Studies of the Comparative Toxicity, Absorption and Elimination of Sulfacetimide and Sulfanilamide

STUDIES OF THE COMPARATIVE TOXICITY, ABSORPTION AND ELIMINATION OF SULFACETIMIDE AND SULFANILAMIDE R

s. FISHER AND H. B. HAAG

From the Department of Pharmacology, Medical College of Virginia, Richmond

Although many reports have appeared from abroad within the last several years bearing on the clinical usefulness of sulfacetimide, the article by Young, Hill, Jewett, and Satterthwaite is the first description of its therapeutic employment in urology to originate in this country. Practically no observations have been reported on its basic pharmacology. The present studies, dealing with the comparative toxicity, absorption and elimination of sulfacetimide1 and sulfanilamide for various laboratory animals, were done with the hope of meeting, at least in part, this deficiency. ACUTE TOXICITY

The acute toxicity of sulfacetimide for mice, rabbits, and dogs was determined with sulfanilamide as the reference control. The mice were albino males weighing from 16-22 gm. The rabbits were of an albino stock, of mixed sexes, and weighed from 1.2 to 2.5 kg. The dogs were male and female mongrels and of widely divergent weights. The mice and dogs were maintained on Purina dog chow; the rabbits on Purina rabbit chow. Administration of the drugs was by stomach tube. In the case of mice and rabbits, the drug was suspended in a mucilage of acacia, whereas all doses given to dogs were suspended in water. For mice, suspensions up to 40 per cent were used when necessary, in order that the total volume per 20 gm. mouse should not exceed 1 cc. In the case of both mice and rabbits, each lot of animals purchased was divided into 2 groups which usually were dosed on the same day; one group receiving sulfacetimide and the other receiving sulfanilamide. All mice were starved 6 hours before being dosed, rabbits 48 hours, and dogs 24 hours. Food was allowed ad libitum two to three hours after administration of the drugs. Water was always allowed freely. Four days was selected as the maximum time limit for observations on mice so as to conform with the experiments of Marshall, Cutting and Emerson. Rabbits and dogs were kept under observation for a period of about a week after administration of either drug. Mice. The L.D. 502 of sulfacetimide for mice by oral administration, using 60 to 80 mice at the critical dose levels, was found to be approximately 16.5 gm. per kg. body weight. Under similar conditions the L.D. 50 of sulfanilamide for mice was found to be 3.7 gm. per kg. body weight, using 20 mice for each level. This figure for sulfanilamide is in close agreement with that found by Marshall and his co-workers. 1 The sulfacetimide (Sulamyd) (p-sulfanil-acetyl-imide) was supplied by the Schering Corporation, Bloomfield, N. J. 2 The dose which kills 50 per cent of the animals.

183

184

R. S. FISHER AND H. B. HAAG

Rabbits. As is illustrated in table 1, the acute toxicity of the 2 drugs for rabbits by oral administration seemed approximately to parallel, but at lower dosage levels, that found in mice. The findings with sulfanilamide are in agreement with those reported by Molitor and Robinson. TABLE

1.-Acute oral toxicity of sulfacetimide and sulfanilamide for rabbits

DOSE

FATALITIES

NUMEER OF RABBITS

I

NUMBER WHICH SHOWED SYMPTOMS OF POISONING*

Sulfacetimide gm. per kg.

per cent

5 10 15

10 9 3

10 33 100

4 6 3

10 90

4 10

Sulfanilamide 2 4

10 10

* The symptoms of poisoning included: diarrhea, loss of righting reaction, dyspnea, marked extensor rigidity, loss of corneal reflexes, and finally, complete paralysis. TABLE

DOSE

2.-Acute oral toxicity of sulfacetimide and sulfanilamide for dogs DEGREE OF INTOXICATION*

NUMBER OF DOGS

FATALITIES

0

+

++

+++

I

Sulfacetimide gm. per kg.

5 8 10 12 15 25

per cent

1 8 2 2 2 2

1 2 1

1

1

5 1 2 1 2

0 50 50 100 100 100

Sulfanilami de 1.6 2.0 3.0 5.0

5 8 2 2

2

3 8 2 2

0 50 100 100

* Gradation of degree of intoxication: 0 = no signs of poisoning. + = mild nausea of short duration; mild excitement. ++ = violent retching; vomiting (occasionally); hyperpnea; muscular incoordination, but not complete loss of ability to stand; intense excitement; whining. +++ = complete loss of ability to stand; loss of righting reaction to ,a greater or less degree; marked extensor rigidity resembling decerebrate rigidity; opisthotonus; paraplegia; blindness; shivering; loss of the placing reaction; convulsive running movements; intense spastic paralysis and in some cases complete coma and death.

Dogs. Because large amounts of either drug frequently caused emesis, the dogs in these experiments were premedicated with 10 mg. per kg. of morphine sulfate intramuscularly some 30 minutes prior to administration of the sulfacetimide or sulfanilamide. Table 2 with its footnote gives the results of these

STUDIES OF SULFACETIMIDE AND SULFANILAMIDE

185

experiments. From these tests it appears that sulfacetimide is about one-fourth as acutely toxic for dogs as is sulfanilamide. These findings are in keeping with the results obtained with mice. Necropsy examinations were done on two mice which died of single doses of 10.0 gm. per kg. of sulfacetimide. These showed no changes from normal except a definite and moderate inflammatory hepatitis. No degenerative changes were found in the liver or kidneys. Examination of tissues from 2 mice which died of single doses of 15.0 gm. per kg. of sulfacetimide showed slight vacuolic and toxic changes of the liver cells but no degenerative changes in the kidneys. The vacuolations apparently indicate some slight tmd.c effect of the larger (15 gm.) dose. On necropsy examination of three rabbits which died after receiving sulfacetimide in amounts of 5, 10, and 15 gm. per kg. and which died on the fifth, seventh and first day respectively, marked degenerative changes probably due to the toxic effects of the drug, were found in the kidney and liver of the first animal. More moderate changes were found in the second animal. No abnormalities whatsoever were found in the animal receiving the largest dose. This might be accounted for by the fact that death occurred within 1 day so that not sufficient time elapsed to allow the development of demonstrable degenerative lesions. Two rabbits which had received sulfanilamide-2.0 and 4.0 gm. per kg.-died in 18 hours and 4 days respectively. The former showed moderate inflammatory infiltrations of the connective tissue of the liver and considerable vacuolic degeneration of the tubule cells of the kidney. In the animal living 4 days, the only finding of pathologic significance was an acute inflammatory myocardial infiltration of nonspecific character and of unexplained origin. With the exception of 2 mice (see below), the following organs were studied in these mice and rabbits: heart, lungs, intestines, liver, spleen, kidneys and adrenals. In the case of 1 mouse dying after 10 gm. per kg. and 1 which died after 15 gm. per kg. of sulfacetimide the heart was not examined. CHRONIC TOXICITY

Rats. Four groups, each consisting of six, 28 day old, male white rats weighing about 50 gm., were selected for feeding experiments which lasted for 75 days. The weights of the animals were recorded at 5-day intervals. The basic diet for all groups consisted of finely ground Purina dog chow to which was added either sulfanilamide or sulfacetimide, except for the control group. Group I received the basic diet only. Group II received the basic diet plus 0.75 per cent sulfanilamide. In the 75-day period each of these tats consumed an average daily amount of food calculated to contain 89.3 mg. of sulfanilamide. Group III received the basic diet plus 0. 75 per cent sulfacetimide. The average daily intake of the drug was 88.5 mg. per rat. Group IV received a diet containing 1.5 per cent sulfacetimide. The average daily consumption of the drug was 174.2 mg. for each animal. Incidentally, this latter figure corresponds to a total intake during the 75-day period of 13.1 gm. of sulfacetimide per rat. The results of the experiments with sulfanila,mide are in general keeping with those of Marshall

R. s.

186

FISHER AND H. B . HAAG

et al. The average growth rates are shown in figure 1. Application of statistical methods to the data on animal weights at the termination of the experiment yielded a "t" value indicating a significant retardation in the growth rate of the sulfanilamide group as compared to the control group. During the course of the experiment there were 2 fatalities; one in the control group and one in the sulfanilamide group; because of extensive tissue destruction no necropsy examinations were made on these animals. At the end of the test period blood smears from each surviving rat were examined. Those from the control group and both groups receiving sulfacetimide showed no abnormalities. Those receiving sulfanilamide showed an increase in 175-

150

125

- - Sulfttcetwude 0.7SZ

50

-

/0

20

40

50

Suzfacetimide t.5 %

60

70

Bo lxlys

FIG. 1. Effect of sulfacetimide and sulfanilamide containing diets on growth rate of

white rats.

polychromatophilic erythrocytes but otherwise app~ared normal. It is interesting to note that one of these animals had a massive infection with Trypanosoma Lewisi in spite of an estimated daily ingestion of approximately 0.5 gm. per kg. of sulfanilamide. For necropsy examination, the animals were killed by rapid decapitation. No gross or microscopical changes which could be related to the experimental procedures were observed in the liver, stomach, intestines, spleen or kidneys of any of the animals. Dogs. Dogs were given daily doses of the drugs, and the red and white blood cell count, hemoglobin content of the blood, and the weight of the animal were recorded weekly. Urinalyses were done from time to time. The concentration of the drug in the blood was also determined weekly at varying intervals from the

STUDIES OF SULFACETIMIDE AND SULFANILAMIDE

187

time of drug administration so as to detect maximum fluctuation. It was established that the method of Marshall and his co-workers for sulfanilamide is satisfactory for determination of the concentration of sulfacetimide in the blood and urL.'le. The color produced on diazotization of sulfacetimide and coupling ·with a-napthyl-amine has a slight purplish character not seen in the color produced by sulfanilamide, but the error introduced by this is negligible if suitable aliquots of the para-toluene sulphonic acid filtrate are used to produce a depth of color in the unknown which is close to the standard. The animals used ,vere of the weight range 6.0 to 10.0 kg. They were maintained on a stock diet of Purina dog chow, and water ad libitum. In all cases the drugs were administered orally in gelatin capsules, the total daily dose being divided into 2 equal parts, one being given at 8 a.m. and the other at 6 p.m. The experiment lasted 11 weeks. Necropsies were done on all animals including those which died or had to be sacrificed during the experimental period. Gross as well as microscopic observation included examination of the liver, spleen, stomach, small intestine, kidneys, and adrenals. Three series of 2 dogs each were experimented on simultaneously. The first group was given 0.2 gm. per kg. of sulfanilamide, the second group 0.4 gm. per kg. sulfacetimide, and the last group 1.0 gm. per kg. sulfacetimide, daily. Because of an early death, an additional animal vvas added to this last series. a. Sulfanilamide: The first dog in this group, a male, showed an increase over its original weight of slightly more than 2 kg. at the end of the experimental period. No abnormalities, qualitative or quantitative, were noted in the red blood cells or the white blood cells. Likewise there were no alterations in the hemoglobin values. Urinalysis done once after 3 weeks and again at the termination of the experiment showed the urine to be entirely normal. Blood sulfanilamide levels ranged during the period from 4.3 mg. to 14.8 mg. per 100 cc. Gross and microscopic studies made when the animal was sacrificed revealed normal tissues, except for a few brownish spots noted macroscopically on the cortex of both kidneys. This dog had received a total of 146 gm. of sulfanilamide during the 11 week period. The second dog of this group, also a male and weighing initially 6.4 kg., had the same weight at the end of the experiment, and except for a period lasting from the thirteenth day of medication to the twenty-second day, showed no abnormalities in the several types of observations made as above. Beginning with the thirteenth day this dog showed signs of anorexia and general depression. The following day it seemed quite ill. The white blood cell count had risen from 13,100 per c. mm. to 31,850, with 96 per cent being of the polymorphonuclear neutrophilic type. The red blood cell count dropped from 5,400,000 to 3,800,000 per c. mm.; the hemoglobin percentage from 92 to 68. The blood sulfanilamide concentrations during this period varied from 9.3 to 18.2 mg. per 100 cc. The sclerae were markedly jaundiced. This condition persisted for several days, at the end of which time however, spontaneous recovery began and was complete within about a week. There had been no interruption in the administration of sulfanilamide. The blood content of sulfanilamide varied during

188

R, S, FISHER AND H, B, HAAG

the entire experiment from 5.0 mg. to 36.4 mg. per 100 cc. When sacrificed at the end of 11 weeks, the necropsy observations were identical with that of the first dog. During the 11 weeks this dog had received a total of 98.5 gm. of sulfanilamide. b. Sulfacetimide: Two dogs, a male of 7.0 kg. and a female of 8.1 kg., were given 0.4 gm. of sulfacetimide per kg. daily. Both gained in weight slightly during the experiment. No deviation from normal was noted in the red blood cell, white blood cell, or hemoglobin estimations. One week before the termination of the experiment the urine of the first animal showed albumin to the extent of 1 plus, and on microscopic examination per high power field, a few hyalin casts, about 8 granular casts, from 1 to 2 red blood cells, and from 2 to 3 pus cells were observed. Sulfacetimide was present in the urine to the extent of 1.68 per cent. One week lat~r the urine was normal except for an occasional cast; no estimation of sulfacetimide content was made then. A blood smear made at this time showed the presence of 16 per cent eosinophilic polymorphonuclear leucocytes. In this dog, as well as in several others, such an eosinophilia was not associated with any evidences of intestinal helminthiasis. The blood sulfacetimide levels of both dogs varied during the period of 11 weeks from 3.3 mg. to 14.3 mg. per 100 cc. The animals were sacrificed at the termination of the experiment; but for the presence of macroscopic brown spots on the cortex of th.e kidneys of both and microscopic inflammatory scars scattered in foci throughout the cortex of the kidneys of one animal, all the tissues examined appeared normal. It was thought that the inflammatory scars were probably not associated with Male dog, 7.6 kg.-1.0 gm. sulfacetimide per kg. daily DATE

12/27/39 1/ 3/40 l/10/40 1/ 17/40 1/24/40 1/31/40 2/ 7/40 2/14/40t 2/21/40 2/28/40 3/ 6/40t 3/13/40

WEIGHT

CONCENTRATION OF DRUG IN BLOOD*

kg.

mg. per 100 cc

millions per c.mm.

thousands per

c.mm.

percentage (17 gm. per 100 cc = 100 per cenl)

7.61 7.64 7.25 7.26 7.42 7.60 7.46 8.40 9.90 10.60 10.96 11.40

0 4.3 12.7 15.3 10.7 14.3 16.5 10.5 34.0 29 .0 19.8

5.32 4.46 4.48 4.76 4.74 5.00 5.40 5.58 6.00 5.64 5.98 5.83

13.70 20.60 18.40 13.30 13.60 14.50 11.75 15.10 17.40 14.35 10 .96 12.50

83 78 74 66 74 70 74 81 83 80 85 85

RED BLOOD CELL

COUNT

WHITE ELOOD CELL COUNT

* All blood concentrations are reported as sulfacetimide.

HEMOGLOBIN

t Denotes urinalysis. February 14, 1940: Except for a specific gravity of 1.033 and the presence of crystals on microscopic study, the urine was normal. The urine was clear when collected (by catheter) but on cooling many drug crystals deposited out; it contained 3.25 per cent sulfacetimide. March 6, 1940: Urinalysis gave essentially the same findings as noted on February 14, 1940.

189

STUDIES OF SULFACETIMIDE AND SULFANILAMIDE

the administration of the sulfacetimide. The first dog received a total of 217 gm. and the second dog received 250 gm. of sulfacetimide during the 11 week period. In the last series of dogs in this chronic experiment, 2 animals, 1 male of 7.61 kg. and 1 female of 8.25 kg. were begun with 1 gm. of sulfacetimide per kg. daily. Because of the death of the female 9 days after the experiment had been started, a third dog, a male of 9.32 kg., was used. Several interesting features of this experiment seem to justify giving the case histories of these 3 animals in some detail. On March 13, 1940 a blood smear showed the following: Polymorphonuclear neutrophilic leukocytes 50 per cent, lymphocytes 34 per cent, eosinophilic leucocytes 13 per cent, monocytes 3 per cent. The white cells appeared normal; the red cells showed a definite hypochromia and there was some tendency for the cell diameter to be less than normal. The dog was sacrificed on March 13, 1940 and necropsy examination showed all structures to be grossly normal except for a few small grayish brown spots in the liver. Microscopic examination of the various tissues showed no abnormalities. This dog had received a total of 585 gm. of sulfacetimide in the 11 week period. Female dog 8.25 kg.-1.0 gm. sulfacetimide per kg. daily DATE

WEIGHT

CONCENTRATION OF DRUG IN BLOOD

12/27/39 1/ 2/40

RED BLOOD CELL

COUNT

WHITE BLOOD CELL COUNT

HEMOGLOBIN

kg.

mg. per 100 cc

millions per c.mm.

thoitsands per c.mm.

percentage (17 gm. per 100 cc = JOO per cent)

8.25 5.75

0

145.0

6.56 6.66

19.40 30.65

95 98

This dog had a high initial white blood cell count but did not appear ill. On January 2, 1940, however, it failed to eat and it became evident that the animal was seriously ill. Opisthotonus and dyspnea developed. The white blood cell count rose to 30,650 with 98 per cent being polymorphonuclear leukocytes and 2 per cent being lymphocytes. The cells showed severe toxic degeneration and there was an abnormal number of immature types. The red blood cells were normal in appearance with respect to size and hemoglobin content. The animal became severely dehydrated, and dyspnea increased; no food was taken after January 3, 1940 and death ensued on January 5, 1940. The drug was continued until death. Many of the symptoms shown by this animal were similar to those seen in dogs in the later stages of acute poisoning from large single doses of sulfacetimide. At necropsy gross inspection revealed general dehydration. The liver was less bloody than normal. The spleen was quite small and the cut surface was dry. The kidneys were pale and showed a few small brownish areas in the cortex. Both lungs showed areas of partial collapse in the lower lobes. The intestinal mucosa was markedly injected and its contents were bloody. Microscopic examination showed the organs to be normal except for some congestion, and foci of

190

R. S. FISHER AND H. B. HAAG

slight inflammatory scarring in the cortex of the kidneys. These latter changes probably cannot be attributed to the drug. This dog had received 74.3 gm. of sulfacetimide in the 9-day period before death. Male dog 9.32 kg.-1.0 gm. sulfacetimide per kg. daily DATE

.1/24/40 1/31/40 2/ 7/40 2/14/40 2/21/40 2/28/40 3/ 6/40 3/13/40 3/20/40

CONCENTRATION OF DRUG IN BLOOD

RED BLOOD CELL COUNT

kg.

mg. per 100 cc of blood

millions per c.1n1n.

thousands per

c.mm.

percentage (17 gm. per 100 cc = 100 per cent)

9.32 9.30 8.86 9.51 9.50 8.70 7.82 7.26

0 26.0 14.6 34.0 14.3 67.5 106.8 105.0 62.4

6.80 6.90 6.76 6.90 6.72 6.88 6.86 6.90 8.95

12.40 14.95 10.70 16.80 13.30 10.15 12.35 14.25 26.00

101 97 100 104 100 110 110 110 135

WEIGHT

WRITE BLOOD CELL COUNT

HEMOGLOBIN

This dog appeared well until March 11, 1940 when it refused to eat and seemed ill. The blood had a non-protein nitrogen content of 54.5 mg. per 100 cc. The dog was given water by stomach tube-1500 cc. in 24 hours-and an abundance of meat was added to the diet. On this regimen it recovered rapidly and by March 15, 1940 it was eating well and regaining weight. Urinalysis on a voided specimen on this date showed a fair number of hyaline casts and a few red cells per high power field. Sulfacetimide was continued, and on March 20, 1940 the animal again appeared ill and was becoming dehydrated. At this time the blood non-protein nitrogen content was 78.8 mg. per 100 cc. The condition grew progressively worse in spite of forced fluids. Examination of the blood on March 23, 1940 showed a red blood cell count of 10,200,000 per c. mm., a hemoglobin percentage of 145, a non-protein nitrogen content of 109 mg. per 100 cc, and a sulfacetimide content of 130 mg. per 100 cc. Administration of the drug was discontinued on March 24, 1940. On March 26, 1940 the animal was moribund and was killed in order to insure tissues suitable for examination. The blood non-protein nitrogen content immediately before sacrifice was 298 mg. per 100 cc and the blood sulfacetimide level was 182 mg. per 100 cc. Gross anatomical examination at death showed that the stomach and intestine were markedly injected and contained a small amount of bloody fluid. The ureters were both patent. No gross changes were evident in the kidneys or liver. Microscopic examination showed a moderate vacuolic and dissociation degeneration of the liver cells and slight but definite degenerative changes in the convoluted tubule cells of the kidney, the glomeruli appearing normal. The nonprotein nitrogen level of the blood of 298 mg. per 100 cc on the day of death could not be accounted for by the relatively slight lesion found in the kidneys; and therefore it must have been due primarily to some physiological alteration in kidney function. This dog had received a total of 559 gm. of sulfacetimide in a period of 60 days.

STUDIES OF SULFACETIMIDE AND SULFANILAMIDE

191

ABSORPTION AND EXCRETION

Mice. Graded doses of sulfacetirnide and of sulfanilamide, suspended in mucilage of acacia, were given to groups of mice by stomach tube. Each group was divided into 5 lots of 2 to 3 mice each and these were killed at 2 hour intervals up to 8 hours and at 24 hours after administration of the drug. The concentration of the drug (both "free" and "total") in the pooled blood of each lot was determined. Figure 2 is a graphic representation of the results based on analysis for the free drugs. From comparison of curve 1 with curve 3 it appears that the mouse can absorb and excrete the greater part of a dose of 3.0 gm. per 175

!50

\,___ '? - - SulfaceUmide -8.oGm per Kg

\

\

------ Sulfan.ilMlide-3.oGmperKg. -

Sulfacetimide -6.0(Jmperl
--- Sulfa.cetimi.de -3.oftmpe,/iq

\,_

--..._

a

,e

__...__.,__ __ .....,

16

~

20

. 24

Ii.ours after edtninistra.tion of drug

Fm. 2. Blood concentrations of sulfacetimide and sulfanilamide in mice after oral administration. "Total" values were only slightly greater than the "free." The curves are designated 1-4 from below up.

kg. of sulfacetimide in 8 hours, whereas the excretion of sulfanilamide occurs at a slower rate, (higher blood levels which persisted for a longer time). Had a slower rate of absorption been responsible for the lower blood level of sulfacetimide it would have been expected that the blood level would remain elevated for a longer period of time than for the similar dose of sulfanilamide. This did not occur. Comparison of curve 2 with curve 3 indicates that in the mouse 6.0 gm. of sulfacetimide per kg. is required to produce a peak blood concentration equivalent to that produced by 3.0 gm. of sulfanilamide per kg. Furthermore, in the case of the sulfacetimide, in the few hours following the peak the blood level fell off rapidly, whereas, with sulfanilamide a high blood level was maintained for a longer period of time. This is interpreted as due to a greater

192

R. S. FISHER AND H. B. HAAG

clearance of sulfacetimide than of sulfanilamide. The values for sulfanilamide are in agreement with those reported by Marshall et al. Dogs. In the course of the mice experiments it was noted that those given 8 gm. of sulfacetimide per kg. did not show as frequent an incidence of severe toxic reactions as those given 3 gm. of sulfanilamide per kg. In further investigation of this observation, a series of dogs was given graded doses of one of the drugs TABLE

3.-Toxicity and blood concentration of sulfacetimide and sulfanilamide in dogs SULFACETIMIDE

Dog number

Dose

SULFANILAMIDE

Degree of poisoning

gm. per kg.

5 6 1

2 3 4 7 8 9 10 11 12* 13 14*, 15* 16 17 18

0.16 0.16 1. 6 1.6 1.6 1.6 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 10.0 10.0

Maximum blood level observed mg. per 100 cc

0 0 0 0

4.0 4.3 67.8 74.6

0 0

90.0 73.4 107.5 83.2 81.2 88.0 103.2 130 .0 65.0t 57.2t 81.3 63.7 113.0

+ 0

+ 0 0 0

0

+ +

Dei:ree_ of p01sonmg

Dose gm.

per

kg.

Maximwn blood level observed mg. per 100 cc

1.6

+++

104.0

1.6

++

40.4

2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0

+++ +++ +++ +++ +++ ++ +++ +++ ++

80.0 83.4 106.0 75.6 89.5t 68 .0 66.3 64.1 58.7

t Died. * In this experiment two dogs died following sulfacetimide administration. Neither showed signs of acute poisoning during the 24 hours after dosing, but became quite ill and weak after from 36 to 48 hours, finally becoming comatose and dying three to four days after receiving the drug. Both had received sulfanilami de two weeks previously. Extremely high blood concentrations of sulfacetimide did not occur, so, although the deaths were undoubtedly related to the administration of the drugs, the exact mechanism is not clear. One animal died 45 hours after dosing with sulfanilamide showing progressive signs of severe poisoning. It had received sulfacetimide two weeks previously. As in the case of the deaths following sulfacetimide, excessively high blood levels of the drug did not occur.

and the blood concentration of the drug determined at intervals-for 24 hours. Usually 2 weeks later the same dogs received the other drug and similar observations were made. In one-half of the dogs sulfacetimide was given first and in the other half sulfanilamide was given first. The severity of the intoxication was recorded as 0, 1 plus, 2 plus or 3 plus in keeping with the observations under "acute toxicity" (table 2). Table 3 represents these results. It will be noted from this table that doses of sulfanilamide, particularly, which caused a rather high percentage of fatalities in the acute experiments previously mentioned,

193

STUDIES OF SULFACETIMIDE AND SULFANILAMIDE

caused few deaths in this experiment. In the experiments dealing with the determination of acute toxicity morphine was given as an antiemetic; it is possible that this premedica+,ion could have influenced the intensity of action of these drugs. Figure 3 shows the blood levels attained in 2 gToups of dogs following doses of 1.6 gm. of sulfacetimide per kg. ( curve 1) and 5.0 gm. of sulfacetimide (curve 2).

I. 6 (im.perf
·----·· represents a»er{¾Je iJaJ,ues from ;,dogs

4

8

16

II!.

20

24-

fiours after ad.mmistrulion of drug

FIG. 3. Blood concentrations of sulfacetimide in dogs after oral administration .

.de (U;-irW
.Sulfacehmt

/~

-G--_____ _

I,'// . ,__ V I

/

,/

--------- ..____ S,ll..lfi:-1 I}

0,.

/

-,-... __

o....... _

i/

3

6

- - ~__

------ ,,tfct0.

~~·

x.

·----- ·'ct"'r !?to0 0!

'

-----. CoRc

··--o-------------------~!1:.Z!::Y:!?_~f!i_~<"!ce__(~li}_c>_
12.

15

18

El

24

fiows after admim.stration of dri.UJ

Fm. 4. Blood concentrations and urinary elimination of sulfacetimide and sulfanilamide in dogs after oral administration.

Comparison of these shows that the blood level reached is not proportional to the dosage. The results with 10.0 gm. of sulfacetimide per kg. (table 3) indicate no further important increase on doubling the amount administered. This observation is in keeping with that of Marshall et al. who reported that in dogs, sulfanilamide in a dose of 2.0 gm. per kg. often produced no increase in the drug concentration in the blood over that noted after administration of 1 gm. per kg.

194

R. S. FISHER AND H. B. HAAG

It seemed advisable to further investigate the question of comparative rates of absorption and excretion of the two drugs. For this purpose 4 female dogs were prepared for catheterization. Each dog was given an amount of one of the drugs equivalent to .5 gm. per kg. of body weight. Food was allowed until 12 to 15 hours before dosing. Water was allowed ad libitum throughout the experimental period. The drug was suspended in water-40 cc per gm. of drug-and administered by stomach tube. Blood was taken, and urine collected by catheter prior to medication, and at the end of 3, 6, 9 and finally 24 hours after administration of the drug. The blood concentration of the drug at each sampling and the amount of drug excreted in the urine during each interval was determined. After a 2 weeks' interval the procedure was repeated with the other drug. The data of this experiment, averaged, are given in figure 4. On 2 different occasions 1 dog vomited a large part of the dose of sulfanilamide within the first hour, thus invalidating the results obtained in that experiment. The fact that it tolerated a similar dose of sulfacetimide is in keeping with results reported elsewhere in this paper, as to the relative toxicity of the 2 drugs. SUMMARY AND CONCLUSIONS

Sulfacetimide by oral administration to mice and dogs appears to be about onefourth as acutely toxic as sulfanilamide similarly administered. In dogs, by oral administration, doses of 5.0 gm. of sulfacetimide per kg. of body weight were better tolerated, i.e., produced less signs of acute poisoning, than were observed in the same dogs following administration of 2.0 gm. of sulfanilamide per kg. of body weight. The growth rate of young rats maintained on a diet containing 1.5 per cent of sulfacetimide showed no significant retardation as compared to controls maintained on a similar diet not containing the drug. After 75 days on this diet no definite pathological changes were found in the liver, kidneys, spleen or intestine of the animals receiving the drug. These rats received on an average of 175 mg. of the drug per day per rat. Two dogs which received 0.4 gm. of sulfacetimide per kg. of body weight daily for 11 weeks showed no loss in weight, no significant changes in white blood cell count, red blood cell count, or hemoglobin, and no demonstrable pathologic changes in the liver, spleen, kidneys, adrenals, stomach or intestines. Of 2 dogs which received 0.2 gm. of sulfanilamide per kg. daily for 11 weeks, 1 showed a transitory hemolytic anemia accompanied by loss of weight from which it recovered entirely, however, before the end of the test period. The other showed no change in the blood picture or loss of weight during the time. At necropsy no tissue abnormalities were found in the liver, spleen, kidneys, adrenals, stomach or intestL.'le of either animal. Daily administration of 1.0 gm. of sulfacetimide per kg. of body weight was not tolerated for 11 weeks in two of three dogs started on this regimen. Although necropsies of these 2 dogs (1 died in 9 days, the other in 60 days) did not reveal causative anatomical changes in the kidneys; blood studies suggest that failure of the excretory function of the kidneys was probably responsible in large degree for

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the death of these 2 animals. The 1 animal living at the end of the 11 week period showed no tissue abnormalities when sacrificed. In mice sulfacetimide appears to be less effective in producing high blood concentrations than is sulfanilamide, there being required approximately 6.0 gm. of sulfacetimide per kg. to produce a concentration similar to that attained after 3.0 gm. of sulfanilamide per kg. of body weight. Again, in dogs given 0.5 gm. per kg. of the drugs it appears that a single dose of sulfacetimide does not produce as high a blood concentration as an equal single dose of sulfanilamide. In mice and dogs a given blood concentration of sulfacetimide is less apt to be accompanied by signs of intoxication regardless of the dose required to produce this level than is a similar blood concentration of sulfanilamide. There is evidence suggesting that sulfacetimide is more readily absorbed and eliminated than is sulfanilamide by the mouse and by the dog. Pathological observations were kindly made by Dr. G. Z. Williams, Associate Professor of Pathology in this institution. REFERENCES MARSHALL, E. K., JR.: Determination of sulfanilamide in blood and urine. Proc. Soc. Exper. Biol. and Med., 36: 422, 1937. - - - : Determination of sulfanilamide in blood and urine. J. Biol. Chem., 122: 263, 1937. - - - , CUTTING, W. C. AND EMERSON, KENDALL, JR.: Toxicity of sulfanilamide. J. A. M. A., 110: 252, 1938. MOLITOR, HANS AND ROBINSON, HARRY: Some pharmacological and toxicological properties of sulfanilamide and benzyl-sulfanilamide. J. Pharmac. and Exper. Therap., 65: 405, 1939. YOUNG, HUGH H., HILL, JUSTINA H., JEWETT, Humi: J. AND SATTERTHWAITE, RICHARD w.: Sulfacetimide: Toxicity and efficacy in gonorrhea and urinary tract infections. J. Urol., 45: 903, 1941.