Application of spinosin in preparation of beta-lactamase inhibitor and antibacterial drug

Abstract

The invention provides an application of spinosin in preparation of a beta-lactamase inhibitor and an antibacterial drug, and belongs to the technical field of research and development of bacterial drug-resistant drugs. Spinosin is creatively used as the beta-lactamase inhibitor, and meanwhile, beta-spinosin is combined with ceftazidime or aztreonam for use, so that the antibacterial activity of the beta-lactamase inhibitor is improved, and the antibacterial activity of the beta-lactamase inhibitor is improved. The sensitivity of various bacteria to the ceftazidime or aztreonam is obviously enhanced. Moreover, compared with existing beta-lactamase inhibitors such as potassium clavulanate, sulbactam and avibactam, the spinosin has the advantage that after the spinosin is combined with the aztreonam, the sensitivity of various bacteria to the aztreonam is more obviously enhanced. Meanwhile, the antibacterial pharmaceutical composition provided by the invention is used as a drug containing a beta-lactamase inhibitor, and is used for resisting drug-resistant bacteria generating beta-lactamase together with beta-lactam antibiotics.

Description

Technical Field

[0001] The invention belongs to the technical field of bacterial drug resistance drug research and development, and more specifically relates to the application of spinosin in the preparation of beta-lactamase inhibitors and antibacterial drugs. Background Art

[0002] Resistance of microorganisms, especially Gram-negative bacteria, to β-lactam antibiotics is often mediated by β-lactamases. β-lactamases are enzymes that catalyze the hydrolysis of the β-lactam ring, which degrades β-lactam antibiotics and renders them ineffective in their antibacterial activity, leading to bacterial resistance to these antibiotics. This resistance mechanism has become an important challenge in global public health, especially in hospital infections and critical care, where the spread of resistant bacteria has made treatment more difficult.

[0003] To address this problem, researchers have developed β-lactamase inhibitors that can slow down or prevent the activity of β-lactamases, thereby protecting β-lactam antibiotics from degradation and restoring the sensitivity of β-lactamase-producing bacteria to these antibiotics. However, some β-lactamase inhibitors currently on the market cannot effectively inhibit all types of β-lactamases, especially some β-lactamases that are widely present in drug-resistant bacteria. For example, KPC (β-lactamase produced by Klebsiella pneumoniae), class C and class D β-lactamases, etc., are all highly resistant to current inhibitors. This further limits the effectiveness of the application of β-lactam antibiotics in clinical treatment.

[0004] Therefore, it is urgent to explore new β-lactamase inhibitors, especially those that can effectively inhibit the currently ineffective β-lactamases in clinical practice, and to combine them with β-lactam antibiotics to treat drug-resistant infections. This is not only of great significance in combating antibiotic resistance, but also provides new strategies and hopes for future clinical treatment. Summary of the invention

[0005] In view of this, the present invention provides the use of spinosin in the preparation of β-lactamase inhibitors. Through the previous in vitro antibacterial experiments, it was found that when spinosin was used in combination with β-lactam antibiotics or other non-β-lactam antibiotics, the activity of the antibiotics against β-lactamase was enhanced, thereby enhancing the antibacterial properties of the antibiotics. Therefore, the spinosin of the present invention is combined with β-lactam antibiotics and can be used to treat bacterial infections in humans or animals.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] In one aspect, an embodiment of the present invention provides the use of spinotoxin in the preparation of a β-lactamase inhibitor, wherein the structure of spinotoxin is shown in formula (1).

[0008]

[0009] The second aspect of the embodiments of the present invention provides the use of spinosin in combination with β-lactam antibiotics in the preparation of antibacterial drugs.

[0010] The third aspect of the embodiments of the present invention provides the use of spinosin in combination with β-lactam antibiotics in the preparation of a drug for reducing the drug resistance of pathogens and reducing the minimum inhibitory concentration of β-lactam antibiotics.

[0011] A fourth aspect of the embodiments of the present invention provides an antibacterial pharmaceutical composition, comprising spinosum and at least one β-lactam antibiotic.

[0012] In a preferred embodiment, the ratio of the concentration of spinosin to the concentration of the β-lactam antibiotic is 1:4-128.

[0013] In a preferred embodiment, the β-lactam antibiotics include aztreonam and ceftazidime.

[0014] Spinosin is one of the active ingredients of Chinese medicine Semen Ziziphi Spinosin, and has multiple physiological activities, including sedation, tranquilizing, hypnotic and other activities. However, there is no report on spinosin as a β-lactamase inhibitor. The present invention creatively uses spinosin as a β-lactamase inhibitor, and at the same time, β-spinosin is used in combination with ceftazidime or aztreonam, which significantly enhances the sensitivity of various bacteria to ceftazidime or aztreonam. Moreover, compared with existing β-lactamase inhibitors such as potassium clavulanate, sulbactam and avibactam, the combination of spinosin and aztreonam more significantly enhances the sensitivity of various bacteria to aztreonam. At the same time, the antibacterial drug composition provided by the present invention is used as a drug containing a β-lactamase inhibitor, and together with β-lactam antibiotics, it fights against drug-resistant bacteria that produce β-lactamase. DETAILED DESCRIPTION

[0015] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0016] The embodiment of the present invention discloses the use of spinosin in the preparation of β-lactamase inhibitors, and records the experimental effect that the combination of spinosin and antibiotics improves the sensitivity of pathogens to β-lactam antibiotics.

[0017] Example 1 Inhibitory effect of spinosin on β-lactamase

[0018] β-lactamase can hydrolyze the amide bond on the β-lactam ring in the structure of nitrocefin, so that the color of the nitrocefin solution changes from yellow to red. Therefore, nitrocefin is used as a substrate, and the mixed reaction solution of spinosin and β-lactamase is added to the nitrocefin substrate solution to observe the change in the color or absorbance value of the substrate solution, so as to judge and measure the inhibitory effect of spinosin on β-lactamase.

[0019] The β-lactamase activity inhibition experiment of spinosum includes the following steps:

[0020] (1) Preparation of spinotoxin and β-lactamase reaction solution: Spinotoxin was prepared into solutions with concentrations of 50 μg / mL, 100 μg / mL, 250 μg / mL, and 500 μg / mL, and β-lactamase solution with a concentration of 1 mg / mL was added thereto to react for 30 minutes, respectively, and then set aside.

[0021] (2) Preparation of test strips containing nitrocefin substrate: Cut the cellulose membrane strips into appropriate sizes (length and width of about 0.5 cm), soak them in a centrifuge tube containing nitrocefin solution (50 μg / mL) for 10 minutes, take them out and set aside.

[0022] (3) Determination of inhibitory effect: The reaction solution of spinosin and β-lactamase of different concentrations which had been reacted for 30 min was added dropwise to the test strip containing nitrocefin substrate. After 1 minute, it was observed that all the solutions changed from yellow to orange or red, and the color of the solution became redder as the concentration of spinosin increased.

[0023] (4) Half inhibitory concentration (IC 50 ) determination: spinosin was prepared into a series of solutions with different concentrations (2.5 μg / mL, 5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL, 75 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL, 800 μg / mL), 200 μL of each solution was added to a 1 mg / mL β-lactamase solution, and after 30 min of reaction, 300 μL of the reaction solution was added to a 50 μg / mL nitrocefin substrate solution, and after 10 min of reaction, the absorbance was measured at 482 nm to establish the IC 50 The IC of spinosum was measured 50 The value is 143.53 μM.

[0024] The above test strip color reaction results and IC 50 The results of the assay showed that spinosad had β-lactamase inhibitory activity.

[0025] Example 2 Antibacterial effect of spinosin combined with β-lactam antibiotics

[0026] This embodiment discloses an antibacterial drug composition, wherein the composition contains a β-lactamase inhibitor and a β-lactam antibiotic, wherein the β-lactamase inhibitor is spinosadine, and the β-lactam antibiotics are ceftazidime and aztreonam, and the concentration ratio of the two is 1:4-64.

[0027] Minimum inhibitory concentration (MIC) determination

[0028] This experimental example adopts the method described by CLSI (Clinical Laboratory Standards Institute) standard, adds 4mg / L of spinosum, and measures the minimum inhibitory concentration (MIC) of β-lactam antibiotics to Gram-negative bacteria. In this embodiment, Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella pneumoniae and Klebsiella pneumoniae are used as test strains, and ceftazidime and aztreonam are used as representatives of β-lactam antibiotics.

[0029] The specific method is as follows:

[0030] The minimum inhibitory concentration (MIC) of spinosin alone and in combination with ceftazidime or aztreonam against Gram-negative bacteria was determined by broth dilution method.

[0031] 1. Experimental Materials

[0032] Ten Gram-negative bacteria were used in the experiment: Escherichia coli ATCC8739, a clinical isolate of Escherichia coli; Pseudomonas aeruginosa ATCC9027, a clinical isolate of Pseudomonas aeruginosa; Enterobacter cloacae ATCC700323, a clinical isolate of Enterobacter cloacae; Klebsiella pneumoniae ATCC700603, a clinical isolate of Klebsiella pneumoniae; and Acinetobacter baumannii ATCC19606, a clinical isolate of Acinetobacter baumannii.

[0033] Antibiotics: Ceftazidime (Mei Lun Biotechnology, purity greater than 94%, batch number J1230A); Aztreonam (China Food and Drug Inspection Institute, purity 96.9%, batch number 130507-201303).

[0034] β-lactamase inhibitors: spinosum (China Food and Drug Inspection Institute, purity 99%, batch number 111869-202406), avibactam (laboratory homemade, purity greater than 95%), sulbactam (McLean, purity 98%, batch number C12084503), clavulanate potassium (Anage, purity 95%, batch number AMP335).

[0035] Other reagents were of analytical grade.

[0036] The required equipment are all conventional experimental equipment and will not be described in detail here.

[0037] 2. Experimental methods

[0038] Preparation of bacterial suspension: Take the 10 kinds of Gram-negative bacteria described in "1. Experimental Materials" and inoculate them into cation-adjusted (MH) broth medium. Culture them at 37°C for 6-8h. After obtaining the bacterial culture, adjust the turbidity with sterile 0.9% sodium chloride injection and adjust the bacterial concentration to 1×10 using a McFarland turbidimeter. 7 CFU / mL, diluted 10-fold to 1×10 with sterile MH broth 6 CFU / mL, for future use.

[0039] Preparation of 10 g / L stock solutions of each test drug: Take appropriate amounts of ceftazidime, aztreonam, spinotoxin, avibactam, clavulanate potassium, and sulbactam, accurately weigh them, and dissolve them in dimethyl sulfoxide (DMSO) to obtain stock solutions with a mother liquor concentration of 10 g / L for use.

[0040] Preparation of 8 mg / L stock solutions of each inhibitor: Take appropriate amount of the above 10 g / L stock solutions of each spinotoxin, avibactam, clavulanate potassium and sulbactam, dilute them with MH broth to 8 mg / L stock solutions of each inhibitor, and set aside.

[0041] Preparation of 64 mg / L stock solutions of each antibiotic: Take appropriate amounts of the above-mentioned 10 g / L ceftazidime and aztreonam stock solutions, dilute them with MH broth to 64 mg / L stock solutions of each antibiotic, and set aside.

[0042] Preparation of working solutions of each test drug series: Take appropriate amount of the above-mentioned 10 g / L stock solutions of ceftazidime, aztreonam, spinotoxin, avibactam, clavulanate potassium and sulbactam, and dilute them with MH broth to concentrations of 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 and 0.0625 mg / L, respectively.

[0043] Preparation of a series of working solutions of antibiotic and inhibitor combinations: Take ceftazidime and aztreonam with a concentration of 64 mg / L respectively, and use the stock solutions of spinoline, avibactam, clavulanate potassium and sulbactam with a concentration of 8 mg / L as solvents, dilute ceftazidime and aztreonam in multiple ratios to obtain a series of working solutions of antibiotic and inhibitor combinations with concentrations of 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 and 0.0625 mg / L, respectively, wherein the concentration of ceftazidime or aztreonam in the combination working solution is 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 and 0.0625 mg / L, respectively, and the concentration of each inhibitor, spinoline, avibactam, clavulanate potassium or sulbactam in the combination working solution is 4 mg / L.

[0044] Determination of MIC of each test drug: Take each test drug series working solution, antibiotic and inhibitor combination series working solution, mix with 10 bacterial suspensions in a 96-well plate, incubate at 37°C for 18-24 hours, set up each bacterial control group without drug addition, and determine the minimum inhibitory concentration (MIC) of each test drug. The results are shown in Table 1.

[0045] Table 1 Minimum inhibitory concentration (MIC, mg / L) of each tested drug against 10 bacteria

[0046]

[0047] It can be seen from the above table:

[0048] 1) Compared with the administration of ceftazidime alone, the MIC of the combination of spinosin and ceftazidime against Pseudomonas aeruginosa ATCC9027, clinical isolates of Enterobacter cloacae, and clinical isolates of Klebsiella pneumoniae showed several-fold reduction, showing a significant synergistic effect.

[0049] 2) Compared with aztreonam alone, the MIC of spinosin combined with aztreonam against Pseudomonas aeruginosa ATCC9027, Enterobacter cloacae ATCC700323, clinical isolates of Enterobacter cloacae, Klebsiella pneumoniae ATCC700603, Acinetobacter baumannii ATCC19606, and clinical isolates of Acinetobacter baumannii all showed several-fold reductions. In particular, the MIC of Pseudomonas aeruginosa ATCC9027 was reduced from 4 mg / L when aztreonam was administered alone to <0.0625 mg / L, and the MIC of Enterobacter cloacae ATCC700323, Klebsiella pneumoniae ATCC700603, and Acinetobacter baumannii ATCC19606 were reduced from 32 mg / L when aztreonam was administered alone to <0.0625 mg / L, and the synergistic effect was significant.

[0050] 3) Compared with clavulanate potassium, sulbactam and avibactam, the combination of spinosum and ceftazidime can more significantly enhance the sensitivity of Pseudomonas aeruginosa ATCC9027 and Acinetobacter baumannii clinical isolates to ceftazidime.

[0051] 4) Compared with clavulanate potassium, sulbactam and avibactam, the combination of spinosum and aztreonam can more significantly enhance the sensitivity of Pseudomonas aeruginosa ATCC9027, Enterobacter cloacae ATCC700323, clinical isolates of Enterobacter cloacae and Klebsiella pneumoniae ATCC700603 to aztreonam.

[0052] The above results fully demonstrate that the combination of spinosin with ceftazidime or aztreonam can significantly enhance the sensitivity of multiple bacteria to ceftazidime or aztreonam. Furthermore, compared with existing β-lactamase inhibitors such as clavulanate potassium, sulbactam and avibactam, the combination of spinosin with aztreonam can more significantly enhance the sensitivity of multiple bacteria to aztreonam.

[0053] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0054] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The use of spinosin in the preparation of β-lactamase inhibitors, characterized in that: The spinosum structure is shown in formula (1).

2. The application of spinosum in combination with β-lactam antibiotics in the preparation of antibacterial drugs.

3. The application of spinosum in combination with β-lactam antibiotics in the preparation of drugs for reducing the drug resistance of pathogens and reducing the minimum inhibitory concentration of β-lactam antibiotics.

4. An antibacterial drug composition, characterized in that: Includes spinosadine and at least one beta-lactam antibiotic.

5. The antibacterial drug composition according to claim 4, characterized in that: The concentration ratio of spinosum to β-lactam antibiotics is 1:4-64.

6. The antibacterial drug composition according to claim 4, characterized in that: The β-lactam antibiotics include aztreonam and ceftazidime.

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