Ronald K.B. Pearce

Combined Use of the Adenosine A2A Antagonist KW-6002 with l-DOPA or with Selective D1 or D2 Dopamine Agonists Increases Antiparkinsonian Activity but Not Dyskinesia in MPTP-Treated Monkeys

The novel selective adenosine A(2A) receptor antagonist KW-6002 improves motor disability in MPTP-treated parkinsonian marmosets without provoking dyskinesia. In this study we have investigated whether KW-6002 in combination with l-DOPA or selective D1 or D2 dopamine receptor agonists enhances antiparkinsonian activity in MPTP-treated common marmosets. Combination of KW-6002 with the selective dopamine D2 receptor agonist quinpirole or the D1 receptor agonist SKF80723 produced an additive improvement in motor disability. Coadministration of KW-6002 with a low dose of L-DOPA also produced an additive improvement in motor disability, and increased locomotor activity. The ability of KW-6002 to enhance antiparkinsonian activity was more marked with L-DOPA and quinpirole than with the D1 agonist. However, despite producing an enhanced antiparkinsonian response KW-6002 did not exacerbate L-DOPA-induced dyskinesia in MPTP-treated common marmosets previously primed to exhibit dyskinesia by prior exposure to L-DOPA. Selective adenosine A(2A) receptor antagonists, such as KW-6002, may be one means of reducing the dosage of L-DOPA used in treating Parkinsons disease and are potentially a novel approach to treating the illness both as monotherapy and in combination with dopaminergic drugs.

Alterations in preproenkephalin and adenosine-2a receptor mRNA, but not preprotachykinin mRNA correlate with occurrence of dyskinesia in normal monkeys chronically treated with L-DOPA.

Chronic treatment with l‐DOPA induces dyskinesia in patients with Parkinsons disease (PD) and 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐treated monkeys, but is not thought to do so in normal humans or primates. However, we have shown that chronic oral high dose l‐DOPA administration, with the peripheral decarboxylase inhibitor, carbidopa and with or without the peripherally acting catechol‐O‐methyl transferase (COMT) inhibitor, entacapone, to normal macaque monkeys for 13 weeks induced dyskinesia in a proportion of animals. In the present study, in situ hybridization histochemistry was used to investigate the effect of chronic l‐DOPA administration on the activity of the direct and indirect striatal output pathways by measuring striatal preprotachykinin (PPT), preproenkephalin‐A (PPE‐A) and adenosine‐2a (A2a) receptor gene expression in these monkeys. Overall there was no significant difference in striatal PPT, PPE‐A and A2a receptor mRNA levels between normal animals and all l‐DOPA (plus carbidopa and/or entacapone)‐treated animals irrespective of whether or not dyskinesia occurred. However, when the level of PPE‐A and A2a receptor mRNA was analysed in eight monkeys displaying marked dyskinesias as a result of l‐DOPA (plus carbidopa with or without entacapone) treatment, there was a significant increase in PPE‐A and A2a receptor mRNA message levels in the striatum compared with animals receiving identical treatment, but displaying few or no involuntary movements, and compared with normal controls. There was no difference in striatal PPT mRNA levels in monkeys exhibiting severe dyskinesia compared with those showing little or no dyskinesia after l‐DOPA treatment or to normal controls. These results suggest that prolonged l‐DOPA treatment alone has no consistent effect on either the direct or indirect pathways, as judged by striatal PPT, PPE‐A or A2a receptor mRNA levels in normal monkeys. However, in monkeys exhibiting marked dyskinesia resulting from chronic l‐DOPA treatment, abnormal activity is detected in the indirect striato‐pallidal output pathway, as judged by striatal PPE‐A and A2a receptor mRNA levels, indicating an imbalance between the direct and indirect striatal pathway which may explain the emergence of dyskinesia in these animals.

Antiparkinsonian activity and dyskinesia risk of ropinirole and L-DOPA combination therapy in drug naïve MPTP-lesioned common marmosets (Callithrix jacchus).

De novo administration of long‐acting dopamine agonists, such as ropinirole, to patients with Parkinsons disease or to 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐treated subhuman primates produces a lower incidence of dyskinesia than occurs with L‐DOPA. This study compares the intensity of dyskinesia produced by combinations of L‐DOPA and ropinirole and by these drugs alone, using the MPTP‐treated common marmoset model of Parkinsons disease. The objective is to determine the optimum therapeutic strategy for the long‐term control of Parkinsons disease with a minimal risk of dyskinesia.

Alterations in striatal neuropeptide mRNA produced by repeated administration of L-DOPA, ropinirole or bromocriptine correlate with dyskinesia induction in MPTP-treated common marmosets

Chronic administration of L-DOPA to MPTP-treated common marmosets induces marked dyskinesia while repeated administration of equivalent antiparkisonian doses of ropinirole and bromocriptine produces only mild involuntary movements. The occurrence of dyskinesia has been associated with an altered balance between the direct and indirect striatal output pathways. Using in situ hybridisation histochemistry, we now compare the effects of these drug treatments on striatal preproenkephalin-A (PPE-A) and adenosine A(2a) receptor mRNA expression as markers of the indirect pathway and striatal preprotachykinin (PPT) mRNA and preproenkephalin-B (PPE-B, prodynorphin) mRNA expression as markers of the direct pathway.The equivalent marked losses of specific [3H]mazindol binding in the striatum of all drug treatment groups confirmed the identical nature of the nigral cell loss produced by MPTP treatment. MPTP-induced destruction of the nigro-striatal pathway markedly increased the level of PPE-A mRNA in the caudate nucleus and putamen and decreased the levels of PPT and PPE-B mRNA relative to normal animals. Repeated treatment with L-DOPA for 30 days produced marked dyskinesia but had no effect on the MPTP-induced increase in PPE-A mRNA in the caudate nucleus and putamen. In contrast, L-DOPA treatment normalised the MPTP-induced decrease in the level of PPT and PPE-B mRNA. Repeated treatment with ropinirole produced little or no dyskinesia but markedly reversed the MPTP-induced elevation in PPE-A mRNA in the caudate nucleus and putamen. However, it had no effect on the decrease in PPT or PPE-B mRNA. Similarly, bromocriptine treatment which induced only mild dyskinesia attenuated the MPTP-induced elevation in PPE-A mRNA in the caudate nucleus and putamen with no effect on reduced striatal PPT or PPE-B mRNA. Neither MPTP treatment nor treatment with L-DOPA, bromocriptine or ropinirole had any effect on adenosine A(2a) receptor mRNA in the striatum. These patterns of alteration in striatal PPE-A and PPT and PPE-B mRNA produced by L-DOPA, bromocriptine and ropinirole show differential involvement of markers of the direct and indirect striatal output pathways related to improvement of locomotor activity and mirror the relative abilities of the drugs to induce dyskinesia.

Both Short- and Long-Acting D-1/D-2 Dopamine Agonists Induce Less Dyskinesia than l-DOPA in the MPTP-Lesioned Common Marmoset (Callithrix jacchus)

The current concept of dyskinesia is that pulsatile stimulation of D-1 or D-2 receptors by L-DOPA or short-acting dopamine agonists is more likely to induce dyskinesia compared to long-acting drugs producing more continuous receptor stimulation. We now investigate the ability of two mixed D-1/D-2 agonists, namely pergolide (long-acting) and apomorphine (short-acting), to induce dyskinesia in drug-nai;ve MPTP-lesioned primates, compared to L-DOPA. Adult common marmosets (Callithrix jacchus) were lesioned with MPTP (2 mg/kg/day sc for 5 days) and subsequently treated with equieffective antiparkinsonian doses of L-DOPA, apomorphine, or pergolide for 28 days. L-DOPA, apomorphine, and pergolide reversed the MPTP-induced motor deficits to the same degree with no difference in peak response. L-DOPA and apomorphine had a rapid onset of action and short duration of effect producing a pulsatile motor response, while pergolide had a slow onset and long-lasting activity producing a continuous profile of motor stimulation. L-DOPA rapidly induced dyskinesia that increased markedly in severity and frequency over the course of the study, impairing normal motor activity by day 20. Dyskinesia in animals treated with pergolide or apomorphine increased steadily, reaching mild to moderate severity but remaining significantly less marked than that produced by L-DOPA. There was no difference in the intensity of dyskinesia produced by apomorphine and pergolide. These data suggest that factors other than duration of drug action may be important in the induction of dyskinesia but support the use of dopamine agonists in early Parkinsons disease, as a means of delaying L-DOPA therapy and reducing the risk of developing dyskinesia.

GDNF reverses priming for dyskinesia in MPTP-treated, L-DOPA-primed common marmosets

Parkinsons disease (PD) is associated with a progressive loss of dopamine neurons in the substantia nigra and degeneration of dopaminergic terminals in the striatum. Although l‐DOPA treatment provides the most effective symptomatic relief for PD it does not prevent the progression of the disease, and its long‐term use is associated with the onset of dyskinesia. In rodent and primate studies, glial cell line‐derived neurotrophic factor (GDNF) may prevent 6‐OHDA‐ or MPTP‐induced nigral degeneration and so may be beneficial in the treatment of PD. In this study, we investigate the effects of GDNF on the expression of dyskinesia in l‐DOPA‐primed MPTP‐treated common marmosets, exhibiting dyskinesia. GDNF or saline was administered by two intraventricular injections, 4 weeks apart, to MPTP‐treated, l‐DOPA‐treated common marmosets primed to exhibit dyskinesia. Prior to GDNF or saline administration, all animals displayed marked dyskinesia when treated with l‐DOPA. GDNF administration produced a significant improvement in motor disability and, following the second injection of GDNF, a significant improvement in the locomotor activity was observed. Following the administration of l‐DOPA there was a greater reversal of disability and a reduction in the intensity of l‐DOPA‐induced dyskinesia in GDNF‐treated animals compared to saline‐treated controls. However, there was no significant difference in l‐DOPAs ability to increase locomotor activity between GDNF‐treated and saline‐treated animals. GDNF treatment caused a significant increase in the number of tyrosine hydroxylase‐positive neurons in the substantia nigra, but no change in [3H]mazindol binding to dopamine terminals was found in the striatum of GDNF‐treated animals compared to saline‐treated controls. In GDNF‐treated animals a small but significant reduction in enkephalin mRNA was observed in the caudate nucleus but not in the putamen or the nucleus accumbens. Substance P mRNA expression was equally reduced in the caudate nucleus and the putamen of the GDNF‐treated animals but not in the nucleus accumbens. Intraventricular administration of GDNF improved MPTP‐induced disability and reversed dopamine cell loss in the substantia nigra. GDNF also diminished l‐DOPA‐induced dyskinesia, which may relate to its ability to partly restore nigral dopaminergic transmission or to modify the activity of striatal output pathways.

Glial cell line-derived neurotrophic factor concentration dependently improves disability and motor activity in MPTP-treated common marmosets

Glial cell line-derived neurotrophic factor (GDNF) has previously reduced motor deficits and preserved nigral dopamine neurones in rhesus monkeys with a unilateral MPTP-induced lesion of substantia nigra. We now report on the ability of GDNF to reverse motor deficits induced by parenteral administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to common marmosets resulting in bilateral degeneration of the nigrostriatal pathway. Prior to GDNF administration, all MPTP-treated animals showed akinesia or bradykinesia, rigidity, postural instability and tremor. Intraventricular injection of GDNF (10, 100 or 500 microg) at 9 and 13 weeks post MPTP treatment resulted in a concentration dependent improvement in locomotor activity and motor disability which became significant after administration of 100 and 500 microg of GDNF. The most prominent improvements were in alertness, checking movements, and posture. It is concluded that intraventricular GDNF administration improves bilateral Parkinsonian motor disability following MPTP treatment and this may reflect an action of GDNF on remaining nigral dopaminergic neurones.

INTRAVENTRICULAR INFUSION OF BASIC FIBROBLAST GROWTH FACTOR (BFGF) IN THE MPTP-TREATED COMMON MARMOSET

Basic fibroblast growth factor (bFGF) prevents damage to the nigrostriatal system in rodents. We now report the effects of bFGF administered by intraventricular infusion to adult common marmosets (Callithrix jacchus) previously rendered parkinsonian by the administration of 1‐methy‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP). Infusion commenced 10 weeks after MPTP treatment and the animals received bFGF in low (1.8 μg/l), medium (18 μg/l), or high (180 μg/l) doses over a 28‐day period. At weekly intervals, automated activity measurements, behavioral disability scoring, and videotape analyses were made. There was no improvement in the motor deficits exhibited by MPTP‐treated common marmosets receiving bFGF infusion compared to vehicle‐treated controls. Three of five high dose animals showed neurological impairment prior to the end of the study. No significant differences were found between control and bFGF‐infused MPTP‐treated common marmosets with respect to nigral tyrosine hydroxylase immunoreactive cell counts and striatal [3H]mazindol binding. All high dose animals showed hydrocephalus which was also observed in four other animals receiving bFGF. Histological examination revealed proliferation of the choroid plexus and ependyma which was most marked in the high dose animals. Adverse effects, in the form of hydrocephalus and neurological deterioration, were presumably secondary to an ependymal and choroid plexus reaction induced by bFGF.

Chronic high dose L-DOPA alone or in combination with the COMT inhibitor entacapone does not increase oxidative damage or impair the function of the nigro-striatal pathway in normal cynomologus monkeys

Summary. Parkinsons disease (PD) is characterised by a loss of pigmented dopaminergic neurones in the zona compacta of substantia nigra. The mechanisms underlying nigral cell death remain unknown but may involve oxidative damage. There has been concern that L-DOPA treatment may accelerate nigral pathology in PD through chemical and enzymatic oxidation to reactive oxygen species. In the present study, we examined tissues from normal macaque monkeys treated for 13 weeks with high doses of L-DOPA (in combination with the peripheral decarboxylase inhibitor, carbidopa) and/or the COMT inhibitor, entacapone. Plasma was analysed for changes in pro-tein carbonyls as a marker of oxidative damage to protein. Cortical tissue was examined for changes in levels of protein carbonyls, lipid peroxidation and oxidative damage to DNA. The integrity of the nigro-striatal pathway was assessed by nigral tyrosine hydroxylase mRNA levels and specific [3H]mazindol binding to dopaminergic terminals in caudate-putamen.No alterations in plasma protein carbonyls were observed in any treatment group. An increase was found in the levels of protein carbonyls, lipid peroxidation and 5-OH uracil, but not other products of oxidative DNA damage, in cerebral cortex of monkeys treated with L-DOPA plus carbidopa or with L-DOPA plus carbidopa and entacapone but this was only statistically significant in the latter group. There was no change in nigral tyrosine hydroxylase mRNA levels or specific striatal [3H]mazindol binding in brain tissue from monkeys treated with either L-DOPA plus carbidopa or L-DOPA plus carbidopa and entacapone. The results show that in the normal monkeys L-DOPA does not provoke marked oxidative damage even at high doses, and that there is little or no potentiation of its effects by entacapone. Neither L-DOPA plus carbidopa nor L-DOPA plus carbidopa and entacapone led to obvious damage to the nigro-striatal pathway.

Actions of the D1 agonists A-77636 and A-86929 on locomotion and dyskinesia in MPTP-treated L-dopa-primed common marmosets

Abstract Common marmosets show parkinsonian motor deficits following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration and develop dyskinesias during chronic L-dopa exposure. The D1 agonists A-77636 [(1R, 3S) 3-(1’-adamantyl)-1-aminomethyl-3, 4-dihydro-5, 6-dihydroxy-1H-2-benzopyran HCl] and A-86929 [(−)-trans 9, 10-hydroxy-2-propyl-4, 5, 5a, 6, 7, 11b-hexahydro-3-thia-5-azacyclopent-1-ena[c]phenanthrene hydrochloride] possess potent antiparkinsonian activity in the MPTP-treated marmoset and we now assess their influence on L-dopa-induced dyskinesias. MPTP-treated marmosets with stable motor deficits were treated with L-dopa plus carbidopa for 28 days to induce dyskinesias. Subsequently, they received A-86929 for 10 days, initially at 0.5 μmol/kg and then at 1.0 μmol/kg for a further 5 days. Several months later, L-dopa 12.5 mg/kg plus carbidopa 12.5 mg/kg was given orally twice daily for 7 days, followed by A-77636 1 μmol/kg for 10 days, and then both A-77636 and L-dopa plus carbidopa were given concurrently for 3 further days. In these L-dopa-primed animals, A-86929 effectively reversed akinesia and produced dose-dependent dyskinesias which were significantly less intense than those produced by L-dopa administration. A degree of behavioral tolerance was encountered, but antiparkinsonian activity was preserved and elicited behaviour was free of hyperkinesis and stereotypy and more naturalistic than that seen with L-dopa. After a week of twice-daily L-dopa dosing, administration of the long-acting D1 agonist A-77636 initially dramatically enhanced locomotion and reproduced dyskinesia with prominent dystonia, but after repeated administration of A-77636, dyskinesia and in particular chorea, gradually disappeared. Tolerance to locomotor stimulation greater than with A-86929 occurred, although activity remained significantly above baseline levels. There was a marked reduction in L-dopa-induced climbing, stereotypy and hyperkinesis and behaviour more closely resembled that of normal unlesioned marmosets. Upon reintroduction of L-dopa concurrently with continued A-77636 administration, dystonic, but virtually no choreic dyskinesias appeared and behaviour was once again free of stereotypy and hyperkinesis, contrasting dramatically with the presence of these behaviours along with abundant chorea when L-dopa is given alone. These results show a lesser liability of A-86929 and A-77636 to reproduce dyskinesia in L-dopa-primed MPTP-lesioned subjects while maintaining effective antiparkinsonian activity and producing a more naturalistic motor response. The differential effects of A-77636 on chorea and dystonia, with suppression of chorea and stereotypy on co-administration with L-dopa, may reflect an altered balance of activity in the direct and indirect striatofugal pathways. These results suggest a possible role for D1 agonists in the treatment of Parkinson’s disease.