Introduction

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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.

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 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 [(±)-trans-10,11-dihydroxy-5,6,6a,7,8,12b-hexahydrobenzo[a]phenanthridine], A-77636, SKF 81297 [(R)-(+)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine], 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 ± 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 (+)-4-propyl-9-hydroxy-2,3,4a,5,6,10b-hexahydro-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.

	Materials and Methods

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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 × 79 × 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 = 3) and to compare the effects of the two MPTP regimens (regimen A, n = 4; regimen B, n = 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 drug-testing period. For a full dose-response curve, overlapping doses were averaged and the mean data were used for analysis.

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 = 4) and compared with normal animals (n = 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 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 (Mini-Mitter), and the average of daylight activity counts was compared.

Statistics. 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. 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 ± S.E.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Appendix 1 References

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 ± 1.5 (n = 4, P < .05) was less than the composite score of 1.5 ± 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 ± 16 counts/min) was 62% of the activity of normal monkeys (70 ± 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.

View larger version (36K): [in this window] [in a new window]   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.

View larger version (45K): [in this window] [in a new window]   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 = 4) consistently displayed more severe impairment than monkeys treated with regimen A (mild, n = 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.

View larger version (22K): [in this window] [in a new window]   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.

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, K_0.5 = 9 nM; D₂, K_0.5 = 3060 nM; Madras, 1993). Baseline behavior and the vehicle injections were similar for all parameters before SKF 81297 administration in normal untreated monkeys (n = 3), in mild parkinsonian monkeys (n = 4), and in advanced parkinsonian monkeys (n = 4; Fig. 4).

View larger version (41K): [in this window] [in a new window]   Fig. 4.   Dose-response effects of the D1 agonist SKF 81297 on behavior of normal animals () and mild () and advanced () parkinsonian MPTP monkeys. Abscissa, cumulative i.m. dose of SKF 81297 on a log scale. Ordinate, mean ± 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 one-way 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).

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, K_0.5 = 27 nM; D₂, K_0.5 = 92 nM; Madras, 1993). Dihydrexidine was administered to normal untreated monkeys (n = 3), mild parkinsonian monkeys treated with two doses of MPTP at 1 month apart (n = 4), and advanced parkinsonian monkeys treated with three consecutive doses of MPTP within 10 days (n = 4).

View larger version (42K): [in this window] [in a new window]   Fig. 5.   Dose-response effects of the D1 agonist dihydrexidine on the behavior of normal animals () and mild () and advanced () 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).

Effects of D₂ Agonists. Quinelorane (D₁, K_0.5 = 52,000 nM; D₂, K_0.5 = 55 nM) and (+)PHNO (D₁, K_0.5 = 16,000 nM; D₂, K_0.5 = 2 nM) are very selective D₂-like agonists in primate striatum (B. K. Madras, in preparation). Quinelorane was administered to normal untreated monkeys (n = 2), mild parkinsonian monkeys (n = 4), and in a single dose to advanced parkinsonian monkeys (n = 3). Quinelorane (3 mg/kg) had little effect on the motor deficits or activity of normal monkeys (n = 2), and the composite score of 2.20 ± 1.80 was slightly elevated compared with vehicle (0.08 ± 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 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 ± 0.5 at 3.0 mg/kg quinelorane) was improved over baseline behavior and vehicle injection scores of 6.2 ± 1.4 and 6.3 ± 1.5, respectively (Fig. 8).

View larger version (35K): [in this window] [in a new window]   Fig. 6.   Dose-response effects of the D2 agonists quinelorane () and (+)-PHNO () on the behavior of mild parkinsonian MPTP monkeys. Quinelorane and (+)-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.

View larger version (29K): [in this window] [in a new window]   Fig. 7.   Dose-response effects of D1 and D2 agonists on imbalance and dyskinesia of monkeys before MPTP administration (), in mild parkinsonism after two doses of MPTP 1 month apart (), and in advanced parkinsonism, after three doses of MPTP within 10 days (). D2 agonists quinelorane and (+)-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).

View larger version (29K): [in this window] [in a new window]   Fig. 8.   Dose-response effects of D1 and D2 agonists on anti-parkinsonian score of mild and advanced parkinsonian MPTP monkeys before MPTP administration (), after two doses of MPTP 1 month apart (), and after three doses of MPTP within 10 days (). 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 (+)-PHNO were effective in alleviating some mild parkinsonism symptoms. ***P < .005 versus control (Ctrl). ¥¥¥P < .005 versus vehicle (Veh).

	Discussion

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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]2-carbomethoxy-3-(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., 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 MPTP-treated 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 antiparkinsonian 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 (+)-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 agonist-induced 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 feasibility 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.