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Psychopharmacologia (Berl.

) 18, 19--25 (1970)


9 by Springer-Verlag 1970

Effect of d-Amphetamine Sulphate


in Combination with CNS Depressants
on Spontaneous Motor Activity of Mice
VIMALA

H. SETHY, P. Y. I~AIK, and U. K. S~ETH

Department of Pharmacology and C.S.I.R. Pharmacological


Research Unit, Seth G. S. Medical College, Parel, Bombay-12, India
Received March 2, 1970

Abstract. The effect of d-amphetamine in combination with pentobarbital


sodium, reserpine, chlorpromazine, meprobamate and chlordiazepoxide has been
studied on spontaneous motor activity in male mice. d-Amphetamine together with
CNS depressants produced a greater effect than amphetamine alone.
Key-Words: d-Amphetamine -- Pentobarbital Sodium -- Reserpine -- Chlorpromazine -- Chlordiazepoxide -- Meprobamate -- Spontaneous Motor Activity.

Introduction
Mixtures of amphetamine and barbiturates have been used in psychiatry for m a n y years in the treatment of anxious or depressed neurotic
patients. Experiments have shown that mixtures of these drugs can be
more effective than either drug alone (Rushton and Steinberg, 1963).
Most of the work has been reported on single acute injections of amphetamine-barbiturate combinations. The present work has been carried out
to discover the effect of chronic treatment with amphetamine and
pcntobarbital sodium on spontaneous motor activity of mice. I n order
to find out whether a beneficial effect is also obtained with other CNS
depressant drugs, the combination of amphetamine with chlorpromazine,
reserpine, chlordiazepoxide and meprobamate has also been studied.

Methods
Male albino mice weighing 20--30 g in groups of 6 were used. Animals
were starved overnight, but were allowed free access to water. All
experiments were carried out between 8 a.m. and 2 p.m.
The effect of chronic treatment with amphetamine sulphate alone or
in combination with drugs like pentobarbital sodium, reserpine, chlorpromazinc (CPZ), chlordiazepoxide and meprobamate, was studied on
spontaneous motor activity (SMA) of mice. The effect of individual drugs
on SMA was also studied; the control group received normal saline.
Three groups were studied with each drug, and drug combination.
2*

20

V. H. Sethy, P. Y. Naik, and U. K. Sheth:


Table 1

Drug

Concentration
mg/ml

Dose
mg/kg

Amphetamine administered-hours
after predrug treatment

Normal saline
Amphetamine sulphate
Pentobarbital sodium
Chlorpromazine
Reserpine
Meprobamate
(suspension in 1~
Gum acacia)
Chlordiazepoxide
(suspension in polyethylene glycol)

0.90/0
0.15
1.2
0.0001
0.01

1 ml/100 g
1.5
12
0.001
0.10

--together
11/2
51/2

2.5

25

1/2

0.1

1/2

Spontaneous motor activity was quantitatively studied daily for


10 days in a photoelectric activity box (Metro Industries). The total
activity of each group (6 mice) was recorded for t0 rain prior to the
administration of the drugs or normal saline. The pre-drug level was
considered as 100~ activity. Thirty minutes after the administration
of amphetamine sulphate or at the time of the peak action of the drugs,
the activity was again recorded for 10 min. This activity was compared
with the pre-drug value and expressed as the mean (3 groups) percentage
change (Sethy and Sheth, 1968).
All the drugs were injected intraperitoneally (i.p.), once daily for
10 days. The concentration of drugs was such that animals received
1 ml/100 g of body weight. The dose, concentration and the time of
administration of amphetamine sulphate are given in Table 1. All the
drugs were dissolved in normal saline except meprobamate and chlordiazepoxide.
Results

Effect of Amphetamine with Pentobarbital Sodium on SMA. Normal


saline decreased activity by 11 to 18~ on daily administration for
10 days. Amphetamine sulphate (1.5 mg/kg) gradually increased SMA
from 18~ to 380/0, the peak effee~ being obtained on the 8th day.
Pentobarbital sodium decreased the activity by 8 to 16~ the effect was
lowest on the 7th and 8th days. Amphetamine in combination with
pentobarbital sodium produced higher activity than that produced by
amphetamine alone. The activity gradually increased and the maximum
effect (96~ was seen on the 9th day (Table 2).

Effect of d-Amphetamine Sulphate

21

Table 2. E]/ect o/ amphetamine sulphate (1.5 mg/kg i.p.) with pentobarbital sodium

(12 mg/kg i.p.) on spontaneous motor activity o/male mice


No. of
day

Normal saline
~ decrease in
activity

Amphetamine
~ increase in
activity

Pentobarbital
~ decrease in
activity

Amphetamine ~pentobarbital ~
increase in
activity

1
2
3
4
5
6
7
8
9
10

14
12
16
16
18
17
15
17
11
14

19
18
23
22
26
29
34
38
31
35

15
15
14
16
12
11
8
8
16
16

21
34
45
65
50
76
57
83
96
90

Table 3. E/]ect o/amphetamine sulphate (1.5 mg/kg i.p.) with chlorpromazine

(0.001 mg/kg) on spontaneous motor activity o/male mice


No. of
day

Normal saline
~ decrease in
activity

Amphetamine
~ increase in
activity

Chlorpromazine Amphetamine -~
~ decrease in chlorpromazine
activity
~ increase in
activity

i
2
3
4
5
6
7
8
9
10

14
11
12
14
15
12
14
17
11
12

17
19
30
36
37
39
42
33
37
47

9
4
7
8
3
8
6
3
12
8

33
55
68
64
71
80
80
82
87
84

E//ect o] Amphetamine with Chlorpromazine on S M A . N o r m a l saline


d e c r e a s e d SMA b y 11 t o 17~ on d a i l y a d m i n i s t r a t i o n . A m p h e t a m i n e
s u l p h a t e g r a d u a l l y i n c r e a s e d t h e a c t i v i t y f r o m 17 t o 470/0 . M a x i m u m
effect was seen on 10th d a y . C h l o r o p r o m a z i n e (0.001 m g / k g ) d e c r e a s e d
t h e a c t i v i t y (3 t o 12~
A m p h e t a m i n e in c o m b i n a t i o n w i t h CPZ p r o d u c e d
m o r e a c t i v i t y t h a n a m p h e t a m i n e alone in t h e s a m e dose. The a c t i v i t y
g r a d u a l l y i n c r e a s e d a n d t h e m a x i m u m effect (870/o) was seen on t h e
9 t h d a y (Table 3).
E/]ect o/ Amphetamine in Combination with Reserpine on S M A .
N o r m a l saline d e c r e a s e d a c t i v i t y b y 14 t o 250/0 . A m p h e t a m i n e increased t h e SMA g r a d u a l l y f r o m 22 t o 73~ T h e m a x i m u m effect was seen
on t h e 9 t h d a y . R e s e r p i n e (0.1 m g / k g ) d e c r e a s e d a c t i v i t y b y 18 t o 40~

22

V . H . SeShy, P. Y. Naik, and U. K. Sheth:

Table 4. Effect o/amphetamine sulphate (1.5 mg/kg i.p.) with reserpine (0.1 mg/kg)

on spontaneous motor activity o/male mice


No. of
day

Normal saline
~ decrease in
activity

Amphetamine
0/9 increase in
activity

Reserpine
~ decrease in
activity

Amphetamine ~reserpine ~
increase in
activity

1
2
3
4
5
6
7
8
9
10

17
14
17
18
20
21
25
18
18
25

33
22
36
41
53
60
67
66
73
73

22
31
18
21
19
23
26
32
40
32

31
28
60
78
85
122
168
232
219
259

Table 5. E//ect o/ amphetamine sulphate (1.5 mg/kg i.p.) with meprobamate

(25 mg/kg i.p.) on spontaneous motor activity o/male mice


No. of
days

Normal saline
~ decrease in
activity

Amphetamine
~ increase in
activity

~r
Amphetamine -b
~ decrease in meprobamate ~
activity
increase in activity

1
2
3
4
5
6
7
8
9
10

11
15
10
16
15
18
18
24
21
24

30
18
40
56
60
53
49
51
60
70

25
30
21
24
18
21
23
21
23
27

33
49
52
66
74
87
72
68
84
84

T h e c o m b i n a t i o n of a m p h e t a m i n e w i t h r e s e r p i n e p r o v e d m o r e effective
t h a n a m p h e t a m i n e alone. T h e effect g r a d u a l l y i n c r e a s e d a n d on t h e
10th d a y a c t i v i t y i n c r e a s e d b y 259~ (Table 4).
E//ect of Amphetamine in Combination with Meprobamate. N o r m a l
saline d e c r e a s e d a c t i v i t y b y 10 t o 24~ o n d a i l y a d m i n i s t r a t i o n . A m p h e t a m i n e i n c r e a s e d a c t i v i t y b y 18 to 70~
M e p r o b a m a t e (25 m g / k g )
d e c r e a s e d SMA b y 18 t o 300/0. A m p h e t a m i n e in c o m b i n a t i o n w i t h
m e p r o b a m a t e p r o d u c e d g r e a t e r effect t h a n t h e a m p h e t a m i n e alone.
H o w e v e r , t h i s increase was n o t s t a t i s t i c a l l y significant (Table 5).
E//ect o/Amphetamine with Chlordiazeproxide on S M A . N o r m a l saline
d e c r e a s e d S M A b y 10 t o 230/0 . A m p h e t a m i n e i n c r e a s e d t h e a c t i v i t y b y
31 t o 50~ t h e m a x i m u m effect being seen on t h e 1st d a y . Chlordiazepo x i d e (1 m g / k g ) d e c r e a s e d a c t i v i t y b y 5 t o 30~ on d a i l y a d m i n i s t r a t i o n

Effect of d-Amphetamine Sulphate

23

Table 6. E]/ect o/ amphetamine sulphate (1.5 mg/kg i.p.) with chlordiazepoxide

(1 mg/kg i.p.) on spontaneous motor activity o/ male mice


No. of
days

Normal saline
~ decrease in
activity

Amphetamine
~ increase in
activity

1
2
3
4
5
6
7
8
9
10

13
10
15
16
18
15
13
16
23
21

50
34
33
33
33
31
47
42
45
~

Chlordiazepoxide ~
decrease in
activity
5
11
10
14
13
15
21
30
28
24

Amphetamine -~
Chlordiazepoxide
~ increase in
activity
60
36
54
63
66
66
65
65
77
68

for 10 days. Amphetamine together with chlordiazepoxide produced


more effect than amphetamine alone. The activity increased b y 36 to
770/0 (Table 6).
Discussion

The results show t h a t a mixture of amphetamine (1.5 mg/kg) and


pentobarbital sodium (12 mg/kg) produced a considerable increase in
SMA in mice as compared to amphetamine alone. The activity gradually
increased and the m a x i m u m effect (960/0) was seen on the 9th day.
Goldstein et al. (1968) reported t h a t barbiturates increase the metabolism of amphetamine. However, our results show t h a t pentobarbital
probably does not increase the metabolism of amphetamine in mice when
given with it for 10 days. Potenbiation of the activity of amphetamine
with small doses of pentobarbital sodium m a y be due to a stimulant
action of subhypnotic dose of pentobarbital. I t has been suggested by
Miller (1961, 1963) t h a t small doses of barbiturate can reduce fear,
including fear of new environment, and m a y so lead indirectly to more
spontaneous activity and thus enhance the action of amphetamine.
An adoptation to behavioural depression induced b y chronically
administered reserpine in rabbits was reported b y Haggendal and Lindqvist (1964) and Pireh and Reeh (1968). Motor activity of rats treated
with reserpine daily for 10 days increased to approximately double the
values observed in saline treated rats (Pitch and Reeh, 1968). I n the
present s t u d y it has been shown t h a t reserpine decreased SMA, but there
was no development of adoptation in ehronicMly treated mice. An
increased excitatory effect of amphetamine on the CNS of reserpine
pretreated animals has been demonstrated (Smith, 1963, 1965; Quinton
and Halliwell, 1963; Stolk and Rech, 1967). I n the present work it has

24

V.H. Sethy, P. Y. Naik, and U. K. Sheth:

been shown t h a t amphetamine, when given together with reserpine


produced more effect on SMA than amphetamine alone. On chronic
treatment with this combination, the effect gradually increased and the
maximum effect was seen on the 10th day. Reserpine depletes both
norepinephrine (NE) and 5-hydroxytryptamine from the peripheral and
central nervous system (Brodie et al., 1956; Brodie and Shore, 1957).
Chronic interruption of normal transmitter function in the peripheral
nervous system results in receptor supersensitivity to certain drugs
(Trendelenburg, 1963; Sharpless, 1964). Similar alterations m a y also
occur in central adrenergic receptors following 'pharmacological denervation' induced by reserpine. This is manifested by the increase in response
to amphetamine in the reserpine pretreated mice. Similar results have
been reported with amphetamine and ephedrine, drugs which presumably
act centrally, as they do in the periphery by releasing N E from adrenergie
nerve terminals (Rech, 1968).
Low doses of chlorpromazine markedly enhanced the central action of
d-amphetamine (Babini et al., 1960; Sulser and Dingell, 1968; Stein,
1962). The measurement of d-amphetamine in brain revealed that both
low and high doses of chlorpromazine cause a marked and prolonged
elevation of d-amphetamlne levels (Sulser and Dingell, 1968 ; Borella et
al., 1969). Amphetamine in combination with chlorpromazinc produced
more effect on SMA than amphetamine alone. On chronic treatment with
this combination the effect gradually increased and was maximum on
the 9th day. The enhanced activity m a y be due to delayed metabolism
of amphetamine in mice pretreated with ehlorpromazine.
Chlordiazepoxide and meprobamate decreased spontaneous motor
activity. In the case of chlordiazepoxide, the percentage decrease in
activity increased with chronic treatment. Amphetamine in combination
with either meprobamate or chlordiazepoxide produced more effect than
the amphetamine alone. The effect gradually increased and was maximum
on the 9th day.
These results show t h a t small dose of CNS depressants can enhance
the action of amphetamine. The increase in amphetamine action b y
CNS depressants m a y be due to inhibition of fear and anxiety thus
leading to smooth and uniform increase in activity. These observations
on combined effects of CNS depressants and amphetamine seem to support the clinically beneficial effects of these drugs, when used in psychiatric practice.
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Effect of d-Amphetamine Sulphate

25

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-

Prof. U. K. Sheth
Department of Pharmacology
Seth G. S. Medical College
Parel, Bombay-12, India