The influence of piribedil (ET495) on components of locomotor activity

European Journal of Pharmacology, 33 (1975) 211--215 © North-Holland Publishing C o m p a n y , A m s t e r d a m -- Printed in The Netherlands

Short communication THE INFLUENCE OF PIRIBEDIL (ET495) ON COMPONENTS OF LOCOMOTOR ACTIVITY P E T E R J E N N E R and C. D A V I D M A R S D E N *

University Department of Neurology, Institute of Psychiatry and King's College Hospital, Medical School, Denmark Hill, London, SE5, U.K. Received 25 April 1975, accepted 21 May 1975

P. J E N N E R and C.D. M A R S D E N , The influence of piribedil (ET495) on components of locomotor activity, E u r o p e a n J. Pharmacol. 33 (1975) 211--215. R e c e n t evidence suggests piribedil affects b o t h cerebral NA neurones and DA neurones and receptors since it increases brain MOPEG-SO4, an observation c o n f i r m e d presently. In the reserpinised mouse piribedil and a p o m o r p h i n e caused a reversal of akinesia, which was significantly enhanced by c o n c u r r e n t administration of clonidine. However, piribedil neither reduced nor increased the m o t o r effects of a p o m o r p h i n e or L-DOPA in the reserpinised mouse, but slightly enhanced that of amphetamine. P h e n o x y b e n z a m i n e reduced the m o t o r action of L-DOPA but had no effect on that of a p o m o r p h i n e or amphetamine. These results suggest piribedil has no significant NA r e c e p t o r blocking or stimulating action in the reserpinised animal. T h e y confirm the view that piribedil increases cerebral NA turnover due to a presynaptic action on NA neurones. Piribedil

MOPEG-SO4

M o t o r activity

1. Introduction Piribedil (ET 495; 1-[3,4-methylenedioxybenzyl]-4-[2-pyrimidyl]-piperazine) has been suggested as potentially useful in the treatment of Parkinson's disease because of its ability to stimulate central dopaminergic (DA) receptors. Evidence for the latter is provided by the capacity of piribedil (a) to cause contraversive turning behaviour in rats with unilateral 6-hydroxydopamine-induced degeneration of one nigro-striatal pathway (Corrodi et al., 1971); (b) to decrease dopamine turnover (Corrodi et al., 1972) and to decrease striatal homovanillic acid levels (Jori et al., 1974); (c) to decrease firing in substantia nigra neurones (Waiters et al., 1975); and (d) to induce stereotypy in rats (Costall and Naylor, 1973). Further, piribedil causes an increase in striatal acetylcholine content that is selectively

* To w h o m all c o r r e s p o n d e n c e should be addressed.

blocked by pimozide (Consolo et al., 1975). These data are interpreted by analogy to results obtained with apomorphine, as indicating that piribedil directly stimulates DA receptors. However, since piribedil does not stimulate the dopamine sensitive adenylate cyclase system in homogenates of rat striatum (Miller and Iversen, 1974) or produce contralateral turning when injected directly into one striatum of reserpine-treated rats (Fuxe et al., 1972), an active metabolite has been suggested. Indeed a metabolite $584 (1-[3,4-dihydroxybenzyl]- 4- [ 2- pyrimidyl]- piperazine) has been shown to be (a) active in stimulating dopamine sensitive adenylate cyclase (Miller and Iversen, 1974); (b) capable of inducing stereotypy (Costall a n d Naylor, 1974) and locomotor activity (Creese, 1974) and turning behaviour (Poignant et al., 1974); and (c) capable of decreasing striatal homovanillic acid concentration (Jori et al., 1974). Other studied metabolites of piribedil do not appear to be responsible for its actions but may have

212

opposite antagonistic effects (Costall and Naylor, 1974). In addition to a direct DA postsynaptic action, evidence for a presynaptic DA action of piribedil (or its metabolites) stems from the finding that interruption of DA pathways inhibits piribedil-induced stereotypy (Costall and Naylor, 1973). Doubt has recently been cast on the relative specificity of action of piribedil as a dopaminergic agonist by the finding of enhanced noradrenaline (NA) turnover in rat (Garattini et al., 1974) as indicated by an increase in brain concentration of 3-methoxy-4~hydroxyphenylethyleneglycol sulphate (MOPEG-SO4), the main metabolite of noradrenaline (Schanberg et al., 1968). In view of this finding we have investigated the effect of piribedil on drug-induced motor behaviour in which both DA and NA components are believed to be involved. We have studied the ability of piribedil to affect the locomotor activity produced by administration of apomorphine, amphetamine, and L-DOPA to reserpinised mice. The effect of clonidine, a NA agonist, on the motor action of piribedil in reserpinised mice has also been investigated.

P. JENNER, C.D. M A R S D E N

or without piribedil (50 mg/kg; Servier Laboratories Ltd.). Activity was also measured following i.p. administration of clonidine (1 mg/kg; Boehringer Ingelheim Ltd.) or piribedil (50 mg/kg) and following administration of either apomorphine (5 mg/kg) or piribedil (50 mg/kg) plus clonidine (1 mg/kg). In some experiments animals were pretreated i.p. with phenoxybenzamine (20 mg/kg; SKF Ltd.) 60 min prior to the start of the experiment. Following injection of drugs, batches of 3 mice were placed in the counting cages and locomotor activity was measured for the time periods indicated in table 1. At the same time, the locomotor activity of control animals injected with 0.1 ml N saline i.p. was measured for the same time period in another counting cage. Whole brain levels of MOPEG-SO4 were measured using the fluorometric technique of Meek and Neff (1972) in normal male Wistar rats (275--325 g). The animals were pretreated with saline (0.2 ml) or piribedil (50 mg/kg) 2.5 hr prior to death and removal of the brain.

3. Results 2. Materials and methods Locomotor activity was measured using Animex activity meters (LKB-Farad L t d . ) w i t h groups of three mice housed in clear perspex cages under conditions of continuous laboratory lighting. Food and water were withdrawn during the test period. Male Swiss Albino mice (20--25 g; Animal Suppliers Ltd.) were pretreated with reserpine (10 mg/kg i.p.; Halewood Chemicals Ltd.) 18-24 hr prior to the experiments. Locomotor activity was recorded during the period of activity following the i.p. administration of L-DOPA (200 mg/kg; Roche Products Ltd.) plus the peripheral decarboxylase inhibitor ~methyldopa hydrazine (MK 486; 25 mg/kg; Merck, Sharp and Dohme Ltd.) or (+)-amphetamine sulphate (4 mg/kg; SKF Ltd.)or apomorphine (2 or 5 mg/kg; Evans Medical Ltd.) with

Piribedil (50 mg/kg) produced a small but significant short-lived reversal of reserpine akinesia in mice (fig. 1; table 1). Clonidine (1 mg/kg) by itself, produced a slight bu insignificant reversal of akinesia. A combination of piribedil plus clonidine, however, produced a marked reversal of reserpine akinesia of 2--3 hr duration which could not be accounted for by a simple summation of activities (fig. 1; table 1). Apomorphine (5 mg/kg) induced reversal of reserpine akinesia was similarly enhanced by clonidine (1 mg/kg) in agreement with the previous observations of And~n et al. (1973). Locomotor activity induced by L-DOPA (200 mg/kg) plus MK 486 (25 mg/kg) and apomorphine (2 mg/kg) was unaffected by the simultaneous administration of piribedil (50 mg/kg). Pretreatment of animals with phenoxybenzamine (20 mg/kg) significantly reduced L-

INFLUENCE OF PIRIBEDIL ON LOCOMOTOR ACTIVITY

ET495 •~= 1400 O (D t-

~ 1000

l

0

lk~

r

-0-

ET 495 plus CLONIDINE

X

213

Fig. 1. Effect of piribedil (ET 495; 50 mg/kg) and clonidine (1 mg/kg) alone and in combination, on reserpine akinesia in mice. Locomotor activity was measured over 10 min intervals for 6 hr. The mean number of counts/10 min is shown for 4 batches of 3 animals in each experiment (-+1 S.E.M). Reserpine (10 mg/kg) was administered 18--24 hr prior to recording. Drugs were administered 2 hr after recording commenced.

-Z 600 <

z<

200 l

1

Drugs admimstered

2

3

4

TIME (h.)

DOPA-induced motor activity b u t had no effect on amphetamine or apomorphine-induced reversal of reserpine akinesia (table 1). Whole brain levels of MOPEG-SO4 in rats were found to be significantly elevated 2.5 hr after piribedil (50 mg/kg) administration (con-

TABLE 1 Effect of piribedil, clonidine, apomorphine, amphetamine and L-DOPA singly and in combination on locomotor activity in reserpinised mice (10 mg/kg i.p. 18--24 hr previously)• All drugs were administered i.p., and motor activity of batches of three mice were measured for the subsequent time period indicated•

Drug and dose (mg/kg)

Period of measurement (min)

Animex counts* (+1 S.E.M.)

Controls (saline 0.1 ml) Controls (saline 0.1 ml) Controls (saline 0.1 ml)

60 120 240

Piribedil 50 Clonidine 1 Piribedil 50 + clonidine 1 Apomorphine 5 Apomorphine 5 + clonidine 1

240 240 240 120 120

2123 968 9454 8082 15297

-+ 224 + 349 -+ 2393 + 339 + 525

Apomorphine 2 Apomorphine 2 + piribedil 50 Amphetamine 4 Amphetamine 4 + piribedil 50 L-DOPA 200 L-DOPA 200 + piribedil 50

120 120 120 120 240 240

7099 8182 9441 10758 21348 21003

+ 402 ( 8 ) , + 480 (8)v + 448 (8).+ -+ 3/35 ( 8 ) v + 1486 (12). -+ 807 (10)T

Apomorphine 2 Apomorphine 2 + PBZ 20 Amphetamine 4 Amphetamine 4 + PBZ 20 L-DOPA 200 L-DOPA 200 + PBZ 20

60 60 120 120 240 240

4341 -+ 333 4193 + 197 10467 + 619 9181 + 1171 13862-+ 2213 8087 + 1752

113 -+ 229 + 342 +

25 (6) 63 (6) 63 (6) (4) (4) (4)** (3) (4)***

(4). (4)t" (4) (4)t ((~)) t"tt

* Number of batches of 3 animals used shown in parentheses. ** p < 0.025 compared to piribedil 50 mg/kg (Student's t-test). *** p < 0.001 compared to apomorphine 5 mg/kg. t p > 0.05 compared to apvmorphine 2 mg/kg, L-DOPA 200 mg/kg or amphetamine 4 mg/kg. t t p < 0.05 compared to amphetamine 4 mg/kg. t t t p < 0.05 compared to L-DOPA 200 mg/kg.

214

trols (6) 154.1 -+ 14.0 ng/g, piribedil-treated 314.1 + 16.7 ng/g; p < 0.001) in agreement with the findings of Garattini et al. (1974).

4. Discussion The ability of clonidine, a central noradrenergic receptor stimulant (And~n et al., 1970) to enhance the reversal of reserpine akinesia by both the DA agonists piribedil and apomorphine indicates that maximal reversal of reserpine akinesia can be obtained by concurrent stimulation of both DA and NA receptors, as suggested by And~n et al. (1973). The fact that phenoxybenzamine reduces the ability of LDOPA to reverse reserpine akinesia is consistent with the view that L-DOPA-induced locom o t o r activity in the reserpinised rodent is due to concurrent stimulation of both DA and NA receptors (Ahlenius and Engel, 1971), and with the finding of a significant increase in brain NA levels as well as DA levels, with L-DOPA in the reserpinised animal (Ahlenius and Engel, 1971). The failure of phenoxybenzamine to attenuate the amphetame reversal of reserpine akinesia may indicate that there is insufficient NA available for release after reserpine. The finding that clonidine enhances the l o c o m o t o r effect of piribedil in the reserpinised mouse suggests that the latter does not significantly block cerebral NA receptors. Further, the inability of piribedil (or its metabolites) to attenuate the l o c o m o t o r activity induced by apomorphine, amphetamine or L-DOPA suggests that this c o m p o u n d does n o t prevent stimulation of either DA or NA receptors by such directly acting or indirectly acting agonists. It is perhaps surprising that piribedil, in view of its ability to stimulate DA receptors, did n o t enhance l o c o m o t o r activity caused by apomorphine and L-DOPA in the reserpinised animal, b u t probably these agonists had already achieved maximum DA receptor stimulation. Piribedil did slightly enhance the l o c o m o t o r effect of amphetamine, possibly by additional direct DA receptor stimulation. The failure of piribedil to enhance the l o c o m o t o r effect of

P. JENNER, C.D. MARSDEN

apomorphine in these experiments also indicates that piribedil does not significantly stimulate NA receptors, in contrast to the effect of clonidine. These results must be interpreted in the light of the discovery that piribedil increases the cerebral formation of MOPEG-SO4 in the rat (Garattini et al., 1974, confirmed in the present study). They suggest that piribedil neither directly blocks nor directly stimulates cerebral NA receptors to any significant degree in the reserpinised mouse. The most reasonable explanation for the effect of piribedil on MOPEGSO4 is that the drug has a pre-synaptic action on NA neurones causing increased NA release and metabolism and consequently a rise in brain MOPEG-SO4 concentration, as suggested by Consolo et al. (1975). Such an effect could have little influence on l o c o m o t o r activity in the reserpinised animal, in which little or no NA is available for release as indicated by the failure of phenoxybenzamine to attenuate the l o c o m o t o r effect of amphetamine. A pre-synaptic action of piribedil on DA neurones, in addition to its direct DA agonist action, has already been proposed (Costall and Naylor, 1973).

Acknowledgements This work was supported by the Medical Research Council, the Research Funds of the Bethlem Royal and Maudsley Hospitals, and the Research Funds of King's College Hospital. We are grateful to Roche Products Ltd. and Servier Laboratories Ltd. for supplies of drugs.

References Ahlenius, S. and J. Engel, 1971, Behavioural and biochemical effects of L-DOPA after inhibition of dopamine-~-hydroxylase in reserpine-pretreated rats, Nauyn-Schmiedeb. Arch. Pharmacol. 270, 349. And~n, N.-E., H. Corrodi, K. Fuxe, B. HSkfelt, T. HSkfelt, C. Rydin and T. Svensson, 1970, Evidence for a central noradrenaline receptor stimulation by clonidine, Life Sci. 9, Part I, 513.

INFLUENCE OF PIRIBEDIL ON LOCOMOTOR ACTIVITY And~n, N.-E., U. Strombom and T.H. Svensson, 1973, Dopamine and noradrenaline receptor stimulation: reversal of reserpine-induced suppression of motor activity, Psychopharmacologia (Berlin) 29, 289. Consolo, S., R. Fanelli, S. Garattini, D. Ghezzi, A. Jori, H. Ladinsky, V. Marc and R. Samanin, 1975, Dopaminergic--cholinergic interaction in the striaturn: studies with piribedil, in: Advances in Neurology, Vol. 9, eds. D. Calne, T.N. Chase and A. Barbeau (Raven Press, New York) p. 257. Corrodi, H., L. Farnebo, K. Fuxe, B. Hamberger and U. Ungerstedt, 1972, ET 495 and brain catecholamine mechanisms: evidence for stimulation of dopamine receptors, European J. Pharmacol. 20, 195. Corrodi, H., K. Fuxe and U. Ungerstedt, 1971, Evidence for a new type of dopamine receptor stimulating agent, J. Pharm. Pharmacol. 23, 989. Costall, B., and R.J. Naylor, 1973, The site and mode of action of ET 495 for the mediation of stereotyped behaviour in the rat, Nauyn-Schmiedeb. Arch. Pharmacol. 278, 117. Costall, B. and R.J. Naylor, 1974, Dopamine agonist and antagonist activities of piribedil (ET 495) and its metabolites, Nauyn-Schmiedeb. Arch. Phai, macol. 285, 71. Creese, I., 1974, Behavioural evidence of dopamine receptor stimulation by piribedil (ET 495) and its metabolite $584, European J. Pharmacol. 28, 55. Fuxe, K., H. Corrodi, L.O. Farnebo, B. Hamberger and U. Ungerstedt, 1972, On the neuropharmacology of ET 495, in: Proc. Intern. Symp. on Trivastal, Monastir.

215

Garattini, S., S.R. Bareggi, V. Marc, G. Calderini and P.L. Morselli, 1974, Effects of piribedil on noradrenaline and MOPEG-SO4 levels in the rat brain, European J. Pharmacol. 28, 214. Jori, A., G. Cecchetti, E. Dolfini, E. Monti and S. Garattini, 1974, Effect of piribedil and one of its metabolites on the concentration of homovanillic acid in the rat brain, European J. Pharmacol. 27, 245. Meek, J.L. and N.H. Neff, 1972, Fluorometric estimation of 4-hydroxy-3-methoxyphenyl-ethyleneglycol sulphate in brain, Brit. J. Pharmacol. 45, 435. Miller, R.J. and L.L. Iversen, 1974, Stimulation of a dopamine-sensitive adenylate cyclase in homogenates of rat striatum by a metabolite of piribedil (ET 495), Nauyn-Schmiedeb. Arch. Pharmacol. 282, 213. Poignant, J.-C., F. Lejeune, E. Malecot, M. Petitjean, G. Regnier and R. Canevari, 1974, Effets compares du piribedil et de trois de ses m~tabolites sur le syst~me extrapyramidal du rat, Experientia 30, 70. Schanberg, S.M., J.J. Schildkraut, G.R. Breese and I.J. Kopin, 1968, Metabolism of normetanephrine-H3 in rat brain. Identification of conjugated 3-methoxy-4-hydroxyphenyl-glycol as the major metabolite, Biochem. Pharmacol. 17, 247. Waiters, J.R., B.S. Bunney and R.H. Roth, 1975, Piribedil and apomorphine: pre- and postsynaptic effects on dopamine synthesis and neuronal activity, in: Advances in Neurology 9, eds. D. Calne, T.N. Chase and A. Barbeau (Raven Press, New York) p. 273.