Abstract:
Pathogens are controlled by concomitant contact with a phosphonomycin antibiotic and a bacteriostatic chemotherapeutic compound by separate or simultaneous administration. The combined action is especially advantageous in contacting resistant microorganisms and other microorganisms which are oridinarily not controlled by contact with the individual chemotherapeutic agents.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This is a continuation-in-part of our earlier filed applications Ser. Nos. 655,757 and 679,165 filed July 25, 1967 and Oct. 20, 1967, respectively, which are now abandoned, application Ser. No. 728,059 filed May 9, 1968, now U.S. Pat. No. 3,639,590 and Ser. No. 59,761 filed July 30, 1970, now abandoned; and U.S. Ser. No. 255,117, filed May 19, 1972, now abandoned. 
    
    
     BACKGROUND OF THE INVENTION 
     Although in the past several decades great strides have been made in the control of pathogenic bacteria by various bacteriostatic chemotherapeutic agents such as the sulfa compounds and various antibiotics, these agents are generally quite specific in their action, being for example active against certain gram-negative or gram-positive bacteria. In addition, the use of these chemotherapeutic agents has resulted in the development of mutant strains of pathogens which are resistant to the known bacteriostats. 
     Accordingly, the search for new agents active against a broad spectrum of pathogens, and in particular against resistant strains, has continued. 
     The bacteriostatic chemotherapeutic agents are a well known group of compounds which are active in inhibiting the growth of pathogenic bacteria. Thus, antibiotics such as tetracyclines, chloramphenicol, erythromycin, novobiocin, oleandomycin and lincomycin, and other chemotherapeutic active substances such as nalidixic acid and the sulfa compounds are well known bacteriostats which are active in inhibiting the growth of various pathogenic bacteria. A number of these bacteriostatic agents, for example chloramplenicol, exhibit untoward side effects at the dosage necessary to control infections, and therefore their use has been limited; and it has been found desirable to find ways of reducing the dosages of such antibiotics. 
     Phosphonomycin, a recently discovered antibiotic, is called (-) (cis-1,2-epoxypropyl)phosphonic acid or (-) (1R, 2S)-1,2-epoxypropylphosphonic acid or the non-proprietary name fosfomycin. This antibiotic, and in particular various derivatives thereof such as salts, labile ester and amide derivatives thereof, are valuable antibiotic substances which are active against various gram-negative and gram-positive pathogens such a Escherichia, Klebsiella, Proteus, Pseudomonas, Salmonella, Staphylococcus and Streptococcus bacteria. Because of their outstanding activity and stability, the salts of phosphonomycin such as those of inorganic as well as organic bases represent preferred forms of the antibiotic. The term &#34;phosphonomycin&#34; as used herein includes not only the free acid but also the various derivatives thereof such as the salts, esters and amides which exhibit antibiotic activity. 
     SUMMARY OF THE INVENTION 
     It is an object of this invention to provide a method of potentiating the effectiveness of bacteriostatic chemotherapeutic agents against various pathogenic microorganisms. Another object is to provide a means of extending the effectiveness of various bacteriostatic agents against species of microorganisms which are ordinarily not inhibited by the chemotherapeutic agent alone. Further, it is an oject of this invention to provide a means of potentiating the effectiveness of bacteriostatic chemotherapeutic agents against resistant strains of pathogens. In addition, it is an object of this invention to provide a means of preventing the emergence of resistant strains of pathogenic bacteria contacted with bacteriostatic agents. Also, this invention provides new compositions containing bacteriostatic agents useful in accomplishing the foregoing objects of this invention. Other objects will be apparent from the detailed description of this invention hereinafter provided. 
     DETAILED DESCRIPTION 
     It has now been found that the foregoing objects of this invention are achieved by concomitantly contacting pathogenic bacteria wtih phosphonomycin and a bacteriostatic chemotherapeutic agent. Thus, in combination with phosphonomycin it is possible to extend the usefulness of the bacteriostats to a broad spectrum of pathogens including this which have mutated and acquired resistance to such chemotherapeutic agents. 
     In accordance with one embodiment of this invention, pathogenic bacteria are controlled by concomitantly contacting these pathogens with phosphonomycin and a bacteriostatic chemotherapeutic agent. The action of the phosphonomycin is to potentiate, augment or synergise the activity of the bacteriostatic agent. This potentiation of the bacteriostats is indeed unexpected since the bacteriostatic agents have an entirely different mode of action than phosphonomycin. Thus, while many of the bacteriostats such as chloroamphenicol and the tetracyclines are effective bacause of their ability to inhibit protein synthesis, phosphonomycin is bactericidal in action and affects the bacterial cell wall as is evident from the detection of spheroplasts when certain bacterial strains are exposed to the antibiotic. That phosphonomycin could act synergistically with bacteriostats is not suggested by their individual modes of action. Thus, phosphonomycin is indeed unique among antibiotics in that it is capable of potentiating the important bacteriostatic chemotherapeutic agents. Even more important is the indication that phosphonomycin does not exhibit any antagonism in various tests with human pathogens. 
     From many in vitro tests with various pathogens, it has been determined that phosphonomycin acts synergistically with a number of bacteriostats such as chloramphenicol, erythromycin, tetracyclines, novobiocin and sulfisoxazole; this synergism being defined as the ability of phosphonomycin to inhibit the growth of the pathogen in the presence of one quarter or less of the MIC (minimum inhibitory concentration) of each of phosphonomycin and the bacteriostat. In an enormous series of tests with almost 200 pathogenic bacteria involving about 15,000 individual tests, the efficacy of phosphonomycin combined with chloramphenicol and erythromycin was determined. These tests indicated that there was no antagonism between phosphonomycin and each of the antibiotics and that in 88% of the tests it was found that phosphonomycin potentiated the effect of chloramphenicol. In a smaller series of trials it was observed that 83% of the tests showed potentiation with a combination of phosphonomycin and erythromycin. Other experimental results have shown that novobiocin, chlortetracycline and sulfisoxazole are potentiated when combined with phosphonomycin and tested with various strains of pathogenic bacteria. 
     The practical advantages of the use of phosphonomycin to potentiate bacteriostats are, therefore, readily apparent. Thus, in potentiating chloramphenicol the combination acts synergistically, making it possible to use less chloramphenicol than would otherwise be necessary to control the pathogens and thereby minimizing the untoward effects sometimes observed with higher dosage levels of this antibiotic. Also, in some instances, for example in the case of novobiocin, the phosphonomycin potentiates the bacteriostat and the combination is effective in controlling pathogens which cannot be effectively controlled by either antibiotic separately. Also, the potentiating effect of the combined action of the bacteriostat and phosphonomycin results in a lower incidence of resistant strains being formed. These upexpected advantages of th effect of phosphonomycin make this an important and valuable advance in the art. Thus, pathogenic bacteria can be controlled by treatment with phosphonomycin and a bacteriostat in the ratio of 1 part of phosphonomycin to 10 parts of the bacteriostat up to 10 parts of the phosphonomycin to 1 part of the bacteriostat. The preferred ratio is 1 part of phosphonomycin to 4 parts of bacteriostat up to 8 parts of phosphonomycin to 1 part of the bacteriostat. 
     This ability of phosphonomycin to potentiate, augment or synergise the effectiveness of bacteriostats provides a means of effectively combating bacterial infections in animals and humans. For this purpose phosphonomycin can be administered enterally in the form of tablets, capsules, syrups or suppositories or parenterally as solutions in suitable liquid mediums such as sterile water or isotonic solutions. These pharmaceutical forms of phosphonomycin can be prepared in accordance with procedures well known in the pharmaceutical art. Thus, for example, in treating human infections caused by pathogenic bacteria with phosphonomycin and chloramphenicol, the phosphonomycin is administered in an amount from about 500-4,000 mg./day and the chloramphenicol in an amount from about 500-2,000 mg./ day. 
     Pursuant to another embodiment of the present invention, phosphonomycin and one or more of the bacteriostatic agents can be combined in a single unit dosage form. For this purpose the chemotherapeutic agents can be combined in suitable unit dosage forms in conjunction with suitable non-toxic solid or liquid diluents or carriers. These dosage unit forms can be used for enteral administration in the form of tablets, capsules, syrups or suppositories or, alternatively, these chemotherapeutic agents can be combined in suitable liquid mediums for parenteral administration. For topical administration the phosphonomycin and the chemotherapeutic bacteriostat can be prepared in the form of salves, ointments and the like in accordance with procedures well known in the art for formulating such compositions. 
     The examples which follow are provided as illustrative embodiments of our invention. 
    
    
     EXAMPLE 1 
     The MIC (minimum inhibitory concentration) of phosphonomycin and each of the other antibiotics by itself was determined before the combination synergy experiment was begun, as follows: 2 ml. of a double strength solution in sterile distilled water of the highest strength to be tested (usually 1000γ) was added to a tube of double strength nutrient broth (NB) and mixed thoroughly. Two-fold serial dilutions were then made by transferring 2 ml. to the next tube (1 × NB) and so on for approximately 12 tubes. The tubes were inoculated with one drop each of a 1:50 dilution of a culture adjusted to a Lumetron 60 reading in NB + 0.2% yeast extract so as to provide a concentration of cells of approximately 25,000/ml. The tubes were then incubated at 37°C. and examined for growth at 24, 48 and 72 hours. 
     In the combination experiment, the MIC value obtained as described above was used as approximately the midpoint of the range to be tested for each of the two antibiotics of the combination. In a manner similar to the above, two-fold dilutions of the first substance in the combination were made except that 1.8 ml. volumes were used and a 2.2 × strength solution of both the antibiotic and the NB were made. The second antibiotic of the combination was added to duplicate rows of serial dilutions of the first substance as follows: 0.2 ml. in NB of a ##EQU1## and a ##EQU2## respectively, of the second substance was added to each tube in a row of serial dilutions of the first antibiotic. The final concentration that resulted enabled the determination of the effect of both MIC/4 and MIC/8 of each substance in the presence of from approximately 32 MIC to ##EQU3## of the other. Inocula were added as in the initial single MIC experiment. The results of these tests are shown in the following table: 
     
                                   Phosphonomycin     MIC of 2nd Subst.                     MIC in Presence                               MIC of   In Presence of                         MIC                MICSecond    Test    Phosphonomycin                     of ≦                            of 2nd      ≦                                               of                         4                  4Substance Organism             MIC     2nd Substance                               Subst.   PHosphonomycin__________________________________________________________________________Novobiocin     Proteus     vulgaris             220     6.8       32       3.9Novobiocin     Pseudomonas     stutzeri             &gt;220    16        32       8Novobiocin     Klebsiella     pneumoniae             750     5.8       32       &lt;2Novobiocin     Staphylococcus     aureus  24      48        0.06+    0.015*Novobiocin     Streptococcus     faecalis             93.6    24*       4        4Novobiocin     Pseudomonas     aeruginosa             186     93.6      250      62.5Erythromycin     Proteus     vulgaris             48      96        1000     500*Erythromycin     Pseudomonas     aeruginosa             376     12        &gt;16000   2000+Erythromycin     Staphylococcus     aureus  &gt;192    24        8        2Erythromycin      Streptococcus     faecalis             96      192       32       16Chlortetracycline     Proteus     vulgaris             48      24*       2        0.25Chlortetracycline     Streptococcus     faecalis             48      48        0.5      0.5__________________________________________________________________________             MIC        MIC*Value obtained in the presence of                instead of ≦             2          4+Resistant growth present 
    
     The foregoing table is significant in showing that the potentiating effect of phosphonomycin is observed in eight of the twelve tests, and that in no case was there any evidence of antagonism by the combination of antibiotics. Also significant is the mutual potentiation of the phosphonomycin-novobiocin combination against Klebsiella. 
     EXAMPLE 2 
     The &#34;criss-cross&#34; method was used to determine potentiation of chloramphenicol by phosphonomycin. This method involved the simultaneous determination of the MIC (minimum inhibitory concentration) of each member of a pair of antibiotics and of combinations of the two in nutrient broth (NB) containing 0.2% yeast extract (YE). Serial two-fold dilutions were made throughout the experiment, and the starting inoculum consisted of approximately 25,000 cells/ml. of Staphylococcus aureus. Incubation was at 37°C. for 24 hours. Identical experiments were performed on two different days. 
     
         ______________________________________Results: MIC&#39;s μg/ml.    MIC    Phosphonomycin CLM* MIC    In Presence of In Presence                MIC        CLM*        MICSecond Phospho-            of          of ≦                                            of                4          MIC         4Subst. nomycin  2nd Subst.         Phosphonomycin______________________________________CLM*  62.5     15.6         7.8   2.0CLM*  31.2     7.8          3.9   0.5______________________________________ *Chloramphenicol 
    
     These results clearly demonstrate the synergistic effect of combinations of phosphonomycin and chloramphenicol against this strain of Staphylococcus aureus. 
     EXAMPLE 3 
     A series of agar plates were prepared containing varying amounts of phosphonomycin and chloramphenicol and mixtures of the two antibiotics which were then inoculated with various test organisms to determine the minimum inhibitory concentrations (MIC) of the individual antibiotics and the effectiveness of the combination of the two antibiotics. 
     In these tests the agar plates were prepared by combining 1 ml. of a sterile aqueous solution of the antibiotic with 9 ml. of sterile Mueller Hinton agar, thoroughly mixing the antibiotic solution with the agar and allowing the resulting mixture to solidify. The amount of chloramphenicol in the plates ranged from 0.2 to 409.6 mcg/ ml., and the amount of phosphonomycin ranged from 2 to 256 mcg/ml. The exact amounts of the antibiotic or antibiotics present in each of the plates are shown in each of the boxes of the following diagrams. 
     
         __________________________________________________________________________Phosphonomycin mcg/mlChloramphenicol meg/ml. 0         2    4    8    16   32   64   128   256__________________________________________________________________________ 0   0/0  2/0  4/0  8/0  16/0 32/0 64/0 128/0 256/00.2  0/0.2     2/0.2          4/0.2               8/0.2                    16/0.2                         32/0.2                              64/0.2                                   128/0.2                                         256/0.20.4  0/0.4     2/0.4          4/0.4               8/0.4                    16/0.4                         32/0.4                              64/0.4                                   128/0.4                                         256/0.40.8  0/0.8     2/0.8          4/0.8               8/0.8                    16/0.8                         32/0.8                              64/0.8                                   128/0.81.6  0/1.6     2/1.6          4/1.6               8/1.6                    16/1.6                         32/1.6                              64/1.63.2  0/3.2     2/3.2          4/3.2               8/3.2                    16/3.2                         32/3.26.4  0/6.4     2/6.4          4/6.4               8/6.412.8 0/12.8     2/12.8          4/12.825.6 0/25.6     2/25.6          4/25.6__________________________________________________________________________ 
    
     
         Phosphonomycin mcg/ml.Chloramphenicol mcg/ml. 0           2    4      8     16    32    64    128   256__________________________________________________________________________ 0    0/0   2/0   4/0   8/0   16/0  32/0  64/0  128/0 256/012.8  0/12.8       2/12.8             4/12.8                   8/12.8                         16/12.8                               32/12.8                                     64/12.8                                           128/12.8                                                 256/12.825.6  0/25.6       2/25.6             4/25.6                   8/25.6                         16/25.6                               32/25.6                                     64/25.6                                           128/25.651.2  0/51.2       2/51.2             4/51.2                   8/51.2                         16/51.2                               32/51.2                                     64/51.2102.4 0/102.4       2/102.4             4/102.4                   8/102.4204.8 0/204.8       2/204.8             4/204.8409.6 0.409.6       2/409.6             4/409.6__________________________________________________________________________ 
    
     The described tests were run in either of above-described series, depending upon the activity of the pathogen in disc plate tests, with discs containing 5 μg and 30 μg of chloramphenicol. When the pathogens were inhibited at both the 5 and 30 μg levels, the pathogen was tested in the first series containing lower concentrations of chloramphenicol. The organisms which were not inhibited at the lower level but were inhibited at the 30 μg level were tested in the second series having a higher concentration of chloramphenicol. 
     Thus, in this series of tests the agar plates contained phosphonomycin and/or chloramphenicol in the amount shown in the numerator and the denominator, respectively, in each of the boxes. (To avoid needless repetition of the concentrations of the antibiotics, only a representative number of the boxes show the concentrations of each of the combinations tested; however, all of the concentrations indicated by each of the boxes were used in tests.) 
     The plates prepared as described above were divided into small sections or boxes so that each section served to test a different species or different organism. Thus, each section of the plate was inoculated with a different strain or species by placing one drop of an aqueous suspension of the microorganism containing about 10,000-20,000 cells. The plates were then incubated for 18 hours at 37°C. and then examined to determine the MIC of the individual antibiotics and the effectiveness of the combinations of the antibiotics against a number of pathogenic organisms. 
     The results of this series of tests are shown in the following table. 
     
         __________________________________________________________________________           High   Weak   No. Strains           Potentiation                  Potentiation                         AdditiveMicroorganism   Tested  Observed                  Observed                         Coaction                              No Effect__________________________________________________________________________Escherichiacoli    81      38     39     2    2Paracoli   2       1      1      0    0Citrobacter   6       1      3      0    2Aerobacter   2       1      1      0    0Klebsiella   27      16     9      0    2Salmonella   4       0      2      0    2Proteus 6       3      2      0    1Pseudomonas   11      6      3      0    2Serratia   2       2      0      0    0Staphylococcuscoagulase+   5       1      2      0    2Staphylococcus   43      17     20     3    3Enterococcus   4       0      3      0    1Total   193     86     85     5    17__________________________________________________________________________ 
    
     EXAMPLE 4 
     In another series of tests carried out following the procedures described in Example 3, the effect of combinations of phosphonomycin (PM) and erythromycin (EM) against ten strains of pathogenic bacteria was determined with the following results: 
     
         No. of Strains Tested                10No Effect            1Potentiation by PM   5Potentiation by EM   4 
    
     EXAMPLE 5 
     To determine the interaction in vivo of phosphonomycin, chloramphenicol, novobiocin and tetracycline, each agent was titrated individually in mice and in the presence of constant quantities (one estimated to be 10% and a second 1% of the expected ED 50 ) of the second antibiotic. For example, using Escherichia coli 2017 as the infecting agent, sodium phosphonomycin was titrated alone and in the presence 5 and also in the presence of 0.5γtetracycline, while tetracycline was titrated alone and in the presence of 50γ, and also in the presence of 5γof sodium phosphonomycin. The two drugs were given by the subcutaneous route at separate sites on the ventral surface of the mouse. Such therapy was given only at the time of infection for the Escherichia coli and Pseudomonas aeruginosa tests but both at the time of infection and 6 hours later for all other tests. Infection was given intraperitoneally using broth suspensions of the test organism. 
     In the first test using Escherichia coli with sodium phosphonomycin and tetracycline, the ED 50  for sodium phosphonomycin was 1000γ, an amount representing, therefore, 100% of the effective sodium phosphonomycin dose. The ED 50  for tetracycline was 50γ, equaling 100% of the effective tetracycline dose. When sodium phosphonomycin was titrated in the presence of 0.5γ tetracycline (here representing 1% of the effective tetracycline ED 50 ), the sodium phosphonomycin ED 50  was again 1000γ or 100% of its effective dose. The combination, therefore, of 100% with 1% or 101% of the combined effective doses was judged to indicate no interaction for this combination. When, however, tetracycline was titrated in the presence of a constant quantity of 50γ sodium phosphonomycin (representing 5% of its effective dose), the tetracycline ED 50  was 13γ or 25% of its effective dose. The combined percent of 30 was as effective as 100% of either drug along, an effect usually termed as synergistic. 
     On Table 1 are listed the test results using Escherichia coli. It can be seen that in two tests the activity of tetracycline has been enhanced when it was titrated in the presence of a constant quantity of sodium phosphonomycin. In the first test, a combination of 25% tetracycline with 5% sodium phosphonomycin for 30% is judged synergistic, but the combination of 54% tetracycline with .5% sodium phosphonomycin is judged to be borderline and not labeled as synergy. In the second test there was synergy with a constant quantity of 2% of the effective sodium phosphonomycin dose but not with 14%. In neither test did a constant quantity of tetracycline enhance sodium phosphonomycin activity. Similarly, it can be seen that synergy has been demonstrated between chloramphenicol and sodium phosphonomycin but that the two tests do not completely repeat each other. 
     On Table 2 are listed results from single tests. It will be noted that against Streptococcus pyogenes only one set of combinations was tested, that is, the titration of tetracycline in the presence of a constant quantity of sodium phosphonomycin. Only additive percentages were demonstrated. In experiments using novobiocin, it is difficult to interpret results because novobiocin itself did not protect against Pseudomonas aeruginosa, nor against Klebsiella pneumoniae at the levels tested. For this latter organism, however, less sodium phosphonomycin was required to protect the mice when 1 mg. of novobiocin was included in the dose than in its absence. This could be considered as potentiation. 
     
                                           Table I__________________________________________________________________________Effect of Combined Antibiotic Therapy of Escherichia coli InfectionTest Organism     s.c. ED.sub.50 μg***                           % ED.sub.50and Challenge    Phosphonomycin*                       CombinedDose     plus     Phosphonomycin*                      Other                           Phosphonomycin*                                    Other %__________________________________________________________________________E. coli 2017    Tetracycline               1000    --  100            1009 LD.sub.50          --     50           100   100                427**  5   43       10    53               1000**  0.5 100       1    101                50     13**                           5        25    30                 5     27**                           ).5      54    55Repeat Test    Tetracycline                350    --  100E. coli 2017         --     31           1007 LD.sub.50          390**  10  111      32    143                380**  1   108       3    111                50     31**                           14       100   114                 0.5   7** 2        23    25E coli 2017    Chloram-    905    --  1009 LD.sub.50    phenicol    --    453           100                500**  30  55        7    63                905**  3   100       1    101                100   107**                           11       24    35                10    250**                           1        55    56Repeat Test    Chloram-    276    --  100E. coli 2017    phenicol    --    120           1007 LD.sub.50          26**   50  9        42    51                92**   5   33        4    37                50     20**                           19       17    36                 5     47**                           2        39    41__________________________________________________________________________  *As sodium salt  **Titrated--other agent constant ***Single dose only, at 0 hour 
    
     
                                           Table 2__________________________________________________________________________Effect of Combined Antibiotic Therapy on Several Experimental Infectionsin MiceTest Organism     s.c. ED.sub.50 μg***                            % ED.sub.50and Challenge    Phosphonomycin*                      CombinedDose     plus     Phosphonomycin*                      Other Phosphonomycin*                                     Other                                         %__________________________________________________________________________Streptococcus    Tetracycline               1400   --    100      100pyogenes 3009        --    55             1006 LD.sub.50         1000   18**  71       33  104                500   35**  64       36  100Pseudomonas    Novobiocin 1420   --    100aeruginosa 3210      --    &gt;1000010 LD.sub.50        2760** 1000  195               1250** 100   88               &gt;200   &gt;10000*               &gt; 20   &gt;10000**Klebsiella    Novobiocin 2130   --    100pneumoniae &#34;B&#34;       --    &gt;25007 LD.sub.50          298** 1000  14       --               1110** 100   52       --               &gt; 100  &gt;2500**               &gt; 10   &gt;2500__________________________________________________________________________  *As sodium salt  **Titrated --other agent constant *** Therapy given at 0 and at 6 hours except P. aeruginosa 3210, 0 hour only 
    
     Various changes and modifications in the procedures herein disclosed will occur to those skilled in the art, and to the extent that such changes and modifications are embraced by the appended claims, it is to be understood that they constitute part of our invention.