Antibacterial drug adjuvant and application thereof

Abstract

The invention discloses an antibacterial drug adjuvant and application thereof, the antibacterial drug adjuvant is amino acid and biotin, and can effectively enhance the inhibition effect of ciprofloxacin on Escherichia coli. The auxiliary agent of the antibacterial medicine is a human body endogenous substance and is small in toxic and side effects, and especially under the condition of low dosage of ciprofloxacin, the medicine effect can be remarkably improved by adding the auxiliary agent. Under the background that the bacterial drug resistance is increasingly severe and the research and development of novel antibiotics are weak, the use effect of the existing mainstream antibiotic ciprofloxacin is effectively improved by the antibacterial drug adjuvant, and a beneficial solution is provided for breaking through the bacterial drug resistance.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmacy, and in particular relates to an antibacterial drug adjuvant and application thereof. Background Art

[0002] Antibiotics are important therapeutic drugs for people to deal with pathogenic bacterial infections and are also an important pillar of modern medicine. Quinolones are antibiotics with a 4-quinolone nucleus that were artificially synthesized in the 1960s (first generation: nalidixic acid). Initially, they were only used for urinary tract infections due to their antibacterial limitations. Today, quinolones (third and fourth generations: ciprofloxacin, gatifloxacin, moxifloxacin, etc.) have become important therapeutic drugs for respiratory infections due to their high concentrations in lung tissue and respiratory secretions, long half-lives, and high bioavailability. With people's continued dependence on antibiotic consumption, antibiotic resistance caused by the adaptive evolution of pathogenic bacteria has become an increasingly serious public health problem worldwide. At present, the two main strategies for solving the antibiotic resistance crisis at home and abroad are to limit the use of antibiotics and to develop new antibiotics.

[0003] However, existing antibiotics are less likely to break through tolerance; and the research and development of new antibiotics is weak. Since 1984, no antibiotics with a completely new skeleton structure have entered clinical research. Summary of the invention

[0004] In view of this, an embodiment of the present invention provides a pharmaceutical adjuvant and application thereof that can enhance and improve the antibacterial activity of existing antibiotics.

[0005] An embodiment of the present invention provides an antibacterial drug adjuvant, wherein the antibacterial drug adjuvant is amino acid and biotin; and the antibacterial drug is ciprofloxacin.

[0006] Furthermore, the amino acid is one of glycyl-L-valine, L-ornithine, L-tryptophan or L-prolylglycine; and the biotin is indole.

[0007] The present invention also provides application of the antibacterial drug adjuvant, which is used in combination with an antibacterial drug to inhibit the activity of Escherichia coli.

[0008] Furthermore, the antibacterial drug adjuvant amino acid is used in combination with ciprofloxacin to inhibit the activity of Escherichia coli.

[0009] Furthermore, one of the antibacterial drug adjuvants glycyl-L-valine, L-ornithine, L-tryptophan or L-prolylglycine is used in combination with ciprofloxacin to inhibit the activity of Escherichia coli.

[0010] Furthermore, the antibacterial drug adjuvant indole is used in combination with ciprofloxacin to inhibit the activity of Escherichia coli.

[0011] The embodiment of the present invention also provides an application method of the antimicrobial drug adjuvant, wherein the antimicrobial drug and the adjuvant are added to an Escherichia coli bacterial solution and treated at a constant temperature for 4 to 24 hours.

[0012] Furthermore, an ELISA instrument was used to measure the OD value of Escherichia coli when the antibacterial drug ciprofloxacin was co-administered with the adjuvant, and the activity inhibition rate of ciprofloxacin on Escherichia coli under the action of the antibacterial drug adjuvant was calculated based on the obtained OD value.

[0013] The present invention also provides a method for detecting the activity inhibition rate of ciprofloxacin on Escherichia coli under the action of an antibacterial drug adjuvant, comprising the following steps:

[0014] (1) Cultivating Escherichia coli in LB liquid medium at a constant temperature until the logarithmic phase;

[0015] (2) Dilute the bacterial solution to a certain OD value;

[0016] (3) Add different concentrations of ciprofloxacin and adjuvants to an equal amount of Escherichia coli culture solution and culture at a constant temperature in a sterile 96-well plate to set as the experimental group;

[0017] (4) After a certain period of incubation, take out the 96-well plate and measure the OD of the solution. 600 ;

[0018] (5) By measuring OD 600 The inhibition rate of ciprofloxacin against Escherichia coli in the presence of antimicrobial adjuvants was obtained by numerical calculation.

[0019] Furthermore, in step (3), a control group and a blank group are set up simultaneously, wherein no auxiliary agent is added to the control group, and the blank group only contains the same amount of culture medium as that of the experimental group.

[0020] Furthermore, the concentration of the antibacterial drug adjuvant is 0.1% to 1%; the concentration of ciprofloxacin is 10 to 1000 ng / mL,

[0021] The beneficial effect brought by the technical solution of the present invention is that the antibacterial drug adjuvant of the present invention is amino acid and biotin, and the antibacterial ability of ciprofloxacin is improved by using amino acid or biotin in combination with the antibacterial drug ciprofloxacin. The antibacterial drug adjuvant of the present invention is a human endogenous substance with small toxic and side effects, which greatly improves the safety of combined antibiotics. The antibacterial drug adjuvant of the present invention can significantly improve the antibacterial effect in the case of low-dose ciprofloxacin. In the context of increasingly severe bacterial resistance and lack of research and development of new antibiotics, the antibacterial drug adjuvant of the present invention effectively improves the use effect of the existing mainstream antibiotic ciprofloxacin, and provides a beneficial solution for breaking through bacterial resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a growth curve diagram of Escherichia coli in Example 1 of the present invention;

[0023] Figure 2 It is the inhibition rate of different antimicrobial adjuvants combined with 10 ng / mL ciprofloxacin on Escherichia coli in Example 2 of the present invention;

[0024] Figure 3 It is the inhibition rate of different antimicrobial adjuvants combined with 100 ng / mL ciprofloxacin on Escherichia coli in Example 3 of the present invention;

[0025] Figure 4 It is the inhibition rate of the antibacterial drug adjuvant indole in Example 4 of the present invention combined with 10 ng / mL ciprofloxacin on Escherichia coli;

[0026] Figure 5 This is the inhibition rate of Escherichia coli when the antibacterial drug adjuvant indole of Example 4 of the present invention is combined with 100 ng / mL ciprofloxacin. DETAILED DESCRIPTION

[0027] To make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be further described below with reference to the accompanying drawings.

[0028] Quinolones are fixed to the complex formed by the target enzyme and broken DNA in the form of non-covalent bonds, which blocks genetic processes such as DNA replication and transcription, leading to DNA degradation and bacterial cell death. Obviously, bacterial tolerance to quinolones is not only related to the mutation of target enzyme-related genes, but also highly dependent on the proliferation and DNA replication of the bacteria themselves. Specifically, cells whose proliferation and DNA replication are inhibited or in a stable period (non-proliferation) with high bacterial density will show tolerance to quinolones, and how to make quinolones break through the tolerance of pathogenic bacteria will require a new interpretation of their antibacterial mechanism. The antibacterial effect of quinolones is closely related to bacterial respiration, that is, the effect of quinolones will accelerate cell respiration to produce ineffective energy supply and lead to cell death. In vitro bacterial culture also shows that increasing the supply of glucose and oxygen to promote cell respiration can effectively break through the tolerance of stable bacteria to quinolones and improve their antibacterial ability. Therefore, enhancing the antibacterial effect of quinolones by promoting bacterial respiration through exogenous supply provides a new breakthrough for solving the tolerance of high-density or non-proliferating bacteria. However, drug response to bacterial infection is an interaction between drugs, bacteria and host cells. The metabolic state and energy substance level of host cells will directly affect bacterial respiration or metabolic state, thereby affecting the antibacterial effect of drugs. Obviously, during the occurrence of bacterial infection diseases, the levels of oxygen and energy substances are determined by the host's intake, which is not an ideal auxiliary supply strategy for quinolone treatment. It is more important to comprehensively characterize and screen metabolites related to bacteria, host cells and respiration under the action of drugs, so as to determine the potential metabolite adjuvants of quinolones to break through bacterial resistance.

[0029] Embodiment 1

[0030] An embodiment of the present invention provides an antibacterial drug adjuvant, wherein the antibacterial drug adjuvant is amino acid and biotin, and the antibacterial drug is ciprofloxacin.

[0031] Preferably, the antimicrobial drug adjuvant amino acid is one of glycyl-L-valine, L-ornithine, L-tryptophan or L-prolylglycine; and the biotin is indole.

[0032] The embodiment of the present invention further provides an application of an antimicrobial drug adjuvant, wherein the antimicrobial drug adjuvant is used in combination with the antimicrobial drug to act on Escherichia coli.

[0033] Preferably, the antibiotic adjuvant amino acid or biotin is used in combination with the antibiotic ciprofloxacin for use in Escherichia coli.

[0034] Preferably, one of the adjuvants glycyl-L-valine, L-ornithine, L-tryptophan or L-prolylglycine is used in combination with the antibacterial drug ciprofloxacin against Escherichia coli.

[0035] Preferably, the adjuvant indole is used in combination with the antibacterial drug ciprofloxacin for Escherichia coli.

[0036] The embodiment of the present invention also provides an application method of the antibacterial drug adjuvant, wherein the antibacterial drug adjuvant and the antibacterial drug ciprofloxacin are added to an Escherichia coli bacterial solution and treated at a constant temperature for 4 hours to 24 hours.

[0037] Preferably, the antibacterial drug adjuvant amino acid and the antibacterial drug ciprofloxacin are added to the Escherichia coli bacterial solution and treated at a constant temperature of 37° C. for 4 h to 24 h.

[0038] Preferably, one of the antibacterial drug adjuvants glycyl-L-valine, L-ornithine, L-tryptophan or L-prolylglycine and the antibacterial drug ciprofloxacin are added to the Escherichia coli bacterial solution and treated at a constant temperature of 37° C. for 4 h to 24 h.

[0039] Preferably, the antibacterial drug adjuvant indole and the antibacterial drug ciprofloxacin are added to the Escherichia coli bacterial solution and treated at a constant temperature of 37° C. for 4 h to 24 h.

[0040] Preferably, an ELISA instrument is used to measure the OD value of Escherichia coli when the antibacterial drug adjuvant and ciprofloxacin are administered in combination, and the activity inhibition rate of ciprofloxacin on Escherichia coli under the action of the adjuvant is calculated based on the obtained OD value.

[0041] The embodiment of the present invention also provides a method for detecting the activity inhibition rate of the antimicrobial drug adjuvant in combination with ciprofloxacin on Escherichia coli, comprising the following steps:

[0042] (1) Refer to the attached Figure 1 , Escherichia coli was cultured in LB liquid medium at a constant temperature until the logarithmic phase;

[0043] Specifically, a single colony was picked from the solid culture medium with a sterile inoculation loop and placed in the LB liquid culture medium. The inoculation loop was gently shaken to disperse the bacteria in the liquid culture medium or transfer the bacterial solution at a ratio of 1:100, and cultured at 37°C for 12 hours; the overnight cultured bacterial solution was then diluted at a ratio of 1:500 and cultured at 37°C for 6 hours;

[0044] (2) diluting the above bacterial solution to a certain OD value;

[0045] Specifically, the OD of the bacterial solution cultured for 6 h was measured. 600 , diluted with sterile LB medium to OD = 0.1;

[0046] (3) The adjuvant and different doses of ciprofloxacin were added to equal amounts of Escherichia coli culture, and cultured at a constant temperature in a sterile 96-well plate. These were set as experimental groups, and each group was repeated 3 times.

[0047] (4) After a certain period of incubation, take out the 96-well plate and measure the OD of the solution. 600 ;

[0048] (5) By measuring OD 600 The inhibition rate of ciprofloxacin against Escherichia coli in the presence of antimicrobial adjuvants was obtained by numerical calculation.

[0049]

[0050] Preferably, in step (3), a control group and a blank group are set up simultaneously, wherein no auxiliary agent is added to the control group, and the blank group only contains the same amount of culture medium as that of the experimental group.

[0051] Preferably, the concentration of the antibacterial drug adjuvant is 0.1% to 1%; the concentration of ciprofloxacin is 10 to 1000 ng / mL.

[0052] Embodiment 2

[0053] The antibacterial drug adjuvant of the first embodiment of the present invention is used, and one of the antibacterial drug adjuvants, glycyl-L-valine, L-ornithine or L-tryptophan, is combined with ciprofloxacin to act on Escherichia coli.

[0054] The activity inhibition rate detection method of Escherichia coli in Example 1 of the present invention is used to determine the activity inhibition rate of 1% glycyl-L-valine, 1% L-ornithine, and 1% L-tryptophan combined with 10 ng / mL ciprofloxacin on Escherichia coli, comprising the following steps:

[0055] (1) Use an inoculation loop to pick up a single colony from the solid culture medium and place it in LB liquid culture medium. Gently shake the inoculation loop to disperse the bacteria in the LB liquid culture medium or transfer the bacterial solution at a ratio of 1:100, and culture at 37°C for 12 hours; then dilute the bacterial solution at a ratio of 1:500 and culture at 37°C for 6 hours until the bacterial solution is diluted to OD = 0.1;

[0056] (2) Weigh 20 mg of glycyl-L-valine, L-ornithine, and L-tryptophan respectively into three 5 mL sterile centrifuge tubes, add 20 μL of 1 μg / mL ciprofloxacin and 2 mL of Escherichia coli with OD=0.1 into each centrifuge tube, and mix well. The final concentrations of glycyl-L-valine, L-ornithine, and L-tryptophan are all 1%, and the final concentration of ciprofloxacin is all 10 ng / mL, which are respectively designated as experimental group 1, experimental group 2, and experimental group 3; set corresponding control groups and blank groups, wherein the control group does not add glycyl-L-valine, L-ornithine, and L-tryptophan, and the blank group only contains the same amount of LB liquid culture medium as the experimental group;

[0057] (3) Incubate the centrifuge tubes of each group of combined drugs at 37°C for 4 h. At the corresponding time, take 200 μL from each centrifuge tube and transfer it to a 96-well plate. Repeat 3 times and measure the OD with an enzyme-labeled instrument. 600 ;

[0058] (4) By measuring OD 600 The inhibition rate of ciprofloxacin against Escherichia coli in the presence of antibacterial adjuvants glycyl-L-valine, L-ornithine or L-tryptophan was obtained by numerical calculation.

[0059] See attached Figure 2 , indicating that the antibacterial effect of 10 ng / mL ciprofloxacin against Escherichia coli was significantly improved under the action of 1% concentration of adjuvant glycyl-L-valine, L-ornithine or L-tryptophan.

[0060] Embodiment 3

[0061] The antibacterial drug adjuvant of Example 1 of the present invention is used, and one of the antibacterial drug adjuvants selected from L-prolyl glycine, 1% glycyl-L-valine, 1% L-ornithine, and 1% L-tryptophan is combined with ciprofloxacin to act on Escherichia coli.

[0062] The activity inhibition rate detection method of Escherichia coli in Example 1 of the present invention is used to determine the activity inhibition rate of 1% L-prolyl glycine, 1% glycyl-L-valine, 1% L-ornithine, and 1% L-tryptophan combined with 100 ng / mL ciprofloxacin on Escherichia coli, comprising the following steps:

[0063] (1) Use an inoculation loop to pick up a single colony from the solid culture medium and place it in LB liquid culture medium. Gently shake the inoculation loop to disperse the bacteria in the LB liquid culture medium or transfer the bacterial solution at a ratio of 1:100, and culture at 37°C for 12 hours; then dilute the bacterial solution at a ratio of 1:500 and culture at 37°C for 6 hours until the bacterial solution is diluted to OD = 0.1;

[0064] (2) Weigh 20 mg of L-prolylglycine, glycyl-L-valine, L-ornithine, and L-tryptophan respectively into 4 5 mL sterile centrifuge tubes, add 20 μL of 10 μg / mL ciprofloxacin and 2 mL of Escherichia coli with OD=0.1 to each centrifuge tube, and mix well. The final concentrations of L-prolylglycine, glycyl-L-valine, L-ornithine, and L-tryptophan are all 1%, and the final concentration of ciprofloxacin is all 100 ng / mL. These are respectively designated as experimental group 1, experimental group 2, experimental group 3, and experimental group 4; set corresponding control groups, in which L-prolylglycine, glycyl-L-valine, L-ornithine, and L-tryptophan are not added, and the blank group contains only the same amount of LB liquid culture medium as the experimental group;

[0065] (3) Incubate the centrifuge tubes of each group of combined drugs at 37°C for 4 h. At the corresponding time, take 200 μL from each centrifuge tube and transfer it to a 96-well plate. Repeat 3 times and measure the OD with an enzyme-labeled instrument. 600 ;

[0066] (4) By measuring OD 600 The inhibition rate of ciprofloxacin against Escherichia coli in the presence of adjuvants was obtained by numerical calculation.

[0067] See attached Figure 3 , indicating that the antibacterial effect of 100ng / mL ciprofloxacin was significantly improved under the influence of 1% adjuvant L-prolylglycine, glycyl-L-valine, L-ornithine or L-tryptophan. At the same time, the combined effect of adjuvants was more significant in the case of low-dose ciprofloxacin.

[0068] Embodiment 4

[0069] The antibacterial drug adjuvant of Example 1 of the present invention is used, and the antibacterial drug adjuvant indole is combined with ciprofloxacin to act on Escherichia coli.

[0070] The method for detecting the activity inhibition rate of Escherichia coli in Example 1 of the present invention was used to determine the activity inhibition rate of indole combined with 10 ng / mL and 100 ng / mL ciprofloxacin on Escherichia coli, comprising the following steps:

[0071] (1) Use an inoculation loop to pick up a single colony from the solid culture medium and place it in LB liquid culture medium. Gently shake the inoculation loop to disperse the bacteria in the LB liquid culture medium or transfer the bacterial solution at a ratio of 1:100, and culture at 37°C for 12 hours; then dilute the bacterial solution at a ratio of 1:500 and culture at 37°C for 6 hours until the bacterial solution is diluted to OD = 0.1;

[0072] (2) Weigh 20 mg of indole in 0.1 g of DMSO and dissolve it completely. Then take 10 μL of indole into two 5-mL sterile centrifuge tubes. Add 20 μL of 1 μg / mL ciprofloxacin to one of the centrifuge tubes and 20 μL of 10 μg / mL ciprofloxacin to the other centrifuge tube. Then add 2 mL of Escherichia coli culture solution with OD=0.1 to each centrifuge tube and mix well. The final concentrations of indole are 0.1% and 10 ng / mL and 100 ng / mL, respectively. No indole is added to the control group. The blank group contains only the same amount of LB liquid culture medium and DMSO as the experimental group.

[0073] (3) Incubate the centrifuge tubes of each group of combined drugs at 37°C for 4 h, 12 h, and 24 h. Take 200 μL from each centrifuge tube and transfer it to a 96-well plate. Repeat 3 times and measure the OD with an enzyme reader. 600 ;

[0074] (4) By measuring OD 600 The inhibition rate of ciprofloxacin activity against Escherichia coli at different doses under the action of indole was obtained by numerical calculation.

[0075] See attached Figure 4 , 5 , indicating that indole can effectively enhance the antibacterial effect of ciprofloxacin. Under low-dose ciprofloxacin conditions, the anti-Escherichia coli effect of ciprofloxacin under the action of indole is more significant.

[0076] Those skilled in the art should understand that the methods and applications described in the present invention are not limited to the embodiments described in the specific implementation manner, and the above specific description is only for the purpose of explaining the present invention, and is not intended to limit the present invention. Those skilled in the art may derive other implementations based on the technical solution of the present invention, which also belong to the technical innovation scope of the present invention, and the protection scope of the present invention is defined by the claims and their equivalents.

Claims

1. An antibacterial drug adjuvant, characterized in that: The antibacterial drug adjuvants are amino acids and biotin; and the antibacterial drug is ciprofloxacin.

2. The antibacterial drug adjuvant according to claim 1, characterized in that: The amino acid is one of glycyl-L-valine, L-ornithine, L-tryptophan or L-prolylglycine; and the biotin is indole.

3. The use of the antibacterial drug adjuvant according to claim 1 or 2, characterized in that: The antibacterial drug adjuvant is used in combination with an antibacterial drug to inhibit the activity of Escherichia coli.

4. The use of the antibacterial drug adjuvant according to claim 3, characterized in that: The antibacterial drug adjuvant amino acid is used in combination with ciprofloxacin to inhibit the activity of Escherichia coli.

5. The use of the antibacterial drug adjuvant according to claim 3, characterized in that: One of the antibacterial drug adjuvants, glycyl-L-valine, L-ornithine, L-tryptophan or L-prolylglycine, is used in combination with ciprofloxacin to inhibit the activity of Escherichia coli.

6. The use of the antibacterial drug adjuvant according to claim 3, characterized in that: The antibacterial drug adjuvant indole is used in combination with ciprofloxacin to inhibit the activity of Escherichia coli.

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