0022-3565/05/3143-1257–1266$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 314, No. 3 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 87379/3049768 JPET 314:1257–1266, 2005 Printed in U.S.A.

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

Diane T. Stephenson, Martin D. Meglasson, Mark A. Connell, Mary A. Childs, Eva Hajos-Korcsok, and Marina E. Emborg 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.) Downloaded from Received April 4, 2005; accepted June 2, 2005

ABSTRACT In this study, we investigated antiparkinsonian activity of the therapies that elicited normalization of PPRS was L-DOPA Ͼ jpet.aspetjournals.org novel, highly selective dopamine D2 receptor agonist suma- sumanirole; did not normalize PPRS in any of the nirole compared with two clinically effective treated monkeys. Dyskinesias were present with L-DOPA treat- therapies in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ment but were not observed in sumanirole-, ropinirole-, or (MPTP) primate model of Parkinson’s disease. Squirrel mon- placebo-treated primates. Pathologically, all MPTP-treated an- keys were rendered parkinsonian by chronic administration of imals displayed neurodegeneration of dopaminergic neurons in MPTP and subsequently dosed with vehicle, L-DOPA plus car- the substantia nigra pars compacta and reactive astrocytosis. bidopa (L-DOPA), ropinirole, or sumanirole over a duration of 8 Neurons immunoreactive with antibodies to the nuclear tran- weeks. Antiparkinsonian effects measured with a parkinsonian scription factor ⌬FosB were most significantly increased in the at ASPET Journals on January 24, 2020 primate rating scale (PPRS) showed that sumanirole elicited striatum of L-DOPA-treated monkeys. These results suggest improved functional outcome compared with vehicle. The do- that sumanirole can exert antiparkinsonian effects similar to pamine D2/D3 agonist ropinirole improved behavioral outcome L-DOPA without the behavioral and morphological conse- similar to sumanirole, whereas L-DOPA treatment yielded the quences of the latter. most significant symptomatic improvement. The relative rank of

Degeneration of the dopaminergic cells in the substantia tages over L-DOPA: 1) they do not require metabolism to an nigra (SN) in Parkinson’s disease (PD) creates a dopamine active form; 2) they do not compete with dietary amino acids (DA) deficiency state that is repleted by administering the for active transport across the intestinal epithelium; 3) ago- DA precursor L-dihydroxyphenylalanine (L-DOPA). L-DOPA nists may have better reproducibility of the dose response is effective in ameliorating the symptoms of bradykinesia, and time action curves; and 4) unlike L-DOPA, may tremors, and muscular rigidity, but it suffers from complica- not generate potentially toxic free radicals and may have tions such as induction of motor fluctuations and dyskine- antioxidant properties (Schapira and Olanow, 2003). sias. As an alternative to L-DOPA, dopaminergic agonists DA receptors exist as five subtypes, each of which may have been developed. Agonists have several potential advan- have different functions based on dissimilar neuroanatomical

expression and pharmacological properties. D1 and D2 recep-

This work was supported by Pfizer Global Research and Development. tors are abundant in the caudate and putamen, whereas D3 M.E.E. is partially supported by National Institutes of Health National Insti- receptors are expressed at lower levels in the basal ganglia tute of Neurological Disorders and Stroke Grant R0I-NS40578 and National Institutes of Health Grant P51RR000167 to the Wisconsin National Primate (Emilien et al., 1999). The D2 receptor may play a key role in Research Center, University of Wisconsin, Madison. the pathophysiology of motor function since it is up-regulated Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. in the striatum in response to DA denervation (Gerfen et al., doi:10.1124/jpet.105.087379. 1990; Gurevich and Joyce, 1999) as well as in human PD and

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, 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. 1257 1258 Stephenson et al. in PD animal models (Joyce et al., 1986; Brooks et al., 1992; tive histopathology of the substantia nigra and quantitative Graham et al., 1993). Furthermore, mice with targeted dele- analysis of ⌬FosB in the basal ganglia. tion of the D2 receptor exhibit parkinsonian locomotor im- pairment (Baik et al., 1995), reduced number of neurons Materials and Methods within the SN (Parish et al., 2001), reduced expression of Animals. Thirty-three male squirrel monkeys (Saimiri sciureus), trophic factors (Bozzi and Borrelli, 1999), absence of axonal approximately 6 years of age, were pair housed with a 12-h light sprouting after 6-OHDA lesion (Parish et al., 2001), and cycle. All animals received water ad libitum. New World Monkey abnormal synaptic plasticity in the striatum (Calabresi et al., Chow was provided fresh each morning (4 h before behavioral test- 1997). By contrast, activity at D3 and D4 receptors may play ing) and supplied ad libitum. Diet was supplemented with fruit juice, a role in the nonmotor symptoms that occur in PD patients, PrimaBurger, Primate Chews, fresh vegetables, wheat bread, based on their localization in limbic regions (Joyce et al., canned or fresh fruit, crackers, and cookies. The study was per- 1986, 2001) and the finding that mice with homozygous null formed in accordance with federal guidelines of proper animal care and with the approval of the Institutional Animal Care and Use mutations of the D3 or D4 receptors exhibit hyperactivity under basal or methamphetamine-stimulated conditions, re- Committee. MPTP Treatment. Twenty-nine monkeys were administered spectively (Accili et al., 1996; Rubinstein et al., 1997). MPTP s.c. one to two times per week to elicit a parkinsonian syn- Currently available DA agonists, e.g., and drome. Four additional animals remained untreated and were used ropinirole, which have activity at both D3 and D2 receptors, as naive controls for biochemistry and brain morphology. Before each produce symptomatic improvement in PD with a reduced dosing, each monkey was evaluated for its tolerance to MPTP and Downloaded from frequency of motor complications compared with L-DOPA motor disability score. Individual doses and the total number of (Lang and Widner, 2002; Wooten, 2003). However, the extent doses administered were titrated to produce similar degrees of par- of reversal of PD signs is somehow limited and inevitably kinsonian 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 after a period of time, L-DOPA supplementation is needed. In ranged from four to 17 doses based on above-mentioned criteria. light of the key role of D receptors in controlling motor 2 Treatment duration ranged over a period of 4 to 5 weeks for the most jpet.aspetjournals.org function, it is plausible that a selective D2 agonist might also sensitive monkeys (four doses) and 18 weeks for the least sensitive be efficacious and even further reduce the incidence of ad- monkeys (17 doses). The total number of MPTP doses was (mean Ϯ verse drug effects, e.g., dyskinesias, motor fluctuations, and S.E.) L-DOPA group, 11.3 Ϯ 1.3; sumanirole group, 12.0 Ϯ 1.6; hallucinations. The beneficial effect of selective stimulation ropinirole group, 11.1 Ϯ 0.9; and placebo group, 12.5 Ϯ 1.6. Cumu- Ϯ L Ϯ of D2 compared with D3 receptors has been recently demon- lative doses were (mean S.E.) -DOPA group, 12.4 2.0 mg/kg; strated in MPTP-treated marmosets. In the study of Silver- sumanirole group, 15.9 Ϯ 3.4 mg/kg; ropinirole group, 12.9 Ϯ 1.6 mg/kg; and placebo group, 12.4 Ϯ 1.8 mg/kg. There were no statis- dale et al. (2004), selective blockade of D3 receptors with the D antagonist S33084 significantly enhanced the antiparkin- tically significant differences between groups for number of doses at ASPET Journals on January 24, 2020 3 (ANOVA, p ϭ 0.92) and total amount of MPTP (ANOVA, p ϭ 0.78). sonian effects of L-DOPA and ropinirole. Hand feeding, s.c. fluid administration, and additional heating were Sumanirole is a highly selective full agonist for the dopa- provided as needed. mine D2 receptor and therefore represents a unique tool to At the end of the MPTP dosing, all animals were stably parkinso- investigate the role of D2 receptors in the control of motor nian and exhibited a narrow range of PPRS scores. The monkeys function. Pharmacological properties of sumanirole include were randomized and placed in different treatment groups. After assignment to study groups, monkeys were allowed to recover for at greater than 200-fold selectivity for the D2 receptor subtype compared with other subtypes in radioli- least 1 month before initiating drug dosing. One monkey assigned to gand binding assays (McCall et al., 2005), potent D receptor the L-DOPA group died during the recovery period and was excluded 2 from the data analysis. pharmacology in rodents in vivo (Durham et al., 1997; Sethy Drug Treatment. Figure 1 outlines the study design for the et al., 1997) and induction of robust rotational behavior in present experiment. The dosing administration schedule for this 6-OHDA-treated rats (McCall et al., 2005). experiment was designed to match clinically relevant dosing strate- Systemic administration of MPTP to squirrel monkeys in- gies. The monkeys received their treatments orally twice a day, and duces a parkinsonian syndrome that is effectively amelio- each was administered in escalating doses to reach rated by treatment with L-DOPA. Furthermore, L-DOPA maximal antiparkinsonian activity. therapy faithfully reproduces the occurrence of abnormal Monkeys were trained to be gently restrained by gloved hand and movements, in particular choreoathetosis dyskinesias (hy- dosed at 12-h intervals by oral administration. Monkeys received ϭ ϩ perkinetic, purposeless dance-like movements; Langston et vehicle (empty gelatin capsule) (n 4), L-DOPA (12.5 mg/kg) (1.5 mg/kg) (n ϭ 9), ropinirole (n ϭ 8), or sumani- al., 2000). Measurements of the animal’s motor function be- role (n ϭ 8). Sumanirole is (5R)-5,6-dihydro-5-(methylamino)-4H- fore and after treatments can be easily quantified using imidazo[4,5,1-ij]quinolin-2(1H)-one (2Z)-2-butenedioate (1:1), also parkinsonian and dyskinesia scales similar to the ones that named PNU-95,666 or U-95,666E. Ropinirole is 4-[2-(dipropylamino) are used clinically. Abnormal movements have been associ- ethyl]-1,3-dihydro-2H-indol-2-one HCl) also named SK&F 101468-A. ated with changes in several neuropeptides such as opioid The dose of L-DOPA ϩ carbidopa was chosen based on prior findings precursors and their upstream transcription factor ⌬FosB that the dose is maximally effective and consistently produces dys- (Sonnenberg et al., 1989; Westin et al., 2001). kinesias in MPTP-treated monkeys (Boyce et al., 1990; Pearce et al., In this study, the effects of the D agonist sumanirole were 1998). The optimal doses of sumanirole and ropinirole were estab- 2 lished for each monkey by titrating each drug concentration until the compared with a nonselective dopamine D /D agonist ropi- 2 3 maximum antiparkinsonian effect was achieved (study weeks 1–4). nirole and to L-DOPA in MPTP-treated primates using be- Such a dosing paradigm was chosen to match the dosing strategy havioral and pathological outcome measures. Behavioral as- used in human clinical studies with dopamine agonists. Each mon- sessment included antiparkinsonian activity and occurrence key received a maximally effective dose of sumanirole or ropinirole of dyskinesias and pathological outcomes included qualita- for 4 additional weeks (study weeks 5–8; Figs. 1 and 2). L-DOPA was Antiparkinsonian Effects of Sumanirole in Primates 1259

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. Downloaded from jpet.aspetjournals.org at ASPET Journals on January 24, 2020

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 Ϯ 0.00 mg/kg b.i.d. during week 3 to 0.12 Ϯ 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 Ϯ 0.15 mg/kg in week 3 to 1.09 Ϯ 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). administered at a fixed dose throughout the duration of the study, tures were centrifuged at 12,500 rpm, and the aqueous phase was similar to clinical treatment strategies. flash frozen by dipping the microcentrifuge tube into a methanol/dry Plasma Drug Assay. For assessment and verification of circulat- ice bath. Approximately 400 ␮l of the nonfrozen organic layer was ing drug levels, pharmacokinetic analysis was performed at specified transferred to autosampler vials and evaporated under a stream of times during the study. Blood samples were taken after an initial N2 gas at 40°C until dry (TurboEvap; Zymark Corp, Hopkinton, MA). dose and were used to determine the time of maximum concentration The residue was reconstituted in 100 ␮l of 25% acetonitrile. Liquid of drug in plasma. This time was then used for the timing of behav- chromatography-mass spectrometry analysis was performed. The ioral scoring. At week 4 after initiation of drug treatment, monkeys lower limit of quantification was 0.085 ng/ml, and accuracy of quality were administered a single dose of L-DOPA, ropinirole, or sumanirole control samples was within 20% of expected values. The calibration (maximal effective dose). Blood was collected into EDTA-containing standards ranged from 0.89 to 1770 ng/ml. tubes at particular time intervals after dosing (1, 2, 4, 8, and 12 h) Primate Parkinsonian Rating Scale (PPRS). Parkinsonian and plasma was analyzed for drug levels. Plasma samples for L- motor disability was assessed in MPTP-treated monkeys two to four DOPA analysis were supplemented with an antioxidant by adding to times per week by two independent raters who were blinded to microfuge tubes 10 ␮l of a 10% sodium metabisulfite solution before treatment group assignments. Naive monkeys were not evaluated freezing. behaviorally. Assessment of inter-rater reliability calculated using L-DOPA concentration was quantified using a modification of an the Spearman correlation coefficient was 0.95. Test-retest variability existing high-performance liquid chromatography and sample prep- for PPRS scores (intrasubject day-to-day variability) calculated us- aration technique (Wikberg, 1991). The lower limit of quantification ing the Spearman-Brown reliability coefficient was 0.80. Assess- for this assay was 0.754 ng/ml for L-DOPA. ments were made by visual observation of monkeys in their home Quantification of sumanirole and ropinirole was performed by cages under peak dose conditions. The severity of parkinsonism was adding 50 ␮l of plasma to individual microcentrifuge tubes. A 5-␮l quantified using the PPRS modified for the squirrel monkey (Lang- aliquot of 50% methanol (MeOH) and 25 ␮l of a working internal ston et al., 2000). In this scale, spatial hypokinesia (0–4), body standard solution (25 ng/ml in 50% MeOH) were added to each, bradykinesia (0–4), manual dexterity (right and left arm, 0–4 each), followed by 400 ␮l of 1:1 hexane/dichloromethane solution. The mix- balance (0–4), and freezing over a 4-min clinical observation period 1260 Stephenson et al. were evaluated. The maximum possible score was 20 points, greater monoclonal antibody ascites (Chemicon International, Temecula, than 12 points corresponded to severe parkinsonism, and a normal CA; 1:800), polyclonal antibody anti-tyrosine hydroxylase (Calbio- control animal scored Յ1.5 points (Langston et al., 2000). A score of chem, San Diego, CA; 1:1000), ⌬FosB (Chemicon International), 6 to 8 corresponds to mild-to-moderate parkinsonian activity by affinity purified rabbit polyclonal antiserum raised against an N- behavioral indices. PPRS assessments were performed consistently terminal peptide, which is common to full-length FosB and ⌬FosB at 1.5 h after drug administration throughout the 8 weeks of treat- (sc-48; Santa Cruz Biotechnology Inc., Santa Cruz, CA; 1:1500); and ment. NeuN (monoclonal antibody; Chemicon International; 1:1000). Each Abnormal Movement Evaluation. A modified time sample neu- of the above-listed antibodies was used to stain the substantia nigra rological observation was used to quantify abnormal movements (GFAP and TH antibodies) or the basal ganglia (TH and ⌬FosB). after drug administration. Data were collected at Tmax (time of Immunohistochemistry was performed using avidin-biotin peroxi- maximum drug concentration in plasma) for 30 min. Briefly, each dase detection kit (ABC; Vector Laboratories, Burlingame, CA) with animal was placed in an isolated large Plexiglas chamber that al- diaminobenzidine (DAB peroxidase substrate kit; Vector Laborato- lowed simultaneous videotaping from two angles. The recording time ries) as chromogen. Experiments were initially conducted by carry- started after a 5-min habituation period. The double videorecordings ing out staining procedures manually on brain sections from naive were obtained once per week and analyzed by two treatment blinded animals to validate each primary antibody by determining the opti- independent raters. Peak-dose dyskinetic movements were scored mum dilution and duration of antibody incubation required to ob- using a modification of the Abnormal Movements Index (Palfi et al., serve maximum signal-to-noise ratio of immunoreaction product. 2000). Orofacial dyskinesia, dystonia and chorea were separately All the treatment groups were stained in parallel as “sets of identified axially and for each limb. Abnormal movements were tissue”. A “set” was constituted by one slide from each level from each rated as being present (n ϭ 1) or absent (n ϭ 0) during each 5-min animal. Slides comprising a complete set were removed from Ϫ80°C Downloaded from time period for a 30-min test session. A dyskinesia index was ob- and allowed to thaw for 10 min before staining. The slides were tained by averaging the incidence of each symptom during the total postfixed in 4% paraformaldehyde on wet ice for 10 min. The staining duration of the test period (maximum score: 11). procedure was run on an automated Dako autostainer (DakoCyto- Tissue Preparation. At 8 weeks after initiation of drug treat- mation California Inc., Carpinteria, CA) as follows. Slides were ments, monkeys were administered a lethal dose of barbiturate and rinsed with PBS and then incubated for 10 min with 0.1% H2O2 in euthanized. The time interval between the final dose of drug and distilled H2O to block endogenous peroxidase, followed by PBS jpet.aspetjournals.org euthanasia was 3 h. Animals were perfused transcardially with rinses. Slides were blocked in 5% denatured goat serum for 10 min, phosphate-buffered saline (PBS) followed by 2% periodate lysine followed by incubating in primary antibody. Antibodies were incu- paraformaldehyde fixative. Brains were fixed overnight at 4°C and bated either 1 h (TH and GFAP) or 48 h (⌬FosB). Sections were then rinsed in PBS. The brains were cryoprotected through a series labeled with anti-mouse or anti rabbit Envision ϩ polymer horse- of graded solutions, 13% sucrose in PBS for 2 days, 15% sucrose in radish peroxidase (DakoCytomation California Inc.) for 30 min fol- PBS for 2 days, and 18% sucrose in PBS for 3 days. Before freezing, lowed by development in DAB (diaminobenzidine). To enhance DAB brain hemispheres were blocked using external landmarks into two reaction, the slides were incubated with DAB Enhancer (DakoCyto-

portions, one containing the basal ganglia and the other the substan- mation California Inc.). For sections containing the SN, immuno- at ASPET Journals on January 24, 2020 tia nigra (Emmers and Akert, 1963). The blocks were then frozen in stained sections were lightly counterstained with hematoxylin to dry ice-cooled isopentane and tissue blocks were stored at Ϫ80°C. delineate cytoarchitecture/cellular structure. For each monkey, one hemisphere was chosen for thin cryosection- One representative section from each level of all monkeys was also ing. Choice of hemisphere was randomized within each treatment stained with cresyl violet for assessment of regions, landmarks and group so that there was an equal number of monkeys represented on overall tissue integrity. One set of sections from the SN levels was the left and on the right sides of the brain within each treatment stained with H&E as well. group. Twenty-micrometer-thick cryosections were collected onto Quantification of ⌬FosB Immunoreactive Neurons. The charged slides (Superfrost Plus; VWR, West Chester, PA) using a number of ⌬FosB-immunopositive neurons in the postcommissural Leica CM3050 cryostat. For each animal, three different coronal basal ganglia was calculated using a profile counting method with levels of the basal ganglia (precommisural, anterior commissure, and ImagePro software, by an investigator blinded to the treatment postcommisural) or the SN (rostral, middle, and caudal portions of group. Representative fields measuring 800 ␮m horizontal ϫ 700 ␮m the SN) were saved with two sections per slide. In total, 32 serial vertical were acquired using a 20ϫ objective from two consecutive sections were collected at each of the three levels and matched immunostained sections. Images were acquired from the caudate between different monkeys using precise anatomical landmarks. and the putamen in sections that were matched coronally between Cryosections were stored at Ϫ80°C until staining. different monkeys. For this determination, a macro was written in Neurochemical Analysis of Cerebrospinal Fluid (CSF). Lev- ImagePro such that each immunoreactive neuron was counted using els of monoamines and metabolites were investigated in CSF sam- a mouse on the computer, and the numbers were automatically ples collected at study termination. CSF was collected via the cis- entered into an Excel spreadsheet. Neurons were assessed by visual terna magna after barbiturate overdose and samples were stored at inspection, and cells that were 10 to 25 ␮m and displayed neuronal Ϫ80°C. At the time of the analysis, samples were centrifuged at morphological features were counted. Representative ⌬FosB-immu- 12,500 rpm for 10 min at 4°C. Aliquots of supernatants, containing nopositive neurons were counted relative to a counting threshold 0.01 N perchloric acid were assayed for monoamines and metabolites based on staining density, target size, and target shape. The param- on an HPLC with Coularray electrochemical detector (ESA Inc., eters of the counting threshold were set based upon a standard Chelmsford, MA), using a Zorbax SB-C18 analytical column (4.6 ϫ control slide from the staining run. In naive brain there were rare, 100 mm, 3.5-␮m particle size) protected by a Zorbax SB-C18 guard lightly stained individual ⌬FosB-positive profiles, and this was used column (4.6 ϫ 12.5 mm, 5-␮m particle size) and maintained at 25°C. to set the threshold. All slides that were quantitatively compared

The mobile phase consisted of 75 mM NaH2PO4, 1.8 mM sodium with each other were stained in the same staining run and counted octanesulfonate, 25 ␮M EDTA, 978 ␮M tetraethylammonium, and using the same thresholding parameters and microscope and digital 9% acetonitrile, pH 3.0. Analytes were monitored on the Coularray camera settings. The number of neurons in each field was counted with applied potentials set at 0 and ϩ300 mV. All peaks were and averaged for each monkey. The mean number of ⌬FosB-ir neu- detected within 15 min. rons for the caudate and putamen was determined by averaging the Immunohistochemistry. The following antibodies were used: values from all the monkeys within each of the treatment groups. anti-glial fibrillary acidic protein (GFAP) monoclonal antibody (In- Statistical Analysis. Statistical analysis of drug effects was per- noGenex, San Ramon, CA; 1:200), anti-tyrosine hydroxylase (TH) formed using the average PPRS values determined by the two raters. Antiparkinsonian Effects of Sumanirole in Primates 1261

Inter-rater reliability calculated using the Spearman correlation co- There were no significant differences in PPRS scores between efficient was 0.95. Test-retest variability for PPRS scores (intra- the different groups at the time of randomization or imme- subject day-to-day variability) was calculated using the Spearman- diately before initiation of dosing (Fig. 3). Brown prediction formula. The Spearman-Brown reliability Clinical Rating after Drug Administration. Over the 8 coefficient was 0.80. Statistical analysis of drug effects was per- weeks of drug treatment, the placebo-treated group showed a formed using the average PPRS values determined by the two raters. mild spontaneous recovery of PD symptoms (PPRS scores 6 Differences in behavioral scores were analyzed using Kruskal- Wallis nonparametric ANOVA followed by Tukey’s multiple compar- to 7 at baseline and 4 to 5 at week 8; Fig. 3). However, the isons test. Statistical significance was set at p Ͻ 0.05. The frequency animals were still considerably impaired, showed a typical with which symptomatic normalization occurred (PPRS Յ 1.5) in PD syndrome that was significantly different from the be- monkeys that received titrated, maximally effective doses of dopa- havior observed with any of the DA replacement treatments. minergic therapies was determined using Pearson’s chi square test. A significant improvement in the PPRS was observed in Analyses of CSF monoamines and metabolites was performed using the first week after L-DOPA treatment. L-DOPA-treated a one-way ANOVA across all treatment groups followed by Dunnett’s monkeys demonstrated continued improvement throughout comparison test for each analyte. the treatment phase (Stephenson et al., 2005). By week 3, ⌬ The percentage of reduction of Fos B neurons as a function of the more than half the animals had reached a PPRS rating of lesion was calculated by comparing the mean values of naive and Յ1.5 points (normalization of PPRS) and remained at that treatment group monkeys within each nucleus. Statistical analysis was performed using Kruskal-Wallis nonparametric ANOVA. If sig- level until the end of the study (week 8). Rating improve- nificant, multiple pairwise comparisons of the means were deter- ments occurred first in spatial hypokinesia (movement Downloaded from mined with t-distribution using a significance level of p Ͻ 0.05. around the cage) and body bradykinesia. Further ameliora- tion occurred when manual dexterity and balance scores Results improved. Abnormal movements were observed within a week after initiation of L-DOPA treatment, consistent with Assessment of the Parkinsonian Syndrome. Squirrel previous reports (Boyce et al., 1990; Pearce et al., 1998).

monkeys were rendered parkinsonian by repetitive systemic For dopamine agonist treatment, individual subjects were jpet.aspetjournals.org administration of MPTP (Fig. 1). Before MPTP treatment, all dose titrated to maximal efficacy, similar to dosing strategy the animals presented a general behavior characteristic of in human PD subjects. L-DOPA treatment was a fixed dose, their age. The animals were active, freely climbing on the as is implemented clinically. The optimal doses of sumanirole cage walls, jumping and exploring the environment. None of and ropinirole were established for each monkey by titrating the monkeys showed parkinsonian signs and their clinical the dose until the maximum antiparkinsonian effect was parkinsonian rating (PPRS) was 0 points. achieved (study weeks 1–4). The final daily dose of ropinirole

During the MPTP treatment, the animals progressively achieved by titration, 0.24 mg/kg/day, was similar to doses at ASPET Journals on January 24, 2020 developed a syndrome characterized by hypokinesia, brady- used in previous studies of MPTP-treated marmosets (0.2– kinesia as well as balance and fine motor skill disturbances. 0.5 mg/kg/day; Fukuzaki et al., 2000). Note the lack of im- There were no statistically significant differences between provement in PPRS between weeks 3 and 4 for both drugs, groups for number of doses (L-DOPA group, 11.3 Ϯ 1.3; suma- after the last dose elevation. Each monkey continued to re- nirole group, 12.0 Ϯ 1.6; ropinirole group, 11.1 Ϯ 0.9; and ceive the final fixed dose of sumanirole or ropinirole for 4 placebo group, 12.5 Ϯ 1.6) or for total amount of MPTP additional weeks (study weeks 5–8; Fig. 2). (L-DOPA group, 12.4 Ϯ 2.0 mg/kg; sumanirole group, 15.9 Ϯ Analysis of antiparkinsonian activity at week 8 showed 3.4 mg/kg; ropinirole group, 12.9 Ϯ 1.6 mg/kg; and placebo statistically significant antiparkinsonian activity of all dopa- group, 12.4 Ϯ 1.8 mg/kg). At completion of MPTP treatment, mine treatments compared with placebo (Fig. 3). Sumanirole- monkeys exhibited a stable parkinsonian syndrome display- treated monkeys presented a significant improvement of ing a PPRS ranking of 6 to 8 without abnormal movements. PPRS scores (compared with placebo) after the first escalat-

Fig. 3. Individual PPRS scores by treatment: predrug and at study termination, week 8. A score of Յ1.5 (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 ϭ 0.004; vehicle versus sumanirole, p ϭ 0.011; vehicle versus ropinirole, p ϭ 0.024; L-DOPA versus ropinirole, p ϭ 0.003. No significant differences were observed between the following groups: ropinirole versus sumanirole (p ϭ 0.20) and L-DOPA versus sumanirole (p ϭ 0.31). Statistical analysis of frequency of normalization was L-DOPA versus sumanirole, p ϭ 0.61; L-DOPA versus ropinirole, p ϭ 0.007; and sumanirole versus ropinirole, p ϭ 0.021. 1262 Stephenson et al. ing dose (Fig. 2). At week 8, there was individual variability and Bass, 1998), ropinirole (Brefel et al., 1998), and sumani- in the response to sumanirole with four of the eight sumani- role (Ware et al., 2002). The maximum plasma concentration Ϯ ␮ role-treated animals showing normalization of PPRS scores of L-DOPA (Cmax) was 6.9 0.5 g/ml and the area under the (Fig. 3). Although ropinirole-treatment produced a signifi- plasma-concentration time curve from 0 to 12 h (AUC0,12) cant improvement of PPRS compared with vehicle, none of was 13 Ϯ 0.7 ␮g⅐mlϪ1⅐h. The apparent time to achieve the the ropinirole-treated animals reached a level of amelioriza- maximum plasma concentration of L-DOPA (Tmax)was1h. tion similar to the L-DOPA-treated monkeys (Fig. 3), i.e., six The time for the plasma L-DOPA concentration to decrease to of eight L-DOPA-treated monkeys and zero of the eight ropi- 50% of the Cmax (herein operationally defined as t1/2) was nirole-treated animals showed normalization of PPRS scores. 2.5 h after Tmax. The pharmacokinetic parameters in mon- Ϯ The doses of ropinirole that exhibited antiparkinsonian ac- keys dosed with ropinirole were Cmax, 14.9 2.6 ng/ml; Ϯ ⅐ Ϫ1⅐ tivity are consistent with that reported in a MPTP marmoset AUC(0,12),51 10 ng ml h; Tmax,1h;andt1/2,3h.The model, which also used a dose titration regime (Eden et al., pharmacokinetic parameters in monkeys dosed with suma- Ϯ Ϯ 1991). Comparisons of the individual groups revealed that nirole were Cmax, 80.4 22.2 ng/ml; AUC(0,12), 389 123 ⅐ Ϫ1⅐ the antiparkinsonian efficacy of sumanirole group was not ng ml h; Tmax,2h;andt1/2, 3 h. These data assured ade- statistically significant different from L-DOPA, whereas ropi- quate drug exposure of monkeys in the various treatment nirole and L-DOPA treatments were significantly different. groups. Both ropinirole and sumanirole improved parkinsonian CSF Levels of Monoamines. Since monoamines in the symptoms, and L-DOPA treatment was most effective in im- CSF can serve as an index of central dopamine levels, mono- Downloaded from proving behavioral outcome among the different dopaminer- amine neurotransmitters and their metabolites were mea- gic treatments. sured in the CSF at the termination of the study. L-DOPA Occurrence of Abnormal Movements. Before MPTP treatment markedly elevated the CSF concentrations of DA, lesion and MPTP ϩ L-DOPA, sumanirole, ropinirole or pla- and the DA metabolites homovanillic acid and dihydroxyphe- cebo treatment none of the animals presented abnormal nylacetic acid compared with placebo-treated MPTP mon- Ϯ Ϯ Ϯ movements. As described previously (Langston et al., 2000), keys (DA: 3.77 0.69 versus 1.33 0.03 ng/ml; HVA: 1028 jpet.aspetjournals.org all the MPTP ϩ L-DOPA-treated monkeys developed abnor- 180 versus 51 Ϯ 11 ng/ml; dihydroxyphenylacetic: 81 Ϯ 22 mal movements (Fig. 4). One week after beginning L-DOPA versus 0.19 Ϯ 0.07 ng/ml; in each case L-DOPA versus pla- administration, animals presented signs of dyskinesia and cebo, respectively; p Ͻ 0.001). By comparison, CSF concen- dystonia. Orofacial dyskinesias were not observed during the trations of DA and metabolites were not significantly differ- first week, but they were evident in the subsequent sessions. ent from placebo in sumanirole- or ropinirole-treated Dyskinetic dance-like movements typical of chorea were ob- monkeys. CSF concentration of serotonin, 5-hydroxyindole

served. Dystonias continued and increased throughout the acetic acid, and norepinephrine did not differ significantly in at ASPET Journals on January 24, 2020 course of L-DOPA treatment, affecting trunk, upper and the various treatment groups in agreement with a previous lower limbs. In comparison, abnormal movements were not report (Russ et al., 1991). However, there was a statistically observed in the placebo-, ropinirole- or sumanirole-treated significant increase in 3-methoxy-4-hydroxyphenylglycol lev- animals at any time throughout the study. els after L-DOPA treatment compared with placebo (75.0 Ϯ Drug Pharmacokinetics. Plasma drug concentration- 17.1 versus 19.6 Ϯ 3.9 ng/ml; p Ͻ 0.001). Therefore, L-DOPA time profiles were determined on day 30 of drug treatment to treatment but not dopamine agonist therapy resulted in el- allow comparison of drug pharmacokinetics with data previ- evated CSF dopamine levels. ously reported for humans. The time course for attaining Pathology of the Substantia Nigra. Routine histopa- plasma peak and trough drug levels were approximately the thology with Nissl and H&E revealed distinctive neurodegen- same in squirrel monkeys and humans for L-DOPA (Harder eration in the substantia nigra pars compacta (SNpc) in all the MPTP-lesioned monkeys. Abnormalities included de- rangement of Nissl substance, presence of swollen neurites, extraneuronal melanin, and reactive astrocytosis. Neurode- generation 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. Pathology of the Basal Ganglia. TH-immunoreactivity (-ir) was dramatically reduced in the basal ganglia of all MPTP-treated monkeys compared with naive controls (Fig. 6). Quantitative image analysis revealed that there was a 90% reduction in TH expression and no significant change in GAD65 or GAD67 expression in basal ganglia as a function of MPTP treatment (Stephenson et al., 2005). No qualitative differences in TH expression were observed as a function of Fig. 4. Effect of vehicle, L-DOPA, sumanirole, and ropinirole on the occurrence of dyskinesias in MPTP-treated monkeys, as measured on the dopaminergic treatment condition. modified Abnormal Movement Index. Minimal ⌬FosB-like immunoreactivity was observed in the Antiparkinsonian Effects of Sumanirole in Primates 1263

Fig. 5. Neuropathology of the substantia nigra in rep- resentative 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 ϩ vehicle-treated monkeys (B and D) dis- play fewer TH-ir neurons, and the remaining neurons have an atrophic morphology (D). E and F, GFAP im- munoperoxidase staining in the SNpc. MPTP-lesioned

SN presents a dense glial fiber network and larger Downloaded from astrocytes that express more intense GFAP staining (F) compared with naive animals (E). Scale bar, 1000 ␮m (A and B) and 50 ␮m (C–F). jpet.aspetjournals.org at ASPET Journals on January 24, 2020

Fig. 6. Immunoperoxidase staining of tyrosine hydroxylase in hemiforebrain sections of a representative naive (A and C) and MPTP ϩ 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 ␮m (C and D).

basal ganglia of naive monkeys. Consistent with previous Quantification of the number of ⌬FosB-ir nuclei revealed reports (Doucet et al., 1996; Perez-Otano et al., 1998), ⌬FosB no significant differences in naive versus MPTP-treated mon- immunoreactivity showed as specific staining of neuronal keys (Fig. 8). Monkeys treated with dopamine agonists nuclei in the caudate and putamen of all MPTP-treated mon- showed a significant increase of ⌬FosB-ir nuclei compared keys (Fig. 7). There was a distinctive increase in the number with naive monkeys. In the caudate nucleus, both ropinirole and intensity of ⌬FosB-ir nuclei in monkeys treated with and sumanirole treatments led to increased ⌬FosB-ir com- MPTP ϩ L-DOPA compared with other treatments (Fig. 7). pared with naive monkeys. In the putamen, ropinirole- but Colocalization of ⌬FosB with NeuN by double immunofluo- not sumanirole-treated monkeys had significantly increased rescence staining on the same tissue section confirmed that number of ⌬FosB-ir neurons. The most profound increases staining was present in neuronal nuclei (not shown). were observed in monkeys treated with MPTP ϩ L-DOPA 1264 Stephenson et al.

Fig. 7. Representative sections of the caudate nucleus showing ⌬FosB-immunoreactivity in the basal ganglia of naive (A), MPTP ϩ placebo- (B), MPTP ϩ sumanirole- (C), and MPTP ϩ L-DOPA (D)-treated monkeys. Scale bar, 50 ␮m. Downloaded from

Fig. 8. Quantification of the number jpet.aspetjournals.org of ⌬FosB-positive neurons in the rep- resentative regions of the caudate and putamen of monkeys as a function of different treatments. Statistical anal- yses shows the following differences: caudate: L-DOPA versus naive, p Ͻ 0.0005 (a); versus vehicle, p Ͻ 0.005 (b); versus ropinirole, p Ͻ 0.005 (c); Ͻ Ͻ

p 0.005, versus sumanirole, p at ASPET Journals on January 24, 2020 0.005 (d); sumanirole versus naive, p Ͻ 0.01 (e); and ropinirole versus na- ive, p Ͻ 0.05 (f); putamen: L-DOPA versus naive, p Ͻ 0.0005 (g); versus vehicle, p Ͻ 0.0005 (h); versus suma- nirole, p Ͻ 0.005 (i); versus ropinirole, p Ͻ 0.05 (j); ropinirole versus naive, p Ͻ 0.05 (k); and versus vehicle, p Ͻ 0.05 (l). All other treatment groups were not statistically different.

where we found a highly significant 3- to 4-fold elevation in sented normalization of the parkinsonian syndrome (PPRS the number of ⌬FosB-ir neurons in the caudate and putamen scores Յ1.5). Differences in the level of improvement be- compared with all other groups of monkeys (Fig. 7). tween groups were not related to the monkeys PD syndrome. Since baseline PPRS scores were matched before drug treat- Discussion ment, there was no preselection bias for assigning monkeys with different levels of impairment to a particular treatment The present study demonstrates that the selective D ag- 2 group. The individual differences in the PPRS scores within onist sumanirole can exert antiparkinsonian effects similar the sumanirole group did not correlate with blood drug lev- to D /D agonist treatment (ropinirole) in MPTP-treated non- 2 3 els, suggesting that variability in drug metabolism does not human primates without eliciting the abnormal movements account for differences in antiparkinsonian effects. Moreover, and pathology associated with L-DOPA therapy. All three circulating levels of drug in the monkeys were similar values dopaminergic drugs evaluated (L-DOPA, sumanirole, and ropinirole) showed antiparkinsonian activity under the con- to those obtained in PD patients that have shown symptom- ditions used in the present study. Antiparkinsonian effects of atic relief after treatment (D’Souza et al., 2002). L-DOPA were significantly better than treatment with ropi- The antiparkinsonian activity of sumanirole may be ex- nirole, similar to clinical findings (Rascol et al., 2000). L- plained by direct stimulation of dopamine D2 receptors. D2 DOPA was able to reduce the PPRS score to normal levels agonist properties of sumanirole have been demonstrated in (score Յ1.5) in six of the eight subjects, whereas none of vivo in several rat models. Pharmacological endpoints includ- ropinirole-treated monkeys achieved that level of improve- ing increase in plasma prolactin secretion and dopamine ment. Four of the eight sumanirole-treated monkeys pre- metabolite concentration in hypothalamus (Durham et al., Antiparkinsonian Effects of Sumanirole in Primates 1265

1997), depression of dopamine neuron electrophysiological enhanced central noradrenergic activity. Interestingly, it has firing rate (Camacho-Ochoa et al., 1995; McCall et al., 2005), been proposed that noradrenergic mechanisms may be in- and elevation of striatal acetylcholine concentrations in nor- volved in L-DOPA-induced dyskinesia since adrenergic recep- mal and denervated rats (Sethy et al., 1997). In several of tor antagonists are effective in improving dyskinetic side these experiments, D2 selectivity of sumanirole was demon- effects of L-DOPA in MPTP-lesioned primates (Henry et al., strated by coadministration of a D2 antagonist (Camacho- 1999; Grondin et al., 2000). The effect of selective D2/D3 Ochoa et al., 1995; Sethy et al., 1997). D2 antagonist treat- receptor agonists on CSF catecholamine markers has not ment was not used in the present study to avoid confounding been widely studied. In contrast to L-DOPA treatment, in this the pathology endpoints. Thus, in MPTP-treated squirrel study, we found no significant change in CSF monoamine or monkeys, we cannot attribute specific components of the metabolite concentrations in ropinirole- and sumanirole-

PPRS score to D2 versus D3 agonist activity. However, in the treated monkeys compared with vehicle treatment. study of MPTP-treated marmosets, administration of a selec- Chronic alterations in dopaminergic neurotransmission tive D3 antagonist significantly enhanced the antiparkinso- have profound effects on gene expression in striatal neurons nian effects of L-DOPA and ropinirole (Silverdale et al., (Gerfen et al., 1990; Robertson et al., 1992; Hope et al., 1994; ⌬ 2004), which supports the beneficial effects of D2 compared Bezard et al., 2001). In that regard, FosB is a transcription with D3 receptors. Future studies using D2 antagonist par- factor upstream to many neuropeptides that have been asso- adigms in combination with sumanirole will be very informa- ciated with the development of abnormal movements (Son- tive to address this issue. nenberg et al., 1989). Increases in ⌬FosB have been reported Downloaded from Antiparkinsonian effects are not necessarily associated in the striatum of MPTP-lesioned monkeys (Doucet et al., with the presence of abnormal movements. It can be argued 1996; Perez-Otano et al., 1998), MPTP-treated mice (Perez- that the presence of dyskinesias in L-DOPA-treated monkeys Otano et al., 1998), 6-OHDA-lesioned rats (Doucet et al., was related to the longer period of L-DOPA administration. 1996), and 6-OHDA-lesioned rats treated with L-DOPA However, 1 week after starting L-DOPA dosing, abnormal (Cenci et al., 1999; Lundblad et al., 2003). Furthermore, ⌬ movements were evident. Abnormal movements were not FosB is elevated in the striatum of human PD cases (Te- jpet.aspetjournals.org observed in monkeys treated with vehicle, sumanirole, or kumalla et al., 2001). In the present study, MPTP treatment ropinirole throughout the study, even after 4 weeks of a resulted in increased number of ⌬FosB-positive neurons in maximally effective antiparkinsonian dose of either agonist. the basal ganglia. The most pronounced elevations were ob- These data suggest that L-DOPA is more likely to induce served with L-DOPA treatment, which represented the only dyskinesias than DA agonist monotherapy in the doses and group that developed dyskinesias. These results substan- duration of treatment used in our studies. Absence of dyski- tially overlap with the study of Doucet and colleagues who

nesias with DA agonist treatment is consistent with previous reported that chronic administration of selective D1 and at ASPET Journals on January 24, 2020 reports showing relative low intensity of abnormal move- D2/D3 receptor agonists in MPTP-treated monkeys produced ments in L-DOPA-naive MPTP-lesioned marmosets treated elevated levels of ⌬FosB-like proteins (Doucet et al., 1996). with antiparkinsonian doses of ropinirole or Furthermore, elevation of ⌬FosB was further enhanced in

(Pearce et al., 1998; Maratos et al., 2003). monkeys treated with a D1 agonist that displayed dyskinetic PD patients treated with L-DOPA develop dyskinesias af- behavior and was not observed in carbegoline-treated mon- ter several years of treatment. However, patients with keys that did not show dyskinesias. These studies suggest MPTP-induced PD quickly develop dyskinesias after L-DOPA that ⌬FosB may be a useful marker to investigate in studies therapy (Langston and Ballard, 1984), similar to what we of therapeutic agents postulated to attenuate L-DOPA-in- observed in parkinsonian monkeys. Several studies (Thorn- duced dyskinesias. burg and Moore, 1975; Zigmond and Stricker, 1980) have In conclusion, our results show that selective D2 agonist suggested that dyskinesias are observed when striatal DA activity with sumanirole shows antiparkinsonian effects loss exceeds 85 to 90%. In that regard, MPTP induces severe comparable with existing dopaminergic therapies without dopaminergic nigral neurodegeneration in a short period of inducing dyskinesias using both behavioral and pathological time compared with the chronic progressive degeneration in assessments. idiopathic PD. This accelerated lesion time course and exten- sive striatal DA loss with MPTP exposure may facilitate the Acknowledgments development of L-DOPA-induced dyskinesias. Whether oc- currence of dyskinesias could be also observed with DA ago- We acknowledge Jennifer A. Boller, Jeffrey Kokmeyer, and John A. Bley for providing expert animal handling and care. We also nists by administering higher doses than the ones used in acknowledge Eric J. Nestler and Linda Perotti for helpful sugges- this study, by combining agonists and L-DOPA or by dispens- tions related to ⌬Fos B staining techniques. We thank Rita Huff, ing agonists to L-DOPA-primed, MPTP-treated monkeys re- Robert McCall, and Kalpana Merchant for numerous helpful discus- mains unanswered and are topics for further investigation. sions regarding pharmacology of sumanirole, Royal John Weaver for Analysis of CSF samples after completion of the study help with statistical analysis of the data, and Alan Opsahl for assis- revealed significant differences of treatment on biochemical tance with preparation of figures. indices of central catecholamine function. In agreement with clinical findings, L-DOPA administration resulted in a References marked increase in CSF dopamine and metabolite levels Accili D, Fishburn CS, Drago J, Steiner H, Lachowicz JE, Park BH, Gauda EB, Lee EJ, Cool MH, Sibley DR, et al. (1996) A targeted mutation of the D3 dopamine (Tohgi et al., 1993). Consistent with being a precursor of receptor gene is associated with hyperactivity in mice. Proc Natl Acad Sci USA catecholamine synthesis, L-DOPA has also induced a marked 93:1945–1949. Baik JH, Picetti R, Saiardi A, Thiriet G, Dierich A, Depaulis A, Le Meur M, and increase in CSF concentrations of 3-methoxy-4-hydroxyphe- Borrelli E (1995) Parkinsonian-like locomotor impairment in mice lacking dopa- nylglycol, the main norepinephrine metabolite, indicating mine D2 receptors. Nature (Lond) 377:424–428. 1266 Stephenson et al.

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