A comparison of the locomotor stimulant properties of amantadine and l- and d-amphetamine in mice

Neuropharmacology,

1972,

I I, 675-682

Pergamon

Press.

Printed

in Cit. Britain

A COMPARISON OF THE LOCOMOTOR STIMULANT PROPERTIES OF AMANTADlNE AND I- AND d-AMPHETAMINE IN MICE” J. E. THORNBURG and K. E. MOORE Department of Pharmacology, Michigan State University, East Lansing, Michigan 48823 (Accepted

15 January 1972)

Summary-Amantadine,

administered intraperitoneally at doses of 20, 40, 80 and 160 mg/kg, increased locomotor activity in mice with peak stimulation at 1 hr; maximal stimulation produced by this drug was only one-tenth of that caused by I- or d-amphetamine. A 4 hr diet containing 0.4% DL-a-methyhyrosine, an inhibitor of catecholamine synthesis, did not alter amantadine-stimulated motor activity but completely blocked the stimulant effects of I- and d-amphetamine. Pretreatment with a monoamine oxidase inhibitor (pheniprazine) did not alter amantadine-stimulated motor activity, and amantadine pretreatment did not influence damphetamine-stimulated locomotor activity. Thus, amantadine, in contrast to the amphetamines, produces a very weak stimulation of locomotor activity which is not dependent upon brain catecholamine synthesis. AMANTADINE(I-adamantanamine hydrochloride, Symmetrel), which appears to be efficacious in ameliorating some symptoms of Parkinson’s disease (SCHWAB et al., 1969; KELLY and ABUZZAHAB, 1971; PARKES et al., 1970), has weak motor stimulant properties (VERNIER et al., 1969). In addition, amantadine inhibits norepinephrine (NE) and dopamine (DA) uptake by rat brain tissue (FLETCHER and REDFERN, 1970; THORNBURG and MOORE, 1971a), and releases DA from rat striatal tissues in vitro (SCATTON et al., 1970) and from cat brain in vivo (VON VOIGTLANDER and MOORE, 1971). Although the mechanism of the anti-parkinsonism effect is unknown, the above findings suggest that amantadine may enhance dopaminergic transmission processes in the corpus striatum. A similar mechanism has been postulated for amphetamine stimulation of motor activity in mice (CARLSSON, 1970) and rats (GROPPETTI et al., 1970; COSTA and GROPPETTI, 1970), although other studies in rats suggest that NE, rather than DA, mediates the stimulant effects of amphetamine (TAYLOR and SNYDER, 1971). The purposes of this study were to determine the dose and time relationships of amantadine effects on motor activity and to test the possibility that brain DA and/or NE are involved

in the stimulant actions of this drug. For this purpose the responses to amantadine are compared to those of d- and I-amphetamine. METHODS

Male albino mice (Spartan Farms) weighing 20-25g were housed 6 per cage in an airconditioned, light-controlled room (light from 7 a.m. to 7 p.m.) and provided standard chow and water ad lib. Four hours prior to behavioral testing, which was conducted between 12 *Presented in part at the Fall Meeting of the American Society for Pharmacology and Experimental Therapeutics at Burlington, Vermont, August 1971 (P/zarmac&gist 13, 202, 1971). Supported by USPHS Grant NS 09174 and MH 13174, 675

616

J. E.

THORNBURG and K. E. MOORE

noon and 5 p.m., food but not water was removed from the home cages. When the effects of DL-a-methyltyrosine (a-MT) were examined, mice were acclimated to eating for only 4 hr each morning. On the day of testing the mice received their normal diet (ground Wayne Lab-Blox) or the same diet containing 0.4 % a-MT. All other drugs were injected i.p. (0.01 ml/g) in saline, and the doses are reported as the respective drug salts. The drugs used were: DL-a-methyltyrosine (Regis Chemical Co., Chicago Ill.); amantadine HCl (from Dr. V. G. Vernier, E. I. du Pont de Nemours and Co., Wilmington, Del.); pheniprazine HCl (from Dr. R. C. Ursillo, Lakeside Laboratories, Milwaukee, Wise.) and d-and l-amphetamine sulfate (from Mr. G. W. French, Smith, Kline and French Laboratories, Philadelphia, Pa.). For motor activity measurements, two mice were placed in a circular actophotometer cage (Woodard Research Corp.) for a 20 min acclimation period. They were then injected with a drug or saline and returned to the same actophotometer cage. Motor activity counts were recorded at 20 min intervals for 2-3 hr and plotted as drug-stimulated activity (the difference between actual recorded counts and the appropriate saline or a-MT control values). Data were analyzed using Student’s t-test (GOLDSTEIN,1964); the level of significance was chosen as P < O-01. RESULTS The time course of effects of 20, 40, 80 and 160 mg/kg amantadine on motor activity is shown in Fig. 1. With each dose, peak stimulation occurred 40-60 min after injection. Although 40 mg/kg amantadine caused maximal peak stimulation, 80 mg/kg had a markedly

TIME

AFTER

AMAHTADINE

lmin)

FIG. 1. Dose and time relationships of amantadine effects on motor activity. Two mice were acclimated to an activity cage for 20 min. Following the injection of amantadine or saline, motor activity was recorded at 20 min intervals for 2-3 hr. Values for drug-stimulated motor activity (n= 11-18) represent differences between total counts/20 min in amantadine-treated animals and the respective activity in saline-treated (control) animals (n=25). Mean values for salinetreated mice in each successive 20 min-period were 835, 297,99, 139, 171 and 121 counts. The respective symbols and doses are: A, 20 mg/kg; 0, 40 mg/kg; q,80 mg/kg; 0,160 mg/kg of amantadine. Solid symbols represent values significantly different from control (P < 0.01).

Amantadine-induced

stimulation

677

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FIG. 2. Dose-response relationships of cumulative amantadine-, d- or I-amphetamine-stimulated motor activity. Drug stimulated motor activities (n= 12-18) represent mean differences between total 20-120 min counts in drug-treated mice and the mean activity in saline-treated mice (835 counts). Solid symbols indicate a significant degree of stimulation (P
1600

0

-100

0

20

40

60 .TlME

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(min

100

120

)

FIG. 3. Comparison of the onset and duration of amantadine- and d-amphetamine-stimulated motor activity. Drug-stimulated motor activities (n= 12-18) represent mean differences between total counts/20 rnin-period in drug-treated mice and respective mean activities in saline-treated mice. Symbols and corresponding doses are: 0, amantadine, 80 mg/kg; 0, d-amphetamine, 1 mg/kg; & d-amphetamine, 2 mg/kg. Solid symbols represent values significantly different from control (PC 0.01).

J. E. THORNBURG and K. E. MOORE

618 longer duration

of action. An initial suppression

mg/kg, a dose approaching

of motor activity was observed after 160

the LD,, of amantadine in mice (VERNIERet al., 1969). The initial

control activity after saline administration

was 835 and 297 counts for the 1st and 2nd 20 min

intervals, respectively. Thus amantadine-induced

stimulation during the initial 40 min could

have been masked by the relatively high basal exploratory

activity. In experiments where the

acclimation period was extended to 100 min the initial post-saline activity was reduced by more than 50 %; nevertheless, the time of onset of significant amantadine-induced stimulation was not altered. Dose-response curves for amantadine and d- and l-amphetamine on cumulative motor activity are depicted in Fig. 2. Maximal motor activity after amantadine is approximately l/IO that elicited by either amphetamine

enantiomer

(Fig. 2). d-Amphetamine

was 5-times

more potent than the I-isomer, a finding in agreement with the previous work of RECH and STOLK (1970) in rats. When compared

with d-amphetamine,

amantadine-stimulated

activity

is slower in onset but longer in duration. This contrast is particularly striking when the effects of 2 mg/kg d-amphetamine and 80 mg/kg of amantadine are compared (Fig. 3). a-MT blocks amphetamine-stimulated motor activity in mice suggesting that amphetamine acts by releasing newly synthesized catecholamines

at central synaptic receptor sites

(Weissman et al., 1966; DOMINICand MOORE, 1969). Catecholamines

have also been implica-

ted in the actions of amantadine (SCATTONet al., 1970; VON VOIGTLANDERand MOORE, 1971), but the results of the experiment summarized in Fig. 4 demonstrate does not alter amantadine-stimulated to minimize nonspecific

irritating effects resulting from the i.p. injection peritoneal

taneous locomotor containing

irritation increases plasma corticosterone

of this insoluble

drug;

this

levels and depresses spon-

activity (THORNBURG and MOORE, 1971b). One hour after a 4 hr diet

0.4 y0 a-MT,

activity recorded

that a-MT-pretreatment

motor activity. a-MT was administered in the diet so as

brain NE and DA contents were significantly

from 20-120

min after saline injection

reduced, and motor

was reduced by 50%

(Table

1).

TABLE1. EFFECT OFA 4-hr, 0.4 % a-METHYL~ROSINE (a-MT)-CONTAINING DIET ON MOUSE BRAWN NE AND DA CONTENTS, PLASMA CORTICOSTERONE AND MOTOR ACTIVITY Diet N

Control

a-MT

Brain NE &g/g)

4

040*0~02

0.22I-to.02

Brain DA @g/g)

4

0~58ztO~O3

0.29kO.02

Corticosterone

4

0~11*0~01

0~09+0~01

18

835&90

(pg/ml)

Motor Activity (counts)

426580 --

Mice (8 per cage) were presented 40 g of ground Wayne Lab-Blox alone or containing 0.4% a-MT for 4 hr. One hour after removing the diet, the animals were sacrificed, and brain NE and DA contents determined according to the method of MOOREand RECH(1967). Plasma corticosterone values measuredusing the procedure of GUILLEMM et al. (1958). Motor activity values representmean 20-120 min counts. Underlined values are statisticallydifferent from control (P< 0.01). Moreover, plasma corticosterone levels were not altered after the a-MT diet (Table 1,) suggesting that administration of a-MT in this manner effectively minimized any nonspecific stressful response. The 4 hr diet of a-MT did not alter motor stimulation elicited by 80 mg/kg of amantadine, although the responses to 1 and 2 mg/kg of d-amphetamine and to 5 mg/kg of

Amantadine-inducedstimulation

679

I-amphetamine were completely blocked (Fig. 4). Note that 80 mg/kg of amantadine produced approximately the same degree of stimulation as 1 and 5 mg/kg of d- and I-amphetamine, respectively. 5400

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AMANTADINE 80

FIG. 4. Effect of a-methyltyrosine (a-MT) on amantadine- and amphetamine-stimulated motor activity. A 0.4% diet of a-MT was presented to mice for the 4 hr-period immediately preceding testing. Drug-stimulated activities in control and a-MT fed mice are indicated by open and solid bars, respectively. The mean 20-120 min values for saline-treated mice on control and a-MT diet were 835 and 426 counts, respectively. Each bar represents the mean Z!ZS.E.M. of 12-18 determinations.

In vitro amantadine blocks the uptake of DA and NE by crude synaptosomal preparations of rat corpus striatum and telencephalon, respectively (THORNBURG and MOORE,1971a). d-amphetamine may act by releasing brain catecholamines (CARR and MOORE, 1971a). or by blocking retrieval of these amines by catecholaminergic nerve terminals (TAYLORand SNYDER,1971). If amantadine blocks reuptake of NE or DA released by amphetamine or adds to an amphetamine blockade of catecholamine reuptake, the two drugs may have additive or supra-additive effects. Nevertheless, as seen in Fig. 5, amantadine at doses of 20 and 40 mg/kg had no significant effects on d-amphetamine-stimulated motor activity. Pheniprazine pretreatment enhances the stimulant actions of d-amphetamine in mice probably by slowing the rate of metabolism of the latter drug (LEW et al., 1971). Amantadine, however, is not extensively metabolized in mice (BLEIDNERet al., 1965), and in the present F

680

J. E. THORNBURGand IL E. MOORE

study 24 hr pretreatment with pheniprazine had no significant effect on spontaneous or amantadine-stimulated motor activity (Table 2).

AMANTADINE A-AMPH

a0 -

1

20 1

40

-

-

11

40

40

-

40

(ms/kg)

-

z

2

(mu/kg)

FIG. 5. EfIect of amantadine pretreatment on d-amphetamine-stim~iated motor activity. Mice were injected with either amantadine or saline, and then accommodated to the actophotometer cages for 20 min. They were then injected with saline or d-amphetamine and returned to the cages for 2 hr. The bars are mean differences between total counts from 20 to 120 min in drugtreated (n= 12) and saline-treated mice (n= 12). There were no significant differences between the activities of mice treated with amantadine-d-amphetamine combinations and with d-amphetamine alone.

TABLE2. EFFECTOF PHEN~~AZINEPRETREATMENT ON A~~ANTADINE-S~~V~ULATE~ MOTORAC~VITY Pretreatment

Saline Pheniprazine

N

Before amantadine (counts/l0 min)

After amantadine (counts/100 min)

8

544132

1370&3f6

12

643+27

1514+266

Mice received i.p. injections of either saline or pheniprazine 24 hr prior to the start of the experiment. Two pheniprazine- or saline-pretreated mice were placed in each actophotometer cage, and after 10 min of acclimation activity was recorded for 10 min. The mice were then removed from the cage, injected with amantadine (80 mg/kg i.p.), and returned to the same actophotometer cage. Amantadine-stimulated motor activity was recorded from 20 to 120 min after injection. DISCUSSION

Comparison of the motor activity stimulant properties of amantadine and I- and damphetamine reveals several major differences. Although each drug caused a dose-related stimulation, maximal amantadine-stimulated activity was only l/l0 that produced by either

Amantadine-induced

stimulation

681

amphetamine enantiomer. A true potency ratio could not be determined due to lack of parallelism of the dose-response curves. In contrast to the immediate onset of amphetamine stimulation, significant amantadine-stimulated activity was not apparent until 20-40 min after the drug. High doses of amantadine caused marked hyperreflexia but never amphetamine-like stereotypy in either mice or rats (unpublished observations; VERNIERet a/., 1969; NAYLORand COSTALL,1971). Moreover, high doses of amphetamine never elicit an initial depression of motor activity as was observed after 160 mg/kg amantadine. d-amphetamine-stimulated motor activity in both rats and mice is blocked by pretreatment with a-MT, an inhibitor of catecholamine synthesis (WEISSMAN et al., 1966; DOMINIC and MOORE,1969). Results from the present study demonstrate that central stimulation by l-amphetamine is also blocked by a-MT pretreatment. Although the relative importance of brain NE and DA in amphetamine-stimulated motor activity remains a point of controversy (TAYLORand SNYDER,1971; CARLSSON, 1970), recent evidence favors a primary role for DA. COSTAand GROPPETTI(1970) reported that d-amphetamine increased the turnover of DA but not NE in rat brain; moreover, the time course ofincreased DA turnover paralleled the period of increased motor activity. In mice, inhibition of dopamine-Shydroxylase with U-14,624 (1-phenyl-2-[2-thiazolyl-2-thiourea]) markedly depletes brain NE contents but does not significantly alter the enhanced motor activity following d-amphetamine (THORNBURG, 1972). Amantadine and d-amphetamine appear to produce motor stimulation by different mechanisms, since a-MT failed to alter amantadine stimulation. FIBIGERet al. (1971) recently stated that a-MT blocked amantadine stimulation of motor activity in rats, although the absence of data supporting their statement precludes further comment. The failure of cc-MT to alter amantadine-stimulated motor activity strongly suggests that newly synthesized brain catecholamines are not required for this action. Nevertheless, there is evidence that amantadine may alter reuptake or release of brain catecholamines. At 1-5 x 1O-6 M concentrations in vitro, amantadine inhibits uptake of NE and DA by brain tissue preparations (FLETCHERand REDFERN,1970; THORNBURG and MOORE,1971a). Striatal tissue from amantadine-pretreated rats shows an increased formation of DA from labeled tyrosine with concomitant enhanced release of DA into the medium (SCATTONet al., 1970). In the cat, amantadine causes enhanced release of DA into a cerebroventricular perfusate from the adjacent brain tissue, primarily caudate nucleus (VONVOIGTLANDER and MOORE,1971). The implication of these data that the effectiveness of amantadine in parkinsonian patients could be due to blockade of reuptake of brain catecholamines is, indeed, tempting in view of the known impairment of the dopaminergic nigrostriatal pathway in parkinsonian patients. Amantadine is much less potent than I- or d-amphetamine in inhibiting DA uptake by striatal tissue (THORNBURG and MOORE,1971a), but it is also a comparatively weaker CNS stimulant -a major advantage in chronic use of a drug in parkinsonian patients. Acknowledgement-The

excellent technical assistance of

Mrs. M. GRAMATMS is gratefully acknowledged.

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682

J. E. THORNBURG

and K. E. MOORE

COSTA,E. and GROPPETTI,A. (1970). Biosynthesis and storage of catecholamines in tissues of rats injected with various doses of d-amphetamine. In: Amphetamines and Related Compounds (COSTAE. and GARATTINI, S., Eds.), pp. 231-255, Raven Press, New York. DOMINIC,J. A. and MOORE,K. E. (1969). Acute effects of a-methyltyrosine on brain catecholamine levels and on spontaneous and amphetamine-stimulated motor activity in mice. Archs int. Pharmacodyn. The?. 178: 166-176.

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effects following inhibition of tyrosine