CN109432107B

CN109432107B - Composition of metformin and doxycycline and application of composition in preparation of medicines for treating bacterial infectious diseases

Info

Publication number: CN109432107B
Application number: CN201811588153.7A
Authority: CN
Inventors: 刘源; 王志强; 李瑞超; 肖霞; 贾瑜倩
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2018-12-25
Filing date: 2018-12-25
Publication date: 2021-04-06
Anticipated expiration: 2038-12-25
Also published as: CN109432107A

Abstract

The invention relates to a composition of metformin and doxycycline and application thereof in preparing a medicament for treating bacterial infectious diseases, clarifies that the human use of metformin which is a hypoglycemic medicament can restore the sensitivity of drug-resistant bacteria to doxycycline, systematically evaluates the effectiveness of the combined use of the metformin and doxycycline in vitro and in vivo, is beneficial to developing a novel antibiotic synergist and relieves the problem of multiple drug-resistant bacteria (MDR) which cause increasingly serious harm.

Description

Composition of metformin and doxycycline and application of composition in preparation of medicines for treating bacterial infectious diseases

Technical Field

The invention belongs to potential application of metformin in bacterial infection diseases, and particularly relates to a synergistic antibacterial effect of metformin and doxycycline in vitro and in vivo.

Background

Antibiotics play a very important role in modern medicine, and their good therapeutic effect on bacterial diseases saves countless lives. However, the large scale and unjustified use of antibiotics in recent years has resulted in the massive development, spread and dissemination of bacterial resistance, which has posed a significant threat to human safety and the healthy development of livestock and poultry farming. New antibacterial strategies are urgently needed to cope with the increasingly severe antibiotic crisis. Antibiotic potentiators are substances that enhance the antibacterial activity of antibiotics by inhibiting bacterial resistance or complementing bactericidal mechanisms. The most classical potentiator is clavulanic acid, which is an inhibitor of beta-lactamases. The combination of clavulanic acid and beta-lactam antibiotics is effective in treating infections caused by beta-lactamase-producing drug-resistant bacteria.

Doxycycline, also known as doxycycline, is a semi-synthetic tetracycline antibiotic prepared by deoxygenation at position 6 of oxytetracycline. It has better antibacterial activity to various gram-positive bacteria and gram-negative bacilli, and belongs to broad-spectrum antibiotics. At present, the traditional Chinese medicine is mainly used for respiratory system and urinary tract infection and skin soft tissue infection caused by sensitive bacteria in human clinical practice. In addition, the compound has the advantages of good tissue permeability, high oral bioavailability and the like, so that the compound is widely applied to veterinary clinic and is used for preventing and treating bacterial infectious diseases of livestock and poultry. However, doxycycline resistance is currently found to be very serious in both human and veterinary clinics. A large number of bacterial drug resistance epidemiological investigations have shown that the doxycycline resistance rate of clinical strains has exceeded 50%. The clinical use curative effect of doxycycline is seriously influenced by the generation and rapid spread of doxycycline drug resistance in large quantities. In the present invention, a potentiator of doxycycline is to be screened by a checkerboard broth dilution method from compounds approved for marketing and non-antibacterial use to restore its antibacterial activity against drug-resistant bacteria.

Disclosure of Invention

In order to overcome the defects, the invention provides a composition of metformin and doxycycline and application thereof in preparing a medicament for treating bacterial infectious diseases so as to restore the antibacterial activity of doxycycline on drug-resistant bacteria.

In order to achieve the purpose of the invention, the invention adopts the technical scheme that: the doxycycline synergist is a composition consisting of metformin and doxycycline, wherein the mass ratio of the metformin to the doxycycline is 1-150: 1.

preferably, the mass ratio of the metformin to the doxycycline is 1-2: 1. the effective dosage of the synergist is more than 50mg/kg (namely more than or equal to 50 mg/kg).

The invention also provides the application of the composition of metformin and doxycycline in the synergistic antibiosis in vivo or in vitro. The organisms refer to livestock and poultry.

The invention also provides application of the composition of the metformin and the doxycycline in preparing a medicament for treating bacterial infectious diseases.

Compared with the prior art, the invention clarifies that the human hypoglycemic agent metformin can restore the sensitivity of drug-resistant bacteria to doxycycline, systematically evaluates the effectiveness of the combined use of the hypoglycemic agent metformin and the drug-resistant bacteria in vitro and in vivo, is beneficial to developing a novel antibiotic synergist and relieves the problem of multiple drug-resistant bacteria (MDR) with increasingly serious harm.

Drawings

Figure 1 is a time sterilization curve for the combined use of doxycycline and metformin; DOX, doxycycline (32 μ g/mL); MET, metformin (5 mg/mL); DOX + MET, doxycycline (32 μ g/mL) + metformin (5 mg/mL);

FIG. 2 shows that doxycycline and metformin are used in combination to treat bacterial infection of pyralid larvas, and that doxycycline and metformin are used in combination to significantly improve the survival rate of yellow rice borer;

figure 3 is a graph of doxycycline and metformin used in combination to treat bacterial infection of the thigh muscle in mice, demonstrating the effectiveness of doxycycline and metformin used in combination in a mouse model of infection.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. In the quantitative experiments in the following examples, three replicates were set up and the results averaged.

ICR female mice were provided to the laboratory animal center at the university of promiscuous.

MHB broth medium is an aqueous solution containing 1.5g/L of beef powder, 1.5g/L of soluble starch and 17.5g/L of acid hydrolyzed casein.

Example 1 synergistic antibacterial action of doxycycline and metformin

The synergistic antibacterial activity of doxycycline and metformin against drug-resistant bacteria was determined using a checkerboard assay with the test strains shown in table 1.

TABLE 1 test strains

Note:^areference 1 is Liu Y, Ding S, Dietrich R,

E，Zhu K.A biosurfactant inspired heptapeptide with improved specificity to kill MRSA[J].Angewandte Chemie International Edition，2017，56(6)，1486-1490.

staphylococcus aureus (Staphylococcus aureus) MRSA T144, Staphylococcus aureus (Staphylococcus aureus)215 (LZD)^R+ cfr) and Enterococcus faecalis (Enterococcus faecalis) VRE A4 in the literature 1 are sequentially named MRSA T144, Staphylococcus aureus 215 (LZD)^R+ cfr) and Enterococcus faecalis VRE a 4.

^bStaphylococcus aureus (Staphylococcus aureus) MRSA T144 is methicillin resistant Staphylococcus aureus. Staphylococcus aureus (Staphylococcus aureus)215 (LZD)^R+ cfr) carries a drug resistance gene cfr and is resistant to linezolid. Enterococcus faecalis (Enterococcus faecalis) VRE a4 is a drug-resistant strain of vancomycin.

The chessboard analysis method comprises the following specific steps:

1) test strains were suspended in MHB broth to a concentration of 1X 10⁸CFU/mL of bacterial suspension.

2) Metformin was taken, dissolved in water and diluted with MHB broth to give a metformin solution with a concentration of 80 mg/mL.

3) Doxycycline was dissolved in water and diluted with MHB broth to give an antibacterial solution at a concentration of 128 μ g/mL.

4) Taking a 96-well plate, adding 100 mu L of MHB broth culture medium into each well, adding 100 mu L of doxycycline solution prepared in the step 2) into each well in the last row, and diluting to the second row from the eighth row in a multiple ratio; adding 100 mu L of the antibacterial drug solution prepared in the step 3) into each well of the first row, diluting the mixture to the tenth row in a double ratio, adding 100 mu L of the bacterial suspension prepared in the step 1) into each well, carrying out standing culture at 37 ℃ for 16-20 h, and observing the lowest concentration combination of the metformin and the doxycycline when the combination is used for inhibiting the growth of bacteria. Positive control wells were set, and 100. mu.L of LMHB broth and 100. mu.L of the bacterial suspension prepared in step 1) were added to each positive control well. The fractional antibacterial concentration FIC index is calculated according to the following formula:

FIC ═ MIC (A combination)/MIC (A alone) + MIC (B combination)/MIC (B alone)

The test results are shown in Table 2. The result shows that the metformin can obviously enhance the antibacterial activity of doxycycline on various drug-resistant bacteria including gram-positive bacteria, gram-negative bacteria and the like, and the synergistic multiple is 8 to 32 times. The combined use of the two has the characteristic that the fractional bacteriostatic concentration index (FIC index) is less than 0.5, which indicates that the combined use of the two has obvious synergistic effect.

Table 2 metformin enhances the antibacterial activity of doxycycline against a variety of drug-resistant bacteria.

^aThe minimum inhibitory concentration of doxycycline to drug-resistant bacteria when used alone;^bdoxycycline couple after addition of metformin

The minimum inhibitory concentration of the drug-resistant bacteria;^cthe antibacterial activity of doxycycline is improved by multiple.

Example 2 time kill curves for doxycycline and metformin

Coli B2 was cultured in BHI broth for 2h (exponential growth phase) and 5h (stationary phase), respectively, before adding equal volumes of 0.01mol/L PBS (pH 7.2), doxycycline, metformin, and a mixture of both, to give final drug concentrations of doxycycline (32 μ g/mL), metformin (5mg/mL), and a mixture concentration (32 μ g/mL doxycycline +5mg/mL metformin). Then, 100. mu.L of the suspension was applied to MHA medium at 4h, 8h, 12h and 24h, respectively, and cultured overnight before colony counting.

The results of the experiment are shown in FIG. 1. The results show that the combination of the two is stable to exponential growth phase compared to doxycyclineThe bacteria have remarkable bactericidal effect, and the number of bacteria is reduced by at least 2 logs₁₀CFU/mL。

Example 3 treatment of bacterial infection in wax moth larvae with doxycycline in combination with metformin

Doxycycline solution: dissolving 15mg of doxycycline in 10mL of sterile water.

Metformin solution: obtained by dissolving 15mg of metformin in 10mL of sterile water.

Mixing the solution: obtained by dissolving 15mg of doxycycline and 15mg of metformin in 10mL of sterile water.

E.coli suspension: resuspending E.coli (Escherichia coli) B2 with PBS buffer to obtain E.coli suspension; the E.coli (Escherichia coli) B2 concentration in the E.coli suspension was 1.0X 10⁸CFUs/mL。

1. Grouping treatment of galleria mellonella larvae

32 larvae of galleria mellonella with a weight of 300mg were randomly divided into a Vehicle group, a doxycycline treatment group, a metformin treatment group and a synergistic treatment group (10 larvae each), and the following treatments were performed, respectively:

vehicle group: injecting 0.01mL of escherichia coli suspension into the lower left second gastropod of the galleria mellonella larvae; injecting 0.01mL PBS buffer solution into the lower right second gastropod after 1 h;

doxycycline treatment group: injecting 0.01mL of escherichia coli suspension into the lower left second gastropod of the galleria mellonella larvae; injecting 0.01mL doxycycline solution into the second lower right gastropod after 1 h;

metformin treatment group: injecting 0.01mL of escherichia coli suspension into the lower left second gastropod of the galleria mellonella larvae; after 1h, injecting 0.01mL of metformin solution into the second lower right gastropod;

the co-treatment group: injecting 0.01mL of escherichia coli suspension into the lower left second gastropod of the galleria mellonella larvae; the second gastropod was injected 0.01mL of the mixed solution after 1 h.

2. Statistic survival rate

And (4) counting the survival rate of the larvae of the galleria mellonella on the 1 st day, the 2 nd day, the 3 rd day, the 4 th day and the 5 th day after the step 1 is finished.

The results of the experiment are shown in FIG. 2. The results show that the survival rate of the galleria mellonella larvae in the synergistic treatment group is obviously improved compared with the doxycycline treatment group; the survival rate of the larvae of the wax moth of the doxycycline treatment group at the 5 th day is only 30 percent, while the survival rate of the larvae of the wax moth of the synergistic treatment group at the 5 th day can reach 80 percent.

Example 4 treatment of bacterial infection of the thigh muscle of mice with doxycycline in combination with metformin

Doxycycline solution: 150mg doxycycline was dissolved in 10mL of sterile water.

Metformin solution: obtained by dissolving 150mg of metformin in 10mL of sterile water.

Mixed solution 1: obtained by dissolving 150mg of doxycycline and 30mg of metformin in 10mL of sterile water.

Mixed solution 2: obtained by dissolving 150mg of doxycycline and 150mg of metformin in 10mL of sterile water.

E.coli suspension: resuspending E.coli (Escherichia coli) B2 with PBS buffer to obtain E.coli suspension; the E.coli (Escherichia coli) B2 concentration in the E.coli suspension was 1.0X 10⁷CFUs/mL。

1. Mouse grouping treatment

48 ICR female mice, weighing 27-30g, were randomized into vehicle, doxycycline, metformin and co-therapy groups (8 per group), and treated as follows:

pre-stream group: injecting 0.1mL of escherichia coli suspension into right thigh muscle of the mouse, euthanizing the mouse after 1h, and taking right thigh muscle of the mouse for colony number determination;

vehicle group: injecting 0.1mL of Escherichia coli suspension into right thigh muscle of the mouse; injecting 0.1mL PBS buffer solution into the abdominal cavity after 1 h;

doxycycline treatment group: injecting 0.1mL of Escherichia coli suspension into right thigh muscle of the mouse; injecting 0.1mL doxycycline solution into the abdominal cavity after 1 h;

metformin treatment group: injecting 0.1mL of Escherichia coli suspension into right thigh muscle of the mouse; injecting 0.1mL of metformin solution into the abdominal cavity after 1 h;

co-treatment group 1: injecting 0.1mL of Escherichia coli suspension into right thigh muscle of the mouse; after 1h, 0.1mL of mixed solution 1 was injected intraperitoneally.

Co-treatment group 2: injecting 0.1mL of Escherichia coli suspension into right thigh muscle of the mouse; after 1h, 0.1mL of mixed solution 2 was injected intraperitoneally.

2. Determination of the number of colonies infected by the thigh of mice

And (3) euthanizing the mice 24h after the step 1, taking right thigh muscle, homogenizing and uniformly mixing meat blocks, then coating a proper amount of the meat blocks on an MHA culture medium, and determining the colony number.

The results of the experiment are shown in FIG. 3. The results show that the synergistic treatment group can effectively reduce the colony number of thigh infection of the mice compared with the doxycycline single treatment group, and reduce the colony number of thigh infection of the mice by at least 2 logs₁₀CFU/thigh。

The foregoing description has described the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention, which is intended to be covered by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. The application of a composition consisting of metformin and doxycycline in preparing a medicament for treating bacterial infectious diseases is characterized in that the mass ratio of metformin to doxycycline is 1-2: 1.

2. the use of claim 1, wherein the bacterial infectious disease is a bacterial infectious disease of livestock and poultry.

3. Use according to claim 1, wherein the bacterium is Escherichia coli.

4. The use of claim 1, wherein the doxycycline is present at a concentration of 1.5 mg/mL and the metformin is present at a concentration of 1.5 mg/mL.

5. The use according to claim 1, wherein the doxycycline is present at a concentration of 15 mg/mL and the metformin is present at a concentration of 15 mg/mL.