Evaluation of the D3 dopamine receptor selective agonist/partial agonist PG01042 on L-dopa dependent animal involuntary movements in rats

Abstract

The substituted 4-phenylpiperazine D3 dopamine receptor selective antagonist PG01037 ((E)-N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)but-2-enyl)-4-(pyridin-2-yl)benzamide) was reported to attenuate L-dopa-associated abnormal involuntary movements (AIMs) in unilaterally lesioned rats, a model of L-dopa-dependent dyskinesia in patients with Parkinson's Disease (Kumar et al., 2009a). We now report that PG01042 (N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)-4-(pyridin-3-yl)benzamide), which is a D3 dopamine receptor selective agonist for adenylyl cyclase inhibition and a partial agonist for mitogenesis, is also capable of attenuating AIMs scores. The intrinsic activity of PG01037 and PG01042 were determined using a) a forskolin-dependent adenylyl cyclase inhibition assay and b) an assay for agonist-associated mitogenesis. It was observed that the in vivo efficacy of PG01042 increased when administered by intraperitoneal (i.p.) injection simultaneously with L-dopa/benserazide (8 mg/kg each), as compared to a 60 min or 30 min pretreatment. PG01042 was found to attenuate AIM scores in these animals in a dose dependent manner. While PG01042 did not effectively inhibit SKF 81297-dependent AIMs, it inhibited apomorphine-dependent AIM scores. Rotarod studies indicate that PG01042 at a dose of 10 mg/kg did not adversely affect motor coordination of the unilaterally lesioned rats. Evaluation of lesioned rats using a cylinder test behavioral paradigm indicated that PG01042 did not dramatically attenuate the beneficial effects of L-dopa. These studies and previously published studies suggest that both D3 dopamine receptor selective antagonists, partial agonists and agonists, as defined by an adenylyl cyclase inhibition assay and a mitogenic assay, are pharmacotherapeutic candidates for the treatment of L-dopa-associated dyskinesia in patients with Parkinson's Disease.

Figure 1. Structure and Pharmacologic Profiles of PG01037 and PG01042

The chemical structure and summary of our current available information on the pharmacological properties of PG01042 is shown. Mean values for the affinity measurements (Ki values) are expressed as nMolar. For the cyclase inhibition assay the intrinsic activity was evaluated using the test ligand and the full D2-like dopamine receptor agonist quinpirole at concentrations approximately 10x the Ki values (PG01042, 200 nM for D2 and 10 nM for D3; PG01037, 1000 nM for D2 and 10 nM for D3; quinpirole, 1000 nM for D2 and 100 nM for D3). The maximum inhibition for quinpirole was 89.3 ± 1.1 (5) for human D2 receptors and 35.0 ± 3.6 (5) for D3 receptors. The percent maximum mitogenesis is based upon dose response curves (one concentration per decade) using a maximum of 10⁻⁵ M test drug performed by the Division of Treatment Research and Development (DTRD) of NIDA. The numerical values are the mean ± the S.E.M. and the number in the parentheses is the number of independent experiments (n). The synthetic methods for these two compounds was previously reported (Grundt et al., 2005).

Figure 2. The Effect of Varying the Pretreatment Time of PG01042 on the Mean L-Dopa Dependent AIMs Scores

The effect of varying the pretreatment time for PG01042 (10 mg/kg) prior to L-dopa administration (8 mg/kg L-dopa and benserazide) on the total AIMs score is shown. Values are expressed as the percent total AIMs relative to vehicle control values. The bar graph corresponds to the following values for the mean ± S.E.M. normalized total AIMs scores: a) 60- minutes pretreatment, 68.0 ± 8.6% and b) 0-minutes pretreatment, 29.4 ± 6.1% with n = 12 in both cases. The asterisk (*) denotes significant difference (defined as p ≤ 0.05) between the effects of a) PG01042 (10 mg/kg) 60 minute pretreatment versus vehicle control (F(1,18) = 10.1, p < 0.05 for n = 10), b) PG01042 (10 mg/kg) 0 minute pretreatment versus vehicle control (F(1, 22) = 86.6, p < 0.001, n = 12) and c) PG01042 (10 mg/kg) 60 minute pretreatment versus PG01042 (10 mg/kg) 0 minute pretreatment (F(1, 20) – 13.2, p < 0.002, n = 10).

Figure 3. Dose Response Curve for the Attenuation of Total AIMs Score by PG01042

Unilaterally lesioned rats were injected (i.p.) with varying doses of PG01042 (0 to 10 mg/kg) followed immediately with a constant dose of L-dopa and benserazide (8 mg/kg each). The normalized mean total AIMs score ± S.E.M. (n = 12; normalized to 100) relative to vehicle controls as a function the dose of PG01042 is shown. A. The variation in the total AIMs score as a function of time (temporal plot) is shown using zero time pretreatment with 10 mg/kg PG01042 (▲), 3 mg/kg PG01042 (■), 1 mg/kg PG01042 (●) and for the vehicle control (❍). Unilaterally lesioned animals were also injected i.p. with 8 mg/kg L-dopa and benserazide. Each point is the summation of total AIMs scores for a total of 12 animals at each observation time point. The calculated IC₅₀ value for this data was obtained using either a (B) linear regression analysis which included a data point at a dose of 0 mg/kg which is essentially equal to the vehicle control (IC₅₀ = 6.6 mg/kg) or (C) a one site fit based upon the law of mass action in which the curve is constrained to values of 0 (infinitely high dose) and 100% (vehicle control) (IC50 = 5.6 mg/kg). For a) 1 mg/kg PG01042, this represents a mean 8.4 ± 7.3% reduction, b) 3 mg/kg PG01042, this represents a mean 25.2 ± 4.4 percent reduction c) 10 mg/kg PG01042, this represents a mean 70.5 ± 6.1% reduction, in the total AIMs score over the observation time.

Figure 4. Effect of Zero Minute Pretreatment of PG01042 on the Temporal Expression of Apomorphine-Dependent AIMs in Lesioned Rats

Unilaterally lesioned rats were injected (i.p.) with vehicle or PG01042 (10 mg/kg) followed immediately with apomorphine (0.05 mg/kg, s.c.). (Left panel) The temporal plots of the summation of total AIM scores is shown as a function of time for animals (n = 12) administered either the vehicle control (❍) or PG01042 (●). (Right panel) The percent of the mean total AIMs score ± S.E.M. (n = 12) relative to vehicle controls (control) as a function the dose of PG01042 (PG42) is shown. The mean value ± S.E.M. for the normalized total AIMs values as a function of PG01042 administration are shown for a) 0 to 60 minutes, b) 0 to 40 minutes and b) 50 to 60 minutes. A significant reduction in total AIM scores for the 0 to 60 minute time period (F(1,22) = 4.4, p < 0.05) and for the 50 to 60 minute time period (F(1,22) = 9.7, p < 0.05) is designated with an asterisk (*).

Figure 5. Effect of Zero Minute Pretreatment of PG01042 on the Temporal Expression of SKF 81297-Dependent AIMs in Lesioned Rats

Unilaterally lesioned rats were injected (i.p.) with PG01042 (●, 10 mg/kg) followed immediately with a constant dose of SKF 81297 (0.05 mg/kg, s.c.). The percent of the mean total AIMs score ± S.E.M. (n = 12) relative to vehicle control (○) as a function of the dose of PG01042 is shown. The mean reduction ± S.E.M. for the normalized total AIMs values using PG01042 was 10 mg/kg, 8.0 ± 6.8% reduction but significance was not achieved (F(1,22) = 0.72, p = 0.40).

Figure 6. Effect of the 5-HT1A Antagonist WAY 100635 on the Activity of PG01042, PG01037 and Buspirone

Unilaterally lesioned animals were used to test the effect of the 5-HT1A antagonist WAY 100635 on the ability of either PG01042 (A left) or PG01037 (A right) to attenuate L-dopa-dependent abnormal involuntary movements. L-dopa was administered at a dose of 8 mg/kg by i.p. injection. PG01042 or PG01037 and L-dopa were administered simultaneously. WAY 100635 was always administered 10 minutes prior to the administration of test compounds (PG01042 or PG01037). Data presented is total AIMs score normalized to 100 ± S.E.M. The conditions of the experiment are as follows: a) L-dopa in the absence of any test compound (control 1); b) L-dopa in the presence of PG01042 (3 mg/kg); c) L-dopa in the presence of both PG01042 and WAY 100635 (1 mg/kg); d) L-dopa in the absence of any test compound (control 2); e) L-dopa in the presence of PG01037 (3 mg/kg); and f) L-dopa in the presence of both PG01037 (3 mg/kg) and WAY 100635 (1 mg/kg). The administration of PG01042 or PG01037 with WAY 100635 resulted in a significant reduction in total AIM score compared to the vehicle control (F(1,22) = 14.4, p < 0.05 and F(1, 22) = 6.4, p < 0.05, respectively with n = 12). While the administration of WAY 100635 (1 mg/kg) resulted in a 10% reduction in the attenuation of total AIMs scores by PG01042, this difference did not achieve significance (F(1, 22) = 1.5, p > 0.05). In addition, the administration of WAY 100635 (1 mg/kg) resulted in no reduction in the attenuation of total AIMs score by PG01037 (3 mg/kg) (F(1, 18) = 0.008, p > 0.5). In Figure 6A the asterisk (*) denotes significant difference between vehicle control and test drugs. (B) Unilaterally lesioned animals were also used to test the effect of WAY 100635 on the ability of buspirone to attenuate L-dopa-dependent abnormal involuntary movements. L-dopa was administered at a dose of 8 mg/kg by i.p. injection. Buspirone was administered 30 minutes prior to administration of L-dopa. WAY 100635 was administered 10 minutes prior to the administration of test compounds (40 minutes prior to L-dopa administration). Data is presented as the total AIMs score normalized to 100 ± the normalized S.E.M. The conditions of the experiment are as follows: a) L-dopa in the absence of any test compound (vehicle control); b) L-dopa in the presence of buspirone (4 mg/kg); and c) L-dopa in the presence of both buspirone (4 mg/kg) and WAY 100635 (1 mg/kg). The administration of buspirone resulted in a significant reduction in total AIM scores compared to vehicle control (F(1,18) = 55.4, p < 0.001, n = 10). The administration of WAY 100635 (1 mg/kg) resulted in a 35% reduction in a significant attenuation of total AIMs scores by buspirone (4 mg/kg) (F(1,18) = 15.4, p < 0.01, n = 10). In Figure 6B the asterisk (*) denotes significant difference (p < 0.001) between the effects of buspirone versus vehicle control. The number symbol (#) denotes a significant difference (p < 0.005) between the effects of buspirone in the absence and presence of WAY 100635.

Figure 7. Effect of PG01042 on Rotarod Performance

A rotarod apparatus was used to assess the effect of PG01042 on motor coordination and agility of unilaterally lesioned rats in the presence or absence of test compound. The data is presented as the mean amount of time the animal remained on the rotarod before falling (Latency to Fall; in seconds) ± S.E.M. The acceleration conditions for the rotarod test was 0 to 44 rpms in 90 seconds. This experiment was performed on 6 consecutive days. Days 1 through 3 were training sessions that were conducted in the morning and afternoon (Training day 1–3, m and a, respectively). After the training session was completed, lesioned rats were administered PG01042 (i.p.) at a dose of 10 mg/kg for the inhibition of total AIMs score and evaluated at 30 minutes (30′) and 60 minutes (60′) post drug administration on days 4 and 6. On day 5 the vehicle was administered and animals were evaluated at 30 minutes (30′) or 60 minutes (60′) post vehicle administration. An ANOVA with repeated measures was used for the statistical comparison of the effect of the mean values of test drug (day 4 and day 6) to its vehicle control (day 5) indicated no statistical significant difference in latency to fall (p ≥ 0.10).

Figure 8. Effects of PG01042 on the Cylinder Test

The data shown is the mean percent preferential usage of either the left (L), right (R) or both (B) forelimbs ± S.E.M. For this experiment the right forelimb is the compromised limb, contralateral to the lesion. Unilaterally lesioned animals were administered either a) saline (control), b) L-dopa and benserazide (L-dopa) at a dose of 8 mg/kg each, c) L-dopa and benserazide with PG01042 (L-dopa/PG01042) at a dose of 10 mg/kg. Animals were evaluated at 12 minutes (left) and (b) 20 minutes (right) post injection. (Left) At the 12 minute time point, animals were evaluated for a total of 5 minutes then returned to their home cage for 3 minutes. At the 12 minute time point there was a significant increase in the usage of the right forelimb following that administration of L-dopa (F(1,12) = 696, p < 0.001, n = 7) or L-dopa with PG01042 (F(1,12) = 36.7, p < 0.001, n = 7), compared to vehicle control animals. In addition, there was a marginal attenuation of right forelimb usage by PG01042 (F(1,12) = 3.85, p > 0.5). (Right) At the 20 minute time point, animals were further evaluated until >13 touches/rat were observed. After administration of L-dopa + vehicle and L-dopa + PG01042 contralateral forelimb usage improved significantly from 2.8% (in presence of saline and vehicle) to 82% (mean percentage use of right paw ± S.E.M.) (F(1,14) = 253, p < 0.001). At 20 minutes post L-dopa/benserazide administration data on forelimb usage (in the absence of PG01042) was not obtainable due to the intensity of the L-dopa-dependent AIMs.