Screening the Novel Dopamine D3 Receptor Agonist SK609 in Rodent
Model of Parkinson’s Disease
1
Huettner ,
2
Simms ,
1
Xu ,
1
Kortagere
Daniel P.
Sherise L.
Wei
and Sandhya
1Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, PA, USA;
2Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, PA, USA
INTRODUCTION
RESULTS
Independent Effects of L-dopa and SK609 on Initiation Time in Stepping Tests
Rotation in PD Rats after 0.25 mg/ml Apomorphine Injection
• Parkinson’s disease (PD) is the second most common age related
neurodegenerative disorder after Alzheimer’s disease. The etiology of PD is
not well understood but the motor symptoms are associated with loss of
dopaminergic neurons in the nigro-striatal regions of the brain.
• However therapy with L-dopa to improve levels of dopamine lead to L-dopa
induced dyskinesia (LID), which is characterized by abnormal involuntary
movements (AIMs). Analysis of brain tissues from rodent and non-human
primate models of PD suggests a key role for Dopamine D3 receptors in PD
and LID (1,2).
• We hypothesize that aberrant expression of D3 receptors coupled with
desensitization of D3 receptors by agonists such as dopamine could
significantly contribute to the development of LID.
• Thus, a selective D3 agonist that does not induce desensitization of D3
receptors in vivo could modulate the motor symptoms of PD and abrogate
the symptoms of LID.
Figure 5: Results from apomorphine-induced tasks, comparing mean outcomes between control (before
surgery), sham, and PD rats (6-OHDA lesioned) (n=4). The PD rats averaged a 27-fold increase in rotation
compared to the controls (*p-value = 0.0006).
Figure 7: Compares the effects of L-dopa and SK609 on initiation time in the stepping tests between left and right paws of
the PD rats tests (n=4). Left paw serves as a control while the right paw exhibits PD phenotype. The ratio of right paw to
left paw (RP:LP) for untreated, L-dopa treated, and SK609 treated, were 4.42, 3.49, and 1.70, respectively (*p-value = 0.02;
**p-value = 0.007; ***p-value = 0.01).
Effects of adjuvant drug therapy (L-dopa + SK609) in AIMs Test
Independent Effects of L-dopa and SK609 on Total Time Time in Stepping Tests
DESIGN AND CHARACTERIZATION OF SK609
• Designed using Hybrid structure based method (3) guided by conformational
sampling (4)
• SK609 had greater than 10,000 fold selectivity for D3 receptors and minimal
effect for D2 receptors (4)
Figure 1: Representative western blots showing 1 μM SK609 induced phosphorylation of
ERK1 and ERK2 proteins in HEK-D2 and HEK-D3 cells. Dopamine (DA) and D3 agonist
PD128907 were used as positive controls.
Figure 8: Compares the effects of L-dopa and SK609 on total stepping time between left and right paws of the PD rats test
(n=4). Left paw serves as a control while the right paw exhibits PD phenotype. The ratio of right paw to left paw (RP:LP) for
untreated, L-dopa treated, and SK609 treated, were 3.15, 2.42, and 1.22, respectively (*p-value = 0.02; **p-value = 0.004;
***p-value = 0.01).
Figure 6: PD rats with treated with a standard dose of L-dopa (6mg/kg) with varying doses of SK609 ranging from
4mg/kg to 20mg/kg. The decreasing AIMs scores with an increasing SK609 dosage reflect a dose-dependent
relationship (*p-value = 0.00009; **p-value = 0.00005), n=4.
CONCLUSION AND FUTURE DIRECTIONS
METHODS
Surgery
Experimental Timeline
• Lesioned rats unilaterally microinjected with 6-OHDA (8µg/2µL in 0.9% sterile normal saline with 0.01% ascorbic acid)
• Sham rats unilaterally microinjected with 0.9% saline with 0.01% ascorbic acid
• Left intra-striatum injection coordinates: AP: 1.0mm; L: 3.2mm; DV: 4.6mm
Rotation Test
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•
•
Two weeks after surgery, the rats were administered apomorphine to be tested in the “apomorphine induced rotation” test
to confirm experimental PD model symptoms
Each rat was administered apomorphine subcutaneously (0.25 mg/kg) and then placed into the rotation testing chamber
(30cm diameter Plexiglas cylinder) for 60 minutes (Figure 3)
Each test was video recorded and analyzed by at least two scorers blinded to the study to count the number of complete
contralateral rotations
.
Stepping Test
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•
•
•
•
•
Intended to model motor deficits in the forelimbs that are analogous to limb akinesia and gait problems seen in PD patients
Adapted the well-established protocol detailed in Olsson, et al. (5)
Animals were pre-trained for 5 days by handling and practicing the stepping test on the wooden board testing surface (30cm
length test with 1.5 cm markings)
L-dopa (6mg/kg) or SK609 (4mg/kg) were administered by intraperitoneal injection (i.p.) to the rat 10 minutes before testing
Rat was held by the experimenter gently with one hand fixing the hind limbs above the test surface (>45° angle) while the
other hand fixed the forelimb that is not being monitored (Figure 4)
Recorded initiation time of the first step by each forelimb and total stepping time to travel the full distance (30 cm)
Figure 2: Schematic Description of the experiments performed in the study, modified from Winkler
et al. (1)
• Pink arrows -> days on which apomorphine induced rotation tests were performed.
• Blue arrows -> days on which dose dependence (increasing from low to high) effects of L-dopa
or the experimental drug (SK609) were evaluated by i.p. injection
• Red hatched arrows -> days on which stepping test and AIMS scores were measured during the
chronic treatment phase of L-dopa either by itself or in combination with SK609.
Abnormal Involuntary Movements (AIMs)
• Designed to evaluate the efficacy of SK609 as an adjuvant to L-dopa to treat symptoms of LID
• Performed as described in Morgese, et al. (6)
• AIMs, consisting of axial, limb, and orolingual dyskinesia, was measured daily between 10:00am and 4:00pm over a 2hour
period after various doses of SK609 i.p. administration (20 minutes prior to testing) followed by L-dopa i.p. administration
(6mg/kg 10 minutes prior to testing )
• Animals were placed in a Plexiglas cylinder (30 cm diameter) for 120 minutes and video recorded for later analysis (Figure 3)
• Each dyskinesia parameter were scored on a severity scaled ranging from 0 to 4 for every 60 second time interval (modified
from Lundblad, et al. (7))
• We have demonstrated that PD rats with akinetic forelimb showed significant
improvement in forelimb stepping test with L-dopa and SK609. Both initiation
time and total stepping time were reduced significantly with SK609 treatment
when compared to either the control or L-dopa.
• In PD rats primed for LID with chronic treatment with L-dopa, SK609
significantly reduced total AIMS associated with L-dopa treatment in a dose
dependent manner.
• These results suggest that the selective D3 agonist SK609 could be used as an
adjuvant with L-dopa to treat symptoms of LID in PD patients without
impairing the positive effects of L-dopa.
• Future directions involve delineating the long term effects of SK609 in
comparison to L-dopa. Furthermore, optimal combination dosages of L-dopa +
SK609 should be researched in order to maximize the positive effects of L-dopa
while maintaining and minimizing LID symptoms with SK609.
ACKNOWLEDGMENTS
Supported by Drexel Ventures Innovation fund grant to SK and the Medical Student Summer
Research Fellowship (MSSRF) at Drexel University College of Medicine.
REFERENCES
Figure 3: Plexiglas chamber used for
rotation tests and AIMs tests.
Figure 4: Stepping Test showing wooden
testing surface and method of handling the rat.
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