0022-3565/00/2922-0714$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics JPET 292:714–724, 2000 Vol. 292, No. 2 Printed in U.S.A. D1 Dopamine Receptor Agonists Are More Effective in Alleviating Advanced than Mild Parkinsonism in 1-Methyl-4phenyl-1,2,3,6-tetrahydropyridine-Treated Monkeys1 MARTIN GOULET and BERTHA K. MADRAS Harvard Medical School, New England Regional Primate Research Center, Division of Neurochemistry, Southborough, Massachusetts Accepted for publication November 10, 1999 This paper is available online at http://www.jpet.org Parkinson’s disease affects primarily the elderly, with the majority of patients diagnosed at 60 years of age or older. As this cohort grows, the incidence and prevalence of Parkinson’s disease will continue to increase, underscoring the importance of early diagnosis and treatment. The degenerative process can be defined by stages and monitored with the Hoehn and Yahr scale (Hoehn and Yahr, 1967). At the onset of clinical symptoms, unilateral tremor, limb stiffness, slowness of movement, and gait disturbances appear but do not interfere with daily life. At stage II/III of disease progression, bilateral tremor appears and disabilities begin to interfere with daily activities. Advanced stages IV/V are characterized by sufficient disability to require living assistance. Current and future strategies for treating patients with Parkinson’s disease depend to some extent on applying the rating scale to tailor therapeutic approaches appropriate to the degree of functional impairment. Received for publication August 17, 1999. 1 This work was supported in part by National Institutes of Health Grants NS30556, DA09462, DA00304, and RR00168. M.G. is the recipient of a postdoctoral training fellowship from the Medical Research Council of Canada. Some results have been presented in abstract form [Goulet M and Madras BK (1998) Efficacy of a dopamine D1 receptor agonist depends on severity of parkinsonism. Soc Neurosci Abstr 24:303.3]. parkinsonism (three doses of 0.6 mg/kg i.v. MPTP within 10 days). In normal monkeys (n 5 3), SKF 81297 and dihydrexidine did not promote increased motor activity. In advanced parkinsonism (n 5 4), D1 and D2 dopamine agonists effectively reversed the motor deficits. In contrast, the therapeutic benefits of D1 agonists SKF 81297 and dihydrexidine were relatively limited in mild parkinsonism (n 5 4). The D2 agonists quinelorane and (1)-PHNO alleviated some symptoms in mild parkinsonism but also reduced balance and induced more dyskinesias than did D1 agonists. Mild and advanced parkinsonism in nonhuman primates can be produced with fixed dosing regimens of MPTP. Based on the therapeutic efficacy and side effect profiles derived from these models, D1 agonists are more promising for the treatment of advanced than of mild Parkinson’s disease. Current drug therapies are largely designed to replace dopamine with either L-dopa or dopamine agonists. Although L-dopa substitution is still the gold standard of antiparkinsonian therapy, motor response oscillations and drug-induced, abnormal involuntary movements (dyskinesia) develop in most patients with Parkinson’s disease after a few years of monotherapy (Marsden and Parkes, 1977). As nigrostriatal nerve terminals degenerate, metabolic conversion of L-dopa to dopamine is impaired. Dopamine agonists bypass this need by acting directly on postsynaptic dopamine receptors. Currently, at least five distinct dopamine receptor subtypes have been identified and grouped into subfamilies, D1-like (D1 and D5) and D2-like (D2, D3, and D4), based on their pharmacological and molecular properties (Neve and Neve, 1997). Although D1- and D2-like agonists are effective antiparkinsonian agents in animal models of Parkinson’s disease (Blanchet et al., 1996a) and in clinical research (Temlett et al., 1989; Gottwald et al., 1997; Rascol et al., 1999), all agonists approved for Parkinson’s disease are preferentially active at D2-type dopamine receptors. Drugs targeted to specific receptor subtypes may offer advantages in terms of efficacy, tolerance, and side effects, but only D2-type agonists ABBREVIATIONS: MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PHNO, 4-propyl-9-hydroxy-2,3,4a,5,6,10b-hexahydro-4H-naphth[1,2b][1,4]oxazine. 714 Downloaded from jpet.aspetjournals.org at ASPET Journals on April 7, 2016 ABSTRACT Selective D1 dopamine receptor agonists exert antiparkinsonian effects in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkey model of Parkinson’s disease and in human Parkinson’s disease. Motor impairment in idiopathic Parkinson’s disease progresses from mild to severe, but the therapeutic potential of D1 dopamine receptor agonists in early and advanced stages of parkinsonism is not known. To compare the effectiveness of D1 agonists at different levels of impairment, we developed a model of mild and advanced parkinsonism in nonhuman primates and a rating scale that differentiated the two models. D1 dopamine receptor agonists (SKF 81297, dihydrexidine) and D2 dopamine receptor agonists [quinelorane, (1)-PHNO were administered to monkeys (Macaca fascicularis) displaying either mild parkinsonism (two doses of 0.6 mg/kg i.v. MPTP 1 month apart) or advanced 2000 D1 Agonist Efficacy in Advanced and Mild Parkinsonism Materials and Methods Animals. The study animals included two female and six male cynomolgus monkeys (Macaca fascicularis) ranging in age from 4.0 to 11.5 years and weighing 3.0 to 7.1 kg at the beginning of the study. Animals were housed individually and fed with a monkey biscuit diet (PMI Nutrition International) supplemented daily with fresh fruit, and had free access to water. They were maintained in humidity- and temperature-controlled climates and exposed to a 12-h light/dark cycle. Antibodies to herpes virus simiae (B virus) were not detectable in these animals. Care and treatment of these monkeys were supervised by veterinarians under the guidelines set forth by the National Institutes of Health and in strict compliance with the American Association of Laboratory Animal Care. All efforts were made to minimize the number of monkeys used for this study and to minimize distress and discomfort. Each subject was evaluated before MPTP administration and served as its own control. MPTP Administration. The neurotoxin MPTP (Aldrich Chemical, Milwaukee, WI) was used to produce a nonhuman primate model of parkinsonism. MPTP (5 mg/ml) in sterile saline was prepared under a safety hood by personnel wearing appropriate protective clothing (respirator mask, gloves, eyewear, sleeve protectors, and apron). After sedation with ketamine hydrochloride (20 mg/kg i.m.; Phoenix Pharmaceuticals Inc., Mountain View, CA), MPTP was administered via an indwelling catheter introduced into the saphenous vein. Two dose regimens of MPTP were used. In regimen A, four monkeys were treated with two injections (0.6 mg/kg i.v.) of MPTP at a 1-month interval. In regimen B, four monkeys were treated with three injections (0.6 mg/kg i.v.) of MPTP within 10 days. Food intake and body weight were carefully monitored after MPTP administration. When necessary, quinelorane (0.1 mg/kg i.m.) was administered to maintain food and water consumption; the resulting behavioral rating from a single dose was used for comparisons between quinelorane treatment in advanced versus mild parkinsonian animals. Drug studies were initiated 8 weeks after the last of two doses of MPTP given 1 month apart and 3 weeks after the last of three doses of MPTP were given within 10 days. Videotaping of Behavior. Monkeys were rated by an observer blinded to the experimental protocol or drug treatments, who had more than 10 years of experience in primate behavior. Animals were videotaped in a filming cage without humans present but with other primates housed in the same room. The cage (85 3 79 3 88 cm) was constructed with a Plexiglas wall and supplied with additional lighting. Each filming session, lasting 2 to 3 h, was divided into 30-min segments. Videotapes were scored 5, 10, 15, 20, and 25 min for 2-min periods of observation after each injection. The average score was used for analysis of each segment of 30 min. Rating Scale. A rating scale was developed to assess the effects of the drugs in normal untreated monkeys before MPTP administration (n 5 3) and to compare the effects of the two MPTP regimens (regimen A, n 5 4; regimen B, n 5 4) (Appendix 1). Spontaneous normal behavior corresponded to 0 on the rating scale. For most of the parameters, impairment was rated on a 1 or 2 scale, to minimize subjectivity. A negative score portrayed hyperactive behavior. Bradykinesia and rigidity were rated as absent (0) or present (1) and therefore could not be use to distinguish mild or advanced degrees of impairment. On the composite scale, a maximum disability score of 14 represented a summation of individual scores for general activity, locomotor activity, posture, imbalance, tremor frequency, body freeze, and feeding ability. Drug-induced dyskinesias were scored as severe, slight, and absent for different segments, face, limb, and trunk (Appendix 1, boxed region). Stereotypy (licking, grooming, scratching, and biting) and frequent head movements with visual scanning were also recorded on the rating form. Drugs. Vehicle injection and a 30-min observation period preceded all drug treatments. At least 7 days after D1 agonist treatment and 14 days after D2 agonist treatment elapsed before another drug or a different dose was introduced in mild parkinsonian animals. The time between treatment with different drugs of advanced parkinsonian animals was lower to limit the time period of the study in the advanced parkinsonian animals. Baseline behaviors (control) were monitored for at least four sessions lasting 30 min each between each drug-testing period. Drugs were prepared fresh daily, administered i.m. within 1 h of preparation, and given on the same morning of the week for each monkey. Graded doses (0.3 ml/kg b.wt. or less) were administered every 30 min, permitting determination of up to a four-point cumulative dose-response curve during a single drugtesting period. For a full dose-response curve, overlapping doses were averaged and the mean data were used for analysis. The D1 agonists SKF 81297 HCl and HBr (SmithKline Beecham, King of Prussia, PA) and dihydrexidine HCl (National Institute on Drug Abuse, Bethesda, MD) were dissolved in 10% ethanol and 0.02% ascorbic acid and diluted with distilled water to achieve doses ranging from 0.01 to 3.0 mg/kg. The D2 agonist quinelorane dihydrochloride (Eli Lilly, Indianapolis, IN) was dissolved in 5% ethanol and Downloaded from jpet.aspetjournals.org at ASPET Journals on April 7, 2016 are efficacious at various stages of the disease (Bergamasco et al., 1990; Gottwald et al., 1997; Rascol et al., 1998). The clinical development of D1 agonist therapies was attenuated by the early failure of a D1 agonist SKF 38393 to reverse parkinsonism in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys and to alleviate symptoms in humans (Braun et al., 1987; Bedard and Boucher, 1989). The conclusions drawn were premature as the short-acting SKF 38393 is a partial agonist with limited capacity to stimulate adenylate cyclase, compared with dopamine, in monkey and rat striatum (Pifl et al., 1991). Subsequently, other D1 agonists, such as dihydrexidine [(6)-trans-10,11-dihydroxy-5,6,6a,7,8,12bhexahydrobenzo[a]phenanthridine], A-77636, SKF 81297 [(R)(1)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3benzazepine], and A-86929, with high intrinsic activity on adenylate cyclase, displayed potent antiparkinsonian effects, similar to those of L-dopa or D2 agonists but with reduced potential to induce dyskinesias (Blanchet et al., 1993; Grondin et al., 1997). Whether D1 agonists are likely to be effective in early or advanced Parkinson’s disease has not been systematically assessed. ABT-431, the prodrug of the D1 agonist A-86929, showed efficacy of similar magnitude to that seen with L-dopa in advanced parkinsonian patients (stage III or IV on the Hoehn and Yahr scale) and reduced dyskinesia (Rascol et al., 1999). In contrast, the short-acting full D1 agonist dihydrexidine showed limited efficacy in parkinsonian patients at Hoehn and Yahr stage of 2.8 6 0.1 (Blanchet et al., 1998). We investigated the therapeutic potential of D1 dopamine in models of mild or advanced parkinsonism, which were developed with two different dosing regimens of the neurotoxin MPTP and distinguishable by a rating scale. The therapeutic potential and side effects of two chemically distinct D1 agonists, the benzazepine SKF 81297 and the phenanthridine dihydrexidine, were investigated in these models. In normal monkeys, SKF81297 and dihydrexidine did not promote increased motor activity. Although D1 receptor agonists showed statistically significant antiparkinsonian effects when administered to monkeys with advanced parkinsonism, efficacy in mild parkinsonism was modest. Two selective D2 agonists, quinelorane and (1)-4-propyl-9-hydroxy-2,3,4a,5,6,10bhexahydro-4H-naphth[1,2b][1,4]oxazine (PHNO), relieved some symptoms in mild parkinsonism, but at effective doses, they also produced more imbalance and dyskinesias than D1 receptor agonists. These studies suggest that D1 agonists may be of therapeutic benefit in the advanced stages of Parkinson’s disease. 715 716 Goulet and Madras Vol. 292 Results Effects of MPTP Treatment. All animals in the study displayed a normal range of motor function before MPTP administration (Fig. 1). All animals treated under regimen A, with two doses of MPTP given 1 month apart, displayed parkinsonian symptoms. According to the rating scale, bradykinesia, tremor, body freeze, rigidity, feeding ability, and stooped posture were statistically significantly different from ratings recorded before MPTP treatment (Fig. 1). Two animals (125-97 and 126-97) displayed more severe tremor, body freeze, and difficulty in feeding than two other monkeys (391-95 and 392-95) that received the same treatment. Despite bradykinesia, these parkinsonian monkeys remained healthy and showed no signs of imbalance. Compared with normal spontaneous activity, animals treated with regimen A were significantly less active, and objectively, the parkinsonian composite score of 5.9 6 1.5 (n 5 4, P , .05) was less than the composite score of 1.5 6 0.6 before MPTP treatment (Fig. 2A). To develop computerized analysis of motor activity in monkeys, a pilot study was conducted in normal and in mild parkinsonian monkeys several months after the study was completed. Subjects were monitored with an accelerometer for 45 days. The activity of mild parkinsonian monkeys (44 6 16 counts/min) was 62% of the activity of normal monkeys (70 6 22 counts/min), but the difference did not achieve statistical significance (Fig. 2, B and C). Computerized monitoring of activity reflected general and locomotor activity more closely than other parameters of the parkinsonian rating scale. A different set of animals (n 5 4) were treated with three consecutive doses of MPTP given within 10 days (regimen B). All animals displayed parkinsonian symptoms, decreased general activity, decreased locomotor activity, bradykinesia, Fig. 1. Comparison of spontaneous activity in cynomolgus monkeys before MPTP administration (open columns); after two doses of MPTP 1 month apart (cross-hatched columns), which produces mild parkinsonism; and after three doses of MPTP within 10 days (solid columns), which produces advanced parkinsonism. Results are composed of four baseline sessions. Differences in activity levels between pre- and post-MPTP treatments were evaluated by paired t test, whereas differences in behavioral scores between regimen A and regimen B of MPTP administration were evaluated by unpaired t test and by a one-way ANOVA with the Fisher post hoc test. Animals treated with two doses of MPTP displayed clear parkinsonian symptoms, without signs of imbalance, and corresponded to Hoehn and Yahr stage I/III for human Parkinson’s disease. Monkeys treated with three doses of MPTP were consistently rated with more severe impairment than monkeys after two doses of MPTP and corresponded to Hoehn and Yahr stage IV/V for human Parkinson’s disease. *P , .05, **P , .01, and ***P , .005 versus pre-MPTP. ¥P , .05 and ¥¥¥ P , .005 versus two doses of MPTP regimen. rigidity, body freeze, loss of feeding ability, and stooped posture, which were statistically different from the pre-MPTP phase (Fig. 1). Compared with normal spontaneous activity, animals treated with regimen B were less active, and the composite score of parkinsonian symptoms of 8.9 6 0.3 was statistically significant compared with the composite score before MPTP: 0.4 6 0.2 (P , .005; Fig. 2A). This set of animals displayed advanced parkinsonian features with no improvement in the parkinsonian rating score during the month after regimen B of MPTP. The composite score remained relatively stable during the drug-testing period (Fig. 3). Monkeys treated with MPTP-regimen B consistently displayed more severe impairment of general activity, locomotor activity, body freeze, and feeding ability than did monkeys treated with MPTP-regimen A (Fig. 1). A profound decrease in locomotor activity and more severe stooping were observed in monkeys treated with regimen B than in monkeys treated Downloaded from jpet.aspetjournals.org at ASPET Journals on April 7, 2016 diluted with distilled water to achieve doses of 0.001 to 3.0 mg/kg. The D2 agonists (1)-PHNO HCl and (2)-PHNO (Merck, Sharp and Dohme Research Laboratories, Essex, England) were dissolved in 10% ethanol and 0.02% ascorbic acid and diluted with distilled water to achieve doses ranging from 0.0001 to 0.1 mg/kg. It should be noted that monkeys used for this study had prior experience with various compounds. Computerized Monitoring of Movement. At the end of the drug-testing period, general motor activity was assessed with an omnidirectional accelerometer (Actiwatch aw4-64K, Mini-Mitter Co., Inc., Sunriver, OR) for the mild parkinsonian monkeys (n 5 4) and compared with normal animals (n 5 3). The animals were sedated (10 mg/kg ketamine) and were fitted to a jacket according to their weight (Lomir, Montreal, Canada). To increase comfort, the jacket was sleeveless and was fabricated with a pocket (3 3 3 inches) in the lower back. The accelerometer unit was placed in the jacket pocket after an accommodation period of at least 2 weeks. The accelerometer was set to record activity counts every minute for a 45-day period. The data were analyzed with the software Rhythmwatch (MiniMitter), and the average of daylight activity counts was compared. Statistics. Differences in activity levels between pre- and postMPTP treatments were evaluated by paired t test, whereas differences in behavioral scores between regimen A and regimen B of MPTP administration were evaluated by unpaired t test and by a one-way ANOVA with the Fisher post hoc test. The dose-response to drugs was analyzed using a one-way ANOVA with the monkey parkinsonism rating score. The Fisher post hoc multiple comparison procedure was used to compare the effects of drug doses with the vehicle injection and baseline activity for each group of monkeys. Data are presented as means 6 S.E. 2000 with regimen A, but these parameters did not reach statistical significance (Fig. 1). Bradykinesia and rigidity, rated as present or absent, were observed after both regimens of MPTP. Although parkinsonian symptoms increased after each MPTP regimen, balance was not affected, and none of the animals displayed dyskinesia (data not shown). Interestingly, tremor frequency was more severe after regimen A compared with after regimen B of MPTP. A significant and persistent difference in behavioral scores was observed for the two sets of animals treated with MPTP regimens A and B and persisted during the investigation of drug effects (Fig. 3). Animals treated with three doses of MPTP given within 10 days were significantly less active and objectively rated, with a parkinsonian composite score of 8.9 6 0.3 (n 5 4), compared with the composite score of animals treated with two doses of MPTP given 1 month apart [5.9 6 1.5 (n 5 4), P , .05] and the average composite score before MPTP treatment [1.0 6 0.4 (n 5 8), P , .0001], as determined by a one-way ANOVA followed by a Fisher PLSD comparison test (Fig. 2A). The two groups of animals that received two and three doses of MPTP 717 Fig. 3. Composite scores (general activity, locomotor activity, posture, imbalance, tremor frequency, body freeze, and feeding ability) from the behavioral rating scale of mild (top) and advanced (bottom) parkinsonism in monkeys during the drug testing sessions. Animals displayed stable motor function during this period. Mean values for monthly and daily time periods are indicated before the drug testing period began (time 0) and during the drug testing period. After two doses of MPTP, all animals showed a constant parkinsonism for at least 3 months after the second MPTP dose. No change in activity was observed in the advanced parkinsonian monkeys for at least 2 months after MPTP was administered. with regimen A and B, respectively, were designated mild and advanced parkinsonian monkeys. Mild parkinsonian monkeys displayed constant parkinsonian symptoms for at least 3 months (Fig. 3). Although the average composite score remained relatively stable for up to 10 months after MPTP, slight improvement in the composite score was found for two animals in contrast to the stable parkinsonism of the other two monkeys. Advanced parkinsonian monkeys displayed severe parkinsonian symptoms for the drug study period that persisted for more than 1 month after the MPTP treatment (Fig. 3). Effects of SKF 81297. The short-acting agonist SKF 81297 is a high-efficacy D1 agonist as measured by its sensitivity to guanine nucleotides in radioligand binding assays and is 343-fold selective for the D1 over the D2 dopamine receptor (D1, K0.5 5 9 nM; D2, K0.5 5 3060 nM; Madras, 1993). Baseline behavior and the vehicle injections were similar for all parameters before SKF 81297 administration in normal untreated monkeys (n 5 3), in mild parkinsonian monkeys (n 5 4), and in advanced parkinsonian monkeys (n 5 4; Fig. 4). In normal monkeys, posture, imbalance, tremor, and feeding ability were unchanged after the administration of SKF 81297 in a dose range of 0.1 to 3.0 mg/kg (n 5 3; Fig. 4). However, in one monkey (009-91), the highest dose of SKF 81297 (3.0 mg/kg) lowered general and locomotor activity and increased bradykinesia, body freeze, and rigidity. In another monkey (200-94), appetite was suppressed and emesis was observed at a high dose of SKF 81297 (3.0 mg/kg). For the highest dose of SKF 81297 (3.0 mg/kg), the summation of individual scores for general activity, locomotor activity, bradykinesia, rigidity, posture, imbalance, tremor frequency, Downloaded from jpet.aspetjournals.org at ASPET Journals on April 7, 2016 Fig. 2. A, composite scores (general activity, locomotor activity, posture, imbalance, tremor frequency, body freeze, and feeding ability) of normal monkeys and monkeys with mild and advanced parkinsonism. Computerized monitoring of normal monkeys (B) and monkeys with mild parkinsonism (C). Cynomolgus monkeys were treated with either two doses of MPTP one month apart (regimen A) or with three doses of MPTP within 10 days (regimen B). In A (top), monkeys treated with regimen B (advanced, n 5 4) consistently displayed more severe impairment than monkeys treated with regimen A (mild, n 5 4). ***P , .005 versus pre-MPTP. ¥¥P , .01. B and C, display examples of 24-h motion monitoring with an accelerometer positioned in a jacket pocket of normal (B) and mild parkinsonian monkeys (C). The y-axis on B and C is set at 140 units. The composite score from 45 days of monitoring motion in mild parkinsonian monkeys was 62% of the composite score of normal monkeys and parallels the findings with general and locomotor activity. Note the depressed activity during nighttime hours. D1 Agonist Efficacy in Advanced and Mild Parkinsonism 718 Goulet and Madras and body freeze was averaged 3.8 6 2.2 (n 5 3). This value exceed normal activity (0.5 6 0.4, n 5 3) but did not reach statistical significance (see Fig. 8). SKF 81297 was relatively ineffective in relieving symptoms of mild parkinsonism in monkeys. In this regard, general activity, locomotor activity, bradykinesia, rigidity, posture, tremor, and feeding did not improve in mild parkinsonian monkeys (Fig. 4). A dose-dependent increase of imbalance and reduction in body freeze resulted from SKF 81297, but the data were not statistically significant (Figs. 4 and 7). Emesis was observed at 3.0 mg/kg SKF 81297 in two monkeys (391-95 and 126-97). Three of the four mild parkinsonian monkeys displayed slight but not statistically significant orofacial dyskinesias with the highest dose of SKF 81297 tested (3.0 mg/kg). The composite score of mild parkinsonian monkeys (5.2 6 0.7 at 3.0 mg/kg SKF 81297) did not differ from prior baseline behavior and vehicle injection scores of 5.9 6 1.6 and 5.7 6 1.6, respectively (Fig. 8). Acute administration of SKF 81297 alleviated symptoms of advanced parkinsonian monkeys in contrast to results observed with mild parkinsonian animals (Fig. 4). Compared with vehicle injection, SKF 81297 (1.0 and 3.0 mg/kg) improved general activity, bradykinesia, rigidity, posture, and body freeze, and these improvements achieved statistical significance as determined by a one-way ANOVA followed by Fisher PLSD comparisons (Fig. 4). SKF 81297 (1.0 and 3.0 mg/kg) also dose dependently improved locomotor activity and tremor frequency in advanced parkinsonian monkeys, but the results were not statistically significant (Fig. 4). Notwithstanding the improvement in motor function and the reversal of stooped posture, feeding ability remained impaired. As with mild parkinsonian monkeys, SKF 81297 promoted slight but not statistically significant imbalance in advanced parkinsonian monkeys (Fig. 7). Three of the four parkinsonian monkeys displayed limb and trunk dyskinesias at 1.0 and 3.0 mg/kg SKF 81297. The composite score of advanced parkinsonian monkeys (3.0 6 1.1 at 3.0 mg/kg SKF 81297) was statistically significant from prior baseline behavior and vehicle injection scores of 9.0 6 0.4 and 9.0 6 0.5, respectively (Fig. 8). Effects of Dihydrexidine. Dihydrexidine is reported to be a full D1 agonist (Lovenberg et al., 1989) and is 3-fold more selective for D1 than D2 receptors in primate striatum (D1, K0.5 5 27 nM; D2, K0.5 5 92 nM; Madras, 1993). Dihydrexidine was administered to normal untreated monkeys (n 5 3), mild parkinsonian monkeys treated with two doses of MPTP at 1 month apart (n 5 4), and advanced parkinsonian monkeys treated with three consecutive doses of MPTP within 10 days (n 5 4). In normal monkeys, general activity, bradykinesia, rigidity, posture, body freeze, tremor, and feeding ability remained unchanged after administration of dihydrexidine in a dose range of 0.01 to 3.0 mg/kg (n 5 3; Fig. 5). However, locomotor activity tended to decrease in a dose-dependent but not statistically significant manner (Fig. 5). As with the other D1 agonist, SKF 81297, dihydrexidine was relatively ineffective in relieving mild parkinsonian symptoms. In this regard, general activity, bradykinesia, rigidity, tremor, and feeding ability did not improve in mild parkinsonian monkeys (Fig. 5). Locomotor activity was reversed, and posture and body freeze also displayed a tendency to improvement but did not reach statistical significance (Fig. 5). Dihydrexidine promoted dose-dependent imbalance and some orofacial dyskinesias, which were not statistically significant (see Fig. 7). The composite score of normal monkeys fluctuated between 0.2 and 1.7 with dihydrexidine treatment and did not differ from prior baseline behavior (0.5 6 0.3) and vehicle (0.7 6 0.5). The composite score of mild parkinsonian monkeys (4.0 6 0.7 at 3.0 mg/kg dihydrexidine) did not differ statistically from prior baseline behavior (6.7 6 1.4) and vehicle (6.3 6 1.6; see Fig. 8). In contrast, dihydrexidine was far more effective in advanced parkinsonian monkeys. Compared with vehicle, dihydrexidine at doses of 3.0 mg/kg improved general activity, locomotor activity, bradykinesia, rigidity, and body freeze, which reached statistical significant as determined by a oneway ANOVA followed by Fisher PLSD comparisons (Fig. 5). Dihydrexidine also dose dependently improved posture and tremor frequency in advanced parkinsonian monkeys, but the results were not statistically significant (Fig. 5). Notwith- Downloaded from jpet.aspetjournals.org at ASPET Journals on April 7, 2016 Fig. 4. Dose-response effects of the D1 agonist SKF 81297 on behavior of normal animals (M) and mild (Œ) and advanced (F) parkinsonian MPTP monkeys. Abscissa, cumulative i.m. dose of SKF 81297 on a log scale. Ordinate, mean 6 S.E. rating score of four monkeys accordingly to the rating scale (right) and the corresponding behavior (left). Dotted line represents normal behavior. A vehicle injection preceded all drug treatments, and animal baseline behaviors (control) were monitored between each treatment. The dose-response to drugs was analyzed using a oneway ANOVA with the monkey parkinsonism rating score. Fisher’s post hoc multiple comparison procedure was used to compare the doses with the vehicle and control for each group of monkeys. SKF 81297 did not change the behavior of normal monkeys and was ineffective in relieving the symptoms of mild parkinsonian monkeys. In contrast, SKF 81297 produced an improvement in advanced parkinsonian monkeys. *P , .05, **P , .01, and ***P , .005 versus control (Ctrl). ¥P , .05, ¥¥P , .01 versus vehicle (Veh). Vol. 292 2000 D1 Agonist Efficacy in Advanced and Mild Parkinsonism 719 Fig. 5. Dose-response effects of the D1 agonist dihydrexidine on the behavior of normal animals (M) and mild (Œ) and advanced (F) parkinsonian MPTP monkeys. Dihydrexidine did not change the spontaneous level of activity levels in normal monkeys and was relatively ineffective in relieving the symptoms of mild parkinsonism in monkeys. In contrast, dihydrexidine alleviated parkinsonism of advanced parkinsonian animals. *P , .05, **P , .01, and ***P , .005 versus control. ¥P , .05, ¥¥P , .01, and ¥¥¥P , .005 versus vehicle (Veh). standing the improvement in motor function, feeding ability remained impaired (Fig. 5). Similar to mild parkinsonian monkeys, dihydrexidine promoted slight imbalance in advanced parkinsonian monkeys (Fig. 7). One (200-94) of the four advanced parkinsonian monkeys displayed orofacial, limb, and trunk dyskinesias at 3.0 mg/kg dihydrexidine. The composite score of advanced parkinsonian monkeys after treatment with 3.0 mg/kg dihydrexidine (3.1 6 1.5) was statistically significantly different from prior baseline behavior and vehicle injection scores of 8.8 6 0.5 and 8.8 6 0.3, respectively (see Fig. 8). Effects of D2 Agonists. Quinelorane (D1, K0.5 5 52,000 nM; D2, K0.5 5 55 nM) and (1)PHNO (D1, K0.5 5 16,000 nM; D2, K0.5 5 2 nM) are very selective D2-like agonists in primate striatum (B. K. Madras, in preparation). Quinelorane was administered to normal untreated monkeys (n 5 2), mild parkinsonian monkeys (n 5 4), and in a single dose to advanced parkinsonian monkeys (n 5 3). Quinelorane (3 mg/kg) had little effect on the motor deficits or activity of normal monkeys (n 5 2), and the composite score of 2.20 6 1.80 was slightly elevated compared with vehicle (0.08 6 0.02; data not shown). Quinelorane dose dependently relieved some symptoms of mild parkinsonism in monkeys (Fig. 6). In this regard, general activity, locomotor activity, posture, body Fig. 6. Dose-response effects of the D2 agonists quinelorane (F) and (1)-PHNO (M) on the behavior of mild parkinsonian MPTP monkeys. Quinelorane and (1)-PHNO were effective in relieving some symptoms of mild parkinsonism in monkeys. *P , .05 and ***P , .005 versus control (Ctrl). ¥P , .05, ¥¥P , .01, and ¥¥¥P , .005 versus vehicle. Downloaded from jpet.aspetjournals.org at ASPET Journals on April 7, 2016 freeze, and feeding tend to improve in mild parkinsonian monkeys (Fig. 6). A significant dose-dependent increase of imbalance and induction of dyskinesias resulted from quinelorane treatments (Fig. 7). The composite score of mild parkinsonian monkeys (4.3 6 0.5 at 3.0 mg/kg quinelorane) was improved over baseline behavior and vehicle injection scores of 6.2 6 1.4 and 6.3 6 1.5, respectively (Fig. 8). (1)-PHNO was also relatively effective in relieving symptoms of mild parkinsonism in monkeys (Fig. 6). In this regard, locomotor activity and bradykinesia significantly improved in mild parkinsonian monkeys (Fig. 6). General activity, rigidity, posture, body freeze, tremor frequency, and feeding also tended to improve in mild parkinsonian monkeys (Fig. 6). A significant dose-dependent increase in imbalance and induction of dyskinesias resulted from (1)-PHNO treatment (Fig. 7). The composite score of mild parkinsonian monkeys [3.5 6 1.1 at 0.03 mg/kg (1)-PHNO] displayed improvement compared with the baseline score of 5.8 6 1.8 or vehicle (6.0 6 1.9), but results were not statistically significant (Fig. 8). The efficacy of PHNO was stereoselective as the (2)-enantiomer of PHNO had no effect on parkinsonian parameters in mild parkinsonian monkeys (data not shown). The D2 agonists quinelorane and (1)-PHNO were well tolerated by the monkeys and induced grooming behavior and penile erection. 720 Goulet and Madras Vol. 292 A single dose of quinelorane (0.1 mg/kg) produced a complete reversal of some parkinsonian symptoms in three advanced parkinsonian monkeys. General activity, bradykinesia, stooped posture, and body freeze returned to normal levels (data not shown). The composite score in these animals fell from 11.0 6 0.2 (vehicle) to 2.6 6 0.7 after 0.1 mg/kg quinelorane (P , .005; Fig. 8). Discussion This study demonstrates the feasibility of producing two relatively stable models of mild and advanced parkinsonism with two different and fixed dosing regimens of MPTP in cynomolgus monkeys. These models, which correspond generally to the classic signs of early or late stages of human Parkinson’s disease, offer important opportunities to evaluate drug therapies and validate experimental neuroprotective and neuroregenerative therapies. The two models differentiated the therapeutic potential of a selective D1 agonist for treatment of Parkinson’s disease. Regardless of the dosing regimen, all monkeys treated with MPTP displayed the principal characteristics of Parkinson’s disease (reduced general and locomotor activity, bradykinesia, rigidity, tremor, and posture disturbance) with varying degrees of impairment. General activity and ability to feed reflected most closely the differences in severity of parkinsonism in advanced animals compared with mild parkinsonian monkeys (P , .0001). The combined behavioral ratings for all the parameters, including those that did not allow distinctions between the two groups (bradykinesia and rigidity), revealed an overall disability one and one-half times more prominent statistically in the advanced than in the mild parkinsonian animals. A positron emission tomography study to image dopamine terminals with the selective probe [11C]2b-carbomethoxy-3b-(4-fluorophenyl)tropane (WIN 35,428) revealed that advanced parkinsonian animals had four times fewer dopaminergic terminals than did mild parkinsonian monkeys (B.K. Madras, in preparation). Monkeys with advanced parkinsonism were generally unable to feed themselves, did not engage in locomotor activity, and displayed marked stooping posture, severe body freeze, and inattention to their environment, which corresponded in severity to stage IV/V of human Parkinson’s disease (Fig. 1). Despite the generalized motor impairment, mild parkinsonian monkeys were more active, had near-normal general activity, displayed slightly stooped posture and infrequent body freeze, and maintained alertness and feeding ability, which corresponded to stage I/III of human Parkinson’s disease Hoehn and Yahr scale. Computerized monitoring of activity revealed the lower activity level of mild parkinsonian monkeys compared with normal animals and closely reflected general and locomotor activity. The accelerometer technology enables continuous (24 h) monitoring of activity for as long as 45 days and circumvents difficulties in monitoring activity during the sleep cycle of primates. Tremor was less prominent in advanced parkinsonian than in mild parkinsonian monkeys (Fig. 1) and parallels the reduced tremor observed in late compared with early stages of human Parkinson’s disease (Fahn et al., 1987). Because some sparing of neurons in the substantia nigra compacta may be required for tremor to develop, massive nigral cell loss by high doses of MPTP may account for the minimal tremor in advanced parkinsonian monkeys (Hantraye et al., Downloaded from jpet.aspetjournals.org at ASPET Journals on April 7, 2016 Fig. 7. Dose-response effects of D1 and D2 agonists on imbalance and dyskinesia of monkeys before MPTP administration (M), in mild parkinsonism after two doses of MPTP 1 month apart (Œ), and in advanced parkinsonism, after three doses of MPTP within 10 days (F). D2 agonists quinelorane and (1)-PHNO induced more severe imbalance and dyskinesias than D1 agonists SKF 81297 and dihydrexidine. *P , .05 and ***P , .005 versus control (Ctrl). ¥P , .05, ¥¥P , .01, and ¥¥¥P , .005 versus vehicle (Veh). 2000 D1 Agonist Efficacy in Advanced and Mild Parkinsonism 721 1993). In MPTP-induced parkinsonism in human, tremor is the only motor symptom less frequently observed than in the idiopathic disease (Tetrud and Langston, 1992), probably because of the rapid and massive nigral cell loss induced by MPTP. The rate of progression of the disease may be slightly less rapid when tremor is the initial symptom (Hoehn and Yahr, 1967), possibly because of the existence of more abundant neurons in the substantia nigra compacta. Imbalance was not observed in both advanced and mild parkinsonian monkeys. In humans, postural instability constitutes an important parameter for evaluation of the degree of severity of Parkinson’s disease and typically emerges in advanced stages of the disease (Bonnet et al., 1987). The absence of imbalance in the nonhuman primate model of Parkinson’s disease may reflect the inadequacy of applying this anthropomorphic measure to nonhuman primates. Nonhuman primates assume a quadrupedal stance for the majority of time, which requires different mechanical strategies to maintain equilibrium than humans with bipedal stances and motion. Hence, normal balance observed in MPTPtreated monkeys may reflect a capacity to compensate with the use of forelimbs as supportive struts. The stability and persistence of the deficits are critical for the evaluation of experimental interventions. Spontaneous behavioral recovery was often reported after i.v. administration of MPTP delivered over a short period of time (Eidelberg et al., 1986; Kurlan et al., 1991). However, stable parkinsonian symptoms were observed after longer intervals between low and chronic doses of MPTP (Hantraye et al., 1993). In the present study, some improvement was noted several months after MPTP treatment. Accordingly, evaluation of antiparkinsonian drugs was completed within 2 and 3 months after MPTP treatment for advanced and mild parkinsonian monkeys. The two models revealed important differences in the therapeutic potential of D1 and D2 dopamine receptor agonists. Parkinson’s disease is currently treated with the dopamine precursor L-dopa and/or dopamine D2 receptor agonists. Although high-efficacy D1 dopamine agonists alleviate parkinsonism in animal models (Mottola et al., 1992; Vermeulen et al., 1993; Goulet et al., 1996; Shiosaki et al., 1996) and in humans (Blanchet et al., 1998; Rascol et al., 1999), comparisons of the efficacy of D1 agonists in mild or advanced parkinsonism have not been reported. The present study is the first to demonstrate that a selective D1 agonist displays limited antiparkinsonian effect in mild parkinsonian monkeys. D1 agonists alleviated parkinsonian signs in monkeys with advanced parkinsonism and confirmed previous studies with SKF 81297, dihydrexidine (Taylor et al., 1991; Domino and Sheng, 1993; Vermeulen et al., 1993; Andringa et al., 1998), and other high-efficacy D1 dopamine agonists (Lovenberg et al., 1989; Mottola et al., 1992; Blanchet et al., 1996b; Goulet et al., 1996; Shiosaki et al., 1996; Grondin et al., 1997). In contrast, SKF 81297 was relatively ineffective in mild parkinsonism and dihydrexidine was moderately effective. Stimulant effects of dihydrexidine were previously reported in mild parkinsonian monkeys (Schneider et al., 1994) and showed a short-lived antiparkinsonian response in one patient of four with mild to moderate signs of parkinsonism (Blanchet et al., 1998). However, the relatively high affinity of dihydrexidine at D2 receptors may contribute to the anti- Downloaded from jpet.aspetjournals.org at ASPET Journals on April 7, 2016 Fig. 8. Dose-response effects of D1 and D2 agonists on anti-parkinsonian score of mild and advanced parkinsonian MPTP monkeys before MPTP administration (M), after two doses of MPTP 1 month apart (Œ), and after three doses of MPTP within 10 days (F). In advanced parkinsonism, D1 and D2 dopamine agonists effectively reversed the motor deficits. SKF 81297 and dihydrexidine effects were relatively limited in mild parkinsonism. Quinelorane and (1)-PHNO were effective in alleviating some mild parkinsonism symptoms. ***P , .005 versus control (Ctrl). ¥¥¥P , .005 versus vehicle (Veh). 722 Goulet and Madras bility of producing and quantifying animal models of Parkinson’s disease that correspond to the signs of early or late stages of human Parkinson’s disease. These models enabled evaluation of the therapeutic potential of D1 agonists for treating mild or advanced Parkinson’s disease. High-efficacy D1 agonists were more effective in advanced than in mild parkinsonism and produced fewer side effects in mild parkinsonism than did D2 agonists. Thus, D1 agonists may be particularly useful as monotherapy for the end stages of the Parkinson’s disease when L-dopa efficacy wanes and dyskinesias are frequently encountered. Appendix 1: Monkey Parkinsonism Rating Scale to Assess Parkinsonian Signs and Effects of Drugs Spontaneous normal behavior corresponded to 0 on the rating scale. For most of the parameters, impairment was rated on a 1 or 2 scale. A negative score portrayed hyperactive behavior. The composite score represents a summation of individual scores for general activity, locomotor activity, bradykinesia, rigidity, posture, imbalance, tremor frequency, and body freeze. Drug-induced dyskinesias were scored as severe, slight, and absent for different segments, face, limb, and trunk. Stereotypy (licking, grooming, scratching, and biting) and frequent head movements with visual scanning were also recorded on the rating form. 1. General Activity 21 Hyperactive (more than two bouts of frenzied, rapid, or sudden movement/2-min observation) 0 Normal (.10 frequent biaxial/triaxial movements/ 2-min observation) 1 Slight (1–10 monoaxial movements or biaxial/triaxial movements/2-min observation) 2 Absent (no movements of any kind) Note: Score on general activity is based on all activity, including locomotion. 2. Locomotor Activity 21 Hyperactive (.2 bouts of exaggerated locomotion/2min observation) 0 Normal (.10 steps/2-min observation) 1 Slight (2–10 steps/2-min observation) 2 Absent (no locomotion) Note: For locomotion to be scored, there must be at least two steps in one direction. Flailing, swaying, and scooting are not considered locomotion. 3. Bradykinesia 0 Normal (normal or usual speed and facility of movement) 1 Present (noticeable slowness of movement) 4. Rigidity 0 Normal (no rigidity or muscle stiffness seen) 1 Present (increased resistance to the passive movement of a limb, obvious difficulty in movement) 5. Posture 21 Head torticollis (head is pulled straight back) 0 Normal (spine does not appear excessively curved) Downloaded from jpet.aspetjournals.org at ASPET Journals on April 7, 2016 parkinsonian effect observed in mild parkinsonian monkeys. Accordingly, selective D1 agonists appear particularly useful as monotherapy for the end stages of the Parkinson’s disease when the effectiveness of L-dopa wanes and the frequency of dyskinesia increases. Side effects are important considerations in assessing the therapeutic potential of dopaminergic drugs. Dyskinesias are a common side effect of L-dopa therapy and are observed in approximately 80% of patients after 5 years of L-dopa treatment (Boyce et al., 1990). At therapeutic doses, D2 agonists quinelorane and (1)-PHNO alleviated some parkinsonian symptoms but produced more severe dyskinesias than D1 agonists. Both D1 and D2 agonist-induced dyskinesias were previously observed in MPTP monkeys (Rupniak et al., 1989; Bedard et al., 1993; Luquin et al., 1994; Blanchet et al., 1996a), but in agreement with our study, dyskinesias were more pronounced with D2 agonists than with D1 agonists (Bedard et al., 1993; Blanchet et al., 1996b). In a human study, the D1 agonist ABT-431 produced less dyskinesia than L-dopa (Rascol et al., 1999). Postural instability, another disabling feature of Parkinson’s disease, typically occurs in the advanced stages of the disease (Bonnet et al., 1987). Imbalance did not occur spontaneously in parkinsonian monkeys but was induced by D2 agonists to a greater extent than by D1 agonists. Taken together, these findings support a more prominent role of D2 agonists in producing dyskinesia and imbalance. These observations highlight advantages of D1 agonists over existing therapies, particularly for advanced parkinsonism and particularly when postural problems emerge. The higher degree of efficacy of D1 agonists in advanced compared with mild parkinsonism may be dose-dependent or reflect differences in underlying biochemical mechanisms at different levels of dopamine depletion. In normal monkeys, dopamine tonically stimulates D1 and D2 receptors. D1 receptors are not appreciably lost with disease progression because no significant down-regulation of striatal dopamine D1 receptors has been demonstrated in treated parkinsonian patients (Raisman et al., 1985; Rinne et al., 1991) and in MPTP-lesioned monkeys (Goulet et al., 1996). Based on behavioral data and results from positron emission tomography imaging (B.K. Madras et al., in preparation), the depletion of dopamine terminals was more pronounced in advanced than in mild parkinsonian monkeys. Hence, in mild parkinsonism, dopamine levels may be sufficient to maximally stimulate D1. In advanced parkinsonism, dopamine depletion may be sufficiently high to enable occupancy and activation of D1 receptors by D1 receptor agonists. This enhanced D1 receptor response may be accounted by a change in D1 receptor availability, receptor-effector coupling, receptor trafficking, disruption of D1-D2 linkage, or other factors. Alternately, the minimal effects of D1 agonists on motor activity in mild parkinsonian or normal animals may result from stimulation of another pool of D1 or other receptors, which diminish motor activity. Such speculation may explain D1 agonistinduced hypokinesia of transgenic mice overexpressing the D1 receptors in the medial prefrontal cortex (Dracheva et al., 1999). Another property that distinguishes D1 and D2 agonists is the rapid tolerance to improved motor function observed with repeated treatment of some (Blanchet et al., 1996a; Goulet et al., 1996), but not all (Asin et al., 1997), D1 agonists in parkinsonian monkeys. In conclusion, the present results demonstrate the feasi- Vol. 292 2000 D1 Agonist Efficacy in Advanced and Mild Parkinsonism 1 Slight stooping (some stooping/hunching of back, head position is forward and down) 2 Severe stooping (extreme hunching of back and shoulders; head position is down at or below knee level when sitting) 6. Imbalance 0 Normal (normal balance) 1 Slight (1–3 episodes of difficulty in stabilizing stance or preventing falling/2-min observation) 2 Severe (.3 episodes of uncontrolled loss of position/ 2-min observation) 7. Body Freeze 8. Tremor Frequency 0 Normal (no visible tremor or a rhythmical shaking of a limb, head, mouth, tongue, or other part of the body) 1 Slight (tremor occurs 1–3 times of short duration for a total duration of 15–30 s/2-min observation) 2 Severe (.3 short duration tremors or tremor of long duration for a total duration of $30 s/2-min observation) 9. Feeding Ability 21 Hyperphagia (ingestion of food at an abnormally rapid rate) 0 Normal (eats with no difficulty) 1 Difficult (able to feed but drops food, misses mouth, difficulty in picking up) 2 Absent (unable to get food to mouth) 10. Orofacial, Limb, and Trunk Dyskinesia 22 Severe (.10 episodes of rapid, jerky, dance like movement of the body, such as tongue darting, lip contracting, grimacing, head scanning, flailing or palsy, and swaying or twisting/2-min observation) 21 Slight (1–10 episodes of involuntary and uncontrolled movements/2-min observation) 0 Absent (normal facial movements/expression) 11. Stereotypy 22 Severe (.6 controlled, coordinated, and repetitive movements serving no apparent function or of duration of .1 min/2-min observation) 21 Slight (stereotypical behavior cluster occurring 3– 6 times or of duration of 10 s to 1 min/2-min observation) 0 Absent Acknowledgments We thank Dr. J. Weinstock (SmithKline Beecham) for generously donating SKF 81297. We also express gratitude to Patricia Matthews, Michele LaReau-Alves, and Tracy Brackett for technical as- sistance and to Sandra Talbot for assistance in graph and manuscript preparation. 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