TOXICITY AND THERAPEUTIC EFFECTS OF
SULPHAPYRIDINE IN ANIMALS*
M. W. JOHANNSEN, AND A. V. ST. GEORGE
From the Department of Pathology, Bellevue Hospital
* Received for publication April 10, 1939.
The following studies were made with the idea of obtaining
(a) information on the toxicity of sulphapyridine ("2-(paraaminobenzenesulphonamido) pyridine"—Whitby, "Dagenan"—
Merck) on mice, (b) its therapeutic effect on type three and six
pneumococcus peritonitis in rats. Sulphapyridine according to
Whitby1 has the following formula:
NH2<
>S02—NH
V
TOXICITY EXPERIMENTS WITH SULPHAPYRIDINE IN WHITE MICE
To determine the toxicity, four groups of white mice, each group consisting
of four animals and each animal weighing approximately 25 grams, were fed
every day with the average therapeutic dose in group A; ten times the therapeutic dose in group B; 50 times in group C, and 100 times in group D. By
assuming that 4 grams of sulphapyridine is the average maximum therapeutic
dose for a human weighing 75 kilograms, we arrived at the therapeutic dose for
mice, as follows: One gram of body weight would receive 4 divided by 75,000
grams of sulphapyridine. As the average mouse weighs 25 grams the therapeutic dose for a mouse would be 100 divided by 75,000 grams, or 1.4 milligrams.
A suspension of 1 gram of sulphapyridine in 50 cc. of distilled water was made
and thoroughly shaken. 7/100 cc. of this suspension contained 1.4 milligrams,
or a therapeutic dose. This amount was administered orally every day to each
of four mice over a period of fourteen days by means of a calibrated pipette. In
a similar manner four mice in group B were treated. Two tablets of sulphapyridine equal to 1000 milligrams were dissolved in 5 cc. of distilled water thus
obtaining 14 milligrams in 7/100 cc. or ten times the therapeutic dose. In group
C, each of four mice received 50 times the therapeutic dose over a period of fourteen days. Two thousand milligrams of sulphapyridine were suspended in 10
414
TOXICITY OF STJLPHAPYRIDINE
415
cc. of distilled water so that 0.35 cc. contained 70 milligrams, i.e., 50 times the
therapeutic dose.
In group A, B, and C, the administration of the drug was simple because the
suspension was not too concentrated and the quantity to be given not too large.
None of the twelve mice thus treated died or appeared to be sick, judging by
their appearance and behavior. Two of each group were sacrificed by means of
chloroform, the remaining six were alive eight weeks after the last dose was
administered.
In group D we tried to administer 140 milligrams per mouse per day. Two
thousand milligrams of sulphapyridine were suspended in 5 cc. of distilled water
so that 0.35 cc. of the suspension contained 140 milligrams.The suspension was
concentrated, nevertheless during the first day all four animals, which had not
received anyfluidfor the twelve hours preceding the administration of the drug
took the entire amount, but we had to give small feedings every three hours.
During the next few days we were not able to force the animals to take the entire
amount; we succeeded, however in giving them not less than sixty times the
therapeutic dose and frequently close to one hundred times per day. One of the
animals expired on the third day after having received three hundred ten times
the therapeutic dose. The animal choked to death in an attempt to feed the
drug. The other three survived for seven days of treatment, no. 2 having received a total of 680 milligrams which is approximately 68 times the therapeutic
dose. No. 3 received 760 milligrams or 76 times the therapeutic dose, animal
4 a total of 720 milligrams or 71 times the therapeutic dose. Animal 2 was
sacrificed by chloroform. The remaining two were alive eight weeks after discontinuance of the drug, and showed no ill effects. None of the three animals
treated showed any signs of toxicity as judged by their behavior and appearance
during the seven days in which the drug was administered.
In group E, three animals were treated with a lethal dose which, according
to Whitby, is 16.6 milligrams per gram or 415 milligrams per mouse. We dissolved 2000 milligrams in 5 cc. of distilled water and tried to give each animal
1 cc. of the suspension. One animal had to be omitted as it would not take any
fluid when held. Animal 1 received a total of 520 milligrams in three days, then
expired. Animal 2 received 600 milligrams in two days and expired. Both
animals appeared drowsy, the fur was rough, and they were unable to keep their
balance. Both these animals were subjected to necropsy.
The six animals in group A, B, and C, the two animals in group D, and the
two animals in group E, showed no naked eye changes at necropsy. Lungs,
heart, liver, kidney, spleen showed no histological changes. Table 1 summarizes
the results.
Effects of sulphapyridine on type three pneumococcus peritonitis in rats
Twelve white rats, each weighing approximately 125 grams, were injected
intraperitoneal^ with a 24 hour broth culture of type three pneumococci. The
pneumococci were obtained from a patient suffering from a severe type three
416
M. W. JOHANNSEN AND A. V. ST. GEORGE
pneumococcus pneumonia. Temperatures were taken every three hours during
the day. When the temperature, taken rectally, reached 102°F. (approximately
24 hours after injection), treatment was instituted, giving to nine of the animals
7 milligrams of sulphapyridine every three hours for three doses per day. We
arrived at 7 milligrams of sulphapyridine as the therapeutic dose for a 125 gram
rat by assuming again that 4 grams is the therapeutic dose for a human weighing
75 kilograms. The drug was given orally in an aqueous suspension and measured with a calibrated pipette. Three animals were not treated. All died thirty
hours after the injection. Necropsy revealed purulent peritonitis. Peritoneal
and heart cultures showed a profuse growth of pure type three pneumococci
which gave specific type swelling of the capsule. Of the treated group, two
TABLE 1
1
1.4
A. 4 animals with
therapeutic
dose
B. 4 animals with 14
10 times the
therapeutic
dose
C. 4 animals with 70
50 times the
therapeutic
dose
D. 4 animals with 140
100 times the
therapeutic
dose
E. 3 animals with 40
220
lethal dose
200
1TXOX
OROOT
2
3
4
s
6
7
8
9
10
11
12
13
14
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4
1.4 19.6
14
14
14
14
14
14
14
14
14
14
14
14
14
196
70
70
70
70
70
70
70
70
70
70
70
70
70
980
90 80
*
80 70
70
80
80 90
60 140
90 80 140 80
Omitted— would not
100 J200
* 1
400 J *
140 100
140 110
100 80
take dose
1
1
310
680
760
710
40
620
600
* Died.
animals were sacrificed and subjected to necropsy two days following the inauguration of treatment. Their temperatures had fallen from 102.8 and 103
respectively to a normal level after four doses of sulphapyridine. Necropsy
revealed a mild peritonitis and a few organisms in the heart's blood and peritoneal cultures, both giving specific type capsule swelling.
On the third day after the commencement of the drug, two animals died.
Their temperatures had remained high, i.e., above 102. Necropsy revealed
purulent peritonitis with fecal material in the peritoneal cavity. Examination
showed perforation of the intestines by the injecting needle. Culture of heart's
blood and peritoneal contents revealed many type three pneumococci and
B. Coli. Some of the pneumococci gave specific type swelling while others
showed degeneration of the capsule. The temperatures of the remaining five
TOXICITY OF SULPHAPYRIDINE
417
animals had reached a constant normal level after the fourth or fifth dose of
sulphapyridine. On the fifth day treatment was stopped and two more animals
were sacrificed the same day. The peritoneum showed no naked eye changes
and culture of the heart's blood and of the peritoneal cavity showed only a few
pneumococci in culture. The heart culture was sterile. The pneumococci no
longer gave specific swelling of the capsule. The remaining three animals
were sacrificed on the eighth day. Heart and peritoneal cultures were sterile.
Throughout the experiments three control animals showed a temperature
ranging between 97 and 98.2.
SUMMARY GROUP I
1. Twelve rats were injected intraperitoneally with 1 cc. of a
24-hour broth culture of type three pneumococci obtained from
a blood culture of a patient.
2. All twelve animals developed fever ten hours after injection.
3. Nine animals were treated with 7 milligrams of sulphapyridine every three hours for three doses per day after the temperature had passed 102.
4. Three animals were untreated. All died approximately
30 hours after injection.
5. In the treated group two animals died. Necropsy performed immediately after death showed secondary invasion by
B. Coli in the peritoneum and heart's blood due to trauma of
the intestines.
6. The remaining animals were sacrificed at different intervals
after instituting treatment. Cultures revealed that the decrease
in the degree of peritonitis and bacteremia until cure was directly
proportionate to the length of time the animal was treated.
Table 2 summarizes the results.
Effects of sulphapyridine on type six pneumococcus peritonitis
The investigation was carried out in identical circumstances as described in
the experiment with type three pneumococci. In this instance 1 cc. of a 24
hour broth culture of type six pneumococci obtained from a patient dead of
pneumococcus meningitis, was injected intraperitoneally. Twelve white rats
each weighing 125 grams were injected. All developed fever approximately 10
hours after injection. Nine animals were treated with 7 milligrams of sulphapyridine orally every three hours after the temperature had passed 102 degrees
(approximately 24 hours after injection of the pneumococci). Three were
untreated. All three died approximately 30 hours after injection. Necropsy
103
X
102«
X
98« 10H 102'
103'
98
98'
98'
98= 10O» 102'
98
98!
98<
98« 100< 103
97
97<
97
97<
97'
98
97
97=
97»
IV
V
VI
VII
VIII
IX
X
XI
XII
xt
xt
xt
103
xt
102'
xt
xt
102'
4 | 10
103'
X
103«
X
102'
X
103
X
102«
X
102»
X
102«
X
101
X
100'
X
101'
X
102»
X
101*
X
101»
X
102
X
103
X
100«
X
100«
X
100
X
100»
X
98'
X
98'
X
99'
X
99s
X
98'
X
103' 103
X
X
100'
X
97'
X
97«
X
97«
X
971
X
97<
X
97'
X
97'
X
103'
X
102' 103'
X
X
100'
X
98
X
97«
X
97
X
97
X
97
X
97
X
104'
X
103'
X
98
X
98'
X
97'
X
97
X
97'
X
104'
X
97'
X
97«
X
97
X
97<
X
98'
X
Animal sacrificed
at 10 a.m.
Animal sacrificed
at 10 a.m.
Animal died 294 hours after the
injection
101' 102
X
X
102
X
101'
X
104' 1
Died at
3:30 p.m.
7TH DAT
8TH DAT
1 | 4 | 10 | 1 | 4 | 10 | 1 | 4 |
6TH DAT
97« 97
97» 98
97i 97' 97'
All 3 animals sacrificed.
Necropsy
revealed no gross
pathology. Heart
97' 97' 97' 97' 97» 97 97 97i 97' 97i 9711 97'
and peritoneal cultures sterile
971 97' 98 97" 98 97< 97« 97' 97' 97' 97« 97«
97i 97' 97< 97' 97
Both animals sacrificed. Necropsy revealed no gross pathology. Culture
of heart's blood and peritoneal cavity show only a few organisms in the
peritoneal culture, pneumococci giving no longer type specific swelling.
Heart culture was sterile
Animals* expired. Necropsy revealed a purulent foul-smelling peritonitis
with fecal material in the peritoneal cavity. Culture of heart's blood and
peritoneal contents reveal many pneumococci and B. coli, some of the pneumococci still gave type specific swelling, while others show degeneration of
the capsule
Necropsy revealed a mild peritonitis, and a few organisms in the heart's blood
and peritoneal cultures, both giving type specific capsule swelling
Culture of heart's blood and peritoneum showed a profuse growth of pneumococci, giving excellent type
specific swelling
4 10
10 | 1
* 1 *Necropsy
revealed purulent peritonitis.
Hour
5TH DAT
Temperature taken on 3 control animals ranged between 97 and 98'.
* All animals given 1 cc. of a 24 hour broth culture of type I I I Pneumococci intraperitoneally at 10 a.m. the first day.
t The animals received at this time 7 mgm. of an aqueous solution of sulphapyridine orally.
xt
98» 100' 103'
xt
xt
99s 102'
100*
99« 102'
101
101'
101'
1
4TH DAT
Animal died 30 hours after the
i njection
10
3RD DAT
103' 1 104« Animal died 31 hours after the
injection
Died at
5 p.m.
98
102«
III
100«
98'
4
103*
Die<l a t
4 p m.
1
97
10
102'
II
4
101«
98'
1
! » D DAT
97«
10»
1ST D A T
I
MUMBER
ANI-
TABLE 2
TEMPERATUBBS IN DEGREES FAHRENHEIT
a
O
H
O
CO
O
>
H
>
m
O
a
g
103'
X
103'
X
98> 101' 102»
99
99< 101
99<
998 101
101' 103
99
98* 101' 102«
100' 102*
99*
99'
99'
97»
98
97«
97»
97'
97«
97'
97«
97«
98
III
IV
V
VI
VII
VIII
IX
X
XI
XII
103'
X
103
X
103'
X
103
X
102«
X
103
X
102'
X
101'
104
102'
X
103
X
102
X
101>
X
101'
X
101«
X
102«
X
103'
X
102
X
104'
4
1
4
10
1
4TH DAT
4
103'
X
92>
X
X
100>
X
98'
X
98'
X
98<
X
98'
X
98'
X
X
100« lOO*
100'
X
100
X
99
X
99
X
100
X
101' 98"
X
X
103
X
98
X
98'
X
98
X
97«
X
97'
X
97'
X
97'
X
97'
X
103«
X
97
X
97'
X
98'
X
97
X
97'
X
97'
X
97'
X
99'
X
97*
X
97'
X
97"
X
98
X
98
X
96»
X
97»
X
97»
X
97'
X
97'
X
Animal died appro*. 30 hrs.
after If was
begun
Animal died 28} hours after the
injection
Animal died 28 hours after the
injection
Animal died 31 hours after the
injection
10
3 R D DAT
1 14
10
1
7TH D A T
1 | 4 |
8TH D A T
4 1 10 | 1 1 4 10 |
&TH DAT
98'
97'
97'
97' 97'
98 97* 97*
97» 97» 97» 98
97« 97« 97» 97» 97«
98' 97' 97« 98
97« 97» 98
98
98
path. Culture of
heart's blood and
peritoneum sterile
Both animals sacrificed.
Necropsy
98' 97' 97» Both animals sacrificed. Necropsy revealed no
gross pathology. Culture of heart's blood and
peritoneum Bterile
97' 97* 97 97'
97" 9 7« 98
Both animals sacrificed. Necropsy revealed no gross pathology. Culture
of heart's blood and peritoneum showed very few organisms in the blood;
peritoneal culture sterile. Pneumococci giving no longer type specific
swelling
Both animals sacrificed. Necropsy revealed a slight peritonitis. Culture of
the heart's blood and peritoneal contents revealed a few pneumococci, many
giving specific swelling of the capsule, while others showed degeneration
of the capsule
Necropsy revealed a purulent foul-smelling peritonitis. Culture of heart's
blood and peritoneal contents showed many pneumococci—some of which
showed degeneration of capsule, while others showed good swelling—and
B. coli
Necropsy revealed purulent peritonitis. Culture of heart's blood and peritoneal contents snowed a profuse growth of pneumococci, giving excellent
type specific swelling of the capsule
10
Hour
5TH D A T
Temperature taken on 3 control animals ranged between 97 and 98s.
* All animals given 1 cc. of a 24 hour broth culture of type VI Pneumococci intraperitoneaUy at 10 a.m. the first day.
t The animals received at this time 7 mgm. of an aqueous solution of sulphapyridine orally.
Xt
Xt
xt
102«
Xt
101' 102«
xt
103
xt
101' 102»
Xt
Xt
100» 102
xt
102«
101' 102»
99
97'
II
101
102'
99»
97'
97
I
103'
10
4
1
2ND DAT
1
1ST DAT
10*
MAD
KVH-
TABLE 3
TEMPERATURES IN DEGREES FAHRENHEIT
CO
h-i
H
h-t
a
>
d
CD
o
Hi
©
420
M. "W. JOHANNSEN AND A. V. ST. GEOKGE
revealed purulent peritonitis. Culture of the heart's blood and peritoneal
contents showed a profuse growth of pneumococci giving specific type swelling
of the capsule. One of the treated group died approximately 30 hours after the
administration of the first dose of sulphapyridine. Necropsy showed injury
of the intestine caused by the injecting needle and peritonitis. Culture of heart's
blood and peritoneal contents showed many B. Coli and pneumococci. Some of
the pneumococci gave good swelling of the capsule with type six serum while
others showed degenerative changes in the capsule. The remaining eight
treated animals showed a drop in temperature to normal after the fourth or fifth
dose of sulphapyridine. They were sacrificed at different intervals after inauguration of treatment and again showed that the decrease in the number of
pneumococci in the peritoneum and heart's blood, until cure, was directly proportional to the length of time the animal was treated. Table 3 demonstrates
the exact results.
CONCLUSIONS
1. We believe the drug to be comparatively harmless and nontoxic in large doses in mice.
2. We believe the drug has definite curative properties in experimentally induced pneumococcus peritonitis types three and
six in rats, when administered sufficiently early and in adequate
amounts.
REFERENCE
(1) WHITBY, LIONEL E. H.: Lancet, p. 1210, 1938, May 28.