Patent Publication Number: US-2022226260-A1

Title: Application of honokiol and magnolol in preparation of mcr-1 enzyme inhibitor

Description:
TECHNICAL FIELD 
     The disclosure relates to the field of medical pharmaceutical technologies, and more particularly to an application/use of honokiol and magnolol in preparation of a mobilized colistin resistance (MCR-1) enzyme inhibitor. 
     BACKGROUND 
     Increasing bacterial resistance has posed a serious threat to global public health, resulting in the emergence of many cases of bacterial infections that are difficult to treat, including those caused by carbapenemase-producing Gram-negative Enterobacteriaceae. With the emergence of super-resistant bacteria, treatment failures and high mortality rates against medicine-resistant bacteria, it has brought greater pressure to treat nosocomial infections, especially ICU infections. Polymyxin is the “last line of defense” for antibiotics therapy for medicine-resistant bacterial infections, but the discovery of MCR-1 has led to the unavailability of medicines for infections caused by polymyxin-resistant and carbapenem-resistant Enterobacteriaceae. At present, there are no reports of effective MCR-1 inhibitors. Therefore, it is of great significance to develop new safe and effective medicines for avoiding bacterial infections. 
     Honokiol and magnolol are mainly derived from the dry bark, root bark and branch bark of Magnolia officinalis Rehd. et Wils. or Magnolia officinalis Rehd. et Wils. var. biloba Rehd. etWils. of Magnoliaceae deciduous arbor plants, which has obvious and lasting pharmacological effects of relaxing central muscles, inhibiting central nervous system, resisting inflammation, resisting pathogenic microorganism, resisting ulcer, resisting oxidation, resisting tumor and the like. Besides the effects, honokiol also has the effects of resisting aging, Cholesterol lowering and other pharmacological effects, both of which play an important role in clinical medicine. 
     After searching, there is no relevant report on honokiol and magnolol in the preparation of MCR-1 inhibitors. 
     SUMMARY 
     The disclosure provides a medical use of honokiol and magnolol in preparation of a mobilized colistin resistance (MCR-1) enzyme inhibitor, and discloses that honokiol and magnolol can inhibit the activity of MCR-1 enzyme and restore the bactericidal activity of polymyxin against MCR-1 positive  Escherichia coli ,  Klebsiella pneumoniae  and other Grain-negative bacteria. 
     Specifically, the honokiol of the disclosure has a molecular formula of C 18 H 18 O 2  and a molecular weight of 266.33. The magnolol has a molecular formula of C 18 H 18 O 2  and a molecular weight of 266.33. 
     The disclosure verifies that magnolol and honokiol can inhibit the activity of MCR-1 enzyme and restore the antibacterial activity of polymyxin against MCR-1 positive Enterobacteriaceae by chessboard minimum inhibitory concentration (MIC) test and time-kill curve method (also referred to as time-sterilizing curve). Furthermore, a mouse thigh muscle infection model is established to prove that honokiol and magnolol combined with polymyxin have a good therapeutic effect on infection caused by MCR-1 positive Enterobacteriaceae. 
     Compared with the related art, the embodiments of the disclosure may mainly have the following beneficial effects. 
     It provides a new medical use of honokiol and magnolol in the preparation of the MCR-1 enzyme inhibitor, and discloses that honokiol and magnolol can inhibit the activity of the MCR-1 enzyme and restore the bactericidal activity of polymyxin against MCR-1 Enterobacteriaceae. In vivo experiments, honokiol and magnolol combined with polymyxin have a good therapeutic effect on bacterial infection expressing MCR-1, especially infections caused by MCR-1 positive Enterobacteriaceae, and have wide medical applications. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1  illustrates a time-kill curve of honokiol combined polymyxin against MCR-1 positive  Escherichia coli  of the disclosure. 
         FIG. 2  illustrates a time-kill curve of magnolol combined polymyxin against MCR-1 positive  Escherichia coli  of the disclosure. 
         FIG. 3  illustrates a colony colonization result of a mouse thigh muscle infection by honokiol combined with polymyxin of the disclosure. 
         FIG. 4  illustrates a colony colonization result of a mouse thigh muscle infection by magnolol combined with polymyxin of the disclosure. 
     
    
    
     DETAILED DESCRIPTION OF EMBODIMENTS 
     The disclosure is further described by the following embodiments without limiting the disclosure in any way. Without departing from the technical solution of the disclosure, any alterations or changes made to the disclosure that are easy to be realized by those skilled in the art will fall within the scope of the claims of the disclosure. 
     Embodiment 1 
     Honokiol and magnolol are used as mobilized colistin resistance (MCR-1) enzyme inhibitors in any pharmaceutically acceptable carrier. 
     Embodiment 2 
     Honokiol and magnolol are used as MCR-1 enzyme inhibitors in preparation of medicines for treating infectious diseases. 
     Embodiment 3 
     Honokiol and magnolol are used as MCR-1 enzyme inhibitors in treatment of bacterial infectious diseases, especially infections caused by MCR-1 positive Enterobacteriaceae. 
     Test Example 1 
     Minimum Inhibitory Concentration (MIC) Test 
     The antibacterial activities of honokiol, magnolol, polymyxin and their combination against MCR-1 producing  Escherichia coli  (also referred to as MCR-1 positive  Escherichia coli ) and  Klebsiella pneumoniae  are tested by chessboard method in 96-well sterile microplates, the MIC values are determined, and the fractional inhibitory concentration (FIC) indexes are calculated. Specifically, FIC=MIC (polymyxin combined)/MIC (polymyxin alone)+MIC (honokiol/magnolol combined)/MIC (honokiol/magnolol alone). The results are shown in Table 1 and Table 2: 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 MIC and FIC values of honokiol combined with polymyxin for different strains 
               
            
           
           
               
               
               
            
               
                   
                 MIC (microgram per milliliter (μg/mL)) 
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Polymyxin B 
                 Polymyxin B 
                 Honokiol 
                 Honokiol 
                   
               
               
                 Strains 
                 alone 
                 co-application 
                 alone 
                 co-application 
                 FIC 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 MCR-1 positive 
                 8 
                 1 
                 &gt;512 
                 32 
                 0.1875 
               
               
                   Escherichia coli  ( E.   
               
               
                 coli DZ2-12R) 
               
               
                 MCR-1 positive 
                 32 
                 2 
                 &gt;512 
                 32 
                 0.125 
               
               
                 
                   Klebsiella 
                 
               
               
                   pneumoniae  ( K.   
               
               
                   pneumoniae  ZJ02) 
               
               
                 MCR-1 engineering 
                 8 
                 0.5 
                 &gt;512 
                 32 
                 0.125 
               
               
                 bacteria  E. coli   
               
               
                 BL21(DE3) 
               
               
                 (pET28a-mcr-1) 
               
               
                   E. coli  BL21(DE3) 
                 1 
                 0.5 
                 &gt;512 
                 32 
                 0.5625 
               
               
                 (pET28a) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 MIC and FIC values of magnolol combined with polymyxin for different strains 
               
            
           
           
               
               
               
            
               
                   
                 MIC (μg/mL) 
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Polymyxin B 
                 Polymyxin B 
                 Magnolol 
                 Magnolol 
                   
               
               
                 Strains 
                 alone 
                 co-application 
                 alone 
                 co-application 
                 FIC 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 MCR-1 positive 
                 8 
                 2 
                 &gt;512 
                 32 
                 0.3125 
               
               
                   Escherichia coli  ( E.   
               
               
                   coli  DZ2-12R) 
               
               
                 MCR-1 positive 
                 32 
                 8 
                 &gt;512 
                 32 
                 0.3125 
               
               
                 
                   Klebsiella 
                 
               
               
                   pneumoniae  ( K.   
               
               
                   pneumoniae  ZJ02) 
               
               
                 MCR-1 engineering 
                 8 
                 1 
                 &gt;512 
                 32 
                 0.1875 
               
               
                 bacteria  E. coli   
               
               
                 BL21(DE3) 
               
               
                 (pET28a-mcr-1) 
               
               
                   E. coli  BL21(DE3) 
                 1 
                 1 
                 &gt;512 
                 32 
                 1.0625 
               
               
                 (pET28a) 
               
               
                   
               
            
           
         
       
     
     In summary, honokiol or magnolol alone have no antibacterial effect, but the combination of honokiol or magnolol with polymyxin can reduce the MIC value of polymyxin to the engineering strain  E. coli  BL21 (DE3) (pET28a -mcr-1) expressing MCR-1, and has an obvious synergistic effect. However, in the engineering strain  E. coli  BL21 (DE3) (pET28a) that does not express MCR-1, the combination of honokiol or magnolol with polymyxin does not have any synergistic effect, and indicating that honokiol or magnolol is an effective MCR-1 inhibitor. Consistent with the above results, the combination of honokiol and polymyxin can reduce the MIC value of polymyxin by 8-fold for MCR-1 positive  Escherichia coli  and 16-fold for MCR-1 positive  Klebsiella pneumoniae . The combination of magnolol and polymyxin can reduce the MIC value of polymyxin by 4-fold for MCR-1 positive  Escherichia coli  and 4-fold for MCR-1 positive  Klebsiella pneumoniae . FIC values indicate that honokiol or magnolol is synergistic with polymyxin in MCR-1 positive strains. 
     Test Example 2 
     Time-Kill Curve (Also Referred to as Time-Sterilizing Curve) Test 
     An overnight culture of MCR-1 positive  Escherichia coli  isolate is taken and adjusted to 1×10 7  colony forming units per milliliter (CFUs/mL) for use. Sterile test tubes are taken and divided into 4 groups (including blank control group, 2 μg/mL polymyxin group B group, 16 μg/mL honokiol or 32 μg/mL magnolol group, and 16 μg/mL honokiol or 32 μg/mL magnolol combined with 2 μg/mL polymyxin B group), each group is marked for 1, 3, 5 and 7 hours. 1 mL of autoclaved Lysogeny broth (LB) medium is added into each of the test tubes, and then 10 microliters (μL) of adjusted bacterial solution are added into each of the test tubes so that the concentration of bacterial solution in each of the test tubes is 1×10 5  CFUs/mL. Among them, 2 μg/mL polymyxin group B group, 16 μg/mL honokiol or 32 μg/mL magnolol group, and 16 μg/mL honokiol or 32 μg/mL magnolol combined with 2 μg/mL polymyxin B group are added with the corresponding amount of antibiotics and inhibitors respectively. Immediately after mixing, the bacterial solution of the antibiotic-free control group is coated and counted as the CFU at 0 H. After that, the bacterial solution in the corresponding test tube is taken every 1, 3, 5 and 7 hours, and the plate is coated for counting, and the time-kill curves are drawn, as shown in  FIG. 1  and  FIG. 2 . 
     In summary, honokiol combined with polymyxin can completely kill MCR-1 positive  Escherichia coli  within 1 hour, and magnolol combined with polymyxin can completely kill MCR-1 positive  Escherichia coli  within 3 hours. 
     Test Example 3 
     Colony Colonization Experiment of Mouse Thigh Muscle Infection 
     Mouse Model of  Klebsiella pneumoniae  Thigh Muscle Infection 
     After BALB/C mice (female, about 20 grains (g)) are restrained, MCR-1 positive  Klebsiella pneumoniae  suspension (2×10 7  CFUs) is injected through the inner thigh to establish a mouse thigh muscle infection model. 
     Colony Colonization Experiment 
     After intramuscular injection of MCR-1 positive  Klebsiella pneumoniae  into the inner thigh of mice, 5 micrograms per kilogram (mg/kg) (50 μL) of polymyxin B dissolved in sterile ultra-pure water (dd H 2 O), 50 mg/kg (50 μL) honokiol or magnolol dissolved in dimethyl sulfoxide (DMSO) solution, and 50 mg/kg of honokiol or magnolol combined with polymyxin B (5 mg/kg) are injected subcutaneously, while the positive control group is subcutaneously injected with 50 μL DMSO blank solvent. All groups of mice are administered subcutaneously every 8 hours for three times. After 36 hours of administration, the mice are sacrificed, and the thigh muscles are weighed, homogenized, diluted and counted. The results are shown in  FIG. 3  and  FIG. 4 . 
     In summary, after treatment of honokiol or magnolol combined with polymyxin B, the number of bacterial colonization in mouse thigh muscle is significantly reduced, and there is no significant effect with magnolol, honokiol, or polymyxin B alone.