Previous studies in Parkinsonian rats and monkeys have shown that β2-selective nicotinic acetylcholine receptor (nAChR) agonists reduce l-Dopa–induced dyskinesias (LIDs), a serious complication of l-Dopa therapy for Parkinson’s disease. Since rodent studies also suggested an involvement of α7 nAChRs in LIDs, we tested the effect of the potent, selective α7 agonist ABT-107 [5-(6-[(3R)-1-azabicyclo[2.2.2]oct-3-yloxy] pyridazin-3-yl)-1H-indole]. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-lesioned monkeys were gavaged with l-Dopa/carbidopa (10 and 2.5 mg/kg, respectively) twice daily, which resulted in stable LIDs. A dose-response study (0.03–1.0 mg/kg) showed that oral ABT-107 decreased LIDs by 40–60%. LIDs returned to control levels only after a 6-week ABT-107 washout, suggesting that long-term molecular changes were involved. Subsequent readministration of ABT-107 decreased LIDs by 50–60%, indicating that tolerance did not develop. ABT-107 had no effect on Parkinsonism or cognitive performance. We next tested ABT-107 together with the β2 agonist ABT-894 [(3-(5,6-dichloro-pyridin-3-yl)-1(S),5 (S)-3,6-diazabicyclo[3.2.0]heptane], previously shown to reduce LIDs in Parkinsonian monkeys. In one study, the monkeys were first given oral ABT-894 (0.01 mg/kg), which maximally decreased LIDs by 50–60%; they were then also treated with 0.1 mg/kg ABT-107, a dose that maximally reduced LIDs. The effect of combined treatment on LIDs was similar to that with either drug alone. Comparable results were observed in a group of monkeys first treated with ABT-107 and then also given ABT-894. Thus, α7 and β2 nAChR–selective drugs may function via a final common mechanism to reduce LIDs. The present results suggest that drugs targeting either α7 or β2 nAChRs may be useful as antidyskinetic agents in Parkinson’s disease.
A critical unmet need for Parkinson’s disease management is a strategy to reduce l-Dopa–induced dyskinesias (LIDs). These abnormal involuntary movements are a serious debilitating side effect of l-Dopa therapy, the gold standard of treatment of Parkinson’s disease, and they develop in most patients with continued l-Dopa use (Obeso et al., 2010; Meissner et al., 2011; Schapira and Jenner, 2011; Wichmann et al., 2011; Iravani et al., 2012; Huot et al., 2013). Currently, the only drug approved for LIDs is amantadine, and it is of only limited effectiveness. There is, therefore, an ongoing search for new therapies. Drugs targeting numerous classes of neurotransmitter receptors, including glutamatergic, serotonergic, cholinergic, and others, reduce LIDs in animal models, suggesting that multiple mechanisms are involved (Fox et al., 2009; Brotchie and Jenner, 2011; Blandini and Armentero, 2012; Duty, 2012; Sgambato-Faure and Cenci, 2012; Huot et al., 2013).
More recent studies have also indicated that the nicotinic cholinergic system is involved in LIDs. Studies in mice, rats, and monkeys showed that the general nicotinic acetylcholine receptor (nAChR) agonist nicotine consistently reduced LIDs in a dose-dependent manner (Quik et al., 2007, 2013d; Bordia et al., 2008; Huang et al., 2011b). Tolerance to the antidyskinetic effect of nicotine did not arise, even with months of treatment. Moreover, nicotine reduced LIDs, whether administered before l-Dopa treatment or once LIDs developed.
Nicotine generally exerts its effect by acting at nAChRs, of which there are multiple subtypes in both the peripheral and central nervous system. These subtypes include the α1β1*, α3β4*, and α7 nAChRs in the periphery, with the α4β2*, α6β2* and α7 nAChRs being the primary ones in the brain (Millar and Gotti, 2009; Quik and Wonnacott, 2011). The asterisk denotes the possible presence of other nAChR subunits in the receptor. Studies with α4, α6, α7, and β2 nAChR subunits null mutant mice suggest that α4β2*, α6β2*, and α7 nAChRs all influence the occurrence of LIDs (Quik et al., 2013b). These findings suggest that drugs targeting β2* and/or α7 nAChRs may yield therapies to reduce LIDs. In fact, pharmacologic studies show that several β2* nAChR agonists reduce LIDs in both rat and monkey models of LIDs (Huang et al., 2011a; Johnston et al., 2013; Quik et al., 2013a; Zhang et al., 2013a, 2014).
In the present study, we tested the effect of the α7 agonist ABT-107 [5-(6-[(3R)-1-azabicyclo[2.2.2]oct-3-yloxy] pyridazin-3-yl)-1H-indole] (from AbbVie, Inc., North Chicago, IL) since studies with α7 nAChR null mutant mice had indicated an involvement of α7 nAChRs in LIDs. We selected ABT-107 because of its potency and selectivity for α7 nAChRs over other available drugs (Briggs et al., 1997; Malysz et al., 2010) and because α7 nAChR drugs have excellent safety and tolerability profiles in clinical studies (Freedman et al., 2008; Othman et al., 2011). The results show that ABT-107 reduced LIDs ∼50% with no worsening of Parkinsonism and no observable side effects. We also tested ABT-107 in combination with an β2* nAChR agonist ABT-894 [(3-(5,6-dichloro-pyridin-3-yl)-1(S),5 (S)-3,6-diazabicyclo[3.2.0]heptane] (AbbVie, Inc.), and this drug combination yielded declines in LIDs similar to those after treatment with either drug along. These data suggest that drugs targeting either α7 or β2* nAChRs have potential as antidyskinetic agents.
Materials and Methods
Squirrel monkeys (n = 21, Saimiri sciureus) ≥5 years of age of either sex were obtained from World Wide Primates (Miami, FL). As required by California state guidelines, they were quarantined for 30 days on arrival. Food consisted of monkey chow, fruits, and vegetables, with water freely provided. The monkeys were singly housed in a humidity- and temperature- controlled room on a 12-hour light/dark cycle. All studies were approved by the Institutional Animal Care and Use Committee and were done according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Induction of LIDs.
The monkeys were injected subcutaneously with 2.0 mg/kg MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Sigma-Aldrich, St. Louis, MO) dissolved in saline to render them Parkinsonian (Quik et al., 2007). After 3 to 4 weeks of recovery from the systemic effects of the MPTP injection, the monkeys were rated for Parkinsonism, which was assessed by evaluating spatial hypokinesia, body bradykinesia, left and right manual dexterity, balance at rest and during movement, freezing during movement, and action tremor; each category was rated from 0 (normal) to 4 (severely Parkinsonian), as described by Quik et al. (2007, 2013d). The maximum Parkinsonian score is 28; however, individual scores are considerably lower because Parkinsonism is variably expressed in monkeys. Any single monkey may have deficits in manual dexterity but exhibit no freezing or tremor and move well, whereas another monkey may exhibit freezing and tremor but have no motor deficits. If the monkeys were not Parkinsonian, MPTP injection was repeated at 1.9 mg/kg. Parkinsonism was rated once weekly on Fridays throughout the study, before and 90 minutes after l-Dopa administration.
Monkeys then underwent gavage with l-Dopa (10 mg/kg) and carbidopa (2.5 mg/kg) twice a day at 8:30 AM and 12:30 PM from Monday to Friday inclusive (Quik et al., 2007, 2013d). Baseline LIDs were recorded from 8:00 to 8:30 AM. The monkeys then underwent gavage with l-Dopa, and recording was continued. The monkeys were given only fruits and vegetables in the morning to allow for optimal l-Dopa absorption, with more fruits and vegetables and monkey chow provided at ∼3 PM. The LID scores provided in the figures represent the ratings averaged over the morning l-Dopa dosing period for Wednesday and Thursday of each week. LIDs were rated for a 1-minute period by a blinded rater from video recordings at the times depicted in the figures. Dyskinesias were assessed on a scale of 0 (no dyskinesias) to 4 (severe dyskinesias) using the following criteria: 1 = subtle dyskinesias that were not sustained (fewer than three trunk movements in a row); 2 = sustained dyskinesias (three or more trunk movements in a row); 3 = moderate dyskinesias that impaired the ability to remain stationary; and 4 = severe dyskinesias that were generalized and incapacitating (Tan et al., 2002; Quik et al., 2007, 2013d).
nAChR Drug Treatments.
The monkeys had previously been treated with other nAChR agonists, followed by a 1-month washout period before initiating the current study. LID scores were at vehicle-treated levels for all monkey groups at the start of the drug treatment regimens. The groups were as follows: a vehicle-treated group (n = 6), a nicotine-treated (n = 5) group, a group of monkeys first treated with ABT-107 and subsequently also given ABT-894 (n = 5), and a group first treated with ABT-894 and then also administered ABT-107 (n = 5). The number of males and females in each group was similar.
All monkeys received 50% diluted orange Gatorade (PepsiCo, Chicago, IL) in the drinking water as a vehicle control for nicotine treatment. The presence of the Gatorade was necessary to mask the bitter taste of nicotine [(−)-nicotine, free base; Sigma-Aldrich] in the nicotine-treated group. The dose of nicotine was started at 50 μg/ml for 2 to 3 days, increased to 150 µg/ml for a further 3 to 4 days and then to 300 μg/ml nicotine, at which level it was maintained for the remainder of the study, as described (Quik et al., 2006, 2013d; Zhang et al., 2013a). Gatorade with and without nicotine was also added to the dry monkey chow. This nicotine dosing regimen yielded monkey plasma nicotine and cotinine levels similar to those in moderate smokers (Quik et al., 2006; Matta et al., 2007). ABT-107 or ABT-894 was administered orally 5 days per week via a small cracker 30 minutes before l-Dopa gavage. Each drug was applied to a small cracker in a 30- to 40-µl aliquot of water (depending on the monkey’s weight) and immediately given to the monkeys. The drug had no effect on body weight; no adverse effects were noted on behavior.
Since nAChR drugs are known to modulate cognitive abilities, we evaluated the effect of ABT-107 on cognition. We used an object retrieval task previously used for squirrel monkeys that measures a component of prefrontal cortex-dependent cognitive control involving inhibition of an initial learned response after subsequent spatial reversals (Lyons et al., 2000, 2004; Zhang et al., 2013a). The monkeys were scored for the average time and number of trials required to retrieve the marshmallow piece within a 30-second period (Lyons et al., 2000; Lyons and Schatzberg, 2003). Cognition was evaluated once weekly on Mondays.
Statistical comparisons were done using GraphPad Prism (GraphPad Software, San Diego, CA). Total dyskinesias were determined by evaluating the area under the curve, with data analyzed by two-way analysis of variance followed by a Bonferroni post hoc test. The data are expressed as percentage of vehicle; that is, it was based on the mean of the vehicle-treated group for the specific day. Differences in Parkinsonian scores, which represent the total of several behavioral parameters, were determined using a t test. The results are the mean ± S.E.M. of the indicated number of monkeys. Differences in LID scores between groups of monkeys were determined using nonparametric tests (Mann-Whitney test) since the scores are integral values, with the median shown. P ≤ 0.05 was considered significant.
The α7 Agonist ABT-107 Reduces Established LIDs.
Monkeys were treated with varying doses of ABT-107 as depicted in the time line (Fig. 1, top panel). ABT-107 (0.03–1.0 mg/kg) was given orally in a cracker 30 minutes before l-Dopa. The results (Fig. 1, middle) show that ABT-107 significantly reduced LIDs by 40% at the 0.03 mg/kg dose and maximally by 50% at the 0.10 mg/dose. The effect of nicotine on LIDs is shown for comparison (Fig. 1, bottom). Nicotine significantly reduced LIDs by 50–70%. Both ABT-107 and nicotine treatment reduced LIDs throughout the 4-hour time course compared with vehicle; median scores are shown in Fig. 2. The dyskinesia scores in the vehicle-treated monkeys (Fig. 2) ranged from 1.0 to 2.0; thus, the monkeys were moderately dyskinetic. The dyskinesia scores decreased to 0–1.0 with drug treatment (Fig. 2). After the 4-week drug treatment regimen, ABT-107 and nicotine were discontinued. The results in Fig. 3 show that LIDs only returned to vehicle-treated levels by 6 weeks of either ABT-107 or nicotine washout.
Combined Effect of the α7 Agonist ABT-107 and the β2* nAChR Agonist ABT-894 on LIDs.
Our previous studies had shown that the β2 nAChR agonist ABT-894 reduced LIDs by 60%, similar to the current results with α7 agonist ABT-107 (Zhang et al., 2014). This raised the question of whether combined treatment with the two different classes of nAChR agonists may yield a greater decline in LIDs than either drug alone. To evaluate this possibility, monkeys were treated with both drugs in combination as follows: one set of monkeys (n = 5) was first treated with 0.10 mg/kg ABT-107, a dose that maximally decreases LIDs (Fig. 4A). The monkeys were given ABT-107 orally for 5 weeks to ensure the drug effect had reached its plateau. A 50–60% decline in LIDs was obtained throughout the entire period. At week 6, the monkeys were also given ABT-894 orally at a dose that optimally reduced LIDs (0.01 mg/kg) (Zhang et al., 2014). The decline in LIDs with combined ABT-107 plus ABT-894 treatment was similar to that observed with ABT-107 alone (Fig. 4A).
Another set of monkeys (Fig. 4B) was first given ABT-894 orally at 0.01 mg/kg, a dose that optimally reduced LIDs (Zhang et al., 2014). The decline in LIDs reached its plateau by 5 weeks of ABT-894 treatment. At week 6, the monkeys were also administered ABT-107 at 0.10 mg/kg (Fig. 4B). No further decline in LIDs was observed with ABT-894 and ABT-107 in combination compared with single drug treatment. Thus, similar results were obtained regardless of the order of treatment of ABT-107 and ABT-894.
The effect of nicotine administration on LIDs in another set of monkeys, treated at the same time as in the studies already described, is shown in Fig. 4C for comparison. A 50–60% decline in LIDs was observed throughout.
The effect of ABT-107, ABT-894, the two drugs in combination, and nicotine on the hourly time course of LIDs is shown in Fig. 5. The average dyskinesia scores in the vehicle-treated monkeys ranged from 1.0 to 2.0 and in the drug-treated monkeys from 0 to 1.5, with the median score shown. The decline in LIDs was similar with all treatments (Fig. 5). The effect of drug washout is shown in Fig. 6. The time required for LIDs to return to vehicle-treated levels with the ABT-107 and ABT-894 drug combination was similar to that for the drugs alone (see Fig. 3).
Effect of the α7 Agonist ABT-107 and the β2* nAChR Agonist ABT-894 on Parkinsonism.
Vehicle-treated monkeys were moderately Parkinsonian. ABT-107 did not significantly affect Parkinsonism either on or off l-Dopa at any time point compared with vehicle (Table 1). The scores shown are for the last 4 weeks of treatment, with similar results throughout the study. In addition, ABT-107 had no effect on cognitive performance at any dose tested (Table 2).
The present results are the first to show that an α7 nAChR agonist reduces LIDs in Parkinsonian monkeys, a model that exhibits many features reminiscent of the dyskinesias that occur in l-Dopa–treated Parkinson’s disease patients. Notably, the ABT-107–induced decrease in LIDs persisted over several months of treatment, indicating tolerance does not develop. The effect of the drug was fairly long-lasting, with 6 weeks of washout required before LIDs returned to values similar to those in vehicle-treated monkeys.
These observations suggest that α7 nAChR agonists may represent a promising class of drugs for the treatment of LIDs in Parkinson disease patients. ABT-107 offers the advantage over other α7 nAChR agonists, such as DMXB [3-(2,4-dimethoxybenzylidene)-anabaseine dihydrochloride], as the former drug is a potent and selective α7 nAChR agonist. Additionally, α7 nAChR drugs have been tested in healthy human volunteers and in schizophrenic patients and were shown to be safe and well tolerated. Side effects were similar to those with placebo treatment (Freedman et al., 2008; Othman et al., 2011), despite the presence of α7 nAChRs in the peripheral nervous system. These characteristics suggest that α7 nAChR drugs may represent good candidates for further development for the treatment of LIDs in patients with Parkinson’s disease.
Our previous data had shown that β2* nAChR agonists reduced LIDs in Parkinsonian monkeys to a similar extent as the α7 agonist ABT-107 in the current study. Varenicline, which acts at β2* and other nAChR subtypes, as well as β2*-selective drugs, including A-85380, sazetidine, TC-2696, TI-10165, TC-8831, and TC-10600, all reduced LIDs to varying extents in rats with a unilateral 6-hydroxydopamine lesion (Huang et al., 2011a; Quik et al., 2013a). Additionally, varenicline and TC-8831 decreased LIDs ∼50% in nonhuman primates with no tolerance, although a limitation of these drugs was the development of emesis (Johnston et al., 2013; Zhang et al., 2013a). We more recently tested two β2* nAChR agonists that had previously been evaluated in phase 2 clinical trials for other indications (Zhang et al., 2014). These included ABT-089, a partial agonist (Decker et al., 1997; Sullivan et al., 1997; Anderson et al., 2009; Marks et al., 2009; Apostol et al., 2012), as well as a full agonist ABT-894 (Ji et al., 2007; Rowbotham et al., 2012; Bain et al., 2013). ABT-089 maximally decreased LIDs by 40% whereas ABT-894 reduced LIDs up to 60% in nonhuman primates (Zhang et al., 2014). None of the agonists worsened Parkinsonism, and there were no detectable side effects and no emesis (Zhang et al., 2014). Moreover, the effects of both the β2* nAChR drug ABT-894, the α7 nAChR drug ABT-107, as well as nicotine, persisted for months with treatment and, in addition, remained for several weeks after drug continuation.
The α7 nAChRs are functionally and pharmacologically distinct from β2* nAChRs. They exhibit a greater calcium conductance and faster desensitization and have a more direct impact on glutamatergic signaling (Albuquerque et al., 2009; Quik and Wonnacott, 2011). These differential characteristics raised the question of whether combination α7 and β2* nAChR drug treatment may yield a greater decline in LIDs. However, when ABT-107 was tested together with the β2* nAChR agonist ABT-894, the improvement in LIDs was comparable to that with either drug alone. In addition, it was similar to the effect of nicotine, a general agonist that acts at both α7 and β2* nAChRs, although with lower affinity for the α7 subtype.
The finding that the β2* and α7 nAChR agonists both reduce LIDs by ∼60% but show no significant additive effect with combined treatments suggests that these classes of drugs either reduce LIDs by a common mechanism or decrease LIDs by affecting different mechanism(s), which ultimately converge to produce parallel downstream changes. A key brain region involved in the generation of LIDs is the striatum, which expresses both β2 and α7 nAChRs. Although not at high density (Quik et al., 2000), α7 nAChRs are located on glutamatergic afferents from the cortex to the striatum to regulate dopamine release (Kaiser and Wonnacott, 2000). In addition, α7 nAChRs are present in the substantia nigra to control striatal dopaminergic function (Quik et al., 2000). β2* nAChR receptors are also expressed in the striatum on striatal dopamine nerve terminals, as well as in other neuronal elements. Lesion studies suggest that the β2* nAChRs on the dopamine nerve terminals are important for the nAChR-mediated reduction in LIDs because nicotine and nAChR drugs most effectively reduced LIDs in Parkinsonian animals with moderate nigrostriatal damage (Huang et al., 2011a,b; Quik et al., 2013c). Thus, a common mechanism by which α7 and β2* nAChR drugs act to reduce LIDs may involve a reduction in striatal dopamine release. Further evidence for this suggestion stems from studies showing that long-term nicotine treatment reduces dopamine release from striatal synaptosomes (Bordia et al., 2013).
In addition to the nigrostriatal dopaminergic system, other neuronal pathways also appear to be involved in the nAChR-mediated decline in LIDs. Evidence for this idea stems from studies showing that nicotine and β2* nAChR drugs still diminished LIDs up to 30% in rats with severe nigrostriatal damage. Possible candidates include nAChRs on nondopaminergic striatal neurons (GABAergic or cholinergic) or nAChRs in other brain regions (Huang et al., 2011a; Quik et al., 2013a). The effects of α7 nAChR drugs may also be mediated by α7 nAChRs in cortex, thalamus, cerebellum, and other brain regions linked to motor control as there is relatively high expression of α7 nAChRs in these regions (Quik et al., 2000).
With respect to the signaling pathways affected by nAChR drugs, activation of the nicotinic acetylcholine system has been linked to alterations in cAMP-linked intracellular signaling and in extracellular signal-regulated kinases (Ding et al., 2011; Feyder et al., 2011; Kawamata and Shimohama, 2011). Changes in the cAMP signaling cascade, including activation of cAMP, increased activity of the cAMP-dependent protein kinase, and of the dopamine- and cAMP-dependent phosphoprotein of 32 kDa have also been implicated in LIDs (Santini et al., 2007, 2010; Calabresi et al., 2008; Rangel-Barajas et al., 2010; Feyder et al., 2011). In addition, LIDs are associated with enhanced activity of extracellular signal-regulated kinase (Santini et al., 2010; Feyder et al., 2011; Wang et al., 2012). These mechanisms and others may thus contribute to the nAChR-mediated reduction in LIDs.
The ability of ABT-894, ABT-107, and nicotine to reduce LIDs weeks after drug discontinuation suggests that they most likely induce their effect via long-lasting molecular adaptations. Indeed, long-term changes in gene expression, intracellular signaling pathways, neuronal plasticity, and morphology have all been linked to the expression of LIDs (Santini et al., 2009; Cenci and Konradi, 2010; Feyder et al., 2011; Huot et al., 2011; Rangel-Barajas et al., 2011; Zhang et al., 2013b). Moreover, these changes may occur via numerous neurotransmitter systems, such as the dopaminergic, glutamatergic, serotonergic, and others, which are all implicated in the development and maintenance of LIDs (Carta and Bezard, 2011; Huot et al., 2011; Blandini and Armentero, 2012; Duty, 2012; Rylander, 2012; Huot et al., 2013).
α7 nAChR agonists offer the advantage that they have also been linked to improvements in cognition in numerous animal models (Levin, 2012; Lendvai et al., 2013) and in various neurologic and psychiatric disorders (Geerts, 2012; Lieberman et al., 2013). These latter observations suggest that α7 nAChR drugs may also be helpful for the memory deficits with Parkinson’s disease, in addition to improving LIDs.
In summary, the present data show that the α7 nAChR agonist ABT-107 reduces LIDs in Parkinsonian monkeys, without affecting Parkinsonism. Our previous work showed similar declines in LIDs with the β2* agonist ABT-894. These data suggest that development of subtype-selective nAChR drugs may offer not only greater specificity with reduced side-effect profiles but also may provide multiple therapeutic options.
The authors thank Tanuja Bordia and Xiomara Perez for helpful comments concerning the manuscript.
Participated in research design: Quik, Zhang, Decker.
Conducted experiments: Zhang, McGregor.
Contributed new reagents or analytic tools: Decker.
Performed data analysis: Zhang, McGregor, Quik.
Wrote or contributed to the writing of the manuscript: Quik, Zhang, McGregor.
- Received May 7, 2014.
- Accepted July 15, 2014.
This work was supported by the National Institutes of Health National Institute of Neurological Disorders and Stroke [Grants NS59910 and NS65851].
- 5-(6-[(3R)-1-azabicyclo[2.2.2]oct-3-yloxy] pyridazin-3-yl)-1H-indole
- (3-(5,6-dichloro-pyridin-3-yl)-1(S),5 (S)-3,6-diazabicyclo[3.2.0]heptane
- 3-(2,4-dimethoxybenzylidene)-anabaseine dihydrochloride
- LIDs, l-Dopa
- induced dyskinesias
- nicotinic acetylcholine receptor
- Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics