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The acute antiparkinsonian and antidyskinetic effect of AFQ056, a novel metabotropic glutamate receptor type 5 antagonist, in l -Dopa-treated parkinsonian monkeys
Parkinsonism and Related Disorders 17 (2011) 270e276
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Parkinsonism and Related Disorders journal homepage: www.elsevier.com/locate/parkreldis
Short communication
The acute antiparkinsonian and antidyskinetic effect of AFQ056, a novel metabotropic glutamate receptor type 5 antagonist, in L-Dopa-treated parkinsonian monkeysq Laurent Grégoire a, b, Nicolas Morin a, b,1, Bazoumana Ouattara a, b,1, Fabrizio Gasparini c, Graeme Bilbe c, Donald Johns c, Ivo Vranesic c, Srikumar Sahasranaman d, Baltazar Gomez-Mancilla c, Thérèse Di Paolo a, b, * a
Molecular Endocrinology and Genomic Research Center, Centre Hospitalier Universitaire de Québec (CHUQ), Pavillon CHUL, Quebec, QC, Canada Faculty of Pharmacy, Laval University, Quebec, QC, Canada Neuroscience Discovery, Novartis Pharma AG, Basel, Switzerland d Novartis Institutes for BioMedical Research, DMPK, East Hanover, NJ, USA b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 August 2010 Received in revised form 10 January 2011 Accepted 14 January 2011
Overactivity of glutamatergic transmission has been implicated in Parkinson’s disease (PD) and levodopa (L-Dopa)-induced dyskinesias. Striatal metabotropic glutamate receptors type 5 (mGluR5) are abundant and provide specific targets to modulate glutamatergic activity. This study investigated the acute effects of the novel mGluR5 antagonist AFQ056 on motor behavior in L-Dopa-treated monkeys with a 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) lesion to model PD. Six Macaca fascicularis MPTP monkeys were treated repeatedly with L-Dopa; this treatment increased their locomotion and reduced their parkinsonian scores, but also induced dyskinesias. When AFQ056 (doses of 5, 25, 125 or 250 mg/kg) was administered one hour prior to a high dose of L-Dopa, the antiparkinsonian activity of L-Dopa was maintained as measured with locomotion and antiparkinsonian scores, whereas dyskinesias were significantly reduced at 25, 125 and 250 mg/kg AFQ056 for peak dyskinesia score and at 125 and 250 mg/ kg for the 1 h peak period of dyskinesia score. Administration of AFQ056 one hour before L-Dopa led to peak or elevated plasma AFQ056 concentrations occurring close to L-Dopa peak-dose dyskinesias. We next investigated AFQ056 25 mg/kg combined with a low dose of L-Dopa. The antiparkinsonian activity of L-Dopa was increased as measured with locomotion, while dyskinesias remained low at these doses. Our results show a beneficial motor effect of AFQ056 with L-Dopa in MPTP monkeys. This supports the therapeutic use of an mGluR5 antagonist to restore normal glutamatergic neurotransmission in PD and decrease dyskinesias. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: mGluR5 Dyskinesia Levodopa Parkinson MPTP monkey AFQ056
1. Introduction The major issue in the treatment of Parkinson’s disease (PD) is the occurrence of abnormal involuntary movements that result from chronic levodopa (L-Dopa) treatment [1] and may affect approximately 80% of L-Dopa-treated patients [2]. These motor complications include L-Dopa-induced dyskinesias [1]. Excessive glutamate release in the basal ganglia motor circuit plays a critical
q The review of this paper was entirely handled by an Associate Editor, R. L. Rodnitzkyi. * Corresponding author. Molecular Endocrinology and Genomic Research Center, Centre Hospitalier Universitaire de Québec (CHUQ), Pavillon CHUL, 2705 Laurier Blvd, Quebec, QC, Canada G1V 4G2. Tel.: þ1 418 654 2296; fax: þ1 418 654 2761. E-mail address: [email protected] (T. Di Paolo). 1 Both contributed equally to this work. 1353-8020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.parkreldis.2011.01.008
role in the expression of PD symptoms and L-Dopa-induced motor complications [3,4]. Thus, it could be expected that antagonizing glutamatergic transmission might alleviate not only PD but also L-Dopa-induced dyskinesias. Indeed, the antiglutamatergic drug amantadine reduces the severity of dyskinesias without worsening parkinsonian symptoms [5]. Ionotropic glutamate receptor antagonists have been tested for their antidyskinetic activity in PD patients [4] and animal models such as the 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) monkey [6]. But, these drugs have significant adverse effects such as cognitive impairment in many patients [5]. N-methyl-D-aspartate (NMDA) antagonists produce a broad range of symptoms, behaviors, and cognitive deficits that resemble aspects of endogenous psychoses, particularly schizophrenia and dissociative states [7]. The action of glutamate is also mediated through a family of eight G-protein coupled receptors that have been found to
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modulate presynaptic neurotransmitter release and the postsynaptic activity of ionotropic glutamate receptors [8]. Among these receptors, the high striatal expression of glutamate metabotropic type 5 receptor (mGluR5) suggests a role for mGluR5 antagonists in movement disorders. Moreover, the description of an antagonistic interaction between mGluR5 and D2 dopamine receptors [9], as well as mGluR5 and NMDA colocalization [10], makes this receptor an interesting pharmacological target [11]. Pharmacological antagonism of mGluR5 with the mGluR5 antagonists 2-methyl-6-(phenylethynyl)pyridine (MPEP) and 3-[(2methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) was shown to inhibit expression of dyskinesias in rodent [12] and primate [13,14] models of PD. Moreover, mGluR5 expression was demonstrated to be enhanced in the basal ganglia of parkinsonian monkeys that developed dyskinesias following chronic L-Dopa treatment [15] and post-mortem brains of PD patients with motor complications [16]. This study suggested that mGluR5 antagonists may be useful in the treatment of L-Dopa-induced dyskinesias in PD, which is recognized to be an area of high medical need, particularly in advanced disease. Therefore, the present study is the first to show that the pharmacological blockade of mGluR5 with an acute treatment of AFQ056, a novel selective mGluR5 antagonist, can attenuate LDopa-induced dyskinesia in MPTP monkeys and also increase the antiparkinsonian activity of L-Dopa. 2. Methods 2.1. Animals Six female ovariectomized cynomolgus monkeys (Macaca fascicularis) weighing between 2.8 and 4.4 kg were used for these experiments. Handling of the primates was performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. All procedures, including means to minimize discomfort, were reviewed and approved by the Institutional Animal Care Committee of Laval University. The animals were rendered parkinsonian by infusion of MPTP (SigmaeAldrich, Canada, Oakville, Ontario) using subcutaneous osmotic minipumps (Alzet, 0.5 mg/24 h) until they developed a stable parkinsonian syndrome. After 1e3 months of stabilization, animals were treated daily with L-Dopa 100/25 capsule (oral administration [p.o.]) (Prolopa, Hoffmann-La Roche; a mixture of 100 mg of L-Dopa and 25 mg benserazide) until clear and reproducible dyskinesias developed. 2.2. Drugs AFQ056 was provided by Novartis Pharma AG, Basel, Switzerland. A fresh suspension of AFQ056 was prepared on every experimental day. AFQ056 was added to a 0.5% (weight/weight) hydroxypropylcellulose aqueous solution (KlucelÒ HF, Aqualon, Hercules Inc, Wilmington, DE), mixed thoroughly with a high speed homogenizer (Polytron, Brinkman Instruments, Westbury) for 15 min and then kept under gentle stirring until administration. A volume between 12 and 20 ml of the AFQ056 suspension was administered by nasogastric gavage. L-Dopa methyl ester and benserazide (SigmaeAldrich Canada, Oakville, ON) were dissolved in sterile 0.9% saline solution and the pH adjusted to 7.
3. Experimental design 3.1. AFQ056 doseeresponse behavioral study with a high dose of L-Dopa Three weeks before beginning the acute study, animals were primed by repeated administration (3 times per week) of L-Dopa capsules (Prolopa 100/25 or 50/12.5 depending on the animals). The animals were first evaluated following vehicle (Klucel 0.5% solution p.o.) administration alone (baseline) and with vehicle and L-Dopa/benserazide subcutaneous (s.c). This treatment was repeated 7 times. L-Dopa doses were adjusted for each monkey and varied between 15 and 35 mg/kg. They were always given simultaneously with a fixed dose of benserazide (50 mg total). Ascending doses of AFQ056 (5, 25, 125, 250 mg/kg) with L-Dopa were tested. AFQ056 was given one hour before L-Dopa administration. AFQ056 alone (25 mg/kg) was also tested. Three days were left between
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each AFQ056 test; moreover each AFQ056 test was two days after the maintenance and comfort administration of a L-Dopa capsule (50 or 100 mg). 3.2. Pharmacokinetic study Blood samplings (4 days, 1 day/week) were performed following administration of AFQ056 (5, 25, 125 mg/kg) and L-Dopa (same dose used in the acute study) or AFQ056 25 mg/kg and saline. AFQ056 dosing was one hour before L-Dopa or saline injections. Blood samples (1 ml) were collected at 15 min, 15 min, 30 min, 1 h, 1 h 30 min, 2 h, 4 h and 24 h after AFQ056 administration in lithium heparin tubes and centrifuged (10 min, 1000 g) at 4 C. Plasma samples were stored at 80 C. AFQ056 plasma concentrations were obtained by plasma partitioning with aqueous solution of saturated sodium chloride and acetonitrile added to each sample before analysis of the supernatant by liquid chromatography tandem mass spectrometry (LC/MS/MS) using atmospheric pressure chemical ionization. The lower limit of quantification of the assay was 10 ng/ml. 3.3. Antiparkinsonian AFQ056 behavioral study with a low dose of L-Dopa Following one week of washout, animals were tested with a low dose of L-Dopa s.c. (5e15 mg/kg) with a fixed dose of benserazide (25 mg), with vehicle or AFQ056 25 mg/kg p.o. Vehicle or AFQ056 were given one hour before L-Dopa injections. 3.4. Behavioral assessment The animals were observed through a one-way screen and were scored “live” for parkinsonian and dyskinetic responses for the total duration of the L-Dopa response. 3.4.1. Parkinsonian score A disability scale developed in our laboratory was used to evaluate the parkinsonian syndrome [6]. Behaviors were scored every 15 min (maximal score: 16): a) Posture: normal ¼ 0, flexed intermittent ¼ 1, flexed constant ¼ 2, crouched ¼ 3; b) Mobility: normal ¼ 0, mild reduction ¼ 1, moderate reduction ¼ 2, severe reduction ¼ 3; c) Climbing: present ¼ 0, absent ¼ 1; d) Gait: normal ¼ 0, slow ¼ 1, very slow ¼ 2, very slow with freezing ¼ 3; e) Grooming: present ¼ 0, absent ¼ 1; f) Vocalization: present ¼ 0, absent ¼ 1; g) Social interaction: present ¼ 0, absent ¼ 1; h) Tremor: absent ¼ 0, mild action tremor ¼ 1, moderate action tremor ¼ 2, resting tremor ¼ 3. 3.4.2. Dyskinetic response Dyskinesias were scored every 15 min for the duration of the treatments. The face, neck, trunk, arms and legs were rated as follows: None ¼ 0; Mild (occasional) ¼ 1; Moderate (intermittent) ¼ 2; Severe (continuous) ¼ 3 for a maximal score of 21 [6]. 3.4.3. Locomotor response Locomotor activity was monitored continuously with an electronic motility monitoring system fixed on each cage (Datascience, St. Paul, Minnesota, USA). Computerized mobility counts were obtained every 5 min. 3.5. Data analysis For each treatment day and for each monkey, a mean parkinsonian score and a mean dyskinetic score (total period) were obtained by averaging all 15 min scores obtained for the duration of
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the response. Moreover, for dyskinesia, values for the one hour peak period and the maximum dyskinesia score were computed. Parkinsonian and dyskinesia scores were analyzed with a Friedman non-parametric test followed by a Dunn’s multiple comparisons test. In the figures, parkinsonian and dyskinesia scores are illustrated with median (horizontal line), interquartile range (box) and range (bars). Values for locomotor activity, duration and delay of responses were analyzed by analysis of variance (ANOVA) for repeated measures followed by Dunnett’s multiple comparisons test. The mean standard error of the mean (SEM) is represented in the graphs illustrating locomotor activity results. Coefficients of correlations and the significance of the linear relationships between variables were determined using a simple regression model using the Statview software. A p 0.05 was considered significant. 4. Results A high dose of L-Dopa administered alone induced a large increase in locomotor activity and a decrease (improvement) in parkinsonian scores of MPTP monkeys that were unchanged by the addition of AFQ056 at 5, 25, 125 or 250 mg/kg (Fig. 1). Moreover,
Fig. 1. Doseeresponse of AFQ056 combined with L-Dopa (15e35 mg/kg) in 6 MPTP monkeys as compared to L-Dopa alone (pre and post AFQ056), and AFQ056 alone with vehicle. A: on locomotion (F7,35 ¼ 8.12, p < 0.0001) and B: on Parkinsonian score (Friedman statistics ¼ 26.11, p ¼ 0.0005).
Fig. 2. Doseeresponse of AFQ056 combined with L-Dopa (15e35 mg/kg) in 6 MPTP monkeys as compared to L-Dopa alone (pre and post AFQ056), and AFQ056 alone with vehicle, on dyskinesia score. A: Total period (Friedman statistics ¼ 6.33, p ¼ 0.27); B: 1 h peak period (Friedman statistics ¼ 14.38, p ¼ 0.01) and C: Maximum dyskinesia (Friedman statistics ¼ 22.08, p ¼ 0.0005).
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Fig. 3. Time-course of L-Dopa-induced dyskinesias in individual L-Dopa-treated monkeys with and without administration of 125 mg/kg AFQ056 compared to plasma AFQ056 concentrations.
AFQ056 alone had no effect on locomotion or on parkinsonian scores (Fig. 1). Addition of AFQ056, at all doses tested, to a high dose of L-Dopa did not delay the elapsed time between L-Dopa administration and behavioral antiparkinsonian effect (16 2 min; F5,20 ¼ 0.59, p ¼ 0.70), but slightly shortened (F5,20 ¼ 7.14, p < 0.001) the duration of the antiparkinsonian effect of L-Dopa alone (206 18 min); this was not dose related: AFQ056 5 mg/kg (207 20 min, N.S.), 25 mg/kg (186 25 min, p < 0.05), 125 mg/kg
(185 23 min, p < 0.01) and 250 mg/kg (189 22 min, p < 0.05). MPTP monkeys administered AFQ056 alone or with L-Dopa had no abnormal behavior at all doses tested. Dyskinesia scores induced by a high dose of L-Dopa with the addition of AFQ056 5, 25, 125 and 250 mg/kg are shown in Fig. 2. Choreic and dystonic dyskinesias were present in these MPTP monkeys. The dyskinesia scores for the total period of L-Dopa effect were not significantly decreased with addition of AFQ056 (Fig. 2A),
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Fig. 4. Acute effect of AFQ056 25 mg/kg with a low dose of L-Dopa (5e15 mg/kg) in 6 MPTP monkeys compared to L-Dopa alone (pre and post AFQ056), AFQ056 alone and vehicle. A: locomotion (F4,20 ¼ 6.37, p < 0.005); B: Parkinsonian score (Friedman statistics ¼ 18.67, p < 0.001); C: delay (colored histograms; F2,10 ¼ 0.869, p ¼ 0.44) and duration of L-Dopa response (hatched histograms; F2,10 ¼ 0.182, p ¼ 0.83); D: dyskinesia score e total period (Friedman statistics ¼ 6.60, p ¼ 0.08); E: dyskinesia score e 1 h peak period (Friedman statistics ¼ 4.80, p ¼ 0.19); F: dyskinesia score e maximum dyskinesia (Friedman statistics ¼ 5.00, p ¼ 0.19).
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whereas the one hour peak dyskinesia scores showed a significant reduction with AFQ056 at 125 and 250 mg/kg (Fig. 2B), and maximal dyskinesias were also reduced by AFQ056 at 25, 125 and 250 mg/kg (Fig. 2C). A pharmacokinetic study of AFQ056 with L-Dopa showed that in these MPTP monkeys, exposure to AFQ056 increased as the AFQ056 dose increased, with maximal concentrations (Cmax SEM) of 42.6 9.5, 268 46 and 1289 303 ng/ml and area under the curve (AUC) of 481 124, 3025 662 and 10,708 2489 ng h/ml for 5, 25 and 125 mg/kg AFQ056, respectively. Administration of AFQ056 one hour before L-Dopa led to peak or elevated plasma AFQ056 concentrations occurring close to L-Dopa peak-dose dyskinesias of the MPTP monkeys (Fig. 3). Our dyskinesia score did not separate choreic from dystonic dyskinesias, thus no clear association between the choreic and/or dystonic nature of dyskinesias and the effect of AFQ056 was noted. The basal parkinsonian score was not clearly related with the AFQ056 antidyskinetic response, but after administration of L-Dopa and 125 mg/kg AFQ056 a significant positive correlation between the Cmax of AFQ056 and baseline parkinsonian scores was observed (Cmax vs. parkinsonian scores R ¼ 0.957, p ¼ 0.0028); this was also observed between AUC during 0e6 h (AUC 0e6H) and baseline parkinsonian scores (R ¼ 0.905, p ¼ 0.013) but not the total 24 h AUC and baseline parkinsonian scores (R ¼ 0.581, p ¼ 0.227). Similar significant correlations with the baseline parkinsonian scores were observed for the 25 mg/kg dose of AFQ056 and L-Dopa, but not for the lower dose of 5 mg/kg of AFQ056 and L-Dopa (data not shown). Administration of a low dose of AFQ056 (25 mg/kg) with L-Dopa (5e15 mg/kg) increased the locomotor activity of the MPTP monkeys compared with these drugs given separately (Fig. 4). A low dose L-Dopa administered alone to MPTP monkeys induced a decrease (improvement) of parkinsonian scores; this was also maintained with the addition of a low dose of AFQ056. Addition of AFQ056 to L-Dopa did not affect the duration and the delay of the onset of the L-Dopa antiparkinsonian effect. The low dose of L-Dopa investigated induced low dyskinesias in these MPTP monkeys that remained low with the addition of a low dose of AFQ056. 5. Discussion Overactivity of glutamate transmission in the basal ganglia is believed to be involved in the pathophysiology of PD and L-Dopainduced dyskinesias [4,17]. The preclinical and clinical use of ionotropic glutamate receptor antagonists confirmed the role of excessive glutamatergic transmission in the pathology of parkinsonian symptoms and dyskinesias [18,19], although psychiatric and cognitive impairments in humans limit their usefulness [7,19]. So, drugs acting at glutamate metabotropic receptors are an alternative therapeutic target to manage motor complications related to L-Dopa treatment [20,21]. In light of our previous biochemical results showing high mGluR5 specific binding in the putamen of parkinsonian and dyskinetic monkeys [15] and published results in a rodent model for L-Dopa-induced dyskinesias [12], we focused on this mGluR subtype. In the present acute experiment, mGluR5 was blocked in MPTP monkeys with the novel antagonist AFQ056. AFQ056 administered alone had no effect on parkinsonian symptoms. All doses of AFQ056 tested with a high dose of L-Dopa, maintained the antiparkinsonian effect as shown by the significant reduction of parkinsonian scores and increased locomotion. At a low dose of L-Dopa, addition of AFQ056 was shown to potentiate the antiparkinsonian effect as shown by the increased locomotion for the combined treatment compared to these drugs administered alone. Moreover, MPTP monkeys treated with both L-Dopa and AFQ056 had reduced dyskinesias compared to L-Dopa treatment
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alone. Of the increasing doses of AFQ056 used, 25, 125 and 250 mg/ kg significantly reduced the maximum scores of dyskinesia. A dose related increase in plasma AFQ056 concentration was measured with the increasing doses administered to MPTP monkeys. During the first 6 h after administration of AFQ056 at 125 mg/kg or 25 mg/kg and L-Dopa, we observed a significant positive correlation between Cmax and baseline parkinsonian scores, as well as between AUC 0e6H and baseline parkinsonian scores. It could be that the more severely motor-impaired MPTP monkeys had higher drug absorption, including MPTP, and thus became more denervated and more parkinsonian. These animals could also have absorbed more AFQ056 and so had higher plasma levels. In rodent models of PD, several studies have investigated the antiparkinsonian [22e27] and antidyskinetic [12,28,29] effects of mGluR5 blockade. Our results show that the antidyskinetic effects of mGluR5 blockade in rodents extend to MPTP monkeys. The development of tolerance to the chronic use of mGluR antagonists [28] may be an obstacle for their clinical use. However, no development of tolerance to the reduction of L-Dopa-induced dyskinesias was observed over 7 days with the mGluR5 antagonist MTEP (5 mg/kg) in 6-hydroxydopamine lesioned (6-OHDA) rats [28]; this was also observed in models of learning and anxiety while for pain, tolerance was observed [28]. Moreover a recent study in 6-OHDA rats, demonstrated the antidyskinetic effect of MTEP (5 mg/kg) in a longer chronic treatment of 3 weeks [30]. The effects of the prototypal mGluR5 antagonists MPEP and MTEP in MPTP monkeys have been recently shown to acutely reduced dyskinesias [13,24]. These results with MPEP and MTEP are similar to ours findings with AFQ056 also showing a reduction of L-Dopa dyskinesias while L-Dopa efficacy was maintained at a high hose dose and increased at sub-optimal doses. Taken together, the results in rodent models and our results in the MPTP-treated monkey model, we can argue in favor of the contribution of mGluR5 in the pathophysiology of L-Dopa-induced dyskinesia and that the blockade of mGluR5 might constitute a novel therapeutic treatment for PD-L-Dopa-induced dyskinesias. To this date, no therapy has been approved for dyskinesias. Since no abnormal behavior was observed in the present experiments with AFQ056, mGluR5 antagonists have the potential to be the first class of drug to be approved for the treatment of L-Dopa-induced dyskinesia in PD. Acknowledgements This work was supported by a grant from the Canadian Institutes of Health Research and Novartis Switzerland who provided the AFQ056 compound. N. Morin holds a studentship from the Parkinson Society Canada and B. Ouattara from the Ivory Coast government. We thank Serge Winter (Novartis) for performing the bioanalysis of AFQ056. Full financial disclosure for the previous 3 years: L. Grégoire, N. Morin and B. Ouattara: none. F. Gasparini, G.Bilbe, I. Vranesic, S. Sahasranaman and B. Gomez-Mancilla are employees of Novartis and have no other source of funding. T. Di Paolo received grants from the Canadian Institutes of Health Research (CIHR) and National Institute of Health (NIH), USA as well as contracts from Novartis Switzerland, Merck Serono S.A.-Geneva, Switzerland and Merck KGaA Germany. References [1] Guigoni C, Aubert I, Li Q, Gurevich VV, Benovic JL, Ferry S, et al. Pathogenesis of levodopa-induced dyskinesia: focus on D1 and D3 dopamine receptors. Parkinsonism Rel Disord 2005;11:S25e9.
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[2] Van Gerpen JA, Kumar N, Bower JH, Weigand S, Ahlskog JE. Levodopa-associated dyskinesia risk among Parkinson disease patients in Olmsted County, Minnesota, 1976e1990. Arch Neurol 2006;63:205e9. [3] Calon F, Rajput AH, Hornykiewicz O, Bedard PJ, Di Paolo T. Levodopa-induced motor complications are associated with alterations of glutamate receptors in Parkinson’s disease. Neurobiol Dis 2003;14:404e16. [4] Chase TN, Oh JD. Striatal dopamine- and glutamate-mediated dysregulation in experimental parkinsonism. Trends Neurosci 2000;23:S86e91. [5] Stocchi F, Tagliati M, Olanow CW. Treatment of levodopa-induced motor complications. Mov Disord 2008;23:S599e612. [6] Hadj Tahar A, Gregoire L, Darre A, Belanger N, Meltzer L, Bedard PJ. Effect of a selective glutamate antagonist on L-dopa-induced dyskinesias in drug-naive parkinsonian monkeys. Neurobiol Dis 2004;15:171e6. [7] Krystal JH, Karper LP, Seibyl JP, Freeman GK, Delaney R, Bremner JD, et al. Subanesthetic effects of the noncompetitive NMDA antagonist, ketamine, in humans. Psychotomimetic, perceptual, cognitive, and neuroendocrine responses. Arch Gen Psychiatry 1994;51:199e214. [8] Meldrum BS. Glutamate as a neurotransmitter in the brain: review of physiology and pathology. J Nutr 2000;130:1007Se15S. [9] Fuxe K, Marcellino D, Rivera A, Diaz-Cabiale Z, Filip M, Gago B, et al. Receptorereceptor interactions within receptor mosaics. Impact on neuropsychopharmacology. Brain Res Rev 2008;58:415e52. [10] Pisani A, Calabresi P, Centonze D, Bernardi G. Enhancement of NMDA responses by group I metabotropic glutamate receptor activation in striatal neurones. Br J Pharmacol 1997;120:1007e14. [11] Conn PJ, Battaglia G, Marino MJ, Nicoletti F. Metabotropic glutamate receptors in the basal ganglia motor circuit. Nat Rev Neurosci 2005;6:787e98. [12] Mela F, Marti M, Dekundy A, Danysz W, Morari M, Cenci MA. Antagonism of metabotropic glutamate receptor type 5 attenuates L-DOPA-induced dyskinesia and its molecular and neurochemical correlates in a rat model of Parkinson’s disease. J Neurochem 2007;101:483e97. [13] Johnston TH, Fox SH, McIldowie MJ, Piggott MJ, Brotchie JM. Reduction of L-DOPA-induced dyskinesia by the selective metabotropic glutamate receptor 5 antagonist 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine in the 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine-lesioned macaque model of Parkinson’s disease. J Pharmacol Exp Therapeut 2010;333:865e73. [14] Morin N, Gregoire L, Gomez-Mancilla B, Gasparini F, Di Paolo T. Effect of the metabotropic glutamate receptor type 5 antagonists MPEP and MTEP in parkinsonian monkeys. Neuropharmacology 2010;58:981e6. [15] Samadi P, Gregoire L, Morissette M, Calon F, Hadj Tahar A, Dridi M, et al. mGluR5 metabotropic glutamate receptors and dyskinesias in MPTP monkeys. Neurobiol Aging 2008;29:1040e51. [16] Ouattara B, Gregoire L, Morissette M, Gasparini F, Vranesic I, Bilbe G, et al. Metabotropic glutamate receptor type 5 in levodopa-induced motor complications. Neurobiol Aging 2009. doi:10.1016/j.neurobiolaging.2009.07.014. Published online 24 December 2009.
[17] Bezard E, Brotchie JM, Gross CE. Pathophysiology of levodopa-induced dyskinesia: potential for new therapies. Nat Rev Neurosci 2001;2: 577e88. [18] Blanchet PJ, Metman LV, Chase TN. Renaissance of amantadine in the treatment of Parkinson’s disease. Adv Neurol 2003;91:251e7. [19] Brown T, de Groote C, Brotchie J. Recent advances in the treatment of L-DOPAinduced dyskinesia. IDrugs 2002;5:454e68. [20] Breysse N, Amalric M, Salin P. Metabotropic glutamate 5 receptor blockade alleviates akinesia by normalizing activity of selective basal-ganglia structures in parkinsonian rats. J Neurosci 2003;23:8302e9. [21] Marino MJ, Williams Jr DL, O’Brien JA, Valenti O, McDonald TP, Clements MK, et al. Allosteric modulation of group III metabotropic glutamate receptor 4: a potential approach to Parkinson’s disease treatment. Proc Natl Acad Sci U S A 2003;100:13668e73. [22] Breysse N, Baunez C, Spooren W, Gasparini F, Amalric M. Chronic but not acute treatment with a metabotropic glutamate 5 receptor antagonist reverses the akinetic deficits in a rat model of parkinsonism. J Neurosci 2002; 22:5669e78. [23] Dekundy A, Pietraszek M, Schaefer D, Cenci MA, Danysz W. Effects of group I metabotropic glutamate receptors blockade in experimental models of Parkinson’s disease. Brain Res Bull 2006;69:318e26. [24] Ossowska K, Konieczny J, Wolfarth S, Pilc A. MTEP, a new selective antagonist of the metabotropic glutamate receptor subtype 5 (mGluR5), produces antiparkinsonian-like effects in rats. Neuropharmacology 2005;49:447e55. [25] Ossowska K, Konieczny J, Wolfarth S, Wieronska J, Pilc A. Blockade of the metabotropic glutamate receptor subtype 5 (mGluR5) produces antiparkinsonian-like effects in rats. Neuropharmacology 2001;41:413e20. [26] Spooren WP, Gasparini F, Bergmann R, Kuhn R. Effects of the prototypical mGlu(5) receptor antagonist 2-methyl-6-(phenylethynyl)-pyridine on rotarod, locomotor activity and rotational responses in unilateral 6-OHDAlesioned rats. Eur J Pharmacol 2000;406:403e10. [27] Spooren WP, Gasparini F, Salt TE, Kuhn R. Novel allosteric antagonists shed light on mglu(5) receptors and CNS disorders. Trends Pharmacol Sci 2001;22: 331e7. [28] Gravius A, Dekundy A, Nagel J, More L, Pietraszek M, Danysz W. Investigation on tolerance development to subchronic blockade of mGluR5 in models of learning, anxiety, and levodopa-induced dyskinesia in rats. J Neural Transm 2008;115:1609e19. [29] Levandis G, Bazzini E, Armentero MT, Nappi G, Blandini F. Systemic administration of an mGluR5 antagonist, but not unilateral subthalamic lesion, counteracts l-DOPA-induced dyskinesias in a rodent model of Parkinson’s disease. Neurobiol Dis 2008;29:161e8. [30] Rylander D, Recchia A, Mela F, Dekundy A, Danysz W, Cenci MA. Pharmacological modulation of glutamate transmission in a rat model of L-DOPAinduced dyskinesia: effects on motor behavior and striatal nuclear signalling. J Pharmacol Exp Therapeut 2009;330:227e35.
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Parkinsonism and Related Disorders journal homepage: www.elsevier.com/locate/parkreldis
Short communication
The acute antiparkinsonian and antidyskinetic effect of AFQ056, a novel metabotropic glutamate receptor type 5 antagonist, in L-Dopa-treated parkinsonian monkeysq Laurent Grégoire a, b, Nicolas Morin a, b,1, Bazoumana Ouattara a, b,1, Fabrizio Gasparini c, Graeme Bilbe c, Donald Johns c, Ivo Vranesic c, Srikumar Sahasranaman d, Baltazar Gomez-Mancilla c, Thérèse Di Paolo a, b, * a
Molecular Endocrinology and Genomic Research Center, Centre Hospitalier Universitaire de Québec (CHUQ), Pavillon CHUL, Quebec, QC, Canada Faculty of Pharmacy, Laval University, Quebec, QC, Canada Neuroscience Discovery, Novartis Pharma AG, Basel, Switzerland d Novartis Institutes for BioMedical Research, DMPK, East Hanover, NJ, USA b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 August 2010 Received in revised form 10 January 2011 Accepted 14 January 2011
Overactivity of glutamatergic transmission has been implicated in Parkinson’s disease (PD) and levodopa (L-Dopa)-induced dyskinesias. Striatal metabotropic glutamate receptors type 5 (mGluR5) are abundant and provide specific targets to modulate glutamatergic activity. This study investigated the acute effects of the novel mGluR5 antagonist AFQ056 on motor behavior in L-Dopa-treated monkeys with a 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) lesion to model PD. Six Macaca fascicularis MPTP monkeys were treated repeatedly with L-Dopa; this treatment increased their locomotion and reduced their parkinsonian scores, but also induced dyskinesias. When AFQ056 (doses of 5, 25, 125 or 250 mg/kg) was administered one hour prior to a high dose of L-Dopa, the antiparkinsonian activity of L-Dopa was maintained as measured with locomotion and antiparkinsonian scores, whereas dyskinesias were significantly reduced at 25, 125 and 250 mg/kg AFQ056 for peak dyskinesia score and at 125 and 250 mg/ kg for the 1 h peak period of dyskinesia score. Administration of AFQ056 one hour before L-Dopa led to peak or elevated plasma AFQ056 concentrations occurring close to L-Dopa peak-dose dyskinesias. We next investigated AFQ056 25 mg/kg combined with a low dose of L-Dopa. The antiparkinsonian activity of L-Dopa was increased as measured with locomotion, while dyskinesias remained low at these doses. Our results show a beneficial motor effect of AFQ056 with L-Dopa in MPTP monkeys. This supports the therapeutic use of an mGluR5 antagonist to restore normal glutamatergic neurotransmission in PD and decrease dyskinesias. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: mGluR5 Dyskinesia Levodopa Parkinson MPTP monkey AFQ056
1. Introduction The major issue in the treatment of Parkinson’s disease (PD) is the occurrence of abnormal involuntary movements that result from chronic levodopa (L-Dopa) treatment [1] and may affect approximately 80% of L-Dopa-treated patients [2]. These motor complications include L-Dopa-induced dyskinesias [1]. Excessive glutamate release in the basal ganglia motor circuit plays a critical
q The review of this paper was entirely handled by an Associate Editor, R. L. Rodnitzkyi. * Corresponding author. Molecular Endocrinology and Genomic Research Center, Centre Hospitalier Universitaire de Québec (CHUQ), Pavillon CHUL, 2705 Laurier Blvd, Quebec, QC, Canada G1V 4G2. Tel.: þ1 418 654 2296; fax: þ1 418 654 2761. E-mail address: [email protected] (T. Di Paolo). 1 Both contributed equally to this work. 1353-8020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.parkreldis.2011.01.008
role in the expression of PD symptoms and L-Dopa-induced motor complications [3,4]. Thus, it could be expected that antagonizing glutamatergic transmission might alleviate not only PD but also L-Dopa-induced dyskinesias. Indeed, the antiglutamatergic drug amantadine reduces the severity of dyskinesias without worsening parkinsonian symptoms [5]. Ionotropic glutamate receptor antagonists have been tested for their antidyskinetic activity in PD patients [4] and animal models such as the 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) monkey [6]. But, these drugs have significant adverse effects such as cognitive impairment in many patients [5]. N-methyl-D-aspartate (NMDA) antagonists produce a broad range of symptoms, behaviors, and cognitive deficits that resemble aspects of endogenous psychoses, particularly schizophrenia and dissociative states [7]. The action of glutamate is also mediated through a family of eight G-protein coupled receptors that have been found to
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modulate presynaptic neurotransmitter release and the postsynaptic activity of ionotropic glutamate receptors [8]. Among these receptors, the high striatal expression of glutamate metabotropic type 5 receptor (mGluR5) suggests a role for mGluR5 antagonists in movement disorders. Moreover, the description of an antagonistic interaction between mGluR5 and D2 dopamine receptors [9], as well as mGluR5 and NMDA colocalization [10], makes this receptor an interesting pharmacological target [11]. Pharmacological antagonism of mGluR5 with the mGluR5 antagonists 2-methyl-6-(phenylethynyl)pyridine (MPEP) and 3-[(2methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) was shown to inhibit expression of dyskinesias in rodent [12] and primate [13,14] models of PD. Moreover, mGluR5 expression was demonstrated to be enhanced in the basal ganglia of parkinsonian monkeys that developed dyskinesias following chronic L-Dopa treatment [15] and post-mortem brains of PD patients with motor complications [16]. This study suggested that mGluR5 antagonists may be useful in the treatment of L-Dopa-induced dyskinesias in PD, which is recognized to be an area of high medical need, particularly in advanced disease. Therefore, the present study is the first to show that the pharmacological blockade of mGluR5 with an acute treatment of AFQ056, a novel selective mGluR5 antagonist, can attenuate LDopa-induced dyskinesia in MPTP monkeys and also increase the antiparkinsonian activity of L-Dopa. 2. Methods 2.1. Animals Six female ovariectomized cynomolgus monkeys (Macaca fascicularis) weighing between 2.8 and 4.4 kg were used for these experiments. Handling of the primates was performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. All procedures, including means to minimize discomfort, were reviewed and approved by the Institutional Animal Care Committee of Laval University. The animals were rendered parkinsonian by infusion of MPTP (SigmaeAldrich, Canada, Oakville, Ontario) using subcutaneous osmotic minipumps (Alzet, 0.5 mg/24 h) until they developed a stable parkinsonian syndrome. After 1e3 months of stabilization, animals were treated daily with L-Dopa 100/25 capsule (oral administration [p.o.]) (Prolopa, Hoffmann-La Roche; a mixture of 100 mg of L-Dopa and 25 mg benserazide) until clear and reproducible dyskinesias developed. 2.2. Drugs AFQ056 was provided by Novartis Pharma AG, Basel, Switzerland. A fresh suspension of AFQ056 was prepared on every experimental day. AFQ056 was added to a 0.5% (weight/weight) hydroxypropylcellulose aqueous solution (KlucelÒ HF, Aqualon, Hercules Inc, Wilmington, DE), mixed thoroughly with a high speed homogenizer (Polytron, Brinkman Instruments, Westbury) for 15 min and then kept under gentle stirring until administration. A volume between 12 and 20 ml of the AFQ056 suspension was administered by nasogastric gavage. L-Dopa methyl ester and benserazide (SigmaeAldrich Canada, Oakville, ON) were dissolved in sterile 0.9% saline solution and the pH adjusted to 7.
3. Experimental design 3.1. AFQ056 doseeresponse behavioral study with a high dose of L-Dopa Three weeks before beginning the acute study, animals were primed by repeated administration (3 times per week) of L-Dopa capsules (Prolopa 100/25 or 50/12.5 depending on the animals). The animals were first evaluated following vehicle (Klucel 0.5% solution p.o.) administration alone (baseline) and with vehicle and L-Dopa/benserazide subcutaneous (s.c). This treatment was repeated 7 times. L-Dopa doses were adjusted for each monkey and varied between 15 and 35 mg/kg. They were always given simultaneously with a fixed dose of benserazide (50 mg total). Ascending doses of AFQ056 (5, 25, 125, 250 mg/kg) with L-Dopa were tested. AFQ056 was given one hour before L-Dopa administration. AFQ056 alone (25 mg/kg) was also tested. Three days were left between
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each AFQ056 test; moreover each AFQ056 test was two days after the maintenance and comfort administration of a L-Dopa capsule (50 or 100 mg). 3.2. Pharmacokinetic study Blood samplings (4 days, 1 day/week) were performed following administration of AFQ056 (5, 25, 125 mg/kg) and L-Dopa (same dose used in the acute study) or AFQ056 25 mg/kg and saline. AFQ056 dosing was one hour before L-Dopa or saline injections. Blood samples (1 ml) were collected at 15 min, 15 min, 30 min, 1 h, 1 h 30 min, 2 h, 4 h and 24 h after AFQ056 administration in lithium heparin tubes and centrifuged (10 min, 1000 g) at 4 C. Plasma samples were stored at 80 C. AFQ056 plasma concentrations were obtained by plasma partitioning with aqueous solution of saturated sodium chloride and acetonitrile added to each sample before analysis of the supernatant by liquid chromatography tandem mass spectrometry (LC/MS/MS) using atmospheric pressure chemical ionization. The lower limit of quantification of the assay was 10 ng/ml. 3.3. Antiparkinsonian AFQ056 behavioral study with a low dose of L-Dopa Following one week of washout, animals were tested with a low dose of L-Dopa s.c. (5e15 mg/kg) with a fixed dose of benserazide (25 mg), with vehicle or AFQ056 25 mg/kg p.o. Vehicle or AFQ056 were given one hour before L-Dopa injections. 3.4. Behavioral assessment The animals were observed through a one-way screen and were scored “live” for parkinsonian and dyskinetic responses for the total duration of the L-Dopa response. 3.4.1. Parkinsonian score A disability scale developed in our laboratory was used to evaluate the parkinsonian syndrome [6]. Behaviors were scored every 15 min (maximal score: 16): a) Posture: normal ¼ 0, flexed intermittent ¼ 1, flexed constant ¼ 2, crouched ¼ 3; b) Mobility: normal ¼ 0, mild reduction ¼ 1, moderate reduction ¼ 2, severe reduction ¼ 3; c) Climbing: present ¼ 0, absent ¼ 1; d) Gait: normal ¼ 0, slow ¼ 1, very slow ¼ 2, very slow with freezing ¼ 3; e) Grooming: present ¼ 0, absent ¼ 1; f) Vocalization: present ¼ 0, absent ¼ 1; g) Social interaction: present ¼ 0, absent ¼ 1; h) Tremor: absent ¼ 0, mild action tremor ¼ 1, moderate action tremor ¼ 2, resting tremor ¼ 3. 3.4.2. Dyskinetic response Dyskinesias were scored every 15 min for the duration of the treatments. The face, neck, trunk, arms and legs were rated as follows: None ¼ 0; Mild (occasional) ¼ 1; Moderate (intermittent) ¼ 2; Severe (continuous) ¼ 3 for a maximal score of 21 [6]. 3.4.3. Locomotor response Locomotor activity was monitored continuously with an electronic motility monitoring system fixed on each cage (Datascience, St. Paul, Minnesota, USA). Computerized mobility counts were obtained every 5 min. 3.5. Data analysis For each treatment day and for each monkey, a mean parkinsonian score and a mean dyskinetic score (total period) were obtained by averaging all 15 min scores obtained for the duration of
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the response. Moreover, for dyskinesia, values for the one hour peak period and the maximum dyskinesia score were computed. Parkinsonian and dyskinesia scores were analyzed with a Friedman non-parametric test followed by a Dunn’s multiple comparisons test. In the figures, parkinsonian and dyskinesia scores are illustrated with median (horizontal line), interquartile range (box) and range (bars). Values for locomotor activity, duration and delay of responses were analyzed by analysis of variance (ANOVA) for repeated measures followed by Dunnett’s multiple comparisons test. The mean standard error of the mean (SEM) is represented in the graphs illustrating locomotor activity results. Coefficients of correlations and the significance of the linear relationships between variables were determined using a simple regression model using the Statview software. A p 0.05 was considered significant. 4. Results A high dose of L-Dopa administered alone induced a large increase in locomotor activity and a decrease (improvement) in parkinsonian scores of MPTP monkeys that were unchanged by the addition of AFQ056 at 5, 25, 125 or 250 mg/kg (Fig. 1). Moreover,
Fig. 1. Doseeresponse of AFQ056 combined with L-Dopa (15e35 mg/kg) in 6 MPTP monkeys as compared to L-Dopa alone (pre and post AFQ056), and AFQ056 alone with vehicle. A: on locomotion (F7,35 ¼ 8.12, p < 0.0001) and B: on Parkinsonian score (Friedman statistics ¼ 26.11, p ¼ 0.0005).
Fig. 2. Doseeresponse of AFQ056 combined with L-Dopa (15e35 mg/kg) in 6 MPTP monkeys as compared to L-Dopa alone (pre and post AFQ056), and AFQ056 alone with vehicle, on dyskinesia score. A: Total period (Friedman statistics ¼ 6.33, p ¼ 0.27); B: 1 h peak period (Friedman statistics ¼ 14.38, p ¼ 0.01) and C: Maximum dyskinesia (Friedman statistics ¼ 22.08, p ¼ 0.0005).
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Fig. 3. Time-course of L-Dopa-induced dyskinesias in individual L-Dopa-treated monkeys with and without administration of 125 mg/kg AFQ056 compared to plasma AFQ056 concentrations.
AFQ056 alone had no effect on locomotion or on parkinsonian scores (Fig. 1). Addition of AFQ056, at all doses tested, to a high dose of L-Dopa did not delay the elapsed time between L-Dopa administration and behavioral antiparkinsonian effect (16 2 min; F5,20 ¼ 0.59, p ¼ 0.70), but slightly shortened (F5,20 ¼ 7.14, p < 0.001) the duration of the antiparkinsonian effect of L-Dopa alone (206 18 min); this was not dose related: AFQ056 5 mg/kg (207 20 min, N.S.), 25 mg/kg (186 25 min, p < 0.05), 125 mg/kg
(185 23 min, p < 0.01) and 250 mg/kg (189 22 min, p < 0.05). MPTP monkeys administered AFQ056 alone or with L-Dopa had no abnormal behavior at all doses tested. Dyskinesia scores induced by a high dose of L-Dopa with the addition of AFQ056 5, 25, 125 and 250 mg/kg are shown in Fig. 2. Choreic and dystonic dyskinesias were present in these MPTP monkeys. The dyskinesia scores for the total period of L-Dopa effect were not significantly decreased with addition of AFQ056 (Fig. 2A),
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Fig. 4. Acute effect of AFQ056 25 mg/kg with a low dose of L-Dopa (5e15 mg/kg) in 6 MPTP monkeys compared to L-Dopa alone (pre and post AFQ056), AFQ056 alone and vehicle. A: locomotion (F4,20 ¼ 6.37, p < 0.005); B: Parkinsonian score (Friedman statistics ¼ 18.67, p < 0.001); C: delay (colored histograms; F2,10 ¼ 0.869, p ¼ 0.44) and duration of L-Dopa response (hatched histograms; F2,10 ¼ 0.182, p ¼ 0.83); D: dyskinesia score e total period (Friedman statistics ¼ 6.60, p ¼ 0.08); E: dyskinesia score e 1 h peak period (Friedman statistics ¼ 4.80, p ¼ 0.19); F: dyskinesia score e maximum dyskinesia (Friedman statistics ¼ 5.00, p ¼ 0.19).
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whereas the one hour peak dyskinesia scores showed a significant reduction with AFQ056 at 125 and 250 mg/kg (Fig. 2B), and maximal dyskinesias were also reduced by AFQ056 at 25, 125 and 250 mg/kg (Fig. 2C). A pharmacokinetic study of AFQ056 with L-Dopa showed that in these MPTP monkeys, exposure to AFQ056 increased as the AFQ056 dose increased, with maximal concentrations (Cmax SEM) of 42.6 9.5, 268 46 and 1289 303 ng/ml and area under the curve (AUC) of 481 124, 3025 662 and 10,708 2489 ng h/ml for 5, 25 and 125 mg/kg AFQ056, respectively. Administration of AFQ056 one hour before L-Dopa led to peak or elevated plasma AFQ056 concentrations occurring close to L-Dopa peak-dose dyskinesias of the MPTP monkeys (Fig. 3). Our dyskinesia score did not separate choreic from dystonic dyskinesias, thus no clear association between the choreic and/or dystonic nature of dyskinesias and the effect of AFQ056 was noted. The basal parkinsonian score was not clearly related with the AFQ056 antidyskinetic response, but after administration of L-Dopa and 125 mg/kg AFQ056 a significant positive correlation between the Cmax of AFQ056 and baseline parkinsonian scores was observed (Cmax vs. parkinsonian scores R ¼ 0.957, p ¼ 0.0028); this was also observed between AUC during 0e6 h (AUC 0e6H) and baseline parkinsonian scores (R ¼ 0.905, p ¼ 0.013) but not the total 24 h AUC and baseline parkinsonian scores (R ¼ 0.581, p ¼ 0.227). Similar significant correlations with the baseline parkinsonian scores were observed for the 25 mg/kg dose of AFQ056 and L-Dopa, but not for the lower dose of 5 mg/kg of AFQ056 and L-Dopa (data not shown). Administration of a low dose of AFQ056 (25 mg/kg) with L-Dopa (5e15 mg/kg) increased the locomotor activity of the MPTP monkeys compared with these drugs given separately (Fig. 4). A low dose L-Dopa administered alone to MPTP monkeys induced a decrease (improvement) of parkinsonian scores; this was also maintained with the addition of a low dose of AFQ056. Addition of AFQ056 to L-Dopa did not affect the duration and the delay of the onset of the L-Dopa antiparkinsonian effect. The low dose of L-Dopa investigated induced low dyskinesias in these MPTP monkeys that remained low with the addition of a low dose of AFQ056. 5. Discussion Overactivity of glutamate transmission in the basal ganglia is believed to be involved in the pathophysiology of PD and L-Dopainduced dyskinesias [4,17]. The preclinical and clinical use of ionotropic glutamate receptor antagonists confirmed the role of excessive glutamatergic transmission in the pathology of parkinsonian symptoms and dyskinesias [18,19], although psychiatric and cognitive impairments in humans limit their usefulness [7,19]. So, drugs acting at glutamate metabotropic receptors are an alternative therapeutic target to manage motor complications related to L-Dopa treatment [20,21]. In light of our previous biochemical results showing high mGluR5 specific binding in the putamen of parkinsonian and dyskinetic monkeys [15] and published results in a rodent model for L-Dopa-induced dyskinesias [12], we focused on this mGluR subtype. In the present acute experiment, mGluR5 was blocked in MPTP monkeys with the novel antagonist AFQ056. AFQ056 administered alone had no effect on parkinsonian symptoms. All doses of AFQ056 tested with a high dose of L-Dopa, maintained the antiparkinsonian effect as shown by the significant reduction of parkinsonian scores and increased locomotion. At a low dose of L-Dopa, addition of AFQ056 was shown to potentiate the antiparkinsonian effect as shown by the increased locomotion for the combined treatment compared to these drugs administered alone. Moreover, MPTP monkeys treated with both L-Dopa and AFQ056 had reduced dyskinesias compared to L-Dopa treatment
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alone. Of the increasing doses of AFQ056 used, 25, 125 and 250 mg/ kg significantly reduced the maximum scores of dyskinesia. A dose related increase in plasma AFQ056 concentration was measured with the increasing doses administered to MPTP monkeys. During the first 6 h after administration of AFQ056 at 125 mg/kg or 25 mg/kg and L-Dopa, we observed a significant positive correlation between Cmax and baseline parkinsonian scores, as well as between AUC 0e6H and baseline parkinsonian scores. It could be that the more severely motor-impaired MPTP monkeys had higher drug absorption, including MPTP, and thus became more denervated and more parkinsonian. These animals could also have absorbed more AFQ056 and so had higher plasma levels. In rodent models of PD, several studies have investigated the antiparkinsonian [22e27] and antidyskinetic [12,28,29] effects of mGluR5 blockade. Our results show that the antidyskinetic effects of mGluR5 blockade in rodents extend to MPTP monkeys. The development of tolerance to the chronic use of mGluR antagonists [28] may be an obstacle for their clinical use. However, no development of tolerance to the reduction of L-Dopa-induced dyskinesias was observed over 7 days with the mGluR5 antagonist MTEP (5 mg/kg) in 6-hydroxydopamine lesioned (6-OHDA) rats [28]; this was also observed in models of learning and anxiety while for pain, tolerance was observed [28]. Moreover a recent study in 6-OHDA rats, demonstrated the antidyskinetic effect of MTEP (5 mg/kg) in a longer chronic treatment of 3 weeks [30]. The effects of the prototypal mGluR5 antagonists MPEP and MTEP in MPTP monkeys have been recently shown to acutely reduced dyskinesias [13,24]. These results with MPEP and MTEP are similar to ours findings with AFQ056 also showing a reduction of L-Dopa dyskinesias while L-Dopa efficacy was maintained at a high hose dose and increased at sub-optimal doses. Taken together, the results in rodent models and our results in the MPTP-treated monkey model, we can argue in favor of the contribution of mGluR5 in the pathophysiology of L-Dopa-induced dyskinesia and that the blockade of mGluR5 might constitute a novel therapeutic treatment for PD-L-Dopa-induced dyskinesias. To this date, no therapy has been approved for dyskinesias. Since no abnormal behavior was observed in the present experiments with AFQ056, mGluR5 antagonists have the potential to be the first class of drug to be approved for the treatment of L-Dopa-induced dyskinesia in PD. Acknowledgements This work was supported by a grant from the Canadian Institutes of Health Research and Novartis Switzerland who provided the AFQ056 compound. N. Morin holds a studentship from the Parkinson Society Canada and B. Ouattara from the Ivory Coast government. We thank Serge Winter (Novartis) for performing the bioanalysis of AFQ056. Full financial disclosure for the previous 3 years: L. Grégoire, N. Morin and B. Ouattara: none. F. Gasparini, G.Bilbe, I. Vranesic, S. Sahasranaman and B. Gomez-Mancilla are employees of Novartis and have no other source of funding. T. Di Paolo received grants from the Canadian Institutes of Health Research (CIHR) and National Institute of Health (NIH), USA as well as contracts from Novartis Switzerland, Merck Serono S.A.-Geneva, Switzerland and Merck KGaA Germany. References [1] Guigoni C, Aubert I, Li Q, Gurevich VV, Benovic JL, Ferry S, et al. Pathogenesis of levodopa-induced dyskinesia: focus on D1 and D3 dopamine receptors. Parkinsonism Rel Disord 2005;11:S25e9.
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[2] Van Gerpen JA, Kumar N, Bower JH, Weigand S, Ahlskog JE. Levodopa-associated dyskinesia risk among Parkinson disease patients in Olmsted County, Minnesota, 1976e1990. Arch Neurol 2006;63:205e9. [3] Calon F, Rajput AH, Hornykiewicz O, Bedard PJ, Di Paolo T. Levodopa-induced motor complications are associated with alterations of glutamate receptors in Parkinson’s disease. Neurobiol Dis 2003;14:404e16. [4] Chase TN, Oh JD. Striatal dopamine- and glutamate-mediated dysregulation in experimental parkinsonism. Trends Neurosci 2000;23:S86e91. [5] Stocchi F, Tagliati M, Olanow CW. Treatment of levodopa-induced motor complications. Mov Disord 2008;23:S599e612. [6] Hadj Tahar A, Gregoire L, Darre A, Belanger N, Meltzer L, Bedard PJ. Effect of a selective glutamate antagonist on L-dopa-induced dyskinesias in drug-naive parkinsonian monkeys. Neurobiol Dis 2004;15:171e6. [7] Krystal JH, Karper LP, Seibyl JP, Freeman GK, Delaney R, Bremner JD, et al. Subanesthetic effects of the noncompetitive NMDA antagonist, ketamine, in humans. Psychotomimetic, perceptual, cognitive, and neuroendocrine responses. Arch Gen Psychiatry 1994;51:199e214. [8] Meldrum BS. Glutamate as a neurotransmitter in the brain: review of physiology and pathology. J Nutr 2000;130:1007Se15S. [9] Fuxe K, Marcellino D, Rivera A, Diaz-Cabiale Z, Filip M, Gago B, et al. Receptorereceptor interactions within receptor mosaics. Impact on neuropsychopharmacology. Brain Res Rev 2008;58:415e52. [10] Pisani A, Calabresi P, Centonze D, Bernardi G. Enhancement of NMDA responses by group I metabotropic glutamate receptor activation in striatal neurones. Br J Pharmacol 1997;120:1007e14. [11] Conn PJ, Battaglia G, Marino MJ, Nicoletti F. Metabotropic glutamate receptors in the basal ganglia motor circuit. Nat Rev Neurosci 2005;6:787e98. [12] Mela F, Marti M, Dekundy A, Danysz W, Morari M, Cenci MA. Antagonism of metabotropic glutamate receptor type 5 attenuates L-DOPA-induced dyskinesia and its molecular and neurochemical correlates in a rat model of Parkinson’s disease. J Neurochem 2007;101:483e97. [13] Johnston TH, Fox SH, McIldowie MJ, Piggott MJ, Brotchie JM. Reduction of L-DOPA-induced dyskinesia by the selective metabotropic glutamate receptor 5 antagonist 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine in the 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine-lesioned macaque model of Parkinson’s disease. J Pharmacol Exp Therapeut 2010;333:865e73. [14] Morin N, Gregoire L, Gomez-Mancilla B, Gasparini F, Di Paolo T. Effect of the metabotropic glutamate receptor type 5 antagonists MPEP and MTEP in parkinsonian monkeys. Neuropharmacology 2010;58:981e6. [15] Samadi P, Gregoire L, Morissette M, Calon F, Hadj Tahar A, Dridi M, et al. mGluR5 metabotropic glutamate receptors and dyskinesias in MPTP monkeys. Neurobiol Aging 2008;29:1040e51. [16] Ouattara B, Gregoire L, Morissette M, Gasparini F, Vranesic I, Bilbe G, et al. Metabotropic glutamate receptor type 5 in levodopa-induced motor complications. Neurobiol Aging 2009. doi:10.1016/j.neurobiolaging.2009.07.014. Published online 24 December 2009.
[17] Bezard E, Brotchie JM, Gross CE. Pathophysiology of levodopa-induced dyskinesia: potential for new therapies. Nat Rev Neurosci 2001;2: 577e88. [18] Blanchet PJ, Metman LV, Chase TN. Renaissance of amantadine in the treatment of Parkinson’s disease. Adv Neurol 2003;91:251e7. [19] Brown T, de Groote C, Brotchie J. Recent advances in the treatment of L-DOPAinduced dyskinesia. IDrugs 2002;5:454e68. [20] Breysse N, Amalric M, Salin P. Metabotropic glutamate 5 receptor blockade alleviates akinesia by normalizing activity of selective basal-ganglia structures in parkinsonian rats. J Neurosci 2003;23:8302e9. [21] Marino MJ, Williams Jr DL, O’Brien JA, Valenti O, McDonald TP, Clements MK, et al. Allosteric modulation of group III metabotropic glutamate receptor 4: a potential approach to Parkinson’s disease treatment. Proc Natl Acad Sci U S A 2003;100:13668e73. [22] Breysse N, Baunez C, Spooren W, Gasparini F, Amalric M. Chronic but not acute treatment with a metabotropic glutamate 5 receptor antagonist reverses the akinetic deficits in a rat model of parkinsonism. J Neurosci 2002; 22:5669e78. [23] Dekundy A, Pietraszek M, Schaefer D, Cenci MA, Danysz W. Effects of group I metabotropic glutamate receptors blockade in experimental models of Parkinson’s disease. Brain Res Bull 2006;69:318e26. [24] Ossowska K, Konieczny J, Wolfarth S, Pilc A. MTEP, a new selective antagonist of the metabotropic glutamate receptor subtype 5 (mGluR5), produces antiparkinsonian-like effects in rats. Neuropharmacology 2005;49:447e55. [25] Ossowska K, Konieczny J, Wolfarth S, Wieronska J, Pilc A. Blockade of the metabotropic glutamate receptor subtype 5 (mGluR5) produces antiparkinsonian-like effects in rats. Neuropharmacology 2001;41:413e20. [26] Spooren WP, Gasparini F, Bergmann R, Kuhn R. Effects of the prototypical mGlu(5) receptor antagonist 2-methyl-6-(phenylethynyl)-pyridine on rotarod, locomotor activity and rotational responses in unilateral 6-OHDAlesioned rats. Eur J Pharmacol 2000;406:403e10. [27] Spooren WP, Gasparini F, Salt TE, Kuhn R. Novel allosteric antagonists shed light on mglu(5) receptors and CNS disorders. Trends Pharmacol Sci 2001;22: 331e7. [28] Gravius A, Dekundy A, Nagel J, More L, Pietraszek M, Danysz W. Investigation on tolerance development to subchronic blockade of mGluR5 in models of learning, anxiety, and levodopa-induced dyskinesia in rats. J Neural Transm 2008;115:1609e19. [29] Levandis G, Bazzini E, Armentero MT, Nappi G, Blandini F. Systemic administration of an mGluR5 antagonist, but not unilateral subthalamic lesion, counteracts l-DOPA-induced dyskinesias in a rodent model of Parkinson’s disease. Neurobiol Dis 2008;29:161e8. [30] Rylander D, Recchia A, Mela F, Dekundy A, Danysz W, Cenci MA. Pharmacological modulation of glutamate transmission in a rat model of L-DOPAinduced dyskinesia: effects on motor behavior and striatal nuclear signalling. J Pharmacol Exp Therapeut 2009;330:227e35.