Patent Publication Number: US-4225588-A

Title: Antibiotic mixtures

Description:
This is a division of application Ser. No. 897,021, filed Apr. 17, 1978. 
    
    
     PATENTABILITY STATEMENT 
     The specification acknowledges that it is known that phleomycins may be potentiated by administration in association with various types of compounds. However, we know of no prior publication whatsoever which would indicate the suitability of compounds of the present application for this purpose. U.S. patent applications corresponding to copending Australian Application No. 84888/75 referred to at page 2, line 7 are Ser. Nos. 613,879 and 828,875. 
     This invention concerns compounds able to potentiate the antibiotics, phleomycin and bleomycin. 
     Phleomycin is a mixture of copper-containing glycopeptides with a wide range of antibiotic and antitumour activity. Unfortunately, insufficient selectivity of action--it is nephrotoxic--has inhibited its adoption for clinical use. Copending application No. 84888/75 describes how the problem may be overcome by administering phleomycin in conjunction with certain compounds of the purine series, which, by amplifying its activity, enable useful antibiotic results to be achieved with less than toxic dosage rates. The present application concerns a further but unrelated range of compounds which display phleomycin-amplifying activity. These compounds are active at much lower concentrations than previously known amplifiers (including purines), and therefore there is correspondingly less possibility of side-effects arising from the amplifying compound itself. In this specification references to phleomycin are to be interpreted as applying equally to bleomycin, although, any particular amplifier may not be found to amplify each antibiotic to an equivalent degree. The basic structures of phleomycins and bleomycins are shown in FIG. 1, from which it will be noted that the essential difference between the two types of compounds resides in the presence of two additional hydrogen atoms in the arrowed thiazole ring of phleomycin. Individual phleomycins (and bleomycins) differ from each other structurally only in respect of the terminal group R, which is a substituted amine moiety. 
     The phleomycin amplifiers according to this invention are characterized as follows: 
     (i) They comprise two or more six-membered rings, at least one of which is an aromatic hydrocarbon or heterocyclic ring, 
     (ii) They include at least one nitrogen atom, either as a member of a ring or as a member of a ring substituent such as an amino, dialkylamino, trialkylammonio or a dialkylaminoalkylamino group. 
     (iii) They carry, or readily acquire, one or two positive charges arising from protonation or quaternization of the aforesaid nitrogen atom(s). 
     (iv) In addition to the substituents referred to in (ii) various other groups may be present, including for example, lower alkyl, lower alkoxy, phenyl, chloro and bromo. 
     Within the broad scheme outlined above, the amplifiers fall into one or other of the following sub-groups: 
     Type 1: Derivatives of aromatic ring systems having three or four linearly or angularly annelated (fused) six-membered rings. Up to two carbon atoms in the ring skeleton may be replaced by heteroatoms such as O, N or S. 
     Type 2: Diarylmethanes, diarylethylenes, diphenyls, diarylamines and azobenzenes, which can adopt spatial conformations corresponding to Type 1 compounds. (In this specification, `aryl` means having carbocyclic or heterocyclic aromatic ring structure). 
     Type 3: Triphenylmethane dyes. 
     FIGS. 2, 3 and 4 show typical ring skeletons according to the types discussed above. It should be understood that these do not represent individual compounds nor is the list exhaustive; specific compounds are illustrated in Table 1. Although, throughout the specification, charges are indicated as attached to particular atoms, it will be appreciated that in practice the charges are not localized but are associated with the systems as a whole. Similarly the diagrams are to be construed as representing all equivalent resonance structures. 
    
    
     The following examples are illustrative of the efficacy of compounds according to this invention in enhancing the bioactivity of phleomycin and bleomycin: 
     In vitro 
     The ability of the compounds to potentiate the cell-killing effects of phleomycin was measured by the following procedure. Stationary phase cultures of E. coli B, with or without the compounds under test, were resuspended in growth medium containing phleomycin (2 ug/ml) and incubated at 37° for 30 minutes. The cells were then membrane filtered (0.45 um pore size; 2.5 cms), washed with growth medium (20 ug/ml) and resuspended in growth medium (20 ug/ml) at 37° C. containing various concentrations, between 10 -10  M and 10 -2  M, of the compound under test. As a control, bacteria were exposed to the same range of concentrations of each compound but without the phleomycin pretreatment. To determine viable cell numbers aliquots were taken from each cell suspension after 120 minutes treatment, plated on agar medium and incubated overnight at 37° C. All operations with amplifying agents were carried out in near dark conditions. 
     The results are set out in Table 1 which shows the concentration of optimum activity of the compounds when used in conjunction with the phleomycin treatment. Compounds were designated as active when the increase in activity with the combined treatment was greater than could be explained by the effects of the two compounds when administered separately. 
     In vivo 
     Walker rat carcinoma 256 as the ascites form was passaged in female Sprague Dawley rats. The ascites fluid was aspirated from the peritoneal cavity of animals bearing 6-7 day old tumours and given two minutes treatment in Boyle&#39;s solution to remove red cells, the supernatant spun off and the tumour cells suspended in Eagle&#39;s Basal Medium to give a final concentration of 10 7  viable cells per ml. Intraperitoneal injections of 0.1 ml (10 6  viable cells) of the suspension were then made into test groups of 6-8 male Srague Dawley rats. 
     Phleomycin dissolved in phosphate-buffered saline was given to animals in the test groups as 0.1 ml intraperitoneal injections twice daily; each injection was followed by a 0.1 ml intraperitoneal injection of the prospective amplifier. The treatments were given for 5-10 days, at varying concentrations of the phleomycin and amplifier. 
     The method of evaluating the antitumour effectiveness of the various treatments was based on those used in the screening programme of the Drug Evaluation Branch, National Cancer Institute, National Institute of Health, Bethesda, Maryland, U.S.A. (Cancer Chemotherapy Reports, Vol. 3, No. 2, September 1972). Thus the effectiveness of the drug treatment was measured by the increased survival time, measured in days, of the treated animals over control ones which received no drugs. Dead animals were counted daily, and median survival times (MST) calculated for all groups. Normally, the MSTs for the buffered saline controls were 8-10 days. The ratios (test group median survival time) divided by (control group median survival time)×100 (T/C) were calculated for all the test groups. A T/C value greater or equal to 125 with a test group size of 6 animals was judged significant and the compound was considered active, if the value was repeatable using a different set of animals and sample of tumour. 
     The results of the experiments are shown in Table 2. From column T/C it will be apparent that an injection rate of 20 ug/kg/day, the phenazine dye Neutral Red was active in that it enhanced the ability of phleomycin at 8, 16 or 32 micrograms/kg/day (MKD) to prolong the survival of rats carrying tumours. Similarly the triphenylmethan dyes, Brilliant Green, Malachite Green and Crystal Violet, as well as the diphenyl methanes Auramin 0, and 4,4&#39;-bis (trimethylamino)diphenylmethane, amplified the antitumour activity of phleomycin. 
     Phleomycin is known to be taken up by most cellular species and when toxic effects can be avoided it becomes available for treatment of bacterial infections, particularly, by E. coli B at a wide range of sites. From present studies it is envisaged that, generally, the administration of phleomycin at a concentration of about 1 mg/l (or mg/kg) in conjunction with one of the amplifying compounds of this invention at a concentration which may be between 10 -3  and 10 -7  molar, depending on the particular compound chosen, should give satisfactory results. For example, a concentration of 10 -7  molar is adequate for Crystal Violet, 10 -4  molar for Ethidium Bromide, and 10 -3  molar for 4,4-bis(trimethylammonio)diphenylmethane. As with the purine amplifiers described in application No. 84888/75, in some cases optimum results might be obtained by supplying the amplifiers separately and at some time after the phleomycin in order to allow the antibiotic to attach to its target sites in cells. 
     The cell-killing activity of phleomycin is ascribed to the interference with the ability of cells to replicate, and also to it causing breakage and degradation of the cell DNA. Essentially the level of phleomycin-potentiation displayed by the compounds of this invention is believed to depend on their ability to bind to DNA, as well as to stimulate DNA breakage in response to phleomycin treatment. The binding mechanisms may vary according to the nature of the compound. For instance, cationic molecules possibly bind to the sugar-phosphate DNA back-bone, in some cases binding may involve stacking interactions (intercalation) between the compounds and the parallel-oriented bases of DNA, and sometimes the binding may occur at the outside of the DNA helix. As a rule, it appears that the efficiency of the interaction is connected with base specificity of binding of the amplifier, higher efficiency being correlated with a preference for interaction at adenine-thymine rich regions of DNA. 
     
                                           TABLE 1                                 
__________________________________________________________________________
Structure and activity of compounds tested for                            
ability to amplify the antibacterial activity of phleomycin               
                                  Optimum                                 
                                  Concen-                                 
                                  tration                                 
                                  Tested                                  
Compound                          (M)                                     
__________________________________________________________________________
ACRIDINES - FREE BASES                                                    
 ##STR1##                                                                 
__________________________________________________________________________
              R.sup.3                                                     
                     R.sup.4                                              
                           R.sup.6                                        
__________________________________________________________________________
Acridine      H      H     H      2.8 × 10.sup.-4                   
3, 6-diaminoacridine                                                      
              NH.sub.2                                                    
                     H     NH.sub.2                                       
                                  9.5 × 10.sup.-5                   
3-aminoacridine                                                           
              NH.sub.2                                                    
                     H     H      1.0 × 10.sup.-4                   
__________________________________________________________________________
ACRIDINIUM CHLORIDES                                                      
 ##STR2##                                                                 
__________________________________________________________________________
           R.sup.2                                                        
               R.sup.3                                                    
                    R.sup.6                                               
                          R.sup.9                                         
                              R.sup.10                                    
__________________________________________________________________________
3,6-bis(dimethyl-                                                         
amino) acridine                                                           
hydrochloride                                                             
(Acridine Orange)                                                         
           H   N(CH.sub.3).sub.2                                          
                    N(CH.sub.3).sub.2                                     
                          H   H   1.0 × 10.sup.-6                   
9-aminoacridine                                                           
           H   H    H     NH.sub.2                                        
                              H   2.6 × 10.sup.-6                   
hydrochloride                                                             
3,6-diamino-N-                                                            
methylacridinium                                                          
chloride                                                                  
(Acriflavine)                                                             
           H   NH.sub.2                                                   
                    NH.sub.2                                              
                          H   CH.sub.3                                    
                                  3.9 × 10.sup.-6                   
Quinacrine OCH.sub.2                                                      
               H    Cl    *   H   3.9 × 10.sup.-5                   
__________________________________________________________________________
  *R.sup.9 = NHCH(CH.sub.3) (CH.sub.2).sub.3.sup.+NH(C.sub.2              
 H.sub.5)Cl.sup.-  -                                                      
 
    
     
         9, 10-DIHYDROANTHRACENES                                                  
 ##STR3##                                                                 
__________________________________________________________________________
2, 7-bis(trimethylammonio)-9, 10-dihydroanthracene                        
dichloride: R = N(CH.sub.3).sub.3 1.4 × 10.sup.-6                   
__________________________________________________________________________
9, 9&#39;-DIANTHRACENES                                                       
 ##STR4##                                                                 
__________________________________________________________________________
2,2&#39;, 7,7&#39;-tetrakis(trimethylammonio)-9,9&#39;-                               
dianthracene tetrachloride: R = N(CH.sub.3).sub.3 +  Cl.sup.-             
                                  5.0 × 10.sup.-5                   
__________________________________________________________________________
ANTHRACENES                                                               
 ##STR5##                                                                 
__________________________________________________________________________
2,7-bis(trimethylammonio)anthracene: R = N(CH.sub.3) .sub.3.sup.+         
Cl.sup.-                          3.0 × 10.sup.-4                   
__________________________________________________________________________
PHENANTHRIDINIUM SALTS                                                    
 ##STR6##                                                                 
__________________________________________________________________________
              R    R.sup.5                                                
                        R.sup.6                                           
                             X                                            
__________________________________________________________________________
Ethidium bromide                                                          
              NH.sub.2                                                    
                   C.sub.2 H.sub.5                                        
                        C.sub.6 H.sub.5                                   
                             Br   1.3 × 10.sup.-5                   
N-methylphenanthri-                                                       
dinium chloride                                                           
              H    CH.sub.3                                               
                        H    Cl   4.0 × 10.sup.-6                   
__________________________________________________________________________
PHENAXINIUM CHLORIDES                                                     
 ##STR7##                                                                 
__________________________________________________________________________
             R.sup.2                                                      
                 R.sup.3                                                  
                     R.sup.5                                              
                          R.sup.7                                         
                              R.sup.8                                     
__________________________________________________________________________
N-methylphenazinium                                                       
chloride     H   H   CH.sub.3                                             
                          H   H   3.0 × 10.sup.-6                   
Neutral Red  CH.sub.3                                                     
                 NH.sub.2                                                 
                     H    NH.sub.2                                        
                              H   3.3 × 10.sup.-5                   
Safranin T   CH.sub.3                                                     
                 NH.sub.2                                                 
                     C.sub.6 H.sub.5                                      
                          NH.sub.2                                        
                              CH.sub.3                                    
                                  1.4 × 10.sup.-5                   
__________________________________________________________________________
PHENOXAZINIUM DYES                                                        
Brilliant Cresol Blue             3.4 × 10.sup.-4                   
 ##STR8##                                                                 
__________________________________________________________________________
Nile Blue                         2.7 × 10.sup.-6                   
 ##STR9##                                                                 
__________________________________________________________________________
PHENAZATHIONIUM DYES                                                      
 ##STR10##                                                                
__________________________________________________________________________
              R.sup.7    R.sup.8                                          
__________________________________________________________________________
Methylene Blue                                                            
              N(CH.sub.3).sub.2                                           
                         H        1.3 × 10.sup.-5                   
Toluidine Blue                                                            
              N(CH.sub.3).sub.2                                           
                         CH.sub.3 1.6 × 10.sup.-5                   
__________________________________________________________________________
XANTHENE DYES                                                             
Pyronin Y                                                                 
 ##STR11##                        1.7 × 10.sup.-4                   
 ##STR12##                                                                
__________________________________________________________________________
         R    X     Y       Z                                             
__________________________________________________________________________
Rhodamine G                                                               
         H    NH.sup.+                                                    
                    NH(C.sub.2 H.sub.5)                                   
                            C.sub.2 H.sub.5                               
                                  1.2 × 10.sup.-4                   
__________________________________________________________________________
PHENOXAZONES                                                              
2-aminophenoxazone                                                        
 ##STR13##                        2.5 × 10.sup.-5                   
__________________________________________________________________________
TRIPHENYLMETHANE DYES                                                     
 ##STR14##                                                                
__________________________________________________________________________
              R         R.sub.1                                           
__________________________________________________________________________
Crystal Violet                                                            
              N(CH.sub.3).sub.2                                           
                        N(CH.sub.3).sub.2                                 
                                  2.4 × 10.sup.-6                   
Methyl Violet N(CH.sub.3).sub.2                                           
                        NHCH.sub.3                                        
                                  1.6 × 10.sup.-6                   
Malachite green                                                           
              N(CH.sub.3).sub.2                                           
                        H         3.0 × 10.sup.-6                   
Brilliant Green                                                           
              N(C.sub.2 H.sub.5).sub.2                                    
                        H         9.6 × 10.sup.-6                   
__________________________________________________________________________
DIPHENYLMETHANE DERIVATIVES                                               
 ##STR15##                                                                
__________________________________________________________________________
               R          X                                               
__________________________________________________________________________
Auramin O      N(CH.sub.3).sub.2                                          
                          NH.sup.+  Cl.sup.-                              
                                  3.3 × 10.sup.-6                   
4,4&#39;-bis(trimethylammonio)                                                
               N(CH.sub.3).sub.3.sup.+  Cl.sup.-                          
                          H.sub.2 2.9 × 10.sup.-5                   
diphenylmethane                                                           
__________________________________________________________________________
OTHERS                                                                    
phenazine                         2.7 × 10.sup.-4                   
 ##STR16##                                                                
__________________________________________________________________________
 ##STR17##                                                                
benzo (b) quinolizinium chloride  2.5 × 10.sup.-5                   
__________________________________________________________________________
4,4&#39; -bis(trimethylammonio)diphenyl-                                      
dichloride                        3.1 × 10.sup.-4                   
 ##STR18##                                                                
__________________________________________________________________________
4-dimethylamino-4&#39;-trimethylammonio                                       
stilbene chloride                 3.1 × 10.sup.-6                   
 ##STR19##                                                                
__________________________________________________________________________
2-p-dimethylaminostyryI-N-ethyl-                                          
quinolinium chloride              3.0 × 10.sup.-7                   
 ##STR20##                                                                
__________________________________________________________________________
 
    
     
                                           TABLE 2                                 
__________________________________________________________________________
                  Amplifier                                               
Phleo             Dose Sched.                                             
MKO Amplifier     MKD  daily                                              
                           MST  T/C  Group Size                           
__________________________________________________________________________
32  Neutral Red   20   ×2                                           
                           &gt;30  &gt;214**                                    
                                     8                                    
32   --           0    ×2                                           
                           11    79  8                                    
16  Neutral Red   20   ×2                                           
                           &gt;30  &gt;214**                                    
                                     8                                    
16   --           0    ×2                                           
                           10    71  8                                    
8   Neutral Red   20   ×2                                           
                           20    143**                                    
                                     8                                    
8    --           0    ×2                                           
                           14.5 103  8                                    
0   Neutral Red   20   ×2                                           
                           11.0  79  8                                    
0   Buffer Saline Control                                                 
                  0    ×2                                           
                           14   (100)                                     
                                     8                                    
20  Brilliant Green                                                       
                  640  ×2                                           
                           11    138**                                    
                                     7                                    
20   --           0    ×2                                           
                           9    112  7                                    
0   Brilliant Green                                                       
                  640  ×2                                           
                           9    112  7                                    
10  Brilliant Green                                                       
                  640  ×2                                           
                           11    138**                                    
                                     7                                    
10   --           0    ×2                                           
                           8    100  7                                    
5   Brilliant Green                                                       
                  640  ×2                                           
                           10    125**                                    
                                     7                                    
5    --           0    ×2                                           
                           8    100  7                                    
0   Buffer Saline Control                                                 
                  0    ×2                                           
                           8    (100)                                     
                                     35                                   
8   Malachite Green                                                       
                  52   ×2                                           
                           16.5  206**                                    
                                     6                                    
8    --           0    ×2                                           
                           8.5  107  6                                    
0   Malachite Green                                                       
                  52   ×2                                           
                           8    100  6                                    
0   Buffer Saline Control                                                 
                  0    ×2                                           
                           8    100  30                                   
4   Crystal Violet                                                        
                  0.5  ×2*                                          
                           &gt;30   250**                                    
                                     8                                    
4    --           0    ×2*                                          
                           13   108  8                                    
0   Crystal Violet                                                        
                  0.5  ×2*                                          
                           12.5 104  8                                    
0   Buffer Saline Control                                                 
                  0    ×2*                                          
                           12.0 (100)                                     
                                     8                                    
*twice daily from days 2 to 10                                            
8   Auramin 0     7.6  ×2                                           
                           18   &gt;180**                                    
                                     6                                    
8    --           0    ×2                                           
                           9.5   95  6                                    
0   Auramin 0     7.6  ×2                                           
                           9.5   95  6                                    
0   Buffer Saline Control                                                 
                  0    ×2                                           
                           10.0 (100)                                     
                                     30                                   
4   4,4&#39;-bis(trimethylammonio)                                            
    diphenylmethane dichloride                                            
                  270  ×2                                           
                           10.0 125**                                     
                                     8                                    
4    --           0    ×2                                           
                           8.0  100  8                                    
0   4,4&#39;-bis(trimethylammonio)                                            
    diphenylmethane dichloride                                            
                  270  ×2                                           
                           8.0  100  8                                    
0   Buffer Saline Control                                                 
                  0    ×2                                           
                           8.0  (100)                                     
                                     32                                   
__________________________________________________________________________
 Explanatory Notes:                                                       
 MK = micrograms/kilogram                                                 
 MKD = micrograms/kilogram animal weight/day                              
 MST = median survival time in days                                       
 ##STR21##                                                                
 **125 for a group size of 6 animals is taken as positive