Toxicology of nialamide, a new psychotherapeutic agent

TOXICOLOGY

AND

APPLIED

Toxicology

PHARMACOLOGY

1, 524-533

(1959)

of Nialamide, a New Psychotherapeutic Agent’ C. S. DELAHUNT

Chas.

Pfizer

Therapeutic

J. M.

AND

Institute,

Received

June

PEPIN

Maywood,

New

Jersey

9, 1959

Several potent monoamine oxidase inhibitors with antidepressant properties have been described recently and their characteristics have been dealt with in detail (Koelle, 1958; Chessin et al., 1958; Horita, 19.59). Rowe et al. (1959) first reported the antidepressant activity of one of the most potent of these compounds, nialamide,2 l- (2 (benzylcarbamyl) ethyl) -2-isonicotinoylhydrazine. From their work this compound appears to be from two to twelve times as potent as iproniazid, depending upon the species and test employed. The formula is as follows: CO-NH-NH-CH,CH,-CO-NH-CH,

Nialamide is a white crystalline powder slightly soluble soluble in one-tenth normal hydrochloric acid.

in

water but very

METHODS The acute toxicity of nialamide was determined in male albino mice and rats. Swiss Webster mice, weight range 15-25 g, and rats of the Wistar Strain, weight range 40-71 g, were employed for the oral and intraperitoneal routes. Subsequent to the administrations, the animals were observed for symptoms and mortality for at least a 7-day period. The Litchfield and Wilcoxon method (1949) was used in calculating the LDBO’s and 19/20 confidence limits. Because certain hydrazines may cause hepatic injury (Bodansky, 1924; 1 Part of

American 2 Niamid,

of

this

material

was

presented

Societies for Experimental Chas. Pfizer & CO.

at

the

Biology.

April

1959

Meeting

of the

Federation

TOXICOLOGY

OF NIALAMIDE

525

Popper, 1958; Zbinden and Studer, 1958), potential antidepressant compounds were screened to detect hepatotoxicity. Mice, rats, rabbits, cats, and dogs were used in combination with all the various routes of administration before selecting the oral route in the dog as the method of choice in appraising early liver damage. Fifty milligrams of iproniazid per kilogram body weight was administered orally to dogs. After 5-7 days of this treatment, jaundice, acute toxic hepatitis and death occurred in most cases. Eighteen out of a total of twenty dogs used evidenced this toxic hepatitis. Twelve of these dogs manifested definite jaundice. These icteric dogs and their livers served as standards against which antidepressant compounds were compared. The chronic toxicity of nialamide was determined in fifteen weanling rats of each sex which were fed diets containing 3, 10, 30, 100, and 300 mg/kg of nialamide. The concentration of this compound in the rats’ feed was adjusted weekly to maintain a constant dosage level. An equal number of rats served as controls. All rats were individually caged and allowed free access to food and water. Food consumption and body weights of individual rats were determined at weekly intervals. Hematologic studies were accomplished after the rats had been on diets for 6, 12, and 26 weeks. Four rats per level were bled for hemoglobin, red blood cell, white blood cell, and differential white cell counts. Subsequent to the hematologic determinations at the twelfth and twenty-sixth week, four rats per level were sacrificed. At necropsy the following tissues were taken for microscopic examination: thyroid, lung, heart, liver, pancreas, spleen? eye, kidney, bladder, adrenals, stomach, small and large intestine, gonads, femur, and pituitary. A six-month canine oral tolerance program was conducted in four groups of six mongrel dogs each, with a similar group of six normal dogs serving as controls. A dosage schedule of 3, 10, 30, and 60 mg/kg of nialamide was employed 6 days a week. The dogs were observed daily for symptoms and weighed at weekly intervals, the dosages being adjusted accordingly. Complete blood counts, urinalyses, blood urea nitrogen, fasting blood sugar, bromosulfalein (30-minute retention), alkaline phosphatase, and serum transaminase determinations were performed prior to and after 2, 4, 12, and 26 weeks following the initiation of the experiment. Two dogs from each level were sacrificed and necropsied at the end of the fourth, twelfth, and twenty-sixth weeks of study. The tissues examined microscopically were as follows: lung, heart, liver, gall and urinary bladders, spleen, pancreas, mesenteric and prescapular lymph nodes. stomach,

526

C. S. DELAHUNT

AND

J. M.

PEPIN

small intestine, large bowel, kidney, adrenals, gonads, uterus or prostate, brain, spinal cord, pituitary, parotid salivary glands, rib bone, skeletal muscle, skin, thyroid, eye, and optic nerve. A second series of beagles were given increased dosesof 70, 80, 90, and 100 mg/kg of nialamide. The same parameters that were employed in series I were used in this study, which is being continued for one year. Three groups of three rhesus monkeys have been receiving daily oral dosagesof 10, 25, and 50 mg of nialamide per kilogram of body weight for three months. Another group of monkeys serve as controls. The compound was administered 6 days per week. Prior to and after one, three, and six months of study the above-mentioned laboratory determinations were made. One monkey per level was sacrificed and necropsied after one month; the remaining two were sacrificed after six months of study. RESULTS

The acute toxicity results are presented in Table 1. The most constant symptoms observed in both mice and rats were excitation, loss of weight, decreased respiration, and depression. TABLE ACUTE

TOXICITY

1 OF

NIALAMIDE 19jzo Confidence

Species

Route

Mouse Mouse Rat

Oral 1.P.U Oral

Rat

I.P.

L%

1000 742 1700 760

limits

(w/k)

( 74 l-1358) ( 548-942 ) (1278-2261) ( 696-830 )

a Intraperitoneal.

In the hepatotoxicity study, the livers from dogs treated with iproniazid had a gross appearancewhich varied from tan to yellow, or that of acute toxic hepatitis (Smith and Jones, 1957). The microscopic appearance was one of hepatic necrosis, as observed in Fig. 1. Nialamide on the other hand did not causeany grosschangesof the liver. This lack of liver damage was noted also in the microscopic examination. The livers from the dogs treated with 60 mg/kg of nialamide for 30 days and for as long as six months (chronic study) had normal architecture and cellular details, as shown in Fig. 2. The survival rate of the chronic treated rats after six months was not significantly different from those of the controls except in the male group

TOXICOLOGY

FIG. I. Phctcmicrograph days. There is destruction stained with hematorylin

OF NIALAMIDE

527

of liver from a dog given jOmg/kg iproniazid for 7 of hepatic architecture and cellular necrosis. Section was and eosin. Magnification: X 60.

528

C. S. DELAHUNT

FIG. 2. Photomicrograph of liver from months. There is normal liver architecture with hematoxylin and eosin. Magnification:

AND

J. M.

PEPIN

a dog given 60mg/kg and cellular detail. X 450.

of nialamide Section was

for six stained

TOXICOLOGY

529

OF NIALAMIDE

on the 300-mg/kg level. These animals had a mortality rate of approximately 40 $. Food consumption was essentially normal at all levels in both sexes during the six-month feeding program. The growth rate was normal up to 30 mg/kg; there some retardation occurred. A severe growth depression was observed at the 300-mg/kg level. Table 2 shows that the hematologic findings were all within the normal limits. The tissues of rats sacrificed after three and six months on test at levels of 3, 10, 30, 100, AVERAGE

HEM.~TOLOGIC

TABLE 2 VALUES OF MALE

NIALAMIDE

FEEDING

FOR

SIX

AND

FEMALE

RATS

ON

MONTHS

Hemoglobin

RBC

WBC

26 Wk

M F

13.2 11.2

13.7

7.4

7.8

17.3

10.2

10.5

8.1

8.3

22.9

10.2

M F

13.1

14.4

7.2

8.2

15.9

10.8

12.2

13.1

8.7

7.9

22.6

9.4

30

M F

13.8

14.7

8.7

11.8

14.0

7.8 8.0

8.6

21.3 25.3

11.6 10.1

10

M F

14.1 14.5

14.3 14.1

7.9 8.4

8.9 8.5

15.4 15.8

11.5 10.8

3

M F

13.5

14.8

7.5

8.8

17.1

11.7

14.0

13.7

8.0

8.6

15.6

10.9

M F

13.8

13.8

7.4

8.8

17.8

14.1

11.8

11.1

8.0

8.2

19.4

10.3

100

Control

26 Wk

26 Wk

6 Wk

300

6 Wk

6 W-k

Sex

and 300 mg/kg were examined microscopically. Except for some incidental findings such as a few pneumonias and parasitic lesions, the tissues of the experimental animals and those of the controls were similar; they were therefore considered to be within normal limits. The remaining rats are being maintained on the original dietary regimen for at least one year. The mongrels on the six-month oral tolerance study (series I) manifested a few insignificant symptoms. Their general over-all physical condition was considered good. The clinical-biochemical findings as shown in Table 3 were all within the normal variations for the mongrel dog. From the hematologic values, a mild reduction in red blood cell count and hemoglobin content is noted at the 60-mg/kg level. Figure 3 shows the reduction graphically. Urinalysis of the dogs on nialamide did not differ from that of the controls. There were no abnormal gross or microscopic

530

C. S. DELAHUNT

AND

J. M.

PEPIN

changes in the tissues obtained from the dogs on test for as long as six months. TABLE EFFECT

OF NIALAMIDE

ON CANINE

3

BLOOD

CHEMISTRY

FOR SIX

MONTHS~ Alkaline phosphatase

Urea

Daily dosage (&kg)

Dog no.

-

B.S.P.* (%)

N aminase

iwz %)

I

F

4 Wk

F

2559 2938

0 0

0 0

35.0 33.5

3093 3094

0 0

1 0

10

3040 3047

0 0

30

3114 2897

60

3101 3196

Control 3

F

I

F

2Wk

F

43.6 38.7

78.1 80.1

96.7 92.3

18.1 11.3

16.8 14.0

4 6

4 8

52.0 35.5

37.2 30.8

75.1

76.4

93.4 88.1

14.8 13.6

15.0 14.3

3 2

4 4

0 0

59.0 51.5

40.3 51.5

81.6 84.8

87.6 95.1

12.5 10.0

10.8 15.1

4 6

5 6

0 0

0 0

53.5 43.5

48.0 50.0

82.4 79.9

94.5 85.0

14.1 13.8

20.0 14.0

6 4

7 6

0 0

0 0

47.0 45.5

33.2 33.5

77.9 80.5

91.5 91.5

11.7 12.7

18.5 16.6

4 4

5 8

a I = initial; F = final. b B.S.P. = bromosulfalein

2 g IO3 0 z i!

retention.

L..-..-.--..-

=--..-

4’

I

*

2

4

I 12

--..-

Conhol

~~~*~~~~~~ Nialamidc,

3 mg I kq

m---m Nialamlde,

IOmghg

1.0

00

(mg %)

I

Nialamide,X)mg/kq

-0.-Nialamide.6Omg/!tg 6 dogs I dose level

, 26

WEEKS

FIG. 3. Average canine hemoglobin area indicated by the arrows represents

content for six months on nialamide. The the range of normal values (Spector, 1956).

TOXICOLOGY

OF N1ALAMIDE

531

The symptomatology in series II was as follows: increase in body temperature; increase in heart and respiratory rates; as well as a “bufoteninlike” syndrome (Himwich et al., 1959). The 70-, 80-, and 90-mg/kg levels were lethal to half the animals on test after approximately 14 days. Some of the toxic effect was enhanced by a respiratory infection that was prevalent shortly after the initiation of this study. The lOO-mg/kg level was lethal to all dogs on an average of 7 days. The laboratory findings after one month of treatment showed a slight elevation in the blood urea nitrogen and alkaline phosphatase levels in a few dogs on 70, 80, and 90 mg/kg dosages. There was also a decrease in hemoglobin content along with a decrease in red blood cell count at all levels after one month. Except for some incidental findings such as pneumonia, the tissues were free of any effects of the experimental material. The monkeys’ general physical condition including appetite and bowel movement was considered good. The laboratory findings were essentially normal with the exception of a slight reduction in hemoglobin and red cell count at the highest dose level. The tissues obtained from the monkeys after one and six months were considered to be within normal limits. DISCUSSION

Rats can tolerate high concentrations (300 mg/kg) of nialamide without abnormal hematologic or histologic findings. Growth depression does occur but this apparently does not cause any deleterious metabolic changes such as those leading to vitamin B deficiencies. The only toxic effect noted in the first series of dogs receiving 3, 10, 30, and 60 mg/kg of nialamide was a mild hypochromic anemia at the highest level. This was not a progressive condition, but a self-limiting one. Fegler (1952) suggests that the glutathione of the red blood cells is very important in maintaining the integrity of the erythrocyte structure. Beutler (1957) states that acetylphenylhydrazine causes a fall in concentration of glutathione in vitro and a hemolytic anemia in V&JO. The cause of the mild hypochromic anemia observed with niaIamide is postulated to be the action of the hydrazine on the glutathione of the red blood cells. This theory is now being explored. Since no histologic changes were produced in the first series of dogs, a second series of beagles was given higher dosages of nialamide. The slight elevations in the blood urea nitrogen and alkaline phosphatase levels noted in the second series of dogs were transient in nature and were not accompanied by any histomorphologic deviations. The excitation syn-

532

C. S. DELAHLJNT

AND

J. M.

PEPIN

drome previously described for the dogs receiving high doses of nialamide is believed to have resulted in the drug’s action on the central nervous system. Free hydrazines, besides possibly causing hemocytolysis, may also produce hepatocellular damage. Bodansky ( 1924) produced hepatic necrosis in dogs by administering p-bromophenylhydrazine hydrochloride. To further explore the lack of hepatotoxicity with nialamide, Bloom et al. (1959) conducted comparative canine metabolic studies with this compound and iproniazid. The most outstanding finding was that very little isonicotinic acid (INA) was excreted following nialamide administration, In contrast, iproniazid-treated animals excreted large amounts of INA. The latter is in agreement with the work published by Randall ( 1958). If one accepts the premise that the more INA in the urine, the more hydrazine available in the body, then this lack of large amounts of free hydrazine will probably account for the absence of hepatotoxicity with nialamide. Randall (1958) states that dogs given iproniazid at doses of 3.5, 7.0, and 14.0 mg/kg by oral, intravenous, and intramuscular routes for 13 weeks produced slight to marked alterations in spleen, bone marrow, and liver. Nialamide was tolerated in dogs at 60 mg/kg for six months without evidence of any histologic aberrations in the tissues examined. SUMMARY 1. The oral LDse’s of nialamide in mice and rats were 1000 and l7OU mg/kg, respectively. 2. No liver damage was evidenced in dogs or rats receiving nialamide, even at toxic and lethal levels up to 100 and 300 mg/kg, respectively. 3. In view of the fact that nialamide is tolerated at 60 mg/kg for at least six months without showing any abnormal histologic changes, a relatively wide margin of safety exists for this type of hydrazine. ACKNOWLEDGMENTS The authors wish to thank Drs, Vincent J. Dardin and Robert Stebbins for reading the chronic toxicity slides; Irma Hinchcliffe, Mary Mulroy, Eleanore Carpenter, and Jean Lassen for their technical assistance; and Carol Mockett for her secretarial assistance. REFERENCES E. (1957). The glutathione instability of drug-sensitive red cells; a new method for the in vitro detection of drug sensitivity. J. Lab. Ch. Med. 49, 84-95. BLOOM, B. M., WAGNER, R. L., JR., DELAHUNT, C. S., and SCHREIBER, E. C. (1959). Metabolism studies with nialamide-A preliminary report. Diseases of Nervous System (in press).

BEUTLER,

TOXICOLOGY

533

(1924). The effect of compounds related to hydrazine in producing and experimental anemia. J. Phurmad. Exfd. Therap. !& 1.27-133. CHESSIN, M. (1958). Biochemical and pharmacological studies of phenylethylhydrazine and selected related compounds. Presented at New York Academy of Science Conference of Amine Oxidase Inhibitors, November 20-22, 1958. FEGLER, G. (1952). Relationship between reduced glutathione content and spontaneous haemolysis in shed blood. Nature 170, 624-625. HIMWICH, H. E., COSTA, E., and HIMWICH, W. A. (1959). Behavior responses of dogs to bufotenin reaching the brain either by the vertebral artery or internal carotid artery. Federation PYOC. 18, 402. HORITA, A. (1959). Pharmacology of monoamine oxidase inhibitors. Con!. on .4 Pharmacologic Approach to the Study of the Mind, Univ. of California, School of Medicine, San Francisco 1959. To be published. KOELLE, G. B. (1958). Pharmacologic significance of inhibition of monoamine oxidase. Quart. Rev. Psych&. and Neurol. 19, 37-50. LITCHFIELD, J. T., JR., and WILXOXON, F. (1949). A simplified method of evaluating dose-effect experiments. J. Pharmacol. Exptl. Theta@. 96, 99-113. POPPER, H. (1958). Hepatic injury in patients who had received iproniazid. Presented at New York Academy of Science Conference of Amine Oxidase Inhibitors, November 20-22, 1958. RANDALL, L. 0. (1958). Toxicology of Marsilid. Quart. Rev. Psych&. and Neurol. 19, 178-182. ROWE, R. P., BLOOM, B. M., P’AN, S. Y., and FINGER, K. (1959). The pharmacological characterization of the antidepressant, nialamide. Federation Proc. 18, 441. SMITH, H. A., and JONES, ‘I’. C., editors. (1957). Veterinary Pathology, p. 763. Lea i? Febiger, Philadelphia. SPECTOR, W. S., editor. (1956). Handbook of Biological Data, p. 175. Saunders, Philadelphia and London. ZBINDEN, G., and STUDER, A. (1958). Experimental pathology of iproniazid and related compounds. Presented at New York Academy of Science Conference of Amine Oxidase Inhibitors, November 20-22, 1958. BODANSKY,

anhydremia

M.

OF NIALAMIDE