Evaluation of D2 and D3 dopamine receptor selective compounds on L-dopa-dependent abnormal involuntary movements in rats

Abstract

A panel of novel D2 and D3 dopamine receptor selective antagonists, partial agonists and full agonists have been evaluated for the ability to attenuate L-dopa-associated abnormal involuntary movements (AIMs) in 6-hydroxydopamine (6-OHDA) unilaterally lesioned male Sprague Dawley rats, which is an animal model of L-dopa-induced dyskinesia (LID). LID is often observed in patients with Parkinson's Disease following chronic treatment with L-dopa. The intrinsic activity of these dopaminergic compounds was determined using a forskolin-dependent adenylyl cyclase inhibition assay with transfected HEK 293 cells expressing either the human D2Long or D3 dopamine receptor subtype. For the initial experiments the 5-HT1A receptor selective partial agonist buspirone was used to verify our ability to quantitate changes in total AIMs and AIMs minus locomotor scores. Two D2 dopamine receptor selective antagonists, SV 156 and SV 293, were evaluated and found to minimally attenuate AIM scores in these animals. Four members of our WC series of D3 dopamine receptor selective compounds of varying intrinsic activity at the D3 dopamine receptor subtype, WC 10, WC 21, WC 26 and WC 44, were also evaluated and found to attenuate AIM scores in a dose dependent manner. The in vivo efficacy of the compounds increased when they were administered simultaneously with L-dopa, as compared to when the compounds were administered 60 min prior to the L-dopa/benserazide. It was also found that the D3 receptor antagonist WC 10 could inhibit the involuntary movements after they had achieved maximum intensity. Unlike the D1-like dopamine receptor selective agonist SKF 81297 and the D2-like dopamine receptor agonist bromocriptine which can precipitate abnormal involuntary movements in these unilaterally lesioned animals, abnormal involuntary movements were not observed after administration of our D3 receptor selective agonist WC 44. In addition, we evaluated the effect of these four D3 dopamine receptor selective compounds for their effect on a) spontaneous locomotion and b) coordination and agility using a rotarod apparatus. We also used a cylinder test to assess the effect of L-dopa on spontaneous and independent use of each of the rat's forelimbs in the presence or absence of test compound. The results of these studies suggest that substituted phenylpiperazine D3 dopamine receptor selective compounds are potential pharmacotherapeutic agents for the treatment of L-dopa-associated dyskinesia in patients with Parkinson's Disease.

Figure 1. Comparison of AIM Scores Before and Sixty Minutes After Feeding Animals

This figure shows a comparison of the effect feeding on the AIM scores of unilaterally lesioned rats administered L-dopa. Animals were administered a mixture of L-dopa (8 mg/kg) and benserazide (8 mg/kg) in sterile saline (9 g/L NaCl in water) by i.p. injection. Rats were either fed approximately 24 hours prior to L-dopa administration (Hungry, ●) or fed (approximately 30–40 grams of rat chow pellets) 1 hour prior to L-dopa administration (Fed, ▲).

Figure 2. Structure and Pharmacological Profile of SV Series D2 Receptor Subtype Selective Compounds

The dissociation constants (nMolar values) for the binding of the D2 receptor selective antagonists, SV 156 and SV 293, to human D2long, D3 and D4.4 dopamine receptors expressed in HEK 293 cells is presented as the mean ± S.E.M. for n > 3 experiments. Values were derived from competitive radioligand binding experiments using ¹²⁵I-IABN as the radioligand. Affinity and log P values are taken from Vangeravong et al., 2006 and the descriptors in parentheses are how these compounds are designated in that publication.

Figure 3. Effect of D2 Dopamine Receptor Subtype Selective Antagonists on L-Dopa-Dependent Abnormal Involuntary Movements

The effect of buspirone (4 mg/kg) and the novel D2 dopamine receptor selective antagonists SV 293 and SV 156 (10 mg/kg) on (A) AIMs minus locomotor score and (B) locomotor score is shown. Each of the test drugs were administered i.p. 60 minutes prior to L-dopa administration. For this experiment L-dopa and benserazide were used at a dose of 8 mg/kg each. Values are the normalized mean scores ± S.E.M. (n ≥ 8). For buspirone the significance level for all three values is p ≤ 0.002. For the SV compounds the significance level is p > 0.01 for the total AIMs score, p > 0.01 for AIMs minus locomotor activity and p > 0.07 for the locomotor score. Data is presented as the summation of the total AIM scores for eight (n = 8) animals as a function of time.

Figure 4. Structure and Pharmacological Profile of the WC Series D3 Receptor Subtype Selective Compounds

The dissociation constants (nMolar values) for the binding of the D3 receptor selective compounds, WC 10, WC 21, WC 26 and WC 44, to human D2long, D3 and D4.4 dopamine receptors expressed in HEK 293 cells is presented as the mean ± S.E.M. for n ≥ 3 experiments. Values were derived from competitive radioligand binding experiments using ¹²⁵I-IABN as the radioligand (Luedtke et al., 2000). Affinity and log P values are taken from Chu et al., 2005 and the descriptors in parentheses are how these compounds are designated in that publication.

Figure 5. Intrinsic Activity of D3 Receptor Selective Compounds Using an Inhibition of Forskolin-Dependent Stimulation Adenylyl Cyclase Assay

The intrinsic activity of the D3 dopamine receptor compounds at both human D2 (solid black) and D3 (hatched) receptors is shown as the mean values ± S.E.M (n ≥ 3). Values were obtained using stably transfected HEK 293 expressing either the human D2 or D3 dopamine receptor subtype. All values were compared to the observed effect using the D2-like dopamine receptor full agonist quinpirole at a concentration ≥ 10x the Ki value obtained using radioligand binding studies. A whole cell adenylyl cyclase assay was used in which quinpirole and the tested compounds were evaluated for the ability to inhibit forskolin-dependent stimulation of adenylyl cyclase activity.

Figure 6. Effect of D3 Dopamine Receptor Subtype Selective Compounds on L-Dopa-Dependent Involuntary Movements with a 60 Minute Pretreatment Time

The effect of buspirone (4 mg/kg) and the novel D3 dopamine receptor selective compounds, WC 10, WC 21, WC 26 and WC 44 (10 mg/kg) on (A) AIMs minus locomotor score and (B) locomotor score is shown. Each of the test drugs were administered i.p. 60 minutes prior to L-dopa/benserazide (8 mg/kg each) administration. Values are the normalized mean scores ± S.E.M. The number of independent experiments used to calculate the mean values is n > 8 for each compound except WC 21 (n = 5). For WC 44 the significance level for the total AIMs and the AIMs minus locomotor activity is p ≤ 0.001 and for the locomotor activity it is p < 0.01. For WC 10, WC 21 and WC 26 the significance level for each of the scores is p < 0.0001.

Figure 7. Comparison of the Effect of Zero and Sixty Minute Pretreatment Time for WC 44, WC 10, WC 26 and WC 21 on the Attenuation of AIM Scores

The effect of novel D3 dopamine receptor selective compounds, WC 44, WC 10, WC 26 and WC 21 (10 mg/kg, i.p.) on total AIMs score is shown. Each of the test drugs were administered i.p. at either 60 minutes prior (solid bars) or simultaneously (zero minutes: hatched bars) to L-dopa/benserazide i.p. administration (8 mg/kg each). Values are the normalized mean scores ± S.E.M relative to vehicle controls (Control) normalized to 100. The number of independent experiments used to calculate the mean values is n > 8 for each condition except for WC 26 at 60 minutes (n = 5). There is a significance level between the vehicle control values and the 60 minute pretreatment is p ≤ 0.01 and the level of significance between the control values and the 0 minute pretreatment time is p ≤ 0.0001.

Figure 8. Dose Response Dependence of WC 10 for the Attenuation of Total AIM Scores

A. Determination of IC50 value: Unilaterally lesioned rats were injected (i.p.) with varying doses of WC 10 (0 to 10 mg/kg) followed immediately with a constant dose of L-dopa and benserazide (8 mg/kg each). The percent of the normalized mean total AIMs score ± S.E.M. (n ≥ 8) relative to vehicle controls as a function of the dose of WC 10 is shown. The mean values ± S.E.M. for the normalized total AIMs values as a function of WC 10 concentrations are as follows: a) 1 mg/kg, 107 ± 11.4, b) 3.0 mg/kg, 92.6 ± 8.2, c) 6 mg/kg, 61.3 ± 9.2 and d) 10 mg/kg, 9.5 ± 4.7. A graph of the percent change in the AIMs score minus the locomotor component as a function of the dose of WC 10 is essentially identical to the one shown in this figure. The calculated IC₅₀ value for this data was found to be 6.6 mg/kg using a linear regression analysis (r² = 0.99). B. Temporal plot: The variation in the total AIMs score as a function of time is shown using zero time pretreatment with an i.p. injection of WC 10 at 10 mg/kg (□), 6 mg/kg (▲), 3 mg/kg (■), 1 mg/kg (●) or vehicle control (❍). 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 8 animals at each observation time point for each dose of WC 10. However, the temporal plot for the vehicle control curve is the mean value for all four experiments.

Figure 9. Ability of WC 10 to Reverse the L-Dopa Associated Abnormal Involuntary Movements in Unilaterally Lesioned Rats

The temporal variation in the total AIMs score as a function of time is shown using 10 mg/kg WC 10 (●) or vehicle control (❍) thirty five minutes after l-dopa/benserazide (8 mg/kg each) administration. Each point is the summation of total AIMs score for a total of 10 animals at each observation time point. The time (minutes) denotes the amount of time after the administration of the L-dopa/benserazide and the arrow marks the time of the administration of WC 10.

Figure 10. Dose Response Dependence of WC 44 for the Attenuation of Total AIM scores

A. Determination of IC50 value: Unilaterally lesioned rats were injected (i.p.) with varying doses of WC 44 (0 to 10 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 ≥ 9) relative to vehicle controls as a function the dose of WC 44 is shown. The mean values ± S.E.M. for the normalized total AIMs values as a function of WC 44 concentrations are as follows: a) 1 mg/kg, 91.4 ± 5.5, b) 3.0 mg/kg, 68.8 ± 10.1, c) 6 mg/kg, 41.4 ± 7.4 and d) 10 mg/kg, 14.6 ± 5.5. The calculated IC₅₀ value for this data was found to be 5.5 mg/kg using a linear regression analysis (r² = 0.98). B. Temporal plot: The variation in the total AIMs score as a function of time is shown using zero time pretreatment with an i.p. injection of WC 44 at 10 mg/kg (□), 6 mg/kg (▲), 3 mg/kg (■), 1 mg/kg (●) or vehicle control (❍). Animals were also injected (i.p.) with 8 mg/kg L-dopa and benserazide. Each point is the summation of total AIMs score for a total of 9 animals at each observation time point for each dose of WC 44. The temporal plot for the vehicle control curve is the mean value for all four experiments.

Figure 11. The Effect of Co-Administration of WC 10 and WC 44 on Total AIM Scores

A comparison of the effect of combining WC 10 and WC 44 in the total AIMs score is shown relative to the administration of each compound alone. Each drug was administered at a dose of 3 mg/kg i.p. L-dopa/benserazide was then immediately administered at a dose of 8 mg/kg, each. The normalized AIM scores ± S.E.M. were a) WC 10, 92.6 ± 8.2 (n = 10; p = 0.6), b) WC 44, 68.8 ± 10.1 (n = 10; p = 0.02) and c) WC 10 plus WC 44, 31.4 ± 7.0 (n = 9; p ≤ 0.0001).

Figure 12. Dose Response Dependence of WC 21 for the Attenuation of Total AIM Scores

A. Determination of IC50 value: Unilaterally lesioned rats were injected (i.p.) with varying doses of WC 21 (0 to 10 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 (n ≥ 8) relative to vehicle controls as a function of the dose of WC 21 is shown. The mean values ± S.E.M. for the normalized total AIMs values as a function of WC 21 concentrations are as follows: a) 1 mg/kg, 81.0 ± 7.7, b) 3.0 mg/kg, 51.2 ± 9.7, c) 6 mg/kg, 24.9 ± 7.0 and d) 10 mg/kg, 1.8 ± 1.5. The calculated IC₅₀ value for this data was found to be 3.8 mg/kg using a linear regression analysis (r² = 0.96). B. Temporal plot: The variation in the total AIMs score as a function of time is shown using zero time pretreatment with i.p. injection of WC 21 at 10 mg/kg (□), 6 mg/kg (▲), 3 mg/kg (■), 1 mg/kg (●) or vehicle control (❍). Animals were also injected (i.p.) with 8 mg/kg L-dopa and benserazide. Each point is the summation of total AIMs score for a total of 8 animals at each observation time point for each dose of WC 21. The temporal plot for the vehicle control curve is the mean value for all four experiments.

Figure 13. Dose Response Dependence of WC 26 for the Attenuation of Total AIM Scores

A. Determination of IC50 value: Unilaterally lesioned rats were injected (i.p.) with varying doses of WC 26 (0 to 10 mg/kg) followed immediately with a constant dose of L-dopa and benserazide (8 mg/kg each). The percent of the mean total AIM score (n ≥ 10) relative to vehicle controls as a function of the dose of WC 26 is shown. The mean values ± S.E.M. for the normalized total AIMs values as a function of WC 26 concentrations are as follows: a) 1 mg/kg, 86.5 ± 4.9, b) 3.0 mg/kg, 69.7 ± 6.3, c) 6 mg/kg, 45.0 ± 9.5 and d) 10 mg/kg 21.8 ± 4.7. The calculated IC₅₀ value for this data was found to be 5.8 mg/kg using a linear regression analysis (r² = 0.99). B. Temporal plot: The variation in the total AIMs score as a function of time is shown using zero time pretreatment with an i.p. injection of WC 26 at 10 mg/kg (□), 6 mg/kg (▲), 3 mg/kg (■), 1 mg/kg (●) or vehicle control (❍). Animals were also injected (i.p.) with 8 mg/kg L-dopa and benserazide. Each point is the summation of total AIMs score for a total of 10 animals at each observation time point for each dose of WC 26. The temporal plot for the vehicle control curve is the mean value for all four experiments.

Figure 14. Effects of WC 10, WC 44, WC 26 and WC 21 on spontaneous locomotor activity of non-lesioned rats

To investigate the effect of the compounds on spontaneous locomotor activity, the mean difference in crossovers (± SEM) from the vehicle-pretreated controls is illustrated. This difference value was calculated by subtracting the average number of crossovers of the respective vehicle group from the number of crossovers of individual subjects in the WC10 (A), WC44 (B), WC26 (C) and WC21 (D) dosage groups. The horizontal line provides a reference point for controls relative to the drug dosage groups. Doses of 3 (□) and 10 (●) mg/kg, IP were examined for each compound. For a more direct comparison to controls, the average number of crossovers of the combined control groups (◇)is shown across time in 10-min periods (E). Asterisks (*) designate a difference from vehicle controls and a plus sign (+) designates a difference from the 3 mg/kg dosage group (p < 0.05).

Figure 15. Rotarod evaluation of WC Series Compounds

A rotarod apparatus was used to assess the effect of the WC series compounds 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 were 0 to 44 rpms in 90 seconds. This experiment was performed on 13 consecutive days. Days 1 through 3 were training sessions that were conducted either in the morning or afternoon (T1m through T3a, respectively). After the training session was completed, lesioned rats were administered a test compound (i.p.) at a dose equal to the IC50 value for the inhibition of total AIMs score and evaluated at 30 minutes (30′) or 60 minutes (60′) post drug administration: a) agonist WC 44, 5.5 mg/kg, day 4 (WC44 30′ and WC 44 60′), b) partial agonist WC 26, 5.8 mg/kg, day 6 (WC26 30′ and WC26 60′), c) partial agonist WC 21, 3.8 mg/kg, day 8 (WC21 30′ and WC21 60′) and d) antagonist WC 10, 6.6 mg/kg, day 10 (WC10 30′ and WC10 60′). Haloperidol (hal) was included as a positive control (5 mg/kg, day 12 (hal 30′ and hal 60′). On the days following the administration of test compound (days 5, 7, 9, 11 and 13), the appropriate matched vehicle (vc) was administered and animals were evaluated at 30 minutes (30′) or 60 minutes (60′) post vehicle administration (vc 30′ and vc 60′). An ANOVA with repeated measures was used for the statistical evaluation of the data using Systat software. An asterisk (*) indicates significance (p ≤ 0.05) when comparing the effect of the mean values for the test drug to either a) its matched vehicle control or b) the mean of the training sessions. A number symbol (#) signifies significance (p < 0.001) for the comparison of the values for the test drug (here haloperidol) compared to values for the matched vehicle control. The dotted line parallel to the x-axis is the mean value (34.60 seconds) for the training session.

Figure 16. Cylinder Test Evaluation of the Effect of the WC Series compounds at a dose equal to the IC₅₀ Values

The cylinder test was performed to assess the independent use of each forelimb in the context of the rat’s natural rearing/exploratory behavior. The data shown is the mean percent preferential usage of either the left (L), right (R) or both (B) forelimbs ± S.E.M. 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 the antagonist WC 10 (L-dopa/WC 10) at a dose equal to the calculated IC₅₀ value (6.6 mg/kg), d) L-dopa and benserazide with the agonist WC 44 (L-dopa/WC 44) at a dose equal to the calculated IC₅₀ value (5.5 mg/kg), e) L-dopa and benserazide with the partial agonist WC 21 (L-dopa/WC 21) at a dose equal to the calculated IC₅₀ value (3.8 mg/kg) or f) L-dopa and benserazide with the partial agonist WC 26 (L-dopa/WC 26) at a dose equal to the calculated IC₅₀ value (5.8 mg/kg). Animals were evaluated at (A) 12 minutes and (B) 20 minutes post injection. At 20 minutes post L-dopa/benserazide administration data on forelimb usage was not obtainable due to the intensity of the L-dopa-dependent abnormal involuntary movements. At the 12 minute time point, animals were evaluated for a total of 5 minutes and then returned to their home cage for 4 minutes. At the 20 minute time point animals were further evaluated for 5 minutes. The number of independent experiments is twelve (n = 12) for all drugs tested, except for WC 21 where n = 11.