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NEUROPHARMACOLOGY

The Effects of a Selective Dopamine D₂ Receptor Agonist on Behavioral and Pathological Outcome in 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-Treated Squirrel Monkeys

Pfizer Global Research and Development, Groton, Connecticut (D.T.S., E.H.-K.) and Kalamazoo, Michigan (M.A.C.); Wisconsin National Primate Research Center, Department of Anatomy, University of Wisconsin, Madison, Wisconsin (M.E.E.); and Ligand Pharmaceuticals, Discovery Research, San Diego, California (M.D.M.)

Received April 4, 2005; accepted June 2, 2005.

	Abstract

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In this study, we investigated antiparkinsonian activity of the novel, highly selective dopamine D₂ receptor agonist sumanirole compared with two clinically effective dopaminergic therapies in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) primate model of Parkinson's disease. Squirrel monkeys were rendered parkinsonian by chronic administration of MPTP and subsequently dosed with vehicle, L-DOPA plus carbidopa (L-DOPA), ropinirole, or sumanirole over a duration of 8 weeks. Antiparkinsonian effects measured with a parkinsonian primate rating scale (PPRS) showed that sumanirole elicited improved functional outcome compared with vehicle. The dopamine D2/D3 agonist ropinirole improved behavioral outcome similar to sumanirole, whereas L-DOPA treatment yielded the most significant symptomatic improvement. The relative rank of therapies that elicited normalization of PPRS was L-DOPA > sumanirole; ropinirole did not normalize PPRS in any of the treated monkeys. Dyskinesias were present with L-DOPA treatment but were not observed in sumanirole-, ropinirole-, or placebo-treated primates. Pathologically, all MPTP-treated animals displayed neurodegeneration of dopaminergic neurons in the substantia nigra pars compacta and reactive astrocytosis. Neurons immunoreactive with antibodies to the nuclear transcription factor FosB were most significantly increased in the striatum of L-DOPA-treated monkeys. These results suggest that sumanirole can exert antiparkinsonian effects similar to L-DOPA without the behavioral and morphological consequences of the latter.

DA receptors exist as five subtypes, each of which may have different functions based on dissimilar neuroanatomical expression and pharmacological properties. D₁ and D₂ receptors are abundant in the caudate and putamen, whereas D₃ receptors are expressed at lower levels in the basal ganglia (Emilien et al., 1999). The D₂ receptor may play a key role in the pathophysiology of motor function since it is up-regulated in the striatum in response to DA denervation (Gerfen et al., 1990; Gurevich and Joyce, 1999) as well as in human PD and in PD animal models (Joyce et al., 1986; Brooks et al., 1992; Graham et al., 1993). Furthermore, mice with targeted deletion of the D₂ receptor exhibit parkinsonian locomotor impairment (Baik et al., 1995), reduced number of neurons within the SN (Parish et al., 2001), reduced expression of trophic factors (Bozzi and Borrelli, 1999), absence of axonal sprouting after 6-OHDA lesion (Parish et al., 2001), and abnormal synaptic plasticity in the striatum (Calabresi et al., 1997). By contrast, activity at D₃ and D₄ receptors may play a role in the nonmotor symptoms that occur in PD patients, based on their localization in limbic regions (Joyce et al., 1986, 2001) and the finding that mice with homozygous null mutations of the D₃ or D₄ receptors exhibit hyperactivity under basal or methamphetamine-stimulated conditions, respectively (Accili et al., 1996; Rubinstein et al., 1997).

Currently available DA agonists, e.g., pramipexole and ropinirole, which have activity at both D₃ and D₂ receptors, produce symptomatic improvement in PD with a reduced frequency of motor complications compared with L-DOPA (Lang and Widner, 2002; Wooten, 2003). However, the extent of reversal of PD signs is somehow limited and inevitably after a period of time, L-DOPA supplementation is needed. In light of the key role of D₂ receptors in controlling motor function, it is plausible that a selective D₂ agonist might also be efficacious and even further reduce the incidence of adverse drug effects, e.g., dyskinesias, motor fluctuations, and hallucinations. The beneficial effect of selective stimulation of D₂ compared with D₃ receptors has been recently demonstrated in MPTP-treated marmosets. In the study of Silverdale et al. (2004), selective blockade of D₃ receptors with the D₃ antagonist S33084 [GenBank] significantly enhanced the antiparkinsonian effects of L-DOPA and ropinirole.

Sumanirole is a highly selective full agonist for the dopamine D₂ receptor and therefore represents a unique tool to investigate the role of D₂ receptors in the control of motor function. Pharmacological properties of sumanirole include greater than 200-fold selectivity for the D₂ receptor subtype compared with other dopamine receptor subtypes in radioligand binding assays (McCall et al., 2005), potent D₂ receptor pharmacology in rodents in vivo (Durham et al., 1997; Sethy et al., 1997) and induction of robust rotational behavior in 6-OHDA-treated rats (McCall et al., 2005).

Systemic administration of MPTP to squirrel monkeys induces a parkinsonian syndrome that is effectively ameliorated by treatment with L-DOPA. Furthermore, L-DOPA therapy faithfully reproduces the occurrence of abnormal movements, in particular choreoathetosis dyskinesias (hyperkinetic, purposeless dance-like movements; Langston et al., 2000). Measurements of the animal's motor function before and after treatments can be easily quantified using parkinsonian and dyskinesia scales similar to the ones that are used clinically. Abnormal movements have been associated with changes in several neuropeptides such as opioid precursors and their upstream transcription factor FosB (Sonnenberg et al., 1989; Westin et al., 2001).

In this study, the effects of the D₂ agonist sumanirole were compared with a nonselective dopamine D₂/D₃ agonist ropinirole and to L-DOPA in MPTP-treated primates using behavioral and pathological outcome measures. Behavioral assessment included antiparkinsonian activity and occurrence of dyskinesias and pathological outcomes included qualitative histopathology of the substantia nigra and quantitative analysis of FosB in the basal ganglia.

	Materials and Methods

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Animals. Thirty-three male squirrel monkeys (Saimiri sciureus), approximately 6 years of age, were pair housed with a 12-h light cycle. All animals received water ad libitum. New World Monkey Chow was provided fresh each morning (4 h before behavioral testing) and supplied ad libitum. Diet was supplemented with fruit juice, PrimaBurger, Primate Chews, fresh vegetables, wheat bread, canned or fresh fruit, crackers, and cookies. The study was performed in accordance with federal guidelines of proper animal care and with the approval of the Institutional Animal Care and Use Committee.

MPTP Treatment. Twenty-nine monkeys were administered MPTP s.c. one to two times per week to elicit a parkinsonian syndrome. Four additional animals remained untreated and were used as naive controls for biochemistry and brain morphology. Before each dosing, each monkey was evaluated for its tolerance to MPTP and motor disability score. Individual doses and the total number of doses administered were titrated to produce similar degrees of parkinsonian disability. Individual doses ranged from 0.2 to 2.3 mg/kg (total dose 12.9–15.9 mg/kg) and the number of individual doses ranged from four to 17 doses based on above-mentioned criteria. Treatment duration ranged over a period of 4 to 5 weeks for the most sensitive monkeys (four doses) and 18 weeks for the least sensitive monkeys (17 doses). The total number of MPTP doses was (mean ± S.E.) L-DOPA group, 11.3 ± 1.3; sumanirole group, 12.0 ± 1.6; ropinirole group, 11.1 ± 0.9; and placebo group, 12.5 ± 1.6. Cumulative doses were (mean ± S.E.) L-DOPA group, 12.4 ± 2.0 mg/kg; sumanirole group, 15.9 ± 3.4 mg/kg; ropinirole group, 12.9 ± 1.6 mg/kg; and placebo group, 12.4 ± 1.8 mg/kg. There were no statistically significant differences between groups for number of doses (ANOVA, p = 0.92) and total amount of MPTP (ANOVA, p = 0.78). Hand feeding, s.c. fluid administration, and additional heating were provided as needed.

At the end of the MPTP dosing, all animals were stably parkinsonian and exhibited a narrow range of PPRS scores. The monkeys were randomized and placed in different treatment groups. After assignment to study groups, monkeys were allowed to recover for at least 1 month before initiating drug dosing. One monkey assigned to the L-DOPA group died during the recovery period and was excluded from the data analysis.

Drug Treatment. Figure 1 outlines the study design for the present experiment. The dosing administration schedule for this experiment was designed to match clinically relevant dosing strategies. The monkeys received their treatments orally twice a day, and each dopamine agonist was administered in escalating doses to reach maximal antiparkinsonian activity.

View larger version (14K): [in this window] [in a new window]   Fig. 1. Experimental design. The dosing administration schedule for this experiment was chosen based on clinically relevant dosing strategies. The monkeys received their treatments orally twice a day and each drug was administered in different doses. PPRS, week 0 to 8, videotape, PPRS, AMI; weeks 0 and 4, pharmacokinetics.

View larger version (13K): [in this window] [in a new window]   Fig. 2. Identification of maximally effective doses of sumanirole and ropinirole by dose titration. A, sumanirole. B, ropinirole. The optimal doses of sumanirole and ropinirole were established for each monkey by titrating the dose until the maximum antiparkinsonian effect was achieved (study weeks 1–4). During the drug titration period the blinded-to-treatment raters evaluated the animals two to three times a week. After week 3, the animals seemed to have reached their maximal improvement, i.e., increasing the ropinirole dose from 0.06 ± 0.00 mg/kg b.i.d. during week 3 to 0.12 ± 0.00 mg/kg b.i.d. during week 4 produced no further improvement in the PPRS score. Similarly, increasing the sumanirole dose from 0.94 ± 0.15 mg/kg in week 3 to 1.09 ± 0.2 mg/kg b.i.d. in week 4 caused no further improvement in parkinsonian symptoms. Each monkey continued to receive the final fixed dose of sumanirole or ropinirole for four additional weeks (study weeks 5–8).

Plasma Drug Assay. For assessment and verification of circulating drug levels, pharmacokinetic analysis was performed at specified times during the study. Blood samples were taken after an initial dose and were used to determine the time of maximum concentration of drug in plasma. This time was then used for the timing of behavioral scoring. At week 4 after initiation of drug treatment, monkeys were administered a single dose of L-DOPA, ropinirole, or sumanirole (maximal effective dose). Blood was collected into EDTA-containing tubes at particular time intervals after dosing (1, 2, 4, 8, and 12 h) and plasma was analyzed for drug levels. Plasma samples for L-DOPA analysis were supplemented with an antioxidant by adding to microfuge tubes 10 µl of a 10% sodium metabisulfite solution before freezing.

L-DOPA concentration was quantified using a modification of an existing high-performance liquid chromatography and sample preparation technique (Wikberg, 1991). The lower limit of quantification for this assay was 0.754 ng/ml for L-DOPA.

Quantification of sumanirole and ropinirole was performed by adding 50 µl of plasma to individual microcentrifuge tubes. A 5-µl aliquot of 50% methanol (MeOH) and 25 µl of a working internal standard solution (25 ng/ml in 50% MeOH) were added to each, followed by 400 µl of 1:1 hexane/dichloromethane solution. The mixtures were centrifuged at 12,500 rpm, and the aqueous phase was flash frozen by dipping the microcentrifuge tube into a methanol/dry ice bath. Approximately 400 µl of the nonfrozen organic layer was transferred to autosampler vials and evaporated under a stream of N₂ gas at 40°C until dry (TurboEvap; Zymark Corp, Hopkinton, MA). The residue was reconstituted in 100 µl of 25% acetonitrile. Liquid chromatography-mass spectrometry analysis was performed. The lower limit of quantification was 0.085 ng/ml, and accuracy of quality control samples was within 20% of expected values. The calibration standards ranged from 0.89 to 1770 ng/ml.

Primate Parkinsonian Rating Scale (PPRS). Parkinsonian motor disability was assessed in MPTP-treated monkeys two to four times per week by two independent raters who were blinded to treatment group assignments. Naive monkeys were not evaluated behaviorally. Assessment of inter-rater reliability calculated using the Spearman correlation coefficient was 0.95. Test-retest variability for PPRS scores (intrasubject day-to-day variability) calculated using the Spearman-Brown reliability coefficient was 0.80. Assessments were made by visual observation of monkeys in their home cages under peak dose conditions. The severity of parkinsonism was quantified using the PPRS modified for the squirrel monkey (Langston et al., 2000). In this scale, spatial hypokinesia (0–4), body bradykinesia (0–4), manual dexterity (right and left arm, 0–4 each), balance (0–4), and freezing over a 4-min clinical observation period were evaluated. The maximum possible score was 20 points, greater than 12 points corresponded to severe parkinsonism, and a normal control animal scored 1.5 points (Langston et al., 2000). A score of 6 to 8 corresponds to mild-to-moderate parkinsonian activity by behavioral indices. PPRS assessments were performed consistently at 1.5 h after drug administration throughout the 8 weeks of treatment.

Abnormal Movement Evaluation. A modified time sample neurological observation was used to quantify abnormal movements after drug administration. Data were collected at T_max (time of maximum drug concentration in plasma) for 30 min. Briefly, each animal was placed in an isolated large Plexiglas chamber that allowed simultaneous videotaping from two angles. The recording time started after a 5-min habituation period. The double videorecordings were obtained once per week and analyzed by two treatment blinded independent raters. Peak-dose dyskinetic movements were scored using a modification of the Abnormal Movements Index (Palfi et al., 2000). Orofacial dyskinesia, dystonia and chorea were separately identified axially and for each limb. Abnormal movements were rated as being present (n = 1) or absent (n = 0) during each 5-min time period for a 30-min test session. A dyskinesia index was obtained by averaging the incidence of each symptom during the total duration of the test period (maximum score: 11).

Tissue Preparation. At 8 weeks after initiation of drug treatments, monkeys were administered a lethal dose of barbiturate and euthanized. The time interval between the final dose of drug and euthanasia was 3 h. Animals were perfused transcardially with phosphate-buffered saline (PBS) followed by 2% periodate lysine paraformaldehyde fixative. Brains were fixed overnight at 4°C and then rinsed in PBS. The brains were cryoprotected through a series of graded solutions, 13% sucrose in PBS for 2 days, 15% sucrose in PBS for 2 days, and 18% sucrose in PBS for 3 days. Before freezing, brain hemispheres were blocked using external landmarks into two portions, one containing the basal ganglia and the other the substantia nigra (Emmers and Akert, 1963). The blocks were then frozen in dry ice-cooled isopentane and tissue blocks were stored at -80°C.

For each monkey, one hemisphere was chosen for thin cryosectioning. Choice of hemisphere was randomized within each treatment group so that there was an equal number of monkeys represented on the left and on the right sides of the brain within each treatment group. Twenty-micrometer-thick cryosections were collected onto charged slides (Superfrost Plus; VWR, West Chester, PA) using a Leica CM3050 cryostat. For each animal, three different coronal levels of the basal ganglia (precommisural, anterior commissure, and postcommisural) or the SN (rostral, middle, and caudal portions of the SN) were saved with two sections per slide. In total, 32 serial sections were collected at each of the three levels and matched between different monkeys using precise anatomical landmarks. Cryosections were stored at -80°C until staining.

Neurochemical Analysis of Cerebrospinal Fluid (CSF). Levels of monoamines and metabolites were investigated in CSF samples collected at study termination. CSF was collected via the cisterna magna after barbiturate overdose and samples were stored at -80°C. At the time of the analysis, samples were centrifuged at 12,500 rpm for 10 min at 4°C. Aliquots of supernatants, containing 0.01 N perchloric acid were assayed for monoamines and metabolites on an HPLC with Coularray electrochemical detector (ESA Inc., Chelmsford, MA), using a Zorbax SB-C18 analytical column (4.6 x 100 mm, 3.5-µm particle size) protected by a Zorbax SB-C18 guard column (4.6 x 12.5 mm, 5-µm particle size) and maintained at 25°C. The mobile phase consisted of 75 mM NaH₂PO4, 1.8 mM sodium octanesulfonate, 25 µM EDTA, 978 µM tetraethylammonium, and 9% acetonitrile, pH 3.0. Analytes were monitored on the Coularray with applied potentials set at 0 and +300 mV. All peaks were detected within 15 min.

Immunohistochemistry. The following antibodies were used: anti-glial fibrillary acidic protein (GFAP) monoclonal antibody (InnoGenex, San Ramon, CA; 1:200), anti-tyrosine hydroxylase (TH) monoclonal antibody ascites (Chemicon International, Temecula, CA; 1:800), polyclonal antibody anti-tyrosine hydroxylase (Calbiochem, San Diego, CA; 1:1000), FosB (Chemicon International), affinity purified rabbit polyclonal antiserum raised against an N-terminal peptide, which is common to full-length FosB and FosB (sc-48; Santa Cruz Biotechnology Inc., Santa Cruz, CA; 1:1500); and NeuN (monoclonal antibody; Chemicon International; 1:1000). Each of the above-listed antibodies was used to stain the substantia nigra (GFAP and TH antibodies) or the basal ganglia (TH and FosB). Immunohistochemistry was performed using avidin-biotin peroxidase detection kit (ABC; Vector Laboratories, Burlingame, CA) with diaminobenzidine (DAB peroxidase substrate kit; Vector Laboratories) as chromogen. Experiments were initially conducted by carrying out staining procedures manually on brain sections from naive animals to validate each primary antibody by determining the optimum dilution and duration of antibody incubation required to observe maximum signal-to-noise ratio of immunoreaction product.

All the treatment groups were stained in parallel as "sets of tissue". A "set" was constituted by one slide from each level from each animal. Slides comprising a complete set were removed from -80°C and allowed to thaw for 10 min before staining. The slides were postfixed in 4% paraformaldehyde on wet ice for 10 min. The staining procedure was run on an automated Dako autostainer (DakoCytomation California Inc., Carpinteria, CA) as follows. Slides were rinsed with PBS and then incubated for 10 min with 0.1% H₂O₂ in distilled H₂O to block endogenous peroxidase, followed by PBS rinses. Slides were blocked in 5% denatured goat serum for 10 min, followed by incubating in primary antibody. Antibodies were incubated either 1 h (TH and GFAP) or 48 h (FosB). Sections were labeled with anti-mouse or anti rabbit Envision + polymer horse-radish peroxidase (DakoCytomation California Inc.) for 30 min followed by development in DAB (diaminobenzidine). To enhance DAB reaction, the slides were incubated with DAB Enhancer (DakoCytomation California Inc.). For sections containing the SN, immunostained sections were lightly counterstained with hematoxylin to delineate cytoarchitecture/cellular structure.

One representative section from each level of all monkeys was also stained with cresyl violet for assessment of regions, landmarks and overall tissue integrity. One set of sections from the SN levels was stained with H&E as well.

Quantification of FosB Immunoreactive Neurons. The number of FosB-immunopositive neurons in the postcommissural basal ganglia was calculated using a profile counting method with ImagePro software, by an investigator blinded to the treatment group. Representative fields measuring 800 µm horizontal x 700 µm vertical were acquired using a 20x objective from two consecutive immunostained sections. Images were acquired from the caudate and the putamen in sections that were matched coronally between different monkeys. For this determination, a macro was written in ImagePro such that each immunoreactive neuron was counted using a mouse on the computer, and the numbers were automatically entered into an Excel spreadsheet. Neurons were assessed by visual inspection, and cells that were 10 to 25 µm and displayed neuronal morphological features were counted. Representative FosB-immunopositive neurons were counted relative to a counting threshold based on staining density, target size, and target shape. The parameters of the counting threshold were set based upon a standard control slide from the staining run. In naive brain there were rare, lightly stained individual FosB-positive profiles, and this was used to set the threshold. All slides that were quantitatively compared with each other were stained in the same staining run and counted using the same thresholding parameters and microscope and digital camera settings. The number of neurons in each field was counted and averaged for each monkey. The mean number of FosB-ir neurons for the caudate and putamen was determined by averaging the values from all the monkeys within each of the treatment groups.

Statistical Analysis. Statistical analysis of drug effects was performed using the average PPRS values determined by the two raters. Inter-rater reliability calculated using the Spearman correlation coefficient was 0.95. Test-retest variability for PPRS scores (intrasubject day-to-day variability) was calculated using the Spearman-Brown prediction formula. The Spearman-Brown reliability coefficient was 0.80. Statistical analysis of drug effects was performed using the average PPRS values determined by the two raters.

Differences in behavioral scores were analyzed using Kruskal-Wallis nonparametric ANOVA followed by Tukey's multiple comparisons test. Statistical significance was set at p < 0.05. The frequency with which symptomatic normalization occurred (PPRS 1.5) in monkeys that received titrated, maximally effective doses of dopaminergic therapies was determined using Pearson's chi square test. Analyses of CSF monoamines and metabolites was performed using a one-way ANOVA across all treatment groups followed by Dunnett's comparison test for each analyte.

The percentage of reduction of Fos B neurons as a function of the lesion was calculated by comparing the mean values of naive and treatment group monkeys within each nucleus. Statistical analysis was performed using Kruskal-Wallis nonparametric ANOVA. If significant, multiple pairwise comparisons of the means were determined with t-distribution using a significance level of p < 0.05.

	Results

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Assessment of the Parkinsonian Syndrome. Squirrel monkeys were rendered parkinsonian by repetitive systemic administration of MPTP (Fig. 1). Before MPTP treatment, all the animals presented a general behavior characteristic of their age. The animals were active, freely climbing on the cage walls, jumping and exploring the environment. None of the monkeys showed parkinsonian signs and their clinical parkinsonian rating (PPRS) was 0 points.

During the MPTP treatment, the animals progressively developed a syndrome characterized by hypokinesia, bradykinesia as well as balance and fine motor skill disturbances. There were no statistically significant differences between groups for number of doses (L-DOPA group, 11.3 ± 1.3; sumanirole group, 12.0 ± 1.6; ropinirole group, 11.1 ± 0.9; and placebo group, 12.5 ± 1.6) or for total amount of MPTP (L-DOPA group, 12.4 ± 2.0 mg/kg; sumanirole group, 15.9 ± 3.4 mg/kg; ropinirole group, 12.9 ± 1.6 mg/kg; and placebo group, 12.4 ± 1.8 mg/kg). At completion of MPTP treatment, monkeys exhibited a stable parkinsonian syndrome displaying a PPRS ranking of 6 to 8 without abnormal movements. There were no significant differences in PPRS scores between the different groups at the time of randomization or immediately before initiation of dosing (Fig. 3).

View larger version (10K): [in this window] [in a new window]   Fig. 3. Individual PPRS scores by treatment: predrug and at study termination, week 8. A score of 1.5 (dotted line) corresponded to "normal" behavior. There were no significant differences in PPRS scores at baseline. At week 8, the following groups had significant differences in PPRS scores: Vehicle versus L-DOPA, p = 0.004; vehicle versus sumanirole, p = 0.011; vehicle versus ropinirole, p = 0.024; L-DOPA versus ropinirole, p = 0.003. No significant differences were observed between the following groups: ropinirole versus sumanirole (p = 0.20) and L-DOPA versus sumanirole (p = 0.31). Statistical analysis of frequency of normalization was L-DOPA versus sumanirole, p = 0.61; L-DOPA versus ropinirole, p = 0.007; and sumanirole versus ropinirole, p = 0.021.

Clinical Rating after Drug Administration. Over the 8 weeks of drug treatment, the placebo-treated group showed a mild spontaneous recovery of PD symptoms (PPRS scores 6 to 7 at baseline and 4 to 5 at week 8; Fig. 3). However, the animals were still considerably impaired, showed a typical PD syndrome that was significantly different from the behavior observed with any of the DA replacement treatments.

A significant improvement in the PPRS was observed in the first week after L-DOPA treatment. L-DOPA-treated monkeys demonstrated continued improvement throughout the treatment phase (Stephenson et al., 2005). By week 3, more than half the animals had reached a PPRS rating of 1.5 points (normalization of PPRS) and remained at that level until the end of the study (week 8). Rating improvements occurred first in spatial hypokinesia (movement around the cage) and body bradykinesia. Further amelioration occurred when manual dexterity and balance scores improved. Abnormal movements were observed within a week after initiation of L-DOPA treatment, consistent with previous reports (Boyce et al., 1990; Pearce et al., 1998).

For dopamine agonist treatment, individual subjects were dose titrated to maximal efficacy, similar to dosing strategy in human PD subjects. L-DOPA treatment was a fixed dose, as is implemented clinically. The optimal doses of sumanirole and ropinirole were established for each monkey by titrating the dose until the maximum antiparkinsonian effect was achieved (study weeks 1–4). The final daily dose of ropinirole achieved by titration, 0.24 mg/kg/day, was similar to doses used in previous studies of MPTP-treated marmosets (0.2–0.5 mg/kg/day; Fukuzaki et al., 2000). Note the lack of improvement in PPRS between weeks 3 and 4 for both drugs, after the last dose elevation. Each monkey continued to receive the final fixed dose of sumanirole or ropinirole for 4 additional weeks (study weeks 5–8; Fig. 2).

Analysis of antiparkinsonian activity at week 8 showed statistically significant antiparkinsonian activity of all dopamine treatments compared with placebo (Fig. 3). Sumanirole-treated monkeys presented a significant improvement of PPRS scores (compared with placebo) after the first escalating dose (Fig. 2). At week 8, there was individual variability in the response to sumanirole with four of the eight sumanirole-treated animals showing normalization of PPRS scores (Fig. 3). Although ropinirole-treatment produced a significant improvement of PPRS compared with vehicle, none of the ropinirole-treated animals reached a level of ameliorization similar to the L-DOPA-treated monkeys (Fig. 3), i.e., six of eight L-DOPA-treated monkeys and zero of the eight ropinirole-treated animals showed normalization of PPRS scores. The doses of ropinirole that exhibited antiparkinsonian activity are consistent with that reported in a MPTP marmoset model, which also used a dose titration regime (Eden et al., 1991). Comparisons of the individual groups revealed that the antiparkinsonian efficacy of sumanirole group was not statistically significant different from L-DOPA, whereas ropinirole and L-DOPA treatments were significantly different. Both ropinirole and sumanirole improved parkinsonian symptoms, and L-DOPA treatment was most effective in improving behavioral outcome among the different dopaminergic treatments.

Occurrence of Abnormal Movements. Before MPTP lesion and MPTP + L-DOPA, sumanirole, ropinirole or placebo treatment none of the animals presented abnormal movements. As described previously (Langston et al., 2000), all the MPTP + L-DOPA-treated monkeys developed abnormal movements (Fig. 4). One week after beginning L-DOPA administration, animals presented signs of dyskinesia and dystonia. Orofacial dyskinesias were not observed during the first week, but they were evident in the subsequent sessions. Dyskinetic dance-like movements typical of chorea were observed. Dystonias continued and increased throughout the course of L-DOPA treatment, affecting trunk, upper and lower limbs. In comparison, abnormal movements were not observed in the placebo-, ropinirole- or sumanirole-treated animals at any time throughout the study.

View larger version (15K): [in this window] [in a new window]   Fig. 4. Effect of vehicle, L-DOPA, sumanirole, and ropinirole on the occurrence of dyskinesias in MPTP-treated monkeys, as measured on the modified Abnormal Movement Index.

CSF Levels of Monoamines. Since monoamines in the CSF can serve as an index of central dopamine levels, monoamine neurotransmitters and their metabolites were measured in the CSF at the termination of the study. L-DOPA treatment markedly elevated the CSF concentrations of DA, and the DA metabolites homovanillic acid and dihydroxyphenylacetic acid compared with placebo-treated MPTP monkeys (DA: 3.77 ± 0.69 versus 1.33 ± 0.03 ng/ml; HVA: 1028 ± 180 versus 51 ± 11 ng/ml; dihydroxyphenylacetic: 81 ± 22 versus 0.19 ± 0.07 ng/ml; in each case L-DOPA versus placebo, respectively; p < 0.001). By comparison, CSF concentrations of DA and metabolites were not significantly different from placebo in sumanirole- or ropinirole-treated monkeys. CSF concentration of serotonin, 5-hydroxyindole acetic acid, and norepinephrine did not differ significantly in the various treatment groups in agreement with a previous report (Russ et al., 1991). However, there was a statistically significant increase in 3-methoxy-4-hydroxyphenylglycol levels after L-DOPA treatment compared with placebo (75.0 ± 17.1 versus 19.6 ± 3.9 ng/ml; p < 0.001). Therefore, L-DOPA treatment but not dopamine agonist therapy resulted in elevated CSF dopamine levels.

Pathology of the Substantia Nigra. Routine histopathology with Nissl and H&E revealed distinctive neurodegeneration in the substantia nigra pars compacta (SNpc) in all the MPTP-lesioned monkeys. Abnormalities included derangement of Nissl substance, presence of swollen neurites, extraneuronal melanin, and reactive astrocytosis. Neurodegeneration of TH-positive neurons in the SNpc was observed in all the MPTP-treated monkeys (Fig. 5). In all MPTP-treated monkeys, GFAP immunoreactivity was profoundly increased in both cell bodies and in the glial fiber network in the SN (Fig. 5) but not in other anatomic regions within the tissue section (e.g., cortex). No qualitative differences in TH or GFAP immunoreactivity was observed in the substantia nigra from animals treated with different therapies.

View larger version (207K): [in this window] [in a new window]   Fig. 5. Neuropathology of the substantia nigra in representative sections from a naive (left) and MPTP-treated monkey (right). A and B, low magnification. C and D, high magnification of TH immunostaining in the SNpc. MPTP + vehicle-treated monkeys (B and D) display fewer TH-ir neurons, and the remaining neurons have an atrophic morphology (D). E and F, GFAP immunoperoxidase staining in the SNpc. MPTP-lesioned SN presents a dense glial fiber network and larger astrocytes that express more intense GFAP staining (F) compared with naive animals (E). Scale bar, 1000 µm (A and B) and 50 µm (C–F).

View larger version (99K): [in this window] [in a new window]   Fig. 6. Immunoperoxidase staining of tyrosine hydroxylase in hemiforebrain sections of a representative naive (A and C) and MPTP + placebo-treated monkey (B and D). The region highlights caudate (Cd) and putamen (Pu) at the precommissural level. Profound reduction of TH fiber and terminal density is observed at both low (A and B) and high (C and D) magnification. More substantial reduction is observed in dorsolateral caudate and putamen than in ventromedial regions. Scale bar, 10 mm (A and B) and 40 µm (C and D).

View larger version (124K): [in this window] [in a new window]   Fig. 7. Representative sections of the caudate nucleus showing FosB-immunoreactivity in the basal ganglia of naive (A), MPTP + placebo- (B), MPTP + sumanirole- (C), and MPTP + L-DOPA (D)-treated monkeys. Scale bar, 50 µm.

Quantification of the number of FosB-ir nuclei revealed no significant differences in naive versus MPTP-treated monkeys (Fig. 8). Monkeys treated with dopamine agonists showed a significant increase of FosB-ir nuclei compared with naive monkeys. In the caudate nucleus, both ropinirole and sumanirole treatments led to increased FosB-ir compared with naive monkeys. In the putamen, ropinirole- but not sumanirole-treated monkeys had significantly increased number of FosB-ir neurons. The most profound increases were observed in monkeys treated with MPTP + L-DOPA where we found a highly significant 3- to 4-fold elevation in the number of FosB-ir neurons in the caudate and putamen compared with all other groups of monkeys (Fig. 7).

View larger version (11K): [in this window] [in a new window]   Fig. 8. Quantification of the number of FosB-positive neurons in the representative regions of the caudate and putamen of monkeys as a function of different treatments. Statistical analyses shows the following differences: caudate: L-DOPA versus naive, p < 0.0005 (a); versus vehicle, p < 0.005 (b); versus ropinirole, p < 0.005 (c); p < 0.005, versus sumanirole, p < 0.005 (d); sumanirole versus naive, p < 0.01 (e); and ropinirole versus naive, p < 0.05 (f); putamen: L-DOPA versus naive, p < 0.0005 (g); versus vehicle, p < 0.0005 (h); versus sumanirole, p < 0.005 (i); versus ropinirole, p < 0.05 (j); ropinirole versus naive, p < 0.05 (k); and versus vehicle, p < 0.05 (l). All other treatment groups were not statistically different.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The present study demonstrates that the selective D₂ agonist sumanirole can exert antiparkinsonian effects similar to D₂/D₃ agonist treatment (ropinirole) in MPTP-treated non-human primates without eliciting the abnormal movements and pathology associated with L-DOPA therapy. All three dopaminergic drugs evaluated (L-DOPA, sumanirole, and ropinirole) showed antiparkinsonian activity under the conditions used in the present study. Antiparkinsonian effects of L-DOPA were significantly better than treatment with ropinirole, similar to clinical findings (Rascol et al., 2000). L-DOPA was able to reduce the PPRS score to normal levels (score 1.5) in six of the eight subjects, whereas none of ropinirole-treated monkeys achieved that level of improvement. Four of the eight sumanirole-treated monkeys presented normalization of the parkinsonian syndrome (PPRS scores 1.5). Differences in the level of improvement between groups were not related to the monkeys PD syndrome. Since baseline PPRS scores were matched before drug treatment, there was no preselection bias for assigning monkeys with different levels of impairment to a particular treatment group. The individual differences in the PPRS scores within the sumanirole group did not correlate with blood drug levels, suggesting that variability in drug metabolism does not account for differences in antiparkinsonian effects. Moreover, circulating levels of drug in the monkeys were similar values to those obtained in PD patients that have shown symptomatic relief after treatment (D'Souza et al., 2002).

The antiparkinsonian activity of sumanirole may be explained by direct stimulation of dopamine D₂ receptors. D₂ agonist properties of sumanirole have been demonstrated in vivo in several rat models. Pharmacological endpoints including increase in plasma prolactin secretion and dopamine metabolite concentration in hypothalamus (Durham et al., 1997), depression of dopamine neuron electrophysiological firing rate (Camacho-Ochoa et al., 1995; McCall et al., 2005), and elevation of striatal acetylcholine concentrations in normal and denervated rats (Sethy et al., 1997). In several of these experiments, D₂ selectivity of sumanirole was demonstrated by coadministration of a D₂ antagonist (Camacho-Ochoa et al., 1995; Sethy et al., 1997). D₂ antagonist treatment was not used in the present study to avoid confounding the pathology endpoints. Thus, in MPTP-treated squirrel monkeys, we cannot attribute specific components of the PPRS score to D₂ versus D₃ agonist activity. However, in the study of MPTP-treated marmosets, administration of a selective D₃ antagonist significantly enhanced the antiparkinsonian effects of L-DOPA and ropinirole (Silverdale et al., 2004), which supports the beneficial effects of D₂ compared with D3 receptors. Future studies using D₂ antagonist paradigms in combination with sumanirole will be very informative to address this issue.

Antiparkinsonian effects are not necessarily associated with the presence of abnormal movements. It can be argued that the presence of dyskinesias in L-DOPA-treated monkeys was related to the longer period of L-DOPA administration. However, 1 week after starting L-DOPA dosing, abnormal movements were evident. Abnormal movements were not observed in monkeys treated with vehicle, sumanirole, or ropinirole throughout the study, even after 4 weeks of a maximally effective antiparkinsonian dose of either agonist. These data suggest that L-DOPA is more likely to induce dyskinesias than DA agonist monotherapy in the doses and duration of treatment used in our studies. Absence of dyskinesias with DA agonist treatment is consistent with previous reports showing relative low intensity of abnormal movements in L-DOPA-naive MPTP-lesioned marmosets treated with antiparkinsonian doses of ropinirole or bromocriptine (Pearce et al., 1998; Maratos et al., 2003).

PD patients treated with L-DOPA develop dyskinesias after several years of treatment. However, patients with MPTP-induced PD quickly develop dyskinesias after L-DOPA therapy (Langston and Ballard, 1984), similar to what we observed in parkinsonian monkeys. Several studies (Thornburg and Moore, 1975; Zigmond and Stricker, 1980) have suggested that dyskinesias are observed when striatal DA loss exceeds 85 to 90%. In that regard, MPTP induces severe dopaminergic nigral neurodegeneration in a short period of time compared with the chronic progressive degeneration in idiopathic PD. This accelerated lesion time course and extensive striatal DA loss with MPTP exposure may facilitate the development of L-DOPA-induced dyskinesias. Whether occurrence of dyskinesias could be also observed with DA agonists by administering higher doses than the ones used in this study, by combining agonists and L-DOPA or by dispensing agonists to L-DOPA-primed, MPTP-treated monkeys remains unanswered and are topics for further investigation.

Analysis of CSF samples after completion of the study revealed significant differences of treatment on biochemical indices of central catecholamine function. In agreement with clinical findings, L-DOPA administration resulted in a marked increase in CSF dopamine and metabolite levels (Tohgi et al., 1993). Consistent with being a precursor of catecholamine synthesis, L-DOPA has also induced a marked increase in CSF concentrations of 3-methoxy-4-hydroxyphenylglycol, the main norepinephrine metabolite, indicating enhanced central noradrenergic activity. Interestingly, it has been proposed that noradrenergic mechanisms may be involved in L-DOPA-induced dyskinesia since adrenergic receptor antagonists are effective in improving dyskinetic side effects of L-DOPA in MPTP-lesioned primates (Henry et al., 1999; Grondin et al., 2000). The effect of selective D2/D3 receptor agonists on CSF catecholamine markers has not been widely studied. In contrast to L-DOPA treatment, in this study, we found no significant change in CSF monoamine or metabolite concentrations in ropinirole- and sumanirole-treated monkeys compared with vehicle treatment.

Chronic alterations in dopaminergic neurotransmission have profound effects on gene expression in striatal neurons (Gerfen et al., 1990; Robertson et al., 1992; Hope et al., 1994; Bezard et al., 2001). In that regard, FosB is a transcription factor upstream to many neuropeptides that have been associated with the development of abnormal movements (Sonnenberg et al., 1989). Increases in FosB have been reported in the striatum of MPTP-lesioned monkeys (Doucet et al., 1996; Perez-Otano et al., 1998), MPTP-treated mice (Perez-Otano et al., 1998), 6-OHDA-lesioned rats (Doucet et al., 1996), and 6-OHDA-lesioned rats treated with L-DOPA (Cenci et al., 1999; Lundblad et al., 2003). Furthermore, FosB is elevated in the striatum of human PD cases (Tekumalla et al., 2001). In the present study, MPTP treatment resulted in increased number of FosB-positive neurons in the basal ganglia. The most pronounced elevations were observed with L-DOPA treatment, which represented the only group that developed dyskinesias. These results substantially overlap with the study of Doucet and colleagues who reported that chronic administration of selective D₁ and D₂/D₃ receptor agonists in MPTP-treated monkeys produced elevated levels of FosB-like proteins (Doucet et al., 1996). Furthermore, elevation of FosB was further enhanced in monkeys treated with a D₁ agonist that displayed dyskinetic behavior and was not observed in carbegoline-treated monkeys that did not show dyskinesias. These studies suggest that FosB may be a useful marker to investigate in studies of therapeutic agents postulated to attenuate L-DOPA-induced dyskinesias.

In conclusion, our results show that selective D₂ agonist activity with sumanirole shows antiparkinsonian effects comparable with existing dopaminergic therapies without inducing dyskinesias using both behavioral and pathological assessments.

	Acknowledgements

 
We acknowledge Jennifer A. Boller, Jeffrey Kokmeyer, and John A. Bley for providing expert animal handling and care. We also acknowledge Eric J. Nestler and Linda Perotti for helpful suggestions related to Fos B staining techniques. We thank Rita Huff, Robert McCall, and Kalpana Merchant for numerous helpful discussions regarding pharmacology of sumanirole, Royal John Weaver for help with statistical analysis of the data, and Alan Opsahl for assistance with preparation of figures.

	Footnotes

 
This work was supported by Pfizer Global Research and Development. M.E.E. is partially supported by National Institutes of Health National Institute of Neurological Disorders and Stroke Grant R0I-NS40578 and National Institutes of Health Grant P51RR000167 to the Wisconsin National Primate Research Center, University of Wisconsin, Madison.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.087379.

ABBREVIATIONS: SN, substantia nigra; PD, Parkinson's disease; DA, dopamine; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; 6-OHDA, 6-hydroxydopamine; ANOVA, analysis of variance; PPRS, primate parkinsonian rating scale; PBS, phosphate-buffered saline; CSF, cerebrospinal fluid; GFAP, glial fibrillary acidic protein; TH, tyrosine hydroxylase; DAB, diaminobenzidine; AUC, area under the plasma-concentration time curve; SNpc, substantia nigra pars compacta; ir, immunoreactivity; SS33084, (3aR, 9bS)-N-[4-(8-cyano-1,3a,4,9b-tetrahydro-3H-benzopyrano[3,4-c] pyrole-2-yl)-butyl]-(4-phenyl) benzamide.

Address correspondence to: Dr. Diane Stephenson, Pfizer Global Research and Development, B274/A1706C; MS8274-1348, Eastern Point Rd., Groton, CT 06340. E-mail: diane.t.stephenson{at}pfizer.com

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