Application of glycine and inosine in the preparation of drugs to improve bacterial susceptibility to antibiotics

Abstract

This invention belongs to the field of biomedical technology, specifically relating to the application of the combined use of glycine and inosine in the preparation of drugs that enhance bacterial susceptibility to antibiotics. This invention is the first to discover that the combination of glycine and inosine as an antibiotic potentiator not only enhances the sensitivity of pathogens to β-lactam antibiotics or their compound preparations, especially ceftazidime and avibactam sodium, exhibiting concentration- and time-dependent gradient effects, but also demonstrates a synergistic effect superior to that of glycine or inosine alone combined with antibiotics. Further experiments show that this combined regimen can effectively eliminate persistent bacteria and biofilms formed by drug-resistant bacteria, increase the intracellular antibiotic concentration of drug-resistant bacteria, and delay the development of antibiotic resistance. This invention provides a new combined drug strategy for overcoming the resistance problem of drug-resistant bacteria, especially carbapenem-resistant bacteria, with advantages such as reduced antibiotic dosage, improved bactericidal efficiency, and delayed resistance development, making it suitable for clinical anti-infective treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to the application of the combined use of glycine and inosine in the preparation of drugs that enhance bacterial susceptibility to antibiotics. Background Technology

[0002] Antimicrobial resistance has become a major challenge in global public health. The emergence and prevalence of multidrug-resistant (resistant to three or more classes of antibiotics), extensively drug-resistant, and even pandrug-resistant bacteria pose a serious threat to human health. Among the many drug-resistant bacteria encountered in clinical practice, carbapenem-resistant Gram-negative bacilli are particularly prominent, including carbapenem-resistant Escherichia coli (CR-ECO), carbapenem-resistant Klebsiella pneumoniae (CR-KPN), carbapenem-resistant Acinetobacter baumannii (CR-AB), and carbapenem-resistant Pseudomonas aeruginosa (CR-PA). Carbapenems are commonly used as last-line drugs in clinical practice, usually only used after other types of antibiotics have failed, and their long-term and widespread use has led to the development of resistance. These bacteria, with multidrug resistance compounded by carbapenem resistance, have extremely low sensitivity to antibiotics; even antibiotic concentrations that might be effective against multidrug-resistant bacteria are ineffective, hence the term "superbugs."

[0003] The development of novel and effective antimicrobial drugs for infections caused by superbugs has been slow, with a severe shortage of existing treatments and very limited clinical trial data for new drugs still in the research and development stage. Therefore, the World Health Organization (WHO) listed the four carbapenem-resistant bacteria mentioned above as key drug-resistant bacteria requiring the development of new antimicrobial drugs for prevention and control in 2017 and 2024, respectively. Existing research has shown that metabolic reprogramming mediated by single-molecule metabolites can improve the bactericidal efficiency of antibiotics; however, while this strategy has a certain promoting effect on susceptible and multidrug-resistant bacteria, its effectiveness against superbugs remains very limited. Therefore, innovation at the conceptual and technological levels is urgently needed to improve the bactericidal effect of antibiotics against superbugs. Summary of the Invention

[0004] The technical problem to be solved by this invention is to overcome the shortcomings and deficiencies of existing technologies in the slow progress of the development of new and effective antibacterial drugs for infections caused by pathogens, especially super-drug-resistant bacteria, and the extreme scarcity of existing therapeutic drugs. The primary objective is to provide the application of glycine combined with inosine in the preparation of antibiotic potentiators.

[0005] A second objective of this invention is to provide the use of glycine combined with inosine in the preparation of a medicament that enhances the sensitivity of pathogens to antibiotics.

[0006] A third objective of this invention is to provide the application of glycine and inosine combined antibiotics in the preparation of antimicrobial drugs.

[0007] The fourth objective of this invention is to provide an antimicrobial drug.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution: This invention protects the use of glycine combined with inosine in the preparation of antibiotic potentiators, wherein the antibiotic is selected from at least one of the following: a first cephalosporin antibiotic or a combination preparation of the antibiotic and a β-lactamase inhibitor; a second cephalosporin antibiotic or a combination preparation of the antibiotic and a β-lactamase inhibitor; a penicillin antibiotic or a combination preparation of the antibiotic and a β-lactamase inhibitor; a carbapenem antibiotic or a combination preparation of the antibiotic and a β-lactamase inhibitor; and aztreonam or a combination preparation of the antibiotic and a β-lactamase inhibitor. The first cephalosporin antibiotic is selected from at least one of ceftazidime, cefepime, and cefoperazone; The second combination of cephalosporin antibiotic and β-lactamase inhibitor is selected from at least one of cefaclor-sulbactam or a pharmaceutically acceptable salt thereof, ceftriaxone-avibactam or a pharmaceutically acceptable salt thereof, ceftriaxone-sulbactam or a pharmaceutically acceptable salt thereof, and cefazolin-sulbactam or a pharmaceutically acceptable salt thereof. The combination preparation of the penicillin antibiotic and β-lactamase inhibitor is selected from at least one of carbenicillin avibactam or a pharmaceutically acceptable salt thereof, carbenicillin sulbactam or a pharmaceutically acceptable salt thereof, ampicillin avibactam or a pharmaceutically acceptable salt thereof, ampicillin sulbactam or a pharmaceutically acceptable salt thereof, amoxicillin avibactam or a pharmaceutically acceptable salt thereof, and amoxicillin sulbactam or a pharmaceutically acceptable salt thereof; The carbapenem antibiotics are selected from meropenem and / or imipenem; The pathogen targeted by the antibiotic potentiator is selected from at least one of Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae.

[0009] This invention also protects the use of glycine combined with inosine in the preparation of a drug that enhances the sensitivity of pathogens to antibiotics, wherein the antibiotic is selected from at least one of the following: a first cephalosporin antibiotic or a combination of the above and a β-lactamase inhibitor; a second cephalosporin antibiotic or a combination of the above and a β-lactamase inhibitor; a penicillin antibiotic or a combination of the above and a β-lactamase inhibitor; a carbapenem antibiotic or a combination of the above and a β-lactamase inhibitor; and aztreonam or a combination of the above and a β-lactamase inhibitor. The first cephalosporin antibiotic is selected from at least one of ceftazidime, cefepime, and cefoperazone; The second combination of cephalosporin antibiotic and β-lactamase inhibitor is selected from at least one of cefaclor-sulbactam or a pharmaceutically acceptable salt thereof, ceftriaxone-avibactam or a pharmaceutically acceptable salt thereof, ceftriaxone-sulbactam or a pharmaceutically acceptable salt thereof, and cefazolin-sulbactam or a pharmaceutically acceptable salt thereof. The combination preparation of the penicillin antibiotic and β-lactamase inhibitor is selected from at least one of carbenicillin avibactam or a pharmaceutically acceptable salt thereof, carbenicillin sulbactam or a pharmaceutically acceptable salt thereof, ampicillin avibactam or a pharmaceutically acceptable salt thereof, ampicillin sulbactam or a pharmaceutically acceptable salt thereof, amoxicillin avibactam or a pharmaceutically acceptable salt thereof, and amoxicillin sulbactam or a pharmaceutically acceptable salt thereof; The carbapenem antibiotics are selected from meropenem and / or imipenem; The pathogen is selected from at least one of Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae.

[0010] Furthermore, the glycine combined with inosine serves as an antibiotic potentiator to enhance the sensitivity of pathogens to the aforementioned specific types of antibiotics.

[0011] Furthermore, the glycine combined with inosine serves as an antibiotic potentiator to enhance the sensitivity of biofilms formed by pathogens to the aforementioned specific types of antibiotics.

[0012] Furthermore, the glycine combined with inosine serves as an antibiotic potentiator to enhance the ability of the aforementioned specific types of antibiotics to kill pathogens.

[0013] Furthermore, the glycine combined with inosine serves as an antibiotic potentiator to enhance the ability of the aforementioned specific types of antibiotics to resist pathogenic bacterial infections.

[0014] Furthermore, the glycine combined with inosine serves as an antibiotic potentiator to enhance the ability of the aforementioned specific types of antibiotics to eliminate pathogens.

[0015] Furthermore, the glycine combined with inosine serves as an antibiotic potentiator to enhance the ability of the aforementioned specific types of antibiotics to enter the intracellular concentration of pathogens.

[0016] Furthermore, the glycine combined with inosine acts as an antibiotic potentiator to delay the development of resistance in pathogens to the aforementioned specific types of antibiotics.

[0017] This invention also protects the use of glycine and inosine combined antibiotics in the preparation of antimicrobial drugs, wherein the antibiotic is selected from at least one of the following: a first cephalosporin antibiotic or a combination preparation of the antibiotic and a β-lactamase inhibitor; a second cephalosporin antibiotic or a combination preparation of the antibiotic and a β-lactamase inhibitor; a penicillin antibiotic or a combination preparation of the antibiotic and a β-lactamase inhibitor; a carbapenem antibiotic or a combination preparation of the antibiotic and a β-lactamase inhibitor; and aztreonam or a combination preparation of the antibiotic and a β-lactamase inhibitor. The first cephalosporin antibiotic is selected from at least one of ceftazidime, cefepime, and cefoperazone; The second combination of cephalosporin antibiotic and β-lactamase inhibitor is selected from at least one of cefaclor-sulbactam or a pharmaceutically acceptable salt thereof, ceftriaxone-avibactam or a pharmaceutically acceptable salt thereof, ceftriaxone-sulbactam or a pharmaceutically acceptable salt thereof, and cefazolin-sulbactam or a pharmaceutically acceptable salt thereof. The combination preparation of the penicillin antibiotic and β-lactamase inhibitor is selected from at least one of carbenicillin avibactam or a pharmaceutically acceptable salt thereof, carbenicillin sulbactam or a pharmaceutically acceptable salt thereof, ampicillin avibactam or a pharmaceutically acceptable salt thereof, ampicillin sulbactam or a pharmaceutically acceptable salt thereof, amoxicillin avibactam or a pharmaceutically acceptable salt thereof, and amoxicillin sulbactam or a pharmaceutically acceptable salt thereof; The carbapenem antibiotics are selected from meropenem and / or imipenem; The pathogen is selected from at least one of Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae.

[0018] Furthermore, the application includes at least one of the following conditions: (1) The β-lactamase inhibitor in the first cephalosporin antibiotic or its combination preparation with a β-lactamase inhibitor is selected from at least one of avibactam or a pharmaceutically acceptable salt thereof, sulbactam or a pharmaceutically acceptable salt thereof, and clavulanic acid or a pharmaceutically acceptable salt thereof; (2) The β-lactamase inhibitor in the carbapenem antibiotic or its combination preparation with a β-lactamase inhibitor is selected from at least one of avibactam or a pharmaceutically acceptable salt thereof, sulbactam or a pharmaceutically acceptable salt thereof, and clavulanic acid or a pharmaceutically acceptable salt thereof; (3) The β-lactamase inhibitor in the combination preparation of aztreonam or a β-lactamase inhibitor is selected from at least one of avibactam or a pharmaceutically acceptable salt thereof, sulbactam or a pharmaceutically acceptable salt thereof, and clavulanic acid or a pharmaceutically acceptable salt thereof.

[0019] Furthermore, the pharmaceutically acceptable salt is a sodium or potassium salt.

[0020] Furthermore, the β-lactamase inhibitor is selected from at least one of sulbactam or a pharmaceutically acceptable salt thereof, avibactam or a pharmaceutically acceptable salt thereof.

[0021] Specifically, the antibiotic is selected from at least one of ceftazidime, ceftazidime avibactam sodium, cefoperazone, cefoperazone sulbactam sodium, cefepime, cefepime sulbactam sodium, cefepime avibactam sodium, ceftriaxone avibactam sodium, ceftriaxone sulbactam sodium, cefaclor sulbactam sodium, cefazolin sulbactam sodium, aztreonam, aztreonam avibactam sodium, imipenem, imipenem avibactam sodium, meropenem, meropenem avibactam sodium, carbenicillin avibactam sodium, carbenicillin sulbactam sodium, ampicillin avibactam sodium, ampicillin sulbactam sodium, amoxicillin avibactam sodium, and amoxicillin sulbactam sodium.

[0022] Furthermore, the application includes at least one of the following conditions: (1) The molar ratio of glycine to inosine is 1:(0.01~20); (2) The mixing ratio of glycine and antibiotic is 1 mol: (0.01-500) g; (3) The concentration of the glycine is ≥5mM; (4) The concentration of inosine is ≥5 mM; (5) The concentration of the antibiotic is ≥5 μg / mL; (6) In the compound preparation, the mass ratio of the antibiotic to the β-lactamase inhibitor is (0.5~8):1.

[0023] Preferably, the molar ratio of glycine to inosine is 1:(0.01~20), more preferably 1:(0.0625~16), even more preferably 1:(0.5~2), and even more preferably 1:(0.8~1.2).

[0024] Specifically, the concentration of glycine can be 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80 mM, or any specific value between the above values, or any range of these values. A suitable concentration can be flexibly selected according to the actual situation.

[0025] Preferably, the mixing ratio of glycine and antibiotic is 1 mol: (0.01–500) g, 1 mol: (0.0625–200) g, and more preferably 1 mol: (1.25–25) g. The appropriate ratio and concentration can be flexibly selected according to the type of antibiotic and specific bactericidal requirements.

[0026] Preferably, the concentration of glycine is 5-80 mM, more preferably 40-80 mM.

[0027] Specifically, the concentration of inosine can be 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80 mM, or any specific value between the above values, or any range of these values. A suitable concentration can be flexibly selected according to the actual situation.

[0028] Preferably, the concentration of inosine is 5-80 mM, more preferably 40-80 mM.

[0029] Specifically, the concentration of the antibiotic can be 5, 10, 20, 40, 50, 100, 150, 160, 200, 300, 400, 500, 600, 700, 800, 1000, 1500 μg / mL, or any value between these specific values, or any range consisting of these specific values. A suitable concentration can be flexibly selected based on the type of antibiotic and specific bactericidal requirements.

[0030] Furthermore, the concentration of the antibiotic is 5~1500 μg / mL, more preferably 50~1500 μg / mL, and even more preferably 50~1000 μg / mL.

[0031] Furthermore, when the antibiotic is selected from any one of a first cephalosporin antibiotic or a combination preparation of it and a β-lactamase inhibitor, or a combination preparation of a second cephalosporin antibiotic and a β-lactamase inhibitor, the concentration of the antibiotic is ≥50 μg / mL, preferably 50~250 μg / mL, and more preferably 60~200 μg / mL.

[0032] Furthermore, when the antibiotic is selected from a compound preparation of penicillin antibiotics and β-lactamase inhibitors, the concentration of the antibiotic is ≥500 μg / mL, preferably 800~1500 μg / mL, and more preferably 1000~1500 μg / mL.

[0033] Furthermore, when the antibiotic is selected from aztreonam or a combination preparation of it and a β-lactamase inhibitor, the concentration of the antibiotic is ≥150 μg / mL, preferably 150~300 μg / mL, and more preferably 180~270 μg / mL.

[0034] Furthermore, when the antibiotic is selected from imipenem or a combination of imipenem and a β-lactamase inhibitor, the concentration of the antibiotic is ≥50 μg / mL, preferably 50~200 μg / mL, and more preferably 80~160 μg / mL.

[0035] Furthermore, when the antibiotic is selected from meropenem or a combination of meropenem and a β-lactamase inhibitor, the concentration of the antibiotic is ≥20 μg / mL, preferably 20~100 μg / mL, and more preferably 40~75 μg / mL.

[0036] Furthermore, in the aforementioned compound preparations containing β-lactamase inhibitors, the mass ratio of the antibiotic to the β-lactamase inhibitor in the compound preparation can be 0.5:1, 1:1, 2:2, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, etc., or any specific ratio between the above-mentioned specific ratios, and may also include any range of these specific ratios. For the sake of simplicity, not all possible point values ​​or range values ​​are listed here.

[0037] Preferably, in the compound preparation, the mass ratio of the antibiotic to the β-lactamase inhibitor is (2~7):1.

[0038] Furthermore, when the β-lactamase inhibitor in the compound preparation is selected from avibactam or a pharmaceutically acceptable salt thereof, the mass ratio of the antibiotic to the β-lactamase inhibitor in the compound preparation is (2~5):1, preferably (2.5~4.5):1, more preferably (3~4):1.

[0039] Furthermore, when the β-lactamase inhibitor in the compound preparation is selected from sulbactam or a pharmaceutically acceptable salt thereof, the mass ratio of the antibiotic to the β-lactamase inhibitor in the compound preparation is (1~3):1, preferably (1.5~2.5):1, more preferably (1.8~2.2):1.

[0040] Furthermore, when the β-lactamase inhibitor in the compound preparation is selected from clavulanic acid or a pharmaceutically acceptable salt thereof, the mass ratio of the antibiotic to the β-lactamase inhibitor in the compound preparation is 1:(6~8), preferably 1:(6.5~7.5), more preferably (6.8~7.2):1.

[0041] Furthermore, the pathogenic bacteria include at least one of sensitive bacteria, drug-resistant bacteria, and persistent bacteria.

[0042] Persisters are phenotypic variants in a microbial population that randomly undergo dormancy and exhibit high antibiotic resistance.

[0043] Furthermore, the drug-resistant bacteria are multidrug-resistant bacteria.

[0044] Furthermore, the multidrug-resistant bacteria are carbapenem-resistant bacteria.

[0045] This invention protects an antimicrobial drug, wherein the active ingredients of the antimicrobial drug include glycine, inosine, and antibiotics; The antibiotic is selected from at least one of the following: a first-class cephalosporin antibiotic or a combination preparation of the antibiotic and a β-lactamase inhibitor; a second-class cephalosporin antibiotic or a combination preparation of the antibiotic and a β-lactamase inhibitor; a penicillin antibiotic or a combination preparation of the antibiotic and a β-lactamase inhibitor; a carbapenem antibiotic or a combination preparation of the antibiotic and a β-lactamase inhibitor; and aztreonam or a combination preparation of the antibiotic and a β-lactamase inhibitor. The first cephalosporin antibiotic is selected from at least one of ceftazidime, cefepime, and cefoperazone; The second combination of cephalosporin antibiotic and β-lactamase inhibitor is selected from at least one of cefaclor sulbactam or a pharmaceutically acceptable salt thereof, ceftriaxone avibactam or a pharmaceutically acceptable salt thereof, ceftriaxone sulbactam or a pharmaceutically acceptable salt thereof, cefazolin avibactam or a pharmaceutically acceptable salt thereof, and cefazolin sulbactam or a pharmaceutically acceptable salt thereof. The combination preparation of the penicillin antibiotic and β-lactamase inhibitor is selected from at least one of carbenicillin avibactam or a pharmaceutically acceptable salt thereof, carbenicillin sulbactam or a pharmaceutically acceptable salt thereof, ampicillin avibactam or a pharmaceutically acceptable salt thereof, ampicillin sulbactam or a pharmaceutically acceptable salt thereof, amoxicillin avibactam or a pharmaceutically acceptable salt thereof, and amoxicillin sulbactam or a pharmaceutically acceptable salt thereof; The carbapenem antibiotics are selected from meropenem and / or imipenem.

[0046] Furthermore, the antimicrobial drug comprises at least one of the following conditions: (1) The β-lactamase inhibitor in the first cephalosporin antibiotic or its combination preparation with a β-lactamase inhibitor is selected from at least one of avibactam or a pharmaceutically acceptable salt thereof, sulbactam or a pharmaceutically acceptable salt thereof, and clavulanic acid or a pharmaceutically acceptable salt thereof; (2) The β-lactamase inhibitor in the carbapenem antibiotic or its combination preparation with a β-lactamase inhibitor is selected from at least one of avibactam or a pharmaceutically acceptable salt thereof, sulbactam or a pharmaceutically acceptable salt thereof, and clavulanic acid or a pharmaceutically acceptable salt thereof; (3) The β-lactamase inhibitor in the combination preparation of aztreonam or a β-lactamase inhibitor is selected from at least one of avibactam or a pharmaceutically acceptable salt thereof, sulbactam or a pharmaceutically acceptable salt thereof, and clavulanic acid or a pharmaceutically acceptable salt thereof; (4) The pathogenic bacteria are selected from at least one of Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae.

[0047] Furthermore, the pharmaceutically acceptable salt is a sodium or potassium salt.

[0048] Specifically, the antibiotic is selected from at least one of ceftazidime, ceftazidime avibactam sodium, cefoperazone, cefoperazone sulbactam sodium, cefepime, cefepime sulbactam sodium, cefepime avibactam sodium, ceftriaxone avibactam sodium, ceftriaxone sulbactam sodium, cefaclor sulbactam sodium, cefazolin sulbactam sodium, aztreonam, aztreonam avibactam sodium, imipenem, imipenem avibactam sodium, meropenem, meropenem avibactam sodium, carbenicillin avibactam sodium, carbenicillin sulbactam sodium, ampicillin avibactam sodium, ampicillin sulbactam sodium, amoxicillin avibactam sodium, and amoxicillin sulbactam sodium.

[0049] Furthermore, the pathogenic bacteria include at least one of sensitive bacteria, drug-resistant bacteria, and persistent bacteria.

[0050] Furthermore, the drug-resistant bacteria are multidrug-resistant bacteria.

[0051] Furthermore, the multidrug-resistant bacteria are carbapenem-resistant bacteria.

[0052] Furthermore, the antimicrobial drug includes at least one of the following parameters: (1) The molar ratio of glycine to inosine is 1:(0.01~20); (2) The mixing ratio of glycine and antibiotic is 1 mol: (0.01-500) g; (3) The concentration of the glycine is ≥5mM; (4) The concentration of inosine is ≥5 mM; (5) The concentration of the antibiotic is ≥5 μg / mL; (6) In the compound preparation, the mass ratio of the antibiotic to the β-lactamase inhibitor is (0.5~8):1.

[0053] The further limitations of the above parameters are the same as before.

[0054] Compared with the prior art, the present invention has the following beneficial effects: This invention is the first to discover that glycine combined with inosine as an antibiotic potentiator not only enhances the sensitivity of pathogens to β-lactam antibiotics or their combination preparations, especially ceftazidime and avibactam sodium, exhibiting concentration- and time-dependent gradient effects, but also demonstrates a synergistic effect superior to that of glycine or inosine alone combined with antibiotics. Further experiments show that this combined regimen effectively eliminates persistent bacteria and biofilms formed by drug-resistant bacteria, increases the intracellular antibiotic concentration of drug-resistant bacteria, and delays the development of antibiotic resistance. This invention provides a novel combination therapy strategy for overcoming the resistance problem of drug-resistant bacteria, especially carbapenem-resistant bacteria, offering advantages such as reduced antibiotic dosage, improved bactericidal efficiency, and delayed resistance development, making it applicable to the field of clinical anti-infective therapy. Attached Figure Description

[0055] Figure 1 Statistical graphs show the data on the combined use of exogenous glycine and inosine to enhance bacterial sensitivity to ceftazidime avibactam sodium (CZA). Graph A shows the bactericidal effects of eight biological target substances on bacteria; Graph B shows the data on the combined use of two key biological target substances to enhance bacterial sensitivity to CZA; Graph C shows the data on the combined use of six biological target substances with inosine or glycine to enhance bacterial sensitivity to CZA; Graph D shows the data on the optimal concentration combination of inosine and glycine to enhance bacterial sensitivity to CZA; and Graph E shows the data on the combined use of inosine and glycine to enhance bacterial sensitivity to CZA. Statistical graphs of synergistic effects: Figure F shows the statistical graph of the optimal concentration combination of fumaric acid and inosine in enhancing bacterial sensitivity to CZA; Figure G shows the statistical graph of the synergistic effect of fumaric acid and inosine in enhancing bacterial sensitivity to CZA; Figure H shows the statistical graph of the optimal concentration combination of fumaric acid and glycine in enhancing bacterial sensitivity to CZA; Figure I shows the statistical graph of the synergistic effect of fumaric acid and glycine in enhancing bacterial sensitivity to CZA; Figure J shows the statistical graph of the effect of glycine in enhancing bacterial sensitivity to CZA by combining it with metabolites of purine metabolism.

[0056] Figure 2 Figure 1 shows the results of the synergistic effect of inosine / glycine on improving the sensitivity of clinically derived drug-resistant Escherichia coli to ceftazidime and avibactam. Figure 2 shows the statistical data of carbapenem-resistant Escherichia coli CR-ECO; Figure 3 shows the statistical data of multidrug-resistant Escherichia coli MDR-ECO; and Figure 4 shows the statistical data of susceptible Escherichia coli S-ECO.

[0057] Figure 3 Statistical graph showing the effects of inosine / glycine synergistic enhancement of bacterial sensitivity to ceftazidime avibactam sodium on antibiotic concentration (A), substance concentration (B), time gradient (C), resident bacteria (D), and biofilm (E).

[0058] Figure 4This section presents statistical charts showing the bactericidal effects of the combined use of glycine and inosine against β-lactam antibiotics. Chart A shows the results of the combined use of glycine and inosine improving bacterial sensitivity to β-lactam antibiotics; Chart B shows the results of the combined use of glycine and inosine improving bacterial sensitivity to β-lactam antibiotics plus β-lactamase inhibitors; Chart C shows the results of the combined use of glycine and inosine improving bacterial sensitivity to meropenem; Chart D shows the statistical chart of the optimal concentration combination of glycine and inosine for improving bacterial sensitivity to meropenem; and Chart E shows the statistical chart of the synergistic effect analysis of glycine and inosine in improving bacterial sensitivity to meropenem.

[0059] Figure 5 This is a statistical chart showing the data on how inosine / glycine synergistically enhances the susceptibility of other carbapenem-resistant Gram-negative bacteria to ceftazidime-avibactam sodium. Chart A shows carbapenem-resistant Klebsiella pneumoniae; Chart B shows clinically carbapenem-resistant Pseudomonas aeruginosa; Chart C shows clinically carbapenem-resistant Acinetobacter baumannii; and Charts D-F show clinically clinical Vibrio alginolyticus, clinically clinical Edwardsiella tarda, and methicillin-resistant Staphylococcus aureus, respectively.

[0060] Figure 6 The graph shows the results of the synergistic enhancement of the bactericidal effect of ceftazidime / avibactam sodium by inosine / glycine compared with that of aztreonam / avibactam sodium, one of the latest combination formulations.

[0061] Figure 7 The results are statistical graphs showing that inosine / glycine combined with ceftazidime and avibactam sodium can improve the survival rate of mice infected with clinically resistant Gram-negative bacteria; among them, Figure A is the statistical graph of CR-ECO; Figure B is the statistical graph of CR-KPN; Figure C is the statistical graph of CR-AB; and Figure D is the statistical graph of CR-PA.

[0062] Figure 8 The results are statistical graphs showing the effects of inosine / glycine combined with ceftazidime and avibactam sodium on improving the clearance ability of mice against clinically resistant Gram-negative bacteria. Among them, Figure A is the statistical graph of CR-ECO; Figure B is the statistical graph of CR-KPN; Figure C is the statistical graph of CR-AB; and Figure D is the statistical graph of CR-PA.

[0063] Figure 9 Statistical graph showing the data on the synergistic effect of inosine / glycine on enhancing the sensitivity of Escherichia coli BW25113, outer membrane protein-deficient strain (A), and dual-regulatory protein gene-deficient strain (B) to ceftazidime avibactam sodium.

[0064] Figure 10 A statistical chart showing the data on how inosine / glycine synergistically increases the concentration of CZA entering bacterial cells.

[0065] Figure 11This is a ceftazidime blood concentration-time curve. Detailed Implementation

[0066] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0067] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0068] Figure 1 A represents Figure 1 Figure A in the text, Figure 1 B indicates Figure 1 The naming meanings of the other figures follow the same pattern as Figure B in the diagram.

[0069] Example 1: The combined use of glycine and inosine significantly improved the susceptibility of bacteria to ceftazidime avibactam sodium. The abbreviation for susceptible Escherichia coli is S-ECO, and it will be referred to as susceptible bacteria from now on; the abbreviation for multidrug-resistant but carbapenem-sensitive bacteria is MDR-ECO, and it will be referred to as multidrug-resistant bacteria from now on; the abbreviation for multidrug-resistant and carbapenem-resistant bacteria is CR-ECO, and it will be referred to as carbapenem-resistant bacteria from now on.

[0070] 1.1 Preparation of bacterial samples for sterilization Pick a single bacterial colony from the plate and transfer it to 50 mL of LB liquid medium. Incubate at 37°C and 200 rpm for 16 hours. Take 50 mL of the bacterial suspension, centrifuge at 8000 rpm for 3 minutes, and discard the supernatant. Wash the bacterial cells three times with an equal volume of 0.85 wt% physiological saline, then suspend the bacterial cells in 1×M9 medium (except for Pseudomonas aeruginosa, which contains 10 mM sodium citrate, the others contain 10 mM acetate). Adjust the bacterial concentration to achieve the desired OD value. 600 The value is 0.2. Dispense 5 mL into test tubes for later use.

[0071] 1.2 Study on the effect of combined use of biological target substances to enhance bacterial sensitivity to CZA Escherichia coli CR-ECO 3 bacterial samples obtained clinically were prepared according to Example 1.1. The drug resistance spectrum of CR-ECO 3 is shown in Table 1.

[0072] Table 1. Drug resistance spectrum of Escherichia coli CR-ECO 3

[0073] Note: R indicates drug resistance, and S indicates drug sensitivity.

[0074] First, the study investigated whether eight biological target substances (glycine (Gly), inosine (Ino), proline (Pro), glucose (Glu), fumaric acid (Fum), uracil (U), ribofuranoose (Rib), and dodecanedioic acid (DDDA)) themselves possessed bactericidal activity. The results are shown in […]. Figure 1 A. As shown in the figure, these eight biological target substances do not have bactericidal effects themselves.

[0075] Next, the effects of the biological targets glycine and inosine on improving bacterial antibiotic susceptibility were investigated. The concentration of the antibiotic cefotaxime avibactam sodium (CAZ:AVI mass ratio 4:1, abbreviated CZA) was 160 μg / mL. Each condition was performed in triplicate. After 6 hours of treatment at 37°C, viable cell counts were performed, and the bacterial survival rate after each treatment condition was calculated using the formula: Survival rate (%) = (Number of viable cells in each group / Number of viable cells in the M9 control group) × 100%. Results are shown below. Figure 1 B. The results showed that adding inosine alone could increase bacterial sensitivity to CZA by 20 times, adding glycine alone could increase bacterial sensitivity to CZA by 7.91 times, however, the combination of the two could increase bacterial sensitivity to CZA by 1006.74 times, which was 50 times higher than adding inosine alone and 126 times higher than adding glycine alone.

[0076] Furthermore, the study investigated how glycine and inosine, in combination with six other biological target substances, could enhance bacterial susceptibility to CZA. Each condition was performed in triplicate. Results are shown below. Figure 1 C. As shown in the figure, the addition of the other six biological targets alone did not increase the sensitivity to CZA. When these six biological targets were combined with glycine or inosine, compared with the addition of inosine alone, only two targets, namely glutamate (Glu) and fumaric acid (Fum), showed an increase in bactericidal multiple of about 9.5 times when combined with inosine. Compared with the addition of glycine alone, only the combination of glutamate and glycine showed an increase in bactericidal multiple of 9.2 times.

[0077] The above studies showed that only the combination of inosine and glycine resulted in the most significant decrease in bacterial survival rate, indicating that the combined use of these two substances was the most effective in improving bacterial susceptibility to antibiotics. The second most effective combination was glutamate with either inosine or glycine, which only increased susceptibility by about nine times. It was also found that fumaric acid, when combined with either inosine or glycine, exhibited different effects.

[0078] Subsequently, we conducted in-depth studies on glycine and inosine, and the interaction mechanisms between fumaric acid and inosine or glycine.

[0079] 1.3 Study on the optimal concentration combination of glycine and inosine to enhance bacterial sensitivity to ceftazidime-avibactam sodium To determine the optimal bactericidal concentration combination of glycine and inosine, prepared *Escherichia coli* CR-ECO 3 bacterial samples were used. Each test tube contained 160 μg / mL CZA, followed by the addition of glycine (final concentrations of 0, 5, 10, 20, 40, and 80 mM) and / or inosine (final concentrations of 0, 5, 10, 20, 40, and 80 mM), forming a 6×6 matrix of experimental groups. Simultaneously, an M9 control group (i.e., a control group without inosine / glycine and CZA) and an antibiotic-only control group (i.e., a control group with 160 μg / mL CZA) were set up. Each combination was performed in triplicate. After incubation at 37°C for 6 hours, viable bacteria were counted. Bacterial counts were recorded, and the bacterial survival rate after treatment with different concentrations of inosine and / or glycine was calculated using the formula: Survival rate (%) = (Number of viable bacteria in the experimental group with antibiotics and / or inosine and / or glycine added / Number of viable bacteria in the M9 control group) × 100%. Finally, the bactericidal multiple increase after the action of inosine and / or glycine was calculated using the following formula: Bactericidal multiple = Bacterial survival rate after action of different concentrations of inosine and / or glycine / Bacterial survival rate of the control group treated with antibiotics alone. The obtained bactericidal multiple represents the effect of the two substances acting alone or in combination at different concentrations to improve bacterial sensitivity to antibiotics. A higher bactericidal multiple indicates a better effect in improving antibiotic sensitivity.

[0080] See results Figure 1 As shown in Figure D, adding inosine alone can increase bacterial sensitivity to CZA by 2.8-21.7 times, while adding glycine alone can increase bacterial sensitivity to CZA by 4.4-10 times. It was also found that when the two substances were used in combination, the sensitivity to CZA was significantly increased compared to the sensitivity of either substance alone, and that when the concentration of both substances was increased to 40 mM, the bactericidal effect could reach up to 1000 times.

[0081] To determine the interaction type of glycine and inosine in combination, the effects of this combination were analyzed using Synergy Finder 3.0 software (with the Bliss reference model selected). The Bliss analysis principle is a rigorous quantitative pharmacological method based on the Bliss independence model. Through systematic experiments, data collection, and calculations, it quantifies the interaction between two substances into a defined Bliss score, thus scientifically and reproducibly determining whether the relationship is antagonistic, additive, or synergistic. A score <0 indicates an antagonistic effect, a score of 0-10 indicates an additive effect, and a score >10 indicates a synergistic effect, with 10-20 indicating moderate synergy, 20-30 indicating strong synergy, and >30 indicating extremely strong synergy. This system provides a precise grading standard for synergistic strength in drug combination studies by quantitatively comparing experimental observations with theoretical predictions. The interaction score is visualized on a dose matrix using a two-dimensional surface: the color depth in the two-dimensional image reflects the strength of inhibition, allowing for comparison of the interaction strength of different drug combinations.

[0082] The glycine and inosine metabolites, along with their different dosage combinations, increased the bactericidal multiple of CR-ECO3 against SCF. The Log10 value was then input into the software. Based on the Bliss model, the software automatically generated an effect score and effect spectrum for the combined effect of the two metabolites. The score was 29.614. The two-dimensional image is shown below. Figure 1 E. The results showed that glycine and inosine had a synergistic effect, with the strongest synergistic effect observed at 40-80 mM.

[0083] Based on the above relationship between concentration and sensitivity, subsequent related experiments were conducted using concentrations of 40 mM glycine and 40 mM inosine, which are abbreviated as 40 mM inosine / glycine (Ino / Gly) in the experiments.

[0084] 1.4 Study on the effect of combining inosine or glycine with fumaric acid to improve bacterial susceptibility to ceftazidime / avibactam sodium. In the results of the study in Example 1.2 ( Figure 1 C) It was found that fumaric acid, when combined with inosine or glycine, showed different results in enhancing sensitivity. Therefore, array experiments were conducted on fumaric acid in combination with inosine or glycine. The combination of fumaric acid and inosine metabolites, and their different dosages, increased the bactericidal multiple of CR-ECO3 against SCF. Figure 1 F) After taking Log10 and inputting it into the software, its score is 9.203. See the two-dimensional graph below. Figure 1 G. This result indicates that fumaric acid and inosine have an additive effect. Combining fumaric acid and glycine metabolites, and their different dosages, increases the bactericidal multiple of CR-ECO3 against SCF. Figure 1H) After taking Log10 and inputting it into the software, its score is -2.456. See the two-dimensional graph below. Figure 1 I. This result indicates that fumaric acid and glycine have an antagonistic effect. The results of these interactions are related to... Figure 1 The sterilization rate results for C were consistent.

[0085] 1.5 Study on the effect of combined use of metabolites from glycine and purine metabolism on improving bacterial sensitivity to ceftazidime-avibactam sodium. Purine metabolism is a crucial metabolic process in bacteria, playing a vital role in bacterial growth, virulence, and antibiotic sensitivity. Inosine, a component of this metabolic process, can synergistically enhance bacterial antibiotic sensitivity with glycine. But what about other substances? To investigate this, bactericidal experiments were conducted using other substances from purine metabolism in combination with glycine. The results are shown below. Figure 1 J indicates that these substances themselves do not kill bacteria; if these substances are added individually on the basis of CZA, they can only increase the sensitivity of bacteria to antibiotics by about 3.59-20 times; if other substances are added on the basis of glycine, in addition to AMP and ATP, IMP (sodium adenosine), adenosine and guanosine can further increase the sensitivity of bacteria to antibiotics by 7.13 times, 18.83 times and 2.42 times respectively, but all are far lower than the combined increase in bacterial sensitivity to antibiotics by glycine and inosine.

[0086] Example 2: Synergistic effect of glycine and inosine to improve the generalizability of clinically derived Escherichia coli to ceftazidime avibactam sodium. Clinically derived *Escherichia coli* samples were prepared according to Example 1.1, including 19 CR-ECO strains, 10 MDR-ECO strains, and 10 S-ECO strains. For CR-ECO strains, each strain was divided into four groups: an M9 control group, a CZA group, a CZA + inosine / glycine group (hereinafter referred to as the synergistic group), and a polymyxin B control group. The concentration of CZA was 160 μg / mL, and the concentration of polymyxin B was 2 μg / mL. For MDR-ECO and S-ECO strains, each strain was divided into three groups: an M9 control group, a CZA group, and a synergistic group. The concentration of CZA was 10 μg / mL for MDR-ECO and 5 μg / mL for S-ECO. The concentrations of inosine and glycine were both 40 mM. Each treatment was performed in triplicate.

[0087] After treatment at 37°C for 6 hours, viable bacteria were counted, and the bacterial survival rate after treatment with inosine / glycine and / or CZA was calculated. The calculation formula is: Survival rate (%) = (Number of viable bacteria after treatment with inosine / glycine and / or CZA / Number of viable bacteria in the M9 control group) × 100%. The bacterial survival rate after treatment with colistin was calculated as follows: Survival rate (%) = (Number of viable bacteria after treatment with colistin / Number of viable bacteria in the M9 control group) × 100%.

[0088] The results of CR-ECO bacteria are shown in Figure 2 A. As shown in the figure, when antibiotics were added alone, the average survival rate of the 20 carbapenem-resistant bacteria was 52.69%, with 10 strains having a survival rate greater than 70% and 5 strains having a survival rate less than 5%. The average survival rate of the synergistic group was 2.67%, with 15 strains having a survival rate less than 1%. The bactericidal multiple increase ranged from 2.57 to 8186 times, indicating that the inosine / glycine synergy can significantly improve the bactericidal efficiency of CZA against carbapenem-resistant bacteria. Meanwhile, a polymyxin B control was performed. The average survival rate of these 20 bacteria was 12.03%, with 3 strains having a survival rate greater than 50% and 7 strains having a survival rate less than 1%. Comparison of the bactericidal results of the synergistic group and polymyxin B revealed that, except for 3 strains where the synergistic group was slightly less effective than polymyxin B, the bactericidal effect of the remaining bacteria was significantly higher than that of polymyxin B, with the bactericidal multiple increase ranging from 2.42 to 2700 times.

[0089] The results for MDR-ECO bacteria are shown below. Figure 2 B. As shown in the figure, the average survival rate of 10 multidrug-resistant bacteria was 55.06% when antibiotics were added alone, while the average survival rate of bacteria in the synergistic group was 0.08%. The bactericidal multiple increase ranged from 290 to 22804 times, indicating that the synergistic effect of inosine / glycine can significantly improve the bactericidal efficiency of CZA against multidrug-resistant bacteria.

[0090] The results for S-ECO bacteria are shown below. Figure 2 C. As shown in the figure, the average survival rate of 10 susceptible bacteria was 50.75% when antibiotics were added alone, while the average survival rate of bacteria in the synergistic group was 0.09%. The bactericidal multiple increase was between 272 and 2671 times, indicating that the synergistic effect of inosine / glycine can significantly improve the bactericidal efficiency of CZA against susceptible bacteria.

[0091] The above results indicate that the inosine / glycine synergy significantly improves the bactericidal efficiency of CZA against clinically derived Escherichia coli, demonstrating effectiveness not only against susceptible bacteria but also against multidrug-resistant and carbapenem-resistant bacteria. Furthermore, this synergistic effect was found to be significantly superior to polymyxin B.

[0092] Example 3: Study on the important conditions for the synergistic enhancement of the susceptibility of clinically derived drug-resistant Escherichia coli to ceftazidime and avibactam sodium by glycine and inosine. 3.1 It exhibits antibiotic concentration-dependent properties: Clinical carbapenem-resistant strain CR-ECO 3 was prepared according to Example 1.1. The samples were divided into an M9 control group, a CZA-only group, and a synergistic group. The CZA-only group consisted of samples with different concentrations of CZA (final concentrations of 0, 40, 80, 120, 160, and 200 μg / mL), while the synergistic group consisted of samples with different concentrations of CZA plus 40 mM inosine / glycine. Each condition was repeated three times. The samples were incubated at 37°C and 200 rpm for 6 hours, and viable cell counts were performed using the plate method to calculate the survival rate. Survival rate (%) = (Number of viable cells with added inosine / glycine and / or CZA / Number of viable cells in the M9 control group) × 100%. The bactericidal multiple increase was then calculated. The formulas were: Bactericidal multiple increase in the synergistic group = Survival rate of the CZA-only group / Survival rate of the synergistic group; Bactericidal multiple increase with different concentrations of CZA = Survival rate of the group without CZA / Survival rate of the group with different concentrations of CZA.

[0093] Survival rate results are shown below Figure 3 As shown in Figure A, the addition of CZA to the inosine / glycine base reduced the number of surviving bacteria. Furthermore, the reduction in the number of surviving bacteria became more significant with increasing CZA concentration.

[0094] The specific findings are as follows: When CZA was added, the antibiotics showed only a weak bactericidal effect. However, the bactericidal effect gradually increased with increasing CZA concentration. When CZA increased from 40 μg / mL to 200 μg / mL, the bacterial survival rate decreased from 98.11% to 32.08%, while the bactericidal multiple increased from 1.02 to 3.12 times. If 40 μg / mL of inosine / glycine was added to the corresponding antibiotic concentration, the bacterial sensitivity to CZA significantly increased, with the bactericidal multiples in the synergistic group increasing by 3.54-1572 times. When the antibiotic concentration gradually increased from 40 μg / mL to 200 μg / mL, the bactericidal multiple in the synergistic group increased from 3.61 times to 4901 times. These results indicate that: 1) the bactericidal effect of a single antibiotic can only be improved by increasing the antibiotic concentration; 2) when antibiotics are used in combination with small molecules, the bactericidal effect can be significantly improved without increasing the antibiotic concentration; 3) the effect of CZA in combination with other substances is related to the CZA concentration, and the higher the CZA concentration, the better the bactericidal effect.

[0095] 3.2 It exhibits inosine / glycine concentration dependence. The prepared CR-ECO 3 samples were divided into an M9 control group, an inosine / glycine group, and a synergistic group. The inosine / glycine group consisted of adding different concentrations of inosine / glycine alone, resulting in final concentrations of 5, 10, 20, 40, and 80 mM. The synergistic group consisted of adding 160 μg / mC CZA to the inosine / glycine group at different concentrations. Each condition was repeated three times. The samples were incubated at 37℃ and 200 rpm for 6 hours. Viable cell counts were determined using plate culture, and the survival rate was calculated using the formula: Survival rate (%) = (Number of viable cells with added inosine / glycine and / or CZA / Number of viable cells in the M9 control group) × 100%. The bactericidal multiple increase was then calculated using the formulas: Bactericidal multiple increase in the synergistic group = Survival rate of the single-substance group / Survival rate of the synergistic group; Bactericidal multiple increase with different concentrations of the substance = Survival rate of the group with different concentrations of the substance / Survival rate of the synergistic group.

[0096] Survival rate results are shown below Figure 3 Figure B shows that the number of surviving bacteria decreased after adding inosine / glycine to the antibiotic regimen. Furthermore, the decrease in surviving bacteria became more significant with increasing inosine / glycine concentration.

[0097] The specific findings are as follows: Adding the substance alone has no bactericidal effect on bacteria; in fact, high concentrations may slightly promote growth. When 160 μg / mL CZA was added alone, the bacterial survival rate was 60.38%, with a bactericidal multiple of only 1.66 times. However, when another 160 μg / mL CZA was added to the corresponding concentration, the bacterial sensitivity to CZA increased significantly, with the bactericidal multiples in the synergistic group increasing by 12.56-3086 times. When the substance concentration gradually increased from 5 mM to 80 mM, the bactericidal multiple in the synergistic group increased from 7.44 times to 1391 times. These results indicate that: 1) a single substance has no bactericidal effect; 2) the bactericidal effect can be significantly improved when antibiotics are used in combination with a substance; 3) the effect of combining antibiotics with a substance is related to the substance concentration; the higher the concentration, the better the bactericidal effect.

[0098] 3.3 It has time dependence Prepared CR-ECO 3 samples were treated with 160 μg / mL CZA and 40 mM inosine / glycine, respectively, with CZA alone as a control, at 37°C for 12 hours. Viable bacteria were counted every 2 hours, with three biological replicates at each time point. Bacterial survival rates were calculated at different time points to investigate the relationship between bactericidal efficiency and time. The survival rate was calculated as follows: Survival rate (%) = (Number of viable bacteria at a time point after adding inosine / glycine and / or CZA) / (Number of viable bacteria in the control group at the same time point) × 100%. The bactericidal multiple increase of the synergistic group at different time points = (Survival rate of the antibiotic group at a time point) / (Survival rate of the synergistic group at that time point).

[0099] Survival results as follows Figure 3 As shown in Figure C, the bacterial survival rate in the synergistic group was significantly lower than that in the single-antibiotic group, and the decrease became more pronounced over time, demonstrating a time effect. Specific details are as follows: For the antibiotic-only group, bacterial survival rate did not decrease significantly within 6 hours, with a fold increase of 1.15-1.66 times. However, bacterial survival rate decreased significantly at 8 hours, with a fold increase of 7.16 times, and reached 44.17 times at 12 hours. For the synergistic group, bacterial survival rate decreased significantly at 2 hours, a 5.75-fold decrease compared to antibiotic-only, and continued to decrease over time, reaching its peak at 6 hours, nearly 700 times. Comparing the bactericidal effects of the synergistic group at different time points, the bactericidal multiple increased by 6.63 times at 2 hours and nearly 1000 times at 6 hours.

[0100] The results of the above-mentioned inosine / glycine concentration gradient, CZA concentration gradient, and time curve experiments show that the bactericidal effect of the inosine / glycine + CZA synergistic group is inosine / glycine concentration-dependent, antibiotic concentration-dependent, and time-dependent. The optimal inosine / glycine combination is 40 mM for both, the optimal antibiotic concentration is 160 μg / mL, and the optimal action time is 6 hours.

[0101] 3.4 Sensitivity of retained bacteria to ceftazidime avibactam sodium Preparation of Persister: Clinical carbapenem-resistant Escherichia coli CR-ECO 3 monoclonal strains were picked and cultured overnight in LB medium. The saturated bacterial culture was then treated with ofloxacin at a final concentration of 5 μg / mL for 4 h. The concentration of ofloxacin was increased to 40 μg / mL and the treatment was continued for another 6 h. The results showed that the bacterial mortality rate did not increase further, thus verifying that the obtained bacteria were Persister.

[0102] The prepared persistent bacteria were adjusted to regulate OD. 600 The value was 0.2, and then 5 mL was dispensed into test tubes for later use. The experiment was divided into 4 groups: M9 control group, 40 mM inosine / glycine group, 160 μg / mL CZA group, and synergistic group. All groups were treated at 37℃ for 6 hours, and viable bacteria were counted and the survival rate was calculated. The calculation formula was: Survival rate (%) = (Number of viable bacteria in each group / Number of viable bacteria in the saline control group) × 100%.

[0103] Survival rate results are shown below Figure 3 As shown in Figure D, the inosine / glycine group and the CZA group had no bactericidal effect on bacteria. The survival rate of bacteria in the synergistic group was significantly reduced, and the bactericidal multiple was increased by 21.56 times compared with antibiotics alone.

[0104] 3.5 Enhance the sensitivity of biofilms to ceftazidime (avibactam) sodium. Biofilm preparation method: Select a single colony of clinical carbapenem-resistant Escherichia coli CR-ECO 3 into a 5 mL LB tube and incubate overnight at 37°C on a shaker. Transfer the culture to 2 mL of fresh LB medium at a ratio of 1:200, add a 6 mm PE-50 biofilm tube sterilized by UV, and incubate at 37°C for 24 hours. Replace the bacterial culture with 1 mL of LB medium daily, and continue incubating the tube for 3 days.

[0105] The prepared drug-resistant bacterial biofilm was washed five times with 1 mL of sterile physiological saline and then placed in a 1.5 mL EP tube. The experiment was divided into four groups: M9 control group, 40 mM inosine / glycine group (i.e., both inosine and glycine concentrations were 40 mM), 160 μg / mL CZA group, and synergistic group. After treating the catheters at 37℃ and 200 rpm for 6 hours, the catheters were ultrasonically cleaned for 10 min to wash off the biofilm and mix thoroughly. After serial dilution, the biofilm was counted by plate spot. The bacterial survival rate was calculated using the formula: Survival rate (%) = Number of viable bacteria in each group / Number of viable bacteria in the physiological saline control group × 100%.

[0106] Survival rate results are shown below Figure 3 As shown in Figure E, the inosine / glycine group and the CZA group had no bactericidal effect on bacteria. The survival rate of bacteria in the synergistic group was significantly reduced, and the bactericidal multiple was increased by 442.17 times compared with CZA alone.

[0107] Example 4: Glycine and inosine synergistically enhance bacterial sensitivity to β-lactam antibiotics with antibiotic specificity. 4.1 It can improve the bactericidal efficiency of some β-lactam antibiotics. Further research was conducted to investigate the synergistic effect of inosine / glycine on enhancing bacterial susceptibility to β-lactam antibiotics. Clinical carbapenem-resistant strain CR-ECO 3 was prepared according to Example 1.1. The samples were divided into an M9 control group, an antibiotic group, and a synergistic group to study the effect of inosine / glycine synergy on the bactericidal efficacy of β-lactam antibiotics. β-lactam antibiotics included penicillins, cephalosporins, monocyclic β-lactams, and carbapenems. In the experiment, the concentrations of inosine and glycine were both 40 mM. The types of antibiotics used and their concentrations are shown in Table 2.

[0108] See results Figure 4A. For penicillin antibiotics: the combination of glycine and inosine did not improve the bactericidal effect of the four antibiotics used in the experiment (carbenicillin, piperacillin, ampicillin, and amoxicillin). For cephalosporin antibiotics: the combination of glycine and inosine increased the sensitivity of bacteria to some antibiotics such as cefoperazone, ceftazidime, and cefepime by 2.86-4.94 times, but did not improve the bactericidal effect of other antibiotics such as cefoxitin, cefaclor, and ceftriaxone. For monolactam antibiotics such as aztreonam, the combination of glycine and inosine increased the sensitivity of bacteria to them by approximately 53.76 times. For carbapenem antibiotics, the combination of glycine and inosine increased the sensitivity of bacteria to imipenem and meropenem by 80.32 times and 145.9 times, respectively.

[0109] The above results indicate that the combination of glycine and inosine can increase the sensitivity of bacteria to some cephalosporins, monocyclic β-lactams, and carbapenems among β-lactam antibiotics, but is ineffective against penicillin antibiotics.

[0110] Table 2. Antibiotic concentrations used in the experiment

[0111] 4.2 It can improve the bactericidal efficiency of some β-lactam antibiotics combined with β-lactamase inhibitors. Further, β-lactam antibiotics were combined with β-lactamase inhibitors (hereinafter referred to as compound antibiotic preparations) to study the synergistic effect of inosine / glycine in enhancing bacterial sensitivity to compound antibiotic preparations. Except for aztreonam and avibactam sodium (mass ratio of 3:1), the other β-lactam antibiotics and β-lactamase inhibitors were prepared at mass ratios of 4:1 (antibiotic: avibactam sodium) and 2:1 (antibiotic: sulbactam sodium). Piperacillin-tazobactam sodium (mass ratio of piperacillin to tazobactam sodium was 1:2) and amoxicillin-clavulanate potassium (mass ratio of amoxicillin to clavulanate potassium was 7:1) were used as the finished formulation.

[0112] CR-ECO 3 bacteria were prepared according to Example 3.1 and subjected to group sterilization. Figure 4The results presented in section B can be categorized into the following types based on the increased bacterial susceptibility to antibiotics: 1) Single antibiotics are ineffective, but combination antibiotics are effective. For example: carbenicillin is ineffective, but carbenicillin-avibactam sodium and carbenicillin-sulbactam sodium are effective; ampicillin is ineffective, but ampicillin-avibactam sodium and ampicillin-sulbactam sodium are both effective; ceftriaxone is ineffective, but ceftriaxone-avibactam sodium and ceftriaxone-sulbactam sodium are effective; ampicillin is ineffective, but ampicillin-avibactam sodium and ampicillin-sulbactam sodium are effective; 2) Single antibiotics are ineffective, and the bactericidal effect of combination antibiotics varies due to different β-lactamase inhibitors. For example: amoxicillin is ineffective, and amoxicillin-clavulanate potassium is also ineffective, while... Amoxicillin-sulbactam sodium is effective, but amoxicillin-avibactam sodium is effective; for example, cefaclor is ineffective, cefaclor-avibactam sodium is also ineffective, but cefaclor-sulbactam sodium is effective; cefazolin is ineffective, cefazolin-avibactam sodium is also ineffective, but cefazolin-sulbactam sodium is effective; 3) Single antibiotics are effective, and compound antibiotic preparations are also effective, such as cefoperazone is effective, cefoperazone-sulbactam sodium, ceftazidime is effective, ceftazidime-avibactam sodium, cefepime is effective, cefepime-sulbactam sodium and cefepime-avibactam sodium are both effective, aztreonam is effective, aztreonam-avibactam sodium is also effective, meropenem is effective, meropenem-avibactam sodium is also effective; imipenem is effective, imipenem-avibactam sodium is also effective.

[0113] 4.3 The combined use of inosine and glycine enhances the susceptibility of carbapenem-containing Gram-negative bacteria to meropenem. Since inosine and glycine also showed good results in enhancing susceptibility to meropenem, they were also studied in depth. Following Example 1.1, 10 clinical carbapenem-resistant Escherichia coli (CR-ECO), 10 clinical carbapenem-resistant Pseudomonas aeruginosa (CR-PA), 10 clinical carbapenem-resistant Klebsiella pneumoniae (CR-KPN), and 10 clinical carbapenem-resistant Acinetobacter baumannii (CR-AB) samples were prepared. Each strain was divided into three groups: M9 control group, meropenem (MEM) antibiotic group, and MEM + inosine / glycine synergistic group. The concentration of MEM was 50 μg / mL, and the concentration of inosine / glycine was 20 mM. After incubation at 37℃ and 200 rpm for 6 hours, the viable bacterial count was detected using plates, and then the bacterial survival rate of different treatment groups and the bactericidal multiple increase of the synergistic group were calculated.

[0114] See results Figure 4C. As shown in the figure, after the addition of MEM antibiotics, the average survival rates of the four bacteria were 87.09% (CR-ECO), 84.82% (CR-KPN), 42.28% (CR-PA), and 99.84% (CR-AB), respectively. However, after the addition of MEM + inosine / glycine, the average survival rates of the four bacteria were 13.08% (CR-EC), 23.92% (CR-KPN), 10.93% (CR-PA), and 3.56% (CR-AB), respectively. Compared with the bactericidal effect of MEM antibiotics, the bactericidal effect of MEM + inosine / glycine was significantly improved, with average increases of 6.66 times (CR-ECO), 3.32 times (CR-KPN), 3.87 times (CR-PA), and 27.64 times (CR-AB), respectively. The results indicate that the combination of inosine / glycine can significantly improve the bactericidal ability of meropenem against carbapenem-resistant Gram-negative bacteria.

[0115] To determine the type of interaction between inosine and glycine, a study was conducted using CR-ECO 3 as the research subject, as described in Example 1.3. The study investigated how the two metabolites, inosine and glycine, and their different dosage combinations increased the bactericidal fold of CR-ECO 3 against MEM. Figure 4 D) After taking Log10, input it into the software. The software automatically generates a combined effect score and effect graph based on the Bliss model. The score is 21.08. See the two-dimensional image below. Figure 4 E. The results showed that inosine and glycine had a synergistic effect, with the strongest synergistic effect occurring at 40-80 mM.

[0116] Example 5: Inosine and glycine synergistically enhance the susceptibility of other clinical Gram-negative bacteria to ceftazidime-avibactam sodium. 5.1 It can improve the susceptibility of clinical Pseudomonas aeruginosa to ceftazidime avibactam sodium. Twenty clinical carbapenem-resistant *Pseudomonas aeruginosa* strains were prepared according to Example 1.1. Each strain was divided into four groups: M9 control group, antibiotic group, synergistic group, and polymyxin B group. The antibiotic CZA concentration was 100 μg / mL, the inosine / glycine concentration was 4 mM, and the polymyxin B concentration was 2 μg / mL. After incubation at 37℃ and 200 rpm for 6 hours, the viable bacterial count was detected by plate testing, and the bacterial survival rate of different treatment groups and the bactericidal multiple increase of the synergistic group were calculated.

[0117] See results Figure 5 A. As shown in the figure, the addition of inosine / glycine significantly improved the bactericidal effect of the antibiotics in these strains, increasing the bactericidal multiple by 4.28-3087 times. Compared with the bactericidal effect of polymyxin B, 11 strains in the synergistic group showed significantly better bactericidal effects than polymyxin B alone.

[0118] 5.2 It can improve the sensitivity of Acinetobacter baumannii in clinical practice to ceftazidime avibactam sodium. Twenty clinical carbapenem-resistant Acinetobacter baumannii samples were prepared according to Example 1.1. Each strain was divided into four groups: M9 control group, antibiotic group, synergistic group, and polymyxin B group. The concentration of antibiotic CZA was 60 μg / mL, the inosine / glycine concentration was 4 mM, and the polymyxin B concentration was 2 μg / mL. After incubation at 37℃ and 200 rpm for 6 hours, the viable bacterial count was detected by plate testing, and the bacterial survival rate of different treatment groups and the bactericidal multiple increase of the synergistic group were calculated.

[0119] See results Figure 5 Figure B shows that the addition of inosine / glycine significantly improved the bactericidal effect of the antibiotics in these strains, increasing the bactericidal multiple by 139 to nearly 10,000 times. Compared with the bactericidal effect of polymyxin B, the bactericidal effect of all strains in the synergistic group was better than that of polymyxin B.

[0120] 5.3 Improve the susceptibility of clinical Klebsiella pneumoniae to ceftazidime avibactam sodium. Twenty clinical carbapenem-resistant Klebsiella pneumoniae samples were prepared according to Example 1.1. Each strain was divided into four groups: M9 control group, antibiotic group, synergistic group, and polymyxin B group. The antibiotic (CZA) was used at a concentration of 160 μg / mL, the inosine / glycine concentrations were 40 mM, and the polymyxin B concentration was 2 μg / mL. After incubation at 37°C and 200 rpm for 6 hours, the viable bacterial count was detected by plate testing, and the survival rate of bacteria in different treatment groups was calculated. The calculation formula was: Survival rate (%) = (Number of viable bacteria after adding inosine / glycine and / or antibiotic, or number of viable bacteria after adding polymyxin B / Number of viable bacteria in the M9 control group) × 100%. Then, the bactericidal multiple increase was calculated using the formula: Bactericidal multiple increase = Survival rate of bacteria in the antibiotic group / Survival rate of bacteria in the synergistic group.

[0121] See results Figure 5 C. As shown in the figure, the addition of inosine / glycine significantly improved the bactericidal effect of antibiotics in these strains, increasing the bactericidal multiple by 3-183 times. Compared with the bactericidal effect of polymyxin B, 11 strains in the synergistic group showed significantly better bactericidal effects than polymyxin B.

[0122] In addition, by Figure 5 As can be seen from D, other Gram-negative bacteria, such as Vibrio alginolyticus (a multidrug-resistant bacterium) and Edwardsiella tarda (a multidrug-resistant bacterium) isolated from fishponds, and Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus from clinical sources, did not show an effect of increasing antibiotic sensitivity with the combination of glycine and inosine.

[0123] 5.4 Comparison of the bactericidal effects of inosine / glycine synergistic enhancement of ceftazidime / avibactam sodium with the bactericidal effects of the latest compound preparation aztreonam / avibactam sodium. Aztreonam-avibactam sodium (AZT+AVI, with a mass ratio of 3:1) is a newly developed combination preparation of a β-lactam antibiotic and a β-lactamase inhibitor. Further studies compared its synergistic effect with inosine / glycine in enhancing the bactericidal activity of aztreonam-avibactam sodium. Following Example 1.1, ten clinically carbapenem-resistant Escherichia coli (CR-EC), ten clinically carbapenem-resistant Pseudomonas aeruginosa (CR-AB), ten clinically carbapenem-resistant Acinetobacter baumannii (CR-KPN), and ten clinically carbapenem-resistant Klebsiella pneumoniae (CR-PA) samples were prepared. Each strain was divided into three groups: an M9 control group, an AZT+AVI antibiotic group, and a CZA+inosine / glycine synergistic group. The concentration of AZT+AVI was 224 μg / mL, the concentration of CZA was 100 μg / mL, and the concentration of inosine / glycine was 40 mM. After incubation at 37℃ and 200rpm for 6 hours, the number of viable bacteria was detected by plate testing, and then the survival rate of bacteria in different treatment groups and the bactericidal multiple of the synergistic group were calculated.

[0124] See results Figure 6 As shown in the figure, after the addition of AZT+AVI antibiotics, the average survival rates of the four bacterial strains were 15.05% (CR-ECO), 39.84% (CR-KPN), 57.02% (CR-PA), and 47.78% (CR-AB), respectively. However, after the addition of CZA+inosine / glycine, the average survival rates of the four bacterial strains were 3.05% (CR-ECO), 0.06% (CR-KPN), 1.41% (CR-PA), and 0.03% (CR-AB), respectively. Compared with the bactericidal effect of AZT+AVI antibiotics, the bactericidal effect of CZA+inosine / glycine was significantly improved, with average increases of 4.93 times (CR-ECO), 622.74 times (CR-KPN), 40.5 times (CR-PA), and 1634.02 times (CR-AB), respectively. The results showed that the synergistic effect of inosine / glycine in enhancing the bactericidal activity of ceftazidime avibactam sodium against carbapenem-resistant Gram-negative bacteria was significantly better than that of aztreonam avibactam sodium, one of the latest approved clinical combination drugs.

[0125] Example 6: Inosine and glycine, in combination with ceftazidime and avibactam sodium, enhance resistance in mice to clinical Gram-negative bacterial infections. 6.1 It can improve the survival rate of mice infected with clinical Gram-negative bacteria. Balb / c mice (6-8 weeks old, approximately 18-20 grams, half male and half female) were housed for one week at a room temperature maintained at 25°C. The housing was artificially controlled for day and night, with 12 hours of light from 8:00 AM to 8:00 PM and 12 hours of darkness for the remaining time. Four types of bacteria were used for challenge, all clinically derived carbapenem-resistant, with resistance profiles shown in Table 3. Mice challenged with each bacterium were divided into 10 groups (n=10 per group): saline group, 200 mg / kg CZA alone group, 125 mg / kg inosine / glycine alone group, 10 mg / kg polymyxin alone group, 100 mg / kg meropenem alone group, and synergistic groups of 200 mg / kg CZA with different concentrations of inosine / glycine. The challenge doses for Escherichia coli CR-ECO 3 were 1 × 10⁻⁶. 7 The challenge dose of CFU for Klebsiella pneumoniae CR-KPN23 is 5.1 × 10⁻⁶. 7 The challenge dose of CFU for Pseudomonas aeruginosa CR-PA 34 is 1.8 × 10⁻⁶. 7 The challenge dose of CFU for Acinetobacter baumannii CR-AB4 was 1.4 × 10⁻⁶. 7 CFU was used for challenge via intraperitoneal injection. One hour after bacterial infection, all mice were administered the drug according to the above grouping and dosage, via intramuscular injection. Mice mortality was observed and recorded daily, and the survival rate of each group was calculated daily for 7 consecutive days. The calculation formula was: Survival rate = (Number of surviving mice in each group / Total number of mice in each group) × 100%.

[0126] The results of the Escherichia coli CR-ECO 3 experiment are shown in the figure. Figure 7 A. As shown in the figure, all mice in the saline control group and the inosine / glycine group died on the first day; the mice in the polymyxin B treatment group had no deaths, with a survival rate of 100%; in the meropenem treatment group, 40% died on the first day, and then no more died, with a survival rate of 60%; in the CZA treatment group, 70% died on the first day, 90% died on the second day, and then no more died, with a survival rate of 10%; in the CZA combined with 25 mg / kg inosine / glycine, 30% died on the first day, 50% died on the second day, and then no more died, with a survival rate of 50%; in the CZA combined with 50 mg / kg inosine / glycine, 20% died on the first day, and then no more died, with a survival rate of 80%; in the CZA combined with 75 mg / kg inosine / glycine, 10% died on the first day, 20% died on the second day, and then no more died, with a survival rate of 80%; in the CZA combined with 100 mg / kg inosine / glycine, 10% died on the first day. After that, the mice did not die, and the survival rate was 90%; in the group where CZA was combined with 125 mg / kg of inosine / glycine, the mice did not die, and the survival rate was 100%.

[0127] The experimental results of Acinetobacter baumannii CR-AB 4 are shown in the figure. Figure 7 B. As shown in the figure, all mice in the saline control group, glycine + inosine group, and meropenem group died on the first day; in the polymyxin B treatment group, 30% died on the first day, and there were no further deaths, with a survival rate of 70%; in the CZA treatment group, 90% died on the first day, and there were no further deaths, with a survival rate of 10%; in the CZA combined with 25 mg / kg inosine / glycine group, 80% died on the first day, and there were no further deaths, with a survival rate of 20%; in the CZA combined with 50 mg / kg inosine / glycine group, 70% died on the first day, and there were no further deaths, with a survival rate of 30%; in the CZA combined with 75 mg / kg inosine / glycine group, 60% died on the first day, 70% died on the second day, and there were no further deaths, with a survival rate of 30%; in the CZA combined with 100 mg / kg inosine / glycine group, 30% died on the first day. The group with CZA and 125 mg / kg of inosine / glycine had a survival rate of 60% after the first day of death, with 40% of the group dying on the second day.

[0128] The experimental results of Pseudomonas aeruginosa CR-PA34 are shown in the figure. Figure 7 C. As shown in the figure, all mice in the saline control group and the inosine / glycine group died on the first day; no mice died in the polymyxin B treatment group, with a survival rate of 100%; in the meropenem treatment group, 40% died on the first day, 50% on the second day, and 60% on the third day, after which no further deaths occurred, with a survival rate of 40%; in the CZA treatment group, 50% died on the first day, 60% on the second day, after which no further deaths occurred, with a survival rate of 40%; in the CZA combined with 25 mg / kg inosine / glycine, 70% died on the first day, after which no further deaths occurred, with a survival rate of 30%; in the CZA combined with 50 mg / kg and 75 mg / kg inosine / glycine, 50% died on the first day, after which no further deaths occurred, with a survival rate of 50%; in the CZA combined with 100 mg / kg inosine / glycine, 20% died on the first day. The group with CZA and 125 mg / kg of inosine / glycine had no deaths on day 1, 10% of which died on day 2, and a survival rate of 70%.

[0129] The results of the Klebsiella pneumoniae CR-KPN23 test are shown in the figure. Figure 7D. As shown in the figure, all patients in the saline control group, inosine / glycine group, and meropenem group died on the first day; in the polymyxin B treatment group, 10% died on the first day, with a survival rate of 90%; in the CZA treatment group, 60% died on the first day, 80% died on the second day, and there were no further deaths, with a survival rate of 20%; in the CZA combined with 25 mg / kg inosine / glycine, 50% died on the first day, 70% died on the second day, and there were no further deaths, with a survival rate of 30%; in the CZA combined with 50 mg / kg inosine / glycine, 50% died on the first day, and there were no further deaths, with a survival rate of 50%; in the CZA combined with 75 mg / kg and 100 mg / kg inosine / glycine, there were no deaths on the first day, 30% died on the second day, and there were no further deaths, with a survival rate of 70%; in the CZA combined with 125 mg / kg inosine / glycine, 10% died on the first day, 20% died on the second day, and there were no further deaths, with a survival rate of 80%.

[0130] The results above indicate that: 1) compared with the saline control group, all groups except for the inosine / glycine (concentration used in the experiment) showed protective effects on mice; 2) compared with the CZA-only group, all synergistic groups at each concentration showed protective effects on mice, and these effects were concentration-gradient dependent. These results suggest that when mice are infected with clinically carbapenem-resistant bacteria, under conditions of weak antibiotic treatment, the synergistic effect of glycine and inosine significantly enhances the mice's resistance to infection by clinically carbapenem-resistant bacteria.

[0131] Table 3. Antimicrobial resistance spectrum of clinical bacteria used in mouse experiments.

[0132] Note: R represents drug resistance, I represents intermediate, and S represents susceptibility.

[0133] 6.2 It can improve the clearance of clinical Gram-negative bacterial infections in mice. Balb / c mice (6-8 weeks old, approximately 18-20 grams, half male and half female) were raised for one week and then divided into four groups according to the infecting bacteria, each group being infected with one of four clinically sourced carbapenem-resistant bacteria. Mice infected with each type of bacteria were further divided into five groups (n=6 per group): a saline group, a group treated with 200 mg / kg CZA alone, a group treated with 125 mg / kg inosine / glycine alone, a group treated with 10 mg / kg polymyxin alone, and a group treated with a combination of 200 mg / kg CZA and 125 mg / kg inosine / glycine. The infecting bacteria and dosage were as described in Example 7.1. Intraperitoneal injection was used for bacterial infection. One hour after bacterial infection, all mice were administered the drugs according to the above grouping and dosage, via intramuscular injection. Six hours later, blood was collected from the orbital cavity or equal-weight internal organs (liver, spleen, kidney, lung), which were thoroughly ground, diluted, and plate-stamped to count the bacterial count within the organs.

[0134] See results Figure 8 As shown in the figure, 1) When mice are infected with drug-resistant bacteria, treatment with inosine / glycine alone does not reduce the number of bacteria in the blood and organs of mice. 2) Treatment with ceftazidime / avibactam sodium reduces the number of bacteria in the blood and organs of mice. For Escherichia coli, the decrease is 2.26-2.89 orders of magnitude; for Klebsiella pneumoniae, the decrease is 2.36-2.9 orders of magnitude; for Acinetobacter baumannii, the decrease is about 1.45 orders of magnitude; and for Pseudomonas aeruginosa, the decrease is 0.5-0.86 orders of magnitude. 3) However, if inosine / glycine is added to the treatment with ceftazidime / avibactam sodium, the number of bacteria in the blood and organs of mice will be significantly reduced. Compared to ceftazidime avibactam, it reduced bacterial counts by 2-3 orders of magnitude for Escherichia coli; by 1.58-2.69 orders of magnitude for Klebsiella pneumoniae; by approximately 2.67-4.29 orders of magnitude for Acinetobacter baumannii; and by 1-1.67 orders of magnitude for Pseudomonas aeruginosa. 4) It was also found that, compared to polymyxin treatment, the number of residual bacteria in blood and organs was comparable to or better than with polymyxin, such as against carbapenem-treated Acinetobacter baumannii.

[0135] The results showed that the synergistic effect of inosine and glycine significantly improved the clearance of Gram-negative bacterial infections in mice, with effects similar to or better than polymyxin.

[0136] Example 7: Study on the mechanism by which inosine and glycine synergistically enhance bacterial sensitivity to ceftazidime avibactam sodium. 7.1 Effects of Adding Inosine and Glycine on the Antibiotic-Based Bactericidal Effects of Bacterial Outer Membrane Protein Gene-Deleted Strains Escherichia coli K12 BW25113 and its 59 outer membrane protein gene deletion strains were prepared according to Example 1.1. Each bacterial strain was divided into three groups: M9 control group, CZA group, and CZA + inosine / glycine synergistic group. The results are shown in […]. Figure 9 A, discovered 4 outer membrane protein genes ( slyB, tsx, yfgL, ompF The loss of these four outer membrane proteins, along with the increased levels of inosine and glycine, led to a decrease in bacterial sensitivity to ceftazidime avibactam sodium, suggesting that these four outer membrane proteins are involved in the entry of ceftazidime avibactam sodium into the bacteria.

[0137] 7.2 Antibiotic bactericidal effects of bacterial dual-regulatory proteins after the addition of glycine and inosine Samples of *Escherichia coli* K12 BW13 and 58 strains with deletions of dual-regulatory protein genes were prepared according to Example 1.1. Results are shown below. Figure 9 B, discovered 3 dual-regulatory genes ( cpxA, rstA, rsrB The deletion of inosine and glycine together increases bacterial sensitivity to CZA, suggesting that these three regulatory genes may be involved in regulating CZA entry into bacteria.

[0138] 7.3 Exogenous addition of inosine and glycine can increase the intracellular concentration of antibiotics. To investigate whether the addition of exogenous inosine and glycine increased the amount of antibiotics entering the bacteria, the seven deletion strains obtained in Examples 7.1 and 7.2 were used as test strains. cpxA, rstA, rsrB, slyB, tsx, yfgL, ompF. in addition, cpxA and cpxR These are a pair of dual-regulated genes in Escherichia coli, therefore... cpxR This study also included (the survival rate also decreased significantly after the gene was deleted, but was weaker than...). cpxA, rstA, rsrB (The decrease in survival rate is due to the reduction in the number of bacteria). Meanwhile, the source strain of the missing strain, K12 BW25113, was used as a control. The intracellular antibiotic concentration detection process is as follows: Bacterial treatment: Nine bacterial strains were cultured according to Example 2.1. The strains were then treated as follows: For BW25113, it was divided into two groups (CZA-only group and inosine / glycine + CZA group). For the other eight deletion strains, inosine / CZA was added to all of them. All bacteria were incubated at 37°C for 6 hours.

[0139] Collect bacterial cells: Centrifuge and collect bacterial cells for each treatment separately, wash and adjust OD600 to 1.0, then take 10 ml of bacterial solution, centrifuge and collect bacterial cells.

[0140] Bacterial cell disruption: Add 300 μL of physiological saline, sonicate to disrupt the bacterial cells (conditions: 35% power, sonication for 2 seconds, pause for 3 seconds, time: 10 min), centrifuge to collect the supernatant, and filter to remove residual bacterial cells from the supernatant.

[0141] Intracellular antibiotic concentration detection: Add 450 μL of K12 strain (overnight saturated, centrifuged, and diluted 2×10⁻⁶ with M9 medium) to an Eppendorf tube. 5 Add 50 μL of the bacterial cell lysis supernatant. After mixing thoroughly, incubate at 37°C and 200 rpm for 6 hours. Detect the number of viable bacteria using plate testing, and calculate the intracellular antibiotic content of each bacterial strain based on the standard curve of CZA versus bacterial count.

[0142] The standard curve for the relationship between antibiotic CZA content and bacterial count is plotted as follows: First, add 450 μL of K12 strain (after overnight saturation and centrifugation, dilute with M9 medium to 2×10⁻⁶) to an Eppendorf tube. 5 First, add 4000, 2000, 1000, 500, 250, and 0 ng of CZA respectively. After mixing thoroughly, incubate at 37°C and 200 rpm for 6 hours, and then detect the viable bacterial count using plate testing. Plot a standard curve of bacterial count versus CZA concentration.

[0143] Intracellular antibiotic content for each strain is shown in the table below. Figure 10 As shown in the figure, the intracellular CZA concentration of BW25113 was 3.2 nM when CZA was added alone. However, when inosine / glycine was added in addition to antibiotics, the intracellular CZA concentration reached 9.66 nM, representing a 6.46-fold increase in intracellular CZA. In contrast, the addition of inosine / glycine to the CZA-added bacterial strains did not increase the intracellular CZA concentration.

[0144] These results suggest that inosine and glycine are regulated by two pairs of dual-regulatory genes in bacteria. cpxA / cpxA and rstA / rsrB Regulation of bacterial outer membrane antibiotic channel proteins slyB, tsx, yfgL and ompF This promotes the entry of CZA into the cell, thereby exerting a synergistic bactericidal effect.

[0145] Example 8: Glycine and inosine synergistically delay bacterial resistance to ceftazidime-avibactam sodium. Effect of successive passages on the MIC value of the drug Using clinical Acinetobacter baumannii and Klebsiella pneumoniae (each bacterium containing three types: susceptible, multidrug-resistant, and carbapenem-resistant) as examples, bacteria were cultured alternately in M9 medium containing antibiotics and fresh LB medium without antibiotics, with / without inosine and glycine. Based on the MIC results of different generations of bacteria against ceftazidime avibactam (CZA), the study investigated whether the synergistic effect of inosine and glycine delayed the development of bacterial resistance to CZA.

[0146] Bacterial passage procedure: Pick a single colony from a plate and incubate overnight at 37°C and 200 rpm until saturation. Centrifuge to collect the bacterial cells and wash twice with physiological saline to remove residual LB medium. Adjust the bacterial culture to OD200. 600 = 0.2 followed by 10 6 CFU / mL concentrations were inoculated into M9 medium with or without 1 / 2 MIC CZA and 40 mM inosine / glycine (Ino / Gly), and cultured at 37°C and 200 rpm for 6 hours. The cultures were then transferred at a 1:1000 ratio to 5 mL of fresh, antibiotic-free LB medium and cultured at 37°C and 200 rpm for 12 hours. The bacterial cultures were preserved as first-generation subcultures. This process was repeated to obtain multiple generations of subcultures. The MIC of CZA in the 1st, 5th, 10th, 15th, and 20th generations of subcultures was determined.

[0147] The results (Table 4) showed that the MIC of CZA gradually increased with increasing passage number for all strains; however, the MIC increase for strains with only CZA was significantly higher than that for strains with CZA + inosine / glycine. This result indicates that inosine / glycine can delay the development of bacterial resistance to CZA.

[0148] Table 4. MIC determination results of Gram-negative bacteria against CZA after passage in CZA with or without inosine / glycine.

[0149]

[0150] Example 9 Pharmacokinetic and Pharmacodynamic Experiments Sample preparation: Ninety-six male SPF-grade BALB / c mice (6–8 weeks old, approximately 20g) were used as experimental animals, divided into four groups (n=24 per group): a blank control group (administered with an equal volume of physiological saline), a CZA monotherapy group, a CZA + inosine group [110 μg / kg], and a CZA + inosine (110 μg / kg) + glycine (220 mg / kg) group. The CZA dosage was 200 μg / kg for all mice. All mice were administered the drug via tail vein injection, with a volume of 100 μL per mouse and an injection time of approximately 10 seconds. Blood samples were collected at eight time points: before administration (0 min) and at 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, and 8 h after administration. At each time point, three mice from each group were sampled, and 200 μL of blood was collected from the orbital venous plexus and placed in pre-heparinized EP tubes. After centrifugation at 4°C and 1500 rpm for 10 min, the plasma was separated, and the pale yellow supernatant was transferred to new EP tubes. Store at 20℃ for later use.

[0151] Before testing, plasma samples were thawed at room temperature, and 100 μL of acetonitrile was added to precipitate proteins. The samples were then centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was collected, rapidly frozen in liquid nitrogen, and then lyophilized in a vacuum freeze-drying apparatus. After lyophilization, 150 μL of pure water was added to reconstitute the samples, and the samples were centrifuged again at 12,000 rpm for 5 min at 4 °C. The supernatant was then analyzed by LC-MS / MS.

[0152] Chromatographic and mass spectrometric data acquisition and peak area integration were performed using SCIEX instrument software. Based on the obtained drug concentration-time data, relevant pharmacokinetic parameters were calculated using DAS pharmacokinetic software, and the drug-time curves were processed using statistical moment analysis to obtain the final pharmacokinetic indices. Statistical analysis was performed using GraphPadPrism 8.0 software. Quantitative data are expressed as mean ± standard deviation. For normally distributed data, unpaired t-tests were used for inter-group comparisons; for non-normally distributed data, Mann–Whitney U tests were used for analysis. A p-value < 0.05 was considered statistically significant.

[0153] Table 5 Pharmacokinetic parameters of ceftazidime

[0154] Ceftazidime blood concentration-time curve as shown Figure 11 As shown, there were significant differences in ceftazidime concentrations among the groups. In the antibiotic-only group, the concentration peaked at 15 min (263.40 mg / L) and decreased to 1.72 mg / L at 8 h. With the addition of inosine, the concentration increased significantly at all time points, also peaking at 15 min (658.39 mg / L) and remaining at 9.78 mg / L at 8 h, indicating that inosine significantly increased ceftazidime plasma concentrations and prolonged their duration of action in vivo. Further addition of glycine to inosine increased the peak concentration to 886.56 mg / L at 15 min; the plasma concentration remained at its highest level from 0.5 to 8 h, reaching 56.92 mg / L at 8 h, significantly higher than both the antibiotic-only group and the antibiotic + inosine group.

[0155] Pharmacokinetic parameters (Table 5) further confirmed the above changes. Compared with ceftazidime avibactam sodium alone, the AUC(0-t) increased from 297.04±27.79 to 1410.77±58.46 mg / L·h (approximately 4.75 times) and Cmax increased from 129.40±16.75 to 608.16±5.93 mg / L after the addition of glycine. With the addition of glycine, the AUC(0-t) continued to increase to 3006.07±74.27 mg / L·h, and the Cmax increased to 830.73±18.41 mg / L. The MRT(0-t) decreased from 1.78 h in the monotherapy group to 1.60 h in the inosine group, but was prolonged to 2.49±0.05 h after the addition of glycine, suggesting that glycine prolongs the drug retention time. The clearance rate (CL) significantly decreased from 0.67±0.07 to 0.14±0.01 in the inosine group, and further decreased to 0.06±0.00 L / h / kg after the addition of glycine; the apparent volume of distribution (Vd) gradually decreased from 1.26±0.36 to 0.18±0.02 L / kg, indicating that more of the drug remained in the bloodstream. The elimination half-life (t1 / 2β) was 2.43±0.11 h, slightly longer than that of the monotherapy group.

[0156] In summary, inosine can significantly increase the blood concentration and exposure of ceftazidime. When combined with glycine, it can further enhance the peak concentration, prolong the residence time and slow down the clearance, resulting in a better overall synergistic effect.

[0157] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of glycine combined with inosine in the preparation of antibiotic potentiators, characterized in that, The antibiotic is selected from at least one of the following: a first-class cephalosporin antibiotic or a combination preparation of the antibiotic and a β-lactamase inhibitor; a second-class cephalosporin antibiotic or a combination preparation of the antibiotic and a β-lactamase inhibitor; a penicillin antibiotic or a combination preparation of the antibiotic and a β-lactamase inhibitor; a carbapenem antibiotic or a combination preparation of the antibiotic and a β-lactamase inhibitor; and aztreonam or a combination preparation of the antibiotic and a β-lactamase inhibitor. The first cephalosporin antibiotic is selected from at least one of ceftazidime, cefepime, and cefoperazone; The second combination of cephalosporin antibiotic and β-lactamase inhibitor is selected from at least one of cefaclor-sulbactam or a pharmaceutically acceptable salt thereof, ceftriaxone-avibactam or a pharmaceutically acceptable salt thereof, ceftriaxone-sulbactam or a pharmaceutically acceptable salt thereof, and cefazolin-sulbactam or a pharmaceutically acceptable salt thereof. The combination preparation of the penicillin antibiotic and β-lactamase inhibitor is selected from at least one of carbenicillin avibactam or a pharmaceutically acceptable salt thereof, carbenicillin sulbactam or a pharmaceutically acceptable salt thereof, ampicillin avibactam or a pharmaceutically acceptable salt thereof, ampicillin sulbactam or a pharmaceutically acceptable salt thereof, amoxicillin avibactam or a pharmaceutically acceptable salt thereof, and amoxicillin sulbactam or a pharmaceutically acceptable salt thereof; The carbapenem antibiotics are selected from meropenem and / or imipenem; The pathogen targeted by the antibiotic potentiator is selected from at least one of Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae.

2. The application of glycine combined with inosine in the preparation of drugs that enhance the sensitivity of pathogens to antibiotics, characterized in that, The antibiotic is selected from at least one of the following: a first-class cephalosporin antibiotic or a combination preparation of the antibiotic and a β-lactamase inhibitor; a second-class cephalosporin antibiotic or a combination preparation of the antibiotic and a β-lactamase inhibitor; a penicillin antibiotic or a combination preparation of the antibiotic and a β-lactamase inhibitor; a carbapenem antibiotic or a combination preparation of the antibiotic and a β-lactamase inhibitor; and aztreonam or a combination preparation of the antibiotic and a β-lactamase inhibitor. The first cephalosporin antibiotic is selected from at least one of ceftazidime, cefepime, and cefoperazone; The second combination of cephalosporin antibiotic and β-lactamase inhibitor is selected from at least one of cefaclor-sulbactam or a pharmaceutically acceptable salt thereof, ceftriaxone-avibactam or a pharmaceutically acceptable salt thereof, ceftriaxone-sulbactam or a pharmaceutically acceptable salt thereof, and cefazolin-sulbactam or a pharmaceutically acceptable salt thereof. The combination preparation of the penicillin antibiotic and β-lactamase inhibitor is selected from at least one of carbenicillin avibactam or a pharmaceutically acceptable salt thereof, carbenicillin sulbactam or a pharmaceutically acceptable salt thereof, ampicillin avibactam or a pharmaceutically acceptable salt thereof, ampicillin sulbactam or a pharmaceutically acceptable salt thereof, amoxicillin avibactam or a pharmaceutically acceptable salt thereof, and amoxicillin sulbactam or a pharmaceutically acceptable salt thereof; The carbapenem antibiotics are selected from meropenem and / or imipenem; The pathogen is selected from at least one of Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae.

3. The application of glycine and inosine in combination with antibiotics in the preparation of antimicrobial drugs, characterized in that, The antibiotic is selected from at least one of the following: a first-class cephalosporin antibiotic or a combination preparation of the antibiotic and a β-lactamase inhibitor; a second-class cephalosporin antibiotic or a combination preparation of the antibiotic and a β-lactamase inhibitor; a penicillin antibiotic or a combination preparation of the antibiotic and a β-lactamase inhibitor; a carbapenem antibiotic or a combination preparation of the antibiotic and a β-lactamase inhibitor; and aztreonam or a combination preparation of the antibiotic and a β-lactamase inhibitor. The first cephalosporin antibiotic is selected from at least one of ceftazidime, cefepime, and cefoperazone; The second combination of cephalosporin antibiotic and β-lactamase inhibitor is selected from at least one of cefaclor-sulbactam or a pharmaceutically acceptable salt thereof, ceftriaxone-avibactam or a pharmaceutically acceptable salt thereof, ceftriaxone-sulbactam or a pharmaceutically acceptable salt thereof, and cefazolin-sulbactam or a pharmaceutically acceptable salt thereof. The combination preparation of the penicillin antibiotic and β-lactamase inhibitor is selected from at least one of carbenicillin avibactam or a pharmaceutically acceptable salt thereof, carbenicillin sulbactam or a pharmaceutically acceptable salt thereof, ampicillin avibactam or a pharmaceutically acceptable salt thereof, ampicillin sulbactam or a pharmaceutically acceptable salt thereof, amoxicillin avibactam or a pharmaceutically acceptable salt thereof, and amoxicillin sulbactam or a pharmaceutically acceptable salt thereof; The carbapenem antibiotics are selected from meropenem and / or imipenem; The pathogen is selected from at least one of Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae.

4. The application according to any one of claims 1 to 3, characterized in that, The application includes at least one of the following conditions: (1) The β-lactamase inhibitor in the first cephalosporin antibiotic or its combination preparation with a β-lactamase inhibitor is selected from at least one of avibactam or a pharmaceutically acceptable salt thereof, sulbactam or a pharmaceutically acceptable salt thereof, and clavulanic acid or a pharmaceutically acceptable salt thereof; (2) The β-lactamase inhibitor in the carbapenem antibiotic or its combination preparation with a β-lactamase inhibitor is selected from at least one of avibactam or a pharmaceutically acceptable salt thereof, sulbactam or a pharmaceutically acceptable salt thereof, and clavulanic acid or a pharmaceutically acceptable salt thereof; (3) The β-lactamase inhibitor in the combination preparation of aztreonam or a β-lactamase inhibitor is selected from at least one of avibactam or a pharmaceutically acceptable salt thereof, sulbactam or a pharmaceutically acceptable salt thereof, and clavulanic acid or a pharmaceutically acceptable salt thereof.

5. The application according to any one of claims 1 to 3, characterized in that, The application includes at least one of the following conditions: (1) The molar ratio of glycine to inosine is 1:(0.01~20); (2) The mixing ratio of glycine and β-lactam antibiotics is 1 mol: (0.01-300) g; (3) The concentration of the glycine is ≥5mM; (4) The concentration of inosine is ≥5 mM; (5) The concentration of the antibiotic is ≥5 μg / mL; (6) In the compound preparation, the mass ratio of the antibiotic to the β-lactamase inhibitor is (0.5~8):

1.

6. The application according to any one of claims 1 to 3, characterized in that, The pathogens include at least one of sensitive bacteria, drug-resistant bacteria, and persistent bacteria.

7. An antimicrobial drug, characterized in that, The active ingredients of the antimicrobial drug include glycine, inosine, and antibiotics; the antibiotics are selected from at least one of the following: a first-class cephalosporin antibiotic or a combination preparation of the antibiotic and a β-lactamase inhibitor; a second-class cephalosporin antibiotic or a combination preparation of the antibiotic and a β-lactamase inhibitor; a penicillin antibiotic or a combination preparation of the antibiotic and a β-lactamase inhibitor; a carbapenem antibiotic or a combination preparation of the antibiotic and a β-lactamase inhibitor; and aztreonam or a combination preparation of the antibiotic and a β-lactamase inhibitor. The first cephalosporin antibiotic is selected from at least one of ceftazidime, cefepime, and cefoperazone; The second combination of cephalosporin antibiotic and β-lactamase inhibitor is selected from at least one of cefaclor-sulbactam or a pharmaceutically acceptable salt thereof, ceftriaxone-avibactam or a pharmaceutically acceptable salt thereof, ceftriaxone-sulbactam or a pharmaceutically acceptable salt thereof, and cefazolin-sulbactam or a pharmaceutically acceptable salt thereof. The combination preparation of the penicillin antibiotic and β-lactamase inhibitor is selected from at least one of carbenicillin avibactam or a pharmaceutically acceptable salt thereof, carbenicillin sulbactam or a pharmaceutically acceptable salt thereof, ampicillin avibactam or a pharmaceutically acceptable salt thereof, ampicillin sulbactam or a pharmaceutically acceptable salt thereof, amoxicillin avibactam or a pharmaceutically acceptable salt thereof, and amoxicillin sulbactam or a pharmaceutically acceptable salt thereof; The carbapenem antibiotics are selected from meropenem and / or imipenem.

8. The antimicrobial drug according to claim 7, characterized in that, The antimicrobial drug includes at least one of the following conditions: (1) The β-lactamase inhibitor in the first cephalosporin antibiotic or its combination preparation with a β-lactamase inhibitor is selected from at least one of avibactam or a pharmaceutically acceptable salt thereof, sulbactam or a pharmaceutically acceptable salt thereof, and clavulanic acid or a pharmaceutically acceptable salt thereof; (2) The β-lactamase inhibitor in the carbapenem antibiotic or its combination preparation with a β-lactamase inhibitor is selected from at least one of avibactam or a pharmaceutically acceptable salt thereof, sulbactam or a pharmaceutically acceptable salt thereof, and clavulanic acid or a pharmaceutically acceptable salt thereof; (3) The β-lactamase inhibitor in the combination preparation of aztreonam or a β-lactamase inhibitor is selected from at least one of avibactam or a pharmaceutically acceptable salt thereof, sulbactam or a pharmaceutically acceptable salt thereof, and clavulanic acid or a pharmaceutically acceptable salt thereof; (4) The pathogenic bacteria are selected from at least one of Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae.

9. The antimicrobial drug according to claim 7, characterized in that, The pathogens include at least one of sensitive bacteria, drug-resistant bacteria, and persistent bacteria.

10. The antimicrobial drug according to claim 7, characterized in that, The antimicrobial drug includes at least one of the following conditions: (1) The molar ratio of glycine to inosine is 1:(0.01~20); (2) The mixing ratio of glycine and antibiotic is 1 mol: (0.01-500) g; (3) The concentration of the glycine is ≥5mM; (4) The concentration of inosine is ≥5 mM; (5) The concentration of the antibiotic is ≥5 μg / mL; (6) In the compound preparation, the mass ratio of the antibiotic to the β-lactamase inhibitor is (0.5~8):1.

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