Abstract:
A substituted pyrazine having the formula ##STR1## is useful for treating and preventing swine dysentery. It may be administered to swine in an effective but non-toxic amount in the form of the drug per se, or a feed composition. Such feeds may be made with the aid of premixes containing the said compound.

Description:
DESCRIPTION 
     This invention relates to the treatment and prevention of swine dysentery, and to products for use therein. 
     Swine dysentery (also known as vibrionic dysentery, bloody scours, or hemorrhagic dysentery) is an enteric disease primarily characterised by muco-hemorrhagig diarrhea with lesions usually restricted to the large intestine. The disease is worldwide and is a major disease problem among swine producers all over the world. 
     The earlier consensus was the Vibrio coli was the primary causative agent. Recent evidence suggests, however, that a spirochete, Treponema hyodysenteriae is involved with the disease and may in fact be the primary etiologic agent. 
     Currently, control measures are based on constant feeding of antibacterial agents with therapy based on use of high levels of these drugs. The drugs used include furazolidone, neomycin, oxytetracycline, tylosin, carbadox, virginiamycin and arsanilic acid. Unfortunately, these drugs give erratic results, even when used at abnormally high levels. 
     Accordingly there is a continuing need for new drugs of low toxicity and high potency to combat swine dysentery. 
     It has now been discovered that a substituted pyrazine of that formula: ##STR2## hereinafter referred to as &#34;D-compound&#34; is useful in veterinary therapy for the treatment and prophylaxis of swine dysentery. This compound selectively combats the swine dysentery-causing organisms without deleteriously affecting the balance of other desired organisms, e.g. in the internal biological system of swine, such as the intestinal flora. 
     The antibiotic substance D-aspergillic acid which has been isolated from cultures of Aspergillus flavus conform to the aforesaid formula. 
     In the method of the present invention, the D-Compound is administered to swine in an amount effective to combat dysentery. It can be advantageously incorporated in a swine feed-stock to provide a swine feed composition for combatting dysentery. It can be incorporated in the swine feed-stock generally at a level of from about 25 g/ton to about 500 g/ton. The preferred level, however, particularly in the absence of the disease, is about 100 to 200 g/ton for prophylaxis, advantageously for a period of 3 to 21 days. However, it there has been an outbreak of the disease, or if new animals whose history is not known have been introduced into a herd, the higher level of 200 to 500 g/ton is preferred until the health of the herd is assured. Generally, however, the prophylactic treatment is continued until the animals are ready for market. The D-Compound can also be administered by incorporation into drinking water provided for swine. 
     The D-Compound is useful for combatting swine dysentery-causing organisms, e.g. dysentery caused by Vibrio or Treponema organisms, or both. The D-Compound is of a low order of toxicity and is suitable for use by oral administration for prophylactic or therapeutic treatment of swine dysentery. It is not nitrogenic. 
     A swine feed-stock for oral administration of D-Compound according to this invention can be readily prepared by intimately admixing the D-Compound alone or as a premix with a conventional swine feed composition to provide a homogeneous feed product. 
     The term feed-stock means any food provided for the swine. Preferably the D-Compound is thoroughly mixed with the feed-stock so that it is uniformly dispersed throughout. However, it may also be sprinkled on the daily food supplies in the form of a powder or as pellets. Thus, the invention is not limited to any particular mode of administration. 
    
    
     The following Example illustrates the invention. 
     EXAMPLE 
     The D-Compound was tested against five strains of Vibrio cholerae at concentrations of 10, 30 and 100 micrograms per milliliter. The results are given in Table 1 below. 
     Tests were also run to see if the compound was effective against Vibrio cholerae El Tor Ogawa 6 in the presence of sewage. Sewage samples were obtained from the sewer system of the city of Modena, Italy. They were centrifuged to separate solids and the supernatant liquid was used in the tests. The results are given in Table 2. 
     At 10 mcg/ml of D, there was no growth of 3 of the organisms after 48 hours, and only marginal growth of the remaining two at 100 mcg/ml. 
     D was tested in vitro against Treponema hyodysenteriae by a known method. The minimum inhibitory concentration (the lowest concentration of compound in a dilution series where growth is inhibited) was 0.1 mcg/ml. The minimum bactericidal concentration (the lowest concentration of compound in which no viable treponemes are observed upon dilution and subculture from the broth onto blood agar plates) was greater than 0.1 mcg/ml but less than 1 mcg/ml. 
     The compound was tested for acute toxicity by several modes of administration in four species, namely mice, rat, guinea pig, and rabbit. The compound was found to be of a low order of toxicity. The results are given below in Tables 3, 4, 5 and 6. 
     In view of the favourable acute toxicity data, the compound was administered orally in sub-acute, but relatively large, doses to mice and rats for 15 days. Data were collected on the effects on death rate, weight, liver, and kidneys. The data are given in Tables 7 and 8. 
     In view of the favourable results on chronic toxicity, a teratogenic study was conducted with male and female mice and rats. The number of young delivered live at birth was comparable with controls. No malformations in either group were observed. The data are given in Table 10. 
     
                       TABLE 1______________________________________Con-       Effect on Various Strains of Vibrio Cholerae centra-  Classical                   Classical                          El Tor                                El Tor                                      El TorCom-  tion     Inaba    Ogawa  Ogawa Ogawa Inabapound μg/ml 35       41     6     8     4______________________________________D     100      -        -      ±  -     ± 30       -        -      -     -     ++ 10       -        -      ++    -     ++______________________________________ -  No growth after 48 hours at 37° C. ±  Just noticeable growth +  Evident growth but to a smaller extent than in untreated control experiments ++  Same degree of growth as in untreated control experiments. 
    
     
                       TABLE 2______________________________________     Concentration              Effect AfterSample      of D       24 hours 48 hours                                  5 days______________________________________Control + Vibrion       --         +++      +++    +++Sewage      --         ---      ---    ---Sewage + Vibrion       --         +++      +++    +++Sewage + Vibrion        5γ/ml                  ---      ---    ---Sewage + Vibrion       10γ/ml                  ---      ---    ---Sewage + Vibrion       20γ/ml                  ---      ---    ---Sewage + Vibrion       30γ/ml                  ---      ---    ---______________________________________ 
    
     
                       TABLE 3______________________________________ Acute Toxicity of D in female MiceDosage   Dead/Treated Animals aftermg/kg    1 day     2 days    4 days  7 days______________________________________  Endoperitoneal Administration2000     6/6                         6/61000               6/6               6/6 500                                 6/12 250                                 0/18  Esophageal Administration0 (x)                              0/64000                     1/12      1/122000                               0/121000                               0/12______________________________________ (x) By gastric lavage and receiving only the vehicle. 
    
     
                                           TABLE 4__________________________________________________________________________ Acute Toxicity of D in the Rat__________________________________________________________________________a.  First Experiment   Route of    Dead/Treated                  Body weight                         (m ± SEM)                                StatisticalSex   Administration      mg/kg          within 21 days                  in g. start                         Termination                                Significance(.sup..)__________________________________________________________________________M  Esophageal      4000          0/4     234.5 ± 13.8                         288.7 ± 13.8                                t 0.05M  Esophageal      0 (x)          1/4     233.7 ± 3.7                         331.0 ± 0.5F  Esophageal      4000          0/4     201.2 ± 4.2                         238.2 ± 12.1                                t 0.05F  Esophageal      0 (x)          1/4     189.2 ± 3.9                         230.0 ± 10.5M  Endoperitoneal       500          1/4     234.0 ± 6.2                         314.3 ± 10.3                                t 0.05M  Endoperitoneal      0 (x)          0/4     230.0 ± 5.7                         324.0 ± 8.7F  Endoperitoneal       500          2/4     206.2 ± 8.7                         286.0 - 272.0                                t 0.05F  Endoperitoneal      0 (x)          0/4     207.5 ± 4.3                         253.5 ± 7.7__________________________________________________________________________ (x) Only the vehicle was administered by the same route. (.sup..) Student&#39;s t test.b.  Second ExperimentRoute of       Dead/Treated                        Body weight                                (±SE)Sex  Administration         mg/kg within 7 days                        in g. start                                Termination__________________________________________________________________________M    Esophageal         4000  0/4      222.5 ± 6.2                                231.7 ± 15.7F    Esophageal         4000  0/4      252.0 ± 16.6                                253.5 ± 12.1M    Intraperitoneal          500  2/4      226.2 ± 6.8                                225.0 - 212F    Intraperitoneal          500  0/4      232.5 ± 5.9                                218.2 ± 7.0__________________________________________________________________________c.  Cumulative Data Regardless of Animal SexRoute ofAdministration         mg/kg Dead/Treated within 7 days__________________________________________________________________________Esophageal    0 (x) 0/8Esophageal    4000  0/16Intraperitoneal         0 (x) 0/8Intraperitoneal          500  4/16__________________________________________________________________________ (x) Only the vehicle was administered. 
    
     
                       TABLE 5______________________________________Acute Toxicity of D in the Guinea PigBy Esophageal AdministrationDosagemg/kg        Dead/Treated within 21 days______________________________________ 500         0/41000         1/42000         5/64000         6/60(x)          0/13______________________________________ (x) Only the vehicle was administered. 
    
     
                       TABLE 6______________________________________Acute Toxicity of D in the RabbitBy Esophageal AdministrationDosage  Dead/Treated Body Weight (m ± SE)mg/kg   within 7 days                in g. start Termination______________________________________2000      0/2.sup.(.)                2250 - 2150 2180 - 21401000    0/4          2037 ± 104.3                            1922.5 ± 71.50(x)    0/4          2135 ± 75                            2262 ± 215 500    0/2          2000 - 2100 1650 - 1550______________________________________ (x) Only the vehicle was administered. (.sup..) There were two dead out of seven treated animals, within 4 days. 
    
     
                       TABLE 7______________________________________Subacute Toxicity of D in the MouseDaily Dose: 500 mg. CO-1 by gastric lavage for 15 days______________________________________          % Body          Weight     Fresh Organ-to-Body   Dead/  Change     Weight RatioOral Treatment     Treated  (m + SE)   Liver   Kidneys______________________________________Vehicle   0/10     20.4 ± 4.2                         5.2 ± 0.2                                 1.4 ± 0.1CO-1,     0/10     -8.1 ± 3.9                         5.9 ± 0.3                                 1.5 ± 0.1500 mg/kg/day______________________________________a. Mortality and Body WeightDaily Dose: 1 g/kg/day for 15 days               Dead/    % BodyOral Treatment      Treated  Weight Change______________________________________Vehicle (H.sub.2 O) 0/12     24.54 ± 0.64CO-1 in H.sub.2 O, 1 g/kg/day               2/12     18.5 ± 0.75Vehicle (adraganth gum) (x)               0/12     25.04 ± 1.18CO-1 in adraganth gum               3/12     16.27 ± 1.31______________________________________b. SGOT and SGPT (24 hrs. after last dose)                        Units/mlOral Treatment      SGOT     SGPT______________________________________Vehicle:Water               116      4Adraganth gum       119      6CO-1 in water       124      9CO-1 in adraganth gum               132      10______________________________________ 
    
     
                       TABLE 8______________________________________Subacute Toxicity of D in Female Rats______________________________________Daily Dose: 22 g/kg/day of D by gastric lavage for 21 days            Body Weight in g (m ± SE)Oral Treatment      Dead/Treated                  Start      Termination______________________________________Vehicle    2/6(x)      200.0 ± 4.1                             233.2 .sub.5/8 5.1D, 2 g/kg/day      1/6(x)      204.1 ± 2.0                             210.6 ± 9.6______________________________________ (x) Death caused by a mistake in esophagus incannalutation. This diagnosi was confirmed at the postmortem examination. 
    
     
         Daily Dose: 2 g/kg/day of D by gastric lavage for 21 daysOral    Average Percent Weight of Fresh Organs (m + SE)Treatment   Lung        Liver        Kidneys______________________________________Vehicle 0.85 ± 0.06               3.45 ± 0.07                            0.95 ± 0.04(3 animals)D,      1.07 ± 0.09NS               4.54 ± 0.10NS(x)                            1.04 ± 0.03NS(5 animals)______________________________________ (x) Death caused by a mistake in esophagus incannalutation. This diagnosi was confirmed at the post mortem examination. 
    
     In view of the favourable sub-acute toxicity, the chronic toxicity in female mice was studied. The results are given in Table 9. 
     
                       TABLE 9______________________________________Chronic Toxicity in the Female MouseDaily treatment by gastric lavage for18 weeks (4.5 months)______________________________________a. Mortality and Body Weight      Dead/   Body Weight in g(m ± SE)Oral Treatment        Treated   Start      Termination______________________________________Vehicle      3/10      28.2 ± 1                             33.0 ± 1.1D, 500 mg/kg/day        2/10      30.4 ± 0.9                             30.0 ± 0.7D, 250 mg/kg/day        0/10      27.3 ± 0.5                             26.7 ± 0.7______________________________________b. Urine excretion.Urine amount excreted by 6 animals in 6 hoursOral Treatment  Urine Amount (ml)______________________________________Controls        6D, 500 mg/kg/day           7D, 250 mg/kg/day           6.5______________________________________c. Blood glucose. Mean values for 6 animals. Bloodsamples were taken 24 hours after the last doseOral treatment   Blood Glucose______________________________________Controls         1.14D, 500 mg/kg/day 1.06D, 250 mg/kg/day 1.10______________________________________d. SGPT and SGOT. Mean values for 6 animals. Blood sampleswere taken 24 hours after the last dose            Units/mlOral Treatment     SGOT    SGPT______________________________________Controls           125     5D, 500 mg/kg/day   159     6D, 250 mg/kg/day   118     5______________________________________Chronic Toxicity of D in the Female Mousee. Fresh Weights of OrgansOral  Fresh Organ-to-Body-Weight RatioTreat- (m ± SE, 4 animals)ment  Kidneys    Heart      Liver   Lungs______________________________________Con-  0.938 ± 0.044            0.481 ± 0.055                       4.57 ± 0.15                               0.674 ± 0.044trolsD, 500mg/   1.07 ± 0.04            0.47 ± 0.02                       4.66 ± 0.91                               1.011 ± 0.110kg/dayD, 250mg/   0.87 ± 0.08            0.60 ± 0.08                       4.57 ± 0.25                               0.731 ± 0.035kg/day______________________________________ 
    
     
                                           TABLE 10__________________________________________________________________________ Teratogenetic study__________________________________________________________________________a. Animal Species: Mouse, Male and female mice housed together for 10days.Oral treatment from 3rd day to 13th days.     Pregnant/            No. of living                      Body Weight                             No. of     Treated           Foetuses per                     of Foetuses                             Foetuses withOral treatment     Animals           Delivery (m ± SE)                     in g (m ± SE)                             malformations__________________________________________________________________________D, 250 mg/kg/day     3/10(x)           10.3 ± 0.6                     1.42 ± 0.05                             0Controls  9/10   9.0 ± 0.9                     1.46 ± 0.07                             0__________________________________________________________________________ (x) On the basis of our wide experience, the above result might be casual The study should be repeated to determine whether CO1 actually prevents pregnancy. 
    
     
         b. Animal Species. Rat. Same experimental conditions as with the mouse.     Pregnant/           No. of Living                     Body Weight                             No. of     Treated           Foetuses per                     of Foetuses                             Foetuses withOral Treatment     Animals           Delivery (m ± SE)                     in g (m ± SE)                             malformations__________________________________________________________________________D, 250 mg/kg/day     7/10  10.8 ± 0.86                     7.08 ± 0.19                             0Controls  6/10  11.3 ± 1.12                     6.82 ± 0.40                             0__________________________________________________________________________