Toxicopathologic and pharmacologic properties of piperacetazine, a potent tranquilizing agent

TOXICOLOGY

AND APPLIED PHARMACOLOGY

Toxicopathologic Piperacetazine,’

6, 49-60

(1963)

and Pharmacologic Properties A Potent Tranquilizing Agent T.

LAWRENCEC.WEAVER,FRANKE.MITCHELL,AND Departments Pitman-Moore

of Biomedical Company,

of

LAMARKERLEY

and Pathological Research, Research Center, Division of The Dow Chemical Company, Indianapolis, Indiana

Received

March

22, 1962

Knapp et al. (1962) and Weaver and Kerley (1962) presented data that characterize piperacetazine (2-acetyl-IO-{ -3 [ -4- (P-hydroxyethyl) piperidino] propyl}-phenothiazine) as a “tranquilizing” agent. Haworth et al. (1961) found this compound effective in psychiatric patients. Knapp and co-workers (1962) showed that piperacetazine possessed pharmacologic actions qualitatively resembling those of chlorpromazine. The profile of activity in their behavioral and pharmacologic tests was such as to suggest certain advantages over chlorpromazine as a neuroleptic agent-particularly a broadening of the scope of therapeutic application, as well as an increase in potency and decrease in toxicity. This report is concerned primarily with detailed toxicology and supplemental pharmacology of piperacetazine. METHODS

Acute Toxicities The 24-hour toxicities of piperacetazine were determined following intragastric (i.g.) administration to BDFr female mice (50) and following i.g. and intraperitoneal (i.p.) administration to male albino Swiss-Webster mice (40 and 50, respectively), male albino Harlan-Wistar rats (40 and 24, respectively), and adult mongrel dogs, unselected as to sex. The data were analyzed by the method of Litchfield and Wilcoxon (1949). Chronic Toxicity Rats. Two hundred weanling rats (40-60 g) were used in this 12-month study. Four groups of 25 males and 2.5 females each were fed diets con1 Available from the Pitman-Moore Company, under the trademark QUIDE.

Company,

49

Division

of

The

Dow

Chemical

50

L. C. WEAVER,

F. E. MITCHELL,

AND

T. L. KERLEY

taining 0.0, 0.005, 0.01, and 0.025% piperacetazine in Harlan Rodent Mash. The food was weighed daily, and the food consumption per rat was computed. The rats were weighed individually once each week. One rat from each cage (5 rats per sex per dose level) was earmarked for hematologic study (packed cell volume, WBC, RBC, differential, and Hb), but if it died during the course of the test, another rat was picked at random to replace it. A gross and microscopic study was made of every rat that survived the test period. Complete gross and microscopic examinations were made of all rats that died during the test period, and any microbiologic studies that were considered necessary to determine the cause of death were made in addition. Organs subjected to histologic study included brain, pituitary gland, salivary gland, lymph node, esophagus, trachea, lungs, liver, kidneys, adrenals, pancreas, spleen, gonads, bone marrow, cartilage, urinary bladder, stomach, intestine, heart, and striated muscle. Tissues were fixed in 10% buffered formalin, embedded in paraffin, cut at 7 y, and, for routine study, stained with azure-eosin. For special study, selected tissues were stained by the PAS method for polysaccharides and Oil Red 0 for lipids (Pearse, 1960). Dogs. Fourteen mongrel dogs were placed in 4 groups, held in metabolism cages, fed Purina Dog Chow supplemented with canned horse meat. and presented with tap water ad libitum. All dogs were deparasitized and vaccinated against canine distemper and infectious canine hepatitis prior to the initiation of the experimental procedure. Group I (2 dogs) served as controls. Groups II, III, and IV (4 dogs per group) received 0.25, 1.0, and 5.0 mg/kg/day of piperacetazine orally in gelatin capsules for about 52 weeks. All dogs were weighed weekly and the dose was readjusted. Appearance and behavior of the dogs were observed daily. Clinicopathologic studies (hematology, liver function, urinalysis) before and after the test period, and a complete gross and histologic study were made of each dog. The scheme of histologic study was identical with that described for the rats.

Anticonvulsant

Effects

The anticonvulsant properties were evaluated in Swiss-Webster mice by the use of the pentylenetetrazol (Metrazol) threshold seizure pattern test (Swinyard, 1949). This tests the ability of a compound to prevent

51

PIPERACETAZINE

convulsions Metrazol.

produced by the subcutaneous

administration

of 85 mg/kg

of

Antihistaminic Effects This activity was determined ( 1) by using isolated guinea pig ileum in smooth muscle baths; (2) by ability to protect guinea pigs from fatal histamine aerosol; and (3) by the inhibition of the vasodepressor effects produced by injected histamine to pentobarbitalized dogs and cats. RESULTS

AND

DISCUSSION

Acute Toxicities The 24-hour LDb,-, values for piperacetazine in male Swiss-Webster mice following i.g. and i.p. administration were 820 (674-996) mg/kg and 120 (105-137) mg/kg, respectively; the latter was not significantly different from the 168-hour i.p. LDno (Knapp et al., 1962). The i,g. LD,,) in female BDF, mice was 552 (426-639) mg/kg. All mice showed marked depression of spontaneous motility and reduced resistance to handling. Ataxia, loss of righting reflex, and severe depression were observed at the higher dosages. The 24-hour LDno for piperacetazine was determined following oral and intraperitoneal administration to male Harlan-Wistar rats weighing 100 * 20 g. It was found to be 390 (354.6-429) mg/kg i.g. and 93 (75.0115.2) mg/kg i.p. No lethal effects were observed following the i.g. administration of piperacetazine at 50 and 100 mg/kg to three dogs at each dose level. However, following intravenous (i.v.) administration, the number of dogs dying/number tested for the various doses (mg/kg) were: O/6 (25), l/2 (32), l/l (40), 2/2 (SO), and l/l, (200). Following intravenous administration, death occurred immediately at the 200 mg/kg dose and within 6 minutes in one dog at 50 mg/kg. Uneventful but slow recovery occurred following a dose of 25 mg/kg. An oral dose of 50 mg/kg in 3 dogs produced profound depression of activity. At the end of 3 days, 1 dog had recovered but 2 were still ataxic. Three days after 100 mg/kg in 3 dogs, 1 had shown partial recovery. At 8 days, 2 of these dogs were ataxic and not eating and were sacrificed. Ctsronic Toxicity

in Rats

The curves depicting weight gain for these rats are presented in Figs. 1 (males) and 2 (females). Female control rats and those on the higher

52

L. C. WEAVER,

F. E. MITCHELL,

AND

T. L. KERLEY

dosage (0.025% diet) showed almost identical weight gains. Female rats on the low level of drug (0.005%) exhibited a final weight significantly greater (t test) than the controls; the group on intermediate level of drug gained intermediately to other groups. Male rats on the low level drug were identical with controls, whereas the average weights

150

ii

y 100-cl!! 50!:::!:!::::::I::!:::!I:::::~ 0 4 6

12

16 TIME

FIG.

20

24 IN

28

32

36

40

44

48

52

WEEKS

1. Average body weights of male rats receiving piperacetazine

in the diet.

of those receiving the largest amount of drug (O.OZS%) were considerably less than controls. There was no appreciable difference in animals or among drug animals at various dosages. The weekly range of drug intake (mg/kg/day) for the period of testing was found to be 2.5-S for the 0.005% diet, 5-9 for the O.Ol%, and 12-26 for the 0.025%. Food consumption varied considerably, and there seemedto be no direct correlation between food intake and weight gains.

53

PIPERACETAZINE

Blood values of the various groups were within limits considered to be normal. No discrepancies were noted in a comparison of the control and treated groups. Each rat was sacrificed with ether and complete postmortem studies were made. Benign mammary gland tumors, murine pneumonia, and 400

1,,

r

FEMALE

I

I

,

I,

I,

I

16 TIME

20

24 IN

,

I

I,

I

I

I1

I

I

I

I

II

RATS

350

300 2 ;f (3 - 250 L: 2; 3 200 :: ; 2

150

100

50 0

FIG.

4

8

12

28

32 WEEKS

36

40

44

48

52

2. Average body weights of female rats receiving piperacetazine in the diet.

low-grade bacterial infection were the principal pathologic findings. These problems may be expected as natural occurrences within a rat colony over an extended period of time. The incidences by comparison of the treated to the control groups further showed no relationship between diseaseand drug intake. From the experimental data, in rats, no toxic effects could be associated with the long-term oral administration of piperacetazine at various dosages.

54

L. C. WEAVER,

Chronic Toxicity

F. E. MITCHELL,

AND

T. L. KERLEY

in Dogs

It is apparent from the results presented in Table 1 that all dogs gained weight during the period of the study. The changes in weight appeared to be unrelated to dose level of the drug. Dogs receiving 0.25 mg/kg/day were quiet and appeared to sleep more during the early part of the study. Dogs receiving 1.0 mgjkg/day showed scleral congestion, slight ataxia, and drowsiness after each dose of drug but always recovered on the following day and consumed their food. Throughout the study, all animals receiving the 5.0 mg/kg/day dose exhibited various degrees of depressed activity and lacrimation. In addition, ataxia and considerable drowsiness from which they could be aroused were frequently observed. During the 5th month of the study, dog no. 368 (0.25 mg/kg/day) was mated with dog no. 561 (5 mg/kg/day) and whelped 3 normal pups ; one died in 24 hours and the remaining two were sacrificed at about 6 weeks. Dog no. 365 (l.Omg/kg/day) after mating with dog no. 602 (0.25 mg/kg/day) on the 1 lth month gave birth to 7 normal pups; 4 days after parturition the bitch and litter were sacrificed. Dog no. 329 (l.Omg/kg/day) was mated just prior to the start of the study and whelped 2 normal pups. The hematologic data of Table 2 show a deviation from normal by low total red blood cell counts at the 7-month interval. An anthelmintic had been administered at 2 months prior to this time. It was believed that adequate time had lapsed to afford recovery from either hookworm infection or effect of the anthelmintic. Control as well as treated groups made spontaneous recovery from the unexplained erythrocyte deficiency. The data on urinalysis correlated with low-grade renal and urinary tract bacterial infection demonstrated by subsequent histopathologic study. Similar alteration is seen in control as in treated groups. Low-grade bacterial type urinary tract infection and obesity were the principal pathologic findings and were attributed to close confinement over a long term of inactivity. Their occurrence in the control animals and a similar severity in each treated group further indicated a lack of relation to piperacetazine. Neurologic granular structures similar to degenerative axis cylinders as described by Newberne et al. (1960) were found principally within the gracilis tract of the medulla, and to less extent in other areas of the brain or spinal cord. Newberne’s data indicated that the structures occur

0.0

0.25

1.0 5.0

2

4

4 4

Number Dose (mg/ of dogs kg/day)

9.7 12.1

10.9 11.1

l/18

9.6 11.3

10.2 10.4

2/15

AVERAGE

9.8 11.2

10.0 10.5

3/14

EFFECTS

TABLE

10.1 11.7

10.6

10.8

4/11

11.8

11.7

10.9

6/14

at various

12.0

10.8

11.4 11.8

IN DOGS

10.9 12.0

10.9 11.6

11.3 11.3

11.2 11.6

9/13

8/16

(month/day)

GAINS

dates 7/12

ON WEIGHT

11.5 10.5

(kg)

1

11.0 10.1

10.8

S/16

Dog weight

OF PIPERCETAZINE

12.3 10.9 -

13.1

12.9

11/16

12.6 11.9

12.4

lop1

-

12.9 11.2

13.6

12/14

z ic

z

z %

F

F

F

F

M

M

249

550

34s

368

602

698

0 7 12

0 7 12

0 7 12

0 7 11

0 7 12

0 7 12

Time (months) _-

a N = Neutrophile,

Sex

Dog. no.

L z

lymphocyte,

8.7 8.7 7.5

21.7 14.1 14.7

11.8 9.7 7.5

lS.2 8.5 13.5

21.9 10.1 15.3

17.9 9.9 10.0

WBC Cl@/ mm3)

Group

13.2 15.3 15.6

14.1 14.7 18.6

15.2 12.3 17.7

16.5 16.5 17.7

II:

15 14.7 17.4

11.8 12.9 14.3

Hb (g/l~ ml)

75 80 56

79 56 71

65 77 78

55

69 49

L

7 8 1

11 2 18

E

23 12 38

20 34 20

19 17 21

30 43 35

2 4 6

1 8 2

16 6 1

1 8 10

-

-

-

-

-

-

4

2 7

2

M

PIPERACETAZINE

0.25 mg/kg/day

27 18 39

8 40 19

I: Controls

Piperacetazine

66 74 60

79 58 63

Group

N

Diff erentiala

TABLE 2 DATA IN DOGS RECEIVING

-~ -E = eosinophile, M = monocyte.

4.91 3.89 5.70

4.97 3.55 7.37

6.45 2.88 5.56

6.66 5.10 7.14

4.77 3.71 7.63

5.57 3.74 6.45

RBC (I@/ mm3)

CLINICOPATHOLOGIC

33 32

36 30 43

26 35

42 33 41

46.5 29 34

33.5 25 40

Hematocrit (%)

1.5 0 0.6

3.5 0 5

6.5

5.5 0 4

2.5 3 11

BSP (% retention)

12 14 13.5

19.5 19 19.5

5

13 13 12.8

12.5 12 12.2

BUN bd 100 ml)

1.7 0.2 0.5

4.6 0.4 0.2

1.3

3.3 13 0.5

0.5

0.8 0.2

Icterus index

z P 2

Sex

F

F

F

F

M

F

M

F

IlO.

317

364

365

329

237

480

561

420

Dog

0

0 7 11

0 7 11

22.1

18.4 8.5 6.4

17.7 14.6 13.4

14.0 10.0

14.4

12

0 7

19.1 10.6

13.0 9.9 5.1

11.0 12.9 10.2

18.2 15.1 8.1

WBC (lOs/ mms)

0 7

0 7 12

0 7 12

0 7 11

Time (months)

6.99

6.32 3.85 6.23

4.48 3.46 5.60

4.29 4.15

5.44

5.62 3.59

6.70 4.41 4.58

7.09 4.08 6.74

6.34 4.60 5.69

RBC (lO+J/ mm”)

14.1

14.2 15.6

16.3 16.2 13.1

17.3 14.4 17.1

16.3

15.6 15.6 15.3

12 12.6 16.8

12.6 13.2

IV:

III: 15.9 16.2 15.3

Group

Group

Hb (g/l00 ml) N

70

59 48 70

63 56 75

87 59

Piperacetazine

55

69 54

65 73 61

70 54 61

64 58 8

20

30 44 22

34 34 20

6 33

32

25 46

28 23 29

29 36 28

31 42 76

L

E

9

11 4 8

3 10 5

6 5

5 mg/kg/day

12

-

6

7 4 10

1 6 1

5 16

1.0 mg/kg/day

Differentiala

2 (Continued)

Piperacetazine

TABLE

-

-

-

-

-

1

4

1 3

1

4 10

-

-

M

51.5

49 30 31

39 25 42

29 29

29

41.5 35

42 38 31

58 26 39

40 31 36

Hematocrit (%)

6

4.4 0 0

2.5 2 0

3 0

0.5

3 0

3.5 0 4

4.5 14 3

3.8 0 0

BSP (% retention)

10.5

7.3 9 16

13.8 15 22

10 16

10.6

18 10

10 12 10.5

14 12 15.5

10.3 13 21

BUN (mg/ 100 ml)

1.8

1.4 5 2.1

2.9 5 1.3

1.6 1

0.7

0 3

1.4 2 0

1.3 15 0

1.7 9 1.3

Icterus index

z

2 R

E

i

58

L. C. WEAVER,

F. E. MITCHELL,

AND

T. L. KERLEY

commonly in dogs, increasing in incidence with age, and no neurologic or pathologic significance has been associated with their presence. It has been suggested that they are probably a nonspecific defect of aging.

Anticonvulsant

Effects

Piperacetazine in doses of 2, 20, 40, and 80 mg/kg i.g. (10 mice per dose) failed to alter significantly the convulsant properties of Metrazol 2 hours after administration.

Antihistaminic

Effects

Piperacetazine was tested in smooth muscle baths using isolated guinea pig ileum for antihistaminic activity in comparison with chlorphenoxamine (Table 3) (see also Kerley et al., 1961). Piperacetazine TABLE In Vitro

ANTIHISTAMINIC

Drug

Piperacetazine Piperacetazine Chlorphenoxamine

ACTIVITY

3

OF PIPERACETAZINE

Concentration of drug (vg/ml of bath) 4.76

AND

CHLORPHENOXAMINE

Numberof experiments

% Change from control

2

-75

23.8

3

23.8

2

-100 -97

was shown to possessan in vitro antihistaminic activity which was at least as potent as that of chlorphenoxamine. The latter drug has been reported to be a more potent antihistaminic than diphenhydramine hydrochloride (Brock et aZ., 19.54). The activities of piperacetazine and chlorphenoxamine to protect guinea pigs from fatal histamine aerosol have been summarized in Table 4. The animals received 2 mg/kg i.p. of piperacetazine or 5 mg/kg of chlorphenoxamine 1 hour before the test. No coughing, convulsions, or death occurred after piperacetazine that were ascribable to histamine, although the animaIs were allowed to remain in the chamber for a period of 8 minutes. The antihistaminic activity of 5 mg/kg of chlorphenoxamine by this test appeared to be less than that of piperacetazine. At the time of test, the pigs that received piperacetazine appeared to be tranquil. They were alert when picked up but did not resist being handled. In dosesas high as 1 mg/kg i.v., piperacetazine failed to completely inhibit the vasodepressorproperties of injected histamine diphosphate.

59

PIPERACETAZINE

Table 5 is a summary of the cardiovascular antihistaminic and antiacetylcholinic properties of piperacetazine. It may be noted that doses of 2 or 4 mg/kg significantly reduced the fall of mean blood pressure for unusually long periods of time but never completely prevented the histamine vasodepression. TABLE ANTAGONISM

OF HISTAMINE

4 AEROSOLS

IN GUINEA

PIGS

No.

Cd

coughing No. tested (time of 1st cough)

S/5 (37 set) O/5

5/S (62 set)

S/5 (204 set)

2.0

327 324

O/5

O/S

-

313

3/5

(36sec)

5.0

306

3/5

(122

5/S (55set) 3/5 (153 set)

5/5 (181 set) l/S (15 min)

Dose (mg/kg i.p.)

Group

Average weight

-

Control Piperacetazine Control Chlorphenoxamine 0 A 1.257~ solution maximum of 8 minutes.

Piperacetazine dose (w/W

ON THE VASODEPRESSOR AND ACETYLCHOLINE

Number of animals

Blood pressure change after piperacetazine (%)

RESPONSES

to histamine

-69

--51/---51

0.50

1 Dog 1 Cat

-17

-61/-69

-43

-28/-37

1 Dog

-

2.0

4 Dogs

-32

8 (2nddose)

1 Dog

-37

OF

to ACh 42/-30 -

-61/-54

-3O/-25

--52/--24

(>60 4.0

a

% Blood pressure change (before/after drug)

1 Dog

1.0

or

5

OF PIPERACETAZINE HISTAMINE

0.25

0.50

No. dead No. tested (time)

of histamine diphosphate was aerosoled until death The test was made 1 hour after drug administration. TABLE

EFFECTS

set)

No. collapsing No. tested (time)

-421-34

min) 1

-m/--2 (>3

hrs)

44/-35

(100 min)

Piperacetazine also reduced but never prevented the fall of blood pressuredue to acetylcholine injections. The higher dosesof piperacetazine did not appear to be more effective against acetylcholine than the lower doses,and the duration of effect was shorter than against histamine. Although complete blockade of histamine was not obtained with doses of piperacetazine up to 4 mg/kg, the degree of protection was good with

60

L. C. WEAVER,

respect to known impressive.

F. E. MITCHELL,

antihistaminics

AND

T. L. KERLEY

and the duration

of action was quite

SUMMARY For one year piperacetazine was administered continuously to rats at 0.005, 0.01, and 0.025% concentrations of their diets, and daily to dogs at 0.25, 1.0 and S.Omg/kg/day dosages by capsules orally. The drug was well tolerated by both species. Laboratory studies of the animals did not reveal any significant pathologic effects that were attributable to the drug. Piperacetazine failed to alter the convulsant properties of Metrazol but did show moderately effective antihistaminic properties. REFERENCES BROCK, N., LORENZ, D., and VIEGEL, H. (1954). Beitrag zur Wirkung von Antihistamin-Substanzen. II. Zur Pharmakologie des Systral. Auzne~mmitteEForscls. 4, 262-268.

K., JONES, L. M., and MANDEL, W. (1961). Clinical experience with a new phenothiazine (piperacetazine). Am. J. Psychiat. 8, 749-750. KERLEY, T. L., RICHARDS,A. B., BEGLEY, R. W., ABREW, B. E., and WEAVER, L. C. (1961). Anticonvulsant and antitremor effects of chlorphenoxamine. I. Phwmacol.

HAWORTH,

Exptl.

Theuap.

I!&

360-365.

KNAPP, D. L., STONE, G. C., HAMBOURGER, W. E., and DRILL, V. A. (1962). Behavioral and pharmacological studies of piperacetazine, a potent tranquilizing agent. Arch. intern. pharmacodynamie l!l6, 152-166. LITCHFIELD, J. T., JR., and WILCOXON, F. (1949). A simplified method of evaluating dose-effect experiments. J. Pharmacol. EzptI. Therap. 96, 99-113. NEWBERNE, J. W., ROBINSON, V. B., ESTILL, L., and BRINKMAN, D. C. (1960). Granular structures in brains of apparently normal dogs. Am. J. Vet. Research 21, 782-786. PEARSE, A. G. (1960). Histochemistry, Theoretical and Applied, 2nd ed. Little, Brown, Boston. SWINYARD, E. A. (1949). Laboratory assay of clinically effective antiepileptic drugs. J. Am. Pharm. Assoc. Sci. Ed. 38, 201-204. WEAVER, L. C., and KERLEY, T. L. (lW2). Strain difference in response of mice to d-amphetamine. J. Phavmacol. Exptl. Therap. 136, 240-244.