Introduction

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A-86929 is a selective ligand for the DA D1 receptor with full agonist activity relative to DA (Shiosaki et al., 1996); the diacetyl derivative, ABT-431, is currently under development for the treatment of PD. One useful model for testing compounds for potential antiparkinsonian properties involves the use of rats or nonhuman primates with unilateral DA depletions. In such animals, direct-acting DA receptor agonists induce rotation away from the lesioned side, presumably because of the increased sensitivity of postsynaptic DA receptors on the denervated side. In rats, unilateral injections of the neurotoxin 6-OHDA reduce forebrain DA levels, whereas in primates, unilateral brain depletions of DA can be achieved through the intracarotid infusion of the neurotoxin MPTP. Results generated in the MPTP primate model have, in particular, proved useful for extrapolation to humans. Although the partial agonist SKF 38393 proved efficacious in rat models of PD, it was unable to ameliorate parkinsonian signs in MPTP-treated primates, perhaps because of its partial agonist activity (Bédard and Boucher, 1989; Braun et al., 1986; Falardeau et al., 1988). Subsequently, D1 receptor agonists with greater intrinsic activity have been demonstrated to relieve parkinsonian signs and symptoms in MPTP-treated primates and in PD patients (Blanchet et al., 1993; Emre et al., 1992; Gnanalingham et al., 1995; Luquin et al., 1994; Taylor et al., 1991; Temlett et al., 1989) and to induce dose-dependent contralateral rotation in monkeys rendered hemiparkinsonian via unilateral, intracarotid infusions of MPTP (Domino and Sheng, 1993a,b; Johnson et al., 1995; Vermeulen et al., 1993; 1995). The behavioral effects of these compounds after repeated daily treatment have not been reported.

The ability of A-86929 to produce rotation in rats bearing unilateral 6-OHDA lesions and in nonhuman primates rendered hemiparkinsonian through intracarotid infusions of MPTP was investigated in the present study. Rotation was examined across a thrice-daily, repeated-treatment regimen across a 10-day period. The results indicate that a D1 receptor agonist with full intrinsic activity may be useful as a chronic treatment for PD.

	Materials and Methods

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Subjects

Twelve adult male Sprague-Dawley rats (Charles River, Portage, MI), weighing approximately 300 g, were used as subjects. Animals were single-housed in hanging wire-mesh cages, with food and water available ad libitum. The light:dark schedule was 12:12 h.

Five adult female Macaca nemstrina (pig-tailed macaque) monkeys weighing 5.4 to 6.9 kg at the time of study were originally obtained from Charles River Co., Port Washington, NY. The animal care and use program conforms to the standards in the Guide for the Care and Use of Laboratory Animals (1985), and we followed the B Virus Working Group Guidelines for Prevention of Herpes Virus Simian Infection in Monkey Handlers (1988). Animals were single housed and maintained on a 12:12 light:dark schedule, with water and monkey chow available ad libitum each morning, and fruit supplements, as warranted.

Surgery

Rats. Twenty minutes after an injection of desmethylimipramine HCl (25 mg/kg ip), rats were anesthetized with sodium pentobarbital (50 mg/kg) and given unilateral injections of 6-OHDA into the medial forebrain bundle, as described previously (Asin et al., 1995). Rats were prescreened for rotation in response to apomorphine about 2 to 3 weeks after lesioning, as also described elsewhere (Asin et al., 1995). Animals were used in the present studies approximately 2 months after surgery. There were six animals in each of the two treatment groups.

Monkeys. The method of Bankiewicz et al. (1986) was used to infuse MPTP unilaterally into one common carotid artery (also see Domino and Sheng, 1993a,b). Animals were anesthetized with ketamine hydrochloride (5-10 mg/kg, base dose) followed by 30 mg/kg i.v. sodium pentobarbital to maintain deep anesthesia. Animals were secured on the operating table, and one common carotid artery was exposed at its bifurcation. MPTP HCl (approximately 3 mg in 60 ml) was infused slowly over a 20-min period, and the wound was then closed. Each animal was given 300,000 U sterile penicillin G benzathine and penicillin G procaine in an aqueous suspension. At the time of the current studies, animals had been displaying hemiparkinsonian symptoms for 3 to 5 years and had been given various selective D1 and D2 DA receptor agonists and other compounds (e.g., amphetamine, cocaine, ketamine, phencyclidine and MK-801) during this time. However, animals had been free of medication for approximately 1 month before the present study.

Apparatus and Procedure

Rats. Animals were tested for rotation in automated rotometers (San Diego Instruments, San Diego, CA), as described previously (Asin et al., 1995). Animals were allowed to habituate to the rotometer bowls for approximately 20 min before the first daily injection of A-86929 (0.11 or 0.22 µmol/kg s.c.); the second and third injections were administered at 3-h intervals. These doses are approximately 50% and 100%, respectively, of the ED₅₀ value for this compound to induce rotation (Shiosaki et al., 1996). During the 10 days of treatment, rotation was measured on days 1, 3, 5, 8 and 10 for 3 h after each injection (total test time of 9 h); on the remaining days, rats were given the three daily injections in their home cages.

Monkeys. Methods were similar to those described elsewhere (see Domino and Sheng, 1993a,b). Briefly, monkeys were placed individually into a standard primate cage modified with a clear plexiglas front. Gross animal behaviors were observed and recorded via three color video cameras (Panasonic VHS model PV-420; Magnavox VR 9344-AV01 and Sears LX-1 model 1934) connected individually to videocassette recorders (Mitsubishi model U-32 VCR). The behavior of the animals was recorded for 30 min after vehicle injection and for 3 h after each drug injection without humans present. Videotapes were scored for the number and direction of complete, 360° turns demonstrated by each animal during each consecutive 5-min period.

Compounds

A-86929 was synthesized at Abbott Laboratories (Michaelides et al., 1995). It was administered to rats in sterile water and to monkeys in sterile 5% glucose in water, purchased from Abbott Laboratories (Abbott Park, IL). 6-OHDA and desmethyl-imipramine HCl were purchased from Sigma Chemical (St. Louis, MO), and MPTP HCl was purchased from Aldrich Chemical Co (Milwaukee, WI).

	Results

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Rats

Separate one-way ANOVAs with repeated measures for each of the two doses of A-86929 were conducted on the total net number of contralateral rotations (contralateral minus ipsilateral) shown by 6-OHDA-lesioned rats. There was a significant effect of Days for both the low [F(4,20) = 4.16; P < .02] and the high [F(4,20) = 5.73; P < .003] doses. Post hoc analysis (Tukey HSD) indicated that for the lower dose, the mean total number of responses on days 3 and 8 differed from each other, whereas for the higher dose, responses on days 8 and 10 both differed significantly from the response on day 1 (P < .05) (see fig. 1).

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Mean (± S.E.) daily (9 h) total net numbers of contralateral rotations (contralateral minus ipsilateral) demonstrated by 6-OHDA-lesioned rats after three daily injections of A-86929. *From Michaelides et al., 1995.

Separate two-way ANOVAs (Injection number × Days) on rotation scores after the three daily injections across the five testing days were also conducted for each dose. For the low dose, there were significant effects of Days [F(4,20) = 4.16; P < .02] and Injection number [F(2,10) = 23.12; P < .001] and a significant Days × Injection number interaction [F(8,40) = 4.75; P < .001]. For the high dose, there were also significant effects of Days [F(4,20) = 5.73; P < .003] and Injection number [F(2,10) = 13.62; P < .001] and a significant Days × Injection number interaction [F(8,40) = 3.72; P < .002]. As can be seen in figure 2, and as borne out in post-hoc analysis (one-way ANOVAs with repeated measures conducted across days for each dose and each injection number), the behavioral response to the first daily injection increased significantly across days for both the low [F(4,20) = 4.28; P < .02] and the high [F(4,20) = 3.85; P < .02] doses. The levels of rotation seen over days in response to the second daily injection remained unchanged at the low dose [F(4,20) < 1.0] but increased significantly at the high dose [F(4,20) = 5.82; P < .004]. The response to the third daily injection tended to decline in rats administered the low dose [F(4,20) = 2.60; p < .07] and remained unchanged at the high dose [F(4,20) < 1.0].

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Mean (± S.E.) net numbers of contralateral rotations across days in rats after each of the three daily injections of A-86929 A) 0.11 µmol/kg. B) 0.22 µmol/kg.

In order to compare the initial and final rotation responses over time after the three daily injections, three-way ANOVAs (Injection number × Time × Days) were also conducted for each dose on rotation scores after each injection on treatment days 1 and 10 (fig. 3). For the low dose, there were significant effects of Injection number [F(2,10) = 10.89; P < .004] and Time [F(5,25) = 12.37; P < .001] but not Days [F(1,5) < 1.0]. Significant interactions were obtained for Days × Injection number [F(2,10) = 5.35; p < .03], Injection number × Time [F(10,50) = 9.03; P < .001] but not Days × Time [F(5,25) < 1.0]. The Days × Injection number × Time interaction was also significant [F(10,50) = 3.13; P < .005]. The results of the ANOVA conducted on the lower dose can be summarized as follows: On the first day of treatment, the response over time to the three injections was similar. By Day 10, post-hoc analysis (Tukey HSD) indicated that the third injection elicited a smaller, shorter-lived response, both within and between days (P < .01), although the overall total number of rotations did not differ significantly between the first and last treatment days.

View larger version (37K): [in this window] [in a new window]   Fig. 3.   Mean (± S.E.) net number of contralateral rotations demonstrated by rats over time after each of the three daily A-86929 injections on the first and last treatment days. A) 0.11 µmol/kg. B) 0.22 µmol/kg.

For the high dose, there were significant effects of Days [F(1,5) = 7.85; P < .04] and Time [F(5,25) = 76.14; P < .001] but not Injection number [F(2,10) = 2.70; P > .10]. Significant interactions terms were obtained for Days × Injection number [F(2,10) = 21.60; P < .001] and Days × Time [F(5,25) = 12.43; P < .001] but not Injection number × Time [F(10,50) < 1.0]. The Days × Injection number × Time interaction was not statistically significant [F(10,50) = 1.08; P > .35]. The results of the ANOVA conducted on data from rats at the higher dose can be summarized as follows: The total number of rotations was greater on day 10 than on day 1, and this was true across each of the three injections (although there was no difference within a day in the behavioral response occurring after each injection). As may be seen in figure 3, response sensitization occurred over the treatment period and was approximately equal in magnitude after each of the three injections.

Monkeys

Dose-response determination. A one-way ANOVA with repeated measures was conducted on the total net number of contralateral rotations (contralateral minus ipsilateral) demonstrated by monkeys at each dose. A-86929 produced significant, dose-dependent increases in the net number of contralateral rotations demonstrated by each monkey (F(2,8) = 9.71; P < .008) (table 1). The duration of action was also dose-dependent (fig. 4). These results indicate that acute administration of A-86929 is able to produce robust contralateral rotation in a primate model of Parkinson's disease.

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Time course of the behavioral response to various doses of A-86929 in MPTP-lesioned primates. Graphed are the mean (± S.E.) net number of contralateral rotations (contralateral minus ipsilateral).

Repeated treatment. On the basis of the initial dose-response study, a dose of 0.30 µmol/kg was chosen for the repeated-administration study. The data generated in the former study indicated that although the duration of the rotation at this dose was approximately 3 h, the duration was reduced to approximately 2 h in the repeated-treatment study after the first day of treatment. Therefore, only data recorded across the 2-h period after each drug injection were analyzed. Initially, the total, net number of contralateral rotations for the first, second and third daily injections were summed and analyzed over days. This one-way (Days) ANOVA with repeated measures failed to indicate a significant effect of Days [F(9,36) = 1.60; P > .15], which indicated that there was no significant change in the effects of A-86929 across the 10-day injection period (fig. 5A). A second ANOVA (Injection number × Days, with repeated measures on both factors) was conducted on the net number of contralateral rotations shown by each monkey during the 2-h period after the first, second and third daily injection (fig. 5B). There was a marginally significant effect of Injection number [F(2,8) = 4.03; P  .06]. As expected, there was no significant effect of Days [F(9,36) = 1.60; P > .15] and no Injection number × Days interaction [F(18,72) = 1.40; P > .15]. A graph of the data (fig. 5B) indicates that although there was no change in drug efficacy over days, the first daily injection tended to elicit greater contralateral rotation than the second and third daily injections. Because this difference in response magnitude appeared to diminish over time, additional data analyses were performed on the data generated on the first and tenth treatment days. For each day, a two-way (Injection number × Time) ANOVA was conducted on 30-min rotation scores after each injection (fig. 6). On day 1 of treatment, there was a significant effect of Time (F(3,12) = 14.16; P < .001) and a marginally significant effect of Injection number (F(2,8) = 3.96; P < .065). The interaction between Injection number and Time was significant (F(2.52; p < .05). These results indicate a significant difference between the response pattern over time after the three injections. In contrast, only a significant effect of Time (F(3,12) = 7.65; P < .005) was obtained from the analysis of data from day 10 [Injection number (F(2,8) = 2.78; P > .10; Injection number × Time (F(6,24) = 1.31; P > .25)]. Thus, by the last treatment day, the responses to the three daily injections were statistically similar. In contrast to the robust contralateral rotation seen after treatment with A-86929, treatment with vehicle elicited ipsilateral rotation during the 30-min period after injection (mean ± S.E.M. = 12.40 ± 5.70).

View larger version (34K): [in this window] [in a new window]   Fig. 5.   A) Mean (± S.E.) daily total numbers of net contralateral rotations across the 10 days of repeated treatment in primates. Total testing time was 6 h. B) Mean (± S.E.) net numbers of contralateral rotations 2 h after the first, second and third daily injections.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Magnitude and duration of the behavioral response in primates to three daily injections of A-86929.1 on the first and last days of the repeated-treatment study.

	Discussion

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In the present study, thrice-daily injections of the full DA D1 receptor agonist A-86929 promoted rotation across a 10-day treatment period in both rats and primates bearing unilateral forebrain DA depletions. The daily total number of rotations did not decline over the 10-day period, and in rats, evidence for response sensitization was obtained with the higher dose (0.22 µmol/kg). These observations are unlike those we reported previously after daily treatment of lesioned rats with other DA D1 receptor agonists (A-77636 and A-85653) (Asin and Wirtshafter, 1993; Asin et al., 1995), which produced rapid development of behavioral tolerance. In contrast, daily treatment with either SKF 82958 or SKF 38393 may lead to either response sensitization (Silverman, 1991, 1992) or, for the case of SKF 38393, tolerance (Engber et al., 1993). Clearly, the behavioral effects of repeated DA agonist treatment rely on a number of factors, including drug doses and treatment regimen, as well as pharmacokinetic and pharmacologic properties of the administered compound (Castro et al., 1985). As we saw in the present study, species differences in the response to repeated treatment may also exist, perhaps as a consequence of differences in drug metabolism and/or distribution. It is notable that both A-77636 and A-85653 have durations of action in 6-OHDA-lesioned rats (measured behaviorally) ranging from 6 to 18 h and that these compounds produce behavioral tolerance after repeated treatment (Asin and Wirtshafter, 1993; Asin et al., 1995). This duration of action is considerably longer than that seen with A-86929 in the present study. Persistent receptor activation contributes to the receptor desensitization produced by A-77636 (Lin et al., 1996).

The hypothesis that a D1 receptor agonist may prove useful for the treatment of Parkinson's disease was tested almost a decade ago using the partial agonist SKF 38393, and, unlike what was seen in rats, the data collected from primates were not encouraging (Braun et al., 1986). However, subsequent studies have indicated that the problem lay not with the hypothesis but with the pharmacology and pharmacokinetics of the compound tested. Thus, although SKF 38393 proved efficacious in rat models of PD, it was unable to ameliorate parkinsonian signs in primates (Bédard and Boucher, 1989; Braun et al., 1986; Falardeau et al., 1988; Nomoto et al., 1985); this compound's partial agonist activity (Anderson and Jansen, 1990; Pifl et al., 1991) and poor blood brain barrier permeability have been offered as possible explanations for its inactivity. Since the original studies with SKF 38393, agonists with greater intrinsic activity (e.g., SKF 82958, CY 208-243 and A-77636) have been demonstrated to relieve parkinsonism in MPTP-treated primates or in humans (Akai et al., 1995; Blanchet et al., 1993; Kebabian et al., 1992; Luquin et al., 1994; Temlett et al., 1989). Additionally, the D1 receptor agonists A-77636, dihydrexidine and SKF 81297, but not SKF 38393 (Barone et al., 1987), promote dose-dependent contralateral rotation in monkeys rendered hemiparkinsonian via unilateral, intracarotid infusions of MPTP (Domino and Sheng, 1993a; Johnson et al., 1995; Vermeulen et al., 1993). The results of these more recent studies using D1 receptor agonists with full intrinsic activity certainly suggest that such compounds may be efficacious in treating PD.

In MPTP-lesioned monkeys given repeated L-dopa treatment, D2 receptor agonists, including quinpirole, bromocriptine and (+)-4-propyl-9-hydroxynaphthoxazine were found to alleviate parkinsonian symptoms but also to induce dyskinesias, as a consequence of L-dopa priming. In contrast, the D1 receptor agonists SKF 82958 and A-77636 also alleviated parkinsonian symptoms but had less propensity for inducing dyskinesias (Blanchet et al., 1993). Although somewhat controversial (Luquin et al., 1994), selective D1 receptor agonists may therefore produce fewer dyskinetic movements than D2 selective agonists in L-dopa-primed animals, and they appear even to potentiate the antiparkinsonian effects of selective D2 agonists (Blanchet et al., 1993; Gagnon et al., 1995; Rouillard et al., 1990) in MPTP-treated primates.

Luquin et al. (1996) recently reported that the behavioral response of MPTP-treated monkeys to the partial D1 agonist CY 208-243 decayed when animals were given four drug injections spaced 3 h apart. The decay was primarily due to a more rapid decline in the motor response to the drug relative to the first injection. A diminution in behavioral efficacy was not seen after (+)-PHNO administered under similar conditions, which suggests that the behavioral tolerance seen in MPTP-treated monkeys after repeated apomorphine injections may involve D1 receptor mechanisms (Luquin et al., 1993). In the present study, the first daily injection of A-86929 tended to elicit a greater and longer-lived response than either the second or the third injection across the 10-day treatment period. In monkeys, this difference was particularly apparent on the first day of treatment, where the magnitude of the rotation during the first 30 min after the first injection was greater than that after the second and third injections. By the tenth treatment day, however, there was no temporal difference in the behavioral response to the first, second or third daily injections, although the response did decay more rapidly after each injection on the tenth day than on the first day. Thus the reduction in the behavioral response seen after short-term, repeated D1 agonist treatment in both this study and the study by Luquin et al. (1996) may subsequently plateau during longer-term treatment. If so, it may be possible to overcome this reduction through dose adjustment.