S-Equol in Alzheimer's Disease 2 (SEAD2) Trial
Phase: PHASE1
Conditions: Alzheimer Disease
Sponsor: Russell Swerdlow

 
 
 
 
 
 
 
 
 
 
S-Equol in Alzheimer’s Disease 2 (SEAD2) Trial 
IND # 122175 
06-09-2020 
 
 
 
 
 
 
 
 
Russell H. Swerdlow, MD 
Principal Investigator 
Professor of Neurology 
 
 
 
 
 
 
 
 
 
 
 
Neurology 
University of Kansas Medical Center 
3901 Rainbow Blvd 
Kansas City, KS 66160 
Phone: 913-945-6632 
Fax: 913-945-5035 
Email: rswerdlow@kumc.edu 
 
 

 
 
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Contents 
 
1.0 INTRODUCTION ....................................................................................................................................... 3 
2.0 OBJECTIVES ............................................................................................................................................. 4 
3.0 BACKGROUND ......................................................................................................................................... 4 
4.0 PARTICIPANT SELECTION ........................................................................................................................ 5 
5.0 TREATMENT PLAN ................................................................................................................................... 8 
6.0 EXPECTED TOXICITIES AND DOSING DELAY/DOSING MODIFICATIONS ................................................ 13 
7.0 DRUG FORMULATION AND ADMINISTRATION ..................................................................................... 13 
8.0 CORRELATIVE STUDIES .......................................................................................................................... 13 
9.0 STUDY CALENDAR ................................................................................................................................. 14 
10.0 MEASUREMENT OF EFFECT ................................................................................................................. 14 
11.0 ADVERSE EVENT REPORTING REQUIREMENTS ................................................................................... 15 
12.0 DATA AND SAFETY MONITORING ....................................................................................................... 16 
13.0 REGULATORY CONSIDERATION........................................................................................................... 16 
14.0 STATISTICAL CONSIDERATION ............................................................................................................ 17 
15.0 PUBLICATION PLAN ........................................................................................................................... 178 
16.0 REFERENCES ........................................................................................................................................ 18 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
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1.0 Introduction 
Introductory Statement 
This study is called the “S-Equol in Alzheimer’s Disease 2” (SEAD2) Study.  The SEAD2 study 
will test whether S-equol can improve a particular energy metabolism deficit that is found in 
persons with Alzheimer’s disease (AD).  Specifically, cytochrome oxidase (COX) activity is 
reduced in both brains and platelets from AD subjects.  S-equol, an estrogen receptor β (ERβ) 
agonist, when administered with other estrogen receptor β (ERβ) agonists, has been shown to 
increase mouse brain COX activity [1].  Subsequently, a pilot clinical study of S-equol generated 
data consistent with a possible increase in AD patient platelet mitochondria COX activity.  In 
that study, the original S-equol in Alzheimer’s Disease (SEAD) trial, S-equol (10 mg twice 
daily) was administered to 15 women with AD.  The platelet mitochondria COX activity before 
initiating S-equol (lead-in), after two weeks of S-equol (active treatment), and two weeks after 
stopping S-equol (wash-out) was determined.  Because the intra-individual variation of this 
enzyme across samples taken at different times was unknown, an anticipated pattern of response 
design was used as the primary outcome.  Participants whose lead-in to active treatment slope 
exceeded their active treatment to wash-out slope were considered to show a positive response 
pattern.  The primary outcome was reached, as 11 positive responses were required to reject the 
null hypothesis and 11 positive responses were observed (p<0.06).   
For the SEAD2 trial, Ausio Pharmaceuticals, LLC will supply S-equol capsules to investigators 
at the Kansas University Medical Center (KUMC).  The KUMC investigators will provide the S-
equol capsules to AD patients, and determine whether the S-equol increases platelet COX 
activity.  Because the original SEAD trial addressed issues of intra-individual variation for this 
enzyme across samples taken at different times, we were able to design SEAD2 so that the intra-
individual differences in COX activity observed when subjects are on S-equol versus placebo 
can serve as continuous values that inform the primary outcome (i.e. does S-equol produce a 
significant increase in mean platelet mitochondria COX activity over placebo).  
As part of an exploratory sub-study, SEAD2 will also evaluate whether platelet mitochondria 
COX activity is lower in AD participants who possess the APOE4 variant of the APOE gene 
than it is in AD participants who do not possess the APOE4 variant of the APOE gene.  This is 
an important question to address because during the original SEAD study, a secondary data 
analysis found platelet mitochondria COX activity was lower in APOE4 carriers than it was in 
non-carriers.  This observation was based on a limited number of participants and therefore we 
currently consider this a preliminary finding.  If validated in a larger number of participants, this 
finding could have important implications for our understanding of AD etiology, biomarker 
development, and therapeutic development.  In addition, the SEAD2 trial will explore if there are 
correlations between platelet mitochondria COX activity and cognitive abilities in both APOE4 
carriers and non-carriers. 

 
 
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2.0 Objectives 
Description of Primary Objective 
The primary trial endpoint is the mean intra-individual difference in platelet mitochondria COX 
activity that is measured when subjects have been treated for one month with S-equol (50 mg 
twice daily) versus one month with placebo.  The study, therefore, uses a crossover design to 
facilitate a paired statistical analysis for data obtained from each of the treated participants. As 
the original SEAD study suggested APOE4 non-carriers may experience a more robust response 
to S-equol than APOE4 carriers, to maximize the power of this study the primary outcome 
measure will focus on APOE4 non-carrier participants.   
Description of Secondary Objectives 
Our secondary objectives are as follows:  
(1) Determine whether S-equol, in a dose of 50 mg twice daily over one month, is safe and 
tolerated when taken by AD participants.  This secondary objective endpoint is a composite 
that takes into account vital signs, standard blood-based safety screens, physical and 
neurological examinations, and ascertainment of participant and caregiver-provided symptom 
complaints.   
(2) Determine whether S-equol, in a dose of 50 mg twice daily over one month, influences 
participant scores on a cognitive assessment test battery. 
(3) Determine whether S-equol, in a dose of 50 mg twice daily over one month, produces 
evidence of increased platelet mitochondria COX activity within the context of an anticipated 
pattern of response analysis.  
 
Description of Exploratory Objectives 
Exploratory objectives in the SEAD2 trial include: 
 
1)  Determine whether platelet mitochondria COX activity is lower in AD participants who 
possess the APOE4 variant of the APOE gene than it is in AD participants who do not possess 
the APOE4 variant of the APOE gene. 
2)  Examine whether there are correlations between platelet mitochondria COX activity and 
cognitive abilities. 
3.0 BACKGROUND 
Prevalence 
The elderly represent a rapidly growing demographic.  This reflects public health initiatives and 
medical advances that reduce risks and provide treatments for many previously terminal 
disorders.  An increasing population of neurodegenerative disease victims is arguably the biggest 
downside to this success.  For several common neurodegenerative diseases incidence rises with 
advancing age and prevalence is quite high [2].  AD, the most common neurodegenerative 
disease, affects 5.4 million Americans and one in every eight Americans over 65 is estimated to 

 
 
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have it [3].  Society is also affected, as families and friends of AD patients provide most day-to-
day care and altogether AD now costs our economy $385 billion annually [4].     
Rationale for Use 
Associations between aging, AD, and mitochondrial function are well-documented [5].  Deficits 
that arise with advancing age tend to exaggerate in AD.  In AD brains various mitochondria-
localized enzymes show reduced activity [6], and in most neurons intact mitochondria are 
numerically reduced [7].  Investigators increasingly agree AD mitochondrial dysfunction is 
disease-relevant and a reasonable therapeutic target [8].  
A consistently demonstrated AD mitochondrial lesion includes reduced COX activity [6].  
Interestingly, COX activity is reduced in both brain and platelet mitochondria obtained from AD 
subjects.  While it is only possible to harvest brain mitochondria from autopsy brains, platelets 
are easily obtained from living subjects and can be serially acquired.  For drugs that may 
enhance mitochondrial function, platelet COX activity offers a unique opportunity for assessing 
mitochondrial target engagement.  
“Mitochondrial medicine” refers to treating disease by therapeutically targeting mitochondria 
[9].  More recently, the term “bioenergetic medicine” was introduced to describe interventions 
that specifically increase cell energy production [10].  For a neurodegenerative disease such as 
AD, the ideal agent must be systemically safe, cross the blood brain barrier, access neurons, 
potentially activate mitochondrial biogenesis, and possibly increase mitochondrial respiration.   
Description of study agent 
S-equol is an ERβ agonist [11, 12].  ERβ is found within mitochondria, and ERβ activation 
reportedly stimulates mitochondrial function.  ERβ has also been implicated in mitochondrial 
biogenesis, the process through which new mitochondria are generated within cells [13], and 
which partly determines a cell’s mitochondrial mass [14].  For these reasons, we hypothesize S-
equol may potentially benefit AD patients.   
4.0 PARTICIPANT SELECTION 
AD participants will be recruited by the University of Kansas Alzheimer’s Disease Center’s (KU 
ADC) Recruitment Division, and will meet current McKhann el al. criteria for probable AD [15]. 
Those qualifying for the trial intervention must be found at screening to not carry a copy of the 
APOE 4 gene.  Each participant must have a study partner.  The study partner will be someone 
who knows the participant well (typically a spouse, relative, or friend of the participant) and is 
able to answer questions about the participant’s changes in health and/or behaviors over the 
course of the study.  In addition, the study partner will oversee the administration of the study 
medication at home and agree to accompany the participant to all study visits. 
Enrollment Information 

 
 
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SEAD2 will recruit and consent approximately 100 participants and the study will include both 
men and women.  Participants will be recruited from various sources including the KU ADC 
Clinical Cohort registry and other research recruitment databases such as the KU Heron 
database, the University of Kansas Medical Center’s Memory Disorders Clinic (KU MDC), and 
persons previously diagnosed with AD who contact the KU ADC Recruitment Division in order 
to enroll in an AD clinical trial.  Our intent is to ultimately identify approximately 40 individuals 
who meet all study entry criteria, including being non-carriers of the APOE4 variant of the 
APOE gene, as power calculations performed on data from the first SEAD study indicate the 
least number of study participants needed to show a mean increase in platelet mitochondria COX 
activity is observed when just APOE4 non-carrier participants are considered.    
Study Recruitment 
Based on historical data, approximately 40-60% of AD participants do not carry an APOE4 
allele.  If we were to consent and screen participants with no a priori knowledge of their APOE 
status, we conservatively estimate we would need to consent approximately 100 participants to 
identify approximately 40 APOE4 non-carrier participants.  Our decision to identify 
approximately 40 APOE4 non-carriers is based on an analysis of the original SEAD study data; a 
power calculation performed using those data determined 35 participants should provide an 80% 
chance of showing an S-equol-associated increase in the mean platelet mitochondria COX 
activity of the group (as compared to the mean platelet mitochondria COX activity of the group 
following a period of placebo treatment) if such an effect indeed exists.  To account for attrition 
and ensure that a minimum of 35 subjects complete the intervention, we plan to enroll 
approximately 40 participants. Additional participants may be enrolled if participants are in 
screening at the time of enrollment of the 40th participant. 
We plan to enrich our recruitment of approximately 40 APOE4 non-carrier study participants by 
recruiting participants through the KU MDC and KU ADC whose APOE genotype status has 
already been determined.  Some individuals followed by the KU MDC have previously 
undergone APOE genotype screening as part of their routine clinical diagnostic evaluation and 
are aware of their APOE genotype.  For individuals with a known APOE genotype (i.e. 
participants for whom the study coordinator, the participant, and the study partner already know 
an APOE4 allele is not present) that are recruited through the KU MDC, genetic counseling will 
still be provided during the consent process.  Repeat APOE genotyping will also be performed to 
confirm the accuracy of the commercial genotype test, although we do not anticipate that the 
commercial determination will be found to be inaccurate.   
We have already determined the APOE status for members of the KU ADC Clinical Cohort.  
However, KU ADC Cohort members were APOE genotyped under a clinical protocol that does 
not permit us to disclose APOE status to these individuals.  Therefore, when recruiting from the 
KU ADC Clinical Cohort, for every 4 APOE4 non-carriers we invite to participate we will also 
invite 1 APOE4 carrier.  During the consenting process we will provide genetic counseling about 
the implications of APOE genotyping and explain to the invited individuals and their care 

 
 
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providers that following informed consent APOE status will be determined and they will be told 
whether the individual does or does not possess an APOE4 allele.  All participants recruited 
through the KU ADC Clinical Cohort will, therefore, undergo repeat APOE genotyping as part 
of the SEAD2 study.   
Additional participants will be recruited from outside the KU MDC or the KU ADC Clinical 
Cohort (i.e. through word of mouth, media appeals, and from individuals who for other reasons 
contact the KU ADC Recruitment Division specifically to learn about potential clinical trial 
options).  For these individuals, we will likely have no a priori knowledge of their APOE 
genotype.  During the consenting process we will provide genetic counseling about the 
implications of APOE genotyping and explain to these individuals and their care providers that, 
following informed consent, their APOE status will be determined and they will be told whether 
the participant does or does not possess an APOE4 allele. 
Intervention Inclusion Criteria 
Individuals are eligible if they meet the following criteria:  
1. Have a diagnosis of AD, per McKhann et al. criteria [15]. 
2. Have a study partner. The study partner must have a close relationship with the participant and 
have sufficient contact, such that observations of any changes in health or behaviors can be 
made.  The study partner must be willing to attend study visits with the participant, be willing to 
answer questions regarding any changes in the participant’s health/well-being and/or changes in 
medications, and help to ensure the study medication is taken as directed. 
3. Do not possess an APOE4 variant of the APOE gene. 
4. Speak English as their primary language.   
5. Are age 50 to 90. 
6. Have not had any medication changes within the past 30 days. 
Intervention Exclusion Criteria 
Individuals are ineligible if they meet any of the following criteria: 
1. Reside in a nursing home or dementia special care unit. 
2. Have a potentially confounding, serious medical risk such as insulin-requiring diabetes, any 
history of cancer that required a systemic chemotherapy or radiation therapy intervention 
within the past 5 years (basal or squamous cell cancer and localized prostate cancer  that has 
been treated successfully within the past 5 years is allowed), or a recent cardiac event (i.e. 
heart attack, angioplasty, etc.).   

 
 
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3. Have any clinically significant abnormal safety laboratory values at the SEAD2 screening 
visit. 
4. Have any clinically significant abnormal findings on vital signs measurements, or on physical 
or neurological examination at the SEAD2 screening visit. 
5. Use any type of systemic estrogen or testosterone replacement therapy. 
6. Has received any investigational drug or investigational therapy within 30 days before the 
Screening Visit. 
 
5.0 TREATMENT PLAN 
Overall Study Design  
SEAD2 is a randomized, double-blind, crossover study whose primary endpoint is platelet 
mitochondria COX activity.  We will test for differences in platelet mitochondria COX activity 
through a paired statistical analysis of the difference in COX activity means observed when 
participants are maintained for 1 month on S-equol versus 1 month on placebo.  It will 
specifically focus on how S-equol affects the primary endpoint in AD participants who do not 
possess an APOE4 allele, since SEAD study data suggest APOE4 non-carriers may have a more 
robust platelet COX activity response to S-equol treatment.   
Study Procedures 
Visit 1 (also referred to as the Screening Visit):  Informed Consent, Study Introduction, Genetic 
Counseling, Demographics, Medical History, Medication Review, Vital Signs, Physical and 
Neurological Examination, Blood Samples for Safety Laboratory Testing, APOE Status, Baseline 
COX Activity.  
For the evening that precedes this visit, participants will be informed to take nothing per oral 
(NPO), other than water or coffee or medications, after midnight.  The visit will occur in the 
morning, and will be conducted at the KU Clinical Translational Science Unit (CTSU).  Prior to 
any study procedures taking place, informed consent will be obtained.  As part of the informed 
consent process, genetic counseling about the implications of APOE genotyping will be provided 
(the implications of APOE genotyping include learning whether one had an increased genetic 
risk of developing AD; family members may also gain insight into their chances of having 
inherited an APOE4 allele).  Participants will be told that only those who do not carry an APOE4 
allele will be able to proceed to the intervention part of the study, and that if they proceed to the 
intervention part of the study at different points they will receive either S-equol or an inert 
placebo.  
Following consent, the participant’s demographic information, full medical history, and current 
medications will be reviewed/recorded with both the participant and study partner.  A diagnosis 
of Alzheimer’s disease will be confirmed by obtaining copies of the participant’s medical 

 
 
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records.  Participants will undergo a measurement of vital signs and a physical and neurological 
examination to ensure he/she meets study entry criteria and does not have any findings of 
concern regarding health.    Participants will then undergo a 48 ml phlebotomy, for the following 
testing:  
• Approximately 8 ml of the blood will be used for safety lab testing, to include 
chemistry, liver function, renal function, and complete blood cell counts 
• Approximately 1 mL of the blood will be used for APOE genotyping 
• Approximately 39 ml of the blood will be used to determine platelet mitochondria COX 
activity 
Visit 2:  APOE Genotype Disclosure, Cognitive Testing, and Randomization.  Visit 2 will occur 
at the KU CTSU.  The outcome of the APOE genotype test will be shared.  For the APOE4 
carrier participants we will not disclose whether they are APOE4 homozygotes or heterozygotes, 
only that they are APOE4 carriers.   
All participants will undergo a cognitive screening battery.  For APOE4 non-carriers, this 
cognitive screen will determine the cognitive baseline performance.  For APOE4 carriers, the 
cognitive performance data will be analyzed to test for correlations between platelet 
mitochondria COX activity and cognitive abilities.  For APOE4 carriers, this will complete their 
participation in the study.   
APOE4 non-carrier participants will be randomized (assignments will be established prior to the 
visit by the study statistician) to receive S-equol between Visit 2 and Visit 3 with placebo 
between Visit 3 and Visit 4, or to receive placebo between Visit 2 and Visit 3 with S-equol 
between Visit 3 and Visit 4.  Participants and other study staff will remain blinded to the 
assignment, allowing this to proceed as a double-blind study. Randomized participants will also: 
1) Undergo vital sign measurements. 
2) Receive two bottles (each bottle contains 35 capsules) of either S-equol 50 mg or placebo 
capsules (the placebo capsules cannot be distinguished from the active treatment), as 
determined by the randomization process.  
3) Commence their initial one-month treatment intervention; the study medication will be 
taken twice daily for approximately one month. 
Visit 3:  Completion of First Intervention: Review of Adverse Events & Concurrent Medications, 
Vital Signs, Blood Samples for Safety Laboratory Testing and COX Activity, Cognitive Battery, 
Assessment of Study Drug Compliance, Physical and Neurological Examination, and 
Intervention Crossover.  At the end of the first one-month treatment period, participants and 
study partners will return to the KU CTSU for Visit 3.  Visit 3 will occur in the morning, and 
participants will be informed to remain NPO, other than water or coffee or medications, after 
midnight of the preceding evening.  At this visit they will: 

 
 
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1) Undergo a review of any changes in health/adverse events since the last visit. 
2) Undergo a review of any changes in concurrent medications since the last visit. 
3) Undergo vital signs measurement.  
4) Undergo a 40 ml phlebotomy (under fasting conditions), which will be used to measure 
platelet mitochondria COX activity and safety labs (CBC, electrolytes, liver function, 
renal function). 
5) Undergo a cognitive battery (Participants will be given a snack/break before proceeding 
to cognitive testing).  
6) Return all used and unused study medication containers. 
7) Undergo determination of study medication compliance. 
8) Be provided two bottles of either S-equol or placebo capsules; participants originally 
randomized to S-equol capsules at Visit 2 will be provided placebo capsules, while 
participants originally randomized to placebo capsules will be provided S-equol capsules. 
Participants and staff (aside from the study statistician) will remain blinded to the 
treatment assignment. 
9) Commence their second one-month treatment intervention. 
Visit 4:  Completion of Second Intervention: Review of Adverse Events & Concurrent 
Medications, Vital Signs, Blood Samples for Safety Laboratory Testing and COX Activity,  
Cognitive Battery, Physical and Neurological Examination, Assessment of Study Drug 
Compliance.  At the end of the second one-month treatment period, participants and study 
partners will return to the KU CTSU for Visit 4.  Visit 4 will occur in the morning, and 
participants will be informed to remain NPO, other than water or coffee or medications, after 
midnight of the preceding evening.  At this point they will: 
1) Undergo a review of any changes in health/adverse events since the last visit. 
2) Undergo a review of any changes in concurrent medications since the last visit. 
3) Undergo vital signs measurement. (Participants will be given a snack/break before 
proceeding to cognitive testing).  
4) Undergo a 48 ml phlebotomy (under fasting conditions), which will be used to measure 
platelet mitochondria COX activity and safety labs (CBC, electrolytes, liver function, 
renal function). 
5) Undergo a cognitive battery.  
6) Return all used and unused study medication containers. 
7) Undergo determination of study medication compliance. 
 
Post-Interventions Phone Call.  Approximately one month after completing Visit 4, participants 
and their study partners will be contacted by phone.  At this time a review of any changes in 
health/adverse events and changes in concurrent medications will be conducted.  At the time of 
the post-intervention telephone call, all previously reported, as well as newly reported adverse 
events, will be assessed.  Open adverse events that are determined not to be related to the study 

 
 
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treatment will not be further followed, while those that are determined to be related to the study 
treatment will be followed until such time as the adverse event has resolved or been declared 
stable, or the participant is lost to follow up, or it is determined by the PI  (or his designee) that 
the adverse event no longer needs to be followed. 
Study Procedure Details 
Mitochondrial Assays.  Approximately forty ml of blood will be collected in tubes containing 
acid-citrate-dextrose as an anticoagulant and kept at room temperature.  Within 24 hours of 
phlebotomy this blood is transferred to the KU ADC Mitochondrial Genomics and Metabolism 
Core for processing.  Upon receipt platelets are isolated by centrifugation and enriched 
mitochondrial fractions are prepared using previously described methods [16]. The protein 
concentrations of the enriched mitochondrial fractions are measured using a DC protein assay kit 
(BioRad, Hercules, CA).  COX Vmax activity is determined as a pseudo first order-rate constant          
(sec-1/mg protein) by measuring the oxidation of reduced cytochrome c at 550 nm [16, 17].   
In addition to measuring COX activity, we will also measure citrate synthase (CS) Vmax activity 
(nmol/min/mg protein). The COX activity will be referenced to its corresponding CS activity to 
correct for potential inter-sample differences in mitochondrial mass.  This assay is performed 
spectrophotometrically by following the formation of 5-thio-2-nitrobenzoate (412 nm) following 
the addition of 100 μM oxaloacetate at 30°C [16].  Therefore, in addition to referencing COX 
activities to total protein, we will also determine the CS activity so that we can correct the COX 
activity for inter-sample differences in mitochondrial mass that arise during the platelet 
mitochondria isolation procedure.  The COX/CS activity will be the activity used in our primary 
analysis and in all secondary analyses except for the secondary analysis comparison of platelet 
mitochondria COX activity between APOE4 carriers and non-carriers.  For that analysis we will 
reference COX activity to mg of protein in the assay sample (COX/mg protein), as the original 
SEAD study suggested CS activity may also vary by APOE genotype.  Regarding our analysis of 
potential COX activity-cognitive test correlations we will consider both the COX/CS and 
COX/mg protein values.  
Future Use of Samples:  If any portion of the blood samples collected for mitochondrial assays is 
left over, it will be placed into a storage freezer  in the KU ADC Mitochondrial Genomics and 
Metabolism Core Lab for future use. The leftover blood sample may be stored for up to 3 years 
and may be used for further studies of metabolism, in order to help better understand results 
found in the main part of the study . 
For all participants, the mitochondrial enzyme measurements will be performed before APOE 
genotype determination.  This will ensure that the study personnel performing the Visit 1 enzyme 
measurements are blinded to the participant’s APOE genotype status.  
Cognitive Battery.  The SEAD2 cognitive battery will consist of the following tests: 
(1) Montreal Cognitive Assessment (MoCA); 

 
 
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(2) Alzheimer’s Disease Assessment Scale-Cognitive Portion (ADASCog), 11 item test; 
(3) Logical memory test (LMT) immediate and delayed recall; 
(4) Stroop testing.   
Vital Signs.  Vital signs measurements will include blood pressure, pulse, temperature, 
respiration rate, and body weight. 
Genetic Counseling.  We will counsel the participant and their study partner that participation in 
this study will not further inform other family members about their risk of developing AD.  The 
lifetime risk of an individual developing AD when they have a first degree relative (a parent or a 
sibling) who has or had sporadic AD (by far the most common type of AD) is about 30%.  If an 
individual has one APOE4 allele, their lifetime risk is similarly about 30%.  Therefore, 
determining whether the APOE genotype of the participant is or is not an APOE4 carrier will not 
further refine the AD risk estimate of the first degree relatives. 
If a participant is found to carry an APOE4 allele, then the chances that a first degree relative 
will also have that same APOE4 allele is 50%.  Therefore, although this study will not further 
inform first degree relatives about their risk of developing AD, they could learn that they have at 
least a 50% chance of inheriting an APOE4 allele. 
If a participant is found to carry two APOE4 alleles (homozygous APOE4 carriers), then 
children of the participant will have a 100% chance of having at least one APOE4 allele.  In 
order to minimize the chances of generating anxiety amongst the children of our genotyped 
participants, we will not disclose whether a participant was an APOE4 homozygote or 
heterozygote, but rather only that the participant “carries an APOE4 allele.”  By participating in 
this study participants, care partners, or other family members will not be able to infer whether or 
not the participant is an APOE4 homozygote or heterozygote. 
It is important to note that the same genetic information provided during our genetic counseling 
process is provided in the KU MDC setting when APOE genotyping is pursued as part of a 
clinical evaluation.  MDC experience reveals providing this information does not cause patient or 
family stress.  Also, we wish to emphasize that the genetic counseling process for SEAD2 will 
differ from the KU MDC process because SEAD2 participants will already carry a diagnosis of 
AD, whereas patients undergoing APOE genotyping in the KU MDC as part of a clinical 
evaluation typically have not already been diagnosed with AD.  For patients genotyped as part of 
a KU MDC clinical evaluation, APOE determination therefore has a far greater diagnostic 
implication than it will in the SEAD2 trial.  As problems with APOE genotype disclosure are not 
observed in KU MDC patients who do not already carry a diagnosis of AD, we do not anticipate 
any problems with APOE genotype disclosure amongst SEAD2 participants who already carry a 
diagnosis of AD.      
Safety Labs.  A complete blood cell count, serum electrolytes, liver function tests, and a screen of 
renal function will be obtained at the start of the study and for those completing each of the two 

 
 
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intervention stages of the study.  These analyses will be performed through the University of 
Kansas Hospital’s clinical laboratory.  These laboratory assessments will allow us to screen for 
adverse effects on electrolyte balance, bone marrow, liver function, and renal function. 
6.0 EXPECTED TOXICITIES  
There are no expected safety issues.  SEAD2 will expose participants to S-equol at a dose of 50 
mg, twice daily, over a one-month time period.  Clinical and safety trials performed to date do 
not indicate appreciable safety issues arise when S-equol is taken at 50 mg BID for periods of up 
to one month.  
7.0 DRUG FORMULATION AND ADMINISTRATION 
Drug Formulation 
S-equol will be provided by Ausio Pharmaceuticals, LLC.  The study drug and matching placebo 
will be provided as 50 mg capsules, which will be taken twice per day approximately 12 hours 
apart.  Bottles will be coded to protect the blinded nature of the study. 
Drug Administration 
Study drug or matching placebo will be dispensed at Visits 2 and 3.  The study drug/placebo is 
taken orally, twice daily, by the study participant.  The study partner serves the role of 
overseeing the administration of the study medication in the home as needed, to ensure it is taken 
as directed.  
Drug Production Site 
Study drug or matching placebo will be produced by Patheon (Toronto Development Centre) for 
Ausio Pharmaceuticals, LLC. 
Drug Transportation 
The study drug and matching placebo will be shipped to the study site by express mail. 
 
8.0 CORRELATIVE STUDIES 
Prior studies show after a single oral dose of 14C S-equol (2 mg/kg in rats, 1 mg/kg in monkeys) 
the radiolabel is rapidly absorbed and is present in the brain [18].  For this reason, we expect 
drug-induced changes in platelet mitochondria will reasonably reflect changes in brain platelet 
mitochondria. The dose and dosing schedule are based on previous work performed by Ausio 
Pharmaceuticals, LLC, which suggests 50 mg BID affects human physiology.   

 
 
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S-equol was tested in AD women as part of the SEAD study.  The exposure in that study was 10 
mg twice daily for 2 weeks, and no serious adverse events occurred.  S-equol has further 
undergone rigorous phase I testing in 101 subjects, Phase 2a testing in 169 menopausal women 
with vasomotor symptoms, and Phase 2a testing in 124 men with benign prostatic hypertrophy.  
In all cases, S-equol has shown an excellent safety profile, and doses up to 150 mg BID for one 
month are well-tolerated.  We do not anticipate any safety issues with the 50 mg BID, one-month 
course the SEAD2 study will use. 
9.0 STUDY CALENDAR 
 
10.0 MEASUREMENT OF EFFECT 
The primary outcome measure will consider the mean intra-individual difference in COX/CS 
activity for participants concluding one month of placebo treatment versus one month of S-equol 
treatment.  If the change in mean activity shows a statistically significant increase following S-
equol treatment, it will be concluded that S-equol increases platelet mitochondria COX/CS 
activity in APOE4 non-carrier individuals.  
Visit 
Visit 1 
Visit 2  
Visit 3 
Visit 4 
Post-
Interventions 
Phone Call 
Timeframe 
Day 0 
Month 1   
(+ 7 days/  
- 14 days) 
Month 2  
(+/- 7 days)  
Month 3 
(+/- 7 days) 
Month 4             
(+/- 7 days) 
Informed Consent 
X 
 
 
 
 
Review medical history 
X 
 
 
 
 
Review concurrent medications 
X 
X 
X 
X 
X 
Review health changes/adverse events 
 
X 
X 
X 
X 
Genetic Counseling 
X 
 
 
 
 
Physical and Neurological Exam 
X 
 
X 
X 
 
Phlebotomy  (Fasting Conditions) 
     For APOE (at V1 only) 
     For COX Activity 
     For Safety Labs 
 
X 
X 
X 
  
 
 
 
 
 
X 
X 
 
 
X 
X 
 
Cognitive Battery  
 
X 
X 
X 
 
Notification of APOE Status and 
Eligibility to Proceed to Intervention 
 
X 
 
 
 
Randomization of APOE4 Non-Carriers 
 
X 
 
 
 
Two bottles of S-equol 50 mg or 
Placebo Capsules (35 capsules per 
bottle) Dispensed* 
 
X* 
X 
 
 
Vital Signs 
X 
X 
X 
X 
 
Pill Count For Compliance 
 
 
X 
X 
 
*During Visit 2, APOE4 carrier participants who are not randomized to the intervention will not 
undergo these procedures. 

 
 
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Secondary outcomes from the intervention part of the study will include safety data, cognitive 
scores, and categorical response patterns defined by platelet mitochondria COX/CS activity 
measurements..  The safety endpoint will consist of a composite of adverse events reported at 
Visit 2, Visit 3, Visit 4, and the post-Interventions Phone Call; any clinically significant 
abnormal vital signs as determined at Visit 2, Visit 3, and Visit 4; any clinically significant 
findings on the physical and neurological examinations performed at Visit 3 and Visit 4; and any 
clinically significant abnormal safety lab values as determined at Visit 3 and Visit 4.  
The cognitive endpoint will include analyses of scores on the MoCA, ADASCog-11, LMT, and 
Stroop tests. 
For the final secondary outcome, we will categorically define participants as responders or non-
responders depending on the slope of COX/CS activity change across their V1, V3, and V4 
measurements.  This anticipated pattern of response analysis will be performed according to the 
rules defined in the original SEAD study.   
As part of our exploratory analysis we will examine whether platelet mitochondria COX activity 
is lower in AD participants who possess the APOE4 variant of the APOE gene than it is in AD 
participants who do not possess the APOE4 variant of the APOE gene.  For this analysis we will 
reference COX to mg protein, in addition to referencing it to CS activity, because data from the 
first SEAD trial suggested platelet mitochondria CS activity may also be influenced by APOE 
genotype.  We will also examine whether there are correlations between platelet mitochondria 
COX activity and cognitive abilities in both APOE4 carriers and non-carriers. 
11.0 ADVERSE EVENT REPORTING REQUIREMENTS 
11.1 Notifying Local Regulatory Authorities 
Adverse event data (injuries/illness) will be presented to the principal investigator (PI) 
throughout the trial as they occur.  The PI (or his designee) will assess adverse events for 
severity, serious criteria, and relatedness to the study medication or study procedures.  Adverse 
events will be reported to the KUMC Human Subjects Committee (HSC) per their guidelines. 
Upon conclusion of the study, all adverse events will be further analyzed and summarized.   
11.2 Notifying the FDA 
The PI/Sponsor will report serious and unexpected adverse events in an expedited fashion, in 
compliance with FDA regulations.  These written notifications of adverse events (IND safety 
reports) will follow the following guidelines:  
• Within 7 calendar days 
Any study event that is: 
– associated with the use of the study drug; 
– unexpected;  
– fatal or life-threatening, and  

 
 
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• Within 15 calendar days 
Any study event that is: 
– associated with the use of the study drug; 
– unexpected, and 
– serious, but not fatal or life-threatening 
-or- 
– a previous adverse event that was not initially deemed reportable but is later found to 
fit the criteria for reporting (reporting within 15 calendar days from when the event 
was deemed reportable). 
12.0 DATA AND SAFETY MONITORING 
Data Collection 
Data Collection will be overseen by the PI.  The PI will routinely review secondary endpoint 
safety data as it accrues.  The study coordinator will ensure transfer of the secondary endpoint 
safety data to the PI in a timely manner.  The PI will also review primary endpoint (biochemical 
assay) data as it is generated, but in a blinded fashion.  The unblinded statistical analyses will be 
conducted following study completion. 
Data Monitoring  
The Data Monitoring plan for this trial focuses on monitoring by the PI and the trial coordinator.  
Review of the rate of participant accrual and adherence to inclusion/exclusion criteria will occur 
during the recruitment phase to assure that participants meet eligibility criteria. 
Safety Monitoring 
The Safety Monitoring plan for this trial focuses on monitoring by the PI and the trial 
coordinator. 
Data will be collected regarding participant and caregiver reports of any changes in health and/or 
behaviors at study visit 3, study visit 4, and the post-interventions phone call (which each occur 
at approximate 1 month intervals).  The frequency and severity of adverse events will be 
examined and their potential causal relationship to the study drug (as determined by the PI or his 
designee) will be assessed at the time of data collection.   
Any emergent medical condition or safety finding that, in the opinion of the PI, may jeopardize 
the participant’s safety if he/she continues in the study will be sufficient reason for participant 
discontinuation from the study. The participant will be followed for the adverse event until it is 
resolved, declared stable, judged to be unrelated to the study drug, or the participant is lost to 
follow up.  
13.0 REGULATORY CONSIDERATION 
Protection of Human Participants and Informed Consent 

 
 
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Regulations in regard to Human Participants Protection and Informed Consent will be followed 
in accordance with 21 CFR 50.  All institutional, FDA, state and federal regulations concerning 
informed consent will be followed.    
Institutional Review Board (IRB) 
Regulations regarding initial and continuing review of the study by an Institutional Review 
Board will be followed.  The University of Kansas Medical Center has a federally approved 
Institutional Review Board, the KUMC Human Subjects Committee (HSC), which will be 
involved in all aspects of study planning and considerations as per 21 CFR 56.  The KUMC HSC 
FWA# is FWA00003411.   
Financial Disclosure 
There are no financial disclosures to be made.  No marketing approval for the study drug is going 
to be pursued by the PI/Sponsor.   
Obligations of Investigators 
The PI/Sponsor, Dr. Russell Swerdlow, will be responsible for all elements of 21 CFR 312 
subpart D.  Dr. Swerdlow will maintain oversight of the clinical trial ensuring relevant regulatory 
requirements are met and necessary guidelines are followed.  The following are co-investigators: 
• Jeffrey Burns, MD, 
• Jonathan Mahnken, PhD. 
The PI has confirmed all co-investigator credentials, experience, and training.  He has also 
ensured additional personnel will follow the appropriate investigational plans and guidelines as 
submitted in the IND.  All changes to the study, including modifications to the IND, will be 
communicated by the PI and he will be the primary liaison to the IRB.   
All detected and newly discovered adverse effects and risks will be reported by the PI at the time 
of the event, per institutional guidelines.  Record-keeping and data is strictly confidential, only to 
be shared between investigators in the study and only those who are directly involved in the care 
of the patient. 
14.0 STATISTICAL CONSIDERATION 
The primary outcome measure will compare the mean COX activity of participants concluding 
their one-month placebo treatment to that of participants concluding their one-month S-equol 
treatment by calculating the difference for each participant.  We will use the intent-to-treat 
principal (with respect to the randomized treatment order) for primary analysis, but if there are 
protocol deviations with respect to treatment order then we will also conduct a per protocol 
analysis as a secondary assessment.  As the outcome measures (difference scores) are continuous 
data values, we will summarize by means and standard deviations, and compare S-equol versus 
placebo using the paired, one-sample t-test with a two-sided p-value of <0.05 considered 
statistically significant.  We will assess normality assumptions through visual assessments of 

 
 
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residual plots, and also a histogram and q-q plots for further residual analyses.  In the event this 
test’s assumptions appear violated we will conduct the nonparametric Wilcoxon rank sum test of 
the difference scores for the primary assessment. 
We will also conduct secondary analyses of COX activity using linear mixed models.  This will 
incorporate all three COX activity measures on each APOE4 non-carrier participant, and will 
also allow inclusion of data for the APOE4 allele carriers with appropriate model structure/ 
specification.  We will use this model as the basis for various contrasts to test our hypotheses 
statistically with respect to secondary objective.  We will assess this model by residual analyses 
(observed versus residual [conditional and unconditional] plots, residual [conditional and 
unconditional] histograms, and q-q plots [conditional and unconditional]).  We will similarly 
assess other study outcomes using this mixed modeling approach, and as needed for non- or 
semi-continuous outcomes we will utilize other exponential family distributions under the 
generalized linear mixed models framework to incorporate dependence structures appropriately.  
Lastly, we will assess for a pattern of response with respect to secondary objective.  Specifically, 
this simplifies to a nonparametric runs test for the difference between the active drug versus 
placebo COX activity measures. 
15.0 PUBLICATION PLAN 
Publications resulting from this study will be prepared by the PI.  Co-authors will include 
personnel from the study site and from Ausio, LLC who have meaningfully contributed to study 
execution or development.  Only co-authors who agree with the final manuscript will be listed as 
authors on the final manuscript.   
16.0 REFERENCES 
1. 
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neurological implications. Trends Neurosci, 2008. 31(10): p. 529-37. 
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3. 
Hebert, L.E., et al., Alzheimer disease in the US population: prevalence estimates using 
the 2000 census. Arch Neurol, 2003. 60(8): p. 1119-22. 
4. 
Alzheimer's Association.  2011 Alzheimer’s disease facts and figures. Alzheimer's and 
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5. 
Swerdlow, R.H., Brain aging, Alzheimer's disease, and mitochondria. Biochim Biophys 
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Swerdlow, R.H., Mitochondria and cell bioenergetics: increasingly recognized 
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7. 
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Swerdlow, R.H., Role and treatment of mitochondrial DNA-related mitochondrial 
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9. 
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Klinge, C.M., Estrogenic control of mitochondrial function and biogenesis. J Cell 
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