Abstract:
Pharmaceutical compositions comprising trimethoprim and a N-ethyl-γ-pyridone-3-carboxylic acid derivative, such as nalidixic, oxolinic or piromidic acid, are more effective than previously known trimethoprim-containing compositions for the treatment of infections affecting the urinary tract. 
     Trimethoprim and the foregoing acid derivatives unpredictably show a noticeable synergistic effect.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to novel trimethoprim-containing pharmaceutical compositions and to the use of such compositions for the treatment of infections affecting the urinary tract. More specifically, the trimethoprim-containing composition of this invention are useful therapeutics in the treatment of acute and chronic infections of the urinary tract due to Gram-negative bacteria. 
     2. Description of the Prior Art 
     Trimethoprim, 2,4-diamino-5-(3,4,5-trimethoxybenzyl) pyrimidine, is a long since known, widely used antimicrobial agent. 
     Generally, trimethoprim has been used as compounded preparations with sulphamethoxazole (Co-trimoxazole). Also trimethoprim/rifampicin compositions have been described. 
     Recently, an increasingly severe criticism has been raised against the foregoing trimethoprim-containing compositions. 
     Co-trimoxazole may cause any of the side-effects typical of sulphonamides. These latter require close supervision of the patient because the onset of serious intoxication is unpredictable. As known, sulphonamide toxic effects range from relatively minor effects such as nausea, vomiting and drowsiness to serious complications, like renal complications (lumbar pain, haematuria etc.), allergic reactions (e.g. skin rashes) and even hepatitis. 
     The trimethoprim/rifampicin composition has been reported to produce abnormalities in liver function caused by the hepatotoxic nature of rifampicin. Alterations in kidney function and renal failure have also been reported. 
     SUMMARY OF THE INVENTION 
     It is, therefore, an object of the present invention to provide a trimethoprim-containing composition which does not have the untoward drawbacks of the prior art compositions. 
     More particularly, it is an object of the present invention to provide a trimethoprim-containing composition, extremely effective for treating infections of the urinary tract, which is free of sulphonamides and rifamicin-type antibiotics and, consequently, of the attendant undesirable side-effects. 
     In accordance with the invention, it has been found a pharmaceutical composition for treating infections of the urinary tract, comprising 
     (a) an effective amount of trimethoprim, 
     (b) an effective amount of a N-ethyl-γ-pyridone-3 carboxylic acid derivative, and 
     (c) an inert excipient therefor. 
     Preferably, the N-ethyl-γ-pyridone-3-carboxylic acid derivative is selected from the group comprising nalidixic acid, oxolinic acid, piromidic acid, their pharmaceutically acceptable salts, amides and esters, and mixtures thereof (briefly referred to hereinbelow acid derivatives A). 
     It has been found that the compositions of the present invention have a powerful synergistic effect, as shown in details in the &#34;Experimental investigations&#34; section of this application. This synergism was totally unpredictable on the grounds of the previously known characteristics of trimethoprim on one hand, and nalidixic, oxolinic and piromidic acid on the other hand. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     It has been found the weight ratio of trimethoprim to nalidixic, oxolinic and piromidic acid should range between 1:2 and 1:10. 
     Preferably, the weight ratio is 1:5. 
     In actual practice, the compositions of the present invention are given orally or parenterally, in any of the usual pharmaceutical forms which are prepared with conventional procedures. These forms include solid and liquid oral unit dosage forms, such as tablets, capsules, suspensions, solutions, syrups and the like as well as saline solutions for in injectable vials or bottles for phleboclysis under conditions of absolute asepticity for administration by the parenteral route. 
     Preferably, an orally administrable, pharmaceutical composition in unit dosage form for treating infections of the urinary tract, comprises: 
     (a) from 45 to 200 mg of trimethoprim; 
     (b) from 200 to 1000 mg of oxolinic, nalidixic or piromidic acid; and 
     (c) an effective amount of an inert excipient therefor. 
     The dose which is administered will be determined by the attending physician having regard to the age, weight and condition of the patient, using sound professional judgment. 
     It has been found, however, that, whereas the trimethoprim dose to be administered is from 2 to 8, preferably 4-6, mg/kg/die, the dose of oxolinic acid is 10-40 mg/kg/die, and the dose of nalidixic acid and piromidic acid is 20-80 mg/kg/die. 
     EXPERIMENTAL INVESTIGATIONS 
     (1) Determination of synergic effect &#34;in vitro&#34; 
     A study carried out to prove the existence of a synergic interaction between trimethoprim and the acids of class (A) gave extremely positive results. In fact, it was possible to show a super-additive effect against most of the strain tested. 
     The experimental work, consisting in the determination of FIC (Fractional Inhibitory Concentration, see S. R. M. Bushby and G. H. Hitchings, Trimethoprim, a sulphonamide potentiator, Br, J. Pharmac. Chemoter. (1968) 33, 72-90) in solid medium for the single anti-bacterials (i.e. the acids of class (A)) and the various compositions was carried out by using 20 strains for each genus of bacteria. 
     In Table 1 there are concisely shown the results obtained, expressed as percentages of strains against which the compositions of this invention exhibit a synergic effect. A value of FIC less than 1 has been regarded as evidence of a synergic effect. 
     It should be noted that there are no significant differences among the various compositions of the present invention, as shown by the percentage range illustrated in Table 1. All the tested compositions contained trimethoprim and either one of the N-ethyl-γ-pyridone-3 carboxylic acid derivatives at a weight ratio of 1:5. 
     In Table 2, the results obtained with the trimethoprim/oxolinic acid composition toward 126 clinically isolated bacteria strains are shown. 
     (2) Studies on resistance development toward nalidixic acid, oxolinic acid and piromidic acid, either alone or in combination with trimethoprim 
     The experimental work was carried out by using the sub-culture technique, i.e. the culture grown in the presence of the highest drug concentration on Clark-Lubs agar plates was used as inoculum in the subsequent sensibility determination. 
     The rate of the resistance development in a series of bacteria strains (3Salmonella thyphimurium-3 Escherichia coli-3 Klebsiella pneumoniae-3 Proteous mirabilis) was shown to be positively affected by the compositions of the present invention. The results were substantially identical to those shown in Table 3 concerning the trimethoprim/nalidixic acid composition, for all of the tested compositions of the present invention. 
     In Table 4, the results obtained with the trimethoprim/oxolinic acid composition are shown. 
     (3) Antibacterial activity &#34;in vivo&#34; 
     A study was carried out in experimentally infected mice. The ratio of ED 50  of the N-ethyl-γ-pyridone-3-carboxylic acid derivatives to the ED 50  of the trimethoprim-containing compositions of the present invention was measured. 
     In Table 5 there are shown the bacteria genuses which were used for evaluating the &#34;in vivo&#34; antibacterial activity as well as the potentiation of the protective effect of the compositions of the present invention, wherein the trimethoprim concentration was exactly one fifth of the ED 50   exhibited by the single acids listed in (A) towards the various bacteria used for experimentally infecting the mice. 
     Table 5 shows that positive results were obtained against all the tested strains, without noticeable differences among the various compositions. 
     (4) Clinical investigations 
     The observations noted above were confirmed in double-blind clinical studies, by treating a number of patients suffering from infections of the urinary tract of comparable seriousness with (1) nalidixic acid, (2) oxolinic acid, (3) co-trimoxazole and (4) the compositions trimethoprim/nalidixic acid and trimethoprim/oxolinic acid of the present invention. 
     At the end of the treatment period (10 days), the following recovery percentages with no relapses were observed: 
     
         ______________________________________nalidixic acid (50 mg/kg/die)                    = 30%oxolinic acid (25 mg/kg/die)                    = 55%co-trimoxazole (320 mg of trimetho-prim + 1600 mg of sulfamethoxazole/die)         = 40%trimethoprim/nalidixic acid                    = 60%trimethoprim/oxolinic acid                    = 92%______________________________________ 
    
     In the Tables 6 and 7, the results obtained with two compositions in accordance with the invention are shown. 
     
                       TABLE 1______________________________________Potentiation of trimethoprim/nalidixic acid, trimethoprim/oxolinic acid and trimethoprim/piromidic acid compositionsagainst 180 bacterial strains.                   Percentage of synergisticOrganisms  No. of strains                   effect______________________________________Staphylococcus      (20)         40-60Escherichia      (20)         60-90Salmonella (20)         70-90Klebsiella (20)         70-90Brucella   (20)         60-80Proteus indol.sup.-      (20)         60-80Proteus indol.sup.+      (20)         50-70Pseudomonas      (20)         10-20Streptococcus      (20)         40-60______________________________________ 
    
     
                       TABLE 2______________________________________Potentiation of the trimethoprim/oxolinic acid compositionagainst 126 isolated strains.   F I C Index     No.     isolatedOrganisms strains  ≧1                     0.99-0.5                            0.49-0.25                                   0.24-0.125______________________________________Enterococcus      8       3      5Staphylococcus     26       12     11     2      1Escherichia     22       4      10     7      1Klebsiella      8       0      4      4Proteus indol.sup.+      9       2      6      1Proteus indol.sup.-     13       6      4      2      1Pseudomonas     10       5      3      2Salmonella      9       5      3Shigella   5       1      1      3Brucella  16       9      5      1______________________________________ 
    
     
                       TABLE 3______________________________________Resistance development to nalidix acid (NA) and to analidixic acid/trimethoprim (NA+T) composition.  Minimum inhibitory concentration (μg/ml)No. of days    NA        NA + T      NA     NA + T______________________________________  Salmonella typhimurium 9                  Escherichia coli K 12                  ATCC 137621        4         4 + 0.25    2      1 + 12        8         4 + 0.25    4      1 + 13         16       4 + 0.25    8      1 + 14         32       8 + 0.25    8      2 + 15         64       8 + 0.25     16    2 + 16        256       32 + 0.25   128    2 + 17        512       32 + 0.25   512    8 + 18        &gt;512      32 + 0.25   &gt;512   32 + 19        &gt;512      64 + 0.25   &gt;512   32 + 110       &gt;512      64 + 0.25   &gt;512   32 + 1______________________________________  Klebsiella pneumoniae                  Proteus mirabilis 70  ATCC 100311        1         0.5 + 1     4      1 + 52        2         0.5 + 1     4      1 + 53        4         1 + 1       8      1 + 54        4         1 + 1       8      4 + 55        8         1 + 1        16    4 + 56        8         2 + 1        32    4 + 57         64       4 + 1        32    8 + 58        256       16 + 1      128    16 + 59        512       16 + 1      128    32 + 510       &gt;512      16 + 1      256    32 + 5______________________________________ 
    
     
                                           TABLE 4__________________________________________________________________________Resistance development to oxolinic acid (ox) and to an oxolinicacid/trimethoprim composition (ox + T).Minimum inhibitory concentration (μg/ml)No. daysOX   OX + T  OX OX + T  OX    OX + T OX    OX + T__________________________________________________________________________Escherichia Coli 19 (&#34;)             Escherichia Coli 6 (&#34;)                        Escherichia Freundii 15 (&#34;)                                     Staphylococcus aureus Leo cc                                     23921    0.097     0.048 + 0.0081             0.195                0.048 + 0.0081                        0.097                            0.048 + 0.0081                                     0.39  0.097 + 0.01622    0.39 0.195 + 0.0081             0.39                0.097 + 0.0081                        0.39                            0.195 + 0.0081                                     0.78  0.195 + 0.01623    0.78 0.195 + 0.0081             0.78                0.195 + 0.0081                        0.39                            0.195 + 0.0081                                     1.56  0.195 + 0.01624    1.56 0.195 + 0.0081             0.78                0.195 + 0.0081                        0.78                            0.195 + 0.0081                                     1.56  0.195 + 0.01625    3.12 0.78  + 0.0081             0.78                0.195 + 0.0081                        1.56                            0.195 + 0.0081                                     1.56  0.78  + 0.01626    3.12 0.78  + 0.0081             0.78                0.195 + 0.0081                        1.56                            0.195 + 0.0081                                     3.12  0.78  + 0.01627    3.12 0.78  + 0.0081             0.78                0.195 + 0.0081                        6.25                            0.195 + 0.0081                                     3.12  0.78  + 0.01628    6.25 1.56  + 0.0081             0.78                0.195 + 0.0081                        6.25                            0.78  + 0.0081                                     6.25  0.78  + 0.01629    6.25 1.56  + 0.0081             3.12                0.78  + 0.0081                        6.25                            1.56  + 0.0081                                     12.5  0.78  + 0.016210   6.25 1.56  + 0.0081             3.12                1.56  + 0.0081                        6.25                            1.56  + 0.0081                                     12.5  1.56  + 0.0162Klebsiella pneumoniae 14 (&#34;)                    -              Klebsiella pneumoniae 62 (&#34;)                            Proteus mirabilis 4 (&#34;)                                       Enterobacteraerogeues 5 (&#34;)1    0.195     0.097 + 0.0162              0.39                  0.195 + 0.0325                            0.39                               0.097 + 0.0162                                       0.39 0.195 + 0.03252    0.78 0.195 + 0.0162              0.39                  0.195 + 0.0325                            0.78                               0.195 + 0.0162                                       0.78 0.195 + 0.03253    1.56 0.195 + 0.0162              1.56                  0.39  + 0.0325                            0.78                               0.195 + 0.0162                                       6.25 0.39  + 0.03254    1.56 0.78  + 0.0162              1.56                  0.78  + 0.0325                            1.56                               0.195 + 0.0162                                       12.5 1.56  + 0.03255    3.12 0.78  + 0.0162              3.12                  1.56  + 0.0325                            1.56                               0.195 + 0.0162                                       12.5 1.56  + 0.03256    12.5 0.78  + 0.0162              6.25                  1.56  + 0.0325                            3.12                               0.195 + 0.0162                                       12.5 1.56  + 0.03257    12.5 0.78  + 0.0162              6.25                  1.56  + 0.0325                            3.12                               0.195 + 0.0162                                       12.5 1.56  + 0.03258    25   1.56  + 0.0162              6.25                  6.25  + 0.0325                            6.25                               0.78  + 0.0162                                       25   6.25  + 0.03259    25   6.25  + 0.0162              12.5                  6.25  + 0.0325                            6.25                               1.56  + 0.0162                                       25   6.25  + 0.032510   25   12.5  + 0.0162              25  12.5  +  0.0325                            12.5                               1.56  + 0.0162                                       25   6.25  + 0.0325__________________________________________________________________________ (&#34;) clinically isolated strains. 
    
     
                       TABLE 5______________________________________Potentiation of ED.sub.50 exhibited by trimethoprim/nalidixic acid,trimethoprim/oxolinic acid and trimethoprim/piromidic acidcompositions.           Average activity increaseOrganisms       (n-Fold)______________________________________Escherichia coli           4.2Klebsiella pneumoniae           3.9Aerobacter aerogenes           5.4Proteus mirabilis           6.1Proteus rettgerii           4.5Pseudomonas aeruginosa           2.2______________________________________ 
    
     
                                           TABLE 6__________________________________________________________________________Clinical evaluation of tripethoprim (4 mg/kg/day) in combination withnalidixicacid (50 mg/kg/day) in 5 patients with complicated urinary infection.Treatment duration: 10 days.                     Outcome of therapyPatientsClinical diagnosis           Infecting organism                     Bacteriologic                            Clinical__________________________________________________________________________1    Chronic cystitis           S. epidermidis                     Eradicated                            Cured2    Chronic cystopyelitis           E. coli   Eradicated                            Cured3    Acute cystoprostatitis           P. mirabilis                     Persisted                            Failure4    Chronic cystopyelitis           P. mirabilis                     Persisted                            Failure5    Chronic cystopyelitis           E. coli   Eradicated                            Cured__________________________________________________________________________ 
    
     
                                           TABLE 7__________________________________________________________________________Clinical evaluation of tripethoprim (4 mg/kg/day) in combination withoxolinic acid(25 mg/kg/day) in 13 patients with complicated urinary infection.Treatment duration: 10 days.                       Outcome of therapyPatientsClinical diagnosis           Infecting organism                       Bacteriologic                              Clinical__________________________________________________________________________1    Pyelonephritis           Escherichia coli                       Eradicated                              Cured2    Acute cystitis           Escherichia coli                       Eradicated                              Cured3    Cysto-prostatitis           Micrococcus sporigenes                       Eradicated                              Cured4    Chronic cysto-pyelitis           Enterobacter agglomerans                       Eradicated                              Cured5    Cysto-prostatitis           Proteus rettgeri                       Eradicated                              Cured6    Acute cystitis           Escherichia coli                       Eradicated                              Cured7    Acute cystitis           Escherichia coli                       Eradicated                              Cured8    Acute cystitis           Escherichia coli                       Eradicated                              Cured9    Acute pyelonephritis           Escherichia coli                       Eradicated                              Cured 10  Acute cystitis           Proteus rettgeri                       Eradicated                              Cured 11  Acute cystitis           Klebsiella pneumoniae                       Eradicated                              Cured 12  Cysto-prostatitis           Pseudomonas aeruginosa                       Persisted                              Failure 13  Acute cystitis           Escherichia coli                       Eradicated                              Cured__________________________________________________________________________