Docosahexaenoic acid reduces levodopa-induced dyskinesias in 1-methyl-4-phenyl-1 2 3 6-tetrahydropyridine monkeys.код для вставкиСкачать
Docosahexaenoic Acid Reduces LevodopaInduced Dyskinesias in 1-Methyl-4-Phenyl1,2,3,6-Tetrahydropyridine Monkeys Pershia Samadi, PhD,1 Laurent Grégoire, BSc,1 Claude Rouillard, PhD,1 Paul J. Bédard, MD, PhD,1 Thérèse Di Paolo, PhD,2 and Daniel Lévesque, PhD1 Objective: The objective of the present study was to investigate the effect of docosahexaenoic acid (DHA), a polyunsaturated fatty acid (omega-3), on levodopa-induced dyskinesias (LIDs) in parkinsonian 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP)–treated monkeys. Methods: We explored the effect of DHA in two paradigms. First, a group of MPTP monkeys was primed with levodopa for several months before introducing DHA. A second group of MPTP monkeys (de novo) was exposed to DHA before levodopa therapy. Results: DHA administration reduced LIDs in both paradigms without alteration of the anti-parkinsonian effect of levodopa indicating that DHA can reduce the severity or delay the development of LIDs in a nonhuman primate model of Parkinson’s disease. Interpretation: These results suggest that DHA can reduce the severity or delay the develoment of LIDs in a nonhuman primate model of Parkinson’s disease. DHA may represent a new approach to improve the quality of life of Parkinson’s disease patients. Ann Neurol 2006;59:282–288 Dopamine replacement therapy using the dopamine precursor L-dopa remains the corner stone of Parkinson’s disease symptom treatment. Unfortunately, the efficacy of L-dopa is seriously hampered by the fact that a large proportion of patients develop dyskinetic involuntary movements.1 The same applies to the 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkey model in which parkinsonian syndrome and L-dopa– induced dyskinesias (LIDs) closely match those observed in human.2,3 Once they have appeared, even if treatment is stopped entirely for several weeks, the first dose will still trigger the same dyskinetic behavior, indicating that treatment with L-dopa has modified the response to dopamine and that this modification is long lasting. The aforementioned observations therefore suggest that denervation and dopaminergic stimulation combine to elicit persistent changes in the basal ganglia, which are consistent with an excessive or poorly regulated response to dopaminergic agents. Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid of the omega-3 class that is enriched in membrane phospholipids of the brain.4 DHA is also recognized as an endogenous activator of nuclear receptors that operate as transcription factors, such as retin- oid X receptors (RXRs).5 Interestingly, DHA increases the number of surviving dopaminergic cells in a process mediated by Nurr1–RXR heterodimers.6 We recently demonstrated that DHA reduced orofacial dyskinesias associated with chronic antipsychotic drug treatment in mice.7 Similar to its role on dopamine neuron survival, the antidyskinetic effect of DHA is dependent on the presence of the transcription factor Nur77, a close homologue of Nurr1, whereas a RXR antagonist exacerbates antipsychotic-induced dyskinesias.7 Because tardive dyskinesias induced by chronic dopamine receptor blockade with conventional antipsychotic drugs and LIDs may share common biological substrates, we therefore undertook a series of experiments to test whether DHA may also reduce LIDs in MPTP monkeys. From the 1Centre de recherche en Neurosciences, Centre Hospitalier Universitaire de Québec (CHUQ), Ste-Foy, Quebec, Canada; and Département de Médecine, Faculté de Médecine, Université Laval, and 2Laboratoire d’Endocrinologie Moléculaire et Oncologie, CHUQ, Faculté de Pharmacie, Université Laval, Quebec City, QC, Canada. Published online Jan 28, 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ana.20738 Received May 26, 2005, and in revised form Sep 7. Accepted for publication Oct 10, 2005. 282 Materials and Methods Animals and Treatments Handling of the primates was performed in accordance to 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. Address correspondence to Dr Lévesque, Neuroscience Unit, RC9800, CHUL Research Center (CHUQ), 2705, Boulevard Laurier, Ste-Foy, Quebec, Canada G1V 4G2. E-mail: firstname.lastname@example.org © 2006 American Neurological Association Published by Wiley-Liss, Inc., through Wiley Subscription Services Cynomolgus (Macaca fascicularis) ovariectomized female monkeys weighing 3 to 5kg were used in this study. For the L-dopa–primed group, monkeys were exposed to the MPTP neurotoxin until they developed a stable parkinsonian syndrome.8,9 Then, each animal was treated orally, once daily, with L-dopa/benserazide (100/25mg, Prolopa, Hoffman-La Roche, Missigauga, ON, Canada) during a few months, until LIDs, with a predominantly choreic nature, developed. Dyskinesias were thereafter reproduced upon subsequent doses of L-dopa. Before the experiments began, the monkeys received an oral dose of L-dopa/benserazide two to three times a week to maintain priming and for their comfort. For the first and second experiments in L-dopa–primed group, the doses of L-dopa (methyl ester form; Sigma-Aldrich Canada, Oakville, ON) varied from 30 to 35mg/kg depending on the threshold of each animal with the aim of having the maximum of dyskinesias and was always accompanied by benserazide (50mg). DHA (100mg/kg, SC or 200mg/kg, PO; Cayman Chemical, Ann Arbor, MI) was administered daily as a suspension in 8% PEG-600 and sterile water (pH was adjusted to 6.0 –7.0) for the duration of the experiment, as indicated in the figures. The dose of 100mg/kg was chosen on the basis of the effect of DHA on tardive dyskinesias and acute parkinsonism induced by the antipsychotic haloperidol in mice.7,10 In the first experiment with primed MPTP monkeys, we had to stop the protocol prematurely because we denoted signs of inflammation and infection at the sites of drug injection. Then, in the second experiment (after several months of washout), we used a per os route of administration for DHA. To compensate for the eventuality of reduced absorption of the drug due to gastrointestinal passage, we increased the dose from 100 to 200mg/kg. For de novo animals, MPTP-treated monkeys were first exposed to DHA (100mg/kg, PO, in a volume of 20 to 25ml according to the weight of the animal) for 3 days before L-dopa therapy was introduced. We used a lower dose of DHA because more chronic treatment was performed in this experiment (several weeks instead of few days). Then, combined oral administration of L-dopa and DHA was performed on a daily basis for 1 month. In these de novo parkinsonian monkeys, a fixed high daily oral dose of 100/25 mg of L-dopa/benserazide was used in every animal. Dyskinesias LIDs were rated according to a behavioral scale including abnormal movements from the face, neck, trunk, arms, and legs, as previously described (scale: none ⫽ 0; mild ⫽ 1; moderate ⫽ 2; severe ⫽ 3).8,9 Dyskinetic scores also included assessment of the amplitude, interference with normal motor activity, and the frequency of abnormal movements. For L-dopa–primed monkeys, mean dyskinetic scores were calculated as the average of all scores obtained during 3 hours after L-dopa administration. For de novo animals, in addition to mean dyskinetic scores, we also showed the peak dyskinetic score, which represents the maximal dyskinetic scores obtained for a period of 15 minutes during the “ON” state of the animal. These nonparametric results are presented as median scores. Parkinsonian Syndrome The parkinsonian scores for each animal were assessed before and after the treatments for a maximal disability score of 16, according to a scale that we have used in several published studies.8,9 In brief, it includes assessment of posture, mobility, climbing, gait, grooming, voicing, social interaction, and tremor. The mean parkinsonian score has been calculated as average of all the scores obtained every 15 minutes during 3 hours after L-dopa administration in primed MPTP monkeys or during the “ON” state of de novo animals. These data are presented as median scores. Locomotor Activity Locomotor activity was quantified using an electronic motility monitoring system (Datascience, St. Paul, MN). Using radio wave frequency, a probe around the neck of each animal transmits the signal to a receiver fixed on each cage. This receiver is connected to a computer and provides a motility count every 5 minutes. Statistical Analysis Comparison between dyskinetic and parkinsonian scores for L-dopa alone and L-dopa ⫹ DHA groups was performed using nonparametric Friedman’s test followed by multiple comparisons based on Friedman rank sums for primed MPTP monkeys and with Mann–Whitney U test for de novo animals. For locomotor activity, total mobility counts were compared using an analysis of variance (ANOVA) for repeated measures followed by Fisher’s probability of least significant difference test (PLSD). The factors described in the Table were compared between the two groups using a Student’s t test. Results The first part of the study was designed to test whether DHA can reduce LIDs once they have appeared. We treated a group of five MPTP monkeys with L-dopa until persistent and stable LIDs developed, as previously described8,9 (Fig 1A, primed #1). Then, we introduced DHA (100mg/kg, SC) in combination with a classic L-dopa treatment. The dose of DHA was chosen on the basis of previous observations showing reduction of antipsychotic-induced tardive dyskinesias and acute parkinsonism of this compound in mice.7,10 The first dose of L-dopa, when reintroduced in these L-dopa–primed monkeys, produced a dyskinetic score similar to previous treatments (DOPA “pre”; see Fig 1A). We observed a significant reduction of LIDs (approximately 35% reduction) the fourth day of concomitant administration of DHA with L-dopa that lasted for the duration of the combined treatment. Five days after DHA washout, LIDs returned to their previous levels (DOPA “post”; see Fig 1A). The addition of DHA to L-dopa had no effect on the antiparkinsonian activity of L-dopa and on L-dopa–induced locomotor activity (see Fig 1A). DHA alone or its vehicle was without effect on parkinsonian and dyskinetic scores (see Fig 1A). In the second experiment, subcutaneous injections Samadi et al: Effects of DHA on LIDs 283 Table. Summary of the Factors That Might Alter the Development of Dyskinesias in De Novo Animals Treatment Group Animal No. Weight (kg) MPTP Dose (mg) Time after MPTP (days) Basal Parkinsonian Score (mean score) L-dopa Mean ⫾ SEM L-dopa ⫹ DHA Mean ⫾ SEM 1 2 3 4 5 3.2 4.0 3.6 4.0 3.2 3.6 ⫾ 0.2 19.1 11.0 19.6 10.0 30.8 18.1 ⫾ 3.8 1 2 3a 4 5 3.6 3.5 3.4 3.7 4.0 3.6 ⫾ 0.1NS 22.2 16.0 12.8 7.0 11.6 13.9 ⫾ 2.5NS 390 124 71 130 30 149 ⫾ 63 150 350 200 129 123 190 ⫾ 42NS 13.7 9.2 10.0 10.0 9.0 10.5 ⫾ 0.8 12.8 13.5 12 10.1 9.7 11.6 ⫾ 0.8NS a This animal was removed from the experiment the first day of L-dopa treatment because it readily develops abnormal movements associated with severe L-dopa intolerance. The data were compared between the two groups using a Student’s t test. MPTP ⫽1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; DHA ⫽ docosahexaenoic acid; NS ⫽ not significant. of DHA was replaced by a per os (gavage) administration (see Fig 1B, primed #2) because we denoted signs of irritation and inflammation at the sites of injection. The same animals, as for the first experiment, were used after a washout period of several months. As for the first experiment, administration of DHA (200mg/ kg, PO) with L-dopa significantly reduced LIDs (see Fig 1B). The effect is somewhat delayed compared with the first experiment, possibly because of the different mode of administration. However, we obtained a similar effect on LIDs (approximately 35% reduction). In both experiments, DHA produced a constant and global reduction of LIDs during all the period of L-dopa effect (see time courses in Fig 2). Again, when we stopped DHA administration (DOPA “post”), LIDs returned to their previous levels (DOPA “pre”; see Fig 1B). Neither antiparkinsonian effect of L-dopa, nor the total locomotor activity of the animals, was altered by the DHA treatment (see Fig 1B). The second part of the study was designed to test if the addition of DHA to L-dopa can prevent or delay the appearance of LIDs. We used 10 monkeys rendered parkinsonian with administration of MPTP, as previously described.8,9 The two groups were matched for factors that may alter the development of LIDs, such as (1) MPTP doses used, (2) time necessary to develop a stable parkinsonian syndrome, and (3) basal parkinsonian scores (see Table). After stabilization of their parkinsonian syndrome, half of them received DHA (100mg/kg, PO) for 3 days, whereas the other half received the vehicle. L-Dopa therapy then was initiated (see Materials and Methods). LIDs started to develop at the first week of treatment and increased over time (see Fig 1C, de novo), as previously reported.8,9 284 Annals of Neurology Vol 59 No 2 February 2006 The administration of DHA with L-dopa reduced both the mean dyskinetic score and the maximal dyskinetic score (peak dyskinesias) induced by L-dopa treatment. Progression of L-dopa alone and L-dopa ⫹ DHA curves over time suggests that DHA is able to delay LID appearance and to reduce the severity of LIDs (see Figs 1C and 3). No modification of the antiparkinsonian effect of L-dopa was observed (see Fig 1C). For the total duration of the experiment (1 month), DHA treatment reduced mean and maximal LID scores by 40 and 46%, respectively (see Fig 1C, insets). The Table summarizes the factors that may have altered the development of dyskinesias in de novo animals. These include weight, cumulative MPTP dose, time to develop sustained parkinsonism, and basal parkinsonian score of animals in L-dopa and L-dopa ⫹ DHA groups. There was no significant difference between the two groups of animals used in the experiment (see Table). Figure 2 displays the time course of the development of mean dyskinesias in primed #1 and #2 groups over time before introducing DHA treatment (DOPA pretreatment) and after different time after L-dopa ⫹ DHA combined treatment. In both experiments DHA produced a constant and global reduction of LIDs during the effect of L-dopa (approximately 3 hours). Figure 3 shows day-to-day evolution of the maximal dyskinetic scores in L-dopa alone and L-dopa ⫹ DHA treated animals with de novo MPTPtreated monkeys. The cumulative LID scores obtained after 1 month of treatment were also reduced by 60% in the L-dopa ⫹ DHA group (see Fig 3, inset). Results from MPTP monkeys treated with de novo L-dopa indicates that DHA may delay or prevent the appearance of LIDs. Note that among de novo animals receiving Fig 1. Effects of docosahexaenoic acid (DHA) on L-dopa–induced dyskinesias (LIDs) in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkeys. (A) Concomitant administration of DHA (100mg/kg, SC) with 30 to 35mg/kg L-3,4-dihydroxyphenylalanine methyl ester plus 50mg benserazide, SC (termed DOPA thereafter) reduces LIDs in DOPA-primed MPTP monkeys. LIDs were scored before, during, and after DHA treatment, as indicated in the figure. The DOPA “post” measure was performed after 5 days of DHA washout. LIDs were rated according to a scale, as previously detailed.8,9 (B) Effect of DHA (200 mg/kg, PO) on LIDs in MPTP monkeys. DOPA-primed animals received daily DHA (orally) for 12 days in combination with their DOPA treatment. (C) Effects of DHA on LIDs in de novo DOPA-treated MPTP monkeys. Ten animals were exposed to MPTP until they developed stable parkinsonian syndrome. Then, half of them received daily DHA (100mg/kg, PO) starting 3 days before daily oral DOPA/ benserazide (100/25mg) administration. The other half received DHA vehicle. Daily administration of DHA and DOPA was maintained for 4 weeks. Dyskinesias were evaluated every day and averaged to obtain weekly dyskinetic scores. (insets) Average dyskinetic scores (mean and maximal) for the entire duration of the experiment (1 month) (*p ⬍ 0.05 vs DOPA group). Mean dyskinetic and parkinsonian scores were obtained from the average score for a total of 3 hours after DOPA administration in A and B and during the “ON” state of the animal under DOPA in (C). The maximal dyskinetic scores were obtained at the dyskinetic peak for a period of 15 minutes during the “ON” state of the animal. The horizontal line represent median score values for dyskinetic and parkinsonian scores. The data analysis for dyskinetic and parkinsonian scores in A and B were done using nonparametric Friedman’s test followed by multiple comparisons based on Friedman rank sums. These data were compared between the two treatment groups in C using Mann–Whitney U test. Locomotor activity is expressed as the sum of motility counts provided by the monitoring system every 5 minutes for a 3-hour period. The values were compared using analysis of variance for repeated measures followed by Fisher’s probability of least significant difference test. Total locomotor activity counts are presented as mean ⫾ SEM (N ⫽ 4-5). ND ⫽ not detected. Samadi et al: Effects of DHA on LIDs 285 Fig 2. Time courses of the development of dyskinesias after L-dopa treatment in L-dopa–primed 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) monkeys. (A) Each curve represents the average mean dyskinetic score for the first experiment with L-dopa –primed animals (primed #1), before starting subcutaneous docosahexaenoic acid (DHA) treatment (DOPA pretreatment) and after 2, 4, and 8 days of DOPA ⫹ DHA combined treatment (100mg/kg, SC) (N ⫽ 5, except for day 8 where N ⫽ 4). (B) Time courses of the development of dyskinesias after L-dopa treatment in the second experiment with L-dopa–primed MPTP monkeys (primed #2). Each curve represents the average mean dyskinetic score of primed #2 animals before starting per os DHA treatment (DOPA pretreatment) and after 6, 8, 10, and 12 days of DOPA ⫹ DHA combined treatment (200mg/kg, PO; N ⫽ 5). individual variability in the response to DHA may exist. Daily observation of the animals also indicated a reduced level of stress in animals receiving DHA. Fig 3. Time courses of the development of the maximum dyskinetic scores after L-dopa treatment in de novo 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkeys. Maximum dyskinetic scores obtained during the “ON” state after given treatments were daily averaged for all monkeys (N ⫽ 5 for DOPA alone and N ⫽ 4 for DOPA ⫹ DHA group). (inset) Cumulative maximal dyskinetic score for the total duration of the treatment (*p ⬍ 0.05 vs DOPA alone, according to a Mann–Whitney U comparison test). DHA was administered per os (gavage) at a dose of 100mg/kg for a period of 1 month. DHA, two developed almost no dyskinesias, and one had dyskinesias of similar intensity than monkeys that received L-dopa alone, whereas the rest of the group had intermediate responses, suggesting that some inter- 286 Annals of Neurology Vol 59 No 2 February 2006 Discussion Management of LIDs represents one of the most important problems encountered with classic dopamine replacement therapy in Parkinson’s disease. The precise biological substrate underlying the appearance of LIDs after dopamine denervation remains obscure. Their delayed appearance and persistence after treatment cessation strongly suggest that long-term and possibly permanent basal ganglia circuitry alterations are involved. Therefore, transcription factors, which regulate gene expression, represent most likely candidates. Indeed, modulation of transcription factors of the Fos family (⌬FosB) is shown to be associated with induction of LIDs.11,12 According to our previous observation in the 6-hydroxydopamine-lesioned rat, we proposed that the orphan member of the nuclear receptor family Nur77 might also be involved.13 Considering that Nur77 and RXRs seem to interact for modulation of dyskinesias after dopamine antagonist treatment,7 we may speculate that the beneficial effect of DHA on LIDs observed in this study might result from interactions with retinoid receptors. However, other mechanisms might also be involved.14 Additional experiments are required to address this issue. These data suggest that DHA reduced LIDs to an extend similar to other more classic drugs (targeting neu- rotransmitter or neuromodulator receptors) previously tested, such as cabergoline, a long-acting D2 agonist,15 OSU6162 compound also a D2 agonist,16 BP897 a dopamine D3 partial agonist,17 and morphine.8 Amantadine is also another potential drug to reduce LIDs18,19 that gives similar (approximately 40%) LID reduction. However, most of these compounds also reduced antiparkinsonian effect of L-dopa, caused tolerance, or displayed their antidyskinetic effects after prolonged administration. In addition, DHA produced a global reduction of LIDs throughout the duration of L-dopa effect, as illustrated in time courses of LID induction in L-dopa–primed monkeys. This is similar to what we previously reported for the opioid agonist morphine.8 Note also that our previous published experiments with MPTP-lesioned monkeys always confirmed a high degree of denervation in animals showing parkinsonian disability scores as shown in this study. For example, biogenic amine levels evaluated by high-performance liquid chromatography demonstrated a decrease of dopamine concentration by approximately 90 to 95% and a decrease of greater than 80% of the metabolites 3,4dihydroxyphenylacetic acid and homovanillic acid compared with intact monkeys in MPTP-treated animals showing similar parkinsonian disability scores as presented here.2,20 –22 Therefore, we may safely assume that the extent of lesions of MPTP-treated monkeys presented here should be comparable with previous studies. Moreover, the mean parkinsonian score of animals distributed amongst the L-dopa alone and L-dopa ⫹ DHA groups were not statistically different (see Table). The absence of effect of DHA on antiparkinsonian property of L-dopa is also indicative that L-dopa absorption or brain bioavailability was not altered by administration of this polyunsaturated fatty acid. Because L-dopa is an amino acid derivative, the possibility that protein-rich meal ingestion alters oral L-dopa absorption and brain bioavailability has been documented.23,24 DHA is an essential polyunsaturated fatty acid that is rapidly taken up by the body and rapidly incorporated into triglycerides in the blood circulation. Although the active transport of DHA into the brain is not well understood, strong evidence indicate that DHA easily enters into the brain. For example, DHA has been used as a prodrug conjugate with the antipsychotic clozapine to increase the brain-to-blood ratio of the drug. It is reported that chemical coupling of clozapine with DHA increased the brain-to-blood ratio of clozapine concentration by a factor of approximately 10.25 In summary, these results demonstrate that DHA administration reduces LIDs in a nonhuman primate model of Parkinson’s disease. If we extrapolate the dose used in this study to humans, we obtain a range of approximately 5 to 10g of DHA per day. This regimen is in the range of doses reported in previous clinical trials using DHA in the context of neuropsychiatric disorders.26 Although these results need to be replicated in humans, it strongly suggests that an adjunct treatment with DHA may represent a new and safe approach27 to improve the quality of life of Parkinson’s disease patients. This work was supported by grants from the Canadian Institutes for Health Research (200303MOP-118040, D.L.), the Parkinson Society of Canada (D.L, C.R.), and the Parkinson’s Disease Foundation of United States. 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