Modulation of GABA-A Receptors and Axial Motor Impairments in Parkinson Disease

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HUM00130361 Mod. of GABA-A Receptors and Axial Motor Imp. in PD Protocol V2.0 9.1.2017

Protocol V 2.0 9.1.2017

University of Michigan IRBMED Approval 9.14.2017 HUM00130361

NCT: Pending

R01 NS099535

Primary Investigator: Nicolaas Bohnen, MD, PhD Professor of Radiology and Neurology University of Michigan

HUM00130361 Mod. of GABA-A Receptors and Axial Motor Imp. in PD Protocol V2.0 9.1.2017

Modulation of GABA-A Receptors and Axial Motor Impairments in Parkinson Disease

Protocol v 2.0 9.1.2017

SPECIFIC AIMS

Axial motor impairments represent a significant cause of disability in Parkinson disease (PD). Dopaminergic treatments typically only provide limited or no relief of these symptoms [1, 2]. Postural instability and gait difficulties (PIGD) motor features are mediated by widespread neural networks that extend well beyond the nigrostriatal system. The role of γ-aminobutyric acid-A (GABA) has received limited attention in human PD research, despite the fact that main outflow structures of the basal ganglia largely employ inhibitory GABA to connect to areas outside the basal ganglia. Abnormally increased GABA activity from these nuclei has been demonstrated previously [3, 4]. We have preliminary in vivo [11C]flumazenil brain PET data that shows that reduced cerebral GABAA receptor availability (likely reflecting increased synaptic or extra- cellular GABA-ergic activity) is associated with more severe PIGD motor features in PD. These observations support the recent notion that excessive GABA-ergic inhibition of the basal ganglia outflow structures may be a critical mechanism underlying axial motor impairments in PD [5]. Flumazenil is a short-acting modulator of the GABAA receptor benzodiazepine binding site, which has shown to rapidly improve motor impairments in PD [6, 7]. Flumazenil, however, can only be reliably given intravenously [7]. Clarithromycin is an FDA-approved and orally available macrolide antibiotic. There is emerging in vitro and in vivo evidence that clarithromycin can act as a negative allosteric modulator of brain GABAA receptors [8]. Our preliminary clarithromycin-[11C]flumazenil PET non-human primate data demonstrate a dose-dependent 11 delay in late dissociation and washout of [ C]flumazenil compatible with the proposed GABAA negative allosteric modulator function [9]. The overarching goal of this project is to perform GABAA receptor biomechanistic and target engagement studies to examine the effect of GABAA receptor modulation on PIGD features in PD. Our main hypothesis is that modulation of cerebral GABAA receptor activity improves dopamine-resistant PIGD motor features in PD, that can be predicted by [11C]flumazenil PET biomarker findings. The use of i.v. flumazenil will allow direct testing of our hypothesis to provide biomechanistic proof of concept. The use of clarithromycin will allow us to explore the possibility that this orally available drug may demonstrate evidence of target engagement of the GABAA receptor resulting in improvement of axial motor impairments in PD.

Specific Aim: To perform a PET biomechanistic GABAA receptor study with detailed quantitative mobility assessments before and 1 hour after the i.v. administration of 1 mg flumazenil and placebo (in a counter- balanced sequence) in PD subjects (n=30, net recruitment) across a range of disease severity (Hoehn and Yahr stages 2-4 [10]). Baseline [11C]flumazenil and vesicular monoamine transporter type 2 (VMAT2) 11 [ C]DTBZ brain PET imaging will be performed prior to drug administration to assess for GABAA receptor availability and the integrity of nigrostriatal dopaminergic nerve terminals, respectively.

Main hypothesis: Flumazenil administration results in improved motor function in PD, including PIGD features, 11 with greater effects present in patients with lower pre-treatment [ C]flumazenil GABAA receptor availability.

Exploratory Aim: To perform a GABAA receptor target engagement with detailed quantitative motor assessment and [11C]flumazenil PET imaging before and after 1 week of oral clarithromycin vs. placebo administration (clarithromycin 250 mg p.o. b.i.d.x 3 days, then 500 mg p.o. b.i.d. x 4 days, n=20; versus placebo p.o. b.i.d., n = 10; total net recruitment =30) in PD subjects across a range of disease severity (Hoehn and Yahr stages 2-4 [10]). Baseline [11C]DTBZ brain PET imaging will be performed to assess for the integrity of nigrostriatal dopaminergic nerve terminals.

Exploratory hypothesis: Clarithromycin modulates GABAA receptors as shown by a delay in dissociation and washout of [11C]flumazenil on post-study PET imaging and results in improved mobility functions in PD patients.

HUM00130361 Mod. of GABA-A Receptors and Axial Motor Imp. in PD Protocol V2.0 9.1.2017

Positive findings in these experiments will establish GABAA receptor system changes as important contributors to axial motor impairments in PD and may potentially open a novel direction for future clinical trials to investigate GABAA negative allosteric modulators, such as clarithromycin, as oral drug therapy for dopamine- resistant motor disability in PD, which currently represents a critically unmet need in the management of PD.

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control muscle tone and mobility functions, which ultimately manifests as akinesia (Figure 1) [3-5, 18]. There is growing, and converging evidence from animal and human studies that confirm cerebral hyperGABA- ergic activity in PD. For example, increased GABA activity from these nuclei has been demonstrated by both electrophysiological and mRNA analysis in parkinsonian animals [3, 4]. It is evident that regional imbalance of the major inhibitory CNS transmitter may have far reaching effects on neuronal network activity underlying motor impairments in PD [21].

In vivo imaging evidence of hyperGABA-ergic state in advancing PD: There are relatively few in vivo imaging reports regarding the impairment of GABAA receptors or GABA concentration in the brain in PD (see Table 1). Kawabata et al. (1997) found a significant correlation between reduced availability of GABAA receptors in the cortex as determined by [123I]iomazenil SPECT and greater motor disability in PD [24]. These findings could not be explained by flow or perfusion data indicating a specific effect of GABAA receptors rather than impaired synaptic or neuronal integrity per se. These authors also found that lower frontal, temporal, parietal and occipital cortical binding correlated with motor disability evaluated by Hoehn and Yahr disease stage [25]. There are also in vivo magnetic resonance [1H] spectroscopy (MRS) imaging studies of the GABA peak in PD. For example, Oz et al. performed 4T-MRS in PD and found elevated GABA ratios in the substantia nigra, which would be consistent with the proposed hyperGABA-ergic state, at least for the pars reticulata [26]. Emir et al. performed 7T-MRS in patients with PD and found that GABA concentrations in the putamen and the pons were significantly higher in patients than in controls [27]. More recent preliminary 3T-MRS data show evidence of elevated thalamic GABA levels that correlate with UPDRS motor ratings in PD [28]. Collectively, these GABAA receptor and GABA concentration in vivo imaging studies provide evidence for a 11 hyperGABA-ergic state in the living PD brain. Our preliminary GABAA receptor [ C]flumazenil PET data are not only supportive of these findings but also provide evidence for a more selective and robust association with axial compared to other motor impairments in PD. These observations provide the scientific rationale that GABAA receptor modulators, such as flumazenil, or negative allosteric modulators, such as clarithromycin, could normalize hyperGABA-ergic activity in PD, and in effect improve the final output flow from the basal ganglia.

Table 1. Overview of in vivo GABA study findings in PD

Study GABAA marker Main findings 123 [25] GABAA [ I]iomazenil SPECT Hoehn and Yahr staging correlated inversely with cortical binding.

Correlation between greater motor symptoms and lower cortical GABAA 123 [24] GABAA [ I]iomazenil SPECT receptors. Findings could not be explained by blood flow effects or changes in neuronal or synaptic integrity. [26] [1H] spectroscopy* (MRS) at 4T Increased GABA/glutamate ratio in the substantia nigra. [27] [1H] spectroscopy *(MRS) at 7T Increased GABA concentrations in the brainstem and putamen.

1 Increased GABA concentrations in the thalamus correlate with UPDRS [28] [ H] spectroscopy* (MRS) at 3T motor severity. *Note that these MRS studies have only examined a small and limited number of pre-set brain regions.

Flumazenil motor studies in PD: Flumazenil is a modulator of the GABAA receptor benzodiazepine binding site that is used clinically to reverse the effects of benzodiazepines. Ondo and Hunter reported open-label treatment results of low dose (0.5 mg) i.v. flumazenil in eight practically defined off PD patients and assessed UPDRS scores, bilateral 1-minute hand-tapping speed, and timed gait tests PD [6]. The timed tapping tests improved over 50 minutes compared to baseline by 4.9 ±16.0 at 10 minutes, 13.5 ± 15.7 at 20 minutes, 16.4 ± 21.0 at 30 minutes, 28.0 ± 19.3 at 40 minutes (P < 0.005) and 23.9 ± 19.1 at 50 minutes (P < 0.01). UPDRS total motor scores also improved (P < 0.05). A closer look at the individual UPDRS motor items (see Table 1, page 684, revealed that PIGD UPDRS sub-scores tended to account for most of this improvement. Changes in tapping scores also correlated very well with changes in the UPDRS motor scores (R= 0.88, P = 0.005) PD [6]. There were no adverse events. Subsequently in 2006, Ondo and Silay reported results of a double-blind, placebo controlled, single dose, crossover trial of 1 mg i.v. flumazenil and placebo in 16 subjects 5

HUM00130361 Mod. of GABA-A Receptors and Axial Motor Imp. in PD Protocol V2.0 9.1.2017 with PD [7]. Flumazenil was again well tolerated in the PD subjects. Subjects were primarily assessed with serial tapping tests (1 minute

for each hand) at 15-minute intervals for 90 minutes after infusion. Secondary assessments included the UPDRS motor part at baseline and 45 minutes after infusion. Subjects then underwent a 90-minute washout and entered

the opposite arm of the crossover. Compared to placebo, change in tapping speed compared to baseline improved throughout the 90-minute period (P < 0.0001) and at each individual time (P < 0.01), except for the first 15 minutes immediately after i.v. administration of flumazenil. There was a trend for improved UPDRS motor scores (+3.44 points) compared to placebo (+1.25 points) in this controlled study. Clinical effects plateaued from 45 to 90 minutes post-injection. These data indicate that flumazenil is well tolerated and results in meaningful motor improvements in PD.

The search for orally available GABAA receptor negative allosteric modulator drugs: Clarithromycin. In vitro electrophysiological studies using whole-cell patch clamped human embryonic kidney cells transfected with α1,β2,γ2 GABAA receptor and AAV-GFP cDNA demonstrated that the administration of clarithromycin in the presence of different concentrations of GABA inhibited GABAA currents at concentrations as low as 3 μM [29]. Nearly 50% inhibition occurred at concentrations of 300μM clarithromycin. These data show that clarithromycin inhibits GABAA currents at clinically relevant concentrations. In humans, there is emerging literature that clarithromycin is a potential treatment for hypersomnia through proposed negative allosteric modulation of GABAA receptors. Enhancement of GABA signaling has recently been demonstrated in a significant proportion of patients with central nervous system hypersomnias. Trotti et al. reported on 53 patients with idiopathic hypersomnia who were prescribed clarithromycin, in whom cerebrospinal fluid demonstrated increased GABAA receptor activity in vitro in excess of controls, suggesting the presence of a novel 'endozepine' [8]. Of these, 34 (64%) reported improvement in daytime sleepiness [8]. In those who reported subjective benefit, objective corroboration of improved vigilance was evident on the psychomotor vigilance task. Subsequently, Trotti and colleagues performed a randomized placebo-controlled cross-over clinical trial in 20 patients with GABAA-related hypersomnia and found that daytime somnolence significantly improved during a 2-week course of clarithromycin 500 mg b.i.d. p.o. [30]. Altered taste perception occurred, but this was the only side effect more common on clarithromycin than placebo. These data indicate that clarithromycin may be useful in the treatment of hypersomnia associated with enhancement of GABAA receptor function. These data posit clarithromycin as an orally available and FDA-approved drug with central CNS effects in humans with intriguing evidence of unique negative allosteric modulation of GABAA receptors.

Impact: Dopamine-resistant motor impairments, such as PIGD features represent a major therapeutic challenge in PD. The impact of elucidating the GABAA-ergic role in dopamine-resistant axial motor impairments would be considerable. Findings that GABAA receptors may play a central role in controlling PIGD motor features augur novel non-dopaminergic treatment approaches not only in PD but also in non-Lewy body types of parkinsonian disorders, such as vascular parkinsonism where excessive GABA-ergic activity has also been shown to be present by in vivo imaging and found to be a determinant of motor parkinsonism, in particular the presence of gait disturbances [31]. In addition, hyperGABA-ergic activity has been demonstrated in progressive supranuclear palsy, which is a dopamine-resistant atypical parkinsonism with severe and disabling axial motor disturbances [32].

METHODS

General overview: The overarching goal of this project is to perform GABAA receptor biomechanistic and target engagement studies to examine the effects of GABAA receptor modulation on motor impairments in PD, including PIGD features. Our main hypothesis is that modulation of cerebral GABAA receptor activity improves axial motor impairments in PD. To achieve the goals of this project, we propose to perform a PET biomechanistic GABAA receptor study with detailed and quantitative motor assessments before and 1 hour after the i.v. administration 6

HUM00130361 Mod. of GABA-A Receptors and Axial Motor Imp. in PD Protocol V2.0 9.1.2017 of 1 mg flumazenil and placebo (in a counter-balanced sequence) in PD subjects (n=30, net recruitment). 11 [ C]flumazenil GABAA receptor PET imaging will be performed prior to drug administration. In an exploratory study we propose to perform a GABAA receptor target engagement with detailed quantitative motor assessment and [11C]flumazenil PET imaging before and after 1 week of oral clarithromycin vs. placebo treatment (clarithromycin 250 mg p.o. b.i.d. x 3 days, then 500 mg p.o. b.i.d. x 4 days, n=20; and placebo p.o. b.i.d., n = 10) in PD subjects (n=30, net recruitment). If tolerability or side effects pose a problem at the higher dose, we would use the lower (500mg/day) dose. As axial motor impairment are typically more prevalent with advancing disease we plan to recruit at least 24 subjects in each study aim who have at least 1 or more PIGD motor features, such as abnormal balance, slow gait or history of falls or freezing of gait based on our sample size calculations. VMAT2 [11C]DTBZ brain PET imaging will be performed in all subjects to assess for the integrity of nigrostriatal nerve terminals.

The use of i.v. flumazenil will allow a direct testing of our main hypothesis to provide proof of concept. The use of clarithromycin in the target engagement study will allow us to investigate evidence of GABAA receptor modulation based on the delay of the [11C]flumazenil PET wash-out phase. Motor assessment for the VMAT2 11 11 [ C]DTBZ and GABAA receptor [ C]flumazenil PET, flumazenil and clarithromycin treatment studies will be performed in the morning in the dopaminergic medication 'off' state.

After obtaining informed consent, study participants will undergo clinical testing, including detailed and quantitative mobility testing, [11C]flumazenil and [11C]DTBZ PET and brain MR imaging studies. Imaging will generally be done on separate days. Patients meeting eligibility criteria will be invited for the i.v. flumazenil/placebo and/or oral clarithromycin/placebo studies. Qualified personnel who have been trained in the use of the instruments and have undergone inter-rater reliability evaluations will perform the assessments. To allow for unbiased assessments, these technicians will be blinded to the treatment status of the subjects. We will perform the neurological exam, motor assessments, and the [11C]DTBZ and [11C]flumazenil brain PET studies in the dopaminergic “off” state.

Inclusion Criteria: 1. PD (M/F; n=68 gross recruitment): PD diagnosis will follow the UK Parkinson's Disease Society Brain Bank Research Center (UKPDSBRC) clinical diagnostic criteria for PD (34). 2. Age 50 years or older 3. Hoehn and Yahr stages 2-4 (10) 4. Abnormal [11C]DTBZ PET study

Exclusion Criteria: 1. PD with Dementia (PDD) or dementia with Lewy bodies (DLB). 2. Other disorders which may resemble PD, such as vascular dementia, normal pressure hydrocephalus, multiple system atrophy, corticobasal ganglionic degeneration, or toxic causes of parkinsonism. Prototypical cases have distinctive clinical profiles, like 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. 3. Subjects on benzodiazepine, GABAB-ergic medications (baclofen, tizanidine), neuroleptic, anticholinergic (trihexiphenidyl, benztropine), or cholinesterase inhibitor drugs. 4. Evidence of a mass lesion on structural brain imaging (MRI). 5. Participants in whom MRI is contraindicated including, but not limited to, those with a pacemaker, presence of metallic fragments near the eyes, spinal cord, chest or cochlear implant. 6. Severe claustrophobia precluding MR or PET imaging. 7. Subjects limited by participation in research procedures involving ionizing radiation. 8. Pregnancy (urine pregnancy test within 48 hours of each PET session) or breastfeeding. 9. History of seizures. 10. Unstable psychiatric disorder including anxiety or panic disorder. 11. History of recent suicide attempt or overdose of tricyclic antidepressants or other medications 12. Any other medical history determined by investigators to preclude safe participation.

HUM00130361 Mod. of GABA-A Receptors and Axial Motor Imp. in PD Protocol V2.0 9.1.2017 Additional exclusion criteria for flumazenil study: 1. Allergy to flumazenil. 2. Significant liver disease. 3. History of alcohol or other substance abuse within past two years. 4. History of regular benzodiazepine use within past year.

Additional exclusion criteria for clarithromycin study 1. Allergy or hypersensitivity to clarithromycin, other macrolide antibiotics, or similar medicines such as azithromycin, erythromycin or telithromycin. 2. History of significant atrial or ventricular arrhythmias. 3. Significant liver or kidney disease. 4. QTc prolongation. 5. Subjects taking digoxin, colchicine, pimozide, cisapride, quetiapine, astemizole, terfendadine, ergotamine, or dihydroergotamine.

Additional non-exclusionary considerations for clarithromycin study: − Possible dose adjustment should be considered for subjects on certain “statin” drugs (e.g. lovastatin, simvastatin, mevastatin, and atorvastatin) due to a risk of rhabdomyolysis from impaired drug metabolism. − Possible dose adjustment should be considered for subjects on calcium channel blockers (e.g. verapamil, amlodipine, and diltiazem) due to the risk of hypotension from impaired drug metabolism, and possible bradycardia with verapamil. − Possible dose adjustment should be considered for subjects taking Class IA (quinidine, procainamide, disopyramide) or Class III antiarrythmics (dofetilide, amiodarone, sotalol) that may prolong the QT interval.

Recruitment & study enrollment targets: Subjects will be recruited from the UM and Ann Arbor VAMC. The UM movement disorders clinic follows a population of about 1,050 clinically well-defined patients with PD and will be the main source of recruitment of patients for this study. Dr. Frey, Co-Director of the Movement Disorders Division at UM, will be in charge of UM patient recruitment. Dr. Bohnen is the Director of the VAMC movement disorders clinic and follows about 240 PD subjects. At present, we recruit 6-8 subjects with PD for imaging studies per month at UM. Therefore, a recruitment requirement of about 1-2 per month for this study is not expected to be challenging.

Additional sources of recruitment are the Michael J. Fox Trial Finder website and the University of Michigan centralized web-based recruitment initiative called UM Health Research that provides an online registry for interested volunteers. This online registry will provide a way for volunteers to let studies know that they are interested in participating in their research. It will also provide study staff with a preliminary screening tool to see who may be interested, and potentially, who is eligible for their study. Volunteers can enter their contact information, basic demographics, study preferences and some medical history. Additionally, volunteers who are UM patients may also grant access to their medical record for screening purposes. Electronic data search tools at the University of Michigan will be used for additional recruitment if necessary. These include Data Direct, Emerse, i2b2, and the electronic medical record (MiChart, CareWeb). Electronic data search tools will be used only with an IRBMED-approved informed consent waiver that is granted for screening purposes. In case of slower than expected recruitment we will increase the number of PD support group lectures in our region. We have found that PD support group lectures are a great tool to recruit subjects for research studies, maximize identification of the most appropriate subjects. Ann Arbor is close to Detroit (45 minutes) and its surrounding metropolitan areas, which host multiple PD support groups. We will also be able to utilize the infrastructure of the Michigan Parkinson Foundation (MPF), the major lay organization supporting PD patients and caregivers in the state of Michigan. The MPF organizes and supports approximately 50 support groups in the state, organizes educational activities related to PD, and has a quarterly newsletter - The Messenger. The MPF mailing list comprises approximately 29,000 individuals, including individuals from northwestern Ohio and northern Indiana. The MPF is strongly supportive of clinical research on PD and related disorders. The MPF will permit us to advertise our research projects in The Messenger and will facilitate mailing of informational materials to MPF affiliated support groups.

HUM00130361 Mod. of GABA-A Receptors and Axial Motor Imp. in PD Protocol V2.0 9.1.2017 This study, to be conducted over a 4-year period, will include a net recruitment total of 60 subjects with PD (30 net subjects for each study). As part of the eligibility screening and during the course of the study we anticipate a combined exclusion and attrition rate of approximately 12%. Therefore we expect to recruit 68 total PD subjects for a net target enrollment of 60 PD subjects. Separate patients groups will be recruited for each study to avoid subject burdening with doubling of drug visits (increasing study burden) and unknown wash-out effects of each drug on motor impairments in PD. We will recruit a total of 17 PD subjects annually.

General testing procedures: Clinical assessment will consist of a general medical and neurological examination. Saliva and blood samples will be obtained for genetic and biomarker analysis.

Our primary balance measures will be assessed with the Opal sensor-based assessment (iSWAY, Mobility Lab, APDM, Inc., Portland, OR) and our primary measures for gait stability will be assessed using an electronic gait mat (GAITRite® system or equivalent Zeno Walkway System). The remainder of motor testing will include general motor assessment, including the MDS-revised UPDRS examination [33], modified Hoehn & Yahr [10], grooved pegboard [34], finger tapping [34], and foot tapping [35] to provide assessment of general motor impairments of PD. The Mini-BESTest assesses dynamic balance, and includes 14 items scored on an ordinal scale and providing sub-scores for anticipatory postural adjustments, reactive postural control, sensory orientation, and dynamic gait domains [36]. We will apply APDM sensor modules (Mobility Lab, APDM, Inc., Portland, OR) incorporated with some of the sub-tests, including the 7-meter iTUG (with sensored subtests), dynamic walking (iWALK), and iSWAY sensory standing test (eyes open/closed/ firm, foam or incline surface). Subjects on dopaminergic drugs will then be asked to take their dopaminergic medications. Dual-energy X-ray absorptiometry (DXA) will be obtained to provide estimates of bone density and lean muscle mass.

For cognitive and functional assessment we will use the PD Cognitive Rating Scale (PDCRS) [37] and Mini- Mental State Examination (MMSE) and Montreal Cognitive Assessment test [38] to provide global cognitive performance scores and also document cognitive status. We will use the PD Cognitive Functional Rating Scale (PDCFRS) to assess functional status [39] and PDQ-39 to assess quality of life [40]. Additional cognitive tasks include CVLT, Stroop task, D-KEFS verbal fluency (COWA-FAS), Sorting task, Trail Making Test, clock copy, and JOLO. Balance confidence [41], freezing of gait [42], physical activity [43], daytime sleepiness [44], apathy, depression (BDI-II), anxiety (Spielberger, [41]), and sleep questionnaires [45] will also be administered. Falls will be assessed prospectively using a 6-month diary. 7-day physical activity will be documented using actigraphy during the daytime with an actigraph device (activPAL or the equivalent Actigraph device).

Flumazenil sub-study: The i.v. flumazenil vs. placebo protocol will be performed as previously reported [7] except for a modified motor outcome test battery focusing on quantitative postural and gait measures. PD patients (net recruitment target n=30) across a range of disease severity (Hoehn and Yahr stages 2-4) will participate in this study. Subjects will be randomly assigned to receive either flumazenil (1 mg pushed intravenously over 5-10 minutes or matching placebo) by means of a computerized random number generator and provided by our statistician Dr. Ivo Dinov in a blinded fashion followed by a counter-balance sequence for the 2 visits as listed below. There will be two visits to administer i.v. flumazenil and i.v. placebo in a counter-balanced sequence: • Visit 1: clinical assessments of flumazenil vs. placebo infusion ("off" PD meds in the morning) & • Visit 2: Reverse drug / placebo ("off" PD meds at same time in the morning).

Although the study by Ondo and Silay [7] showed that flumazenil demonstrated a significant benefit starting from 30 and lasting at least through 90 minutes, we will choose the 60-85 minute time period for primary analysis of the motor variables given the apparent plateau function in the time-activity curve of the [11C]flumazenil and i.v. flumazenil infusion PET data. The test battery and time points of administration are listed in Table 2 below. The difference in performance between the outcome measure will be the 60 min test performance relative to t=0 min performance using a within-subject design [7]. The study will be performed in with on-site nursing and ACLS-certified physician and BLS-certified research staff. Vital signs will be monitored throughout the study. The study will be performed under IND 132,314 with assistance by Michigan Institute for Clinical and Health Research (MICHR) IND/IDE Investigator Assistance Program (MIAP) services. Flumazenil 9

HUM00130361 Mod. of GABA-A Receptors and Axial Motor Imp. in PD Protocol V2.0 9.1.2017 and matching placebo (saline) will be prepared and dispensed by the University of Michigan Research Pharmacy.

Table 2. Clinical test protocol for the i.v. flumazenil/placebo infusion study. Test measure & duration Pre-infusion t 0 min t 30 min t 60 min t 70 min t 90 min iSWAY- Mobility Lab ✓ ✓ ✓ ✓ Electronic gait mat ✓ ✓ ✓ ✓ Mini-BESTest (APDM sensored) ✓ ✓ PIGD-UPDRS ✓ ✓ Finger tapping ✓ ✓ ✓ ✓ ✓ ✓ Somnolence visual analog scale ✓ ✓ ✓ ✓ ✓ ✓ Pegboard testing ✓ ✓ Adverse event assessment ✓ ✓ ✓ ✓ ✓ ✓ [11C]DTBZ PET ✓ [11C]flumazenil PET ✓

Clarithromycin sub-study: Clarithromycin is approved to be given to adults in doses ranging from 250 mg–500 mg orally twice per day although doses are often given up to 1000 mg p.o. b.i.d. The 500 mg b.i.d. dosage is based on maximum FDA- approved dose and found to be also successful to reduce excessive daytime somnolence in patients with hyperGABA-ergic hypersomnolence syndromes [30]. Pharmacokinetic analysis of the parent drug and the active 14-hydroxy metabolite data suggests complete (or nearly complete) absorption of the drug after oral administration [46]. The half-life of the active 14-hydroxy clarithromycin metabolite after single oral dosing of 250 mg is 3.9-4.2 hours but increases to 6 hours after higher dose (1,200 mg) [46, 47]. There will be two groups of PD subjects participating in this study: i) clarithromycin 250 mg p.o. b.i.d. x 3 days, then uptitrated to 500 mg p.o. b.i.d. x 4 days (n=20) and ii) placebo p.o. b.i.d.(n = 10), for a net recruitment target of 30 subjects. Subjects will be randomly assigned to one of the two groups by means of a computerized random number generator and provided by our statistician Dr. Ivo Dinov in a blinded fashion. Subjects randomized to clarithromycin will take 250 mg p.o. b.i.d. for 3 days and then -if no significant side-effects - the dose will increased to 500 mg p.o. b.i.d. If the PI decides that the patient should stay on (or return to) the 250 mg p.o. b.i.d. dose, the assessments will be done as if they were on the 500mg p.o. b.i.d. dose. Apart from the baseline clinical visit for the overall study, which will also serve at the day 1 pre-treatment visit, there will be 2 separate visits for the sub-study: i) day 7 repeat testing on treatment with repeat [11C]flumazenil PET after completion of which the study drug will be discontinued and ii) day 14 visit for non-treatment follow-up to assess for possible persistent effects. The test battery and time points of administration are listed in Table 3 below. If the patient cannot undergo a repeat [11C]flumazenil PET study on Day 7 of the treatment then clarithromycin or placebo will be continued for at least 1-2 days to allow the repeat PET study. The study will be performed under IND 132,314 with assistance by Michigan Institute for Clinical and Health Research (MICHR) IND/IDE Investigator Assistance Program (MIAP) services. Clarithromycin and matching placebo (lactose in a gel capsule) will be prepared and dispensed by the University of Michigan Research Pharmacy. The 90-62 min dynamic PET time- activity curve difference in [11C]flumazenil concentration (μCi/cc), i.e., after stopping the radioligand infusion at 62 minutes, will be used for the complementary analysis to evaluate possible dose-dependent effects of clarithromycin on delayed dissociation and wash-out of the radioligand.

HUM00130361 Mod. of GABA-A Receptors and Axial Motor Imp. in PD Protocol V2.0 9.1.2017

Table 3. Clinical test protocol for the p.o. clarithromycin (250 mg bid p.o., 500 mg bid p.o., and placebo bid p.o. groups, n=10 each, net total n=30). Pre-Rx** Pre-Rx Rx Post-Rx

(> -1 day) day 1*** day 7 (day 14) iSWAY, iWALK, iTUG - Mobility Lab ✓ ✓ ✓ Electronic gait mat ✓ ✓ ✓ Mini-BESTest (APDM Sensored) ✓ ✓ ✓ MDS-UPDRS ✓ ✓ ✓ Finger/foot tapping & pegboard testing ✓ ✓ ✓ Epworth sleepiness scale & somnolence VAS ✓ ✓ ✓ Spielberger State Anxiety scale ✓ ✓ ✓ Adverse event assessment (incl. ECG and blood sample) ✓ ✓ ✓ [11C]DTBZ PET ✓ [11C]flumazenil PET (late dissociation curve)* ✓ ✓

MR imaging: MRI will be obtained for anatomic volume-of-interest (VOI) definition and partial volume correction required for the PET analysis, include unexpected pathology, and get measures of connectivity. MR studies will be acquired in all subjects on a 3 Tesla Philips Achieva. Standard T1-weighted, proton-density-weighted, and T2- weighted sequences will be obtained at isotropic spatial resolution. Following patient positioning and a sagittal survey, the following scans will be performed: T1-weighted SPGR, T2-weighted FLAIR, DTI, and rs-fcMRI.

PET Imaging: PET will be performed in 3D imaging mode using a Biograph 6 TruPoint PET/CT scanner (Siemens Molecular Imaging, Inc., Knoxville, TN), which acquires transaxial slices. A head-holder/shielding unit will be attached to the patient bed to reduce the contribution of detected photon events originating from the body outside the scanner field-of-view. Images will be corrected for scattered events also as implemented by the vendor.

10 mCi [11C]flumazenil and 15 mCi [11C]DTBZ will be prepared as described in the RDRC reviewed and approved Master Formula Card and Detail File which are on file in the University of Michigan cyclotron facility PET Chemistry laboratory. Standard procedures will be followed for [11C]flumazenil and [11C]DTBZ acquisition. Arterial and/or venous blood samples may be collected during [11C]flumazenil acquisition. A repeat DTBZ or FMZ PET scan may be obtained in case of technical failure during acquisition.

Statistical analysis: Main hypothesis: Flumazenil administration results in improved motor function in PD, including PIGD features, 11 with greater effects present in patients with lower pre-treatment [ C]flumazenil GABAA receptor availability.

For analysis of our main hypothesis we will compute the 60-min vs. time 0 flumazenil treatment scores for repeated measures ANOVA of the motor variables and correlate this with regional cerebral GABAA receptor density based on pre-treatment [11C]flumazenil PET. We will supplement this analysis by using mixed model analysis of variance (MM-ANOVA) to determine possible effects of baseline motor function and other variables in the drug vs. placebo response (see also table 4). 11

HUM00130361 Mod. of GABA-A Receptors and Axial Motor Imp. in PD Protocol V2.0 9.1.2017

For analysis of exploratory hypothesis we will perform MM-ANOVA by studying the effect of the clarithromycin groups (active drugs versus placebo) on the delayed wash-out of the radioligand (90 vs. 62 min difference in radiotracer concentration) to test the exploratory hypothesis that clarithromycin causes a delay in dissociation and late washout of [11C]flumazenil (see also table 4). For the analysis of the clinical motor effect, we will perform repeated measures ANOVA of 7-day vs. day 1 clarithromycin administration scores for the quantitative postural and gait motor variables and then regress this with regional cerebral GABAA receptor densities based on pre- treatment [11C]flumazenil PET. We will complement this analysis by using MM-ANOVA to determine possible effects baseline motor function, striatal VMAT2 nerve terminals and other variables in the drug vs. placebo response.

Table 4. Study analytics summary Hypotheses Proposed method Primary parameters Other covariate(s) Main Hy: Within-subjects repeated-measures ANOVA to Dependent: APDM Mobility Lab Striatal DTBZ determine the effect of flumazenil administration mediolateral postural sway & stride VMAT2 binding, on primary quantitative mobility measures time variability using an electronic age, compared to placebo administration, with gait mat (GAITRite® system, CIR gender, duration of baseline cerebral GABAA receptor density and Systems, Inc) [48, 49]. disease. striatal DTBZ VMAT2 binding as key covariates. Exploratory We will study the effect of clarithromycin on the 90-62 min time-activity curve Striatal DTBZ Hy: delayed wash-out of the radioligand (90 vs. 62 difference in [11C]flumazenil VMAT2 binding, min difference in radiotracer concentration) concentration (μCi/cc). age, gender, using MM-ANOVA. We will also use MM- APDM Mobility Lab mediolateral duration of disease ANOVA to determine the effect of clarithromycin postural sway & stride time administration on primary quantitative mobility variability using an electronic gait measures compared to PD patients in the mat (GAITRite® system, CIR placebo group with baseline cerebral GABAA Systems, Inc) [48, 49]. receptor availability and striatal DTBZ VMAT2 Independent: Clarithromycin binding as key covariates. administration group vs. placebo group

Post hoc analysis: we will explore alternative clinical mobility measures (total MDS-UPDRS motor score and PIGD sub-score, Mini-BESTest score) and regional cerebral GABAA receptor availability associations. Co- variate effects of levodopa-equivalent dose (LED), daytime sleepiness, cognition, function, mood, sleep, physical activity, fall and freezing of gait status and inflammatory markers (clarithromycin study).

Sample size considerations and randomization: With respect to the main hypothesis and based on prior data from i.v. flumazenil studies in PD [6] existing data are based on clinical measures, including the motor UPDRS examination. Based on these data and using the PIGD sum of the UPDRS items #27 'arising from chair', #28 'posture', #29 'gait', #30 'postural instability' and #31 'body bradykinesia and hypokinesia' where flumazenil treatment resulted in 62.5% improved scores, 37.5% stable scores and no subjects who declined, our sample size of 24 subjects with at least one PIGD motor feature will have a power of β >0.90 and α = 0.05 to find a significant flumazenil effect for improved PIGD UPDRS motor scores. These power calculations are conservative as these are based on prior clinical UPDRS ratings that suffer from linear quantitative biometric properties. Therefore, the use of quantitative postural and gait measures in this study will provide statistically more robust and reliable data for more precise analysis. Furthermore, our study population will also include 6 subjects without evidence of PIGD motor features to better study the biomechanistic effects of the drug across a wider range of PD symptomatology. Based on prior finger tapping data [6, 7] our sample will have a β >0.99 and α = 0.05 to find a significant flumazenil effect. With respect to the regression analysis between the flumazenil treatment difference scores and pretreatment GABAA receptor availability, we have no preliminary

HUM00130361 Mod. of GABA-A Receptors and Axial Motor Imp. in PD Protocol V2.0 9.1.2017 data but the general statistical goal is to keep the regression model at a level where 8-10 observations are used for each parameter estimate in the model. Our net sample size of 30 subjects fits well within this goal. Randomization will occur by computerized random number generator and will be provided by our statistician Dr. Ivo Dinov who is blinded to the study participants and is not part of the clinical assessment team.

Protection of Human Subjects: The known or expected risks will be defined as: Likely - occurring in more than 25% of people (more than 25 out of 100 persons); Common – occurring in 10% - 25% of people (in 10 to 25 out of 100 persons); Infrequent - occurring in 1 - 10% of people (1 to 10 out of 100 people); Rare - occurring in less than 1% of people (fewer than 1 out of 100 persons); Very Rare - occurring in less than 0.1% of people (fewer than 1 in 1,000 persons).

- The known or expected risks will be described in normal script. The actions that the researchers take to minimize these risks will be described in italic script, as demonstrated in this paragraph.

General risks: - There is a very rare risk of breach of confidentiality, which may affect privacy, self-esteem, social standing, employability, and insurability. Section 9.1 will provide more detailed information on how we protect your privacy. In general, study records will be kept in databases maintained by the investigators. These databases are kept separate from medical records, are protected by passwords, and only accessible to personnel involved in the study. If you withdraw from the study at any time, a record of the withdrawal and the reasons given for withdrawing may be kept as part of the study record.

- There is a rare risk that you may experience some minor anxiety (‘test anxiety’), become worried, or have an anxiety reaction in response to any of these tests and procedures. For example, you become worried about your health, or you may experience a sudden fear of the confined space while in the scanner. Trained research staff will conduct all tests and procedures. The staff will be prepared to respond to your anxiety, concerns and behavioral changes, by temporarily suspending testing, breaking up testing sessions into several brief visits if needed, and/or answering your questions. During the PET and MRI scans you will be able to talk to technologists throughout the scan and indicate right away if you wish to stop the study and leave the scanner.

- There is an infrequent chance that you may develop "freezing" symptoms when you do not take your dopaminergic medication, which is caused by stiffening of your muscles making movements more difficult. You may require additional assistance from your caretaker during this time. Any risk of adverse effects will be minimized by careful supervision during test procedures that are being conducted when are withdrawn of your dopaminergic medications. You will be instructed to resume taking your dopaminergic medications after the completion of the testing. If the withdrawal of medication is intolerable, you may resume taking medication and withdraw from this study at any time.

- During the course of the study you may receive several injections to draw blood samples (during clinical testing or the PET scan) or inject the flumazenil medication or the PET radiotracers through intravenous catheters. There is an infrequent risk of bruising, bleeding, infection, or soreness at the injection site. There is a very rare risk for infection. There is a rare risk that you may feel dizzy, lightheaded, or faint after an injection. Blood drawing or injections will be performed by a certified and experienced research technician who is also trained in blood borne pathogens control. Aseptic techniques will be used in accordance with University of Michigan guidelines. You can lie down if you feel dizzy, lightheaded or faint after an injection.

- None of the test results, brain images, and procedures in this study will be reviewed or interpreted for making a medical diagnosis. For example, there is the potential that the MRI scan may reveal an abnormality that is already in your body, such as a cyst or tumor. Any result or abnormality that would 13

HUM00130361 Mod. of GABA-A Receptors and Axial Motor Imp. in PD Protocol V2.0 9.1.2017 be indicative of current or future disease will most likely not be discovered. Many such abnormalities are not clinically significant, but you may need or want to investigate them further. Such a finding might require additional studies, and maybe even treatment, which would not be paid for by the investigators, the sponsor, or the University of Michigan. The research results of the brain images and genetic testing will NOT be communicated back to you. You should consult your personal doctor if you have any health concerns

Clinical tests: - There is an infrequent risk of physical fatigue during the clinical examination. Trained research staff will conduct all the tests and administer all the questionnaires. The staff will be prepared to respond to your concerns by temporarily suspending testing and/or breaking up testing sessions into several brief visits if needed.

- There is a common risk that you may have a dry mouth after providing the saliva sample. You may drink some water after providing the sample.

- Risks associated with the DXA scan are described below under the header “PET scans”

Motor testing: - Many of the tests are comparable to normal standing and walking conditions that you may experience in everyday-life. Nonetheless, there is an infrequent risk of falling or near-falling during these tests which may result in fall-related injuries. Trained research staff will remain in close proximity to you at all times, and observe (‘spot’) you to prevent you from falling. - There is a very rare risk that the sensors to measure overall movement and balance may become detached and that you may trip. You may also trip on the pressure sensitive mat. We will regularly check the sensors for appropriate attachment and you will be closely monitored.

Cognitive and neurobehavioral tests: - There is an infrequent risk of boredom, frustration, and/or mental and physical fatigue during the neuropsychological and neurobehavioral testing. Trained research staff will conduct all the tests and administer all the questionnaires. The staff will be prepared to respond to your concerns by temporarily suspending testing and/or breaking up testing sessions into several brief visits if needed.

For participants participating in the flumazenil sub-study: - There is a rare risk of having a seizure (which can be serious) or feeling anxious after injection with flumazenil. There is an infrequent risk of feeling “dizzy,” “lightheaded,” “woozy” or nausea. Subjects with a history of seizures or significant anxiety should not participate in this study. Patients with significant repeated exposure to benzodiazepine medications should not participate as well. Please inform the study team of any history of seizures or anxiety that you have. Dizziness, lightheadedness and nausea will disappear rapidly. If necessary, medication to treat a seizure will be given to you by a physician. You may also be asked to stay in the laboratory until it is deemed safe to leave.

For participants participating in the clarithromycin sub-study: - There is a rare risk of having a seizure or experiencing a heart rhythm abnormality which can be serious. There is also a rare risk of feeling anxious or confused. Infrequent side effects include diarrhea, nausea, abnormal taste, dyspepsia (stomach upset), abdominal pain/discomfort, and headache. Subjects with a history of seizures or significant anxiety should not participate in this study. Please inform the study team of any history of seizures or anxiety that you have. Additionally, subjects with a history of certain heart arrhythmias or “Long QT syndrome” or taking certain medications should not participate in this study. We will carefully review your medication and health history with you, and perform a baseline ECG, to decide if this study is appropriate for you. A blood sample may be taken as well. The majority of side-effects of clarithromycin when given to patients with infection are mild and short lasting. However, please let the study team know if these side-effects are intolerable and we may lower the dose. 14

HUM00130361 Mod. of GABA-A Receptors and Axial Motor Imp. in PD Protocol V2.0 9.1.2017

MRI scan: - There is a minor risk of discomfort or anxiety from being in the confined space of the MRI scanner. We will provide pads and blankets to make you as comfortable as possible. You will be able to talk to us throughout the study, and you will be able let us know right away if you want to stop the study and get out of the scanner At your request, you may be provided with a mild sedative; however, you must have made prior arrangements to be driven home by an accompanying adult.

- The MRI scanner makes loud, vibrating noises. You will wear foam earplugs to reduce the loud noises made by the scanner and prevent any hearing damage.

- - - Some studies, like this one, have the potential to cause "peripheral nerve stimulation" (PNS). PNS is a light touching sensation on the skin surface, lasting only for a few seconds. It may cause mild discomfort, but is not harmful to you. The MRI machine is operated within FDA guidelines so the potential for inducing PNS is low.

- Sometimes, subjects report a temporary, slight dizziness, light-headedness or nausea during or immediately after the scanning session. If you feel dizzy or light-headed, we will have you get up slowly from the scanner.

- Because the strong electromagnetic fields can move metal objects and cause heating, there is a risk that loose objects (jewelry, keys) outside your body could be accelerated by the magnetic field and strike you, causing you injury. There is also a risk that the magnetic fields could disturb a metal fragment in your body, interfere with an implanted device, such as a pacemaker or neurostimulator, or cause metal (including foil-backed medication patches) on or in your body to heat up, causing you harm. We keep the environment around the MRI scanner completely free of loose metal objects that could be moved by the magnetic field, and we will make sure that you have no metal on your body that could be affected by the MRI scanner. We will also ask you questions and have you complete an MRI screening form to make sure that you have no metal inside your body that would cause you harm during the MRI scan. The radiologist may order an X-ray to make sure there are no metallic fragments in your eyes or chest.

PET scans: - There is a very rare risk that you could experience an allergic reaction to the PET tracer. This could involve itching, skin rash or shortness of breath shortly after injection. However, because of the very small tracer amounts used in PET imaging, the risk is very rare. The use of [11C]DTBZ and [11C]flumazenil 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 [11C]DTBZ or [11C]flumazenil PET radiotracer doses used in this study have not been reported. Certified staff will be in attendance at all times during the study. A physician will be available and an emergency cart is located in the PET Facility for treatment of any adverse reactions that may occur.

- During the course of this study, you will be exposed to radiation from the CT scan (embedded in the PET scanner) and the [11C]DTBZ (dopamine scan) and [11F]flumazenil (GABA receptor) PET radiotracers. Subjects participating in the clarithromycin sub-study will be exposed to radiation from an additional [11C]flumazenil PET scan. The risks associated with the amount of radiation exposure participants receive in this study are considered very rare and comparable to every day radiation exposure risks. Please inform the investigators if you have had any major radiation exposure in the past, particularly in the past year, such as medical treatment with X-rays or radioactivity, or diagnostic X-rays, CT-scans or nuclear medicine scans. The biological effect of radiation in humans is measured in terms of Sieverts (Sv) or mSv (1/1000 Sv), which is a unit of uniform whole body exposure. The radiation exposure you will receive from the CT, and DTBZ and flumazenil PET scans is equivalent to a uniform whole body dose of 4.7 mSv, which is approximately 10% of the annual radiation exposure (50 mSv) permitted to radiation workers by federal regulations. For subjects 15

HUM00130361 Mod. of GABA-A Receptors and Axial Motor Imp. in PD Protocol V2.0 9.1.2017 participating in the clarithromycin sub-study, an additional flumazenil PET study would add 2.0 mSv to the total exposure resulting in a maximum of 6.7 mSv, which is approximately 13% of the annual radiation exposure (50 mSv) permitted to radiation workers by federal regulations. The radiation you will be exposed to from the DXA scan is 0.0159 mSv, which is significantly less than 1% of the permitted annual radiation exposure. You will be instructed to use the bathroom and urinate as soon as possible after the PET scans in order to minimize bladder exposure. There is a small chance of technical failure of one of the PET scans, which can be repeated but may result in additional exposure (DTBZ scan 2.5 mSv or flumazenil PET scan 2.0 mSv). 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.

- No PET or DXA studies will be performed on pregnant, nursing, or potentially pregnant women. A urine pregnancy test will be performed on all women of childbearing potential within 48 hours prior to the PET/DXA scanning session.

Fall diary study: - There is a very rare risk that confidentiality will be breached when the diary, fall-event information sheet, or the activity questionnaire are returned to us by mail. You will be asked not to write your name on neither the envelope nor on any of these test materials. We will write a code on the test materials that will allow us to connect these test materials with your name. This code is securely monitored and behind lock and key, only accessible by the principal investigator of this study (see also 9.1).

Assessment of daily life activity: - There is a very rare risk of the movement monitor (actigraph) detaching, which may result in tripping during the daily life monitoring of overall movement (actigraphy). It should be noted that the actigraph only measures overall movement. It does not record your geographical location or specific activities that you were performing, nor can this be derived at a later point from the data that is stored in the actigraph. You will receive instruction for proper attachment of the actigraph.

As with any research study, there may be additional risks that are unknown or unexpected.

Data safety and monitoring board (DSMB): University of Michigan neurologists (Dr. Vikas Kotagal and Peter Todd) who are not involved with any of the research procedures will serve as chairs for the DSMB for this study. They will review outcomes with the PI on an annual basis. Review of study procedures and adverse effects will be performed on a monthly basis. The PI will be responsible for the day-to-day monitoring any potential breach of confidentiality and for reporting any adverse events (AE) 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. 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 PI will be in daily contact with the study research staff. The research staff will test the participants, score and enter the data and will monitor their procedures to ensure that confidentiality is maintained. Research staff will be responsible for reporting any significant events to the PI. The PI will ensure that IRBMED is notified of any adverse event following IRBMED guidelines. At the time of renewal, IRBMED 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.

HUM00130361 Mod. of GABA-A Receptors and Axial Motor Imp. in PD Protocol V2.0 9.1.2017

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