Introduction factors may contribute to the progression of PD such as Material and methods

Brain Research 883 2000 216–223 www.elsevier.com locate bres Research report The dopamine agonist pramipexole scavenges hydroxyl free radicals induced by striatal application of 6-hydroxydopamine in rats: an in vivo microdialysis study a,b , b c Boris Ferger , Peter Teismann , Joachim Mierau a Behavioural Neurobiology Laboratory , Swiss Federal Institute of Technology Zurich, Schorenstrasse 16, CH-8603 Schwerzenbach, Switzerland b Institute of Pharmacology and Toxicology , Faculty of Pharmacy, Philipps-University of Marburg, Ketzerbach 63, 35032 Marburg, Germany c Department of CNS Research , Boehringer Ingelheim Pharma KG, Binger Straße 173, 55216 Ingelheim, Germany Accepted 29 August 2000 Abstract Hydroxyl free radical production seems to play an important role in the pathogenesis of Parkinson’s disease. In the present study, we investigated the dopamine agonists pramipexole and pergolide as well as the nitrone compound S-PBN N-tert-butyl-a-2-sulfophenylnit- rone to reduce hydroxyl radical formation. Microdialysis experiments were carried out in non-anaesthetized Wistar rats. Salicylate was incorporated into the perfusion fluid to measure indirectly hydroxyl radicals indicated by 2,3-dihydroxybenzoic acid 2,3-DHBA. Local perfusion with 0.2 or 2 nmol 2 ml min 6-hydroxydopamine 6-OHDA via the microdialysis probe significantly increased 2,3-DHBA levels 14-fold and 47-fold, respectively. Systemic application of either pergolide 0.05 mg kg or pramipexole 1 mg kg failed to significantly reduce 6-OHDA-induced hydroxyl radical production. In contrast, a 40 min pretreatment with pramipexole 2 and 10 nmol 2 ml min via the probe before onset of 6-OHDA perfusion, significantly attenuated 2,3-DHBA levels compared with vehicle controls. S-PBN pretreatment 2 nmol 2 ml min was not effective to reduce 2,3-DHBA levels. In conclusion, pramipexole was able to reduce hydroxyl radical levels induced by 6-OHDA in vivo after local application. This property of pramipexole may be beneficial under conditions of enhanced hydroxyl radical formation in parkinsonian brains and may add to its well known dopamine D -like receptor 2 agonistic effects.  2000 Elsevier Science B.V. All rights reserved. Theme : Disorders of the nervous system Topic : Neurotoxicity Keywords : Pramipexole; 6-Hydroxydopamine; Parkinson’s disease; Salicylate assay; Radical

1. Introduction factors may contribute to the progression of PD such as

oxidative stress, excitotoxicity, mitochondrial DNA dam- Parkinson’s disease PD is characterized by the pro- age, glial and inflammatory processes and apoptosis of gressive loss of dopaminergic neurons originating in the nigral neurons [24]. Potential protective strategies aim to substantia nigra pars compacta and the sustained decrease develop drugs which reduce the influence of these patho- in striatal dopamine content [3,10,32]. The malfunction of genic factors and most of them are interrelated. the basal ganglia circuits is responsible for the cardinal We focused our interest on the investigation of oxidative motor symptoms of PD such as tremor at rest, muscular stress in PD. There are many lines of evidence that rigidity, bradykinesia akinesia as well as stooped posture oxidative stress takes place in PD. The dopamine loss leads and instability [36]. to a compensatory increase in dopamine turnover including Although the cause of PD is still unknown [18], many enhanced dopamine metabolism and radical formation, because the enzymatic dopamine metabolism via mono- amine oxidase B MAO-B not only produces 3,4- dihydroxyphenylacetic acid DOPAC but also hydrogen Corresponding author. Tel.: 141-1-655-7371; fax: 141-1-655-7203. E-mail address : fergertoxi.biol.ethz.ch B. Ferger. peroxide. Hydrogen peroxide itself is not a radical but 0006-8993 00 – see front matter  2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 9 2 9 - 2 B . Ferger et al. Brain Research 883 2000 216 –223 217 reacts with iron ions to form hydroxyl radicals the most tracellular dopamine levels were measured in the third reactive oxygen species. experiment. The ‘gold standard’ for treatment of Parkinson’s disease today is levodopa, the precursor of dopamine, in combina- tion with a peripheral decarboxylase inhibitor. For many

2. Material and methods

years the safety of levodopa and its long-term benefit have been topics of discussion. Levodopa significantly increased 2.1. In vitro Fenton system hydroxyl radical formation after systemic injection in combination with the peripheral decarboxylase inhibitor Hydroxyl radicals were generated according to a previ- [37] and many in vitro studies reported toxic effects of ously published method [12,39]. In brief, a mixture of 0.3 levodopa for review see [19] and more recently the mM FeCl 0.3 mM Na EDTA and 3 mM H O in 5 ml of 3, 2 2 2 induction of apoptosis [41]. Furthermore, long-term Tris-buffer adjusted to pH 7.4 in the presence of 0.5 mM levodopa therapy is accompanied with psychiatric and salicylic acid was incubated for 15 min at 378C. S-PBN, motor side effects dyskinesias and the efficacy of pramipexole or vehicle were co-incubated in this Fenton levodopa medication decreases after some years of treat- system in order to assess their possible radical scavenging ment. Therefore, delaying the onset of levodopa therapy or effects using the salicylate hydroxylation assay. adjunctive medication with dopamine agonists or MAO inhibitors may be beneficial in the early phase of PD. 2.2. Animals Dopamine agonists act on dopamine receptors to mimic the effects of dopamine. In contrast to levodopa they do Adult male albino Wistar rats Hsd Cpb:WU, Fa. Har- not need surviving presynaptic dopaminergic neurons for lan-Winkelmann GmbH, Borchen, Germany weighing uptake and metabolism. Pramipexole is a nonergot dopa- about 300 g at the time of microdialysis probe implanta- mine agonist with an azepine structure and exerts full tion, were used in this study. An ambient room tempera- intrinsic activity on D subfamily receptors, especially the ture was maintained at 23628C, the relative humidity at 2 D receptor, with little interaction to adrenergic and 5565. The animals were kept in a 12 h light–dark cycle 3 serotoninergic receptors [26,30,31]. The mechanism of D lights on at 07.00 a.m.–07.00 p.m. and housed indi- 2 autoreceptor mediated reduction of extracellular dopamine vidually in Plexiglas cages 40320324 cm with light  levels was postulated to protect nigral neurons against metal covering and fed with standard food Altromin , Fa. associated oxidative stress [23]. Altromin, Lage, Germany and tap water. All efforts were Indeed, pramipexole reduced extracellular dopamine made to minimize animal suffering, to reduce the number levels [4] and more recently neuroprotective effects of of animals used, and to utilize alternatives to in vivo pramipexole by additional mechanisms such as antioxidant techniques. The experimental protocols were approved by effects and induction of a trophic factor were claimed [5]. the appropriate institutional governmental agency Re- ¨ To study the effects of pramipexole on hydroxyl free gierungsprasident Gießen, Germany. radicals in vitro, we used a cell-free Fenton system. Under these conditions the potential antioxidant effects are direct- 2.3. In vivo microdialysis experiments ly related to the chemical structure of the compound and not to indirect effects such as interaction with biological The microdialysis experiments were carried out as antioxidant mechanisms such as induction of antioxidant described earlier [13,38]. A guide cannula was implanted enzymes or reduction of the radical generating dopamine under chloral hydrate anaesthesia 400 mg kg i.p. and metabolism. aimed at the head of the caudate nucleus coordinates from In vivo, two routes of administration systemic and bregma: AP: 11.25, ML: 12.6, DV: 22.5 according to the local of pramipexole were applied to investigate the atlas of Paxinos and Watson [29]. On the following day a potential effect on hydroxyl radical levels. In the first microdialysis probe CMA 12, membrane diameter 0.5 experiment, systemic application of pramipexole was mm, membrane length 4 mm, Carnegie Medicin, Stock- compared with pergolide on the reduction of basal hy- holm, Sweden was introduced and the rats were placed in droxyl radical levels without any exogenous stimulation of a microdialysis system with balance arm for freely moving hydroxyl radical formation. In the second experiment, animals. The perfusion fluid contained 5 mM salicylic acid 1 1 systemic application of pramipexole was compared with dissolved in a modified Ringer solution Na 147 mM, K 21 21 2 pergolide on the reduction of hydroxyl radical levels 4 mM, Ca 1.3 mM, Mg 1 mM, Cl 155.6 mM, flow induced by striatal reverse dialysis with 6-OHDA. In the rate: 2 ml min. After an equilibration period of approxi- third experiment, local application of pramipexole was mately 180 min a stable baseline was obtained and the compared with S-PBN instead of pergolide, because of intraperitoneal injection of pramipexole 0.05, 0.2, and 1 the poor solubility of pergolide in the perfusion fluid on mg kg, pergolide 0.05 mg kg or saline was carried out. the reduction of hydroxyl radical levels again induced by The mean of three 20 min basal dialysate fractions were striatal reverse dialysis with 6-OHDA. Furthermore, ex- set as 100. 218 B In experiments with exogenous hydroxyl radical stimu- column 12533 mm, pre-column 533 mm filled with lation the perfusion fluid was changed for 60 min to a fluid Nucleosil 120-3 C18 Knauer, Berlin, Germany. Data additionally containing 0.2 or 2 nmol 2 ml min of 6- were calculated by an external standard calibration. hydroxydopamine for reverse dialysis to deliver 6-OHDA The detection limit for 2,3-DHBA was approximately into the surrounding tissue of the microdialysis probe. 0.1 nmol l and its in vitro recovery microdialysis probes Then, the perfusion fluid was changed back again to the CMA 12 was 15–20. The values of 2,3-DHBA and perfusion fluid of the baseline recording, which was dopamine are expressed as percentages of three predrug without 6-OHDA. In control experiments modified Ringer dialysates and were not corrected for the recovery of the solution was used instead of 6-OHDA. probes. Samples were collected every 20 min in vials containing 10 ml 0.4 M perchloric acid and were directly analyzed by HPLC or stored at 2708C until analysis.

3. Results