Application of magnoflorine in preparation of beta-lactamase inhibitor

Abstract

The invention provides application of magnoflorine in preparation of beta-lactamase inhibitors and antibacterial drugs, and belongs to the technical field of research and development of bacterial drug-resistant drugs. According to the invention, magnoflorine is creatively used as a beta-lactamase inhibitor; meanwhile, the magnoflorine is combined with the ceftazidime or the aztreonam for use, so that the sensitivity of various bacteria to the ceftazidime or the aztreonam is obviously enhanced; in addition, 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 to the application of magnolia alkaloids in the preparation of beta-lactamase inhibitors and antibacterial drugs. Background Art

[0002] Microbial resistance, especially in Gram-negative bacteria, to β-lactam antibiotics is mainly achieved through the action of β-lactamases. β-lactamases are enzymes that catalyze the hydrolysis of the β-lactam ring, which destroys the structure of β-lactam antibiotics and makes them lose their antibacterial activity, thus causing bacteria to develop resistance to these drugs. This resistance mechanism has become a major challenge in global public health, especially in hospital infections and intensive care settings, where the spread of resistant bacteria makes 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, protect β-lactam antibiotics from degradation, and restore the sensitivity of β-lactamase-producing bacteria to these antibiotics. However, the 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, such as KPC (β-lactamase produced by Klebsiella pneumoniae), C and D β-lactamases, etc., which show strong resistance to existing β-lactamase inhibitors. This further limits the clinical application of β-lactam antibiotics.

[0004] Therefore, there is an urgent need to develop new β-lactamase inhibitors to inhibit β-lactamases that cannot be effectively inhibited clinically, and to use them in combination with β-lactam antibiotics to treat resistant bacterial 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 magnolia alkaloids in the preparation of β-lactamase inhibitors. Through the previous in vitro antibacterial experiments, it was found that magnolia alkaloids can enhance the activity of the antibiotics against β-lactamase when used in combination with β-lactam antibiotics or other non-β-lactam antibiotics, thereby enhancing the antibacterial properties of the antibiotics. Therefore, the magnolia alkaloids of the present invention combined with β-lactam antibiotics can be used to treat bacterial infections in humans or animals.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions: The first aspect of the embodiment of the present invention provides the use of magnolia alkaloids in the preparation of β-lactamase inhibitors. The structure of magnolia alkaloids is shown in formula (1): (1) The second aspect of the embodiments of the present invention provides the use of magnolia alkaloids in combination with β-lactam antibiotics in the preparation of antibacterial drugs.

[0007] The third aspect of the embodiments of the present invention provides the use of magnolia alkaloids 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.

[0008] A fourth aspect of the embodiments of the present invention provides an antibacterial pharmaceutical composition, comprising magnolia alkaloids and at least one β-lactam antibiotic.

[0009] In a preferred embodiment, the ratio of the concentration of magnolamine to the concentration of β-lactam antibiotics is 1:8 to 64:1.

[0010] In a preferred embodiment, the β-lactam antibiotics include aztreonam and ceftazidime.

[0011] Magnolia alkaloids are quaternary ammonium alkaloids derived from traditional Chinese medicine. There is no report on magnolia alkaloids as β-lactamase inhibitors. The present invention creatively uses magnolia alkaloids as β-lactamase inhibitors. At the same time, magnolia alkaloids are used in combination with ceftazidime or aztreonam to significantly enhance the sensitivity of various bacteria to ceftazidime or aztreonam. At the same time, the antibacterial drug composition provided by the present invention is a drug containing a β-lactamase inhibitor, which is used together with β-lactam antibiotics to fight against drug-resistant bacteria that produce β-lactamase. DETAILED DESCRIPTION

[0012] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in combination with the data lists in 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.

[0013] The embodiment of the invention discloses the use of magnolia alkaloids in the preparation of β-lactamase inhibitors.

[0014] Example 1: An antibacterial drug composition 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 magnolidine.

[0015] Example 2: Minimum Inhibitory Concentration (MIC) Determination This experimental example adopts the method described by the CLSI (Clinical Laboratory Standards Institute) standard, adds 4 mg / L of magnolamine, and determines the minimum inhibitory concentration (MIC) of β-lactam antibiotics against Gram-negative bacteria. In this example, Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella pneumoniae and Acinetobacter baumannii are used as test strains, and ceftazidime and aztreonam are used as representatives of β-lactam antibiotics.

[0016] The specific method is as follows: The minimum inhibitory concentration (MIC) of magnolamine alone and in combination with ceftazidime or aztreonam against Gram-negative bacteria was determined by broth dilution method.

[0017] 1. Experimental Materials Ten Gram-negative bacteria were used in the experiment: Escherichia coli ( Escherichia coli )ATCC8739, clinical isolates of Escherichia coli, Pseudomonas aeruginosa ( Pseudomonas aeruginosa )ATCC9027, Pseudomonas aeruginosa clinical isolates, Enterobacter cloacae ( Enterobacter cloacae )ATCC700323, clinical isolates of Enterobacter cloacae, Klebsiella pneumoniae ( Klebsiella pneumoniae )ATCC700603, Klebsiella pneumoniae clinical isolate, Acinetobacter baumannii ATCC19606, Acinetobacter baumannii clinical isolate.

[0018] 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).

[0019] β-lactamase inhibitors: magnolamine (China Food and Drug Inspection Institute, purity 99%, batch number 111869-202406), avibactam (laboratory-made, purity greater than 95%), sulbactam (McLean, purity 98%, batch number C12084503), clavulanate potassium (Anage, purity 95%, batch number AMP335).

[0020] Other reagents were of analytical grade.

[0021] The required equipment are all conventional experimental equipment and will not be described in detail here.

[0022] 2. Experimental methods 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 6-8 hours. 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.

[0023] Preparation of stock solutions of each test drug: Take appropriate amount of each test drug, ceftazidime, aztreonam, magnolamine, avibactam, clavulanate potassium, and sulbactam, weigh accurately, and dissolve them in dimethyl sulfoxide (DMSO) to obtain stock solutions with a concentration of 10 g / L for use.

[0024] Preparation of stock solutions of each inhibitor Take appropriate amount of the above-mentioned stock solutions of 10 g / L of magnolia alkaloid, avibactam, clavulanate potassium and sulbactam, dilute them with MH broth to a stock solution of 8 mg / L of each inhibitor, and set aside.

[0025] Preparation of each antibiotic stock solution: Take appropriate amount of the above-mentioned 10 g / L ceftazidime and aztreonam stock solutions, dilute them with MH broth to a concentration of 64 mg / L of each antibiotic stock solution, and set them aside.

[0026] Preparation of working solutions of each test drug series: Take appropriate amounts of the above-mentioned 10 g / L stock solutions of ceftazidime, aztreonam, magnolamine, 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.

[0027] Preparation of a series of working solutions of antibiotic and inhibitor combinations Ceftazidime or aztreonam with concentrations of 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 mg / L were taken, respectively, and diluted twice by equal volume with 8 mg / L stock solutions of magnolia alkaloid, avibactam, clavulanate potassium, and sulbactam as solvents to obtain a series of working solutions of each antibiotic and inhibitor combination, wherein the concentration of ceftazidime or aztreonam in the combination working solution was 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, and 0.0625 mg / L, respectively, and the concentration of magnolia alkaloid, avibactam, clavulanate potassium, and sulbactam inhibitors in the combination working solution was 4 mg / L.

[0028] Determination of MIC of each test drug The working solutions of each test drug series and the working solutions of the antibiotic and inhibitor combination series were mixed with 10 bacterial suspensions in a 96-well plate and incubated at 37°C for 18-24 hours. A control group of bacteria without drug addition was set up to determine the minimum inhibitory concentration (MIC) of each test drug. The results are shown in Table 1.

[0029]

[0030] As shown in Table 1, 1) compared with the administration of ceftazidime alone, the MIC of magnolialine combined with ceftazidime against ATCC standard strains of Escherichia coli, ATCC standard strains of Pseudomonas aeruginosa, clinical isolates of Enterobacter cloacae and clinical isolates of Klebsiella pneumoniae were significantly reduced. 2) compared with the administration of aztreonam alone, the MIC of magnolialine combined with aztreonam against ATCC standard strains of Escherichia coli, clinical isolates of Pseudomonas aeruginosa, ATCC standard strains of Pseudomonas aeruginosa and clinical isolates of Enterobacter cloacae were significantly reduced. 3) Compared with the marketed β-lactamase inhibitors clavulanate potassium, sulbactam and avibactam, the combination of magnolia alkaloids and ceftazidime can more significantly enhance the sensitivity of ATCC standard strains of Escherichia coli and clinical isolates of Enterobacter cloacae to ceftazidime; 4) Compared with clavulanate potassium, sulbactam and avibactam, the combination of magnolia alkaloids and aztreonam can more significantly enhance the sensitivity of ATCC standard strains of Pseudomonas aeruginosa to aztreonam.

[0031] The above results fully demonstrate that the combination of magnolia alkaloids with ceftazidime or aztreonam can significantly enhance the sensitivity of multiple bacteria to ceftazidime and aztreonam. Furthermore, compared with existing β-lactamase inhibitors such as clavulanate potassium, sulbactam and avibactam, the combination of magnolia alkaloids with ceftazidime or aztreonam can more significantly enhance the sensitivity of multiple bacteria to aztreonam.

[0032] 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.

[0033] 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 magnolia alkaloids in the preparation of β-lactamase inhibitors, characterized in that: The structure of the magnolia alkaloid is shown in formula (1), （1）。 2. The application of magnolia alkaloids in combination with β-lactam antibiotics in the preparation of antibacterial drugs.

3. The application of magnolia alkaloids 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 magnolamine and at least one beta-lactam antibiotic.

5. The antibacterial drug composition according to claim 4, characterized in that: The ratio of magnolamine concentration to β-lactam antibiotics is 1:8 to 64:

1.

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

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