Clinical trial NCT03461861
Conditions: APOE 4
Keywords: Healthy Adult Normal Memory APOE 4 Brain Magnetic Resonance Imaging MRI Alzheimer's disease Hippocampus Genetic testing Dementia AGB101
Sponsor: Medical College of Wisconsin
Start date: 2019-04-11; Completion date: 2021-03-31
Enrollment: 26 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
The purpose of this project is to test the hypothesis that AGB101 low dose levetiracetam extended release formulation can reduce abnormal hyperfunctional activity in the hippocampus in normal, healthy adults. The investigators will compare the functional connectivity results after taking AGB101 or placebo.

Detailed description:
In this study the investigators want to find out whether the use of a perturbation, such as AGB101 low dose of levetiracetam extended release formulation, in healthy adults can reduce abnormal hippocampal network activity. The investigators also want to study whether this low dose of LEV can improve memory function.

Generic levetiracetam is a type of drug called an anti-epileptic or anti-seizure medication. It is FDA approved worldwide for adults and children as young as one month with seizures. It is a generic drug used in long-term epilepsy treatment. It is relatively safe and has an acceptable side-effect profile.

AGB101 has been developed as a novel extended release formulation of low dose levetiracetam (below clinically marketed doses for epilepsy) for slowing the progression of amnestic mild cognitive impairment.

It is known that age and the APOE 4 gene are important risk factors for late-onset Alzheimer's disease. Further studies have shown that cognitively normal, older adults have more hyperfunctional brain network activity, increased alpha beta accumulation, decreased memory function, and decreased brain volume, which is consistent with Alzheimer's disease patterns.

Arms: arm 1: AGB101 220 mg/day capsule, once daily dosing for 2 weeks. After a 4 week washout, to be followed by Placebo, given as a capsule, once daily dosing for 2 weeks. arm 2: Placebo, given as a capsule, once daily for 2 weeks. After a 4 week washout, to be followed by AGB101 220 mg/day capsule, once daily dosing for 2 weeks.
Interventions: intervention 1: AGB101 220 mg intervention 2: Placebo
Intervention descriptions: intervention 1: AGB101 oral dose of 220 mg/day capsule, given as once-daily dosing. intervention 2: Placebo, oral capsule given once-daily.

Protocol documents:
SAP_002.pdf: 
Network-Level Mechanisms for Preclinical  
Alzheimer’s Disease Development 
 
 
Principal Investigator: Shi-Jiang Li, PhD (Department of Biophysics) 
 
Co-investigator: Piero G. Antuono, MD (Department of Neurology) 
 
Address 
Medical College of Wisconsin 
8701 Watertown Plank Road 
Milwaukee, WI 53226 
 
Funding Sponsor: National Institutes of Health, National Institute on Aging (R21 AG 056882) 
 
Version: 5 
 
Version Date:   April 1, 2019 
 
Replaces Version:  4, September 21, 2018 
 
 


13 
 
  Protocol v5 01Apr2019 
spin-labeling (ASL) cerebral blood flow (CBF) perfusion images. The data acquisition scans, plus patient 
handling time in and out of scanner, will result in an approximately 1-hour scanning session. 
 
Prior to fMRI scans, subjects will undergo MRI safety screening and mock scanner training, as detailed in 
sections 7.3. and 7.4., respectively. 
 
5.4.6. Blood Draws 
All subjects will undergo a blood draw at the last day of each treatment period and on the last day of the 
washout period. Samples will be labeled with the study name, study visit number, contents of each collection 
vial, and the date of collection. Tubes may also be labeled with a barcode for sample management.  
 
5.4.7. Randomization, Study Drug Dispensation, Administration, and Compliance 
The Investigational Drug Service at Froedtert Hospital will randomize participants to the treatment conditions. 
 
In Period 1, all study participants will receive treatment with either AGB101 (220 mg, orally, QD) or placebo; in 
Period 2, all subjects will crossover and receive the other treatment. This is a double-blind study, so both the 
participants and study team will be blind to the treatment received. 
 
All study treatments (drug and placebo) are dispensed in identical nondescript tablets by the Investigational 
Drug Service at Froedtert Hospital. Tablets are provided for daily morning doses. Study drugs will be 
dispensed at baseline and on the last day of the 4-week (-/+ 1 week) washout period. Subjects will be asked to 
self-administer treatment QD, once in the morning. 
 
Treatment compliance will be monitored by participant self-report at the end of each treatment phase, bottle 
return and pill count, and analysis of AGB101 blood values at each visit during the study protocol. 
 
Data Management  
6.1. The Froedtert Hospital Investigational Pharmacy system will be used to enroll patients and obtain the 
randomly assigned treatment group. Data forms will be printed, completed, and entered into a database. The 
database will be monitored for completeness, consistency, accuracy, and timeliness. No patient-identifying 
information will be stored in the database. To ensure that data are secure from external violation, computer 
systems containing study data will be password protected, and physical access to the computer systems will 
be limited.  
 
6.2. Determination of Changes in Network Activity and Concurrent Improvement in EM. The cognitive 
and MRI/rfMRI datasets will be obtained at baseline (before placebo/AGB101 administration) and on the last 
day of each 2-week treatment period. These two time points will be denoted t = 1, 2. The two 25-subject 
groups, both of which will receive both placebo and AGB101 at different treatment phase, will be denoted g = 
1, 2. The set of EM scores for subject s, from group g, at time t will be denoted Cog(s,g,t), where s = 1, …, 25; 
g = 1, 2; and t = 1, 2. The HFC calculated between the hippocampus and the bilateral sensorimotor cotex for 
subject s, from group g, at time t will be denoted HFC(s,g,t).  
 
6.3. Functional-Connectivity Changes Due to Perturbation. We employed age, years of education, gender, 
and gray matter (GM) volume as time-invariant covariates to obtain residual variables to determine the HFC 
changes due to perturbation. We will model the HFC as a function of these nuisance regressors, using the 
following multiple linear regression equation, and retain the residuals as 𝑯𝑭𝑪𝑹𝒆𝒔
ା(𝒔, 𝒈, 𝒕) for further analysis: 
 
 
where 𝑯𝑭𝑪ା
෫(𝒔, 𝒈, 𝒕) ≡ 𝑹𝒆𝒔(𝒔, 𝒈, 𝒕). This regression will be performed using all of the data (i.e., all subjects, all 
groups, and all times). The GM measurement will be conducted with GM concentration and hippocampal 
volume measurement, as described in our publication and others (71-74). We will test the residuals 
𝑯𝑭𝑪ା
෫(𝒔, 𝒈, 𝒕) using three-factor crossed-nested analysis of variance (ANOVA), with fixed factors of group and 
𝑯𝑭𝑪(𝒔, 𝒈, 𝒕) = 𝜷𝟎+ 𝜷𝟏𝑨𝒈𝒆(𝒔)+ 𝜷𝟐𝑬𝒅𝒖(𝒔) + 𝜷𝟑𝑮𝑴(𝒔) + 𝜷𝟒𝑪𝑺𝑭(𝒔) + 𝜷𝟓𝑮𝒆𝒏𝒅𝒆𝒓(𝒔) + 𝑹𝒆𝒔(𝒔, 𝒈, 𝒕) , 


14 
 
  Protocol v5 01Apr2019 
time and a random factor of subject nested within group (i.e., all subjects will be tested at all times, but the 
placebo and AGB101groups will contain different treatment phases). We will test for both group and time main 
effects, as well as for the group × time interaction. If we find significant main effects or interactions, we will 
perform post hoc procedures to determine which time and group (i.e., placebo vs. AGB101) combinations are 
significantly different. In addition to the HFC analysis, we will conduct a DMN analysis 75).  
 
6.4. EM Changes Due to AGB101Perturbation. The two EM scores (AVLT and LMT) will be normalized to Z-
score by transforming individual raw test scores according to the mean and standard deviation of the scores for 
all subjects. Then, Z-scores of each test will be averaged to obtain individual composite memory Z-score, as 
we have previously published (76). Similar to the above model for HFC changes due to AGB101perturbation, 
we will model the Cog score, using the time-invariant covariates as nuisance regressors:  
 
 
Define 𝜟𝑬𝑴
෫(𝒔, 𝒈, 𝒕) ≡ 𝑹𝒆𝒔(𝒔, 𝒈, 𝒕). We will test the residuals 𝜟𝑬𝑴
෫(𝒔, 𝒈, 𝒕) using a three-factor crossed-nested 
ANOVA, with fixed factors group and time, and random factor subject nested within group. We will test for 
group and time main effects, as well as the group × time interaction. If we find significant main effects or 
interactions, we will perform post hoc procedures to determine which time and group combinations are 
significantly different.  
 
6.5. Prediction of Changes in Correlation Between HFC and EM Before and After AGB101Perturbation. 
A multiple linear regression is used to model EM as a linear function of HFC and test the difference in slope 
and intercept between the AGB101and placebo groups. If the subject is in the AGB101group, I(s,g) = 1; if the 
subject is in the placebo group, I(s,g) = 0.  
𝑬𝑴(𝒔, 𝒈) = 𝜷𝟎,𝟎+ 𝜷𝟎,𝟏𝑰(𝒔, 𝒈)+ 𝜷𝟏,𝟎𝑯𝑭𝑪(𝒔) + 𝜷𝟏,𝟏𝑯𝑭𝑪(𝒔)𝑰(𝒔, 𝒈) + 𝑹𝒆𝒔(𝒔) 
Here, HFC(s) represents baseline HFC for subject s (for simplicity, nuisance regressors are not shown).  
 
6.6. Expected Results, Deliverable, and Problems  
Through this study, we expect to realize two goals: Scientifically, we will demonstrate that aberrant HFC 
represents a primary upstream mechanism that may contribute to cognitive deficits in CN APOE 4 carriers. 
Further, we will demonstrate that changes in the dysfunctional hippocampal network upon AGB101perturbation 
can be quantified as a biomarker for measuring network responses. Clinically, we expect that the short-term 
changes in network activity after the 2-week perturbation period will lead to a decrease in HFC, and possibly to 
cognitive improvement. This study can provide conceptual proof for preventive intervention. Using the 
predictive relationship between HFC and the EM changes, individuals carrying the APOE 4 allele can then be 
stratified into high and low HFC activity subgroups to further refine the subpopulation. Also, this study will 
provide information and address the power of adequate enrollment, protocol adherence, subject retention, 
and safety for future pilot trials. 
 
We may be able to detect HFC changes in the 2-week period after AGB101perturbation as a primary 
outcome, but we may not be able to detect significant EM changes as the secondary outcome in this short 
R21 funding mechanism. In a future study, we could extend follow-up to 26 weeks or a year to enable us to 
detect the slowly declining EM functions.  
 
7.DATA SAFETY MONITORING PLAN 
An appointed DSMB will discuss the ethical conduct of the trial, review event definitions and the subject ICF 
form, and further develop plans for monitoring the data and safety of the trial. The DSMB will meet at least 
every six months to review the course of the trial and interim data. The DSMB agrees to communicate with 
institutional review board (IRB)/human subject committees to provide reassurances, as appropriate. There will 
be three DSMB members with the following areas of expertise: 
 
 
Neural mechanism underlining learning and memory in aging and neurological disease 
 
Aging and memory loss 
 
Psychology and neuroimaging 
 
𝜟𝑬𝑴(𝒔, 𝒈, 𝒕) = 𝜷𝟎+ 𝜷𝟏𝑨𝒈𝒆(𝒔)+ 𝜷𝟐𝑬𝒅𝒖(𝒔) + 𝜷𝟑𝑮𝑴(𝒔) + 𝜷𝟒𝑪𝑺𝑭(𝒔) + 𝜷𝟓𝑮𝒆𝒏𝒅𝒆𝒓(𝒔) + 𝑹𝒆𝒔(𝒔, 𝒈, 𝒕) 

Prot_000.pdf: 
Network-Level Mechanisms for Preclinical  
Alzheimer’s Disease Development 
 
 
Principal Investigator: Shi-Jiang Li, PhD (Department of Biophysics) 
 
Co-investigator: Piero G. Antuono, MD (Department of Neurology) 
 
Address 
Medical College of Wisconsin 
8701 Watertown Plank Road 
Milwaukee, WI 53226 
 
Funding Sponsor: National Institutes of Health, National Institute on Aging (R21 AG 056882) 
 
Version: 5 
 
Version Date:   April 1, 2019 
 
Replaces Version:  4, September 21, 2018 
 
 


2 
 
Protocol v5 01Apr2019 
PROTOCOL SUMMARY 
 
Title 
Network-Level Mechanisms for Preclinical Alzheimer’s 
Disease Development 
Principal Investigator  
Shi-Jiang Li, PhD 
Study Site 
Medical College of Wisconsin 
Clinical Trial Phase 
Phase II 
Study Disease 
Alzheimer’s disease 
Main Eligibility 
Criteria 
• Fluent in English 
• At least 8 years of education 
• Healthy APOE 4 carriers and noncarriers 
• Age 55–75 years  
• Normal cognitive function 
• Free of neurological or psychiatric comorbidities 
Study Rationale 
Levetiracetam (LEV) is a generic drug used in long-term epilepsy 
treatment, with known pharmacokinetic and pharmacodynamic 
properties. It is relatively safe and has an acceptable side-effect 
profile. A novel extended release formulation of AGB101 (220 mg 
of levetiracetam) was developed to allow for once-daily dosing in 
amnestic mild cognitive impairment (aMCI). So far, no studies have 
been conducted to see if a low dose of AGB101can effectively 
reduce the network dysfunction in cognitively normal subjects who 
are APOE 4 carriers. The pre-dementia state among APOE 4 
carriers can be identified by measuring abnormal brain connectivity 
with functional MRI. Considering this, use of AGB101 to reduce the 
network dysfunction in cognitively normal subjects who are APOE 
4 carriers should be explored. 


3 
 
Protocol v5 01Apr2019 
Primary Objectives 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
To determine whether normalization of resting-state connectivity 
can be achieved in APOE 4 carriers with 2 weeks AGB101 (220 
mg, QD) treatment compared with APOE 4 noncarriers receiving 
the same treatment 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Secondary Objectives 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
To measure cognitive function in APOE 4 carriers and noncarriers 
at the beginning and end of treatment with placebo 


4 
 
Protocol v5 01Apr2019 
Endpoints 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
• Primary outcome measurements: Network changes in the 
bilateral sensory motor cortex regions of the hippocampal 
functional connectivity after AGB101perturbation (time frame: 2 
weeks) 
• Secondary outcome measurements: Episodic memory 
performance and neuropsychological test battery as assessed at 
baseline (Visit 1) and at the post-perturbation procedures 
(time frame: 2 weeks) 
Study Design 
 
 
 
 
 
 
 
 
This is a randomized, double-blind, placebo-controlled crossover 
study in which AGB101 (220 mg, QD) or placebo will be 
administered to 50 healthy 55–75-year-old subjects (comprising 25 
APOE 4 carriers and 25 noncarriers), with the order of treatments 
counterbalanced in a within-subject crossover design.  
 
The study consists of prescreening (Visit −1), baseline/screening 
(Visit 1), a 2-week treatment period (Visit 2), a 4-week washout 
period (Visit 3), and a 2-week treatment period (Visit 4).  
Study Agent/
Intervention 
Description 
AGB101 Levetiracetam Extended Release 220 mg and placebo 
will be administered orally, QD 
Number of Subjects 
50, of which 25 are APOE 4 carriers and 25 are noncarriers 
Subject Participation 
Duration 
8 weeks: 2 weeks of AGB101/placebo, followed by a 4-week 
washout, followed by 2 weeks of AGB101/placebo  
Duration of Follow up 
None 
Estimated Time to 
Complete Enrollment 
18 months 
Statistical 
Methodology 
 
We will employ the multivariate regression model to determine the  
correlational changes between the hippocampal functional 
connectivity and the episodic memory due to AGB101perturbation; 
we will assume age, years of education, gender, and gray matter 
volume as time-invariant covariates.  
Safety Assessments 
Regular data safety monitoring board meetings will be scheduled 
to monitor progression of the study and review drug adverse 
events 
Efficacy Assessments 
Statistically significant changes between the hippocampal 
functional connectivity and episodic memory relationship at 
baseline and at the end of the trial 
Unique Aspects of this 
Study 
This is the first study to evaluate the safety and efficacy of AGB101 
to regulate brain connectivity in cognitively normal subjects at risk 
for dementia. 


5 
 
  Protocol v5 01Apr2019 
Table of Contents 
1. 
Study Objectives ........................................................................................................................................ 6 
2. 
Background and Significance ..................................................................................................................... 7 
3. 
Rationale .................................................................................................................................................... 9 
4. 
Study Design .............................................................................................................................................. 9 
5. 
Study Visits and Procedures ..................................................................................................................... 11 
6. 
Data Management .................................................................................................................................... 13 
7. 
Data Safety Monitoring Plan ..................................................................................................................... 14 
8. 
References ............................................................................................................................................... 22 
 
 


6 
 
  Protocol v5 01Apr2019 
1. STUDY OBJECTIVES 
Neuropathological, structural, and functional changes related to Alzheimer’s disease (AD) may begin 
insidiously, 20 to 30 years before the manifestation of clinical symptoms. This long, preclinical phase of AD 
provides a critical window of opportunity to conduct early disease intervention. In this study, we will test our 
hypothesis that, during the preclinical AD developmental phase in cognitively normal (CN) older 
subjects with the apolipoprotein E ε4 (APOE 4) allele, decreased abnormal hyperfunctional 
connectivity can be correlated with improved episodic memory (EM) using a perturbation, such as 
AGB101 low dose levetiracetam extended release formulation The major pieces of foundational evidence 
that support our hypothesis are described below. 
 
1.1. Justification of Selecting CN Subjects with the APOE 4 Allele  
It is well recognized that age and the APOE 4 allele are the most important risk factors for late-onset AD. It is 
known that APOE 4 allele carriers have a higher risk of converting from CN to mild cognitive impairment (MCI) 
than noncarriers (1-3). In fact, more than 50% of AD patients are carriers of the APOE 4 allele (4-6). 
Converging evidence shows that CN, older individuals with the APOE 4 allele manifest functional network 
disruption (7-13), increased Aβ accumulation (14), decreased memory function (7, 8, 12), and decreased brain 
volume (15-18), consistent with AD patterns. It is likely that these patterns are detectable at the preclinical AD 
stage. If we are able to elucidate the mechanisms linking preclinical network disruption and the emergence of 
clinical symptoms, we can use this critical window of time for potential intervention with disease-modifying 
therapy. It is evident that selecting CN subjects with the APOE 4 allele will facilitate such a preventive strategy. 
 
1.2. Objective  
A low dose of LEV (125 mg, twice daily [BID]) can reduce task-induced hippocampal hyperactivity and improve 
fMRI task performance in human subjects with MCI. Pioneering studies have demonstrated that, among 
several antiepileptic drugs, LEV suppresses neural hyperactivity and reverses synaptic and cognitive deficits in 
AD mice models (19-21). Recent research further demonstrates that a low dose of LEV can reduce task-
induced hippocampal hyperactivity in human MCI subjects (22, 23). This proof-of-principle human study 
suggests that abnormal neural activity may deteriorate memory function (24) and that AD-related neural 
network dysfunctions are an early and potentially treatable disease component (25-31). So far, no data are 
available to demonstrate if LEV perturbation can reduce abnormal network activity and concurrently improve 
memory decline in CN, older subjects with the APOE 4 allele. To fill this gap, our specific aim follows:  
 
Specific Aim 1. Determine network-level changes in hippocampal functional connectivity (HFC) after a low-
dose AGB101 levetiracetam extended release perturbation (220 mg, QD) for a 2-week period in a 
randomized, double-blind, and placebo-controlled study with 50 cognitively normal (CN) older subjects 
(ranging in age from 55 to 75 years), of which 25 are APOE 4 carriers and 25 are noncarriers, and correlate 
the network changes with the EM before and after a 2-week perturbation with a low dose of AGB101 or 
placebo.  
 
1.3. Outcome Measurements  
The following outcome measurements will be used to test our proposed hypothesis:  
 
• 
Primary outcome measures: Network changes in the bilateral sensory motor cortex regions of the 
HFC after AGB101 perturbation (time frame: 2 weeks) 
• 
Secondary outcome measures: EM performance and neuropsychological test battery as assessed at 
baseline and the post-perturbation procedures (time frame: 2 weeks) 
 
1.4. Impact of the Proposed Study  
Currently, no FDA-approved therapy exists for CN, older subjects who have abnormal functional connectivity 
and an increased risk for progression to dementia. If this mechanistic approach is successful, we will lay a 
conceptual framework for future clinical preventive trials in CN subjects with abnormal functional connectivity. 
Analogous to the use of baby aspirin to prevent the risk of cerebrovascular stroke, we hope a daily low dose of 
AGB101 will effectively prevent or slow memory decline. 
 
 
 


7 
 
  Protocol v5 01Apr2019 
2. BACKGROUND AND SIGNIFICANCE 
2.1. Background  
Substantial evidence supports our network dysfunction hypothesis that pathophysiological events may be 
“upstream” of amyloid-β1-42 protein (Aβ) accumulation: 1) Early network dysfunction has been reported before 
detectable Aβ formation (9) in certain subjects, such as young APOE 4 carriers (10, 11, 32), supporting the 
antagonistic pleiotropic trajectory and insidious network dysfunction in Alzheimer’s disease (AD) progression 
(33). 2) Abnormal hyperactivity stimulates Aβ formation and toxicity (26-31, 34), as evidenced by the fact that 
the Aβ-binding regions overlap the default mode network (DMN) regions(12, 13, 35-37). 3) Subjects with AD 
risk factors, including APOE 4 carriers (13, 38) and individuals with amnestic mild cognitive impairment (aMCI) 
(39-41), manifest network dysfunction abnormalities. 4) Network dysfunctions have been found in the DMN, 
salience network, executive control network, dorsal attention network, etc. (12, 42), consistent with AD 
pathology. 5) Abnormal network activity impairs memory function (24), and different dementia types target 
specific neural network sets (43). 6) Most importantly, recent pioneering studies have demonstrated that 
antiepileptic drugs, such as LEV, target network dysfunctions by suppressing neural hyperactivity and 
reversing synaptic and cognitive deficits in AD rodent models (19-21) and in aMCI subjects (22). Both 
hyperactive (44) and hypoactive (45) circuits can have a negative impact on the disease process. In the 
present study, we will robustly identify and measure the dysfunctional networks as biomarkers in CN older 
subjects. 
 
Abnormal hyper-HFC at the network level may be upstream of the underlying neurodegenerative processes. 
Recently, we published an article that discusses use of the rigorous event-based probability model to 
comprehensively demonstrate that, during the decade-long progression from the preclinical phase of AD to 
overt AD dementia, certain neuropathological, structural, and functional events (measured by various 
biomarkers) occur sequentially. Abnormal functional connectivity occurs first, followed by a decrease in the 
level of Aβ and an increase in the level of phosphorylated tau proteins in the cerebrospinal fluid; in turn, there 
is a decrease in structural brain volume, MCI, and the full manifestation of AD dementia. These results indicate 
that abnormal hippocampus hyperfunctional connectivity at the network level could be upstream of the 
underlying neurodegenerative processes, even before the level of Aβ is detectable in the brain (46, 47).  
 
2.2. Significance  
If this mechanistic study succeeds, we will be able to 1) provide conceptual proof that AD development can be 
prevented or delayed by targeting dysfunctional neural networks at the preclinical phase, thus facilitating early 
intervention, even in CN, older subjects (48); 2) identify subject groups that have no clinical symptoms but 
have hyperfunctional connectivity (e.g., APOE 4 allele carriers who are at high risk for AD development), which 
may prove to be a powerful strategy to decrease or halt cognitive decline; 3) monitor whether drug intervention 
reaches specific neural network targets and assess the network level responses; 4) predict if the network 
activity modulation can concurrently improve memory decline; and 5) provide essential information needed to 
prepare future clinical preventive trials. The long preclinical phase provides a critical window of time for 
potential intervention with disease-modifying therapy.  
 
This study provides network-level mechanisms that emphasize that dysfunctional networks at the preclinical 
phase can be targeted for secondary prevention at the asymptomatic stage before significant cognitive 
impairment. Specifically, we will focus on the following: 
 
• 
Preclinical AD Development Phase. Currently, three clinical trials are being conducted with LEV in the 
AD research field. Two are testing whether LEV can help AD subjects control seizure-like activity and 
provide cognitive benefits to AD patients, particularly in individuals with silent epileptic activity. The other is 
testing whether the daily use of low-dose LEV (125 mg, BID) administered to MCI subjects can improve 
memory function. Details of these three trials are presented on the clintrials.gov website 
(https://clinicaltrials.gov/ct2/show/NCT01554683, 
https://clinicaltrials.gov/ct2/show/NCT02002819, 
and 
https://clinicaltrials.gov/ct2/show/NCT01044758, respectively). In contrast, we propose studying CN 
subjects with the APOE 4 allele to reveal if the reduced hyper-HFC and improved EM decline are 
correlated. If this study is successful with CN subjects, we can take full advantage of this critical window of 
time (i.e., the long preclinical phase before significant cognitive impairment) and potentially intervene with 
disease-modifying therapy.  


8 
 
  Protocol v5 01Apr2019 
• 
Enriched Testing of Subjects with APOE 4 Allele. As stated, more than 50% of AD patients are carriers 
of the APOE 4 allele (4-6). The APOE 4 allele is confirmed as the most important genetic risk factor for 
late-onset AD. The APOE polymorphism modulates the risk of late-onset AD in an isoform-dependent 
manner (ε4>ε3>ε2) (49, 50). The APOE 4 allele accentuates cerebral amyloid deposition, structural and 
functional abnormalities, and memory decline in cognitively healthy adults. By treating APOE 4 allele 
carriers at high risk for AD development with a daily low dose of AGB101 (220 mg, QD) at the preclinical 
phase, it will be possible to determine if hyper-HFC is reduced and the memory decline is improved. 
 
 
3. RATIONALE 
3.1. General Information Regarding LEV  
LEV (Keppra®) is FDA approved worldwide. It is the most commonly approved drug for adjunctive treatment of 
partial, primary generalized tonic-clonic, and myoclonic seizures for use in adults and children as young as one 
month. LEV has a novel structure and unique mechanisms of action, although detailed mechanisms for its 
antiseizure activity are still unclear (51). Unlike other antiepileptic drugs, LEV’s action mechanisms appear to 
involve neuronal binding to synaptic vesicle glycoprotein SV2A (52), inhibiting calcium release from 
intraneuronal stores, opposing the activity of negative modulators of GABA- and glycine-gated currents, and 
inhibiting excessive synchronized activity between neurons (53). In addition, LEV inhibits N-type calcium 
channels. This is believed to impede impulse conduction across synapses (54). LEV is associated with rapid 
and complete absorption, high oral bioavailability, minimal metabolism, and primarily renal elimination. It lacks 
cytochrome P450 isoenzyme-inducing potential and is not associated with clinically significant pharmacokinetic 
interactions with other drugs, including other antiepileptic drugs (55). AGB101 has been developed as a novel 
extended release formulation of low dose levetiracetam (below clinically marketed doses for epilepsy) for 
slowing the progression of amnestic mild cognitive impairment. AGB101 is formulated at a strength of 220 mg 
consisting of Levetiracetam, Hypromellose (Methocel K15M Premium CR), Colloidal Silicon Dioxide, Silicified 
Microcrystalline Cellulose (ProSolv HD90), Magnesium Stearate and coated with Opadry. 
 
3.2. Justification of Treatment Selection  
So far, no studies have been conducted to see if a low dose of LEV can effectively reduce the network 
dysfunction in CN subjects who are APOE 4 carriers. As described above, LEV is a generic drug used in long-
term epilepsy treatment, with known pharmacokinetic and pharmacodynamic properties. It is relatively safe and 
has an acceptable side-effect profile. In addition, we have seven specific and fundamental reasons to select 
LEV as the perturbation in this study. 1) Pioneering research has tested the concept that excess activity in the 
hippocampus is a dysfunction condition that may be key in underlying, age-related impairment in hippocampal-
dependent memory processes. With a rat model, Koh et al. showed that a low dose of LEV can improve 
memory in aged rats (19). 2) A comprehensive study showed that, with the human APP mice model, acute 
LEV treatment reduces abnormal activity spikes. Chronic LEV improves learning and memory, and reverses 
behavioral abnormalities and synaptic deficits in the hippocampus (20). With a transgenic mouse model of AD 
(5XFAD mice), Devi et al. recently suggested that suppressing overexcitation with acute LEV treatment around 
the time of acquisition or early consolidation of memory may be sufficient to reverse memory decline 
associated with aging (21). 3) A recent human study showed that LEV treatment (125 mg, BID) for two weeks 
suppressed task-induced hyper-blood oxygen level dependent (BOLD) signals in aMCI subjects (22). 4) LEV 
has minimal interactions with other drugs. This is significant because older subjects often take many other 
drugs. 5) Most importantly, because LEV may impede impulse conduction across synapses and suppress 
neural hyperactivity (23), we can employ the resting-state functional magnetic resonance imaging (rfMRI) 
method to detect and determine how LEV modulates or normalizes dysfunctional networks, and whether the 
normalization of network functions can stop or delay EM decline or even improve memory. 6) The selection of 
low LEV doses. Within a low dose range between 5–10 mg/kg in a rat model, LEV effectively suppressed 
hyperactivity and improved memory performance (19-21, 55). Further findings demonstrated that a low dose of 
LEV improves memory in aged rats with cognitive impairment, but it does not at high doses (19). Many studies 
showed that LEV treatment at high doses creates a loss of antiepileptic efficacy during sustained treatment (20, 
56-58) in rodents and in a small portion of patients with epilepsy (59, 60). For the present study, we have 
selected a 220 mg, QD dose of AGB101 levetiracetam extended release which is about 10 times less than the 
clinical antiepileptic dose, for CN subjects carrying the APOE 4 allele. 7) Selection of duration of LEV 
perturbation. According to an animal model (20), although acute LEV can effectively suppress hyperactivity, 
the transient reduction of the abnormal spikes did not reverse abnormal behaviors until the chronic LEV 


9 
 
  Protocol v5 01Apr2019 
reversed the abnormalities in synaptic activity-related proteins. This result suggests that long-term LEV 
perturbation is necessary to provide the time for network adaptation and normality. As such, we designed a 2-
week, low-dose LEV perturbation study to address two questions: One, can a low dose of LEV decrease 
abnormal hyper-HFC? Two, will the decreased functional connectivity ameliorate EM decline? 
 
3.3.  Preliminary Results 
Higher HFC activity correlates with greater EM decline in CN APOE 4 carriers. To investigate if high HFC 
activity is detrimental to EM in CN APOE 4 carriers, 21 CN subjects with APOE 4 (aged 66.62 ± 5.84 years) 
and 27 CN subjects with APOE ε3ε3 (APOE 3) (aged 70.11 ± 5.23 years) were recruited in a two-year 
longitudinal study. Structural and rfMRI data were obtained, and changes in EM scores were recorded. The EM 
scores included the auditory verbal learning test (AVLT) and logical memory test (LMT), which are the two 
most common EM assessments and are used in various multicenter studies. These tests also can detect 
accelerated EM decline in cognitively normal APOE 4 carriers (61). As shown in Fig. 1a, CN APOE ε3ε3 
subjects exhibited neural correlates of EM function in the Papez circuit regions. However, the baseline HFC 
activity does not correlate with the rate of EM decline (data not shown).  
 
Conversely, CN APOE 4 carriers exhibited neural correlates of EM function in the bilateral sensory motor 
cortex regions (Fig. 1b) and the baseline HFC activity in the sensory motor cortex negatively correlated with 
the annual rate of EM change (Fig. 1c). These results suggest that APOE ε3ε3 and APOE 4 alleles encode 
different network systems for EM functions, demonstrating genetic regulation. Further, higher HFC activity 
predicts a faster annual rate of EM decline in APOE 4 carriers but not in APOE ε3ε3 subjects. The baseline 
HFC activity in the sensory motor cortex predicts the rate of EM decline: the higher the activity, the faster the 
rate of decline. Most importantly, these results have been validated with independent datasets from ADNI 2 
(Alzheimer’s Disease Neuroimaging Initiative) with 14 CN APOE 4 carriers (aged 71.95 ± 4.67 years), as 
shown in Fig. 1d. These results suggest that activity in Papez circuit commonly mediates EM function without 
causing EM decline in APOE ε3ε3 subjects, whereas, in APOE 4 carries, the higher activity in sensory motor 
cortex is detrimental and can be used to predict the annual rate of EM decline (Figs. 1c and 1d). These 
preliminary results demonstrate the feasibility of detecting abnormal network activity and correlating it with EM 
decline. We will test our hypothesis that decreased hyperfunctional connectivity can be correlated with 
improved EM using a daily, low-dose LEV perturbation (AGB101 levetiracetam extended release).  
 
4.  
STUDY DESIGN 
4.1. Overall Design  
This study will test the hypothesis that decreased abnormal hyperfunctional connectivity can be 
correlated with improved EM using a daily, low-dose LEV perturbation (AGB101 levetiracetam 
extended release). In this study, there are two study groups: APOE 4 carriers (n=25) and APOE4 noncarriers 
(n=25). The functional connectivity changes in HFC will be determined in a randomized, double-blind, placebo-
controlled crossover manner, in which a low dose of AGB101 levetiracetam extended release (220 mg, QD) or 
placebo is administered to 50 healthy 55-75-year-old subjects (comprising 25 APOE 4 carriers and 25 
noncarriers), with the order of treatments counterbalanced in a within-subject crossover design, as illustrated in 
Fig. 1. APOE polymorphisms and their mediation for 
EM functions. a) Neural correlates of EM functions in 
APOE ε3ε3 subjects in the Papez circuit region. b) 
Neural correlates of EM functions in APOE 4 carriers 
in the bilateral sensory motor cortex regions. c) 
Associations between baseline HFC connectivity in 
the bilateral sensory motor cortex and the annual rate 
of EM decline in APOE 4 carriers are significant. d) 
The relationship in APOE 4 carriers described in c 
was validated with an independent dataset from ADNI 
2. To calculate the rate of EM change, we converted 
AVLT and LMT scores to a composite standardized 
Z-score and then divided by the follow-up duration.  


10 
 
  Protocol v5 01Apr2019 
Fig. 2 below. The study procedures are almost identical to those described in Refs (77, 78). In brief, study 
participants will receive placebo during one treatment period and AGB101 levetiracetam extended release 
during the other treatment period, with the order of treatments counterbalanced (randomized, double-blind).  
  
 
 
 
 
Visit 1 (baseline) and Visits 2 and 4 (which are separated by a 4-week washout period, -/+ 1 week) include a 
physical and neurological examination, a neuropsychological assessment, and fMRI scans. At baseline visit 
and the third visit, each subject will be provided with the study medication. At the end of the period 1, period 2 
and washout period, each participant will have a blood draw. Treatment compliance will be assessed by 
reviewing participant self-reports at the end of each period, bottle return and pill count, and analyzing of 
AGB101 levetiracetam blood values at each study visit. The study team is blind to the APOE status of the 
participants and AGB101 levetiracetam blood levels until the final group analysis of the data. The company 
Agenebio, Inc. will provide AGB101 levetiracetam extended release tablets as well as placebo. The Froedtert 
Hospital Investigational Drug Service will randomize participants to the treatment conditions, dispense the 
study medication, and control blinding and unblinding of study data according to standard clinical trial 
procedures. Data safety is monitored by three data safety monitoring board (DSMB) members who are not 
related to the study, in collaboration with the Froedtert Hospital Investigational Drug Service. 
 
4.2. Inclusion and Exclusion Criteria 
Inclusion Criteria 
• 
Fluent in English  
• 
At least eight years of education 
• 
A Geriatric Depression Scale (GDS) (62) score < 6 and Hachinski Ischemic Score ≤ 4 
• 
Normal general cognitive function as well as 1) normal memory function, documented by MOCA score 
of 23 or greater, and a RBANS Delayed Memory Index score of 85 or greater. 
 
 
 
Exclusion criteria 


11 
 
  Protocol v5 01Apr2019 
• 
Neurological diseases, such as Parkinson’s disease, multi-infarct dementia, Huntington’s disease, 
normal pressure hydrocephalus, brain tumor, progressive supranuclear palsy, seizure disorder, 
subdural hematoma, multiple sclerosis, or a history of significant head trauma followed by persistent 
neurologic deficits or known structural brain abnormalities 
• 
Presence of major psychiatric disease or chronic unstable medical conditions  
• 
History of drug abuse 
• 
History of alcohol abuse (4 or greater drinks per day on average) 
• 
Contraindication to MRI 
• 
Known clinically significant abnormalities in B12 or thyroid function tests 
• 
End Stage Renal Disease (ESRD) 
• 
Hemodialysis (HD)  
 
4.4. Subject Recruitment 
We will screen about 150 CN subjects ranging in age from 55–75 years to obtain 25 APOE 4 carriers and 25 
noncarriers, according to a 20–25% prevalence of the APOE 4 allele among this population (66). Recruitment 
efforts will be conducted in the metropolitan Milwaukee, WI, area, which has more than 1 million inhabitants. 
The Medical College of Wisconsin (MCW) Dementia Research Center has extensive experience in recruiting 
from the clinic and the Milwaukee community, using flyers, radio announcements, press releases, and 
community presentations. We also will contact the University of Wisconsin-Madison based Alzheimer 
Research Center to recruit to potential volunteers registered with that center and residing in the southeastern 
counties of Wisconsin.  
 
Study Visits and Procedures 
5.1. Schedule of Visits 
At screening (Visit −1) informed consent (IFC), blood (or buccal swab) for APOE genotyping, demographics, 
medical history, MRI safety screening, and MOCA/RBANS will be obtained in one or more visits. After a 
subject’s APOE genotype is known, the subject will then be invited to return for the baseline visit. At baseline 
(Visit 1), the subject will undergo a physical and neurological exam, and neuropsychological assessments. 
Neuropsychological assessments will be reviewed by the clinician neurologist as part of the inclusion and 
exclusion criteria. Those subjects who meet the inclusion criteria and have none of the exclusion criteria will be 
enrolled in the study. Enrolled subjects will undergo fMRI scans, be randomized to a treatment group, and 
receive the study medication for Period 1. Visit 2 will be scheduled on the last day of Period 1; at this visit, a 
brief medical and neurological examination, a neuropsychological assessment, a blood draw, and fMRI scans 
will be administered. Visit 3 will be scheduled at the end of the washout period. At this visit, each subject will 
have a blood draw and be provided with the study medication for Period 2. Finally, Visit 4 will be scheduled 
for repeat cognitive tests, blood draw and fMRI scans as Visits 1 and 2. Treatment compliance will be 
monitored by participant self-report at the end of each treatment phase, bottle return and pill count, and 
analysis of AGB101 levetiracetam blood values at each visit during the study protocol. 
 
Refer to Table 1 for a schedule of study procedures. 
 
5.2. Subject Retention and Compensation 
The study staff will monitor treatment compliance as well as potential adverse events (AE). The study staff will 
apply the standard operating procedure currently in place to monitor AEs. Subjects will be compensated for 
their participation in the study.  
 
5.3. Early Termination Visit  
If a subject decides to exit the study, a termination visit will be scheduled. This will include all assessments 
normally performed at Period 2 visit. In addition, any incidental imaging findings will be dealt with in 
accordance with the standard operating procedures. 
 
5.4. Study Procedures 
5.4.1. Informed Consent 


12 
 
  Protocol v5 01Apr2019 
Participants will provide written informed consent twice: first at the screening phase for collection of blood (or 
buccal swab) for APOE testing as well as for other screening procedures; the second consent is for qualified 
individuals to participate in the main intervention phase and all related study procedures. The consent process 
will involve distributing a full consent form to the potential study participant and a thorough presentation on the 
purpose and risks of the study, and an explanation of study procedures by the staff member obtaining consent. 
Sufficient time for questions, discussion, and the subject’s decision will be allowed for proper informed consent 
by the participant. Once the participant fully understands the study protocol as outlined in the consent form, 
he/she will acknowledge his/her consent with a signature that is placed directly below the text on the consent 
form. Each participant will receive a complete copy of the signed consent form for his/her personal records. 
The study coordinator will keep the original signed copy for study records.  
 
5.4.2. Blood Sample (or Buccal Swab) 
A blood sample (or buccal swab) will be collected at the prescreening period to determine whether the subject 
is a carrier or noncarrier of the APOE 4 gene.  
 
5.4.3. Demographics, Medical History, and Physical and Neurological Exam  
Demographic information, a comprehensive medical history and neurological examination with vital signs occur 
at the screening visit, and vital signs occurs on the last day of each study period. (see table 1) Data are 
collected from participants using HCP and National Alzheimer's Coordinating Center (NACC) procedures and 
forms, including demographic information (age, gender, race, ethnicity), medical and surgical history, 
hospitalization history, medications and supplements, and family history of Alzheimer’s disease. We will also 
ask questions pertaining to diet, sedentary/active lifestyle, sleep history, fluency in languages, marital status, 
socioeconomic status, education, and work history, and also obtain information on smoking, alcohol use, 
caffeine use, and drug use.  
 
5.4.4. Neuropsychological Assessments  
AD is characterized by progressive EM deficit at the predementia stage. Current AD diagnosis guidelines 
recognize EM deficit as a core clinical criterion for typical AD diagnosis (67, 68) and recommend objective 
decline in EM performance as the starting point of AD clinical onset (69). Practically, AVLT and LMT are the 
two most common EM assessments that are used in various multicenter studies, including the Alzheimer’s 
Disease Neuroimaging Initiative and the Australian Imaging, Biomarker and Lifestyle Study of Ageing. These 
tests also can detect accelerated EM decline in cognitively normal APOE 4 carriers (61). Therefore, this study 
will employ AVLT as objective measurements to evaluate the EM decline and the changes to LEV perturbation. 
For comprehensive cognitive measurements of CN subjects, all enrolled subjects will be administered the 
WAIS-IV Digit Span and Coding, RAVLT, Boston Naming Test, and Verbal Fluency: FAS & Animal Fluency.  
 
5.4.5. Neuroimaging Assessments  
We will conduct imaging on our research-dedicated GE 3T MRI scanner (Signa Premier) equipped with a 48-
channel receive-only head coil, which is FDA-approved, or in situations where a back-up scanner is needed, 
on our research-dedicated GE MR750 3T MRI scanner with 32-channel receiver coil and software version 
DV26. We will use adapted multiecho image sequence to conduct this project. Below, we briefly describe the 
imaging acquisition, parameters, and image types. The first type of measurement is the T1-weighted high-
resolution anatomical images. The second type of measurements is task-driven and resting-state fMRI 
scans. The 3T fMRI data will be acquired following the adapted General-electric multiecho image sequence, 
the acquired multiecho images can be effectively denoised with 3.75x3.75x3.75 mm spatial resolution (70). For 
fMRI scans, one is for EM and the other is for finger tapping. We will utilize a memory task, which is being 
employed currently for the Alzheimer’s Disease Connectome Project in healthy older adults, including 
participants with memory disorders. The task involves serial presentation of novel (NV) and previously learned 
(PL) pictures obtained from a published normative set. During the fMRI scan, NV or PL items are presented 
sequentially for the duration of the scan, and the task is to decide whether the present item is new or 
previously learned. The task elicits activation in the bilateral ventral and medial temporal lobes, bilateral 
hippocampal, and fusiform regions. For the Finger tapping task, we will utilize a task developed at MCW. The 
task follows a block design where participants alternate between finger tapping and rest, in response to on-
screen instructions. This task robustly activates the motor network. For the resting-state fMRI scans, two 8-min 
scans will be acquired with phase up and phase down encoding, respectively. The third type is the arterial 


13 
 
  Protocol v5 01Apr2019 
spin-labeling (ASL) cerebral blood flow (CBF) perfusion images. The data acquisition scans, plus patient 
handling time in and out of scanner, will result in an approximately 1-hour scanning session. 
 
Prior to fMRI scans, subjects will undergo MRI safety screening and mock scanner training, as detailed in 
sections 7.3. and 7.4., respectively. 
 
5.4.6. Blood Draws 
All subjects will undergo a blood draw at the last day of each treatment period and on the last day of the 
washout period. Samples will be labeled with the study name, study visit number, contents of each collection 
vial, and the date of collection. Tubes may also be labeled with a barcode for sample management.  
 
5.4.7. Randomization, Study Drug Dispensation, Administration, and Compliance 
The Investigational Drug Service at Froedtert Hospital will randomize participants to the treatment conditions. 
 
In Period 1, all study participants will receive treatment with either AGB101 (220 mg, orally, QD) or placebo; in 
Period 2, all subjects will crossover and receive the other treatment. This is a double-blind study, so both the 
participants and study team will be blind to the treatment received. 
 
All study treatments (drug and placebo) are dispensed in identical nondescript tablets by the Investigational 
Drug Service at Froedtert Hospital. Tablets are provided for daily morning doses. Study drugs will be 
dispensed at baseline and on the last day of the 4-week (-/+ 1 week) washout period. Subjects will be asked to 
self-administer treatment QD, once in the morning. 
 
Treatment compliance will be monitored by participant self-report at the end of each treatment phase, bottle 
return and pill count, and analysis of AGB101 blood values at each visit during the study protocol. 
 
Data Management  
6.1. The Froedtert Hospital Investigational Pharmacy system will be used to enroll patients and obtain the 
randomly assigned treatment group. Data forms will be printed, completed, and entered into a database. The 
database will be monitored for completeness, consistency, accuracy, and timeliness. No patient-identifying 
information will be stored in the database. To ensure that data are secure from external violation, computer 
systems containing study data will be password protected, and physical access to the computer systems will 
be limited.  
 
6.2. Determination of Changes in Network Activity and Concurrent Improvement in EM. The cognitive 
and MRI/rfMRI datasets will be obtained at baseline (before placebo/AGB101 administration) and on the last 
day of each 2-week treatment period. These two time points will be denoted t = 1, 2. The two 25-subject 
groups, both of which will receive both placebo and AGB101 at different treatment phase, will be denoted g = 
1, 2. The set of EM scores for subject s, from group g, at time t will be denoted Cog(s,g,t), where s = 1, …, 25; 
g = 1, 2; and t = 1, 2. The HFC calculated between the hippocampus and the bilateral sensorimotor cotex for 
subject s, from group g, at time t will be denoted HFC(s,g,t).  
 
6.3. Functional-Connectivity Changes Due to Perturbation. We employed age, years of education, gender, 
and gray matter (GM) volume as time-invariant covariates to obtain residual variables to determine the HFC 
changes due to perturbation. We will model the HFC as a function of these nuisance regressors, using the 
following multiple linear regression equation, and retain the residuals as 𝑯𝑭𝑪𝑹𝒆𝒔
+ (𝒔, 𝒈, 𝒕) for further analysis: 
 
 
where 𝑯𝑭𝑪+
̃ (𝒔, 𝒈, 𝒕) ≡ 𝑹𝒆𝒔(𝒔, 𝒈, 𝒕). This regression will be performed using all of the data (i.e., all subjects, all 
groups, and all times). The GM measurement will be conducted with GM concentration and hippocampal 
volume measurement, as described in our publication and others (71-74). We will test the residuals 
𝑯𝑭𝑪+
̃ (𝒔, 𝒈, 𝒕) using three-factor crossed-nested analysis of variance (ANOVA), with fixed factors of group and 
𝑯𝑭𝑪(𝒔, 𝒈, 𝒕) = 𝜷𝟎+ 𝜷𝟏𝑨𝒈𝒆(𝒔)+ 𝜷𝟐𝑬𝒅𝒖(𝒔) + 𝜷𝟑𝑮𝑴(𝒔) + 𝜷𝟒𝑪𝑺𝑭(𝒔) + 𝜷𝟓𝑮𝒆𝒏𝒅𝒆𝒓(𝒔) + 𝑹𝒆𝒔(𝒔, 𝒈, 𝒕) , 


14 
 
  Protocol v5 01Apr2019 
time and a random factor of subject nested within group (i.e., all subjects will be tested at all times, but the 
placebo and AGB101groups will contain different treatment phases). We will test for both group and time main 
effects, as well as for the group × time interaction. If we find significant main effects or interactions, we will 
perform post hoc procedures to determine which time and group (i.e., placebo vs. AGB101) combinations are 
significantly different. In addition to the HFC analysis, we will conduct a DMN analysis 75).  
 
6.4. EM Changes Due to AGB101Perturbation. The two EM scores (AVLT and LMT) will be normalized to Z-
score by transforming individual raw test scores according to the mean and standard deviation of the scores for 
all subjects. Then, Z-scores of each test will be averaged to obtain individual composite memory Z-score, as 
we have previously published (76). Similar to the above model for HFC changes due to AGB101perturbation, 
we will model the Cog score, using the time-invariant covariates as nuisance regressors:  
 
 
Define 𝜟𝑬𝑴
̃ (𝒔, 𝒈, 𝒕) ≡ 𝑹𝒆𝒔(𝒔, 𝒈, 𝒕). We will test the residuals 𝜟𝑬𝑴
̃ (𝒔, 𝒈, 𝒕) using a three-factor crossed-nested 
ANOVA, with fixed factors group and time, and random factor subject nested within group. We will test for 
group and time main effects, as well as the group × time interaction. If we find significant main effects or 
interactions, we will perform post hoc procedures to determine which time and group combinations are 
significantly different.  
 
6.5. Prediction of Changes in Correlation Between HFC and EM Before and After AGB101Perturbation. 
A multiple linear regression is used to model EM as a linear function of HFC and test the difference in slope 
and intercept between the AGB101and placebo groups. If the subject is in the AGB101group, I(s,g) = 1; if the 
subject is in the placebo group, I(s,g) = 0.  
𝑬𝑴(𝒔, 𝒈) = 𝜷𝟎,𝟎+ 𝜷𝟎,𝟏𝑰(𝒔, 𝒈)+ 𝜷𝟏,𝟎𝑯𝑭𝑪(𝒔) + 𝜷𝟏,𝟏𝑯𝑭𝑪(𝒔)𝑰(𝒔, 𝒈) + 𝑹𝒆𝒔(𝒔) 
Here, HFC(s) represents baseline HFC for subject s (for simplicity, nuisance regressors are not shown).  
 
6.6. Expected Results, Deliverable, and Problems  
Through this study, we expect to realize two goals: Scientifically, we will demonstrate that aberrant HFC 
represents a primary upstream mechanism that may contribute to cognitive deficits in CN APOE 4 carriers. 
Further, we will demonstrate that changes in the dysfunctional hippocampal network upon AGB101perturbation 
can be quantified as a biomarker for measuring network responses. Clinically, we expect that the short-term 
changes in network activity after the 2-week perturbation period will lead to a decrease in HFC, and possibly to 
cognitive improvement. This study can provide conceptual proof for preventive intervention. Using the 
predictive relationship between HFC and the EM changes, individuals carrying the APOE 4 allele can then be 
stratified into high and low HFC activity subgroups to further refine the subpopulation. Also, this study will 
provide information and address the power of adequate enrollment, protocol adherence, subject retention, 
and safety for future pilot trials. 
 
We may be able to detect HFC changes in the 2-week period after AGB101perturbation as a primary 
outcome, but we may not be able to detect significant EM changes as the secondary outcome in this short 
R21 funding mechanism. In a future study, we could extend follow-up to 26 weeks or a year to enable us to 
detect the slowly declining EM functions.  
 
7.DATA SAFETY MONITORING PLAN 
An appointed DSMB will discuss the ethical conduct of the trial, review event definitions and the subject ICF 
form, and further develop plans for monitoring the data and safety of the trial. The DSMB will meet at least 
every six months to review the course of the trial and interim data. The DSMB agrees to communicate with 
institutional review board (IRB)/human subject committees to provide reassurances, as appropriate. There will 
be three DSMB members with the following areas of expertise: 
 
• 
Neural mechanism underlining learning and memory in aging and neurological disease 
• 
Aging and memory loss 
• 
Psychology and neuroimaging 
 
𝜟𝑬𝑴(𝒔, 𝒈, 𝒕) = 𝜷𝟎+ 𝜷𝟏𝑨𝒈𝒆(𝒔)+ 𝜷𝟐𝑬𝒅𝒖(𝒔) + 𝜷𝟑𝑮𝑴(𝒔) + 𝜷𝟒𝑪𝑺𝑭(𝒔) + 𝜷𝟓𝑮𝒆𝒏𝒅𝒆𝒓(𝒔) + 𝑹𝒆𝒔(𝒔, 𝒈, 𝒕) 


15 
 
  Protocol v5 01Apr2019 
7.1. Safety Plan 
The safety issues in this study are related to use of LEV and acquisition of fMRI data. This study uses the 
AGB101 levetiracetam extended release at a dose that is 10 times smaller than the doses typically used to 
treat epilepsy, the main indication for standard LEV. LEV is associated with rapid and complete absorption, 
high oral bioavailability, minimal metabolism, and primarily renal elimination. The LEV plasma half-life in adults 
is 7 ± 1 hour and is unaffected by either dose or repeated administration. LEV is eliminated from the 
systemic circulation by renal excretion as an unchanged drug, which represents 66% of administered dose. 
The total body clearance is 0.96 mL/min/kg and the renal clearance is 0.6 mL/min/kg. The mechanism of 
excretion is glomerular filtration with subsequent partial tubular reabsorption. The metabolite ucb L057 is 
excreted by glomerular filtration and active tubular secretion with a renal clearance of 4 mL/min/kg. LEV 
elimination is correlated to creatinine clearance. LEV clearance is reduced in patients with impaired renal 
function. It lacks cytochrome P450 isoenzyme-inducing potential and is not associated with clinically significant 
pharmacokinetic interactions with other drugs, including other antiepileptic drugs (55). Among AEs reported 
over placebo at standard therapeutic doses of 1000–2000 mg daily are pharyngitis (9%), mood swings (4%), 
dizziness (5%), infection (5%), asthenia (6%), and somnolence (7%). Serious dermatological reactions, 
including Stevens-Johnson syndrome and toxic epidermal necrolysis, have been reported in at 14–17 days 
from initiation of treatment.  
 
7.1.1 Adverse Event Reporting 
 
Throughout the course of the study, all AEs will be monitored and recorded on an AE CRF, including the AE’s 
description, start and end date, outcome, seriousness, severity, action taken, and relationship to the study 
drug. If AEs occur, the first concern is the safety of the study subjects. All AEs will be followed until resolved or 
stable and the outcome documented on the CRF. 
 
7.1.2 Definitions and Criteria 
 
7.1.3 Adverse Events 
 
Per International Conference on Harmonisation (ICH) E2A: An AE is any untoward medical occurrence in a 
subject or clinical investigation subject administered a pharmaceutical product and which does not necessarily 
have to have a causal relationship with this treatment. An AE can therefore be any unfavorable and unintended 
sign (including an abnormal laboratory finding, for example), symptom, or disease temporally associated with 
the use of a medicinal product, whether or not considered related to the medicinal product. 
 
Events meeting the definition of an AE include: 
 
• 
Exacerbation of a chronic or intermittent pre-existing condition including either an increase in frequency 
and/or intensity of the condition 
• 
New conditions detected or diagnosed after investigational product administration even though it may 
have been present prior to the start of the study 
• 
Signs, symptoms, or the clinical sequelae of a suspected drug interaction 
• 
Signs, symptoms, or the clinical sequelae of a suspected overdose of either investigational product or a 
concomitant medication 
 
Clinically significant abnormal findings (laboratory test results, vital signs, physical examination findings, ECGs, 
radiologic exams, or other studies) should be recorded as AEs. A “clinically significant” finding is one that 
affects clinical management, including additional visits, monitoring or referrals, diagnostic tests or alteration of 
treatment, or that is considered clinically significant by the investigator. A clinically significant finding may be a 
change in a test that has previously been abnormal but now requires additional action. 
 
When a medical or surgical procedure is performed, the condition that leads to the procedure should be 
recorded as the AE. 
 


16 
 
  Protocol v5 01Apr2019 
Events that do not meet the definition of an AE include: 
 
• 
Anticipated day-to-day fluctuations or expected progression of pre-existing disease(s) or condition(s) 
present or detected at the start of the study unless judged by an investigator to be more severe than 
expected for the subject’s underlying condition 
• 
Abnormal laboratory, ECG, or vital sign measurements that are not labelled clinically significant (see 
definition above) 
• 
Situations where an untoward medical occurrence did not occur (social and/or convenience admission 
to a hospital) 
• 
Pregnancy 
• 
Overdose in the absence of other AEs will not be reported as an AE in its own right 
• 
Changes in C-SSRS during the course of the study indicating worsening should be evaluated by the 
investigator for clinical significance, and if clinically significant (eg, alteration in medical care or 
intervention is required), an associated AE should be recorded, if present. The AE should be the 
primary underlying clinical manifestation assessed as clinically significant, and not the change in score 
itself. 
• 
Change in cognitive functioning (improvement or worsening) is also not considered to be an AE. 
 
7.1.4    Serious Adverse Events 
 
An SAE or reaction is any untoward medical occurrence that at any dose: 
• 
Results in death 
• 
Is life-threatening 
• 
Requires inpatient hospitalization or prolongation of existing hospitalization, with the exception of: 
o Visits to the emergency room or hospital department that do not result in an overnight hospital 
admission 
o Elective surgery for a pre-existing condition that has not worsened 
o Routine health assessments requiring admission not associated with any deterioration in 
condition 
o Social admission (lack of housing, family circumstances, etc.) 
• 
Results in persistent or significant disability or incapacity 
• 
Is a congenital anomaly or birth defect 
• 
Is an important medical event (eg, intensive treatment in an emergency room or at home for allergic 
bronchospasm, blood dyscrasias or convulsions that do not result in hospitalization, development of 
drug dependency or drug abuse, or malignancy tumors [histologically different from the primary tumor]) 
 
Note: The term “life-threatening” in the definition of “serious” refers to an event in which the subject was at risk 
of death at the time of the event; it does not refer to an event that hypothetically might have caused death if it 
were more severe.  
 
Medical and scientific judgment should be exercised in deciding whether expedited reporting is appropriate in 
other situations, such as important medical events that may not be immediately life-threatening or result in 
death or hospitalization, but may jeopardize the subject or may require intervention to prevent one of the other 
outcomes listed in the definition above. These should also usually be considered serious. 
 
Seriousness (not severity) serves as a guide for defining regulatory reporting obligations. An SAE is not 
necessarily severe, eg, a hospitalization for a diagnostic procedure must be reported as an SAE even though 
the occurrence is not medically serious. By the same token, a severe AE is not necessarily serious: nausea of 
several hours’ duration may be rated as severe but may not be considered serious. 
 
7.1.5     Unexpected Adverse Drug Reactions 
 


17 
 
  Protocol v5 01Apr2019 
An unexpected adverse drug reaction (ADR) is a reaction for which the nature or severity is not consistent with 
the applicable product information (see current Investigator’s Brochure). Until product information is amended, 
expedited reporting is required for additional occurrences of the reaction. Reports that add significant 
information on specificity or severity of a known, already documented SAE constitute unexpected events. For 
example, an event more specific or more severe than described in the Investigator's Brochure would be 
considered "unexpected.” Specific examples would be (a) acute renal failure as a labeled ADR with a 
subsequent new report of interstitial nephritis and (b) hepatitis with a first report of fulminant hepatitis. 
 
Assessing Intensity and Relationship 
 
All AEs will be assessed on 2 descriptive parameters: intensity and relationship to the study drug: 
1. Intensity refers to the severity of an event and references the impact on a subject’s functioning. 
2. Relationship refers to the likelihood that the event being assessed was caused by the study drug. 
 
Intensity 
Each AE will be classified according to the following criteria: 
Mild: 
The AE does not interfere in a significant manner with the subject’s normal level 
of functioning. 
Moderate: 
The AE produces some impairment of functioning but is not hazardous to the 
subject’s health. 
Severe: 
The AE produces significant impairment of functioning or incapacitation and is a 
definite hazard to the subject’s health. 
 
When changes in the intensity of an AE occur more frequently than once a day, the maximum intensity for the 
experience should be noted. If the intensity category changes over a number of days, those changes should be 
recorded separately (with distinct onset dates). 
 
Relationship 
 
Each AE will be assessed as to its relationship to the study drug based on the following criteria. Although the 
attribution by the investigator will be collected for reported events, for analytic purposes a temporal association 
with the use of the study drug will be assumed sufficient for at least plausible association. 
 
Not related: 
No causal relationship exists between the study drug and 
the AE, but an obvious alternative cause exists, eg, the 
subject’s underlying medical condition or concomitant 
therapy. 
Related: 
There is a reasonable/plausible possibility that the AE may 
have been caused by the study drug. 
When assessing the relationship to the study drug, the following criteria will be considered: 
• 
Temporal relationship 
• 
Positive rechallenge 
• 
Positive dechallenge (resolution upon stopping the study drug, in absence of other intervention or 
treatment) 
• 
Known class effect 
• 
Biological plausibility 
• 
Lack of alternative explanation—concomitant drug or disease 
 
7.1.6     Reporting Procedures and Requirements 
 
Adverse Events 
 


18 
 
  Protocol v5 01Apr2019 
AE recording will begin after the first dose of study drug is administered the morning after baseline. Thereafter, 
AEs will be ascertained by asking the subject how he/she has been since the last visit. Any AEs occurring 
before the start of treatment (ie, before the first dose of the study drug) will be recorded in the medical history 
only. If the investigator detects an AE in a study subject within 1 month (4 weeks, -/+ 1 week) of the last 
scheduled follow-up visit this will also be collected. All AEs will be followed until resolved or stable and the 
outcome documented on the CRF. 
 
Also, the sign, symptom, or disease present before starting the treatment period are only considered AEs if 
they worsen after starting the treatment period.  
 
The investigator should report all information about AEs on the AE/SAE section of the Case Report Form 
(CRF). Whenever possible, an AE will be reported using a diagnostic term (eg, “common cold” or “upper 
respiratory infection” rather than “runny nose, cough, mild fever”) and should be described with the attributes. 
 
Serious Adverse Events 
 
Each AE will be assessed to determine whether it meets seriousness criteria (Section 7.1.2.2). If the AE is 
considered serious, the investigator should report this event to Agene Biotech as outlined below and to the 
Institutional Review Board (IRB) & Data Safety Monitoring Board (DSMB) according to their standard operating 
procedures. 
 
All information about SAEs will be collected and reported by entering the SAE information in the AE CRF. The 
investigator should send the initial report to the Site PI and to Agene Biotech within 24 hours of becoming 
aware of the SAE and followed by follow-up reports as soon as possible, whether the events are deemed 
related to the study drug or not. The information provided in the AE system should be as complete as possible, 
but minimally it must contain the following: 
• 
Subject number 
• 
Brief description of the SAE (diagnosis or signs/symptoms) 
• 
Serious criteria 
• 
Causality assessment 
• 
Assessment of the intensity of the event 
 
Agene Biotech will receive notification of the initial SAE via an e-mail from MCW Study Team. Site personnel 
will complete the paper SAE report form, scan and e-mail it within 24 hours to the following address: 
safety@agenebio.com 
  
SAEs that are ongoing should be followed until resolved or stabilized to a level acceptable to the investigator. 
Information not available at the time of the initial report (eg, an end date for the AE, laboratory values received 
after the report, or hospital discharge summary) must be documented on a follow-up form. All follow-up 
information must be reported in the same timelines as initial information.  
 
Any SAEs considered related to the study drug and discovered by the study team at any interval after the study 
must also be reported to the Agene Biotech within 24 hours following knowledge of the event. 
 
Sponsor Reporting of Suspected Unexpected Adverse Reactions to Regulatory Authorities (SUSAR)  
Per 21 CFR 312.32[c](1)(i)) the Sponsor (MCW) must report in an IND Safety Report any suspected adverse 
reaction to study treatment that is both serious and unexpected. Before submitting an IND safety report, the 
Sponsor needs to ensure that the event meets all three of the definitions: 
• 
Suspected adverse reaction 
• 
Serious 
• 
Unexpected 
 


19 
 
  Protocol v5 01Apr2019 
The Sponsor/MCW Study Team will submit a FDA Form 3500A MedWatch for expedited reporting of all 
events that qualify for expedited reporting. Refer to the Form FDA 3500A Supplemental Manual for instructions 
and timelines on mandatory reporting. 
 
Unblinding Treatment Allocation 
Generally, only SUSARs for which the treatment allocation of the subject is unblinded should be reported by 
the sponsor to the pertinent regulatory authorities.  
 
When an event may be a SUSAR, the blind should be broken only for that specific subject, and only if 
knowledge of the study drug assignment is material to the medical management of the AE. When unblinded to 
assist in the medical management, the blind for that subject should be maintained for individuals responsible 
for the ongoing conduct of the study (eg, management, monitors, and investigators) and those responsible for 
data analysis and interpretation of results at the conclusion of the study (eg, biometrics personnel).  
 
Unblinded information should only be accessible to those who need to be involved in the safety reporting to 
pertinent regulatory authorities, independent ethics committees/independent review boards (IRBs) and 
DSMBs, or individuals performing ongoing safety evaluations during the study. 
 
Reporting to Institutional Review Board and Data Safety Monitoring Board 
The IRB/DSMB will be notified of any SUSARs according to local regulations and within the designated 
timeframe. Refer to MCW IRB Reporting Policy. These will be reported to the DSMB in parallel. 
 
7.2. MRI Safety 
The MRI scanner device is a U.S. Food and Drug Administration (FDA)-cleared device for safe and 
noninvasive imaging of the interior of the human body. The GE MR750 MRI uses a 32-channel receive-only 
head coil, which is an investigational device. See below for further information on the 32-chanel receive-only 
head coil. The pulse sequences employed in this study were developed based on multiecho sequence by GE 
Healthcare, which constrains the pulse sequences to operate within the FDA limits for safe operation with 
respect to gradient switching (dB/dt) and RF power (specific absorption rate [SAR]). The proposed MRI scans 
are NOT for a use of substantial importance in diagnosing, curing, mitigating, or treating disease or otherwise 
preventing impairment of human health, and they do not present a potential for serious risk to the health, 
safety, or welfare of a subject. The scanner also restricts the software from exceeding FDA safety levels. The 
scanner monitors the SAR for research scans just as it does for all other scans. Thus, the scanner with the 
software fully engaged operates, from a technical design and functional standpoint, as a nonsignificant risk 
device in accordance with 21 CFR 812. By staying below these limits, the operating conditions of the MRI 
device are generally deemed, in and of themselves, to make the MRI device a nonsignificant risk device.  
 
The Nova Medical 32-channel head coil (model number NMSC075-32-3GE-MR750) is an investigational 
device that is not approved by FDA for clinical use. This coil device includes multiple features for safe 
operation involving human studies. The 32-channel coil is designed and constructed as a receive-only detector 
of RF signals that are emitted by the brain following the RF excitation generated by the GE Healthcare MRI 
scanner, which is FDA approved. During the RF excitation by the scanner, the coil device is decoupled (made 
inactive) through redundant circuitry; thus, the coil device never transmits RF to the subject and, therefore, it 
has no impact on subject risk or safety.  
 
More specifically, the coil design and construction include the following safety features: 1) High-voltage 
breakdown (>2 kV) UL-94V0 flame retardant housing. 2) Rugged construction to ensure safe operation in case 
of rough handling. 3) Active detuning circuitry providing greater than 35 db isolation per element. 4) High power 
passive detuning circuits in case primary detuning circuitry fails. 5) Multiple common mode traps in all receive 
coil cables. 6) Minimum of 5 mm spacing between coil conductors and patient contact. 
 
Additionally, the coil was designed and manufactured under an ISO 13485 certified quality management 
system. As part of this quality system, Nova Medical has conducted a failure means and effects analysis 


20 
 
  Protocol v5 01Apr2019 
(FEMA) of this product, and we feel that it is a nonsignificant risk under foreseeable normal conditions when 
used on the 3T GE X750 MRI scanners at MCW. 
 
The Medical College of Wisconsin has installed a new GE 3T MRI scanner (Signa Premier) equipped with a 
48-channel receive-only head coil, which is FDA-approved. We will use the new GE 3T Signa Premier MRI 
scanner and 48-channel head coil for study subjects. The GE750 MRI scanner with the 32-channel head coil 
will be used as a back-up for scheduling purposes. 
 
7.3. MRI Safety Screening 
MCW-specific magnetic resonance (MR) safety screening procedures will be followed. All participants will be 
screened for medical devices, implants, and metal prior to undergoing MRI, first at the screening visit (Visit -1) 
and again prior to entering the scanner room on Visits 1, 2 and 4. If it is necessary to review medical records to 
confirm contraindication, a review of medical records (e.g., previous surgeries) will take place prior to the 
scheduled visit. During the screening process, participants will also be questioned about their ability to 
temporarily remove transdermal patches (such as birth control or nicotine patches). Women of child-bearing 
potential will be asked to confirm that they are not pregnant when signing the ICF. If a woman has concerns or 
is uncertain of her pregnancy status, she will be excluded, as the risks of an MRI scan to pregnant women are 
currently unknown. The risks related to an MRI are minimal for a properly administered visit. The MR 
technicians are trained and prepared to deal with any problems that may arise.  
 
7.4. Mock Scanner Training  
As part of the study, participants will engage in a mock scanner training session. The purpose of this session is 
to acclimate participants to the scanner and to assess their comfort level, minimize any anxiety, and provide 
practice and training on all tasks that will be conducted in the real MRI environment. Training in the mock 
scanner will take approximately 20 minutes and will occur prior to a subject’s first imaging session. After the 
session, any questions or concerns will be discussed. Participants may be excused from the mock scanner 
training at subsequent study visits.  
 
7.5. Incidental Findings by MRI 
The study subjects to be recruited for this study are cognitively healthy. Sometimes, however, a few incidental 
abnormalities may be found in these study subjects, such as brain tumors, vascular lesions, moderate to 
severe white matter lesion load, and other neuroradiological abnormalities that would preclude subjects from 
being included in the analysis, or that are clinical abnormalities requiring follow-up. These subjects will be 
advised to see their physicians for formal assessment. Dr. Piero Antuono is a board-certified neurologist at 
MCW. He or a Co-I (MD) will consult with the subjects in such cases.  
 
7.6. Subject Safety 
Due to the duration and the low doses of AGB101administered in this study, we anticipate this study to be at 
minimal risk to the volunteer. We do not expect frequent or severe AEs to be present. Subjects will be 
instructed to call the study site for intercurrent illnesses. All AEs will be monitored according to the Safety Plan 
(see Section 7.1). The DSMB board will review the initiation frequency and severity of any AEs. Stopping rules 
will be invoked for the subject and study if AE are reported above the 5% threshold. Subject safety will be 
ensured during the study period, and the study may be closed with regular visits to the resolution of the AE. 
Safeguards to ensure subject confidentiality will consist of maintaining records in a locked cabinet in a locked 
room in the Department of Neurology research offices. All computers are password protected to further ensure 
safety. 
 
7.8. Data  
AE data analysis will be performed every 2 months. Plans for unexpected problems involving risk to 
participants or others will consist in notification of the principal investigator in order to activate appropriate 
medical or professional intervention as needed.  
 
7.9. Efficacy  
Evaluation of efficacy will consist of measurement of brain connectivity at the interim point when recruitment 
has reached 50% of subject with a second fMRI session. Secondary outcome measure of cognitive 


21 
 
  Protocol v5 01Apr2019 
assessment will also be reviewed. It is anticipated, however, that with the short, 2-week treatment period, 
statistical significance may not be reached. 
 
7.10. Feedback Mechanism  
Evaluation and response to subjects’ complaints will be routinely addressed by the DSMB. The IRB and 
principal investigator will be notified of significant protocols violations and emergence of unexpected AEs at 
any time during the study. In addition, a report by the DSMB will be provided to the IRB at the end of the study. 
 
 
Table 1. Schedule of study procedures. 
 
 
 
 
 
Screeningh 
Baseline 
Period 1b 
Washout c 
Period 2 b,d 
Visit 
−1 
1 
2e 
3i 
4e 
Duration 
 
 
2 weeks 
4 weeks 
2 weeks 
Informed Consent #1a 
X 
 
 
 
 
Blood (or Buccal Swab) for APOE 
X 
 
 
 
 
Demographics 
X 
 
 
 
 
Medical History 
X 
 
 
 
 
Informed Consent #2a 
 
X 
 
 
 
Inclusion/Exclusion Criteria 
 
X 
 
 
 
NeuroExam 
X 
 
 
 
 
Vital Signs 
X 
X 
X 
X 
X 
C-SSRS 
X 
 
X 
 
X 
MOCA, RBANS 
X 
 
 
 
 
Neuropsychological Battery 
 
X 
X 
 
X 
Neuroimaging Assessments (fMRI)f 
 
Xg 
X 
 
X 
Blood Draw for AGB101 level 
 
 
X 
X 
X 
Randomization 
 
X 
 
 
 
Dispense Medication 
 
X 
 
X 
 
Administer Study Medication 
 
 
X 
 
X 
Medication Compliance  
 
 
X 
 
X 
Adverse Events 
X 
X 
X 
X 
Xf 
a The ICF administered at the screening visit will address the blood (or buccal swab) for APOE and other screening 
procedures. If the study subject is eligible to enroll in the study, a separate ICF administered at the baseline visit will 
address the remainder of the study. 
b Study procedures will take place on the last day of each 2-week treatment period. 
c Medication is dispensed and other procedures obtained on the last day of the 4-week (-/+ 1 week) washout period. 
d In the case of early termination, the Period 2 procedures will be performed. 
e Visit takes place on the last day of the study period which may be a minimum of 14 days and a maximum of 21 days. 
f Prior to each fMRI scan, subjects will undergo MRI safety screening. 
g Subjects will undergo mock scanner training. At subsequent visits, the subject may be excused from mock scanner 
training. 
h Screening activities may be split into 2 or more visits as needed (i.e. separate visit for APOE testing only and 
subsequent screening visit(s) for other screening procedures). Informed Consent #1 will be obtained prior to all 
activities at the first screening visit. With the exception of APOE genetic testing, all other screening procedures must 
be repeated if greater than 3 months elapse from the date of the first screening procedure. 
i Start of Period 2 will be a minimum of 21 days and a maximum of 35 days post the date of the last dose of Period 1. 
(i.e. Washout period of 4 weeks, -/+ 1 week window permitted for scheduling purposes) 
f Telephone call to subject at 4 weeks post last dose in Period 2 (-/+ 1 week) to assess for AEs and AE outcomes. 


22 
 
  Protocol v5 01Apr2019 
8. REFERENCES 
1. Boyle PA, Buchman AS, Wilson RS, Kelly JF, Bennett DA. (2010) The apoe epsilon4 allele is associated 
with incident mild cognitive impairment among community-dwelling older persons. Neuroepidemiology. 
34(1):43-9.  
2. Jefferson AL, Beiser AS, Seshadri S, Wolf PA, Au R. (2015) Apoe and mild cognitive impairment: The 
framingham heart study. Age and ageing. 44(2):307-11.  
3. Tyas SL, Salazar JC, Snowdon DA, Desrosiers MF, Riley KP, Mendiondo MS, Kryscio RJ. (2007) 
Transitions to mild cognitive impairments, dementia, and death: Findings from the nun study. American 
journal of epidemiology. 165(11):1231-8.  
4. Corder EH, Saunders AM, Strittmatter WJ, Schmechel DE, Gaskell PC, Small GW, Roses AD, Haines JL, 
Pericak-Vance MA. (1993) Gene dose of apolipoprotein e type 4 allele and the risk of alzheimer's disease 
in late onset families. Science (New York, N.Y.). 261(5123):921-3.  
5. Crean S, Ward A, Mercaldi CJ, Collins JM, Cook MN, Baker NL, Arrighi HM. (2011) Apolipoprotein e 
epsilon4 prevalence in alzheimer's disease patients varies across global populations: A systematic 
literature review and meta-analysis. Dementia and geriatric cognitive disorders. 31(1):20-30.  
6. Ward A, Crean S, Mercaldi CJ, Collins JM, Boyd D, Cook MN, Arrighi HM. (2012) Prevalence of 
apolipoprotein e4 genotype and homozygotes (apoe e4/4) among patients diagnosed with alzheimer's 
disease: A systematic review and meta-analysis. Neuroepidemiology. 38(1):1-17.  
7. Sperling RA, Laviolette PS, O'Keefe K, O'Brien J, Rentz DM, Pihlajamaki M, Marshall G, Hyman BT, 
Selkoe DJ, Hedden T, Buckner RL, Becker JA, Johnson KA. (2009) Amyloid deposition is associated with 
impaired default network function in older persons without dementia. Neuron. 63(2):178-88.  
8. Hedden T, Van Dijk KR, Becker JA, Mehta A, Sperling RA, Johnson KA, Buckner RL. (2009) Disruption of 
functional connectivity in clinically normal older adults harboring amyloid burden. The Journal of 
neuroscience : the official journal of the Society for Neuroscience. 29(40):12686-94.  
9. Sheline YI, Morris JC, Snyder AZ, Price JL, Yan Z, D'Angelo G, Liu C, Dixit S, Benzinger T, Fagan A, Goate 
A, Mintun MA. (2010) Apoe4 allele disrupts resting state fmri connectivity in the absence of amyloid 
plaques or decreased csf abeta42. The Journal of neuroscience : the official journal of the Society for 
Neuroscience. 30(50):17035-40.  
10. Reiman EM, Chen K, Alexander GE, Caselli RJ, Bandy D, Osborne D, Saunders AM, Hardy J. (2004) 
Functional brain abnormalities in young adults at genetic risk for late-onset alzheimer's dementia. 
Proceedings of the National Academy of Sciences of the United States of America. 101(1):284-9.  
11. Filippini N, MacIntosh BJ, Hough MG, Goodwin GM, Frisoni GB, Smith SM, Matthews PM, Beckmann CF, 
Mackay CE. (2009) Distinct patterns of brain activity in young carriers of the apoe-epsilon4 allele. 
Proceedings of the National Academy of Sciences of the United States of America. 106(17):7209-14.  
12. Sheline YI, Raichle ME, Snyder AZ, Morris JC, Head D, Wang S, Mintun MA. (2010) Amyloid plaques 
disrupt resting state default mode network connectivity in cognitively normal elderly. Biological psychiatry. 
67(6):584-7.  
13. Li W, Antuono PG, Xie C, Chen G, Jones JL, Ward BD, Singh SP, Franczak MB, Goveas JS, Li SJ. (2014) 
Aberrant functional connectivity in papez circuit correlates with memory performance in cognitively intact 
middle-aged apoe4 carriers. Cortex; a journal devoted to the study of the nervous system and behavior. 
57:167-76.  
14. Morris JC, Roe CM, Xiong C, Fagan AM, Goate AM, Holtzman DM, Mintun MA. (2010) Apoe predicts 
amyloid-beta but not tau alzheimer pathology in cognitively normal aging. Annals of neurology. 67(1):122-
31.  
15. Li B, Shi J, Gutman BA, Baxter LC, Thompson PM, Caselli RJ, Wang Y. (2016) Influence of apoe genotype 
on hippocampal atrophy over time - an n=1925 surface-based adni study. PloS one. 11(4):e0152901.  
16. Manning EN, Barnes J, Cash DM, Bartlett JW, Leung KK, Ourselin S, Fox NC. (2014) Apoe epsilon4 is 
associated with disproportionate progressive hippocampal atrophy in ad. PloS one. 9(5):e97608.  
17. Peter J, Scheef L, Abdulkadir A, Boecker H, Heneka M, Wagner M, Koppara A, Kloppel S, Jessen F. (2014) 
Gray matter atrophy pattern in elderly with subjective memory impairment. Alzheimer's & dementia : the 
journal of the Alzheimer's Association. 10(1):99-108.  
18. Striepens N, Scheef L, Wind A, Meiberth D, Popp J, Spottke A, Kolsch H, Wagner M, Jessen F. (2011) 
Interaction effects of subjective memory impairment and apoe4 genotype on episodic memory and 
hippocampal volume. Psychological medicine. 41(9):1997-2006.  


23 
 
  Protocol v5 01Apr2019 
19. Koh MT, Haberman RP, Foti S, McCown TJ, Gallagher M. (2010) Treatment strategies targeting excess 
hippocampal activity benefit aged rats with cognitive impairment. Neuropsychopharmacology : official 
publication of the American College of Neuropsychopharmacology. 35(4):1016-25.  
20. Sanchez PE, Zhu L, Verret L, Vossel KA, Orr AG, Cirrito JR, Devidze N, Ho K, Yu GQ, Palop JJ, Mucke L. 
(2012) Levetiracetam suppresses neuronal network dysfunction and reverses synaptic and cognitive 
deficits in an alzheimer's disease model. Proceedings of the National Academy of Sciences of the United 
States of America. 109(42):E2895-903.  
21. Devi L, Ohno M. (2013) Effects of levetiracetam, an antiepileptic drug, on memory impairments associated 
with aging and alzheimer's disease in mice. Neurobiology of learning and memory. 102:7-11.  
22. Bakker A, Krauss GL, Albert MS, Speck CL, Jones LR, Stark CE, Yassa MA, Bassett SS, Shelton AL, 
Gallagher M. (2012) Reduction of hippocampal hyperactivity improves cognition in amnestic mild cognitive 
impairment. Neuron. 74(3):467-74.  
23. Yassa MA, Stark SM, Bakker A, Albert MS, Gallagher M, Stark CE. (2010) High-resolution structural and 
functional mri of hippocampal ca3 and dentate gyrus in patients with amnestic mild cognitive impairment. 
NeuroImage. 51(3):1242-52.  
24. Vossel KA, Beagle AJ, Rabinovici GD, Shu H, Lee SE, Naasan G, Hegde M, Cornes SB, Henry ML, Nelson 
AB, Seeley WW, Geschwind MD, Gorno-Tempini ML, Shih T, Kirsch HE, Garcia PA, Miller BL, Mucke L. 
(2013) Seizures and epileptiform activity in the early stages of alzheimer disease. JAMA neurology. 
70(9):1158-66.  
25. Baxter MG. (2012) Quieting the overactive hippocampus restores memory in aging. Trends in cognitive 
sciences. 16(7):360-1.  
26. Palop JJ, Chin J, Roberson ED, Wang J, Thwin MT, Bien-Ly N, Yoo J, Ho KO, Yu GQ, Kreitzer A, 
Finkbeiner S, Noebels JL, Mucke L. (2007) Aberrant excitatory neuronal activity and compensatory 
remodeling of inhibitory hippocampal circuits in mouse models of alzheimer's disease. Neuron. 55(5):697-
711.  
27. Palop JJ, Mucke L. (2010) Amyloid-beta-induced neuronal dysfunction in alzheimer's disease: From 
synapses toward neural networks. Nature neuroscience. 13(7):812-8.  
28. Verret L, Mann EO, Hang GB, Barth AM, Cobos I, Ho K, Devidze N, Masliah E, Kreitzer AC, Mody I, Mucke 
L, Palop JJ. (2012) Inhibitory interneuron deficit links altered network activity and cognitive dysfunction in 
alzheimer model. Cell. 149(3):708-21.  
29. Busche MA, Chen X, Henning HA, Reichwald J, Staufenbiel M, Sakmann B, Konnerth A. (2012) Critical 
role of soluble amyloid-beta for early hippocampal hyperactivity in a mouse model of alzheimer's disease. 
Proceedings of the National Academy of Sciences of the United States of America. 109(22):8740-5.  
30. Busche MA, Eichhoff G, Adelsberger H, Abramowski D, Wiederhold KH, Haass C, Staufenbiel M, Konnerth 
A, Garaschuk O. (2008) Clusters of hyperactive neurons near amyloid plaques in a mouse model of 
alzheimer's disease. Science (New York, N.Y.). 321(5896):1686-9.  
31. Putcha D, Brickhouse M, O'Keefe K, Sullivan C, Rentz D, Marshall G, Dickerson B, Sperling R. (2011) 
Hippocampal hyperactivation associated with cortical thinning in alzheimer's disease signature regions in 
non-demented elderly adults. The Journal of neuroscience : the official journal of the Society for 
Neuroscience. 31(48):17680-8.  
32. Dennis NA, Browndyke JN, Stokes J, Need A, Burke JR, Welsh-Bohmer KA, Cabeza R. (2010) Temporal 
lobe functional activity and connectivity in young adult apoe varepsilon4 carriers. Alzheimer's & dementia : 
the journal of the Alzheimer's Association. 6(4):303-11.  
33. Tuminello ER, Han SD. (2011) The apolipoprotein e antagonistic pleiotropy hypothesis: Review and 
recommendations. International journal of Alzheimer's disease. 2011:726197.  
34. Cirrito JR, Yamada KA, Finn MB, Sloviter RS, Bales KR, May PC, Schoepp DD, Paul SM, Mennerick S, 
Holtzman DM. (2005) Synaptic activity regulates interstitial fluid amyloid-beta levels in vivo. Neuron. 
48(6):913-22.  
35. Buckner RL, Snyder AZ, Shannon BJ, LaRossa G, Sachs R, Fotenos AF, Sheline YI, Klunk WE, Mathis CA, 
Morris JC, Mintun MA. (2005) Molecular, structural, and functional characterization of alzheimer's disease: 
Evidence for a relationship between default activity, amyloid, and memory. The Journal of neuroscience : 
the official journal of the Society for Neuroscience. 25(34):7709-17.  
36. Sheline YI, Raichle ME. (2013) Resting state functional connectivity in preclinical alzheimer's disease. 
Biological psychiatry. 74(5):340-7.  


24 
 
  Protocol v5 01Apr2019 
37. Vlassenko AG, Vaishnavi SN, Couture L, Sacco D, Shannon BJ, Mach RH, Morris JC, Raichle ME, Mintun 
MA. (2010) Spatial correlation between brain aerobic glycolysis and amyloid-beta (abeta ) deposition. 
Proceedings of the National Academy of Sciences of the United States of America. 107(41):17763-7.  
38. Bookheimer  SY, Strojwas  MH, Cohen  MS, Saunders  AM, Pericak-Vance  MA, Mazziotta  JC, Small  GW. 
(2000) Patterns of brain activation in people at risk for alzheimer's disease. New England Journal of 
Medicine. 343(7):450-6.  
39. Dickerson BC, Salat DH, Bates JF, Atiya M, Killiany RJ, Greve DN, Dale AM, Stern CE, Blacker D, Albert 
MS, Sperling RA. (2004) Medial temporal lobe function and structure in mild cognitive impairment. Annals 
of neurology. 56(1):27-35.  
40. Celone KA, Calhoun VD, Dickerson BC, Atri A, Chua EF, Miller SL, DePeau K, Rentz DM, Selkoe DJ, 
Blacker D, Albert MS, Sperling RA. (2006) Alterations in memory networks in mild cognitive impairment and 
alzheimer's disease: An independent component analysis. The Journal of neuroscience : the official journal 
of the Society for Neuroscience. 26(40):10222-31.  
41. Ewers M, Sperling RA, Klunk WE, Weiner MW, Hampel H. (2011) Neuroimaging markers for the prediction 
and early diagnosis of alzheimer's disease dementia. Trends in neurosciences. 34(8):430-42.  
42. Goveas JS, Xie C, Chen G, Li W, Ward BD, Franczak MB, Jones JL, Antuono PG, Li SJ. (2013) Functional 
network endophenotypes unravel the effects of apolipoprotein e epsilon 4 in middle-aged adults. PloS one. 
8(2):e55902.  
43. Seeley WW, Crawford RK, Zhou J, Miller BL, Greicius MD. (2009) Neurodegenerative diseases target 
large-scale human brain networks. Neuron. 62(1):42-52.  
44. Mohajeri MH, Saini K, Schultz JG, Wollmer MA, Hock C, Nitsch RM. (2002) Passive immunization against 
beta-amyloid peptide protects central nervous system (cns) neurons from increased vulnerability 
associated with an alzheimer's disease-causing mutation. The Journal of biological chemistry. 
277(36):33012-7.  
45. Tampellini D, Capetillo-Zarate E, Dumont M, Huang Z, Yu F, Lin MT, Gouras GK. (2010) Effects of synaptic 
modulation on beta-amyloid, synaptophysin, and memory performance in alzheimer's disease transgenic 
mice. The Journal of neuroscience : the official journal of the Society for Neuroscience. 30(43):14299-304.  
46. Chen G, Shu H, Chen G, Ward BD, Antuono PG, Zhang Z, Li SJ. (2016) Staging alzheimer's disease risk 
by sequencing brain function and structure, cerebrospinal fluid, and cognition biomarkers. Journal of 
Alzheimer's disease : JAD. 54(3):983-93.  
47. Young AL, Oxtoby NP, Daga P, Cash DM, Fox NC, Ourselin S, Schott JM, Alexander DC. (2014) A data-
driven model of biomarker changes in sporadic alzheimer's disease. Brain : a journal of neurology. 137(Pt 
9):2564-77.  
48. Cavedo E, Lista S, Khachaturian Z, Aisen P, Amouyel P, Herholz K, Jack CR, Jr., Sperling R, Cummings J, 
Blennow K, O'Bryant S, Frisoni GB, Khachaturian A, Kivipelto M, Klunk W, Broich K, Andrieu S, de 
Schotten MT, Mangin JF, Lammertsma AA, Johnson K, Teipel S, Drzezga A, Bokde A, Colliot O, Bakardjian 
H, Zetterberg H, Dubois B, Vellas B, Schneider LS, Hampel H. (2014) The road ahead to cure alzheimer's 
disease: Development of biological markers and neuroimaging methods for prevention trials across all 
stages and target populations. The journal of prevention of Alzheimer's disease. 1(3):181-202.  
49. Seripa D, Panza F, Franceschi M, D'Onofrio G, Solfrizzi V, Dallapiccola B, Pilotto A. (2009) Non-
apolipoprotein e and apolipoprotein e genetics of sporadic alzheimer's disease. Ageing research reviews. 
8(3):214-36.  
50. Corder EH, Saunders AM, Risch NJ, Strittmatter WJ, Schmechel DE, Gaskell PC, Jr., Rimmler JB, Locke 
PA, Conneally PM, Schmader KE, et al. (1994) Protective effect of apolipoprotein e type 2 allele for late 
onset alzheimer disease. Nature genetics. 7(2):180-4.  
51. Hilal-Dandan R, Brunton LL, Goodman LS. Goodman and gilman's manual of pharmacology and 
therapeutics, editors randa hilal-dandan, laurence l. Brunton. New York: McGraw-Hill; 2014. 
52. Lynch BA, Lambeng N, Nocka K, Kensel-Hammes P, Bajjalieh SM, Matagne A, Fuks B. (2004) The 
synaptic vesicle protein sv2a is the binding site for the antiepileptic drug levetiracetam. Proceedings of the 
National Academy of Sciences of the United States of America. 101(26):9861-6.  
53. Vogl C, Mochida S, Wolff C, Whalley BJ, Stephens GJ. (2012) The synaptic vesicle glycoprotein 2a ligand 
levetiracetam inhibits presynaptic ca2+ channels through an intracellular pathway. Molecular 
pharmacology. 82(2):199-208.  
54. Rogawski MA. (2006) Diverse mechanisms of antiepileptic drugs in the development pipeline. Epilepsy 
research. 69(3):273-94.  


25 
 
  Protocol v5 01Apr2019 
55. Lyseng-Williamson KA. (2011) Spotlight on levetiracetam in epilepsy. CNS drugs. 25(10):901-5.  
56. Loscher W, Honack D. (2000) Development of tolerance during chronic treatment of kindled rats with the 
novel antiepileptic drug levetiracetam. Epilepsia. 41(12):1499-506.  
57. Glien M, Brandt C, Potschka H, Loscher W. (2002) Effects of the novel antiepileptic drug levetiracetam on 
spontaneous recurrent seizures in the rat pilocarpine model of temporal lobe epilepsy. Epilepsia. 43(4):350-
7.  
58. van Vliet EA, van Schaik R, Edelbroek PM, da Silva FH, Wadman WJ, Gorter JA. (2008) Development of 
tolerance to levetiracetam in rats with chronic epilepsy. Epilepsia. 49(7):1151-9.  
59. Loscher W, Schmidt D. (2006) Experimental and clinical evidence for loss of effect (tolerance) during 
prolonged treatment with antiepileptic drugs. Epilepsia. 47(8):1253-84.  
60. Ben-Menachem E, Edrich P, Van Vleymen B, Sander JW, Schmidt B. (2003) Evidence for sustained 
efficacy of levetiracetam as add-on epilepsy therapy. Epilepsy research. 53(1-2):57-64.  
61. Caselli RJ, Dueck AC, Osborne D, Sabbagh MN, Connor DJ, Ahern GL, Baxter LC, Rapcsak SZ, Shi J, 
Woodruff BK, Locke DE, Snyder CH, Alexander GE, Rademakers R, Reiman EM. (2009) Longitudinal 
modeling of age-related memory decline and the apoe epsilon4 effect. The New England journal of 
medicine. 361(3):255-63.  
62. Sheikh JI, Yesavage JA. (1986) Geriatric depression scale (gds): Recent evidence and development of a 
shorter version. Clinical Gerontologist: The Journal of Aging and Mental Health. 5(1-2):165-73.  
63. Wechsler D. Wms-r : Wechsler memory scale-revised : Manual. San Antonio [u.a.]: Harcourt Brace 
Jovanovich; 1987. 
64. Folstein MF, Folstein SE, McHugh PR. (1975) "Mini-mental state". A practical method for grading the 
cognitive state of patients for the clinician. Journal of psychiatric research. 12(3):189-98.  
65. Morris JC. (1993) The clinical dementia rating (cdr): Current version and scoring rules. Neurology. 
43(11):2412-4.  
66. Raber J, Huang Y, Ashford JW. (2004) Apoe genotype accounts for the vast majority of ad risk and ad 
pathology. Neurobiology of aging. 25(5):641-50.  
67. Dubois B, Feldman HH, Jacova C, Hampel H, Molinuevo JL, Blennow K, DeKosky ST, Gauthier S, Selkoe 
D, Bateman R, Cappa S, Crutch S, Engelborghs S, Frisoni GB, Fox NC, Galasko D, Habert MO, Jicha GA, 
Nordberg A, Pasquier F, Rabinovici G, Robert P, Rowe C, Salloway S, Sarazin M, Epelbaum S, de Souza 
LC, Vellas B, Visser PJ, Schneider L, Stern Y, Scheltens P, Cummings JL. (2014) Advancing research 
diagnostic criteria for alzheimer's disease: The iwg-2 criteria. The Lancet. Neurology. 13(6):614-29.  
68. Albert MS, DeKosky ST, Dickson D, Dubois B, Feldman HH, Fox NC, Gamst A, Holtzman DM, Jagust WJ, 
Petersen RC, Snyder PJ, Carrillo MC, Thies B, Phelps CH. (2011) The diagnosis of mild cognitive 
impairment due to alzheimer's disease: Recommendations from the national institute on aging-alzheimer's 
association workgroups on diagnostic guidelines for alzheimer's disease. Alzheimer's & dementia : the 
journal of the Alzheimer's Association. 7(3):270-9.  
69. Dubois B, Hampel H, Feldman HH, Scheltens P, Aisen P, Andrieu S, Bakardjian H, Benali H, Bertram L, 
Blennow K, Broich K, Cavedo E, Crutch S, Dartigues JF, Duyckaerts C, Epelbaum S, Frisoni GB, Gauthier 
S, Genthon R, Gouw AA, Habert MO, Holtzman DM, Kivipelto M, Lista S, Molinuevo JL, O'Bryant SE, 
Rabinovici GD, Rowe C, Salloway S, Schneider LS, Sperling R, Teichmann M, Carrillo MC, Cummings J, 
Jack CR, Jr. (2016) Preclinical alzheimer's disease: Definition, natural history, and diagnostic criteria. 
Alzheimer's & dementia : the journal of the Alzheimer's Association. 12(3):292-323.  
70. Kundu P, Brenowitz ND, Voon V, Worbe Y, Vertes PE, Inati SJ, Saad ZS, Bandettini PA, Bullmore ET. 
(2013) Integrated strategy for improving functional connectivity mapping using multiecho fmri. Proceedings 
of the National Academy of Sciences of the United States of America. 110(40):16187-92.  
71. Xie C, Li W, Chen G, Douglas Ward B, Franczak MB, Jones JL, Antuono PG, Li SJ, Goveas JS. (2012) The 
co-existence of geriatric depression and amnestic mild cognitive impairment detrimentally affect gray 
matter volumes: Voxel-based morphometry study. Behavioural brain research. 235(2):244-50.  
72. Petersen RC, Aisen P, Boeve BF, Geda YE, Ivnik RJ, Knopman DS, Mielke M, Pankratz VS, Roberts R, 
Rocca WA, Weigand S, Weiner M, Wiste H, Jack CR, Jr. (2013) Mild cognitive impairment due to alzheimer 
disease in the community. Annals of neurology. 74(2):199-208.  
73. Fischl B, Dale AM. (2000) Measuring the thickness of the human cerebral cortex from magnetic resonance 
images. Proceedings of the National Academy of Sciences of the United States of America. 97(20):11050-
5.  


26 
 
  Protocol v5 01Apr2019 
74. Jack CR, Jr. (1994) Mri-based hippocampal volume measurements in epilepsy. Epilepsia. 35 Suppl 6:S21-
9.  
75. Fox MD, Raichle ME. (2007) Spontaneous fluctuations in brain activity observed with functional magnetic 
resonance imaging. Nature reviews. Neuroscience. 8(9):700-11.  
76. Xie C, Bai F, Yu H, Shi Y, Yuan Y, Chen G, Li W, Chen G, Zhang Z, Li SJ. (2012) Abnormal insula functional 
network is associated with episodic memory decline in amnestic mild cognitive impairment. NeuroImage. 
63(1):320-7.  
77. Bakker A, Krauss GL, Albert MS, Speck CL, Jones LR, Stark CE, Yassa MA, Bassett SS, Shelton AL, 
Gallagher M. (2012) Reduction of hippocampal hyperactivity improves cognition in amnestic mild cognitive 
impairment. Neuron. 2012 May 10;74(3):467-74.  
78. Bakker A, Albert MS, Krauss G, Speck CL, Gallagher M. (2015) Response of the medial temporal lobe 
network in amnestic mild cognitive impairment to therapeutic intervention assessed by fMRI and memory 
task 
performance. 
Neuroimage 
Clin. 2015 Feb 
21;7:688-98. 
doi: 
10.1016/j.nicl.2015.02.009. 
eCollection 2015.