Clinical trial NCT04403399
Conditions: Parkinson's Disease
Sponsor: University of Michigan
Start date: 2017-06-29; Completion date: 2019-06-26
Enrollment: 34 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
With an appropriate oral dose of Varenicline (VCN) identified from experiments 1 \& 2 of the study (see NCT02933372), the investigators will administer VCN to Parkinson Disease (PD) participants to determine if VCN improves walking speed and measures of balance. PD participants will receive VCN or a placebo (fake drug) for 3 weeks to assess the effects of VCN administration on gait speed and balance. Participants will undergo examinations to assess the intensity of their Parkinsonism and asked questions to assess their mood and thinking.

Detailed description:
To demonstrate that α4β2\* nAChRs are appropriate therapeutic targets in Parkinson's Disease (PD), it is necessary to study key pharmacokinetic-pharmacodynamic features of α4β2\* nAChR in the context of the PD brain with loss of nerve cells that produce the neurotransmitter acetylcholine, a pathologic environment in which they may exhibit unique features. This personalized medicine approach focuses our studies on the subgroup of PD subjects with loss of nerve cells that produce the neurotransmitter acetylcholine identified by Project II and the Clinical Resource Core. The investigators will assess α4β2\* nAChR features using PET imaging with the α4β2\* nAChR ligand \[18-Fluorine\]flubatine, subacute administration of the α4β2\* nAChR partial agonist Varenicline (VCN), and laboratory measures of gait, balance, and attention. The investigators will use \[18-Fluorine\] flubatine PET to assess VCN occupancy of brain α4β2\* nAChRs (experiments 1 \& 2). VCN will be administered to both PD participants (experiment 1) and healthy controls (experiment 2) and both populations will undergo a flubatine PET scan to assess VCN occupancy. Using this PET data to select an appropriate VCN dose, the investigators will perform a pharmacodynamic study (experiment 3) with subacute VCN administration to determine if α4β2\* nAChR stimulation improves laboratory measures of gait function, postural control, and attentional function in PD subjects with loss of nerve cells that produce the neurotransmitter acetylcholine.

Arms: arm 1: Varenicline 0.5 mg BID orally for 3 weeks, followed by a 3-week washout period, then placebo BID orally for 3 weeks arm 2: Placebo BID orally for 3 weeks, followed by a 3-week washout period, then Varenicline 0.5 mg BID orally for 3 weeks
Interventions: intervention 1: Varenicline intervention 2: Placebo
Intervention descriptions: intervention 1: Initial 0.25 mg oral dose of varenicline administered with monitoring over 4 hours, then total daily dose escalated over the next 2 days until final 0.5 mg BID oral varenicline administered for the remaining 3 weeks. intervention 2: Initial placebo oral dose administered with monitoring over 4 hours, then total daily dose escalated over the next 2 days until final placebo BID dosing administered orally for the remaining 3 weeks.

Protocol documents:
Prot_001.pdf: 
Version Date: April 17, 2018 
       
 ClinicalTrials.Gov NCT Number: 04403399 
 
 
 
Cholinergic Mechanisms of 
Gait Dysfunction in 
Parkinson’s Disease 
ROGER L. ALBIN, MD 


 
1 
 
Udall Project 3- Cholinergic Mechanisms of Gait Dysfunction in PD (HUM00093188) 
PI: Roger Albin, MD 
Version: 9.0 
Dates Modified: 3/31/2017, 3/7/2017, 10/9/2017, 4/17/2018 
 
Project III: Specific Aims 
Falls and other abnormalities of gait and balance are common in PD, are largely levodopa unresponsive, 
and are major contributors to morbidity and mortality. The central theme of the U-M Udall Center is that 
degeneration of basal forebrain (BF) and pedunculopontine (PPN) cholinergic projections, in the context of 
degraded striatal motor control, contributes significantly to PD gait and balance deficits (3 Hit model; detailed in 
Overview). Our preclinical work (Project I) identifies attentional deficits, secondary to loss of cortical cholinergic 
afferents, as a key mechanism through which cholinergic deficits contribute to impaired gait and balance. The 
goal of this pilot target engagement/pharmacodynamic clinical study is to assess α4β2* nicotinic cholinergic 
receptors (nAChRs) as a therapeutic target for improving gait, balance, and attentional capacity in PD.   This   
proposal   is   directly   responsive   to NINDS PD 2014 Research Report “highest priority recommendations” 
calling for studies aimed at understanding mechanisms of and developing treatments for balance and gait 
disorders in PD (Clinical recommendations 2 & 3; Basic recommendation 3; Translational recommendation 6) 
and the Udall RFA request for pilot target engagement/pharmacodynamics studies. 
PD exhibits heterogeneity of cholinergic degeneration (Overview, Project II), indicating that cholinergic 
interventions should be targeted selectively to the subgroup of patients with cholinergic deficits. α4β2* nAChRs 
are key mediators of CNS cholinergic neurotransmission. Our preclinical data (Project I) indicates that α4β2* 
nAChR agonists reduce fall risk by improving attentional  function in our model of combined dopaminergic- 
cholinergic degeneration. The central hypothesis of this clinical target engagement/pharmacodynamic study is 
that deficient activation of α4β2* nAChRs is a key contributor to gait and balance abnormalities in PD, and that 
restoring activity of this target will reduce these debilitating levodopa unresponsive symptoms. 
To demonstrate that α4β2* nAChRs are appropriate therapeutic targets in PD, it is necessary to study 
key pharmacokinetic-pharmacodynamic features of α4β2* nAChR in the context of the degenerating, 
hypocholinergic PD brain, a pathologic environment in which they may exhibit unique features. This personalized 
medicine approach focuses our studies on the subgroup of hypocholinergic PD subjects identified by Project II 
and the Clinical Resource Core. We will assess α4β2* nAChR features using PET imaging with the α4β2* nAChR 
ligand [18F]flubatine, subacute administration of the α4β2* nAChR partial agonist varenicline (VCN), and 
laboratory measures of gait, balance, and attention. We will use [18F]flubatine PET to assess VCN occupancy of 
brain α4β2* nAChRs. Using this PET data to select an appropriate VCN dose, we will perform a 
pharmacodynamic study with subacute VCN administration to determine if α4β2* nAChR stimulation improves 
laboratory measures of gait function, postural control, and attentional function in hypocholinergic PD subjects. 
Specific Aim 1: To use [18F]flubatine PET to assess VCN occupancy of brain α4β2* nAChRs after subacute VCN 
administration to the subgroup of PD subjects with cholinergic deficits in comparison with normal control subjects. 
Hypothesis  1a:  Low  dose,  subacute  VCN  administration  produces  high  α4β2*   nAChR  occupancy  in  PD 
subjects with cholinergic deficits. 
Hypothesis 1b:  Low  dose,  subacute  VCN  administration  produces  equivalent  α4β2*   nAChR  occupancy  in  
PD subjects with cholinergic deficits and normal control subjects. 
Specific Aim 2: To assess the effects of pharmacologic α4β2* nAChR activation on laboratory measures of gait 
function and postural control in PD subjects with cholinergic deficits. 
Hypothesis  2:  Pharmacologic α4β2*   nAChR  activation  in  PD  subjects  with  cholinergic  deficits  will  improve 
laboratory measures of gait function and postural control. 
Specific Aim 3: To assess the effects of pharmacologic α4β2* nAChR activation on a measure of attentional 
function (SAT; sustained attention test) in PD subjects with cholinergic deficits. 
Hypothesis  3:  Pharmacologic α4β2*   nAChR  activation  in  PD  subjects  with  cholinergic  deficits  will  improve 
attentional performance on the SAT. The improvement in SAT performance will correlate with improvement in 
laboratory measures of gait function and postural control assessed in Specific Aim 2. 
 
 


 
2 
 
Our group possesses unique expertise in preclinical models, nAChR pharmacology, cholinergic imaging, 
and physiological measures of gait function, postural control,  and  attention.  This  project  interacts directly with 
Project II, depends on the Clinical Resource Core, and is strongly connected  to  Project  I preclinical 
experiments. The potential impact of the proposed experiments is considerable. This work would provide strong 
support for our central hypothesis, establish therapeutic validity of the novel  animal  model employed in Project 
I, and implement new methodologies for the evaluation of treatments to improve gait and balance deficits in PD. 
These experiments would identify α4β2* nAChRs as a therapeutic target in a clinically definable PD subgroup, 
setting the stage for trials of α4β2*  nAChR  agonists  as  adjunctive  treatments  to mitigate balance and gait 
abnormalities in these patients. 
 
Project III: Research Strategy 
 
Significance 
While the major clinical features of PD are associated historically with dopaminergic nigrostriatal 
projection degeneration, several important and disabling clinical features of PD are caused by the combined 
degeneration of dopaminergic and non-dopaminergic systems and are resistant to  dopamine  replacement 
therapy. Among the most troubling dopamine resistant phenomena are falls and associated gait abnormalities 
(1-7). Of the cardinal features of PD, gait and balance disorders are the least responsive to dopaminergic 
replacement, even in subjects with early disease (8-10). In longitudinal studies,  dopamine  non-responsive 
features, including postural control deficits, falls, and gait  disorders,  dominate  the  clinical  picture  (11-13). 
Hoehn and Yahr stage 3 (HY3) marks increased risk for fall-related disability in PD, as up to 40% of HY3 patients 
experience multiple falls, often with injury, including a high fracture incidence (6). In one PD cohort study, 
approximately one-third experienced a new hip fracture over a 10 year period (14). Even when falling is avoided 
by self-imposed activity reductions, fear of falling impairs function significantly and degrades quality of life. There 
are no effective therapies to prevent falls and only limited understanding of the non-dopaminergic systems that 
impair gait and postural control in PD. 
Project III will tackle this critical clinical problem by pursuing pilot target engagement studies to identify 
a novel treatment strategy in PD. Our selected target, α4β2* nicotinic  acetylcholine  receptors  (nAChRs),  is 
based on compelling preliminary data implicating degeneration of cholinergic systems in postural control and gait 
dysfunction in PD, and the critical role of α4β2* nAChRs in mediating relevant aspects of cholinergic 
neurotransmission (see below, Overview, Project I, Project II). Beyond its potential clinical impact, this work 
will improve scientific knowledge of gait and postural control dysfunction  in  PD.  Together  with  the  other 
projects in the U-M Udall Center, Project III will contribute to testing the 3 Hit Model of gait dysfunction in PD 
(Overview). This model posits the PD-defining posterior putaminal dopaminergic deficit as the first  “hit” 
disrupting execution of habitual motor actions such as gait, placing increased demand for compensation on other 
brain systems. The model hypothesizes that degeneration of basal forebrain (BF) and pedunculopontine (PPN) 
cholinergic projections (second and third “hits”) are significant causes of  the  progressive  levodopa- resistant 
gait abnormalities in PD. Consistent with this model, degeneration of cholinergic terminals/neurons of the BF and 
the PPN are documented well in PD (15,16). PD patients with a history of falls, moreover, have significantly 
reduced cholinergic projections compared to  non-falling  patients,  whereas  these  groups  do  not differ in the 
degree of dopaminergic denervation (see Approach below; Overview and Project II) (17,18). 
α4β2*  nAChRs are expressed highly in the terminal fields of BF and PPN cholinergic projections (cortex 
and thalamus/cerebellar cortex/striatum, respectively), highlighting their  potential  utility  as  a  pharmacologic 
target for gait and balance abnormalities in PD. These receptors exhibit complex pharmacology, however, 
necessitating pharmacokinetic-pharmacodynamic studies, as proposed here, to determine their value as a 
therapeutic target. It was believed previously, for example, that α4β2* nAChRs upregulated by chronic agonist 
treatment are desensitized, but recent in vivo experiments indicate that chronic nicotininc agonist exposure 
induces functional α4β2* nAChRs (see Project  I  for  further  discussion)  (19-25).  Critically,  α4β2*  nAChRs 
might also show unique alterations in the context of the degenerating, hypocholinergic PD brain, 
necessitating  assessment  of  this  target  in  this  particular  environment. For example, in PD there are 
significant post-transcriptional  alterations in  receptor  subunit  composition  and  function  for  N-methyl-D- 
aspartate receptors, another important ionotropic receptor family (reviewed by Picconi et al. [26]). Regions with 
little intrinsic pathology could be affected by changes in cholinergic afferents and PD pathologies could induce 
significant changes in α4β2* nAChR composition and function. Our studies are highly significant because they 
will assess the function of α4β2* nAChRs in the hypocholinergic degenerating  PD  brain,  establishing  the 
potential of these receptors as a therapeutic target. 


 
3 
 
 
Innovation 
The proposed experiments are innovative in pursuing in vivo  pharmacokinetic-pharmacodynamic studies 
to evaluate engagement of a specific molecular target in the subgroup of hypocholinergic PD subjects – defined 
by PET imaging in Project  II. These innovative experiments will use PET methods to assess a key 
pharmacokinetic variable, receptor occupancy, to select the dose for  pharmacodynamic  studies.  Our  PET 
studies will employ a novel nAChR ligand, [18F]flubatine, with favorable kinetic features. 
Our approach differs considerably from previous work and the unique design proposed will 
overcome key limitations of those studies. A pilot study by Dr. Fay Horak (consultant for this Center) and 
colleagues  explored  the  acetylcholinesterase  inhibitor  (AChEI)  donepezil  for  PD-related  falls,  with  modest 
positive results (27). But, AChEIs are non-specific activators of nAChRs and muscarinic AChRs, including 
presynaptic autoreceptors inhibiting  ACh  release. Several features of cholinergic neurotransmission suggest 
that such non-specific agonism is unlikely to be effective (28). In addition, imaging data indicates that AChEI 
treatment produces at most 20% inhibition of brain cholinesterase, with many subjects not achieving significant 
cholinesterase inhibition (29). One α4β2* nAChR agonist, SIB-1508Y, was tested in PD (30). In contrast to several 
aspects of our proposed work, this study did not identify a hypocholinergic subgroup, did  not specifically assess 
gait and postural control, and all subjects were levodopa naive. Our  3  hit  model  and preliminary data (Project 
I) suggests that an effect of cholinergic agonists may require simultaneous treatment with levodopa. The α4β2* 
nAChR partial agonist varenicline (VCN) has not been tested in PD, but there is relevant experience in 
Spinocerebellar Ataxia Type 3 (SCA3) (31). Zesiewicz et al. reported that VCN was 
tolerated well and improved axial but not appendicular function in SCA3 subjects (31). This finding is intriguing 
in view of our [18F]FEOBV data demonstrating significant cholinergic innervation of the cerebellar vermis and 
archicerebellum, but not the cerebellar hemispheres (Project II Preliminary Data). 
Results confirming  our predictions would strongly support our central hypothesis that α4β2*  nAChRs 
possess pharmacokinetic-pharmacodynamic characteristics appropriate for future trials aimed at ameliorating 
gait and postural control dysfunction secondary to cholinergic  deficits.  These  experiments  will  expand  the 
clinical pharmacology of α4β2* nAChRs in the context of PD and  implement  new  methods  for  evaluating 
potential new therapeutic targets in PD. This work will also help to establish therapeutic validity of the novel 
rodent model further developed in Project I (32). Validation of the rodent model will greatly enhance the future 
research on treatments to improve gait and postural control disorders in PD. 
 
Interaction with Other Projects and Cores 
The experiments proposed here are strong analogues of critical  Project  I  experiments.  Subjects 
studied in this Project will undergo characterization of brain cholinergic pathways in Project II. Subject clinical 
characterization, general cognitive assessments, assessment of gait function and postural control, attention, and 
other relevant assessments will be performed in the Clinical Resource Core (Core B). Data management and 
biostatistical support for data analyses will be provided by the Biostatistics and Data Management Core (Core 
C). This project will be assisted indirectly by Education and Outreach Core (Core D) for subject recruitment, and 
relies on the Administrative Core (Core A) for budgetary oversight and project management. 
 
Approach 
 
Background & Preliminary Data 
Cholinergic Neurotransmission & PD Gait Dysfunction: 
Numerous studies indicate that cognitive dysfunction affects gait 
and balance functions (reviewed by 33, 34, and in Overview), 
implicating BF cholinergic degeneration in gait dysfunction in 
PD. Impaired attention and executive control are  risk  factors  for 
falling and  simultaneous  cognitive  task  (dual-task)  demands 
affect biomechanical parameters of balance and gait in PD 
patients (35-38). In controls and PD subjects, individuals with 
attentional/executive deficits are more prone to falls and gait 
abnormalities (see Project II) (35-39). Degeneration of BF 
corticopetal projections is well established in PD and  the 
magnitude of BF terminal loss is greater in PD than in AD 
(15,16,40,41). BF cholinergic projections  are  crucial  for 
 
cholinergic  innervation. 


 
4 
 
neocortical  modulation  of  attention  by  enhancing  detection  
of relevant cues and filtering distractors (28). As described in 
considerable work by one participant in this Udall Center 
application, Martin Sarter, BF  mediated  cholinergic  
neurotransmission  regulates  attention  via modulation of 
thalamocortical afferent effects on cholinergic presynaptic 
heteroreceptors (see Project I) (28,42-45). Dr. Sarter’s and Dr. 
Albin’s recent studies in a novel rodent model of combined 
dopaminergic and cholinergic pathway lesions indicate that this 
dual lesion paradigm, mimicking the  pathology  of  many  PD 
patients, produces gait and balance abnormalities analogous to 
those found in PD (Project I Preliminary Data and below) 
(32,46). 
The mesopontine PPN complex has wide projections to many regions, including other brainstem 
structures,  the  cerebellum,  and  dense  connections  with  the  basal  ganglia  and  thalamus  (47-50).  The  
PPN 
contains heterogeneous cholinergic, glutamatergic, and GABAergic neuron subpopulations with important 
cholinergic projections to the basal ganglia, thalamus, and brainstem/cerebellar locomotor regions (49). BF and 
PPN cholinergic projections may overlap in the mediodorsal and reticular thalamic nuclei (51). A recent post- 
mortem study correlated PD fall propensity with magnitude of PPN pathology (52). PD fallers had greater loss of 
PPN cholinergic neurons. A parallel “experiment of nature”  supports  a  crucial  role  for  PPN cholinergic neuron 
integrity for maintenance of normal posture. Progressive Supranuclear Palsy is a neurodegenerative tauopathy 
with profound loss of PPN cholinergic neurons (see Project II)(53), and falls are an early, prominent feature of 
that illness. Recent non-human primate experiments further highlight the importance of PPN function in the 
maintenance of postural control and gait. Karachi  et  al  demonstrated  that  isolated  cholinergic  PPN lesions 
produce dopamine non-responsive gait slowing and postural control abnormalities (52,54). Consistent with our 
“3 Hit” model of PD gait and balance deficits, impairments of postural control and gait were more pronounced in 
older MPTP lesioned primates with PPN cholinergic neuron loss. 
Our previously published work 
associates decrements in cholinergic BF 
and PPN projections with fall  propensity, 
and provides a strong  rationale  for 
exploring  cholinergic  neurotransmission  
as a potential target to ameliorate falls in 
PD (17,18). These PET imaging studies 
demonstrate considerable heterogeneity 
of cholinergic system changes in PD 
subjects (Figure 1, previous page), 
allowing for correlation with important 
clinical features (17,18,55). These  data  
suggest  the presence of PD subgroups 
that may exhibit unique      responses      to      
pharmacologic 
interventions. Using the cholinergic terminal 
Figure 2: Cortical and thalamic cholinergic innervation in 
healthy 
elderly controls (HC) and non-demented PD 
subjects (from reference 17). PD subjects exhibit significant 
but widely variable cortical cholinergic denervation and a 
smaller fraction of PD subjects exhibit thalamic denervation. 
Y-axis scales different between cortical and thalamic regions 
of interest. 


 
5 
 
ligand [11C]PMP, we quantified neocortical (index of BF system integrity) and thalamic (index  primarily  of  PPN  
integrity) cholinergic terminal density in PD subjects with  and   without  fall  histories  (18).  PD fallers had thalamic 
deficits, while those without fall histories were comparable to controls. PD fallers exhibited cortical cholinergic deficits 
of significantly greater magnitude than those without fall histories. In contrast, falling and non-falling PD subjects did 
not differ in striatal dopaminergic deficits. In a  subsequent  large  (N=101) [11C]PMP study, we correlated cholinergic 
projection changes with measures of gait function, cognition, and postural control (17). We identified significant 
decreases in cortical cholinergic innervation in the PD group with substantial overlap with controls (Figure 2). One 
third of the PD group exhibited cortical cholinergic denervation (5th percentile or below of controls) and approximately 
one sixth of the PD  group  exhibited equivalent thalamic cholinergic denervation. PD subjects with cortical cholinergic 
deficits exhibited significantly slower gait speeds, even after adjustment for striatal dopaminergic deficits (17,56). 
These subjects exhibited significant impairments of executive-attentional function. PD subjects with thalamic 
cholinergic deficits were significantly more likely to have a fall history (Project II Preliminary  Data).  Striatal  
dopaminergic  terminal changes were comparable between PD subjects with and without cholinergic deficits. 
Considered together, our results implicate both BF and PPN deficits in abnormal postural control and gait in PD, and 
associate these problems with cognitive, particularly attentional, dysfunction. 
 
Nicotinic Cholinergic Receptors (nAChRs) as a Therapeutic Target in PD: The great majority of CNS nAChRs 
are α4β2*  heteropentamers (* for potential presence of other subunits), a significant minority are  〈7 
homopentamers, and small  populations  of  other  subtypes  are  expressed  (57).  α4β2*  nAChRs  may  be  pre- 
and/or post-synaptic. Presynaptic nAChRs are located on both cholinergic (homosynaptic) and non-cholinergic 
(heterosynaptic)  terminals.  α4β2*   nAChRs  are  expressed  in  the  terminal  fields  of  BF  (cortex)  and  PPN 
(thalamus,  cerebellar  cortex)  cholinergic  projections.  Considerable  evidence  indicates  that  BF  cholinergic 
projections    modulate    attention    by    activating    presynaptic    thalamocortical    terminal α4β2* nAChRs 
(heteroreceptors; Project I) (28,46). In the primate MPTP model, Schneider and colleagues demonstrated that a 
α4β2* nAChR agonist, SIB-1508Y, improves attention and executive function (58). There is little evidence of 
thalamic pathology in PD and thalamocortical projections are not known to degenerate in PD. These facts open the 
door to selective modulation of attention via neocortical mechanisms by α4β2* nAChR agents; enhancing attention 
by activating these receptors with α4β2* nAChR agonists may improve gait and postural dysfunctions in PD patients 
with cholinergic deficits. 
Postmortem and imaging studies demonstrate reduced α4β2* nAChR expression in several regions in PD 
brain and likely reflect reduced expression of both presynaptic homoreceptors and postsynaptic receptors (59,60,61). 
One recent study, for example, of mildly to moderately affected PD subjects with the α4β2* nAChR PET ligand [18F]FA-
85380 described varying (20%-40%) reductions of α4β2* nAChR binding in many regions (61). Our understanding, 
however, of the expression of α4β2* nAChRs in PD is limited. Some prior studies used less resolute SPECT imaging, 
and [18F]FA-85350 α4β2* nAChR imaging has disadvantageous kinetics, with an up to 7 hour scan acquisition time – 
quite difficult for PD  subjects.  Moreover,  in  contrast  to  our proposed study focused on hypocholinergic subjects, 
prior work was in unselected cohorts that  certainly examined PD subjects with varying cholinergic deficits, probably 
increasing variation in estimates of regional α4β2*  nAChRs. 
We will overcome existing shortcomings by imaging α4β2* nAChRs in two ways. First, we will use (−)- 
[18F]norchloro-fluoro-homoepibatidine (NCFHEB; flubatine), a newly developed and highly specific  α4β2* nAChR 
ligand with favorable binding kinetics, which allows scanning in 90 minutes (62-66). Our PET Center is now 
performing human [18F]flubatine imaging routinely (see SA1) (66). Second, we  will  only  assess  PD subjects with 
documented loss of cholinergic projections, as determined by [18F]FEOBV imaging, a ligand for the vesicular 
acetylcholine transporter (VAChT) that provides previously unattainable resolution of cholinergic terminals (Project 
II). This innovative approach is likely  to  provide  information  on  α4β2*  nAChRs  in  PD uniquely valuable for the 
assessment of this target in PD-related gait abnormalities. 
 
α4β2* nAChR Partial Agonists: A number of nAChR partial agonists are under development and one, varenicline 
(VCN; “Chantix”), is in clinical use for smoking cessation (67,68). High levels of receptor occupancy can be achieved 
with partial agonists. This feature is useful for pharmacokinetic imaging  experiments,  as proposed in SA1, as a large 
percentage of the total receptor population can be imaged. VCN is a potent α4β2* nAChR partial agonist (Ki  of 0.4 
nM) with good brain penetration, high oral bioavailability, an elimination t1/2 of 
~24 hours, low plasma protein binding, and primarily  renal excretion without metabolism (69). Low plasma protein 
binding and minimal hepatic metabolism reduce adverse drug interactions.   VCN has some 〈7 nAChR 
partial agonist activity, though at 200-fold higher concentrations (Ki > 84 nM). Recent PET data indicates that VCN 
binds human CNS α4β2* nAChRs robustly at relevant and relatively low oral doses. Lotfipour et al. used [18F]FA-
85350 PET to show that a single, low oral VCN dose (0.5 mg; usual treatment dose = 1 mg po bid) produced near 


 
6 
 
total occupancy of brain α4β2* nAChRs in acutely abstinent smokers (70). α4β2* nAChR partial agonists have 
additional properties that are reassuring in the context of PD. α4β2* nAChRs are located on dopaminergic nigrostriatal 
perikarya and terminals.   These compounds may promote dopamine release from 
nigrostriatal terminals (67). The contribution of stimulating these dopamine neuron located α4β2* nAChRs to their 
beneficial effects on cognition is unclear (see Project I), but there is speculation that use of α4β2* nAChR agonists 
may allow lower L-dopa doses. Zhang et al. studied the effects of the α4β2* nAChR partial agonist ABT-089 (to be 
evaluated in Project I) in the non-human primate  MPTP  model  (71).  ABT-089  treatment reduced levodopa related 
peak dose dyskinesias without altering parkinsonism or cognition. The dopamine releasing properties of nigrostriatal 
α4β2* nAChRs will not be a confounding factor in our studies as we are assessing dopamine non-responsive features 
of PD in subjects on L-dopa. There  is speculation  also  that nAChR stimulation may have neuroprotective effects 
as nicotinic agonists reduce dopaminergic neuron death in PD toxin models (72). There is no reason to think that 
α4β2* nAChR partial agonists will interfere with dopaminergic treatment for PD, impair cognition, or worsen underlying 
neurodegenerative processes. 
 
Safety is a critical issue. Following reports of suicidal behavior and suicide associated with VCN, the FDA 
issued a “black box warning”. Confounding  assessment  of  possible  side  effects  is  the  fact  that these problems 
occur during nicotine withdrawal and tobacco abusers have a high prevalence of relevant psychiatric comorbidities. 
Recent comprehensive reviews of VCN trial experience and other large datasets, such as the UK General Practice 
Research Database, and a recently completed trial specifically examining this issue, do not support a connection 
between worsening mood disorders, suicidality, and VCN (73-78). In a recent subacute VCN treatment study in 
normal adults, there was no change in mood, impulsivity, or emotional processing related to depressive features, 
though  VCN  did  enhance  cognitive  performance  (79).  Concerns have been raised about VCN and rare but 
serious cardiovascular events.  Recent analyses of trial data did not show a significant relationship and a recent very 
large cohort study of  VCN  use  in  Denmark  did  not  find evidence of increased cardiovascular events (80,81). 
Indeed, in a recent preventive cardiology trial, VCN use was associated with better control of cardiovascular risk 
factors (82). Considered together, these data argue strongly that VCN is safe, and suggest that earlier concerns, to 
the extent they are accurate, apply to the subgroup of acutely abstinent smokers. This subgroup will be excluded 
from our studies (see Clinical Resource Core). In a very large (~8000 participants), recently published trial examing 
psychiatric symptoms accompanying VCN use for smoking cessation, there was no increased risk of mood disorder 
or suicidality (Athenelli et al., Lancet,2016 Jun 18;387(10037):2507-20. doi: 10.1016/S0140-6736(16)30272-0).  Moreover, we 
emphasize that these studies seek  to  establish  α4β2*   nAChR  as  a  viable target, not to promote a specific 
compound as a therapeutic - which would require a clinical trial. In this context, we employ VCN as a representative 
agonist to explore the pharmacodynamics of α4β2* nAChR stimulation in the context of the hypocholinergic 
degenerating PD brain. Regardless of whether VCN is ultimately tested in a clinical trial - a real possibility considering 
the considerable new data indicating it’s safety and tolerability (73 - 
81) - these data are critical for the evaluation α4β2* nAChR as a therapeutic target in PD-related gait disturbances. 
 
The FDA recently (3/9/15) updated the labeling for VCN (http://www.fda.gov/Drugs/DrugSafety/ucm436494.htm).  
There are some reports of seizures following VCN use.  In addition, there are some reports of alcohol intolerance in 
individuals using VCN.  Again, these have occurred in acutely abstinent smokers.  These are isolated reports and 
whether VCN is actually associated with seizures and alcohol intolerance is unknown.  We will take precautions, 
however, to guard against these potential side effects (see below). 
 
Preclinical Data: nAChR Partial Agonists and PD-related Falls: In experiments described in Project I, we present 
a new model of combined dopaminergic-cholinergic deficits that recapitulates key features of gait and balance 
disorders in PD (32). In this model, Dr. Sarter piloted adjunctive treatment with ABT-089, an α4β2* nAChR partial 
agonist. ABT-089 produced attentional improvements and showed excellent tolerability in phase II clinical trials for 
AD and ADHD (83-89). Preliminary studies show  that  ABT-089  administration  improves balance in the dual lesion 
model (Project I Preliminary Data). Moreover, there is a strong correlation between fall propensity and performance 
on a measure of attention capacity, the sustained attention test (SAT; r = 0.61 [p < 0.05]). We will perform parallel 
experiments with VCN in hypocholinergic PD subjects and Dr. Sarter will examine VCN in the novel rodent model in 
Project  I. This parallel design will help to establish therapeutic 
validity of the rodent model and, in turn, provide mechanistic insight into the pharmacodynamic properties of 
α4β2*  nAChR explored in these clinical studies. 
 
Summary of Preliminary Data: The foregoing discussion establishes our capacity to recruit and characterize 
appropriate types and numbers of PD subjects, demonstrates a link between cholinergic system changes in gait and 
balance dysfunction in PD – including preclinical data (Project I) supporting our central hypothesis – and  shows  


 
7 
 
capacity  to  use  multi-tracer  PET  imaging.    Our  group  has  also  developed  a  novel,  specific 
cholinergic terminal tracer ([18F]FEOBV; Project II Preliminary Data) that will facilitate PD participant selection. 
 
Overall Rationale and Organization 
These clinical pharmacokinetic-pharmacodynamic experiments aim to establish α4β2* nAChRs as 
appropriate targets for treatment of levodopa resistant gait and balance deficits in hypocholinergic PD subjects. 
Subjects will be drawn from Project II and the Clinical Resource Core. In SA1, we will use [18F]flubatine PET 
imaging to determine the lowest VCN dose producing high α4β2* nAChR occupancy. In SA2, we will use this dose to 
perform pharmacodynamic studies of VCN effects on laboratory measures of gait and postural control. These studies 
will determine if α4β2* nAChR stimulation produces the predicted effects on  laboratory measures of balance and gait 
– directly addressing our central hypothesis. In SA3, we will study the effects of subacute VCN administration on a 
measure of attentional capacity sensitive to cholinergic deficits, testing our hypothesis that improvement in attention 
capacity will correlate with improvements in gait and postural control. These data will evaluate a likely physiological 
mechanism mediating the effects of stimulating α4β2*  nAChRs in the context of the hypocholinergic PD brain, and 
provide important validation data for the rodent model used in Project I. 
We propose subacute (days) VCN administration and focus on hypocholinergic PD subjects. Because 
repeated α4β2* nAChR stimulation, as would be experienced by chronically treated patients, induces functional 
receptor upregulation, assessing α4β2* nAChR occupancy with a single VCN dose in VCN naïve subjects will be 
misleading. Moreover, a VCN dose that produces high percent receptor occupancy in a VCN naïve subject may 
exhibit considerably less α4β2* nAChR occupancy in subjects exposed repeatedly to VCN and with α4β2* nAChR 
upregulation. It might be scientifically interesting to include normocholinergic PD subjects, but safety concerns hinder 
this approach. In our model (Overview; Project I data), dopaminergic striatal deficits result in compensatory “loading” 
of other brain  systems.  Normocholinergic PD  subjects  depend  on  increased cholinergic neurotransmission to 
compensate for striatal dysfunction, placing them at increased risk of adverse events resulting from reduced 
cholinergic signaling (32,46). VCN is a partial agonist with an efficacy of approximately 40% of ACh. In normal 
individuals, VCN produces measurable improvements on cognitive tests 
(79,88), but in normocholinergic PD subjects, VCN may interfere sufficiently with α4β2* nAChR mediated 
compensation to produce functional impairments,  including cognitive impairment  and/or  diminished balance, 
including damaging falls. It is not feasible to recruit sufficient subjects to do these experiments in an inpatient setting, 
which would be required to mitigate these potential side effects. For these reasons, we will not expose 
normocholinergic PD subjects to VCN. Beyond these important safety concerns, adding study groups to the proposed 
experiments would reduce statistical power for a fixed sample size, increasing the Ns required for a valid study 
beyond that which can be supported by funds available for Udall Center studies. 
 
We will investigate the possibility that regulation of α4β2* nAChRsmight be different in PD brain versus brains of 
normal individuals.  We will investigate this possibility with a focused study of α4β2* nAChR receptor occupancy in a 
small group of normal subjects.  Following the conclusion of receptor occupancy studies in hypocholinergic PD 
subjects, we will perform a more limited, parallel, dose-receptor occupancy study to determine if there are major 
differences in VCN receptor occupancy between PD and control subjects.  
 
Subject Selection: PD subjects will be drawn from Project II  subjects who have undergone [18F]FEOBV imaging to 
identify cholinergic deficits. 
We will study subjects with cortical innervation deficits below the  5th percentile  cutoff  of  controls as defined 
previously (17). General inclusion and exclusion  criteria  are  specified  in  the  Clinical 
Resource Core and Project  II.  Current  or previous (within last 6 months) use of any product or medication  
containing  nicotinic  agents, including use of tobacco products such as cigarettes, cigars, pipes, chewing tobacco, 
etc., e- cigarettes, OTC nicotine patches, chewing gum containing nicotine, or varenicline is an exclusion. While most 
data does not indicate that VCN causes suicidality or cardiovascular problems (73 - 
81), we will exclude subjects with depression, active cardiovascular disease, history of seizures, or alcohol use 
disorders. (see Human Subject Protection). 
The Clinical Resource Core and Project II project initial enrollment of 95 PD subjects with longitudinal follow-
up of 75 PD subjects. Based on prior results, we project that 30% (32) of the initially enrolled subjects will be 
hypocholinergic. Based on preliminary data (Project II), 25% (20) of the longitudinally followed PD subjects will 
convert from normo- to hypocholinergic during the project period. This gives a total of 52 eligible subjects (Table 1). 
Our long-standing experience is that a significant fraction of subjects participate in multiple experiments, so we 
anticipate little difficulty in recruiting the projected 55 PD subject-equivalents required.  In our recently concluded 
Program Project Grant (NS15655), we studied 151 PD subjects.  All underwent at least 2 PET studies with 112 


 
8 
 
undergoing 3 or more scans (69 - 3 studies; 43 - 4 studies). We are basing our estimates on a stringent 5th  percentile 
cut-off relative to our normal control database. We will carefully monitor subject recruitment to enroll the target number 
of hypocholinergic PD subjects. If needed, Core B will preferentially recruit an enriched sample of PD subjects with 
longer duration disease  and/or  mild  cognitive changes, subjects more likely to exhibit cholinergic deficits. Core B 
will be aided in recruitment by the Core D (Education and Outreach Core).  Appendix 1 contains a detailed schedule 
of study activities. 
 
IND Exemption: We obtained an IND exemption from the FDA.  
 
Specific Aim 1: To use [18F]flubatine PET to assess VCN occupancy of brain α4β2* nAChRs after subacute VCN 
administration to the subgroup of PD subjects with cholinergic deficits. 
Rationale and Predictions: We will use [18F]flubatine PET to select an appropriate VCN dose for SA2 and 
SA3 studies. Application of [18F]flubatine PET  will  allow  selection  of  the  lowest  dose  producing  high (>90%) 
receptor occupancy. Measuring VCN displacement of [18F]flubatine obviates the need for clinical dose escalation 
studies in hypocholinergic  PD  subjects.  α4β2*  nAChRs  are  complex  heteromeric  proteins  with highly regulated 
expression and function. These studies are essential, as the PD brain is a very different environment from a normal 
brain. We predict that that these studies will identify a VCN dose at the lower end of the clinically used range producing 
high (>90%) α4β2*  nAChR occupancy.  A recent acute VCN dose study in acutely abstinent smokers (upregulated 
α4β2*  nAChRs) with [18F]FA-85380 reported that a single 0.5 mg 
 
 
oral VCN dose (typical treatment dose = 1 mg bid) produced ~95% receptor occupancy with imaging several hours 
after ingestion (70). This result suggests  strongly  that  lower  clinical  VCN  doses  will  produce  high (>90%) 
receptor occupancy. All subjects will undergo baseline [18F]flubatine scanning after subacute VCN treatment to 
induce upregulation and following VCN washout to allow measurement of per cent VCN occupancy of upregulated 
α4β2*  nAChRs. 
Preliminary Data: Our PET Center is performing 
[18F]flubatine imaging on a routine basis (Figure 3, Table 2). 
[18F]Flubatine PET has regional distribution expected for α4β2* 
nAChRs, excellent signal to noise properties, and imaging can 
be completed in 90 minutes. Notably, regional binding values 
are highly reproducible across subjects with low  coefficients  
of  variation  (Table  2).  Procedures:  α4β2* 
nAChR expression will be measured in vivo with [18F]flubatine. Subjects will receive varying VCN doses. 
[18F]Flubatine imaging will be done 10 days after initiation of VCN administration and 5 days after VCN 
discontinuation (Figure 4). The difference between [18F]flubatine binding before and after VCN discontinuation will 
be measured to assess α4β2* nAChR occupancy. Receptor occupancy will be  assessed  in  regions exhibiting high 
α4β2* nAChR expression; thalamus, striatum, and cerebellum, as results are likely to be more variable in regions 
with lower α4β2*  nAChR expression. Based on prior animal and human studies, 7-10 days 
is adequate to induce receptor upregulation (91-95). VCN has a t1/2 of ~24 hours and 10 days will be adequate to 
attain equilibrium and induce upregulation. VCN pharmacokinetics (PK) do not change with age  and absorption is 
unaffected by food (96). Five days is adequate to clear VCN. Tmax is 2-4 hours post oral administration. The first scan 
will occur in the afternoon of day 10 after the morning VCN dose to avoid peak dose effects which could increase 
variance. 
The recommended clinical VCN dose schedule is 0.5 mg per day for 3 days, followed by 0.5 mg bid for 4 
days, followed by 1 mg bid. Mocking et al. recently reported positive neuropsychological results of subacute VCN 
 
 
 
 


 
9 
 
treatment (0.5 mg/day for 3 days, then 1 mg/day for 4 days) in normal controls (79). Because we are administering 
VCN to a clinical population and not young, healthy controls, we propose lower initial doses than Mocking et al. We 
will start with 0.25 mg/day on a subacute treatment schedule of 10 treatment days. A potential acute side effect of 
VCN is nausea, though this is unusual in non-smokers with doses below 1 mg per day. To reduce the chance of 
nausea or other side-effects, we will use a series of escalating dose schedules (Appendix 2). VCN will be prepared 
by the U-M Health System Research Pharmacy. VCN pills are small, easily encapsulated in gelatin capsules, and 
will be dispensed in blister packs to facilitate administration and compliance monitoring. The initial maximum dose 
studied will be 0.25 mg/day. Subsequent, projected maximum doses are 0.25 po bid, 0.5 mg po bid, and 1 mg po 
bid (if needed). Twice daily dosing is used to minimize  peak  dose  effects.  We  anticipate  3  dose  points,  each  
consisting  of  3  participants  (total  of  9 
 
 
 
 
 
participants), to establish the required oral dose for SA2 and SA3 experiments. The results of the initial (0.25 mg/day 
maximum dose) study will be used to select the next dose point. If there is a substantial effect on [18F]flubatine binding  
(>50% reduction), we will use the 0.25 mg  bid maximum dose schedule for the next experiment. If the effect on 
[18F]flubatine binding is <50% reduction, we will use 0.5 mg bid maximum dose schedule for the next experiment. 
Selection of the subsequent dose schedule will be based on the results of 
the 0.5 mg bid maximum dose experiment. We will increment dose schedules until there is no additional reduction in 
[18F]flubatine binding, i.e., remaining binding is non-specific binding, and the penultimate maximum dose defines our 
SA2-SA3 study dose.  Based on the work of Lotfipour et al., who found that a single oral 0.5 
mg VCN dose produced ~100% receptor occupancy (70), we don’t anticipate using the highest (1 mg po bid) dose 
schedule). We will measure plasma VCN levels at the time of the initial scan (Pharmacokinetics Core, U- M College 
of Pharmacy; see attached letter from Dr. Duxin Sun, Core Director). These measurements are not for formal PK 
studies as VCN PK is well established, but rather to confirm study drug compliance. We aim to establish the lowest 
dose producing high ~100%) receptor occupancy and if we achieve this goal after studying 2 dose schedules (6 
participants total), we will stop and proceed to SA2 experiments. 
PD Subjects will continue their customary PD treatments. Subjects will be evaluated in the University of 
Michigan Functional Neuroimaging, Cognitive and Mobility Laboratory (see Facilities & Resources).  All subjects 
will undergo a standard clinical and brief psychometric evaluation immediately prior to VCN treatment initiation, at 
the time of initial scan and at the time of the second scan. Subjects will undergo a MDS-UPDRS, Geriatric Depression 
Scale, Montreal Cognitive Assessment, WAIS Digit Span and Digit Symbol coding tests, and D-KEFS executive 
function/working memory tests. Subjects will be screened for suicidality with the Columbia Suicide Severity Rating 
Scale (C-SSRS; http://www.cssrs.columbia.edu/) before VCN treatment and at the time of each PET study. Dr. Albin 
is certified in use of the C-SSRS (Appendix 3) and Dr. Dauer and other relevant members of the Clinical Resource 
Core will also become certified.  Subjects will be  directly observed for 3 hours after their initial VCN dose, with phone 
follow-up the next day and weekly thereafter.  Subjects will be screened for history of seizures and alcohol abuse.  
The latter will be assessed with the WHO Alcohol Use Disorder Identification Test (AUDIT).  Per the WHO guidelines, 
individuals with scores >8 (ages 18-65) or >7 (ages >65) will be excluded.  Individuals with a history of seizures will 
be excluded.  Participants will be advised to refrain from alcohol use during study participation. An investigator (Dr. 
Albin or Dr. Dauer) will be on-call for subject concerns at all times.   Data will be recorded on standard CRFs and 
entered into the database for management and analysis by the Biostatistics and Data Management Core. Adverse 
events will be classified by organ system and assessed for causality. All potential possible adverse events will be 
discussed by the Udall Center Executive Committee acting as DSMB, and reported promptly to the IRB and NINDS.\ 
Following completion of the PD subject study experiment 1, we will perform a limited experiment to assess 
α4β2*  nAChR occupancy in age-matched controls.  Controls will receive VCN (no placebo) for 10 days.  We will 
select a subset of the doses used for the PD subjects in experiment 1 to assess receptor occupancy in normal 
controls.  Assuming we don’t detect very high (>90%) occupancy with our initial dose (0.25 mg/day), we will expose 
controls to one of three different dosages of varenicline to construct a dose-occupancy relationship for comparison 
with the results in PD subjects.  Based on the results of experiment 1, experiment 2 will include the following dosages 
 


 
10 
 
with a goal of 3 patients per cohort: .25mg QD, .25mg BID and .5mg BID.  All procedures will be identical to 
experiment 1.  Control subjects will be drawn from the normal control pool characterized concurrently in the Clinical 
Core of the Udall project, much as the PD subjects are drawn from the pool of PD subjects. Control subjects will also 
be recruited from the UM Health Research website if needed.  We anticipate studying 9 subjects, 3 at each dosage 
point.  To adjust for potential dropouts, we will budget 12 control subjects.  
Potential Problems and Alternatives: The results of Loftipour et al. suggest that low doses will produce high 
α4β2*  nAChR occupancy (70). Commercially available VCN pills are small and division beyond bisection is likely to 
be inaccurate. 0.25 mg/day is the lowest feasible dose and if we find this dose produces high per cent receptor 
occupancy, no further testing will be necessary. VCN tolerability may be a problem as this agent has not been tried 
specifically in PD subjects previously. To avoid any problems, we are starting with low doses and using an 
incrementing schedule. Nausea was infrequent in a recent trial in AD which  used significantly higher doses (89). If 
tolerability is an issue, then we will alter the incrementing schedule to more 
gradually “ramp” up to the desired dose. 
 
Specific Aim 2: To assess the effects of pharmacologic α4β2* nAChR activation on laboratory measures of gait 
function and postural control in PD subjects with cholinergic deficits. 
Rationale and Predictions:   The central hypothesis of this project is that deficient activation of α4β2* 
nAChRs consequent to the degeneration of cholinergic projections causes balance and gait abnormalities in PD. 
Consequently, restoration of α4β2* nAChR stimulation should improve these deficits. In this target engagement 
pharmacodynamic experiment, we will evaluate the effects of the α4β2* nAChR partial agonist VCN on laboratory 
measures of gait function and postural control in hypocholinergic PD subjects. We predict that subacute VCN 
administration will improve gait function and postural control in  hypocholinergic  PD subjects.  We chose a pair of 
relevant physiologic endpoints; gait speed and Horak’s JERK (97,98,99).  Slow gait is a cardinal sign of PD and we 
demonstrated recently that slowed gait is  strongly  associated  with neocortical cholinergic denervation (56). Similarly, 
Rochester et al. found that gait speed in PD correlated with 
an indirect clinical electrophysiologic measure of cortical cholinergic function  (100).  Slowed  gait  is  also  a feature 
of our combined dopaminergic-cholinergic rat model (32). We will  apply  the  Instrumented  test  of postural Sway 
(ISway; APDM  Mobility  Lab;  http://apdm.com/gait-and-posture/Mobility-Lab/index.html),  a relatively simple and 
robust method for evaluating postural control with a belt-worn inertial sensor (97,98,99). ISway was developed 
specifically to assess postural control in PD subjects  and  is  validated  against  more complex methods. 
Abnormalities of these types of measures are associated in both cross-sectional and prospective studies with 
increased fall risk in the elderly, and measure relevant postural control phenomena in 
PD (101-104). JERK is one of several parameters measured by ISway, captures postural sway irregularity, and 
possesses strong validity, good test-retest behavior, and good correlation with UPDRS III PIGD scores (97). 
Preliminary Data: Our group implemented the ISway system in the 
Functional Neuroimaging, Cognitive and Mobility Laboratory (Table 2). We 
studied 11 similar PD subjects, 6 with a history of falls and 5 without any 
fall history.  ISway  measures  of  postural  control differentiated fallers and 
non-fallers (Table 2). 
Procedures: The SA1 experiment will select the  VCN  dose. Based 
on discussions with the proposed Biostatistical and Data Management 
co-Core leaders, we will employ a randomized double- blind, placebo-
controlled, 2-period, 2-sequence crossover design to assess the effects of 
subacute VCN administration on gait function, postural control, and 
attention in PD subjects with cholinergic deficits (Figure 5). PD participants 
will be on stable doses of dopaminergic therapy for 3 months prior to 
enrollment and continue  on their usual 
schedule of dopaminergic medications for the duration of this study. Individuals with depression and unstable cardiac 
disease will be excluded. Within-subjects crossover designs were employed successfully in recent VCN trials for AD 
and mood disorders during smoking cessation (89,90). 
We will administer VCN or placebo for 3 weeks, followed by a 3 week washout period, and crossover to 
placebo or VCN for the 3 week second treatment period. Study visits will be completed within a five business day 
window before or after the target date.  The duration of the treatment and washout periods is based partly on prior 
human experimental and  trial experience (89-95). Mocking et al. detected improved cognitive effects of VCN with a 
week of treatment (79). We chose a longer period to ensure stable receptor upregulation and because initiation of 
treatment may require gradual dose incrementing.   The washout period is based partly on work by Zhang et al. with 
another α4β2* nAChR partial agonist in MPTP lesioned non-human primates (71). This group reported an effect of 
 


 
11 
 
ABT-089 on levodopa induced dyskinesias close to 2 weeks after dose cessation, well beyond the t1/2 of this agent. 
This wash-out interval was used also in the recent VCN AD trial (89). The randomization schedule and process will 
be prepared by the Biostatistics and Data Management Core. Blinded, encapsulated study drug (VCN and 
placebo) will be supplied by  the  U-M Research Pharmacy to the study coordinator. The latter will dispense the study 
medication. Subjects will be evaluated in the University of Michigan Functional Neuroimaging, Cognitive and Mobility 
Laboratory (see Facilities and Resources). Subjects will be directly observed for 3 hours after initial VCN dose and 
with phone follow-up calls 1-2 business days after starting the medication treatment period, and weekly thereafter. 
Follow-up phone calls will be completed within a two business day window before or after the target date.  VCN 
plasma levels will be evaluated at end of both treatment periods on days 22 and 64 through a blood draw to monitor 
compliance. Blood samples will be stored at the University of Michigan Functional Neuroimaging, Cognitive & Mobility 
Laboratory until the study is finished. Data will be recorded on standard eCRFs  and  entered  into  the database for 
management and analysis by the Biostatistics  and Data  Management Core. Adverse events will be classified by 
organ system and assessed for causality. All potential possible adverse events will be discussed by the Executive 
Committee, and reported promptly to the IRB and NINDS. 
Gait speed and postural control will be assessed at baseline, at the end of the first VCN/placebo 
administration period, at the end of the washout period, and at the end of the second VCN/placebo administration 
period. Gait and postural control will assessed by the Clinical Resource Core (Core B) using the Mobility Lab system 
that is already established (see Table 3; methodology for Mobility  Lab  will  be  as described in detail in Project II). 
MDS-UPDRS, Geriatric Depression Scale, Montreal Cognitive Assessment, WAIS Digit Span and Digit Symbol 
coding tests, and D-KEFS executive function/working memory tests will be administered as part of the evaluations. 
Subjects will be screened for suicidality with the C-SSRS before VCN treatment, weekly during phone follow-up, and 
at the end of treatment-washout periods. 
Sample Size: The main basis for the sample size for this 2-sequence, 2-period, 2-treatment crossover study is based 
on gait speed and the postural control parameter, ACC JERK. Both endpoints are considered primary and so a 
Bonferroni adjustment will be made to control the Type I error for the study at the one-sided 
1.5 level (i.e., each endpoint will be tested at the 0.025 level). Published (56) and preliminary data in PD subjects 
(Table 2) provide estimates of effect and variability for these parameters in the placebo group. We powered our study 
based on conservative estimates derived from these data (i.e., using the 95%  upper confidence bound of the SD as 
the estimate of variability in the sample size calculations) and the desire to detect  at  least  a  10%  relative  treatment  
effect  in  gait  speed  and  60%  relative  treatment  effect  in  ACC 
JERK. With a total of 33 subjects, we have at least 80% power to detect a treatment difference between VCN and 
placebo of 0.112 m/s and -0.131 m2/s5 with a Bonferroni-adjusted two-sided Type I error  of  2.5%, assuming within-
subject correlation of at least 0.64 and 0.72, respectively (a conservative estimate given that 
test-retest correlation of ACC JERK in PD subjects was 0.86[97]). We will enroll 42 subjects to allow for a 10% attrition 
rate. We will examine some pre-defined secondary endpoints; RMS (root mean square of center of pressure 
variability), gait speed variability, and anterior-posterial sway velocity. These secondary endpoints are chosen on the 
basis of published data with ISway (97), and preliminary data (Table 2), and  additional preliminary data suggesting 
association of gait speed variability and  anterior-posterior  sway  velocity  with deficits of specific cholinergic pathways 
(Project II preliminary data). We will use a gatekeeper testing strategy to control the familywise error for the 
secondary endpoints that play a supportive role in interpreting the effects of VCN (105,106). If at least one of the 
primary endpoints is significant (at least one of the hypotheses is rejected), hypotheses for the secondary endpoints 
will be tested using Hochberg’s step-up method (107). This approach ensures the overall Type I error rate is no 
greater than 0.05. 
Analysis Method. For all aims, we will conduct exploratory analyses to examine the distributions of outcomes 
under each treatment, as well as individual and mean profiles over time. Graphical approaches such as boxplots and 
scatterplots with linear or non-linear (e.g., loess) methods will be used, allowing identification of outliers, linearity, and 
correlation of measurements within person and across time. If outcome data do not appear normally distributed, 
transformations will be employed. Because participants’ outcomes will measured at more than one time point per 


 
12 
 
person, our analyses will be based on linear mixed effects models (108-110). We expect no carryover since 
treatments will be spaced three weeks apart. Tests to compare treatments will be based on Wald and chi-squared 
tests based on maximum likelihood or restricted maximum likelihood methods. Alternative covariance structures 
(other than compound symmetry) will also be explored and model fit examined using the Akaike Information Criteria 
(AIC). 
Potential Problems and Alternatives: All measures proposed for this experiment are well established in the 
University of Michigan Functional Neuroimaging, Cognitive and Mobility Laboratory and the Clinical Resource Core. 
The major foreseeable difficulties are subject recruitment and retention. We’ve had considerable success recruiting 
and retaining subjects in protocols involving up to 4 PET studies, serial evaluations, long-term follow-up (months to 
years),  and  subject  clinical-psychometric-gait  &  balance evaluations as intensive as those proposed. We budget 
a 10% drop out rate in this study, which is typical of our experience in prior prospective studies and identical to the 
dropout rate in the recent AD study (89). A related concern would be unexpected difficulty with VCN tolerability, 
leading to excessive dropouts.   The goal of the SA1 experiments is to find the lowest dose providing high α4β2* 
nAChR occupancy, which provides protection against this possibility. In the recent AD study, VCN was tolerated well 
at higher doses than we expect to employ. If tolerability is a problem, we would use a slower dose escalation regimen. 
It is possible that there will be differential effects of VCN on gait speed and postural control. We will be drawing 
these subjects from Project II, where all participants will receive anatomically resolute [18F]FEOBV imaging. As 
discussed in Project II, there may be correlates between specific pathway deficits, such as BF projection versus PPN 
projection degeneration, or even more specific pathway changes such as PPN  to cerebellar vermis projection 
deficits, and specific gait or postural control measures (see Project II Preliminary 
Data). We will have a rich dataset to examine these possibilities, including not only imaging data but also the 
considerable gait and balance data generated by the Mobility Laboratory system for exploratory analyses. It is 
possible that VCN will have no effect on gait, balance, or cognition in these subjects. This would  be disappointing 
but important as it would steer the field towards other types of cholinergic receptors as therapeutic targets. 
Comparison of these results with parallel Project I experiments will also be important for evaluating the novel model 
developed in that Project. 
 
 
Specific Aim 3: To assess the effects of pharmacologic α4β2* nAChR activation on a measure of attentional function 
(SAT; sustained attention test) in PD subjects with cholinergic deficits. 
Rationale and Predictions: Our preclinical work with a novel rodent model of cholinergic-dopaminergic gait 
deficits indicates that diminished attentional function following loss of corticopetal BF afferents is a key mediator of 
the effects of cholinergic system degeneration on gait and balance (Overview; Project 1). A large body of prior 
research indicates attentional function impairments contribute significantly to impaired gait and postural control, 
including falls, in normal elderly and PD patients (33-39). In a recent large prospective study, for example, attentional 
function deficits predicted falls in older adults (39).   Dr. Sarter’s extensive prior work 
indicates  that  α4β2*   nAChRs  are  crucial  actors  in  cholinergic  modulation  of  attention  (Project  1)  (42-46). 
Consistent with this work, our preliminary preclinical data suggests that stimulation of α4β2* nAChRs improves gait 
and balance in the novel dual lesion model (Project 1). These combined results lead to an important prediction: 
subacute α4β2* nAChR administration to hypocholinergic PD subjects will improve attentional performance and 
improved attentional performance will correlate with improved gait  and  postural  control induced by α4β2* nAChR 
stimulation. Evaluating this prediction is important for assessing the mechanisms by which cholinergic projection 
system degeneration impairs gait and balance and is important also for assessing the validity of the novel rodent 
model presented in Project 1. 
Preliminary Data: Dr. Sarter’s  group  developed  a  robust  test  of attentional 
function, the Sustained Attention Test (SAT), for use in rodent experiments, and 
subsequently adapted and validated the SAT for use in humans (Project 1) (111-
113). Briefly the SAT consists of a randomized sequence of cued and non-cued (or 
signal and nonsignal) trials. Responses are hits and misses, and correct rejections 
and false alarms, respectively. Hits and correct rejections are rewarded. SAT 
performance reflects frontal  cortical cholinergic  signaling  through  α4β2*    nAChRs.     
We  evaluated  a  particularly 
relevant version of the SAT including a distractor component (dSAT), in PD subjects 
who had previously undergone [11C]PMP imaging to assess cholinergic projection 
systems integrity. There was considerable variation in dSAT performance, which was 
a linear function of cortical cholinergic terminal integrity 
(Figure 6). These results indicate that dSAT performance in PD directly reflects cortical 
cholinergic signaling, likely mediated by α4β2*  nAChRs. 
 
cortical
 
cholinesterase 
ith
dSAT


 
13 
 
Procedures: This experiment will be conducted in parallel with those for SA2. 
Attention will be evaluated with the dSAT at all evaluation points in the double-blind 
crossover design VCN intervention study described above. We will correlate dSAT 
changes in response to VCN with gait speed and postural control changes in 
response to VCN. The sample size is determined from the goals of SA2. With this 
sample size, an effect size of 0.4 can be detected with at least 
80% power and a two-sided Type I error of 0.05 assuming a within-subject  correlation  of  0.69.  Similar statistical 
methods as those used for SA2 will be employed to test the impact of VCN on dSAT. An exploratory analysis will be 
conducted to examine medication of treatment effects on gait and other postural outcomes by dSAT.  In the presence 
of a treatment effect, a variable is declared a mediator if it complies with three things: it is affected by the treatment, 
it predicts the endpoint of interest, and the treatment effect is attenuated when the potential mediating factor is 
adjusted for. Mediation analysis will be conducted by including mediating factors in the mixed model regression 
analysis outlined above. Structural equation models will be used to estimate the direct effect of VCN and the indirect 
effects of the VCN due to mediators, as has been recommended for intervention studies of disease prevention 
(114,115). 
Potential Problems and Alternatives: All procedures, including the dSAT, are well established in our center. 
Most of the potential problems are discussed above (SA2 Potential Problems and Alternatives). Our prediction is that 
improved dSAT performance will correlate with improved gait speed and postural  control measures. There may be 
dissociations between results of dSAT testing and  gait  speed-postural  control outcome measures. Attentional 
performance may improve but not gait speed and postural control. Gait speed and postural control may improve 
without commensurate improvements in dSAT  performance.  These outcomes would be important for evaluating our 
understanding of how cholinergic deficits degrade gait and postural control. Lack of correlation would direct future  
research  away  from  attentional  mechanisms  and towards  alternative  mechanisms,  such  as  PPN  projection  
roles  in  sensorimotor  integration.    We  will  have 
[18F]FEOBV  data  on  cholinergic  projection  systems  integrity  for  all  these  subjects  and  secondary  analyses 
looking at specific functional deficits and specific pathway changes may be revealing.   Whether or not dSAT 
performance response correlates with improvements in gait speed and postural control measures is  also important 
for evaluating the validity of the dual lesion model. It is  crucial  to  know  the  limitations  of  this interesting model 
prior to using it extensively for preclinical evaluations of potential therapies. 
 
Standard Methods: 
MRI and VMAT2 PET Imaging, Standard PET Methods and Image Processing: See Clinical Resource 
Core.  Flubatine  PET  Imaging:  [18F]Flubatine  will  be  prepared  in  high  radiochemical  purity  (>95%)  with  a 
method developed by our group (68). PET scans will be acquired using a protocol consisting of 18 frames acquired 
over 90 min following bolus injections of 8 mCi [18F]flubatine. Individual image frames within each scan  will  be  co-
registered  to  frame  10  (10-15  min  post-injection)  to  account  for  patient  motion.Full  kinetic 
modeling requires arterial blood sampling and >two hours of imaging, impractical for this patient population and we 
will use simpler analyses. Our primary analysis will use an average of the 70-90 min scan data, when tissue TACs 
are relatively constant, approaching equilibrium (Figure 3). Venous blood samples will be drawn at 70 and 90 min, 
plasma extracted and counted in a NaI well-counter. Plasma metabolites will be assessed to determine the 
radioactivity concentration of authentic flubatine. Radiotracer binding measures will be obtained by normalizing the 
PET tissue concentrations to the authentic venous plasma radiotracer concentration averaged over the  70 and  90 
min  samples. This  measure of tracer binding  approximates  the total tissue volume of distribution relative to plasma, 
VT. This method is well suited to quantification of flubatine binding as flubatine undergoes minimal catabolism, has 
only moderate plasma protein binding, and equilibrates instantaneously between blood and plasma (116). A 
secondary analysis will use the simplified reference tissue model (sRTM) (117). SRTM assumes a reference tissue 
devoid of specific binding sites, used in lieu of an arterial plasma input function to estimate tissue binding potentials. 
Since no gray matter tissue is devoid of specific binding sites for flubatine, we will test white matter  as  a  reference  
tissue.  White  matter  reference regions will be defined on co-registered MR volumes, and segmented with FreeSurfer 
to produce white matter masks. Binary masks, smoothed to PET resolution with white matter defined by all brain 
voxels with a mask value >0.9 after smoothing, will assure that reference region voxels have >90% contribution from 
white matter. 
Gait Speed and Postural Control Methods: Gait and postural control will be assessed in the Functional 
Neuroimaging, Cognitive and Mobility Laboratory with Mobility Lab (APDM, Portland, OR) systems  now standard in 
our center (see Project II, SA2 Preliminary Data). The postural control and gait assessments are usually completed 
in approximately 45 minutes. These studies will be performed in the Clinical Resource Core using methodology 
described in detail in Project II. 


 
14 
 
dSAT:  The dSAT will be administered as described previously (108-110).  A PC with E-Prime software is 
used to present either a visual signal or no cue in the middle of the screen followed after a constant delay by an 
auditory signal to indicate via key pushing whether or not a visual signal was presented. Using different keys, 
participants indicate both signal presentation and non-presentations. Participants receive feedback on response 
accuracy with a small financial reward for correct responses on each trial. Signal durations and inter- trial intervals 
are varied.  In the distractor condition, the background screen alternates between silver and black at 10 Hz.  This 
task requires minimal training and reliable data is obtained in a single session.  The SAT score is calculated from all 
outcomes. 
Future Directions: These experiments are likely to lead in several useful directions. Demonstration that α4β2* 
nAChR stimulation improves physiological measures of gait function and postural control in hypocholinergic PD 
subjects will spur investigation of α4β2* nAChR agonists for treatment of gait and postural control problems in PD. 
nAChR pharmacology is an active area of industrial R&D with compounds going to phase II trials and coming to 
market. With the preclinical methods exemplified by the novel model featured in Project I and methods used in this 
project, we would be well positioned to evaluate other α4β2* nAChR agonists or other cholinergic agents as adjunctive 
treatments in PD. We would aggressively evaluate additional promising compounds. Our group would move an α4β2* 
nAChR agonist into clinical trials for PD, preferably in phase IIa trials. Tthe development and validation of convenient 
methods to identify the subset of  PD  patients  with cholinergic deficits (see Project II and Clinical Resource  Core), 
and the relatively non-invasive physiologic methods used to evaluate α4β2* nAChR stimulation in this study  would  
be  valuable  components  of  such studies. Our group, with large clinic populations, strong clinical  expertise,  an  
institutional CTSA  (Michigan Institute for Clinical & Health Research; MICHR), and strong expertise in both the basic 
and clinical pharmacology of cholinergic agents would be well positioned to lead the development  of  novel  
cholinergic therapies for PD. We would pursue these studies outside the Udall Center mechanism with 
complementary R01, U01, translational R21, or NeuroNext applications. 
 
 
Project III: Protection of Human Subjects 
 
i) Human Subjects 
Description of subject populations: Subjects will include 57 PD subjects and 12 control subjects. 
 
Recruitment: Subjects will be recruited from the UMHS and VAAAHS Movement Disorders Clinics, the Clinical 
Resource Core and with the assistance of the Education and Outreach Core. The UMHS Movement 
Disorders clinic  follows a population of approximately 2,000 clinically well-defined patients with PD and/or 
parkinsonism and will be the main source of recruitment of patients for this study. Dr. Albin, Co-Director of the 
UMHS Movement Disorders Clinic, will be in charge of UM patient recruitment. In addition, subjects will be 
recruited from the VAAAHS Movement Disorders clinic, directed by Dr. Bohnen, and currently follows 
approximately 250 well-defined subjects with PD. At present, we  recruit  4-6 subjects with PD for imaging 
studies at UM per month. We anticipate that about some participants for this study can be recruited from the 
concluding P01 NS15655 (Kirk Frey, PI) that studied about 4 subjects with PD per month, most of whom have 
longer duration of disease. Further information about recruitment and retention are described in the Clinical 
Resource Core and the Education and Outreach Core. 
 
Inclusion Criteria: 
1. PD diagnosis will be based on the United Kingdom Parkinson's Disease Society Brain Bank Research Center 
(UKPDSBRC) clinical diagnostic criteria. We will enrich the cohort using the following strategies:  recruiting 
subjects at modified Hoehn and Yahr stages 2 or higher (from data collected in a medication “OFF” state in 
UDALL project 2), recruiting subjects with a duration of motor disease 5 years or longer, recruiting subjects 
that are age >65 years, or recruiting subjects with the PIGD phenotype. Duration of motor disease will be 
defined as the time between onset of motor symptoms and time of entry into the study. The PIGD phenotype 
is defined as described previously. PD subjects with defined cholinergic deficits will be recruited as described 
in Project II. PD subjects will have cortical cholinergic deficits based on 5th percentile cutoff of the normal 
controls as defined previously. 
2. PD Patients with stable dopaminergic replacement therapy for 3 months prior to enrollment and expected to 
maintain stable dopaminergic therapy for duration of study participation. 
 
OR 
 


 
15 
 
3. Normal healthy control individuals age 45 years and older without any history of neurological disease. 
 
 
Exclusion Criteria: 
1. Other disorders which may resemble PD with or without dementia, such as vascular dementia, normal 
pressure hydrocephalus, progressive supranuclear palsy, multiple system atrophy, corticobasal ganglionic 
degeneration, or toxic causes of parkinsonism. Prototypical cases have distinctive clinical profiles, like 
vertical supranuclear gaze palsy, early and severe dysautonomia or appendicular apraxia, which may 
differentiate them from idiopathic PD. The use of the UKPDSBRC clinical diagnostic criteria for PD will 
mitigate the inclusion of subjects with atypical parkinsonism and all participants will undergo [11C]DTBZ PET 
to confirm striatal dopaminergic denervation. 
2. Subjects on neuroleptic, anticholinergic (trihexiphenidyl, benztropine), or cholinesterase inhibitor drugs. 
3. Current or previous (within last 6 months) use of any product or medication containing nicotinic agents, 
including use of tobacco products such as cigarettes, cigars, pipes, chewing tobacco, etc., e- cigarettes, 
OTC nicotine patches, chewing gum containing nicotine, or varenicline. 
4. Evidence of a stroke or mass lesion on structural brain imaging (MRI). 
5. Participants in whom magnetic resonance imaging (MRI) is contraindicated including, but not limited to, 
those with a pacemaker, presence of metallic fragments near the eyes or spinal cord, or cochlear implant. 
6. Severe claustrophobia precluding MR or PET imaging 
7. Subjects limited by participation in research procedures involving ionizing radiation. 
8. Pregnancy (test within 48 hours of each PET session) or breastfeeding. 
9. Significant risk of cardiovascular event. 
10. Active, significant mood disorder. 
11. History of seizures. 
12. Active alcohol abuse.  
 
ii) Sources of Materials 
Information gathered specifically for this research project includes test results from clinical test scales, 
standardized neuropsychological measures and from experimental cognitive tests, neurobehavioral rating 
scales, clinical motor data, questionnaires, standardized interview, and data derived from the PET studies that 
reflect the amount of dopaminergic nerve terminals, cholinergic nerve terminals, and  nicotinic receptors in the 
brain, and from structural MRI. 
 
iii) Potential Risks 
Confidentiality of Research Information: The research data to be collected from subjects will consist of 
confidential information relating to clinical, neuropsychological, mood, partial genotype, neuroanatomical, and 
neurochemical functions. These research data are not intended for entry into the subjects’ clinical medical 
records. However, the data remain potentially discoverable. This may lead to violation of privacy and 
embarrassment of subjects. 
 
Clinical, neuropsychological and Behavioral Testing: Risks in regard to the neuropsychological, behavioral, and 
motor assessments are limited to fatigue, frustration and momentary embarrassment that may occur when one 
experiences difficulty performing a task or learning a new skill. 
 
PET scans & Venipuncture: Insertion of a catheter for intravenous injection of the PET radiopharmaceutical may 
be commonly associated with slight pain or bruising at the puncture site and rare chance of infection. 
Participation in this research study will involve exposure to radiation associated with the PET transmission scans 
and the administration of the radioactive drugs. Subjects participating in experiments involving PET imaging will 
undergo 2 [18F]Flubatine PET studies. These subjects will have previously undergone [11C]DTBZ and 
[18F]FEOBV PET and in some subjects undergoing [18F]Flubatine PET may be studied in the same year as they 
undergo DTBZ and FEOBV PET, for a total of 4 PET studies in 1 year. Maximum total dosimetry for any subject 
would be 15 mCi for DTBZ, and 8 mCi for each FEOBV and flubatine study. Total exposure will be well below 
the annual radiation exposure (5 rem) permitted to radiation workers by federal regulations. Participants will be 
instructed to void two hours post injection to minimize bladder exposure. This radiation dose is not expected to 
produce any harmful effects, although there is no known minimum level of radiation exposure for non-radiation 
workers considered to be totally free of the risk of causing genetic defects or cancer. The risk associated with 
the amount of radiation exposure participants receive in this study is considered low and comparable to every 
day risks. In the event of incomplete imaging or technical failure, it may be necessary to repeat the [18F]Flubatine 


 
16 
 
PET scan.  We will not exceed radiation dose limits.  No PET studies will be performed on pregnant or potentially 
pregnant women, as confirmed by pregnancy testing. The use of these PET tracers is considered to be generally 
safe and effective as approved by the University of Michigan Radioactive Drug Research Committee in 
accordance with Food and Drug Administration regulations (21 CFR 361.1). Adverse reactions to tracers used 
in this study have not been reported. However, the possibility exists for a rare reaction to any of the drugs or 
procedures to which the participant will be exposed. ACLS-certified staff will be in attendance at all times during 
the study and an emergency cart will be in close proximity. Other physical risks involve possible muscle aches 
from lying still. Medical intervention will be furnished by UMHS if serious adverse effects would occur. 
 
Participants will be asked to provide a blood sample on two separate visits.  There is an infrequent risk of 
bruising, bleeding, infection or soreness associated with the venipuncture, similar to the risks associated with 
routine blood testing.  Participants may feel dizzy or lightheaded or may rarely even faint when the needle is put 
in or taken out. 
 
Mood Disorder: In anecdotal reports, VCN has been associated with increased risk of significant mood disorder, 
including worsening mood, suicidal ideation, suicide attempts, and completed suicide. The FDA issued a “Black 
Box” warning for VCN because of concerns about these possible psychiatric side-effects of VCN. Though 
present data do not substantiate a connection between VCN administration and serious mood problems (see 
Approach), we will exclude patients with active mood disorders. 
 
Cardiovascular Risk: In anecdotal reports, VCN has been associated with increased risk of serious 
cardiovascular events, such as myocardial infarction or stroke. Though systematically collected data do not 
substantiate a connection, subjects with considerable risk for cardiovascular events will be excluded. 
 
Seizures:  In anecdotal reports, VCN has been associated with seizures.  Potential subjects with a history of 
seizures will be excluded.   
 
Alcohol Intolerance:  In anecdotal reports, VCN has been associated with intolerance of alcohol use.  Potential 
subjects with active alcohol abuse will be excluded.  
 
Sleep Disorders:  VCN use is associated with sleep disorders, including insomnia and vivid dreams.  Participants 
will be counseled about these potential side effects and instructed to contact the investigators for any concerns.  
 
Gastrointestinal Symptoms:  The primary known side effect of VCN is gastrointestinal symptoms.  Nausea, 
vomiting, and constipation are reported with VCN use. 
 
Risk of Multiple Study Participation: Being in more than one research study at the same time, or even at different 
times, may increase the risks to you.  It may also affect the results of the studies. You should not take part in 
more than one study without approval from the researchers involved in each study.   
 
Other Symptoms:  Lightheadedness has been reported with VCN use. 
 
 
iv) Adequacy of protection against risks 
Recruitment and Informed Consent: 
Subjects will be recruited from the UM and VAAAHS Movement Disorders clinics. Individuals willing to 
participate will be scheduled for the research procedures at which time the nature and risks of the procedures 
will again be reviewed with the subjects and a written informed consent form will be obtained by one of the study 
investigators. 
One copy of the signed consent form will be given to the subject, one will be placed in the patient’s medical 
record and a third will be kept in the patient’s study binder kept at the patient’s research site. Study visits will be 
conducted in the UM Functional Neuroimaging, Cognitive & Mobility Laboratory. Imaging procedures will be 
performed at the University of Michigan Health System. 
 
Protection Against Risk: 
Confidentiality of Research Information: The possibility of unintended disclosure of medical or research data is 
minimal, but not entirely impossible. We will employ stringent safeguards against unintended and inappropriate 
discovery and dissemination of personal medical and research data in our subjects  by a  multi-layered 
approach.   All data bearing potential subject identifiers will reside solely in locked files in the offices of the study 


 
17 
 
investigators or study coordinator. Original data collection documents will be maintained in secure files under 
the control of the investigators or study coordinator. Entries regarding details of the research project and its 
results will not be submitted to clinical medical databases. Electronic databases in the project will employ subject 
codes that cannot be linked directly to participants without a “key”, possessed only by the study investigators in 
a secure location, and maintained separately from the databases. Primary databases will not be housed on 
systems with Internet access, preventing unauthorized intrusions. Personal information that would directly 
identify study subjects will not be used in any publications or presentations resulting from this research study, 
unless separate written permission is given by the subject (or proxy). Any superfluous records will be shredded. 
 
Clinical, neuropsychological and Behavioral Testing: Care will be taken to minimize distress. In addition to a 
lunch and afternoon rest break, subjects are given multiple short breaks during the clinical testing day when 
needed. Subjects will be addressed in a courteous manner that does not infringe the patient’s dignity. Physicians 
and trained personnel with considerable experience working with older individuals and patients with PD will 
perform all evaluations and administer tests. These individuals are prepared to respond to patient anxiety, 
concern and other behavioral changes as appropriate to the situation. Offering breaks and reassuring subjects 
will further minimize risks when necessary. If needed for some subjects, the clinical testing protocol can be split 
over 2 days. The UM Functional Neuroimaging, Cognitive and Mobility laboratory has facilities to allow rest  
breaks when needed. The investigators presently have similar ongoing studies with 1-day clinical testing and 1-
day imaging protocols in similar PD populations. Excepts when subjects are severely demented, our PD 
participants who are typical septuagenarians or octogenarians have no difficulties completing these protocols. 
 
PET Scan & Venipuncture: A nuclear medicine technologist will perform the PET studies under the supervision 
of a physician who is a specialist in nuclear medicine. A physician or nurse will be available at all times during 
the study, and any adverse reactions will be treated immediately. A fully equipped medical cart is located in the 
Nuclear Medicine suite. We will use aseptic techniques and highly trained personnel to minimize the risks 
associated with venipuncture. No PET studies will be performed on pregnant or potentially pregnant women, as 
confirmed by pregnancy testing within 48 hours prior to each PET imaging session. In order to lessen the pain 
and fatigue of lying still, the subject will be given a break outside of the scanner midway through the procedure. 
 
Mood Disorders: All potential participants will be screened formally for mood disorder and suicidal ideation with 
the Geriatric Depression Scale (GDS) and Columbia Suicide Severity Rating Scale (C-SSRS). Individuals with 
GDS scores > 5 will be excluded at the discretion of the PI. Similarly, any potential participants describing 
significant suicidal ideation (scores of 4 or 5 on the relevant questions of the C-SSRS) will be excluded. Mood 
and suicidality will be evaluated regularly during the study (see Approach; and Schedule of Activities, Appendix) 
with the GDS and C-SSRS. Individuals with worsening GDS scores will be evaluated by the investigators 
promptly and referred for psychiatric evaluation if clinical interview substantiates concerns about significantly 
worsening mood disorder. Significant suicidal ideation will result in emergent psychiatric evaluation. VCN 
administration will  be stopped immediately in cases of worsening mood or suicidal ideation. Mood disorder risks 
will be discussed with potential subjects and the “Black Box” labeling disclosed explicitly.  All subjects will be 
instructed to page the investigators with concerns at any time. 
 
Cardiovascular Disease Events: Individuals with history of stroke (excluded under general exclusion criteria) or 
myocardial infarction within 1 year, unstable angina, or active PVOD will  be  excluded. Subjects will be 
counseled about risk of cardiovascular events and instructed to page the investigators with any concerns. Any 
suggestions of cardiovascular disease – chest pain, claudication type pain, TIA – will result in VCN 
discontinuation and emergent evaluation. 
 
Seizures:  Individuals with a history of seizures will be excluded.  Participants will be counseled about possible 
seizure risk and instructed to page the investigators with any concerns.  Any suggestions of seizures will result 
in VCN discontinuation and emergent evaluation. 
 
Alcohol Interactions:  Individuals with active alcohol abuse will be excluded.  The Alcohol Use Disorders 
Identification Test (AUDIT) will be used to screen for alcohol abuse.  Individuals scoring over the recommended 
cutoffs (>7 for >65 years; >8 for 18-65 years) will be excluded.  Participants will be counseled to refrain from 
alcohol during study participation.  
 
Sleep Disorders:  VCN use is associated with sleep disorders, including insomnia and vivid dreams.  Participants 
will be counseled about these potential side effects and instructed to contact the investigators for any concerns.  


 
18 
 
 
Gastrointestinal Symptoms:  VCN will be initiated at a very low dose and the dose increased over 2-3 days.  
Individuals who are not able to tolerate the full dose of VCN will be reduced to the next lowest dose as needed.  
If they are still not able to tolerate the VCN then the VCN will be stopped and the patient will be withdrawn from 
the study. 
 
Study Monitoring Plan:  (please see section vii below). 
 
 
v) Potential Benefits of the Proposed Research to the Subjects and Others 
In the course of this study patients will receive clinical evaluations including neuropsychiatric testing and imaging 
studies. No direct immediate benefit of these studies is anticipated. Although generally the results of these 
examinations will not be made available to their treating  physician, if a significant, unexpected abnormality is 
detected this will be reported to the patient and his/her physician. 
 
vi) Importance of the Knowledge to be Gained 
Data obtained from this research may become important for the development of improved methods of reducing 
gait and balance impairments in PD and other disorders. Such mechanisms would provide a rational basis for 
targeted intervention of gait and balance impairments in PD and other disorders. 
 
vii) Study Monitoring Plan 
We obtained an IND exemption from the FDA.   Study safety will be monitored in 2 complementary ways.  A 
study monitor from the MICHR IND/IDE Investigator Assistance Program (MIAP) will monitor the study 
according to standard procedures once experiment 2 begins. We impaneled an independent DSMB board; Dr. 
Stephan Taylor (Dept. of Psychiatry), Dr. William Gehring (Dept. of Psychology), and Dr. Wen Ye (Dept. of 
Biostatistics).  The DSMB will review the study annually during experiment one, then every 6 months during 
experiment two.  The PIs will be responsible for monitoring any break in confidentiality and for reporting any 
adverse events following University of Michigan IRB guidelines. For purposes of this study, an AE is defined as 
any unfavorable or unintended change in structure, function, signs, or symptoms temporally associated with 
participation in this study, whether or not a causal relationship with the study has been established. Clinically 
significant abnormalities may be considered an AE if deemed appropriate by the PIs.  Unexpected worsening 
of a pre- existing condition is also considered an AE, as is the discovery of an abnormal finding during physical 
exam that was not included in the medical history. Breaches of confidentiality will be considered related to the 
research whenever they occur and will be reported. Withdrawals from the study and the reason for these 
withdrawals will also be reported. The PIs are in daily contact with the Project research staff testing the  
participants,  scoring  and entering data, and will monitor their procedures to ensure that confidentiality is 
maintained. The PIs will ensure that the DSMB and IRB are notified of any adverse event following the IRB 
guidelines. Expected and unexpected serious (including fatal) adverse reactions and major unresolved disputes 
between the research investigator(s) and the research participant or between research investigator(s) will be 
expeditiously reported to the IRB of the University of Michigan. At the time of renewal, the IRB will be provided 
with a summary indicating the frequency of the monitoring, cumulative adverse event data, information regarding 
participant safety or ethics changes, confidentiality issues, benefit-to-risk changes and recommendations on 
continuing, changing or terminating the study. The sponsor, NINDS, will also be notified of any adverse events 
simultaneously with the DSMB and IRB. 
 
Costs and Payments: Subjects will not be charged for their participation in this study. Subjects will be 
compensated for time and effort with $200 for completing the initial clinical evaluations and $400 upon 
completion of the imaging or VCN administration procedures. Subjects will receive $100 to defray housing and 
meal costs (for subject and caregiver) as we anticipate a number of subjects will have at least one overnight 
stay, and $50 to defray travel costs (fuel and parking costs). 
 
 
Project III: Inclusion of Women and Minorities 
 
Inclusion of Women: 
Women will be included in this research project. Males and females will be given equal priority in recruitment. 
However, because PD affects males more than women we expect to recruit a higher percentage of males. The 
PI will monitor recruitment  of  women  to  this  project  throughout  the  study,  and  institute procedures to 


 
19 
 
enhance the enrollment of women, if numbers are not adequate. See the Targeted/Planned Enrollment Table. 
 
Inclusion of Minorities: 
See the Targeted/Planned Enrollment Table for this project. Enrollment targets are based on population 
estimates of the 2010 US census for the state of Michigan and Washtenaw County (location of UMHS) with 
some modifications. For Michigan; 78.9% White, 14.2% African-American, 0.6% Native American, 2.4% Asian, 
0% Hawaiian & Pacific Islander, 1.5% Other Race, 2.3% Two or More Races; 4.4% Hispanic/Latino, 95.6% Not 
Hispanic or Latino. For Washtenaw County; 74.5% White, 12.7% African- American, 0.3% Native American, 
7.8% Asian, 0% Hawaiian & Pacific Islander, 1.5% Other Race, 2.3% Two or More Races; 4.0% Hispanic/Latino, 
96% Not Hispanic or Latino. Hispanic/Latino residents tend to be recent immigrants and younger than the 
population average, so we project enrollment of fewer Hispanic/Latino participants than the Hispanic/Latino 
proportion of state/county population. Similar considerations apply to Asians. Some epidemiologic evidence 
suggests that PD is less prevalent among African-Americans, so we project fewer African-American enrollees 
than the proportion of African-Americans in our state/county. The Education and Outreach Core will assist with 
Minority recruitment. 
 
 
Project Ill:  Inclusion of Children 
 
Justification for Exclusion of Children: 
No children will be included. PD occurs predominantly in late-life and, indeed, the risk of developing PD 
increases with increasing age. In rare cases with  clear familial inheritance,  the onset  of the  disease may occur in 
the 30's and 40's, but there is no evidence of clinical PD  in  children.  Therefore, the research  topic to be studied is 
not relevant to children. 


Contact PD/PI: Dauer, William, T 
Project-003 (613) 
 
OMB Number: 0925-0002 
 
Planned Enrollment Report 
 
 
Study Title: 
Project III: a4b2* nAChRs, Gait, and Balance in PD 
 
Domestic/Foreign: 
Domestic 
 
Comments: 
 
 
Racial Categories 
Ethnic Categories 
Total 
Not Hispanic or Latino 
Hispanic or Latino 
Female 
Male 
Female 
Male 
American Indian/Alaska Native 
0 
0 
0 
0 
0 
Asian 
0 
0 
0 
0 
0 
Native Hawaiian or Other Pacific Islander 
0 
0 
0 
0 
0 
Black or African American 
1 
0 
0 
0 
1 
White 
28 
38 
1 
1 
68 
More than One Race 
0 
0 
0 
0 
0 
Total 
29 
38 
1 
1 
69 
Study 1 of 1 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Tracking Number:                    Funding Opportunity Number: RFA-NS-14-003. Received Date                                                                        20


 
21 
 
 
 
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SAP_000.pdf: 
Udall P3E3 SAP version 1.0 
01AUG2019 
Page 1 of 11 
 
Udall P3E3 Statistical Analysis Plan 
 
TRIAL FULL TITLE 
Cholinergic Mechanisms of Gait Dysfunction in Parkinson’s 
Disease – Proj#3, Experiment 3 
CLINICALTRIALS.GOV 
NCT NUMBER 
04403399 
SAP VERSION 
1.0 
SAP VERSION DATE 
01AUG2019 
TRIAL STATISTICIAN 
Cathie Spino, DSc 
PROTOCOL VERSION 
(SAP ASSOCIATED 
WITH) 
Protocol Version 9.0 
April 17, 2018 
TRIAL PRINCIPAL 
INVESTIGATOR 
Roger L. Albin, MD 
SAP AUTHOR(s) 
Cathie Spino, DSc 
 
 


Udall P3E3 SAP version 1.0 
01AUG2019 
Page 2 of 11 
1 
Table of Contents 
1 
Table of Contents 
2 
2 
Introduction 
3 
2.1 
Preface 
3 
2.2 
Scope of the analyses 
3 
3 
Study Objectives and End points 
3 
3.1 
Study Objectives 
3 
3.2 
End points 
3 
4 
Study Methods 
4 
4.1 
General Study Design and Plan 
4 
4.2 
Inclusion-Exclusion Criteria and General Study Population 
5 
4.2.1 
Inclusion Criteria 
5 
4.2.2 
Exclusion Criteria 
5 
4.3 
Randomization and Blinding 
6 
4.4 
Study Assessments 
6 
5 
Sample Size 
7 
6 
General Analysis Considerations 
7 
6.1 
Timing of Analyses 
7 
6.2 
Analysis Populations 
7 
6.2.1 
Intention to Treat Population 
7 
6.2.2 
Compliant Population 
7 
6.2.3 
Safety Population 
7 
6.3 
Covariates and Subgroups 
7 
6.4 
Missing Data 
7 
6.5 
Interim Analyses and Data Monitoring 
8 
6.6 
Multiple Testing 
8 
7 
Summary of Study Data 
8 
7.1 
Subject Disposition 
8 
7.2 
Protocol Deviations 
8 
7.3 
Demographic and Baseline Variables 
8 
7.4 
Treatment Compliance 
9 
8 
Efficacy Analyses 
9 
8.1 
Primary, Secondary and Exploratory Efficacy Analyses 
9 
9 
Safety Analyses 
9 
9.1 
Adverse Events 
9 
9.2 
Deaths, Serious Adverse Events and other Significant Adverse Events 
10 
9.3 
Other Safety Measures 
10 
10 References 
10 
 
 
 


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2 
Introduction 
 
2.1 
Preface 
Falls and other abnormalities of gait and balance are common in Parkinson’s disease (PD), are largely 
levodopa unresponsive, and are major contributors to morbidity and mortality. The central theme of 
the University of Michigan Udall Center is that degeneration of basal forebrain (BF) and 
pedunculopontine (PPN) cholinergic projections, in the context of degraded striatal motor control, 
contributes significantly to PD gait and balance deficits. Our preclinical work identifies attentional 
deficits, secondary to loss of cortical cholinergic afferents, as a key mechanism through which 
cholinergic deficits contribute to impaired gait and balance. The goal of this pilot target 
engagement/pharmacodynamic clinical study is to assess α4β2* nicotinic cholinergic receptors 
(nAChRs) as a therapeutic target for improving gait, balance, and attentional capacity in PD.   
 
To demonstrate the α4β2* nAChRs are appropriate therapeutic targets inPD, it is necessary to study 
key pharmacokinetic-pharmacodynamic features of α4β2* nAchR in the context of the degenerating, 
hypocholinergic PD brain, a pathologic environment in which they may exhibit unique features.  This 
personalized medicine approach focuses our studies on hypocholinergic PD subjects.  We will assess 
α4β2* nAChR features using Positon Emission Tomography (PET) imaging with the α4β2* nAChR 
ligand [18F]flubatine, subacute administration of the α4β2* nAChR partial agonist varenicline (VCN), 
and laboratory measures of gait, balance, and attention.  We will perform a pharmacodynamics study 
with subacute VCN administration to determine if α4β2* nAChR stimulation improves laboratory 
measures of gait function, postural control, and attentional function in hypocholinergic PD subjects. 
 
2.2 
Scope of the analyses 
These analyses will assess the efficacy and safety of varenicline in comparison with placebo, 
addressing the primary and secondary objectives of the study.   
 
3 
Study Objectives and End points 
 
3.1 
Study Objectives 
 
Primary Objective 
• 
To assess the effects of pharmacologic α4β2* nAChR activation on laboratory measures of 
gait function and postural control in PD subjects with cholinergic deficits. 
 
Secondary Objectives 
• 
To assess the effects of pharmacologic α4β2* nAChR activation on a measure of attentional 
function (SAT; sustained attention test) in PD subjects with cholinergic deficits. 
 
Exploratory Objectives 
• 
To assess the effects of pharmacologic α4β2* nAChR activation on additional measures of 
motor, cognition, and behavior in PD subjects with cholinergic deficits. 
 
 
3.2 
End points 
All efficacy end points are assessed at the end of each treatment period (days 22 and 64). 
 
3.2.1 
Primary Efficacy End points 
• 
JERK (postural measure based on time-based derivative of lower trunk accelerations) 
• 
Gait speed under normal pace and no dual task conditions  
 
3.2.2 
Secondary Efficacy End points 
• 
Sustained attention test (SAT) 
 


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3.2.3 
Exploratory Efficacy End points 
• 
Movement Disorder Society Unified Parkinson’s Disease Rating Scale, part III (MD-
UPDRSIII; “on” state) 
o Total score 
o Postural instability and gait disorder (PIGD) subscore 
• 
Gait measures 
o Gait speed under fast pace and no dual task conditions 
o Gait speed under normal pace and dual task conditions minus gait speed under normal 
pace and no dual task conditions 
o Gait speed under fast pace and dual task conditions minus gait speed under fast pace 
and no dual task conditions 
o Cadence under normal pace and no dual task conditions 
o Cadence under fast pace and no dual task conditions 
o Mean stride length under normal pace and no dual task conditions 
o Mean stride length under fast pace and no dual task conditions 
o % coefficient of variation (CV) stride length under normal pace and no dual task 
conditions 
o %CV stride length under fast pace and no dual task conditions 
o  Mean stride time under normal pace and no dual task conditions 
o  Mean stride time under fast pace and no dual task conditions 
o  %CV stride time under normal pace and no dual task conditions 
o  %CV stride time under fast pace and no dual task conditions 
o Mean stride width under normal pace and no dual task conditions 
o  Mean stride width under fast pace and no dual task conditions 
o  %CV stride width under normal pace and no dual task conditions 
o  %CV stride width under fast pace and no dual task conditions 
• 
Postural measures 
o Mean sway velocity 
o Root mean square sway distance (Sway RMS) 
o Timed Up and Go (iTUG) 
• 
Cognitive measures 
o Montreal Cognitive Assessment (MoCA) score 
o Wechsler Adult Intelligence Scale (WAIS-III) Digit Symbol score 
o California Verbal Learning Test (CVLT) Short Term Memory score 
o CVLT Long Term Memory score 
o CVLT Recognition score 
o Delis-Kaplan Executive Function System (D-KEFS) Stroop III (interference) score 
o D-KEFS Sorting total score 
o D-KEFS Verbal Fluency – Letters total score 
o D-KEFS Verbal Fluency – Animal score 
o Judgment of Line Orientation (JOLO) score 
o Trail Making Test (TMT) 4 score 
o Distractor Sustained Attention Test (dSAT) score 
• 
Behavioral measures 
o Geriatric Depression Scale (GDS) 
o Questionnaire for Impulsive-Compulsive Disorders in PD (QUIPs)  
 
 
4 
Study Methods 
 
4.1 
General Study Design and Plan 
This study is a single-center, randomized placebo-controlled double-blind crossover study of VCN in  
participants with PD to assess its effects on measures of gait, balance, and cognition.  Participants 
were randomized 1:1 to one of two treatment sequences:  placebo followed by VCN 0.5 mg b.i.d., or 


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VCN followed by placebo.   Participants received an initial 0.25 mg dose or equivalent placebo 
following baseline evaluations and were monitored for 4 hours after initial study medication 
administration with total daily dose or equivalent placebo escalated over the next 2 days.  Treatment 
periods were 3 weeks in duration and interrupted by a 3-week washout period.  Participants 
underwent a standard evaluation at baseline, at the end of the first treatment period (week 3 [day 22]), 
at end of the washout period-beginning of the second treatment period (week 6 [day43]), and at the 
end of the second treatment period (week 9 [day 64]). 
 
The schema below describes the main elements of the study design: 
 
 
 
 
4.2 
Inclusion-Exclusion Criteria and General Study Population 
 
4.2.1 
Inclusion Criteria 
1. PD diagnosis will be based on the United Kingdom Parkinson's Disease Society Brain Bank 
Research Center (UKPDSBRC) clinical diagnostic criteria. We will enrich the cohort by 
recruiting subjects at modified Hoehn and Yahr stages 2 or higher, duration of motor disease 5 
years or longer, age >65 years, or the PIGD phenotype. Duration of motor disease will be 
defined as the time between onset of motor symptoms and time of entry into the study. The 
PIGD phenotype is defined as described previously. PD subjects with defined cholinergic 
deficits will be recruited as described in Project II. PD subjects will have cortical cholinergic 
deficits based on 5th percentile cutoff of the normal controls as defined previously. 
2. Stable dopaminergic replacement therapy for 3 months prior to enrollment and expected to 
maintain stable dopaminergic therapy for duration of study participation. 
 
4.2.2 
Exclusion Criteria 
1. Other disorders which may resemble PD with or without dementia, such as vascular dementia, 
normal pressure hydrocephalus, progressive supranuclear palsy, multiple system atrophy, 
corticobasal ganglionic degeneration, or toxic causes of parkinsonism. Prototypical cases have 
distinctive clinical profiles, like vertical supranuclear gaze palsy, early and severe 
dysautonomia or appendicular apraxia, which may differentiate them from idiopathic PD. The 
use of the UKPDSBRC clinical diagnostic criteria for PD will mitigate the inclusion of subjects 
with atypical parkinsonism and all participants will undergo [11C]DTBZ PET to confirm striatal 
dopaminergic denervation. 
2. Subjects on neuroleptic, anticholinergic (trihexiphenidyl, benztropine), or cholinesterase 
inhibitor drugs. 
3. Current or previous (within last 6 months) use of any product or medication containing nicotinic 
agents, including use of tobacco products such as cigarettes, cigars, pipes, chewing tobacco, 
etc., e- cigarettes, OTC nicotine patches, chewing gum containing nicotine, or varenicline. 
4. Evidence of a stroke or mass lesion on structural brain imaging (MRI). 
5. Participants in whom magnetic resonance imaging (MRI) is contraindicated including, but not 
limited to, those with a pacemaker, presence of metallic fragments near the eyes or spinal cord, 
or cochlear implant. 
6. Severe claustrophobia precluding MR or PET imaging 
7. Subjects limited by participation in research procedures involving ionizing radiation. 
8. Pregnancy (test within 48 hours of each PET session) or breastfeeding. 
9. Significant risk of cardiovascular event. 


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10. Active, significant mood disorder. 
11. History of seizures. 
12. Active alcohol abuse.  
 
4.3 
Randomization and Blinding 
Participants were randomized 1:1 to one of two treatment sequences: placebo followed by VCN 0.5 
mg b.i.d., or VCN followed by placebo.  A statistician prepared the randomization list using permuted 
blocks with random block sizes.  The list with randomization number and treatment allocation was 
sent to the research pharmacy and a blinded list of randomization numbers was sent to the study 
coordinator.  After patient consent was completed and eligibility confirmed, the coordinator assigned 
the next randomization number to the participant, and sent a prescription with participant ID and 
randomization number to the research pharmacist who dispensed the appropriate study medication. To 
mask drug, VCN pills or placebo were encapsulated in gelatin sheaths. 
 
4.4 
Study Assessments 
 
4.4.1 
Primary Efficacy Assessments 
Two co-primary end points were selected:   a measure of postural stability and gait function. 
JERK is the time-based derivative of lower trunk accelerations during standing spontaneous sway.  
JERK was chosen because it tracks postural instability in PD [1]. Normal pace gait speed with no 
distractors was chosen as prior studies indicated that neocortical cholinergic denervation is associated 
with slower gait speed in PD [2].  We hypothesized that VCN treated participants would ambulate 
faster and that VCN treatment would reduce JERK.   
 
Postural stability was assessed with the Ambulatory Parkinson's Disease Monitoring (APDM) 
wearable sensor system (APDM Wearable Technologies, Inc.) using the iSWAY protocol, with 
participants standing on a foam pad with eyes open and eyes closed. Standard postural measures, 
including JERK, were assessed and calculated using the manufacturer’s software (Mobility Lab 
Version 1).  Gait analysis was performed on an 8-meter GAITRite pressure sensitive walkway (CIR 
Systems, Inc.) and standard parameters were analyzed using ProtoKinetics Movement Analysis 
Software (GAITRite version 5.09C; ProtoKinetics, LLC). Gait assessments were repeated with a dual-
task protocol in which participants counted backwards by three starting at a random number (under 
100) provided by the examiner.   
 
4.4.2 
Secondary Efficacy Assessments 
We assessed attentional function with a Sustained Attention Test (SAT), established to reflect CNS 
cholinergic systems function in humans [3, 4, 5].  The SAT is performed with 2 conditions: without 
and with a distractor (dSAT).  SAT and dSAT results are reported as the vigilance index, a measure 
that corrects estimates of accurate detection with penalties for false detections and not confounded by 
errors of omission [6].  SAT was selected as the second end point.  
 
4.4.3 
Exploratory Efficacy Assessments 
Additional assessments of postural control, gait function and attentional function were measured as 
exploratory end points.  Cognitive assessments were also measured for exploratory analyses.  
 
Motor Assessments: Movement Disorder Society Unified Parkinson’s Disease Rating Scale, part III 
(MD-UPDRSIII; “on” state); MDS-UPDRSIII postural instability and gait subscore (PIGD) subscale 
score (sum of items 3.1, 3.9-3.13); Gait Speed (normal pace); Gait Speed (fast pace); Gait Speed 
(normal pace - dual task); Gait Speed (fast pace – dual task); Postural stability measures – mean sway 
velocity, JERK, root mean square sway distance (RMS).  To assess the effects of attentional loading, 
normal pace and fast pace gait were performed under dual task conditions.   Dual task conditions 
typically lead to slower gait speed.  Differences in gait speed between dual task and no dual task 
conditions are a measure of the attentional burden imposed by the dual task.  To assess the effects of 
VCN on this aspect of gait performance, we compared the differences between no dual task and dual 
task gait speed between VCN and placebo treatment periods.  We used the APDM system’s iTUG 


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(Timed Up and Go) protocol to collect additional exploratory data.   
 
Cognitive Assessments: For cognition, we used a general cognitive measure, the MoCA and selected 
tests to examine major cognitive domains but focusing on attention and executive function.   MoCA; 
Wechsler Adult Intelligence Scale-III Digit Symbol modalities test; CVLT short term memory test; 
CVLT long term memory test; CVLT recognition test; D-KEFS Stroop III; D-KEFS sorting total; D-
KEFS verbal fluency letters total; D-KEFS verbal fluency animals; D-KEFS Trail Making Test 4; 
JOLO test.   
 
Behavioral Assessments:  GDS  
 
5 
Sample Size  
The sample size (planned initially at four participants per dosing group) for the VCN- α4β2* nAChR 
occupancy study was based on logistical considerations.  For the Crossover study, we calculated that 
33 participants would provide at least 80% power to detect within-patient treatment differences of 
0.122 m/s in gait speed and -0.131 m2/s5 for JERK, assuming within-participant correlation of > 0.64 
and > 0.72, respectively, using a paired t-test and a two-sided Type I error of 0.025 (Bonferoni 
adjustment for co-primary endpoints).  This approach is conservative given our analysis method uses 
mixed effects models. Estimates for treatment differences were based on Bohnen et al. [2] for normal 
pace gait speed and Mancini et al. for JERK [7]. 
 
6 
General Analysis Considerations 
 
6.1 
Timing of Analyses 
The final analysis will be performed after all randomized participants have completed their 9-week 
visit or dropped out prior to their 9-week visit, all corresponding data have been entered, cleaned, 
locked and unblinded as per the data coordinating center’s standard operating procedures. This 
statistical analysis plan document was finalized prior to the database lock and unblinding. 
 
6.2 
Analysis Populations 
All randomized participants will be used in the analyses of subject disposition. 
 
6.2.1 
Intention to Treat Population 
The main analysis set for efficacy is the intention to treat (ITT) population, defined as all participants 
randomized.  
 
6.2.2 
Compliant Population 
A compliant population is used for sensitivity analyses of the efficacy end points.  It is defined as the 
ITT population, excluding all participants who have major protocol deviations.  Major protocol 
deviations are defined as eligibility criteria violations for which no exemption was granted, and 
without evidence of significant increases in VCN plasma levels between placebo and VCN treatment 
periods. 
 
6.2.3 
Safety Population 
The Safety Population is defined as all participants who are randomized and receive at least one dose 
of study drug.  The Safety Population will be used for all safety analyses, as well as demographic and 
baseline analyses.  
 
6.3 
Covariates and Subgroups 
There are a limited number of covariates that will be incorporated in statistical models in our analyses 
because of the relatively small sample size in each treatment group:  the baseline outcome measure.  
We will not impute missing values for baseline covariates. 
  
6.4 
Missing Data 
We will summarize the extent of missing data over time for the primary end points.  We will 


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investigate the missing data mechanism (missing at random, not missing at random), which is 
important for the validity of our analytic approaches, through exploratory analysis. Exploratory 
analyses will include plots of the mean profile of end points at weeks 0, 3, 6, and 9 by treatment 
sequence for those who have complete data throughout the study and those who don’t. 
 
The primary analysis of the primary end points assumes a missing at random mechanism.  If data are 
not missing at random, we will use a multiple imputation approach within the pattern-mixture model 
framework [8].  The imputation models, applied sequentially for each missing data pattern, will 
include baseline value, treatment group, and demographic variables (age and gender), allowing for the 
dependence of later time points on earlier time points.  If there are issues with over-fitting, the 
demographic variables will be omitted. The analysis model will include the same covariates used in 
the primary analysis of the primary end points and will incorporate the uncertainty due to imputation 
in the calculation of the standard error, as described by Rubin [9]. 
 
6.5 
Interim Analyses and Data Monitoring 
No formal interim analyses were planned nor carried out for this study.  The study was overseen by a 
Data and Safety Monitoring Board (DSMB) that reviewed the pooled and by-treatment subject 
disposition, study conduct and safety data approximately every 6 months.   
 
6.6 
Multiple Testing 
Two-sided p-values will be reported.  For the co-primary endpoints, a Bonferonni adjustment for 
multiplicity will be applied.  For all other endpoints, no adjustments for multiplicity will be made.  
Thus, p-values for secondary and exploratory outcomes will be interpreted with caution (note that this 
is a departure from the protocol).  Means and standard errors will be provided to summarize treatment 
differences for efficacy end points. 
 
7 
 Summary of Study Data 
Descriptive summary statistics will be derived for all data at baseline, separately by treatment 
sequence and overall.  For efficacy, exploratory and safety data, data will be presented by 
treatment group.  Treatment group will be characterized as “Varenicline” and “Placebo”; for 
pooled summaries, “Overall” will be used as the column heading.  All tables will be annotated 
with the total population size relevant to that table/treatment, including any missing observations. 
 
For continuous variables, mean, standard deviation, median, interquartile range, minimum and 
maximum will be reported.  For categorical variables, number and percentages will be reported 
(excluding missing values).  Graphical methods will be heavily used in this pilot study to assess the 
pattern of response over time for key variables and to assess the relationships among variables. 
 
7.1 
Subject Disposition 
The number of participants approached for study participation, the number consented and the number 
who did not consent (including reasons:  screen failures, refusals) will be summarized in a CONSORT 
diagram.  The number of participants who dropped out prior to randomization, and the reasons for 
dropout, will be summarized.  The number randomized and treated and the number who dropped out 
by weeks 3, 6, and 9 will be provided, as well as the number in each of the analysis populations (i.e., 
ITT, Compliant, Safety).  Reasons for post-treatment dropout will be provided.    
 
7.2 
Protocol Deviations 
Major protocol deviations that exclude a patient from the Compliant Per Protocol Population are 
described in section 8.3.2.  A listing of protocol deviations that exclude participants from the 
Compliant Population will be provided. A listing of participants who receive exemptions for study 
eligibility will also be provided. 
 
7.3 
Demographic and Baseline Variables 
Demographic variables include:  age at consent (defined as a continuous variable, e.g., 52.6 years), 
sex, race and ethnicity.  


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Baseline is defined as pre-treatment measures.  Baseline variables include:   
• 
Age at diagnosis (years) 
• 
MDS-UPDRS III total score 
• 
GDS score 
• 
MoCA score 
 
7.4 
Treatment Compliance 
A listing of compliance with study medication (# of doses taken by day) was provided by treatment 
and period.   
 
8 
Efficacy Analyses 
 
8.1 
 Primary, Secondary and Exploratory Efficacy Analyses 
For the Crossover study, linear mixed models containing treatment sequence, treatment period, 
treatment group, and dependent-variable baseline value, with participant within treatment sequence as 
a random effect, will be used for analysis of continuous outcomes.  To compare differences between 
VCN and placebo, a test for carryover based on the sequence effect will be conducted using patient 
with sequence as the error term.  Results will be presented as least squares (LS) mean and standard 
error (SE).  The co-primary endpoints will be tested at the 2-sided, 0.025, significance level. All other 
tests will be based on a 2-sided significance level of 0.05; no adjustments will be made for additional 
multiple comparisons.  Primary, secondary and key efficacy endpoints will be analyzed in all 
randomized participants (intention-to-treat [ITT] population) and in the Compliant Population.  
 
Additional sensitivity analyses will be performed for UPDSR-III total and PIG subscale scores, where 
baseline degree of total cortical cholinergic denervation (from PET scans) and baseline degree of 
striatal dopamine denervation (from PET scans) were incorporated as covariates in separate models.  
These models will also be run for gait speed end points. 
 
9 
Safety Analyses 
Safety endpoints will be analyzed in all randomized participants who received at least one dose of 
study medication (Safety Population). We will include adverse events that occurred in the washout 
period with the treatment given in period one.   
 
Safety data, including adverse events (AEs) and the Columbia-Suicide Severity Rating Scale (C-
SSRS), will be summarized descriptively by treatment group for the Safety Population. 
 
9.1 
 Adverse Events 
Treatment-emergent adverse events are AEs that start on or after the first study day treatment is 
administered. The causal relationship of the AE to the study drug is determined by the site 
investigator as “not related” or “related”.  Adverse event severity grades are reported according to the 
Common Terminology Criteria for Adverse Events (CTCAE) Version 4.0. If the CTCAE does not 
have a grading for a particular adverse event, the severity of the event is reported by the investigator 
as mild, moderate, severe, or very severe.  In the case of multiple occurrences of the same AE within 
the same subject, AEs will be summarized according to the maximum severity reported for each body 
system and overall.  
 
Descriptive summary statistics for treatment-emergent AEs will be reported.  The number of 
treatment-emergent AEs and the frequencies (number and percentage) of participants with one or 
more treatment-emergent AE will be summarized by treatment group, overall, by severity, and by 
body system.  Coding of adverse events into body system was performed by the study chair for 
adverse events and by the medical monitors for serious adverse events.  All treatment-emergent AEs 
related to study drug will be summarized, as will the frequencies of participants with one or more 
treatment-emergent AE related to study drug.  Similarly, all treatment-emergent AEs causing study 


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discontinuation, and frequencies of participants experience these, will be summarized. 
 
A subject listing of all treatment-emergent AEs and treatment-emergent AEs causing study 
discontinuation will be presented.  
 
In accordance with clincaltrial.gov reporting requirements, the following table summarizing adverse 
events is required and will be provided: 
● Other (Not Including Serious) Adverse Events: A table of anticipated and unanticipated 
events (not included in the serious adverse event table) that exceed 5% within either treatment 
group, grouped by organ system, with number and frequency of such events in each treatment 
group. 
 
AEs that occurred after consent and before treatment will be listed. 
 
The summary statistics will be produced in accordance with section 9. 
 
9.2 
Deaths, Serious Adverse Events and other Significant Adverse Events 
Descriptive summary statistics for treatment-emergent serious adverse events (SAEs) will be reported.  
The number of treatment-emergent SAEs and the frequencies (number and percentage) of participants 
with one or more treatment-emergent SAE will be summarized by treatment group, overall and by 
body system.  Coding into body system was performed by the data coordinating center for SAEs.  All 
treatment-emergent SAEs related to study drug will be summarized, as will the frequencies of 
participants with one or more treatment-emergent SAE related to study drug.  Similarly, all treatment-
emergent SAEs causing study discontinuation, and frequency of participants experiencing these, will 
be summarized.  
 
A subject listing of all treatment emergent SAEs, SAEs causing study discontinuation, and deaths will 
be presented. 
 
In accordance with clinicaltrials.gov reporting requirements, the tables below summarizing deaths and 
SAEs are required: 
● All-Cause Mortality:   A table of all anticipated and unanticipated deaths due to any cause, 
with number and frequency of such events in each treatment group. 
● Serious Adverse Events: A table of all anticipated and unanticipated serious adverse events, 
grouped by organ system, with number and frequency of such events in each treatment group. 
 
The summary statistics will be produced in accordance with section 9. 
 
9.3 
 Other Safety Measures 
Listing of C-SSRS significant suicidal ideation (suicidal ideation >2) will be provided. 
 
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