E. coli disease. Polymyxin has become the last line of defense for the treatment of MDR Gram-negative bacteria.

[0004] The large-scale and irrational use of polymyxins and the widespread spread of the plasmid-mediated polymyxin resistance gene mcr-1 have led to the gradual aggravation of the problem of polymyxin resistance. The research and development cycle of new antimicrobial drugs is long and costly. The development of polymyxin enhancers is a strategy that can effectively extend the service life of polymyxins and alleviate bacterial resistance. Screening compounds that have synergistic effects with polymyxins from the FDA drug library can effectively accelerate the drug development cycle and reduce the risk of drug development failure. The development of polymyxin enhancers to restore the sensitivity of polymyxin-resistant Enterobacteriaceae to polymyxins is of great significance. Summary of the invention

[0005] The invention provides a medical use of dronedarone hydrochloride in preparing a polymyxin synergist, and discloses that dronedarone hydrochloride can effectively restore the antibacterial activity of polymyxin against drug-resistant strains of Enterobacteriaceae.

[0006] The present invention verifies that dronedarone hydrochloride can effectively enhance the in vitro antibacterial activity of polymyxin through chessboard method-minimum inhibitory concentration test, growth curve test, bactericidal curve test and ITC test. Further, by establishing a mouse ExPEC resistant strain peritoneal infection model, it is verified that dronedarone hydrochloride can effectively restore the in vivo therapeutic effect of colistin.

[0007] The dronedarone hydrochloride of the present invention has the molecular formula: 31 H 45 ClN2O5S, molecular weight: 593.22, structural formula is as follows:

[0008] The present invention finds that dronedarone hydrochloride can effectively restore the antibacterial activity of polymyxin against drug-resistant strains of Enterobacteriaceae.

[0009] The present invention finds that dronedarone hydrochloride can destroy the integrity of cell membrane structure by combining with cardiolipin and phosphatidylglycerol, thereby exerting a synergistic antibacterial effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 :The chessboard method-minimum inhibitory concentration test of dronedarone hydrochloride combined with colistin against three drug-resistant strains of Enterobacteriaceae; Figure 2 :Growth curve of dronedarone hydrochloride against ExPEC resistant strains; Figure 3 :Bacterial killing curve of dronedarone hydrochloride combined with colistin against ExPEC resistant strains; Figure 4 :Isothermal titration calorimetry (ITC) of dronedarone hydrochloride and cardiolipin; Figure 5 :ITC binding study of dronedarone hydrochloride and phosphatidylglycerol; Figure 6 : The therapeutic effect of dronedarone hydrochloride combined with colistin on the ExPEC resistant strain infection model. DETAILED DESCRIPTION

[0011] The present invention is further described by way of examples through the following implementations, which do not limit the present invention in any way. Without departing from the technical solution of the present invention, any modification or change made to the present invention that is easily implemented by a person of ordinary skill in the art will fall within the scope of the claims of the present invention.

[0012] Example 1 The role of dronedarone hydrochloride as a polymyxin synergist in the preparation of antibiotic adjuvants.

[0013] Example 2 Dronedarone hydrochloride can be used as a polymyxin synergist to prepare drugs for treating MDR Gram-negative bacteria infections.

[0014] Test Example 1 Synergistic antibacterial effect of dronedarone hydrochloride and colistin in vitro 100 μL of medium containing different concentrations of colistin and 100 μL of medium containing different concentrations of dronedarone hydrochloride were added to the columns and rows of a 96-well plate, and 1 μL of bacterial solution (final concentration 5 × 10 5 cfu / well). After oscillation and mixing, culture at 37℃ for 16-24 h, observe the results and calculate the partial inhibitory concentration index FICI. Figure 1 .

[0015] Results: Dronedarone hydrochloride combined with colistin showed good synergistic antibacterial activity against ExPEC-resistant strains, Salmonella HYM-2 and K. pneumoniae kc-2 (FICI were 0.05 ± 0.00, 0.14 ± 0.00 and 0.02 ± 0.00, respectively).

[0016] Test Example 2 Effects of dronedarone hydrochloride on the growth of ExPEC resistant strains Pick a single colony of the ExPEC resistant strain into an LB tube and culture it overnight. The next day, transfer the bacterial solution to fresh sterile LB medium and culture it until the bacterial solution OD 600nm = 0.1~0.3, and different concentrations of drugs (0, 16, 32 μg / mL) were added to the samples. The absorbance of the samples was measured every 1 h using an ELISA reader until the bacteria grew to the plateau phase. The results were statistically analyzed and a growth curve was drawn. Figure 2 .

[0017] Results: Dronedarone hydrochloride did not affect the growth of ExPEC resistant strains at the experimental concentrations.

[0018] Test Example 3 Bactericidal effect of dronedarone hydrochloride combined with colistin against ExPEC resistant strains The ExPEC resistant strains cultured overnight were transferred to fresh LB medium and grown to the logarithmic growth phase. The bacterial solution was added to the positive control group, the dronedarone hydrochloride single treatment group (8 μg / mL), the colistin single treatment group (1 μg / mL), and the combined treatment group to make the final concentration of 5 × 10 5cfu / well. The cultures of each group were taken out at 0, 6, 12 and 24 h, and the dilution plates were counted. The number of bacteria in each group at each time point was counted, and the bactericidal curve was drawn. Figure 3 .

[0019] Results: The combination of 8 μg / mL dronedarone hydrochloride and 1 μg / mL colistin could kill all bacteria within 24 h. Dronedarone hydrochloride or colistin alone could not exert bactericidal effect.

[0020] Test Example 4 Binding of dronedarone hydrochloride to cardiolipin or phosphatidylglycerol The binding capacity of 0.8 mM dronedarone hydrochloride with 0.1 mM cardiolipin or phosphatidylglycerol at 25°C was determined by isothermal calorimetric titration. The titration was performed every 120 s for a total of 20 measurements. The data were processed using software to calculate the equilibrium dissociation constant (K D The results are shown in Figure 4 and Figure 5 .

[0021] Results: Dronedarone hydrochloride had strong binding ability with cardiolipin or phosphatidylglycerol, and the binding constants were 2.1 × 10 -5 and 1.3 × 10 -6 .

[0022] Test Example 5 Therapeutic effect of dronedarone hydrochloride combined with colistin on the mouse ExPEC resistant strain infection model Female BALB / c mice (18-22 g) aged 6-8 weeks were fed for 3 days and allowed free access to food and water. They were infected with the drug-resistant strain of ExPEC (6 × 10 7 cfu / mouse), establish the mouse ExPEC resistant strain intraperitoneal infection model. Two hours after infection, the mice were randomly divided into four groups: infection group, dronedarone hydrochloride treatment group (10 mg / kg), colistin treatment group (0.25 mg / kg) and combination group. Dronedarone hydrochloride and colistin were administered once by intraperitoneal injection. Mice were killed 48 hours after infection, and the liver was taken for colonization. The results are shown in Figure 6 .

[0023] Results: Combination therapy significantly reduced the bacterial load in the livers of infected mice compared with treatment with dronedarone hydrochloride or colistin alone.

Claims

1. Application of dronedarone hydrochloride in the preparation of polymyxin synergist.

2. The use according to claim 1, wherein dronedarone hydrochloride is used in the preparation of a polymyxin synergist, characterized in that The application is the enhancement effect of dronedarone hydrochloride on the antibacterial activity of polymyxin.

3. The use according to claim 1, wherein dronedarone hydrochloride is used in the preparation of a polymyxin synergist, characterized in that The application is the application of dronedarone hydrochloride in the preparation of medicines for treating polymyxin-resistant bacterial strain infections.

4. The application as claimed in claim 1, any easily achievable modification or change made to the present invention will fall within the scope of the claims of the present invention.

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