Evaluation of the D3 dopamine receptor selective antagonist PG01037 on L-dopa-dependent abnormal involuntary movements in rats

Abstract

The D3 dopamine receptor selective antagonist PG01037 has been evaluated for the ability to attenuate L-dopa-associated abnormal involuntary movements (AIMs) in unilaterally lesioned male Sprague-Dawley rats, which is a model of L-dopa-dependent dyskinesia in patients with Parkinson's Disease. The intrinsic activity of PG01037 was determined using a) a forskolin-dependent adenylyl cyclase inhibition assay with transfected HEK 293 cells expressing either the human D2Long or D3 dopamine receptor subtype and b) an assay for agonist-associated mitogenesis. For the initial experiments, the 5-HT1A receptor selective partial agonist buspirone was used as a positive control to verify our ability to quantitate changes in total AIMs and AIMs minus locomotor scores. Subcutaneous (s.c.) administration of PG01037 was found to have minimal effect on AIMs score. However, it was observed that the in vivo efficacy of PG01037 increased when administered by intraperitoneal (i.p.) injection 15 min after L-dopa/benserazide administration, as compared to a 60 min, 30 min or 0 min pretreatment. It was also found that i.p. administration of PG01037 could inhibit involuntary movements after they had achieved maximum intensity. PG01037 was found to attenuate AIM scores in these animals in a dose dependent manner with IC(50) value equal to a) 7.4 mg/kg following L-dopa/benserazide administration (8 mg/kg each, i.p.) and b) 18.4 mg/kg following the administration of apomorphine (0.05 mg/kg, s.c.). However, PG01037 did not effectively inhibit SKF 81297-dependent abnormal involuntary movements. Rotarod studies indicate that PG01037 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 PG01037 did not dramatically attenuate the beneficial effects of L-dopa. These studies suggest that D3 dopamine receptor selective antagonists are potential pharmacotherapeutic candidates for the treatment of L-dopa-associated dyskinesia in patients with Parkinson's Disease.

Figure 1. Structure and pharmacologic profile of PG01037

The chemical structure and summary of our current available information on the pharmacological properties of PG01037 is shown. Mean values for the affinity measurements (Ki values) are expressed as nM. Mean values ± S.E.M. are provided for the human D2 and D3 dopamine receptor with n ≥ 3. The remaining affinity values were obtained from Novascreen through the NIDA Contract N01-DA-8-8839. PG01037 was tested at a concentration of 10 μM in 61 binding assays. For the assays in which PG01037 inhibited binding >50%, mean Ki values were obtained (n=2). Ki values for the binding to the low affinity state of human D2 and D3 dopamine receptors were obtained using competitive radioligand binding experiments with ¹²⁵I-IABN as the radiotracer (Luedtke et al., 2000). Maximal intrinsic activity was achieved using the test ligand and the full D2-like dopamine receptor agonist quinpirole at concentrations approximately 10× the Ki values (PG01037, 1000 nM for D2 and 10 nM for D3; quinpirole, 1000 nM for D2 and 100 nM for D3). 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).

Figure 2. AIM scores on day 20 of the 21 day dyskinetic induction protocol

The bar graph shows the total AIMs score (closed bars) and the AIMs minus locomotion scores (hatched bars) for 11 rats (A-K) on day 20 of the 21 day dyskinetic induction protocol. Rats received daily doses of L-dopa and benserazide at 8 mg/kg each. AIM scores were determined using the scoring protocol described by Dekundy and co-workers (2007). Scores are plotted in the increasing order of the total AIMs score for each animal. The mean and S.E.M. values for the total AIMs score are 41.2 ± 6.8 and for the AIMs minus the locomotor component 32.5 ± 7.0, which is 16.5% and 21.5% variability, respectively.

Figure 3. Dose response curve for the rat AIM scores with varying doses of L-dopa or benserazide

After the 21-day induction and stabilization of L-dopa-dependent involuntary movement was completed, a dose dependent evaluation of AIM scores was performed using the scoring protocol described by Dekundy and co-workers (2007). The left panel (A) shows the mean total AIM scores (●) ± S.E.M. and the total AIMs score minus the locomotor component (○) ± S.E.M. for varying dose of L-dopa. Mean values are for n = 11 animals. L-dopa (0 to 10 mg/kg) was administered by i.p. injection with a constant amount of benserazide (8 mg/kg). The right panel (B) shows the mean total AIM scores (■) ± S.E.M. and the total AIMs score minus the locomotor component (○) ± S.E.M. for varying dose of benserazide. Mean values are for n = 12 animals. Benserazide (0 to 16 mg/kg) was administered by i.p. injection with a constant amount of L-dopa (8 mg/kg).

Figure 4. Effect of administration of PG01037 or buspirone 60 minutes prior to L-dopa administration on rat AIMs score

(A) A comparison was made of the ability of PG01037 (10 mg/kg; n = 9) and buspirone (4 mg/kg; n = 11) to attenuate L-dopa-dependent AIMs in rats. Both drugs were administered i.p. 60 minutes prior to the L-dopa (8 mg/kg). This graph shows the mean total AIM scores (solid bars) ± S.E.M. and the total AIMs score minus the locomotor component (hatched bars) ± S.E.M. for PG01037 (65.9 ± 2.7 and 64.7 ± 2.9) and buspirone (57.9 ± 8.5 and 58.9 ± 8.1), respectively. Under these conditions both drugs statistically decreased the abnormal involuntary movements (p < 0.0001). (B) The variation in the total AIMs minus the locomotor component score as a function of time is shown using a 60 minute pretreatment time with 10 mg/kg PG01037 (●) or vehicle control (○). Each point is the summation of total AIMs minus the locomotor scores for 11 animals at each observation time point. This represents a mean 34.1 ± 8.0 (S.E.M.) percent reduction in the AIMs minus locomotor score over the observation time. Plots of the summation of the total AIMs score are essentially identical in shape with a 35.5 ± 8.4 percent reduction in the mean score.

Figure 5. The effect of varying the pre-administration time of PG01037 on the mean L-dopa-dependent AIM scores

(A) The effect of varying the pretreatment time for PG01037 (10 mg/kg) prior to L-dopa administration (8 mg/kg L-dopa and benserazide) on both the total AIMs score (solid bars) and the AIMs score minus the locomotor component (hatched bars) is shown. Values are expressed as the percent of the corresponding vehicle control values. The bar graph corresponds to the following values for the mean ± S.E.M. normalized total AIM scores and the AIM scores minus the locomotor component, respectively: a) 60 minutes pretreatment, 65.9% ± 2.7 and 64.4% ± 2.9 with n = 9, b) 30 minutes pretreatment, 51.3% ± 5.7 and 51.2% ± 4.8 with n = 8 and c) 0 minutes pretreatment, 42.4% ± 7.0 and 44.2% ± 7.0 with n = 10. PG01037 statistically decreased the abnormal involuntary movements (p < 0.0001) at all time points. (B) The variation in the total AIMs score as a function of time is shown using zero time pretreatment with 10 mg/kg PG01037 (●) or vehicle control (○). Animals were also injected (i.p.) with 8 mg/kg L-dopa and benserazide. Each point is the summation of total AIM scores for 11 animals at each observation time point. This represents a mean 59.0 ± 21.8 (S.E.M.) percent reduction in the total AIMs score over the observation time. Plots of the summation of the AIMs score minus the locomotor component are essentially identical in shape with a 56.9 ± 22.3 percent reduction in the AIMs minus locomotor component.

Figure 6. Concentration dependence analysis to determine the effect of doses of PG01037 on AIM scores

Unilaterally lesioned rats were injected (i.p.) with varying doses of PG01037 (0 to 25 mg/kg) followed immediately with a constant dose of L-dopa and benserazide (8 mg/kg each). The percent of the mean total AIMs score ± S.E.M. (n ≥ 8) relative to vehicle controls as a function of the dose of PG01037 is shown. The mean values ± S.E.M. for the normalized total AIMs values as a function of PG01037 concentrations are as follows: a) 1 mg/kg, 88.6 ± 9.1, b) 2.5 mg/kg, 86.6 ± 5.9, c) 5 mg/kg, 55.2 ± 7.6, d) 10 mg/kg, 42.4 ± 7.1 and e) 25 mg/kg, 17.3 ± 8.9. A graph of the percent change in the AIMs score minus the locomotor component as a function of the dose of PG01037 is essentially identical to the one shown in this figure. At doses of 1 and 2.5 mg/kg there is not a significant attenuation of the total AIMs score. However, at doses of 5, 10 and 25 mg/kg there was a significant reduction (p < 0.0001) of total AIM scores relative to the vehicle controls. The calculated IC₅₀ value was found to be 7.4 mg/kg using a reversible one site fit model constraining the maximum and minimum mean values to 100% (no test drug) and 0%, respectively.

Figure 7. Effect of post treatment of PG01037 on the temporal expression of L-dopa dependent AIMs in lesioned rats

(A) The variation in the total AIMs score as a function of time is shown using 10 mg/kg PG01037 (●) or vehicle control (○) fifteen minutes after L-dopa/benserazide (8 mg/kg each) administration. Each point is the summation of total AIM scores for 11 animals at each observation time point. This represents a mean 86.5 ± 3.9 (S.E.M.) percent reduction in the total AIMs score over the total observation time. The time (minutes) denotes that amount of time following the administration of the L-dopa/benserazide and the arrow marks the time of the administration of PG01037. Plots of the summation of the AIMs score minus the locomotor component are essentially identical in shape. (B) The variation in the total AIMs score as a function of time is shown using 10 mg/kg PG01037 (●) or vehicle control (○) thirty five minutes after L-dopa/benserazide (8 mg/kg each) administration. Each point is the summation of total AIM scores for 11 animals at each observation time point. This represents a mean 47.6 ± 8.2 (S.E.M.) percent reduction in the total AIMs score over the total observation time. The time (minutes) denotes the amount of time following the administration of the L-dopa/benserazide and the arrow marks that time of the administration of PG01037. Plots of the summation of the AIMs score minus the locomotor component are essentially identical in shape.

Figure 8. Effect of thirty five minute post-treatment of PG01037 on the L-dopa dependent individual AIMs in lesioned rats

A scatter gram of the summation of locomotor activity (Loco), axial dystonic movements (Axial), forelimb movement (Forelimb) and orolingual movements (Oro) for n = 12 animals is shown. Open circles (○) designate vehicle control values and solid circles (●) designate animals administered PG01037 35 minutes after the L-dopa administration and then scored at 50, 70, 90 and 100 minutes post L-dopa treatment. There was a 66.6%, 69.5%, 61.9% and 55.0% reduction in the locomotor, axial, forelimb and orolingual movements, respectively. The overall mean reduction in total AIMs score was 64.5 ± 35.1 (S.E.M.).

Figure 9. Effect of pretreatment times for PG01037 on apomorphine-dependent AIMs in lesioned rats

(A) Unilaterally lesioned rats were treated with PG01037 (10 mg/kg) at varying times (30, 20, 10 and 0 minutes) prior to the administration of apomorphine (0.05 mg/kg, s.c.). The mean normalized values for the total AIM scores ± S.E.M. are shown relative to the values obtained from animals injected with vehicle. The number of animals (n) for each pretreatment time is as follows: a) 0 minutes, n = 12, b) 10 minutes, n = 6, c) 20 minutes, n = 5 and d) 30 minutes, n = 6. Similar plots for the total AIMs score are essentially identical to those shown in this figure. Although there was no significant reduction of the total AIMs score at the 30 and 20 minute pretreatment times, there was significant reduction when PG01037 was administered at 10 minutes (p < 0.01) and 0 minute (p < 0.0001) prior to the administration of L-dopa. The normalized mean total AIMs activity for animals administered PG01037 at zero minutes prior to apomorphine administration is 61.9 ± 4.4% of vehicle control values. (B) The variation in the total AIMs score as a function of time is shown using zero time pretreatment with 10 mg/kg PG01037 (●) or vehicle control (○). Animals were also injected (s.c.) with 0.05 mg/kg apomorphine. Each point is the summation of total AIM scores for a total of 12 animals at each observation time point. This represents a mean 38.1 ± 4.4 (S.E.M.) percent reduction in the total AIMs score over the observation time. Plots of the summation of the AIMs score minus the locomotor component are essentially identical in shape with a 38.8 ± 4.1 percent reduction in the AIMs minus locomotor component.

Figure 10. Concentration dependence analysis to determine the effect of doses of PG01037 on apomorphine-induced total AIM scores

Unilaterally lesioned rats were injected (i.p.) with varying doses of PG01037 (0 to 25 mg/kg) followed immediately with a constant dose of apomorphine (0.05 mg/kg, s.c.). The percent of the mean total AIMs score ± S.E.M. (n ≥ 11) relative to vehicle controls as a function of the dose of PG01037 is shown. The mean values ± S.E.M. for the normalized total AIMs values as a function of PG01037 concentrations are as follows: a) 2.5 mg/kg, 90.8 ± 3.1, b) 10 mg/kg, 61.9 ± 4.4 and c) 25 mg/kg, 47.4 ± 8.4. The calculated IC₅₀ value was found to be 18.5 mg/kg using a reversible one site fit model constraining the maximum and minimum mean values to 100% (no test drug) and 0%, respectively.

Figure 11. SKF 81297 dependent induction of abnormal involuntary movements and the effect of PG01037 on SKF 81297 dependent abnormal involuntary movements in unilaterally lesioned animals

(A) The temporal plots for the induction of abnormal involuntary movements in unilaterally lesioned rats is shown. Summation of total AIM scores (n = 6) were determined at 10 minute intervals starting at t = 5 minutes. SKF 81297 was administered s.c. at the following doses: a) 0.5 mg/kg (●), b) 0.25 mg/kg (▲), c) 0.125 mg/kg (■) and d) 0.0625 mg/kg (○). (B) The effect of PG01037 (10 mg/kg, i.p.) on SKF 81297 (0.1 mg/kg, s.c.) on the total AIMs (tAIMs), AIMs minus locomotor activity (AIMs-loco) and locomotor activity (Loco) scores is shown. PG01037 or vehicle was administered 10 minutes prior to the administration of SKF 81297. The data are the normalized mean values ± S.E.M. for n = 9 animals. The normalized mean scores for each is as follows: a) total AIMs, 84.7 ± 7.3; b) AIMs-locomotor, 84.2 ± 7.0; c) locomotor score, 86.4 ± 9.0. The significance level for each of these scores compared to the vehicle control (control) is p ≥ 0.09.

Figure 12. Effect of the 5-HT1A antagonist WAY 100635 on the activity of PG01037

Unilaterally lesioned animals were used to test the effect of the 5-HT1A antagonist WAY 100635 on the ability of either buspirone or PG01037 to attenuate L-dopa-dependent abnormal involuntary movements. L-Dopa was administered at a dose of 8 mg/kg by i.p. injection. PG01037 and L-dopa were always administered simultaneously. WAY 100635 was always administered 10 minutes prior to the administration of test compounds (PG01037 or buspirone). Buspirone was administered 30 minutes prior to the administration of L-dopa. Data are 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 #1); B. L-dopa in the presence of buspirone (4 mg/kg); C. L-dopa in the presence of both buspirone (4 mg/kg) and WAY 100635 (1 mg/kg); D. L-dopa in the absence of any test compound (vehicle control #2); E. L-dopa in the presence of 3 mg/kg PG01037; F. L-dopa in the presence of both PG01037 (3 mg/kg) and WAY 100634 (1 mg/kg); G. L-dopa in the absence of test compound (vehicle #3); H. L-dopa in the presence of PG01037 (6 mg/kg); I. L-dopa in the presence of both PG01037 (6 mg/kg) and WAY 100634 (1 mg/kg). The administration of WAY 100635 (1 mg/kg) resulted in a 35% reduction in the attenuation of total AIM scores by buspirone (4 mg/kg). The administration of WAY 100635 (1 mg/kg) resulted in no reduction in the attenuation of total AIM scores by PG01037 at a dose of 3 mg/kg. The administration of WAY 100635 (1 mg/kg) resulted in a 15% reduction in the attenuation of total AIM scores by PG01037 at a dose of 6 mg/kg. The asterisk (*) denotes significant difference (p < 0.05) between the effects of a) buspirone versus vehicle control (A vs. B) (p <0.0001) and buspirone with WAY 100635 with the vehicle control (A vs. C) (p < 0.01), b) buspirone in the absence and presence of WAY 100635 (B vs. C) (p = 0.001), c) PG01037 at 6 mg/kg and vehicle (G vs. H) (p < 0.0001) and d) PG01037 and WAY 100635 and the vehicle control (G vs. I) (p =0.001). There was no significant effect of WAY 100635 on PG01037 at either 3 or 6 mg/kg (p > 0.1).

Figure 13. Effect of PG01037 on rotarod performance

The effect of PG01037 (10 mg/kg, i.p. injection) is shown for n = 12 animals. The data are presented as the mean values ± S.E.M. At the time of this experiment the lesioned animals were approximately 10 months old. Animals were acclimated to the rotarod apparatus once per week in the previous two weeks prior to training and testing. For this experiment the animals were fed approximately 1 hour prior to the morning session. The rotarod apparatus was accelerated from 0 to 44 rpm/minute in 90 seconds. For the training session (days 1 through 3), two test sessions were performed per day. One session was conducted in the morning (m) and one session was conducted in the afternoon (a). On days 1, 2 and 3 animals were not injected with test drug or vehicle. On days 4 and 6 rats were administered 10 mg/kg PG01037 i.p. and then evaluated at 30 and 60 minutes (30′ and 60′, respectively) post drug administration. On day 5 rats were tested after administration with a similar volume of vehicle (vehicle). No significant differences (p < 0.05) were found for a comparison of a) training session with vehicle controls, training session with test drug or b) matched vehicle controls with test drug.

Figure 14. Cylinder Test for PG01037

Unilaterally lesioned rats were tested for the percentage of forelimb preference (either right (R), left (L) or both (B) forelimbs) using the cylinder test. Animals were injected with either vehicle (control) or L-dopa/benserazide (L-dopa) (8 mg/kg each) in the absence or presence of PG01037 (L-dopa/PG01037). Animals were injected with L-dopa and test drug or vehicle simultaneously, then evaluated at 12 minutes (A) and 20 minutes (B) post injection. It was not possible to evaluate the animals at 20 minutes post drug administration when animals were given L-dopa/benserazide in the absence of PG01037 due to the intensity of the abnormal involuntary movements. Data are plotted as the mean values ± S.E.M. for seven animals (n = 7).