Acute Effect of Lemborexant on CSF Amyloid-Beta and Tau
Phase: PHASE2
Conditions: Alzheimer Disease
Sponsor: Brendan Lucey

 
 
 
 
 
 
Protocol 
Acute Effect of Lemborexant on CSF Amyloid-
Beta and Tau 
 
PI: Brendan Lucey, MD, MSCI 
 
NCT05728736 
Protocol Version: 3.0 
Last Updated: 06/08/2023 
IRB Approved: 06/21/2023 
 

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Protocol Title: Acute Effect of Lemborexant on CSF Amyloid-Beta and Tau 
(Lemborexant Trial) 
 
 
Principal Investigator:  
Brendan P. Lucey, MD, MSCI 
Associate Professor of Neurology 
Washington University School of Medicine 
660 South Euclid Avenue, Campus Box 8111 
St. Louis, MO 63110 
Office: 314-747-3805 
E-mail: luceyb@wustl.edu 
 
Investigational New Drug:  
Lemborexant 
Governing IND:                                 
163516 
Cross-Reference Letter of Authorization:  Eisai, IND 111871 
 
Date of Original FDA Submission:              August 23, 2022 
Sponsor-Investigator IND 
Serial Number 0000-Initial Submission 
 
Protocol Version Date, Number: 
June 8, 2023 Version 3.0 
 
Approval Date: 
FDA: September 23, 2022 
IRB:  December 07, 2022 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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IRB SUMMARY OF REVISIONS 
 
Date 
Protocol 
Version  
Regulatory 
Submission  
Revision History 
10/10/2022 
2 
IRB initial 
review 
Protocol V2 (9.28.2022) 
- 
Added additional information to 
section 3.4 Interim Analysis 
- 
Updated section 8.1.4 Physical 
Exam 
- 
Updated the safety related criteria 
for stopping study under section 
13.2 Plan for Adverse Events 
- 
Changed safety labs listed on the 
summary of events table.  
- 
Blood sampling change from 6 to 12 
ml.   
06/08/2023 
3 
IRB 
Amendment 1 
Protocol V3 (06.08.2023) 
- 
Clarified bedtime and study drug 
administration time at V4 
- 
Moved APOE sample collection 
from V3 to V1 due to processing 
time constraints  
- 
Included intent to share participant 
data with ADRC/SEABIRD studies 
- 
Included data shared with study 
team by SEABIRD and ADRC at 
time of referral 
- 
Updated exclusion criteria to include 
marijuana use at PI’s discretion 
- 
Added information on participant 
referrals/recruitment from ADRC 
- 
Changed time point for PSQI 
administration to occur at prescreen 
or Visit 1 
- 
Updated lemborexant drug 
interactions 
- 
Informed consent form (ICF) V3 
(06.08.2023) 
 Updated the ICF’s purpose to 
include the current FDA 
approved dosages for 
lemborexant the dose used in this 
research. 
 Added ADRC data sharing 
language  

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 Moved APOE sample collection 
from V3 to V1 
 Included a Schedule of Events to 
enhance participant 
understanding of what each visit 
entails 
 Updates to correct spelling & 
formatting to improve readability  
 
 
 
 

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Protocol Title: 
Acute Effect of Lemborexant on CSF Amyloid-Beta and Tau 
(Lemborexant trial) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Confidentiality Statement: 
 
The information in this document is privileged or confidential and may not be disclosed 
unless such disclosure is required by applicable laws and regulations. It is the property of 
Washington University and should not be copied by or distributed to persons not involved in 
the study. Persons to whom the information is disclosed must be informed that the 
information is privileged or confidential and may not be further disclosed by them. These 
restrictions on disclosure will apply equally to all future information supplied to you, which 
is indicated as privileged or confidential. Any unauthorized review, use, disclosure, copying 
or distribution is strictly prohibited. 
 
Signature:  
 
Date:  
 
Brendan P. Lucey, MD, MSCI  
 
Principal Investigator 
 

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SUB-INVESTIGATORS 
None 
ABBREVIATIONS 
AD- Alzheimer’s Disease  
ADL- Activities of Daily Living 
ADRC- Knight Alzheimer’s Disease Research Center 
AE- Adverse event  
AIC- Akaike information criterion 
APOE- Apolipoprotein Epsilon 
APP- Amyloid Precursor Protein  
Aβ- Beta Amyloid  
CNS- Central Nervous System  
CSF- Cerebral Spinal Fluid 
C-SSRS- Columbia Suicide Severity Rating Scale 
CTRU- Clinical Translational Research Unit  
DORA- Dual Orexin Receptor Antagonist 
DSMC- Data Safety Monitoring Committee  
DSM- Data Safety Monitoring 
ECG- Electrocardiogram 
EEG- Electroencephalogram 
FDA- Food and Drug Administration 
GABA- Gamma-Aminobutyric Acid  
GUID- Global Unique Identifier 
HRPO- Human Research Protection Office  
ICTS- Institute of Clinical and Translational Sciences 
IMEs-Important Medical Events  
IND- Investigational New Drug 
IRB- Institutional Review Board 
ISI- Insomnia Severity Index  
LEM- Lemborexant 
LME- Linear Mixed Effect 
MAD- Multiple Ascending Dose 
MEQ- Morning/Eveningness Questionnaire 
MMRM- Mixed Model for Repeated Measures 
MMSE- Mini Mental Status Examination  
NCRU- NeuroClinical Research Unit 
NCT- National Clinical Trial 
NIH- National Institute of Health 
OSA- Obstructive Sleep Apnea  
OXRI- Orexin Receptor 1 
OXR2- Orexin Receptor 2 
PBO-Placebo 
PD- Pharmacodynamic 

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PET- Positron Emission Tomography 
PHI- Protected Health Information 
PI- Principal Investigator 
PK- Pharmacokinetics 
PSG- Polysomnography 
PSQI- Pittsburgh Sleep Quality Index 
PVT- Psychomotor Vigilance Testing 
SAE- Serious Adverse Event 
SAAMII- Simulation Analysis and Modeling 
SEABIRD- Study to Evaluate Amyloid in Blood and Imaging Related to Dementia 
SILK- Stable Isotope Labeling Kinetics 
SOP- Standard Operating Procedures 
SSS- Stanford Sleepiness Scale 
TEAE Treatment Emergent Adverse Event 
UP- Unanticipated Problem  
VFH- Volunteers for Health 
WU- Washington University 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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TABLE OF CONTENTS 
 
IRB SUMMARY OF REVISIONS ..................................................................................................... 2 
SUB-INVESTIGATORS ....................................................................................................................... 5 
None 5 
ABBREVIATIONS ................................................................................................................................. 5 
GUID- Global Unique Identifier .......................................................................................................... 5 
1. 
BACKGROUND AND SIGNIFICANCE ........................................................................... 11 
1.1 Background: .............................................................................................................................. 11 
1.2 The Orexin System: ................................................................................................................ 11 
1.3 Orexin and Alzheimer’s Disease: ....................................................................................... 11 
1.4 Preliminary Data: .................................................................................................................... 12 
1.5 Study Rationale:....................................................................................................................... 14 
2. SPECIFIC AIMS: ............................................................................................................................. 15 
3. APPROACH: ..................................................................................................................................... 15 
3.1. Experimental Design:............................................................................................................ 15 
3.2 Screening for amyloid-positivity with plasma Aβ:........................................................ 17 
3.3 Power Calculation: .................................................................................................................. 17 
3.4 Interim Analysis: ..................................................................................................................... 18 
3.5 Data Analysis Plan: ................................................................................................................. 18 
3.6 Study Outline and Timeline: ................................................................................................ 19 
4. 
SCIENTIFIC RIGOR, TRANSPARENCY, AND DATA SHARING: ..................... 19 
5. 
POTENTIAL PROBLEMS AND ALTERNATIVE APPROACHES: ...................... 19 
6. 
FUTURE DIRECTIONS .......................................................................................................... 20 
7. 
STUDY POPULATION AND ENROLLMENT .............................................................. 20 
7.1 Justification for human subject involvement: ................................................................. 20 
7.2 Subject population characteristics: ..................................................................................... 21 
7.3 Eligibility criteria: ................................................................................................................... 21 
7.3.1 Inclusion criteria: ................................................................................................................. 21 
7.3.2 Exclusion criteria: ................................................................................................................ 21 
7.4 Description of Study Visits: ................................................................................................. 23 
7.4.1 Visit 1: .................................................................................................................................... 23 
7.4.2 Visit 2: ..................................................................................................................................... 23 
7.4.3 Visit 3: ..................................................................................................................................... 23 

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7.4.4 Visit 4: ..................................................................................................................................... 24 
8. 
STUDY PROCEDURES AND METHODS ...................................................................... 24 
8.1 Specimens, records, or data:................................................................................................. 24 
8.1.1 Cerebrospinal fluid (CSF) specimens: ........................................................................... 24 
8.1.2 Blood specimens: ................................................................................................................. 24 
8.1.3 Polysomnography: ............................................................................................................... 25 
8.1.4 Physical Exam: ..................................................................................................................... 25 
8.2 Data from human subjects: ................................................................................................... 25 
8.2.1 Personal identifiers: ............................................................................................................. 25 
8.2.2 Sleep log: ................................................................................................................................ 26 
8.2.3 Mini Mental State Exam (MMSE): ................................................................................ 26 
8.2.4 Columbia- Suicide Severity Rating Scale (C-SSRS): ............................................... 26 
8.2.5 STOP-Bang Questionnaire: .............................................................................................. 26 
8.2.6 Pittsburgh Sleep Quality Index (PSQI): ........................................................................ 26 
8.2.7 Stanford Sleepiness Scale (SSS): .................................................................................... 26 
8.2.8 Morningness/ Eveningness Questionnaire (MEQ): ................................................... 26 
8.2.9 Psychomotor Vigilance Testing (PVT): ........................................................................ 26 
8.2.10 Electrocardiogram (ECG): .............................................................................................. 27 
8.2.11 Physical and Neurological Examination: ................................................................... 27 
8.2.12 Blood Collection: .............................................................................................................. 27 
8.3  Scale to Inform the Disclosure of Amyloid Results..................................................... 27 
8.4 Disclosure of Amyloid Results ........................................................................................... 27 
9. 
Potential risks ............................................................................................................................... 27 
9.1 Lumbar Catheter: ..................................................................................................................... 27 
9.2 Intravenous Catheter: ............................................................................................................. 28 
9.3 Lemborexant: ............................................................................................................................ 28 
9.4 Polysomnogram (PSG): ......................................................................................................... 29 
9.5 Questionnaires: ........................................................................................................................ 29 
9.6 Bedrest:....................................................................................................................................... 29 
9.7 Privacy/confidentiality: ......................................................................................................... 29 
10. Adequacy of Protection Against Risks ................................................................................. 29 
10.1 Recruitment: ........................................................................................................................... 30 
10.2 Consent: ................................................................................................................................... 30 

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10.3 Lumbar catheter: ................................................................................................................... 31 
10.4 Intravenous catheter: ............................................................................................................ 31 
10.5 Lemborexant: ......................................................................................................................... 31 
10.6 Polysomnogram (PSG): ...................................................................................................... 32 
11. Definitions ..................................................................................................................................... 32 
11.1 Adverse Event:....................................................................................................................... 32 
11.2 Suspected Adverse Reaction: ............................................................................................ 32 
11.3 Adverse Reaction: ................................................................................................................. 33 
11.4. Serious: ................................................................................................................................... 33 
11.5 Life-threatening: .................................................................................................................... 33 
11.6. Important Medical Events (IMEs): ................................................................................. 33 
11.7 Unexpected: ............................................................................................................................ 33 
11.8 Causality and Severity Assessment: ................................................................................ 34 
11.9 Severity/Intensity: ................................................................................................................. 34 
11.10 Attribution: ........................................................................................................................... 34 
11.11 Documenting Adverse Events: ....................................................................................... 35 
11.12 Pre-existing Conditions: ................................................................................................... 35 
12. Food and Drug Administration Mandatory Reporting ..................................................... 35 
12.1 Initial Safety Reporting: ...................................................................................................... 35 
12.2 Follow-up Reporting: ........................................................................................................... 36 
12.3 Annual Report:....................................................................................................................... 37 
12.4  FDA Reporting Process - Written and Facsimile: ...................................................... 37 
12.5  Eisai (IND holder of Lemborexant): .............................................................................. 37 
12.6 Institutional Review Board Mandatory Reporting: ..................................................... 37 
12.7 Unanticipated Problem (UP) Criteria: ............................................................................ 37 
12.8 Exception Criteria: ................................................................................................................ 37 
12.9 IRB process of reporting events: ...................................................................................... 38 
12.10 Data Safety Monitoring (DSM) Reporting: ................................................................ 38 
12.11 Other Reportable Events: ................................................................................................. 38 
12.11.1. Non-Compliance and Serious Non-Compliance: ................................................. 39 
12.11.2. Protocol Exceptions and Deviations: ....................................................................... 39 
12.11.3. Timeframe for Reporting Required Events ............................................................ 39 
13. Privacy/confidentiality .................................................................................................................. 40 

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13.1 Breach of privacy/confidentiality: ................................................................................... 40 
13.2 Plan for Adverse Events (AE):.......................................................................................... 41 
14. Potential Benefits of the Proposed Research to Human Subjects and Others ........... 42 
14.1 Potential benefits to participants: ..................................................................................... 42 
14.2 Potential benefits to others: ................................................................................................ 42 
14.3 Risks vs. benefits: ................................................................................................................. 43 
15. Importance of the Knowledge to be Gained ........................................................................ 43 
16. Data and Safety Monitoring Plan ........................................................................................... 43 
17. ClinicalTrials.gov Requirements ............................................................................................ 44 
18. APPENDICES ............................................................................................................................... 45 
18.1 Appendix 1: Lemborexant 25 mg Safety Data ......................................................... 45 
19. REFERENCES: ........................................................................................................................... 57 
 
 

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1. BACKGROUND AND SIGNIFICANCE 
1.1 Background:  
Alzheimer’s disease (AD) is characterized by deposition of amyloid-β (Aβ) as insoluble 
plaque (i.e., amyloid-positive), tau aggregation and hyperphosphorylation, increased immune 
response, neuronal degeneration, synaptic loss, and eventual cognitive dysfunction, dementia, 
and death. Soluble forms of Aβ and tau, proteins critical to Alzheimer’s disease pathogenesis, 
change in cerebrospinal fluid (CSF) with sleep-wake activity: 1) CSF Aβ and tau increase 
during wakefulness and decrease during sleep in both mice1 and humans2; 2) overnight sleep 
deprivation increases CSF Aβ and tau levels by ≥30% via increased production/release3-5; 3) 
tau hyperphosphorylation (p-tau), an early step in tau-mediated neurodegeneration, is also 
affected by sleep deprivation depending on the specific site of phosphorylation5. Based on 
these findings, sleep disturbances are recognized as a potentially modifiable risk factor for 
Alzheimer’s disease6, 7.   
 
1.2 The Orexin System:  
Orexin-A and orexin-B (also known as hypocretin-1 and hypocretin-2) are two neuropeptides 
of 33 and 28 amino acids encoded by a common precursor polypeptide, prepro-orexin8. 
Neurons producing orexin are exclusively localized to the perifornical area and the lateral 
and posterior hypothalamic area and project to the brainstem nuclei, amygdala, hippocampus, 
and cerebral cortex9-12. Orexins bind to two G protein-coupled receptors, orexin receptor 1 
(OXR1) and orexin receptor 2 (OXR2)8. The orexin system regulates sleep-wake activity, 
feeding behavior, energy homeostasis, and the reward system8. Orexins are wake-promoting 
neuropeptides and orexin deficiency causes narcolepsy, a sleep disorder resulting in 
excessive daytime sleepiness, sleep paralysis, sleep-related hallucinations, and cataplexy. 
Further, blockade of orexin function with a dual orexin receptor antagonist (DORA) 
increases sleep. Based on its role in narcolepsy pathophysiology, DORAs have been pursued 
as a treatment of insomnia. 
  
1.3 Orexin and Alzheimer’s Disease:   
Substantial evidence supports a role for the orexin system in the development of amyloid 
deposition. Knocking out the orexin gene in amyloid precursor protein (APP) transgenic 
mice led to a marked decrease in amyloid pathology in the brain while over-expression of 
Figure 1.  Marked reduction of amyloid pathology in mice when orexin is reduced.  The difference in amyloid pathology between APP/PS1-21 
mice (A) and APP/PS1-21 orexin knock out mice (B) is shown.  A dual orexin receptor antagonist, almorexant, administered to mice daily for 8 
weeks via intraperitoneal injection significantly decreased amyloid deposition in the brain (C).  Panels A and B from:  Roh JH, et al., J Exp Med. 
2014; 211: 2487. Panel C from:  Kang JE, et al., Science. 2009; 326: 1005. 

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orexin in the hippocampus did not alter 
amyloid pathology13 (Fig 1A-B). 
Studies in APP transgenic mice that 
develop amyloid deposition found that 
treatment with a DORA, almorexant, 
decreased soluble Aβ concentrations 
while intra-cerebroventricular 
administration of orexin increased 
them1. Further, prolonged treatment 
with almorexant for 8 weeks decreased 
amyloid deposition (Fig 1C); this 
effect was recently replicated in mice 
with suvorexant14. In humans, patients 
with narcolepsy (i.e., with orexin 
deficiency) have reduced CSF Aβ, tau, 
p-tau, and amyloid deposition on 
amyloid PET compared to age- and 
sex-matched controls15, 16. Finally, 
there are no preclinical studies in 
animal models or human clinical 
studies showing that other sleep-
inducing drugs (e.g., zolpidem, sodium 
oxybate) affect CSF AD biomarkers. 
These findings strongly suggest that 
blocking orexin will modulate amyloid 
pathology in the brain. 
 
1.4 Preliminary Data:   
Our preliminary data shows that a 
first-in-class DORA, suvorexant, 
acutely decreases tau phosphorylation 
and Aβ in human CSF within hours. In 
this study, cognitively normal adults 
aged 45-65 years old were screened 
for poor sleep quality (defined by 
sleep efficiency <85% as measured by 
actigraphy) and then randomized to 
suvorexant 10 mg, suvorexant 20 mg, or placebo.  After admission to our Clinical 
Translational Research Unit (CTRU) on Day 1, an in-dwelling lumbar catheter was placed at 
~8pm and CSF sampled every 2 hours for 36 hours. Participants received their blinded tablet 
at 9pm and the lights were turned off so they could sleep as they are able. Upon waking on 
Day 2 (~6-7am), participants remained awake until ~9pm when they received their second 
Figure 2:  Effect of suvorexant on cerebrospinal fluid (CSF) Aβ.  Participants 
received placebo, suvorexant 10 mg, or suvorexant 20 mg.  In all graphs, Aβ 
concentrations at each time are normalized to the average concentration of hours 0-
6.  Aβ38 (A), Aβ40 (B), and Aβ42 (C) show that the suvorexant 20 mg group is 
decreased compared to placebo.  Blue: placebo; Red: suvorexant 10 mg; Green: 
suvorexant 20 mg.  Error bars indicate standard error.  The vertical dashed lines 
show when placebo/drug was administered. The horizontal dashed line is at 100% 
of baseline.  Shaded area is 08:00-20:00. *p<0.05 after Bonferroni correction.  
Unpublished data. 

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blinded tablet; there is no change in 
treatment with the second tablet. At 
hour 36 on Day 3 (~8am), the 
lumbar catheter was removed and 
the study ended. There was no sleep 
manipulation other than the 
intervention conditions. Thirteen 
participants completed the placebo 
group, thirteen participants 
completed the suvorexant 10 mg 
group, and twelve participants 
completed the suvorexant 20 mg 
group. We normalized the average 
Aβ concentration to hours 0-6 
because these time points do not 
represent the effect of the 
intervention due to the ~5 hour 
transit time from the brain to the 
lumbar catheter2, 3.  We found that 
CSF Aβ38, Aβ40, and Aβ42 
concentrations decreased in the 
suvorexant 20 mg group compared 
to the placebo group (Fig 2). For 
Aβ42, the suvorexant 20 mg group 
was significantly decreased after 
Bonferroni correction for multiple 
comparisons at hour 24 (17.33% 
mean difference, 95% confidence 
intervals 1.852-32.8, p=0.0253) and 
36 (22.12% mean difference, 95% 
confidence intervals 0.79-43.45, 
p=0.041). 
 
We also measured different forms of tau and phosphorylated tau.  The unphosphorylated tau 
forms of threonine-181 (T181), serine-202 (S202, and threonine-217 (T217) as well as the 
phosphorylated forms of T181 (pT181), S202 (pS202), and T217 (pT217) were normalized 
to the average concentration of hours 0-6 as described above.  There were no significant 
differences between groups after Bonferroni correction for multiple comparisons (Fig 3, 4, 
5).  However, there was a significant decrease of the pT181/T181 ratio in the suvorexant 20 
mg group compared to placebo (Fig 3C).  The pTau/Tau ratios indicate the occupancy rate of 
tau phosphorylation17 and are a measure of the phosphorylation rate.  pS202/S202 shows a 
trend toward decreasing in suvorexant group compared to placebo but is not significant.  
pT217/T217 shows no difference between groups.  pS202/S202 has a lower occupancy rate 
than pT181/T181 and pT217/T217 has a lower occupancy rate than pS202/S202 suggesting 
that prolonged treatment or increase occupancy such as is seen in amyloidosis would result in 
a larger effect.  
Figure 3:  Effect of suvorexant on cerebrospinal fluid (CSF) tau threonine-181 (T181) 
phosphorylated T181 (pT181).  Participants received placebo, suvorexant 10 mg, or 
suvorexant 20 mg.  In A and B, concentrations at each time are normalized to the 
average concentration of hours 0-6.  There is no difference between groups for T181 
(A) or pT181 (B).  The pT181/T181 ratio was normalized to the hour zero 
concentration (C) shows that the suvorexant 20 mg group is decreased compared to 
placebo.  Blue: placebo; Red: suvorexant 10 mg; Green: suvorexant 20 mg.  Error bars 
indicate standard error.  The vertical dashed lines show when placebo/drug was 
administered. The horizontal dashed line is at 100% of baseline.  Shaded area is 08:00-
20:00. *p<0.05; **p<0.01; ***p<0.001 after Bonferroni correction.  Unpublished data.

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1.5 Study Rationale:  
Prior studies showed that sleep loss increased CSF Aβ and tau in humans3-5, 18, 19, however 
treatment with sodium oxybate (a 
GABA-B receptor agonist) did not 
decrease CSF Aβ or tau compared to 
controls3. Our preliminary data 
shows that treatment with 
suvorexant, a DORA, will decrease 
the CSF pT181/T181 
phosphorylation rate and Aβ 
concentration acutely over hours. 
These findings and the studies 
performed in animal models suggest 
that the beneficial effects measured 
by orexin receptor blockade are not 
simply due to sedation or sleep and 
depends on specific neurotransmitter 
networks. Treatment with DORAs 
may lead to lower CSF levels of Aβ 
and tau phosphorylation, and 
therefore decreased deposition of Aβ 
as insoluble plaques, decrease 
development of tauopathy, and 
ultimately lower the risk of AD. 
However, we do not know if the 
effect on CSF tau phosphorylation 
and Aβ is specific to suvorexant. A 
second DORA, lemborexant, was 
recently approved by the Food and 
Drug Administration20 and has not 
been shown to decrease CSF 
pT181/T181 and Aβ. We 
hypothesize that the effect of 
lemborexant 25 mg on CSF 
pT181/T181 and Aβ will be similar 
to or greater than suvorexant 20 
mg compared to placebo. 
Understanding if lemborexant has a 
similar effect on CSF Alzheimer 
biomarkers is critical to designing 
future clinical trials with the ultimate 
goal of designing a phase III clinical 
trials using lemborexant to prevent or delay AD.  
 
Figure 4:  Effect of suvorexant on cerebrospinal fluid (CSF) tau serine-202 (S202) 
phosphorylated S202 (pS202).  Participants received placebo, suvorexant 10 mg, or 
suvorexant 20 mg.  In A and B, concentrations at each time are normalized to the 
average concentration of hours 0-6.  There is no difference between groups for S202 
(A) or pS202 (B).  The pS202/S202 ratio was normalized to the hour zero 
concentration (C) shows that the suvorexant 20 mg group is decreased compared to 
placebo.  Blue: placebo; Red: suvorexant 10 mg; Green: suvorexant 20 mg.  Error bars 
indicate standard error.  The vertical dashed lines show when placebo/drug was 
administered. The horizontal dashed line is at 100% of baseline.  Shaded area is 08:00-
20:00. Unpublished data. 

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2. SPECIFIC AIMS: 
To determine the acute effect of lemborexant on CNS tau phosphorylation in individuals with 
poor sleep quality.  Cognitively normal amyloid-positive adults aged 60-80 years old with a 
Pittsburgh Sleep Quality Index (PSQI) >5 will be randomized to lemborexant 25 mg (N=20) 
or placebo (N=10). All participants will have indwelling lumbar catheter placed to sample 
CSF every 2 hours for 48 hours.  Each participant will be infused with 13C6-leucine for future 
analyses of Aβ stable isotope labeling kinetics. 
Aim 1:  To test the hypothesis that treatment with lemborexant 25 mg will decrease the 
CSF pT181/T181 ratio compared to placebo (primary outcome). 
Aim 2:  Measure the effect of lemborexant on other CSF AD biomarkers such as 
pS202/S202, pT217/T217, and Aβ (secondary outcome). 
 
3. APPROACH:   
3.1. Experimental Design:   
To investigate the acute (over hours) 
effect of lemborexant on CSF 
pT181/T181, we will recruit 
participants who are cognitively 
normal amyloid-positive.  
Participants age 60-80 years will be 
screened for normal cognitive 
function (Mini-Mental Status 
Examination21 score ≥27), poor 
sleep (PSQI>5), and will be 
evaluated for sleep disorders by 
history, questionnaires (STOP-
Bang22), and an attended 
polysomnogram performed at the 
Washington University Sleep 
Medicine Center. Please see Table 
1 for the full inclusion/exclusion 
criteria. 
Initially, twelve participants with 
a PSQI >5 will be randomized to 
receive placebo (N=4) or 
lemborexant 25 mg (N=8).  Amyloid 
status will be determined by a 
plasma Aβ test.  All participants will undergo attended polysomnography (PSG) during the 
screening period to assess for moderate or severe obstructive sleep apnea (OSA), which is 
exclusionary. Randomized participants will be admitted to the CTRU in the early afternoon 
(Night 1). All participants will have their sleep monitored with unattended full-montage PSG 
(TrackItTM; Lifelines, Troy, IL) that will allow for sleep staging according to the gold 
standard American Academy of Sleep Medicine criteria23 and has already been used in 
Figure 5:  Effect of suvorexant on cerebrospinal fluid (CSF) tau threonine-217 
(T217) phosphorylated T217 (pT217).  Participants received placebo, 
suvorexant 10 mg, or suvorexant 20 mg.  In A and B, concentrations at each 
time are normalized to the average concentration of hours 0-6.  There is no 
difference between groups for T217 (A) or pT217 (B).  The pT217/T217 ratio 
was normalized to the hour zero concentration (C) shows that the suvorexant 
20 mg group is decreased compared to placebo.  Blue: placebo; Red: 

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similar studies to monitor sleep for 36-48 hours2, 3. Participants will be monitored with PSG 
throughout the CTRU admission. 
At approximately 20:00, a lumbar catheter and two IVs will be placed for collecting 6 ml 
of CSF every 2 hours for 48 hours. Sampling start time will begin ~1 hour prior to the typical 
bedtime defined by sleep logs for each participant in order to allow for 13C6-leucine infusion 
and frequent sampling of blood prior to bedtime. Up to 12 ml of blood will be collected at the 
following time points: 0, 5 minutes, 10 
minutes, 15 minutes, 30 minutes, 1 hour, 1.5 
hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 
hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 
hours, 16 hours, 18 hours, 20 hours, 22 hours, 
24 hours, 26 hours, 28 hours, 30 hours, 32 
hours, 34 hours, 36 hours, 38 hours, 40 hours, 
42 hours, 44 hours, 46 hours, and 48 hours 
(Table 2). Approximately 1 hour before the 
habitual bedtime on Day 1 (t=0), all 
participants will start an infusion of 800 mg 
labeled 13C6-leucine to label proteins in vivo 
during intracellular translation in order to 
monitor Aβ kinetics3, 24, 25. Aβ stable isotope 
labeling kinetics (SILK) is an innovative 
approach to measure Aβ production and 
clearance rates, and we have used the above 
protocol in the Sleep Quality study with 
acceptable labeling of Aβ (Suppl Fig 1).   
 Participants will be allowed to sleep as they are able immediately with the lights turned 
off at their habitual bedtime (bedtime is at hour 1, approximately 22:00-00:00). Nursing staff 
will use dim red lights (safelights) when collecting CSF and blood in the dark. The lumbar 
catheter ports will be placed on the outside of the gown sleeve for easy access to minimize 
disturbance during fluid collection. Participants will then sleep until final awakening in the 
morning. During Day 2, all participants will be in well-lit rooms with regular monitoring for 
staying awake and naps will not be permitted. At ~22:00 on Day 2 (hour 25), all participants 
will follow the same sleep routine except that there will be no infusion of labeled 13C6-
leucine.  However, the time of hour 25 will depend on the participant’s regular bedtime.  
Participants will receive the same placebo or lemborexant dose as the previous night (Fig 6). 
The study will end at ~20:00 on Day 3 (hour 48) when the IVs and lumbar catheters will be 
removed. All participants will then sleep overnight and will be monitored for at least 8 hours 
after catheter removal and then discharged. CSF tau, phosphorylated tau, and Aβ kinetics will 
be quantified by mass spectrometry as was done for our previous work3, 5. 
 
 
 
 
 
 
 
 
Table 1:  Inclusion/Exclusion Criteria 
Inclusion criteria 
1.  Age 60-80 
2.  Mini-Mental score ≥ 27 
3.  Pittsburgh Sleep Quality Index >5 
4.  Positive plasma Aβ test (i.e., amyloid-positive)  
Exclusion criteria 
1.  Sleep disorders other than insomnia and mild 
obstructive sleep apnea 
        -STOP-Bang score >5 
        -ex. restless legs syndrome, narcolepsy 
2.  Cardiovascular or cerebrovascular disease 
3.  Hepatic or renal impairment 
4.  Pulmonary disease 
5.  Psychiatric disorder requiring medication 
6.  Alcohol or tobacco use (PI discretion) 
7.  Use of sedating medications 
8.  Abnormal movement of the non-dominant arm 
9.  Contraindications to lumbar catheter 
          -Anticoagulant 
10.  Body Mass Index <35 

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C:  catheter placement; B:  bedtime; X: CSF/blood sampling; R:  catheter removal 
 
3.2 Screening for amyloid-
positivity with plasma Aβ:   
We plan to screen 100 participants 
to enroll 30 participants in this 
trial.  Cognitively normal 
participants who are amyloid-
positive on the plasma Aβ test are eligible for the study.  The plasma Aβ test has an 80% 
sensitivity, 83% specificity for amyloid-positivity after accounting for age and ApoE 
genotype.  Assuming that 25% of the cognitively normal population will be amyloid-positive, 
we plan to screen 100 cognitively normal participants with an PSQI>5 to obtain ~20 
amyloid-positive cognitively normal individuals (Table 3).  We expect to recruit the 
remaining participants from the Knight Alzheimer’s Disease Research center (ADRC) and 
on-going studies at Washington University such as the SEABIRD study. 
  
3.3 Power Calculation:   
A maximum of 30 participants will be recruited for this study and will be randomized in a 
2:1 (treatment:placebo) ratio with lemborexant 25 mg (N=20) and placebo (N=10).  Our 
preliminary data was used to estimate the effect size between the placebo arm and suvorexant 
20 mg arm.  Effect sizes were calculated based on two-sided independent two-sample t test 
with type I error of 0.05 and 80% power.  G*power 3.1 was used to calculate effect sizes and 
Table 2:  Timeline of Catheter Placement and Removal, Bedtime, and Sampling 
Hour 
0 
1 
2 
4 
6 
8 
10 12 14 16 18 20 
22 
24 
Event 
C, X 
B 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Hour 
25 
26 28 
30 
32 34 36 38 40 42 44 46 
48 
49 
Event 
B 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X, R 
B 
Table 3:  Recruitment Using Plasma Aβ 
 
Amyloid+ Amyloid- 
Total 
Tests positive 
20 
13 
33 
Tests negative 
5 
63 
68 
Total 
25 
76 
101 
Table 4.  Power Analysis and Change from Baseline Detected with Different Sample   
Sizes 
 
pT181/T181 
[norm t=0; 
average t=14-18] 
pS202/S202  
[norm t=0; 
average t=36] 
Aβ42 
[norm 
average t=0-
6; average 
t=22-26] 
Placebo Group Mean (SD) 
1.56 (1.41) 
-2.83 (1.37) 
123.97 
(13.16) 
Suvorexant 20 mg Group Mean 
(SD) 
-1.09 (2.20) 
-4.52 (3.01) 
110.21 
(16.66) 
Effect Size of Suvorexant 20 mg 
Compared to Placebo 
1.43 
0.72 
0.92 
Power (10 participants/group) 
86% 
33% 
50% 
Power (15 participants/group) 
95% 
47% 
65% 
Power (20 participants/group) 
99% 
60% 
80% 

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perform the power calculation.  Table 4 shows the power analysis for CSF pT181/T181, 
pS202/S202, and Aβ42.   
 
Effect sizes were calculated based on the preliminary data in Figures 2-4 and assume equal 
intervention groups.  For CSF pT181/T181, the average of hours 14-18 in Figure 3C was 
used to determine means, standard deviations, and effect size.  For CSF pS202/S202, the 
average of hour 36 in Figure 4C was used to determine means, standard deviations, and 
effect size.  For CSF Aβ42, the average of hours 22-26 in Figure 2C was used to determine 
means, standard deviations, and effect size. Table 4 shows the power to detect these effect 
sizes with 10 participants/group, 15 participants/group, and 20 participants/group.  Note, CSF 
Aβ42 may increase in amyloid-positive participants with amyloid plaque accumulation is 
decreased.   
  
3.4 Interim Analysis:   
After 12 participants have completed the study (placebo N=4; lemborexant N=8), an interim 
analysis will be performed for efficacy to see if there is a significant difference in CSF 
pT181/T181 between lemborexant 25 mg and placebo groups. If there is a statistically 
significant (p<0.05) or trend towards statistically significant (p<0.1) reduction in the primary 
outcome (CSF pT181/T181 ratio) or secondary outcome (CSF amyloid-beta), then the study 
may be continued at lemborexant 25 mg.  If there is not a trend towards a statistically 
significant (p>0.1) reduction in the primary outcome (CSF pT181/T181 ratio) or secondary 
outcome (CSF amyloid-beta), or power analysis suggests that statistical significance will not 
be achieved by the fully enrolled study, then the study will be terminated. 
 
3.5 Data Analysis Plan: 
For each participant, AD biomarkers (Aβ38, Aβ40, Aβ42, T181, S202, T217, pT181, pS202, 
pT217) will be normalized to the average of the first 6 hours (t=0-6, 20:00-02:00) before 
changes from the intervention will be seen in CSF.  We will also normalize to the hour 0 
concentration. Phosphorylated tau ratios (pT181/T181, pS202/S202, pT217/T217) will be 
normalized to the first time point (hour 0).  The trajectory of changes from these baselines 
over time will then be plotted by treatment arms to examine whether the change in 
concentrations or ratios is linear. If the linearity assumption is valid, (linear mixed effects) 
LME models with random intercept and slope, similar to our previous work3, 5, will be used 
for analysis, otherwise mixed model for repeated measure (MMRM) will be used.  Fixed 
effect in the model will include treatment group, time and their interaction. Baseline will be 
included as a covariate. The normality assumption will be examined using residual plot and 
appropriate transformation (e.g., log) will be considered.  Unstructured covariance matrix 
will be used and if there is convergence issue, various other covariance matrix structure (e.g., 
compound symmetry, First‐order Autoregressive) will be compared and the best fit structure 
will be selected for final analysis based on Akaike information criterion (AIC). 
 

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3.6 Study Outline and Timeline:   
Figure 6 shows an overview of the study design with a habitual bedtime of 22:00.  Table 5 
shows the study timeline.  We anticipate that this study will take 2 years to complete. 
 
 
4. SCIENTIFIC RIGOR, TRANSPARENCY, AND DATA SHARING:   
 
We will ensure scientific rigor by working closely with the biostatisticians collaborating on 
this project to implement adequate database control, statistical procedures, data analysis 
methods, and unbiased and accurate interpretation of findings from all publications of this 
project. We will consider all relevant biological variables by performing statistical analyses 
on sex, race, age, and other variables as covariates in statistical models. We will assess their 
effects in association with other main predictors on main outcomes through appropriate 
interactions in the models.  We will register the study with ClinicalTrials.gov and will follow 
CONSORT Guidelines in reporting the study findings. Selected data will be shared with 
the ADRC and SEABIRD for participants referred to our study; specifically, this will 
consist of amyloid status, CSF tau and Aβ at hour 0, and screening MMSE. Finally, we 
will share data and biospecimens with qualified investigators.   
 
5. POTENTIAL PROBLEMS AND ALTERNATIVE APPROACHES:  
 
We plan to recruit participants from the community, the ADRC, and from an ongoing study 
testing the plasma Aβ test (SEABIRD, PI: Randall Bateman).  We have received permission 
Table 5:  Proposed Timeline for Trial 
 
Year 1 
Year 2 
Months 
Q1 
Q2 
Q3 
Q4 
Q1 
Q2 
Q3 
Q4 
Start-Up 
  
Enrollment 
  
 
IP/MS 
 
 
 
Analysis 
 
  
Manuscript 
Prep/Submission to 
Conferences 
 
 
Figure 6:  Overview of Study Design.  Overnight periods are shaded. 

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to recruit participants from the ADRC and SEABIRD study and expect to recruit the majority 
of the participants from the community.  We have budgeted to screen 100 participants with 
plasma Aβ tests and to perform 50 polysomnograms to evaluate for moderate/severe 
obstructive sleep apnea from a database of >10,000 research volunteers maintained at 
Washington University (Volunteers for Health). We have successfully recruited from the 
database for our previous studies. We plan on 1 participant/month completing the study and 
have performed at most 15-18 lumbar catheter admissions per year in previous studies. The 
COVID-19 pandemic may result in delays both recruiting participants and bringing them into 
the CTRU as well as processing samples. 
 
6. FUTURE DIRECTIONS 
 
This project tests the hypothesis that CSF pT181/T181 is decreased with lemborexant, a 
DORA, compared to placebo.  The proposed study may suggest innovative AD prevention 
and treatment approaches that involve sleep therapies such as DORAs in older individuals 
with poor sleep quality. If successful, future studies will include an interventional trial to 
prevent amyloidosis (or slow amyloid growth) and tauopathy in those identified by clinical 
criteria.  Such a prevention trial could dramatically impact the incidence and prevalence of 
AD.   
 
SUPPLEMENTAL MATERIAL  
 
7. STUDY POPULATION AND ENROLLMENT  
7.1 Justification for human subject involvement:  
Alzheimer Disease (AD) is a current and growing public health problem characterized by 
progressive cognitive impairment resulting in dementia.  AD is estimated to afflict millions 
of people in the coming decades.  Multiple lines of evidence suggest a role for sleep 
disturbances in the development of AD.  Further, there is evidence in mice that improving 
sleep parameters such as sleep efficiency (i.e. sleep quality) may prevent the initial 
pathological changes seen in the brain with AD.  These findings need to be replicated in 
humans if sleep interventions are to be deployed to prevent or delay AD.  In this study, I 
propose to translate the findings to humans that improving sleep efficiency has similar effects 
Supplementary Figure 1:  Aβ labeling curves for stable isotope labeling kinetics (SILK) for A38 (A), Aβ40 (B), and Aβ42 (C).  800 mg of 
13C6-leucine is infused over 10 minutes at 20:00 (t=0) in participants treated with placebo (N=11), suvorexant 10 mg (N=11), and 
suvorexant 20 mg (N=6).  Note, this data has not been normalized to the plasma free leucine concentration and Aβ kinetics cannot be 
determined.  This data is presented to demonstrate feasibility. Standard error bars are shown. Unpublished data. 

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as in mice. The specific objective of this study is to translate basic science findings to 
humans for the long-term improvement in human health, therefore the proposed study must 
be carried out in human subjects.   
 
7.2 Subject population characteristics:  
The goal is to enroll healthy, cognitively normal amyloid-positive adults 60-80 years old with 
no medical comorbidities that would affect the risk of AD and with no conditions, 
medications, or substance use that may affect outcome measures or contraindicate study 
procedures (e.g. cerebrospinal fluid (CSF) amyloid-β (Aβ) levels obtained by lumbar 
catheter).  Participants will be recruited from the Volunteers for Health registry of >10,000 
research study volunteers maintained at Washington University, the ADRC, as well as an 
ongoing study of a plasma Aβ test (SEABIRD, PI: Randall Bateman).  Individuals from both 
sexes and any race or ethnicity will be included.  Minors will not be included because AD 
does not occur in individuals <18 years old, even if predisposed to develop AD through an 
autosomal dominant mutation.  Further, it is not known if the diurnal Aβ pattern is present in 
children.  The relevance of determining if sleep alters Aβ in children to the pathogenesis of a 
neurodegenerative disorder that will potentially develop decades later is also unknown at this 
time. As a result, we will not include any children in this study.   
 
7.3 Eligibility criteria: 
7.3.1 Inclusion criteria: 
o Age 60-80 years 
o Any sex 
o Any race/ethnicity 
o Mini-Mental Status Examination score (MMSE) ≥ 27 
o Positive plasma Aβ test (i.e., amyloid-positive) 
o Pittsburgh Sleep Quality Index >5 
 
7.3.2 Exclusion criteria: 
o Cognitive impairment as determined by history of MMSE < 27 
o Inability to speak or understand English 
o Any sleep disorders other than insomnia 
 No history of moderate-to-severe sleep-disordered breathing and 
STOP-Bang score > 5 
 History or reported symptoms suggestive of restless legs syndrome, 
narcolepsy or other sleep disorders 
 No more than mild sleep apnea (AHI <16) on PSG 
o Sleep schedule outside the range of bedtime 22:00-midnight  
o Contraindication to lumbar catheter (anticoagulants; bleeding disorder; allergy 
to lidocaine or disinfectant; prior central nervous system or lower back 
surgery) 
o Cardiovascular disease requiring medication except for controlled 
hypertension  (PI discretion) 
o Stroke 
o Hepatic or renal impairment 

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o Pulmonary disease (PI discretion) 
o Type 1 diabetes 
o HIV or AIDS 
o Neurologic or psychiatric disorder requiring medication (PI discretion) 
o Suicidal ideations 
o Alcohol, tobacco or marijuana use (PI discretion) 
o Use of sedating medications (PI discretion) 
o Inability to get out of bed independently 
o In the opinion of the investigator, the participant should be excluded due to an 
abnormal physical examination.  
o Current pregnancy 
o Body Mass Index >35 
o History of migraines (PI discretion) 
o History of drug abuse in the last 6 months 
o History or presence of any clinically significant medical condition, behavioral 
or psychiatric disorder (including suicidal ideation), or surgical history based 
on medical record or patient report that could affect the safety of the subject or 
interfere with study assessments or in the judgment of the PI participant is not 
a good candidate. 
o Urinary or fecal incontinence 
o Concurrently enrolled in another trial of an investigational drug or device 
 
We will enroll up to 30 participants to complete the study. Participants will receive either 
placebo or lemborexant 25 mg, a dual orexin receptor antagonist. Lemborexant is 
approved by the Food and Drug Administration (FDA) for the treatment of insomnia 
(approved doses 5-10 mg). Lemborexant 25 mg will be used in this study to see if there is 
an effect on CSF pT181/T181 and Aβ. The use in this study will be considered 
investigational and is not approved by the Food and Drug Administration (FDA).  
 
Participants that screen fail may be invited back to rescreen at a later date. 
 
Sampling plan and justification:  
• Power calculations are shown in “C3. Power Calculation” portion of the research plan 
above.  Due to concerns about recruitment if participants have to repeat the study, the 
study power calculation is based on each participant completing the study once 
without repeating. 
• Children will be excluded and age range will be limited to 60-80, as detailed in the 
previous section “Subject population characteristics”. 
 
Vulnerable populations: We will not enroll participants from vulnerable populations. 
 
Assignment to a study group: Participants will be randomized to receive placebo or 
lemborexant 25 mg at the Clinical Translational Research Unit (CTRU) admission. We will 
randomize 8 participants to receive 25 mg of lemborexant for 2 nights and 4 participants to 
receive placebo for two nights. After an interim analysis for safety and futility, a decision 
will be made to either: 1) continue the study with lemborexant 25 mg, 2) continue the study 

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with a lower dose of lemborexant, or 3) stop the study. If the study is continued, then 12 
participants will be randomized to lemborexant and 6 participants will be randomized to 
placebo. In addition to being a FDA approved drug for the treatment of insomnia, 
lemborexant is also a drug with the same mechanism of action as was used in experiments to 
decrease Aβ with sleep in mice, therefore this will be a direct translation of this finding.  The 
drug’s mechanism of action is to block orexin-A and orexin-B receptors.  The orexin 
neuropeptide acts in the brain to promote wakefulness.  Through antagonism of this system, 
sleep is induced.   
 
Collaborating sites: None 
 
7.4 Description of Study Visits: 
At all study visits, safety assessments will consist of monitoring and recording all adverse 
events (AEs) at each visit.  
 
7.4.1 Visit 1:  
Participants must provide informed consent prior to any study related assessments 
take place. Screening assessments include review of demographics, medical history, 
and medications.  Vital signs and a physical and neurological exam are performed. 
Blood is collected for the amyloid blood test and APOE testing (APOE processing 
will not occur until later in the screening period and may not be processed at all 
should participants screen fail) and questionnaires are administered. If the participant 
was referred by SEABIRD, the amyloid blood test may not need to be repeated. 
However, if the participant was referred by the ADRC, the amyloid blood test will be 
repeated. 
 
Additional safety assessment at this visit include an electrocardiogram (ECG), routine 
blood tests, and administration of the Columbia Suicide Rating Scale (CSSRS). 
 
Participants will be sent home with a sleep log to complete for two weeks.  
   
7.4.2 Visit 2:  
Participants will undergo polysomnography (attended sleep study) at the Washington 
University Sleep Medicine Center for sleep apnea screening.  Participants will have 
the option to have the sleep study results added to their medical record. All 
participants will receive a generic letter stating if the results were normal or 
abnormal.  Participants with abnormal results will be encouraged to follow up with 
their primary care physician.  
  
7.4.3 Visit 3:  
This visit will occur one week prior to the Clinical Translational Research Unit 
(CTRU) admission. Eligibility criteria, and changes to medical conditions or 
medications will be reviewed. Weight will be measured, blood will be collected for a 
coagulation panel and participants will be sent home with a sleep log.   
 

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7.4.4 Visit 4:   
Participants will be admitted to the Clinical Translational Research Unit (CTRU) for 
a 3-night stay.  Questionnaires and psychomotor vigilance testing (PVT) will be 
administered at admission and discharge.   
 
Throughout the CTRU admission, participants will be monitored with a full 10-20 
EEG montage as well as bilateral EOG and chin EMG to stage sleep.  EEG 
monitoring will occur throughout the CTRU stay while CSF and blood sampling 
occur.   
 
The participant’s habitual bedtime is based on the sleep log completed after visit 1. 
The leucine infusion (T=0) will begin approximately one hour prior to the habitual 
bedtime (~21:00-23:00).   
 
Lemborexant 25mg or placebo will be administered ~ the participant’s habitual 
bedtime on day 1 (hour 1) and day 2 (hour 25), and the lights will be turned off to 
allow for sleep.    
 
Blood and CSF samples will be collected over 48 hours as specified below. CSF 
safety labs will be collected at the end of the sampling period. The nursing staff will 
monitor participants throughout the stay, and all food and water intake will be 
recorded during the sampling period.  
 
Participants stay 8-12 hours after the study is completed for observation to monitor 
for adverse events.  Participants will contact the study team after discharge if they 
develop any physical complaints. In addition, the physician or a member of the 
research staff will contact participants by phone 24 and 48 hours after discharge.  
8. STUDY PROCEDURES AND METHODS   
8.1 Specimens, records, or data:  
We will obtain the following from our participants at visit 4 
8.1.1 Cerebrospinal fluid (CSF) specimens:  
Each participant regardless of study aim or group will have an indwelling lumbar 
catheter placed for the collection of CSF every 2 hours for 48 hours (hours 0, 2, 4, 6, 
8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48).  At 
each time point, 6 ml of CSF will be collected.  At the end of the study an additional 
2 ml of CSF (when available) will be sent to the lab to check for any signs of 
infection. Therefore, a total of 152 ml of CSF will be collected from each participant 
over 48 hours. It is expected that the catheter will fail to draw CSF during the study 
for some participants. This will result in missing CSF samples and missing CSF 
safety labs.   
 
8.1.2 Blood specimens:  
Each participant will have an intravenous (IV) catheter placed for collection of blood.  
At the start of the study, blood will be collected 0, 5, 10, 15, and 30 minutes after the 

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lumbar catheter is placed.  Then, blood will be collected every 30 minutes until hour 
4. After hour 4, blood will be collected every 2 hours until hour 48. Approximately 1-
3 teaspoons of blood will be collected at 34 time points over 48 hours. The total 
amount of blood taken over 24 hours is 120-265 ml (half of a standard blood 
donation).   
 
8.1.3 Polysomnography: 
Polysomnogram recordings to determine sleep stages will be obtained from all 
participants.  This includes standard electrodes for electroencephalography, 
electromyogram, and electrooculogram. These recordings are not identifiable unless a 
participant requests the recordings to be placed in his/her medical record.  
 
8.1.4 Physical Exam: 
Prior to discharge, a medically qualified professional will perform a brief physical 
examination that consists of a review of the major body systems (i.e., skin, 
head/ears/eyes/nose/throat (HEENT), cardiovascular, pulmonary, abdomen, 
musculoskeletal, and extremities).  
 
8.2 Data from human subjects:  
Our participants will provide the following data: 
 
8.2.1 Personal identifiers: 
• Name, date of birth, phone number, mailing address, and email (if they want). 
These are for the purposes of proper identification for the study and 
communication about research activities. Additionally, social security number 
is collected only for purposes of participant reimbursement.  
• GUID IDs will be obtained for the purpose of data sharing.  Additional 
information collected to create GUID IDs include the place of birth and legal 
last name at birth.  
• Answers to screening questions for inclusion/exclusion criteria. 
• Demographic information: date of birth, sex, race/ethnicity, education, 
handedness 
• Medical history: Comorbidities, surgeries, medications, allergies, alcohol use 
• Medical record numbers may be documented on medical records obtained for 
the study. 
 
All personal identifying data are stored separately from all other data.  Only team members 
trained in the protocol and approved by the Human Research Protection Office (HRPO) will 
have access to private identifiable information, including the Principal Investigator, research 
coordinator/polysomnographic technicians, and the mentor.  
 
Data that do not include PHI will be coded with a study ID number not derived from personal 
identifiers.  This data will contain dates of assessments.  
 

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8.2.2 Sleep log: 
The sleep log is a daily record that logs sleep patterns over time.  The sleep log will 
be used to assess habitual bedtimes.  
 
8.2.3 Mini Mental State Exam (MMSE):  
The MMSE is a brief, frequently used screening instrument to assess cognitive 
function28. The MMSE scale evaluates orientation, memory, attention, concentration, 
naming, repetition, comprehension, and ability to create a sentence and to copy two 
overlapping pentagons. The MMSE is scored as the number of correctly completed 
items with a lower score indicative of poorer performance and greater cognitive 
impairment. The total score ranges from 0 (worse) to 30 (perfect performance). 
 
8.2.4 Columbia- Suicide Severity Rating Scale (C-SSRS):  
Consistent with FDA regulatory guidance26, occurrence of suicide-related thoughts 
and behaviors will be assessed. The C-SSRS is a scale that captures the occurrence, 
severity, and frequency of suicide-related thoughts and behaviors during the 
corresponding assessment period27. The scale includes suggested questions to elicit 
the type of information needed to determine if a suicide-related thought or behavior 
occurred.  If a suicide-related thought or behavior is identified at any time during the 
study, a thorough evaluation will be performed by a study physician, and appropriate 
medical care will be provided, such as referring the participant to their primary 
physician or therapist, or getting the participant to a medical facility for safety.  The 
number to a suicide prevention hotline is also provided in the informed consent 
document.  
 
8.2.5 STOP-Bang Questionnaire: 
The STOP-Bang Questionnaire is a screening instrument used to assess risk for 
Obstructive Sleep Apnea (OSA).  
 
 
8.2.6 Pittsburgh Sleep Quality Index (PSQI): 
This is an assessment tool of overall sleep quality and collects information about 
multiple sleep disorders to generate an overall sleep quality score. 
 
8.2.7 Stanford Sleepiness Scale (SSS): 
A self-assessment that documents how alert an individual is feeling. 
 
8.2.8 Morningness/ Eveningness Questionnaire (MEQ): 
A self-assessment to measure whether a person’s circadian rhythm produces peak 
alertness in the morning, in the evening or in between.  
 
8.2.9 Psychomotor Vigilance Testing (PVT): 
An assessment that measures speed responses to visual stimuli via a thumb operated 
device.  
 

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8.2.10 Electrocardiogram (ECG): 
An appropriately qualified individual will conduct a standard 12-lead resting ECG. 
The ECG report must be reviewed, signed, and dated by the site PI (or a medically-
qualified individual delegated by the site PI). 
 
8.2.11 Physical and Neurological Examination: 
A medically qualified professional will perform a brief physical examination that 
consists of a review of the major body systems (i.e., skin, head/ears/eyes/nose/throat 
(HEENT), cardiovascular, pulmonary, abdomen, musculoskeletal, and extremities) 
and a brief neurological examination which will include an assessment of cranial 
nerves, strength, coordination, reflexes, sensation, tremor, gait and mental status. 
Assessments of height, weight, and vital signs (systolic and diastolic blood pressure, 
pulse, temperature, and respiration) are included 
 
8.2.12 Blood Collection: 
All participants will be screened with a blood test for Aβ status unless they have been 
referred by the SEABIRD study.  The plasma Aβ test has been shown to be very 
sensitive and specific for Aβ deposition in the brain with an area under the curve of 
0.8829,30. 
 
Blood will also be obtained for genetic testing, plasma AD biomarkers and other 
blood-based biomarkers, and routine labs for safety purposes. 
   
8.3  Scale to Inform the Disclosure of Amyloid Results 
The Concern’s About AD Scale31 allows participants to indicate their level of agreement with 
six statements about developing AD.  The scale will be used only to inform the participant’s 
concerns about AD during the disclosure of amyloid results. 
 
8.4 Disclosure of Amyloid Results 
Participants will receive implied amyloid results.  Eligible participants are cognitively normal 
and amyloid-positive.  Risk of having elevated brain amyloid will be screened using a plasma 
amyloid-beta blood test.   
 
Using a standard operating procedure (SOP) for the disclosure of amyloid results, the study 
team will convey the results of the plasma amyloid-beta test.  Disclosure of amyloid results 
will be informed by the Concern’s About Alzheimer’s Disease Scale,31 This scale will not 
affect participant eligibility and is not part of the inclusion/exclusion criteria.  The purpose of 
the scale is to inform disclosure of the amyloid results.  Please see the SOP for the Disclosure 
of Amyloid Results. 
9. Potential risks 
9.1 Lumbar Catheter: 
• Discomfort during the procedure is common and mild.  Some people may have 
discomfort sitting in the same position for the duration of the lumbar catheter 

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placement. Lidocaine will be used for local anesthesia, which feels like a small pinch 
followed by burning sensation (<10 seconds). There is pressure but not pain during 
the insertion of the lumbar puncture needle. During the lumbar catheter procedure, 
participants may experience momentary (<1 second) cramping or pain in a leg, due to 
the needle or the catheter briefly touching a floating nerve ending.  
• Back soreness is common and mild.  Some people have soreness of the area where the 
needle and catheter were inserted, especially when the lidocaine wears off. This 
soreness resolves by itself over 2-3 days. 
• Feeling faint is common and mild.  As with any medical procedure, some people feel 
faint. This is a normal response and is not a physical effect of the needle.  
• Post-lumbar catheter headache occurs frequently (50-80% of participants) and is mild 
to moderate. Another possible symptom is ringing in the ears.  Individuals may have a 
headache following placement of a lumbar catheter, due to the decreased volume of 
CSF. For the majority of individuals, the headache resolves when the catheter is 
removed.  Occasionally (30% or less), this headache persists after removal of the 
catheter and requires treatment with a “blood patch.”   
• Bleeding is extremely rare, but is moderate to severe when it occurs. In individuals 
with problems of blood clotting, a lumbar catheter may cause a large amount of 
bleeding.  
• Infection is an extremely rare risk of a lumbar catheter and can be moderate-to-
severe.  Very rarely, the lumbar puncture needle and catheter may introduce 
pathogens internally, leading to infection. 
 
9.2 Intravenous Catheter: 
• Discomfort from minor pain, bleeding, bruising, or swelling caused by the needle or 
intravenous catheter are common and mild. 
• Bruising at the phlebotomy site is infrequent and mild.  Some individuals may have 
bruising due to leakage of a small amount of blood from the vein into the surrounding 
tissue. This will spontaneously resolve. 
• Feeling faint is common and mild.  As with any medical procedure, some people feel 
faint. This is a normal response and is not a physical effect of the needle. 
 
9.3 Lemborexant: 
• This study will use lemborexant 25 mg, a dose greater than the 10 mg dose approved 
by the FDA for the treatment of insomnia.  Treatment-emergent adverse events for 
lemborexant 25 mg were similar to lemborexant 10 mg, however.  See Appendix 1 
for a summary of treatment-emergent adverse events for different doses of 
lemborexant.   
• Somnolence is a common, generally mild side effect of lemborexant.  This is an 
expected effect of this drug given its mechanism of action.  Participants will be 
monitored in the Clinical Translational Research Unit prior to discharge and will be 
assessed for level of alertness with a Stanford Sleepiness Scale testing and 
Psychomotor Vigilance Testing performed upon admission and at the time of 
discharge. The principal investigator will evaluate the participant and review these 
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• Additional mild and common reactions to lemborexant include: headache, and 
abnormal dreams.  
• Abnormal sleep behaviors, such as sleep walking, are rare with lemborexant (2 
episodes in the registration program).  All participants will take lemborexant on the 
Clinical Translational Research Unit at Washington University that has 24 hours a 
day nursing coverage monitoring the participants. 
• Additional uncommon or rare reactions to lemborexant include heart palpitations, 
tachycardia, sleep paralysis (the inability to move or speak for up to several minutes 
upon sleep-wake transitions), hypnagogic/hypnopompic hallucinations (visualizations 
upon sleeping and awakening), and mild cataplexy-like symptoms (muscle weakness 
lasting from seconds to a few minutes but no confirmed cataplexy).  
• 
Lemborexant may interact with multiple medications including alcohol, antifungals (e.g., 
itraconazole, fluconazole), antibiotics (e.g., clarithromycin, rifampin), antiretrovirals (e.g., 
efavirenz, etravirine), verapamil, carbamazepine, St. John’s wort, bosentan, and modafinil. 
• The principal investigator will evaluate participants for potentially interacting 
medications and participants will be excluded from the study if the interacting drug(s) 
cannot be discontinued. 
• Very rare:  For all individuals, but primarily in depressed participants treated with 
hypnotics, worsening of depression and suicidal tendencies have been reported. 
 
9.4 Polysomnogram (PSG): 
• Irritation of skin from electrodes used during study is rare and mild.  Skin needs to be 
cleaned before adhesive is applied for the electrodes. Some individuals with sensitive 
skin may have irritation of their skin.  
• Worse sleep quality is common and mild.  People usually do not sleep as well in an 
unfamiliar environment. Therefore, participants may be more tired than usual the next 
day.  
 
9.5 Questionnaires: 
• Participants may experience emotional discomfort when answering some questions in 
the questionnaires. If any particular question makes the participant uncomfortable, the 
participant may discuss it importance and the need to answer it with the specially- 
trained interviewer. Participants have the right to refuse to answer any question for 
any reason.  
 
9.6 Bedrest: 
• There is risk of feeling dizzy, lightheaded, or woozy upon standing after 60 hours of 
bed rest.  This is common and mild.   
 
9.7 Privacy/confidentiality: 
• Breach of privacy/confidentiality (very rare, severity varies): A risk in any research 
study is that confidential information about participants may be accidentally 
disclosed.  
10. Adequacy of Protection Against Risks 

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Recruitment and Informed Consent  
10.1 Recruitment:  
Human subjects will be recruited from the ADRC, the ongoing SEABIRD study, or 
Volunteers for Health, a community-based registry of >10,000 individuals.  We developed 
advertisements targeting poor sleep quality to be sent to this registry.  Potential participants 
can indicate their interest in this study through an online system, in which case their name 
and contact information is sent to the principal investigator, or they can call the phone 
number that is on the advertising materials.  Referral sources may provide additional 
information to the study team at the time of the referral, including name, age, date of birth. 
CDR scores, imaging data, CSF and plasma biomarker data (including amyloid status).   
Additional data, biofluid markers, and genetic information may be obtained from the referral 
sources after study enrollment.   
 
10.2 Consent:  
Two separate consent processes are required, one for an initial phone screen to assess if 
participants are likely to meet inclusion/exclusion criteria for the study, and a second formal 
written consent. No waivers of elements of consent will be sought. 
• Phone screen consent: Because the initial phone screen contains questions that 
involve protected health information, verbal consent is obtained prior to going 
through the screen. The Washington University HRPO will review and approve a 
phone script for verbal consent, which includes (1) A statement that the phone screen 
is for research, (2) purpose of the phone screen, (3) expected duration of phone 
screen, (4) description of the phone screen, (5) identification of any procedures which 
are experimental, (6) description of risks and benefits, (7) disclosure of alternatives 
(i.e. not doing phone screen), (8) how confidentiality of records will be maintained, 
(9) a statement that participation is voluntary and not participating will involve no 
penalty or loss of benefits to which the person is otherwise entitled. 
• Consent for enrollment in study: Potential subjects are mailed a copy of the consent 
document to review and discuss with other individuals (if desired) prior to an in-
person consent process. Consent will take place in a private exam room at the 
Washington University Sleep Medicine Center or the Neurology Clinical Research 
Unit (NCRU). Only members of the research team who have had training and are 
approved by the Washington University HRPO (including PI) may participate in the 
consent process. Information in the consent document includes: (1) A statement that 
the study is for research, (2) purpose of the research, (3) expected duration of study 
participation, (4) description of the research procedures, (5) identification of any 
procedures which are experimental, (6) description of risks and benefits, (7) 
disclosure of alternatives, (8) how confidentiality of records will be maintained, (9) a 
statement that participation is voluntary and not participating will involve no penalty 
or loss of benefits to which the person is otherwise entitled. Potential participants may 
ask questions either beforehand by phone or during this face-to-face visit, before 
deciding to enroll in the study. After all questions have been answered, and if the 
person wants to enroll in the study, the consent process will be finalized by both 
participant and research team member signing the consent document. Every 
participant is provided with a copy of the signed consent document. 
 

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10.3 Lumbar catheter:  
• Discomfort during the procedure: To minimize discomfort, participants will sit at the 
edge of the bed with supports and pillows to keep them in an ergonomic position. 
Lidocaine will be used for local anesthesia. Only physicians experienced in 
performing lumbar catheters will perform this procedure. 
• Back soreness: Participants are provided with information sheets on using heat, 
stretching, and acetaminophen if back soreness occurs. 
• Feeling faint: If a participant feels faint, s/he will be assisted in lying down in the bed 
until the feeling has passed. The lumbar catheter can be performed with participants 
lying down on their side, in this case.  
• Post-lumbar catheter headache: This risk will be minimized by having the participants 
lie in bed with their heads down (the Trendelenburg position) for one hour after the 
catheter is removed.  Participants will remain in bed for 6-8 hours after the catheter is 
removed.  Participants are also informed they can minimize this risk by having 
caffeine after the lumbar catheter is removed, and by drinking plenty of water 
throughout the study while the catheter is in place. If a post-lumbar catheter headache 
does occur, participants will be offered treatment with a “blood patch.” 
• Bleeding: Individuals with this risk (blood clotting problems or taking anticoagulants) 
are excluded from participation in the study. 
• Infection: Proper sterile technique will be used during all lumbar catheter placements.  
At the end of the study immediately prior to the removal of the catheter, an additional 
2 ml of CSF will be drawn, when available, and sent to the lab to check for any signs 
of infection.  Further, participants are given a 24/7 phone number for a member of the 
research team after discharge from the Clinical Translational Research Unit in case of 
complications. 
 
10.4 Intravenous catheter: 
• Discomfort during the procedure: Only physicians and nurses experienced in placing 
intravenous catheters will perform the procedure. 
• Bruising at phlebotomy site: Discomfort from bruising can be alleviated by moist heat 
pack, which can be provided in the Clinical Translational Research Unit.  
• Feeling faint: If a participant feels faint, s/he will be assisted in lying down in the bed 
until the feeling has passed. Participants will be encouraged to drink plenty of water 
during the study, to minimize the chance of feeling faint. 
 
10.5 Lemborexant:  
All participants will take lemborexant on the Clinical Translational Research Unit at 
Washington University which has 24 hours a day nursing coverage monitoring the 
participants.  Participants will only take lemborexant in this monitored setting.  Nurses will 
be available to assist all participants if they develop these symptoms.  Further, the 
participants will be under constant video monitoring while in the Clinical Translational 
Research Unit; this video is not recorded and is only viewable by the nursing staff. 
Lemborexant will be discontinued if reactions are distressing to the participant or severe.  
Participants will be monitored in the Clinical Translational Research Unit prior to discharge 
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Vigilance Testing performed upon admission and at the time of discharge.  The principal 
investigator will evaluate the participant and review these measures prior to approving 
discharge.  Potential adverse events from lemborexant include: 
• Somnolence:  Somnolence may be severe in participants with compromised 
respiratory function; therefore, obstructive sleep apnea and chronic obstructive 
pulmonary disease are exclusion criteria.  Lemborexant will be discontinued if 
somnolence is distressing to the participant or severe. 
• Next day drowsiness 
• Additional mild and common reactions to lemborexant include headache, and 
abnormal dreams 
• Abnormal sleep behaviors 
• Additional uncommon and rare reactions include heart palpitations, tachycardia, sleep 
paralysis, hypnagogic/hypnopompic hallucinations, and mild cataplexy-like 
symptoms.   
• Lemborexant may interact with multiple medications:  Participants taking 
medications that interact with lemborexant will be evaluated by the principal 
investigator.  If the potentially interacting medications cannot be discontinued, then 
the participants will be excluded from the study. 
• Depressed mood and/or suicidal ideations:  All participants will be screened for 
suicidal ideations and psychiatric disorders.  All participants with psychiatric disease 
requiring medications will be excluded both to control this risk and also because 
psychiatric medications may interfere with sleep.  Participants with suicidal ideations 
will be excluded.  This risk is also controlled because participants will only take 
lemborexant for 2 nights in the monitored setting of the CTRU with 24 hours a day 
nursing coverage. 
 
10.6 Polysomnogram (PSG): 
• Irritation of skin from electrodes used during study: When possible, an alternative 
cleaning agent or adhesive will be used. If irritation is more than mild, that electrode 
will not be used for the study. 
11. Definitions  
For this study, the following standard definitions for reporting will be used:  
 
11.1 Adverse Event: 
Adverse Event (AE) is any untoward medical occurrence associated with the use of the study 
drug in humans, whether or not considered related to the drug. An AE (also referred to as an 
adverse experience) may include any unfavorable and unintended sign (i.e., abnormal lab test 
finding), symptom, or disease temporally associated with the use of the drug, and does not 
imply any judgement about causality.   
 
11.2 Suspected Adverse Reaction: 
A suspected adverse reaction is any adverse event that is determined for which there is a 
reasonable possibility that the study drug caused the adverse event. For IND safety reporting, 

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'reasonable possibility' means there is evidence to suggest a causal relationship (reasonable 
possibility) between the drug and the adverse event. A suspected adverse reaction implies a 
lesser degree of certainty about causality than an adverse reaction.  
 
11.3 Adverse Reaction: 
Adverse Reaction means any adverse event caused by a study drug. Adverse reactions are a 
subset of all suspected adverse reactions where there is a reason to conclude that the drug 
caused the event.  
 
11.4. Serious: 
An adverse event or suspected adverse reaction is considered serious if the event or reaction, 
in the view of the PI, results in any of the following serious outcomes:  
  
• Death;   
• A life-threatening adverse event as defined as placing the participant at immediate 
risk of death from the AE as it occurred;   
• Initial or prolonged inpatient hospitalization (hospital admission defined as ≥ 24 
hours or prolongation of a hospital stay due to adverse event);   
• Persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions;  
• Congenital anomaly/birth defect.   
 
11.5 Life-threatening: 
An adverse event or suspected adverse reaction is considered “life-threatening” if, in the 
view of the PI, its occurrence places the participant at immediate risk of death. It does not 
include an adverse event or suspected adverse reaction that, had it occurred in a more severe 
form, might have caused death.  
 
11.6. Important Medical Events (IMEs): 
IMEs are not immediately life-threatening, events that places a participant at immediate risk 
of death or require hospitalization may be considered serious when, based upon appropriate 
medical judgment that jeopardizes the participant and may require medical or surgical 
intervention to prevent one of the above serious outcomes. Examples of such medical events 
include allergic bronchospasm requiring intensive treatment in the emergency room or at 
home, blood dyscrasias or convulsions that do not result in inpatient hospitalization, or the 
development of drug dependency or drug abuse.  
  
11.7 Unexpected: 
An adverse event or suspected adverse reaction is considered unexpected if it is not listed in 
the Investigator's Brochure (IB) if one exists or is available or is not listed at the specificity 
or severity that is currently observed, or the event is not consistent with the risk information 
described in the protocol, informed consent form, general investigational plan or elsewhere in 
the current IND application when an IB is not required or available. The PI will determine if 
an adverse event is unexpected.  
  

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Expected adverse reactions are adverse events that are known to occur for the investigational 
drug and should be collected in a standard, systematic format using a severity grading scale. 
Expectedness is assessed based on the awareness of AEs previously observed, not on the 
basis of what might be anticipated from the properties of the study drug. For risk related to 
this IND protocol, see “Expected Risk” section below.   
 
11.8 Causality and Severity Assessment: 
A clinical determination will be made, as to the likelihood that an AE is related to the 
administration of the study drug or research visit procedure. The PI or designee will 
determine the criteria below for the severity/intensity of the AE and its relationship to the 
administration of the investigational study drug. Other etiologies such as pre-existing 
conditions, concomitant therapy, study-related procedures, accidents that occur while on 
study, and other external factors will be considered during the evaluation of relatedness. The 
adverse event assessment report will be submitted with any reportable adverse event.  
 
11.9 Severity/Intensity: 
An adverse event will be classified as one of the following to describe the severity (intensity) 
and any necessary action taken in an attempt to resolve the event:   
  
• Mild:  A mild AE is usually transient in nature and generally does not interfere with 
normal activities. Participants are asymptomatic or have mild symptoms; intervention 
is not indicated.  
• Moderate:  A moderate AE is sufficiently discomforting to interfere with normal daily 
activities. Participants may need minimal, local, or noninvasive intervention;   
• Severe:  Also referred to as medically significant but not immediately life-
threatening; hospitalization or prolongation of hospitalization is indicated; disabling; 
limiting self-care activities of daily living (ADL). Note that a severe event is not 
necessarily a serious event.  
• Life Threatening:  Any AE that places the participant, in the view of either the PI or 
designee, at immediate risk of death from the AE as it occurred. It does not include an 
AE that, had it occurred in a more severe form, might have caused death.  
 
11.10 Attribution: 
The degree of certainty about causality will be graded using the following relationship 
categories:  
 
• Unrelated:  An adverse event is clearly not related to the drug or procedure.   
• Unlikely Related:  An adverse event is doubtfully related to the drug procedure.   
• Possibly Related:  An adverse event is may be related to the drug or procedure.   
• Probably Related:  An adverse event is likely related to the drug or procedure.   
• Definitely Related:  An adverse event is clearly related to the drug or procedure.   
• For the purposes of regulatory reporting, a causality assessment of unlikely related 
will be managed as “unrelated”. An adverse event that is definitely, probably, or 
possibly related will be managed as “related” and considered a “reasonable 

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possibility” that the drug caused the adverse event and, therefore, meets the definition 
of a suspected adverse reaction.  
 
11.11 Documenting Adverse Events: 
All serious and non-serious adverse events as well as abnormal test results/findings, 
regardless of suspected causal relationship will be documented in the subjects’ research 
record. Adverse events will be reported to the IRB per university guidelines.  
  
Adverse events that are classified as reportable will be followed until resolution or 
stabilization as evaluated by a physician. Clinically significant changes from the subjects’ 
baseline will be considered noteworthy. All clinically significant changes, relevant medical 
history and concomitant medications and action taken will be documented in the subjects’ 
case report forms (source document forms). Events that which do not meet the requirements 
of SAE reporting will be reviewed and confirmed by the Investigator and reported with the 
IRB annual continuing review, Data Safety and Monitoring report and the FDA annual 
report.   
 
11.12 Pre-existing Conditions: 
Pre-existing conditions that are present at the time of informed consent will be considered 
participant baseline. All baseline conditions will be recorded on the medical history in the 
source document forms. Worsening of the severity or frequency of a pre-existing condition in 
a research participant which does not necessarily have a causal relationship with the 
investigational drug is a reportable adverse event. Any changes, i.e., deterioration or 
worsening in these condition(s) will be documented and evaluated by the PI or designee. AEs 
with signs and symptoms that are believed to be due to the pre-existing condition(s) will not 
be reported as an AE unless there is an increase in frequency and severity. The investigator’s 
clinical determination of any adverse event will be recorded on the AE form in the source 
document forms and maintained in the subjects’ research record. All AEs will be documented 
on the adverse event log and maintained in the regulatory binder.   
12. Food and Drug Administration Mandatory Reporting  
12.1 Initial Safety Reporting: 
The Sponsor-Investigator must report to the Food and Drug Administration (FDA) observed 
or voluntarily reported adverse events that meet criteria according to the following 
timeframe:  
  
• 7-day IND Safety Report” – Unexpected Fatal or Life-threatening    
Report any unexpected fatal or life-threatening adverse reactions associated with the 
use of the study drug. Reporting will occur as soon as possible but no later than seven, 
(7) calendar days following the Sponsor-Investigator’s initial receipt of the 
information. Preferably by facsimile or secure email addressed to the Regulatory 
Project Manager followed by a formal official submission.   
Any study event that is:  
o associated with the use of the study drug  
o serious (fatal or life-threatening)  

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o unexpected  
 
• “IND Safety Report” – 15-day Written Reports  
Written IND safety reports may be submitted electronically on an FDA Form 3500A 
(MedWatch) or in a narrative format in accordance with 21 CFR 312.32. Report any 
of the following study events to the FDA as soon as possible but no later than 15 
calendar days.  
Any study event that is:  
o associated with the use of the study drug  
o serious, but not fatal or non-life-threatening  
o AND unexpected  
Any previous or increased rate of events:  
o A previous adverse event that was not initially deemed reportable but is later 
found to fit the criteria for reporting.  The event must be reported within 15 
calendar days from when the event was deemed reportable.  
o Findings from other clinical, animal, or in-vitro studies, including reports of 
mutagenicity, teratogenicity, or carcinogenicity that suggest significant risk to 
human subjects.  
o A clinically important increase in the rate of any serious suspected adverse 
reaction. The Sponsor-Investigator will identify previously reported similar 
adverse events in IND safety reports and analyze the significance of the 
current event in light of the previous reports.  
Any of the following adverse events as a suspected adverse reaction only if there is 
evidence to suggest a causal relationship between the drug and the adverse event.  
o A single occurrence of an uncommon event and known to be strongly 
associated with the drug.  
o One or more occurrences of an event that is not commonly associated with the 
drug, but is otherwise uncommon in the population being studied.  
 
12.2 Follow-up Reporting: 
The Sponsor-Investigator will report any additional information that pertains to an IND 
safety report previously submitted or a previous adverse event that was not initially 
deemed reportable but is later found to fit the criteria for reporting.  
• 
“Follow-up IND Safety Report”– Follow-up Information  
Any relevant follow-up information that pertains to an IND safety report previously 
submitted will be submitted within 15 calendar days from day zero, date of the 7-day 
report.   
 
A previous adverse event that was not initially deemed reportable but is later found to 
fit the criteria for reporting must be submitted as a “Follow-up IND Safety Report”. 
Reports should be submitted without delay, as soon as the information is available but 
no later than 15 calendar days after the Sponsor-Investigator receives the 
information.  
 

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12.3 Annual Report: 
A summary of all IND safety reports, including the most frequent and most serious AEs, will 
be submitted each year within 60 days of the anniversary of the date that the IND became 
active, the date clinical studies were permitted to begin.  
 
12.4 
FDA Reporting Process - Written and Facsimile: 
The FDA prefers IND “7-day” safety reports be submitted via secure email or by facsimile. 
Submit IND “15-day” written and follow-up safety reports and all other reports to the FDA. 
 
12.5    Eisai (IND holder of Lemborexant): 
The study team shall notify Eisai within twenty-four (24) hours of receiving notification of 
any serious adverse events experienced by a participant that could be reasonably related to 
lemborexant. For the purpose of this requirement, “serious” means: (1) death; (2) in-patient 
hospitalization or prolonged hospitalization; (3) life-threatening; (4) persistent or significant 
disability or incapacity; (5) congenital anomaly or birth defect; (6) other serious events that 
may jeopardize the patient and may require medical or surgical intervention to prevent one of 
the other five listed outcomes.  Serious adverse events attributed to lemborexant will be 
reported using the Eisai serious adverse event form.  Serious adverse events may be reported 
via one of the following: 
 
Fax: 732-791-1111 
Email:  ESI_Safety@eisai.com 
 
12.6 Institutional Review Board Mandatory Reporting: 
The PI or designee will report, to the Institutional Review Board (IRB) at Washington 
University (WU), the Human Research Protection Office (HRPO) in accordance with WU 
IRB policies and procedures. The PI or designee will report immediately (within 10 working 
days unless it results in death), to the IRB, any observed or volunteered self-reported adverse 
event that meets the unanticipated problem (UP) criteria or if the Investigator believes the 
information meets the exception criteria as described below:  
  
12.7 Unanticipated Problem (UP) Criteria: 
The PI or designee will report, to the IRB, any observed or volunteered self-reported adverse 
event that is determined to meet all three criteria listed below:  
• Must be unexpected in terms of (nature, severity, and/or frequency) given the 
research procedures that are described in the protocol and consent form and the 
characteristics the subject population being studied;   
• Must be reasonably related to the research; and   
• Must place participants or others at a greater risk of harm than previously known or 
recognized or approved by the IRB.   
 
12.8 Exception Criteria: 
The Investigator believes the information identifies a change to risks or potential benefits of 
the study or if the Investigator believes information indicates a change to the risks or benefits 
of the study.   

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12.9 IRB process of reporting events: 
Events that do not meet the unanticipated problem (UP) criteria or the exception criteria, 
should be reported with the IRB annual continuing review, i.e., IND external safety reports. 
Federal regulations do not require immediate reporting to the IRB of IND/outside safety 
reports for events that do not meet the criteria outlined in 21 CFR 312.32(c). Thus, IND 
Safety reports that do not meet the regulatory requirement for immediate reporting may only 
be reported in summary format at the time of IRB continuing review.  
  
Adverse events that meet the UP or exception criteria will be reported as follows:  
  
• 1-day Reportable Events  
Report the death of a research participant to the IRB within one (1) working day of 
the occurrence of the event or notification of the event to the PI, or to a member of 
the research team.  
• 10-day Reportable Events  
Report any unanticipated problems (UP) involving risks to participants or others 
which occur at WU or that impacts participants or conduct of the study, 
noncompliance, or receipt of new information that may impact the willingness of 
subjects to participate or continue participation in the research study, i.e., an updated 
package insert with a change to the risks or benefits resulting in a modification to the 
informed consent form and/or protocol. The occurrence of the event or notification of 
the event to the Investigator, or to a member of the research team must be reported to 
the IRB within ten (10) working days.  
  
12.10 Data Safety Monitoring (DSM) Reporting: 
In compliance with the Washington University School of Medicine Institutional Data Safety 
Monitoring (DSM) Plan, the PI will provide a DSM report to the IRB annually. The PI, 
research coordinator and members of the research team will review all study data as it 
becomes available. Investigators not involved with the study will independently review the 
safety data and state if the study should be continued or not. The report will include the 
protocol title, IRB protocol number, IND number, the activation date of the study, the 
number of subjects enrolled to date, the date of first and most recent subject enrollment, a 
summary of all adverse events regardless of severity or relationship to subject dropouts and 
corresponding reason for dropout, the investigational plan for the coming year, any new 
information related to the investigational drug or a summary of any recent literature that may 
affect the safety and ethics of the study.  
 
12.11 Other Reportable Events: 
Major deviations or series of minor deviations having the potential to adversely impact the 
health, safety, or welfare of participants or others or adversely impact the study’s ability to 
produce scientifically valid results must be reported to the IRB within 10 working days of the 
occurrence of the event or notification to the Principal Investigator of the event. Major 
deviations resulting in death of a participant must be reported within one (1) working day of 
the occurrence of the event or the notification to the Investigator of the event. Any non-

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compliance and protocol exceptions or deviations will be documented and maintained in the 
regulatory binder and participant’s research chart.  
 
12.11.1. Non-Compliance and Serious Non-Compliance: 
•  Noncompliance may occur due to lack of knowledge or due to deliberate choice to 
ignore regulations, institutional policies, or determinations of the IRB. 
Noncompliance is defined as failure to follow any applicable regulation or 
institutional policies that govern human subject’s research or failure to follow the 
determinations of the IRB.   
• Serious noncompliance is defined as noncompliance that materially increases risks 
that results in substantial harm to subjects or others, or that materially compromises 
the rights or welfare of participants.  
  
12.11.2. Protocol Exceptions and Deviations: 
• Protocol exceptions apply only to a single participant or a singular situation. Pre-
approval of any protocol exceptions must be obtained by the IRB prior to the event. 
In the event a situation occurs which requires deviation from the protocol, the PI will 
make the final judgement on whether or not a participant study is prematurely 
completed.   
• Protocol deviations such as less than expected tracer production can be accounted for 
during data analysis and will not necessarily result in cancellation of the scan. In the 
event of any deviations from the investigational plan/protocol that affects the life or 
physical well-being of a participant in any emergency, the PI will promptly notify the 
FDA and IRB as required.  
• HRPO definitions of minor and major deviations  
• 
A minor or administrative deviation is one that does not have the 
potential to negatively impact the rights, safety, or welfare of participants 
or others or the scientific validity of the study.    
• 
A major deviation is one that does have the potential to negatively 
impact the rights, safety, or welfare of participants or others or the 
scientific validity of the study.    
 
12.11.3. Timeframe for Reporting Required Events  
Table 1. Reporting Events  
The table below summarizes the timeframe for reporting required events after initial 
receipt of the information by the PI or member of the research team as described 
above.   
 
Serious, fatal life-threatening adverse 
events  
IRB 
FDA reporting for 
lemborexant 
Any death and immediately life-
threatening events, whether related or 
unrelated and any adverse event that 
meets the definition of IRB unanticipated 
Within one (1) 
working day to the 
IRB 
As soon as possible 
but no later than 7 
calendar days   

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problem or FDA unexpected and serious 
suspected adverse reaction.  
Non-fatal/Non-life-threatening adverse 
events   
 
 
Any unexpected and serious suspected 
adverse reaction, findings from other 
clinical or animal studies (internal or 
external) and clinically important 
increases in severely suspected adverse 
reactions.  
Within 10 working 
days to the IRB  
As soon as possible 
but no later than 15 
calendar days   
Annual reportable adverse events  
 
 
Adverse events that do not meet the 
definition of unanticipated problem or 
serious and unexpected suspected adverse 
reaction should be submitted 
cumulatively.   
IRB Annual 
Continuing Review; 
DSM report   
FDA Annual 
Report  
Other reportable adverse events  
 
 
New Information that may impact the 
willingness of subjects to participate in 
the research study – interim analysis, 
safety monitoring report, published paper, 
change in labeling, revised Investigator’s 
Brochure.   
Within 10 working 
days to the IRB  
As soon as possible 
but no later than 15 
calendar days  
Noncompliance and Protocol Deviations   
 
A serious noncompliance, major protocol 
deviation or series of minor deviations 
which do not meet the above criteria.  
Within 10 working 
days to the IRB  
FDA Annual 
Report.   
Non-serious, noncompliance issue  
IRB Annual 
Continuing Review  
None unless 
requested by IRB  
13. Privacy/confidentiality 
13.1 Breach of privacy/confidentiality:  
• All data will be labeled with a random number rather than with any identifiable 
information. All physical materials (such as paper, data storage disks, and 
biospecimens) will be stored in locked cabinets/freezers in a locked research area, 
accessible only to the research team. All electronic information will require a 
password and will be accessible only to the research team. Any paper or electronic 
information that could link a participant to the study (such as consent documents) will 
be stored separately from all other study materials, also in a secure manner that is 
accessible only to the research team. If a report or article is written about this study or 
the study data are shared with other researchers, it will be done in such a way that 
participants cannot be identified. 
• For individuals who give verbal consent for the Phone screen, and then do not “pass” 
the screening questions, their phone screen will be shredded, and no information is 

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kept about them. 
 
Protections for research involving vulnerable populations: This project does not involve 
vulnerable populations. 
 
13.2 Plan for Adverse Events (AE):  
Response to an adverse event or risk will differ based on the type and severity of the event. A 
Data Safety Monitoring Committee (DSMC) will serve to mitigate these risks and ensure a 
formal process for dealing with adverse events. A DSMC consisting of experts in the study 
procedures and the effects of sleep disorders on cardiac and psychiatric morbidities has been 
established for the proposed study.  
o Qualified individuals monitoring the study: The DSMC members have 
substantial experience and expertise in this area of research and in clinical 
research, and are qualified to monitor the study. 
o The DSMC will meet prior to enrollment of any participants, to review study 
procedures and potential adverse events, and then provide guidance on HRPO 
application and consent/informational materials.  
• Web-Based Instruction on Conducting Human Research: In accordance with the NIH 
policy effective October 1, 2000, this is to certify that Washington University key 
personnel involved in the design and conduct of the human subjects research aspect 
of this proposal have been educated on the protection of human research participants. 
An interactive web-based program that provides information on conducting human 
research and the informed consent process is organized into five modules. These 
modules include detailed information, examples and exercises related to basic 
principles, history, consent form process, and after approval requirements such as 
continuing review and adverse events. 
• Review of adverse events: All personnel who will be in contact with participants 
and/or data are prepared to identify adverse events and have been instructed to report 
their occurrence immediately to the principal investigator. 
• Definition: An adverse event will be defined as any negative change in health related 
to study-related procedures. 
• Classification of Events: A Safety Documentation form will assess for details of any 
adverse events, severity, and relatedness to study procedures. 
• Data Collection Procedures for Adverse Events: At each visit and with each 
interaction/phone call, adverse events will be screened for and recorded. If any 
adverse events have occurred, these will be logged in the Safety Documentation 
form. 
• Reporting Procedures: The Safety Documentation form will be discussed during the 
DSMC meetings. The DSMC will meet according to participant completion goals 
(e.g., 5 participants complete the study, 10 participants complete the study, 15 
participants complete the study, etc.). If a serious adverse event (SAE) occurs, the 
DSMC will meet as soon as possible to determine the course of action. If the DSMC 
deems changes to the study are necessary for safety reasons, the study will halt 
immediately until the changes have been approved through the Washington 
University HRPO. 

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• Safety-related criteria for stopping the study: The trial will be stopped if there is an 
unexpected serious adverse event (SAE). Otherwise, circumstances that would 
warrant stopping the study are not anticipated. However, should any circumstances 
arise that compromise the safety of the participants, they will be reported to the 
DSMC who will suspend research until appropriate safeguards allow continuation of 
the study.  
• Adverse Event Reporting Period: Serious adverse events and Adverse events, 
regardless of the causal assessment to the study interventions (lemborexant, lumbar 
catheter, etc.), are collected from the time the subject signs the study informed 
consent through 28 days after the participant’s last dose of lemborexant.  Serious 
adverse events will reported directly to Eisai.   
o Expected adverse events (i.e., are listed above and on consent forms) will be 
discussed at DSMC meetings. Also, they will be reported to the HRPO during 
annual renewals. 
o Unexpected serious adverse events will be reported within 10 days, per 
HRPO requirements. 
Anticipated adverse events that would require medical intervention are: 
• A post-lumbar catheter headache that does not resolve with conservative measures such 
as rest, hydration, and caffeine intake. This will be treated with “blood patch” in the 
Clinical Translational Research Unit, at no cost to the participant. 
• Side effects to lemborexant such as somnolence and confusion upon waking.  Risks from 
lemborexant are minimized with the inclusion/exclusion criteria and that participants will 
only receive the drug for 2 nights in the monitored setting of the Clinical Translational 
Research Unit at Washington University.  If any severe adverse events occur after the 
first dose of lemborexant, then the drug will be discontinued and the participant 
monitored closely. 
14. Potential Benefits of the Proposed Research to Human Subjects and Others 
14.1 Potential benefits to participants: 
The only direct benefit to the participant is that they will be screened for sleep apnea with the 
STOP-Bang questionnaire and other sleep disorders by the history and examination of the 
principal investigator.  This may result in a diagnosis that may have otherwise been 
undetected.  Participants who screen fail due to concerns for a sleep disorder like sleep apnea 
will be informed of the reason for exclusion, withdrawn from the study, and encouraged to 
follow-up with their primary care physician.  
 
14.2 Potential benefits to others: 
Potential benefits to society will be knowledge of whether or not sleep modification can 
change CSF Aβ production and concentrations.  If CSF Aβ production and concentrations 
can be modified by improving sleep efficiency, then this opens up a new potential treatment 
option for the prevention or treatment of Alzheimer's disease from which they, or future AD 
patients, may benefit. 
 

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14.3 Risks vs. benefits:  
The benefit of the knowledge to the gained in the study, as well as the personal benefit to 
participants, outweighs the risks in the study. Risks have been minimized to the greatest 
possible extent, while still achieving the scientific aims of the research study. 
15. Importance of the Knowledge to be Gained 
The knowledge to be gained in this study is very important because it is the first study 
directly assessing the relationship of poor sleep efficiency and Aβ in a relevant human 
population. If this study is successful, it may lead to treatments targeting sleep for possibly 
delaying or reducing risk of AD. AD is a devastating neurodegenerative disorder 
characterized by progressive cognitive decline.  The public health impact of dementia is 
enormous with the number of individuals with AD projected to increase from 5 million 
Americans in 2010 to 13.5 million Americans in 2050.  Even a modest reduction in the risk 
of AD would have a tremendous public health impact.  Demonstrating that sleep alters Aβ 
may suggest innovative AD treatment approaches that involve sleep therapies. 
16. Data and Safety Monitoring Plan 
This study is a clinical trial, however it is not an NIH-defined phase III clinical trial.  
Lemborexant is FDA-approved for the treatment of insomnia.  This study will use 
lemborexant 25 mg, which is higher than the FDA approved dose. Comparisons are being 
made between poorly-sleeping older adults treated (lemborexant) and untreated individuals 
(placebo). There is a Data Safety Monitoring Committee and monitoring plan as described in 
section “2.B Protections Against Risk” above.  In compliance with the Washington 
University School of Medicine Institutional Data Safety Monitoring (DSM) Plan. The PI, 
research coordinator and members of the research team will review all study data as it 
becomes available. Investigators not involved with the study will independently review the 
safety data after 5 participants have completed the study and state if the study should be 
continued or not. The report will include the protocol title, IRB protocol number, IND 
number, the activation date of the study, the number of subjects enrolled to date, the date of 
first and most recent subject enrollment, a summary of all adverse events regardless of 
severity or relationship to subject dropouts and corresponding reason for dropout, the 
investigational plan for the coming year, any new information related to the investigational 
drug or a summary of any recent literature that may affect the safety and ethics of the study. 
 
Given that the study requires significant involvement from the participants, we have taken 
steps to make the study less taxing for participants. These steps include having nurses 
available in the CTRU for assisting the research participants at all times and providing 24 
hour/7 day/week access to study staff and physicians.  
 
All participants will be identified by a de-identified study number to protect patient 
confidentiality. Participants may be linked to the “Lemborexant Sleep” study in Epic if they 
already have a record in the Epic system.  Spreadsheets listing the participants’ name and 
corresponding study number will be managed by the study coordinator, and will be stored in 
a password protected file behind the Neurology firewall. Only the research team will have 

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access to this spreadsheet. Data monitoring and analysis will be performed by the principle 
investigator. Data will also be collected on any adverse events related to the study 
procedures.   
 
All data from mass spectrometry analyses are electronically uploaded and locked in the 
Bateman laboratory’s database (Firebird 2.1.4) blinded to clinical status and other variables. 
Future studies may involve a collaboration with Dr. Donald Elbert, an Aβ stable isotope 
labeling kinetics (SILK) expert in modeling at Washington University, the Aβ SILK data will 
be analyzed with the compartmental model using the SAAM II modeling program blinded to 
clinical status and other variables. All statistical analyses will be conducted in collaboration 
with Dr. Chengjie Xiong, an expert in statistics at Washington University, or the ICTS 
biostatistics core. All data is compared in a pre-specified fashion.  
17. ClinicalTrials.gov Requirements 
This study will be registered at ClinicalTrials.gov and the final study results will be reported 
on the website within 12 months of study completion.  This will enable registration of 
primary and secondary aims and allow for broadest publication in journals.   
 

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18. APPENDICES 
18.1 Appendix 1: Lemborexant 25 mg Safety Data 
Introduction 
Lemborexant (LEM) is a dual orexin receptor antagonist approved in the US, Japan, Canada, 
Australia, and other Asian countries for the treatment of adult patients with insomnia.  Doses 
of 2.5 (LEM 2.5; Japan only), 5 mg (LEM5) and 10 mg (LEM10) are currently approved for 
these patients.  Across the clinical program, a total of 150 subjects in 7 studies were exposed 
to lemborexant (E2006) 25 mg (LEM25). Multiple doses of LEM25 were evaluated in three 
multiple dose studies, E2006-A001-002, E2006-A001-003, and E2006-G000-201, which are 
described in more detail below.  Other studies not included in this overview employed single 
dose paradigms. 
Study E2006-A001-002 (NCT01673451) 
Study Design 
This was a single-center, randomized, double-blind, placebo-controlled, sequential, multiple-
dose study. The study was conducted in 2 parts: Part A (6 cohorts of healthy adults receiving 
evening dosing) and Part B (1 cohort of elderly adults receiving evening dosing).  
In Part A, it was planned for a total of 48 healthy adult subjects (18 to 55 years) to be 
enrolled into 1 of 6 cohorts sequentially in a gradual dose escalation manner, randomized to 
receive either LEM or matching placebo in the evening 30 minutes before habitual bedtime, 
and after 3 hours fasting, each night for 14 days. Each cohort was comprised of 6 LEM- and 
2 placebo-treated subjects. Blood samples were collected for PK analysis at prespecified 
timepoints, and PD assessments were conducted (not reported here). 
During Part B, one cohort of 8 elderly subjects (≥65 to 80 years) were randomized and 
treated for 14 days in the evening with LEM25 or matching placebo 30 minutes before 
habitual bedtime, and after 3 hours fasting. This cohort comprised 6 LEM- and 2 placebo-
treated subjects. Blood samples were collected at prespecified timepoints, and PD 
assessments were conducted. 
Overall, 55 subjects were randomized, and all received study drug (placebo, lemborexant 2.5 
mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg). A total of 11 subjects (6 adult, 5 elderly) were 
randomized to LEM25.   
Summary of Adverse Events 
There appeared to be more treatment-related TEAEs with increasing doses of LEM, and the 
overall incidence of TEAEs was higher in LEM (all doses) (92.7%) compared to placebo 
(78.6%). There were no serious TEAEs. In the LEM groups, 1 subject (2.4%) in the LEM25 
group had a TEAE leading to study drug dose adjustment and withdrawal from the study. 

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This subject discontinued study drug due to an AE (pyrexia) that was not considered to be 
related to study drug and was withdrawn from the study (Table 1). 
 

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Table 1 
Overview of Treatment-Emergent Adverse Events – Safety Analysis Set, Study E2006-A001-002 
Category 
Placebo 
(N=14) 
E2006 
2.5mg 
(N=6) 
5mg 
(N=6) 
10mg 
(N=6) 
25mg 
(N=6) 
25mg 
Elderly 
(N=5) 
50mg 
(N=6) 
75mg 
(N=6) 
E2006 
Total 
(N=41) 
TEAEs 
11 (78.6) 5 (83.3) 
5 (83.3) 
6 (100) 
5 (83.3) 
5 (100) 
6 (100) 
6 (100) 
38 (92.7) 
Treatment-related TEAEsa 
6 (42.9) 
3 (50.0) 
3 (50.0) 
4 (66.7) 
4 (66.7) 
5 (100) 
6 (100) 
6 (100) 
31 (75.6) 
Severe TEAEs 
0 
0 
0 
0 
0 
0 
0 
0 
0 
Serious TEAEs 
0 
0 
0 
0 
0 
0 
0 
0 
0 
Deaths 
0 
0 
0 
0 
0 
0 
0 
0 
0 
Other SAEs 
0 
0 
0 
0 
0 
0 
0 
0 
0 
Life Threatening 
0 
0 
0 
0 
0 
0 
0 
0 
0 
Requires inpatient 
hospitalization or prolongation 
of existing hospitalization 
0 
0 
0 
0 
0 
0 
0 
0 
0 
Persistent or significant 
disability or incapacity 
0 
0 
0 
0 
0 
0 
0 
0 
0 
Congenital anomaly / birth 
defect 
0 
0 
0 
0 
0 
0 
0 
0 
0 
Important medical events 
0 
0 
0 
0 
0 
0 
0 
0 
0 
TEAEs leading to study drug 
dose adjustment 
0 
0 
0 
0 
1 (16.7) 
0 
0 
0 
1 (2.4) 
TEAEs leading to study 
treatment withdrawal 
0 
0 
0 
0 
1 (16.7) 
0 
0 
0 
1 (2.4) 

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Table 1 
Overview of Treatment-Emergent Adverse Events – Safety Analysis Set, Study E2006-A001-002 
Category 
Placebo 
(N=14) 
E2006 
2.5mg 
(N=6) 
5mg 
(N=6) 
10mg 
(N=6) 
25mg 
(N=6) 
25mg 
Elderly 
(N=5) 
50mg 
(N=6) 
75mg 
(N=6) 
E2006 
Total 
(N=41) 
Placebo group includes placebo subjects pooled from all cohorts in the study. Percentages are based on the total number of subjects in 
relevant treatment group. 
For each row category, a subject with 2 or more adverse events in that category is counted only once. MedDRA = Medical Dictionary 
for Regulatory Activities; TEAE = treatment-emergent adverse event, SAE = serious adverse event. 
a:  Includes TEAEs considered by the investigator to be possibly or probably related to study drug or TEAEs with missing causality. 
MedDRA Version 16.0. 
Source:  Table 14.3.1.2.  
 

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Serious Adverse Events – Safety Analysis Set 
No TEAEs in any treatment group were severe. The adult LEM groups that reported only 
mild TEAEs were 2.5, 5, 10, and 25 mg.  In the elderly LEM25 group, there was one TEAE 
of somnolence and one TEAE of hypoxia, both of which were rated moderate.  
Overall Safety Conclusions 
LEM was safe and well-tolerated following dosing in adult subjects up to 75 mg per day for 
14 days, and in elderly subjects receiving 25 mg per day for 14 days. There was a higher 
incidence of treatment-related TEAEs among LEM subjects (all doses) (31/41, 75.6%) 
compared to placebo subjects (6/14, 42.9%). The incidence of treatment-related TEAEs 
appeared to be dose-related with higher rates occurring at higher doses, the highest incidence 
(100%) occurring in the LEM25 elderly, LEM50, and LEM75 dose groups. However, most 
TEAEs were mild in severity. No SAEs were reported during the study. 
Study E2006-A001-003 (NCT02039089) 
Study Design 
 
This was a single-center, multiple-dose, randomized, double-blind, placebo-controlled, 
parallel-group study in healthy male and female subjects. The study consisted of 2 parts: Part 
A (3 cohorts of healthy Japanese subjects dosed in the evening) and Part B (1 cohort of 
healthy white subjects dosed in the evening). The cohorts were conducted sequentially. Part 
A was started first with the 2.5-mg dose cohort, followed by the 10-mg dose cohort, and then 
the 25-mg dose cohort. Part B was conducted in parallel with the 10-mg cohort of Part A, 
with overlap. Subjects received treatment with study drug for 14 days. 
 
Summary of Adverse Events 
 
The number of subjects with TEAEs and the incidence of TEAEs were low and similar 
across dose groups. No trends with dose or race were seen. Most TEAEs were judged by the 
investigator to be treatment related (i.e., related or not related as described in the clinical 
study protocol to study drug). All TEAEs were mild. There were no severe TEAEs, serious 
TEAEs, TEAEs leading to withdrawal, or deaths during the study. All TEAEs resolved by 
the end of the study. 
 
Somnolence and abnormal dreams were the most frequently reported treatment-related 
TEAEs, with 4 Japanese subjects (1 each in the placebo and 2.5-mg groups, and 2 in the 25-
mg group) and 1 white subject (10-mg group) reporting somnolence and 1 Japanese subject 
(25-mg group) and 2 white subjects (10-mg group) reporting abnormal dreams (Table 2).  
 

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Table 2 
Summary of Treatment-emergent Adverse Events (Safety Analysis Set, Study E2006-A001-003) 
 
Japanese 
White 
Characteristic 
Placebo 
N = 6 
n (%) E 
2.5 mg 
N = 6 
n (%) E 
10 mg 
N = 6 
n (%) E 
25 mg 
N = 6 
n (%) E 
Overall 
Japanese 
N = 18 
n (%) E 
Placebo 
N = 2 
n (%) E 
10 mg 
N = 6 
n (%) E 
TEAEs 
2 (33.3%) 3 
1 (16.7%) 2 
0 
2 (33.3%) 4 3 (16.7%) 6 
1 (50.0%) 1 
3 (50.0%) 3 
Serious TEAEs 
0 
—0 
0 
0 
0 
0 
0 
Severe TEAEs 
0 
0 
0 
0 
0 
0 
0 
Related TEAEs 
2 (33.3%) 2 
1 (16.7%) 2 
0 
2 (33.3%) 3 3 (16.7%) 5 
0 
3 (50.0%) 3 
TEAEs Leading to 
Withdrawal 
0 
0 
0 
0 
0 
0 
0 
TEAEs Leading to Death 0 
0 
0 
0 
0 
0 
0 

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Overall Safety Conclusions – Study 003 
Multiple ascending doses of LEM were safe and well tolerated in healthy Japanese and white 
subjects in this study. The incidence of TEAEs were low and similar across dose groups, and 
the most common TEAE was somnolence. There were no significant patterns in laboratory 
parameters to suggest a relationship to dose group. No TEAEs were related to laboratory or 
hematology measurements. Urinalysis test results were mostly negative or normal; no 
abnormal test results were considered to be of clinical significance. Vital signs, ECG 
parameters, and high-precision QT analyses were unremarkable, and an effect on the 
placebo-corrected change-from-baseline QTcI (ΔΔQTcI) exceeding 10 msec could be 
excluded at the observed peak plasma level after multiple dosing up to 25 mg/day for 14 
days.  
Study E2006-G000-201 (NCT01995838) 
Study Design 
E2006-G000-201 was a multicenter, multiple dose, randomized, double-blind, placebo-
controlled, parallel-group, Bayesian adaptive, dose-response study in subjects with chronic 
insomnia. Subjects were randomized to 1 of 6 doses of LEM (1 mg, 2.5 mg, 5 mg, 10 mg, 15 
mg, or 25 mg) or placebo according to the randomization scheme. 
The study had 2 phases: Prerandomization and Randomization. The Prerandomization Phase 
lasted up to 21 days and consisted of a Screening Period (Days –21 to –2) and a Baseline 
Period (Day –1). After the Baseline Period, all eligible subjects were randomized in a double-
blind manner to receive LEM or placebo for 15 nights during the Treatment Period (Days 1 
to 15). All subjects then received placebo in a single-blind manner, for 2 nights (Days 16 to 
17) during the Rebound Insomnia Assessment Period (Days 16 to 18). Subjects did not 
receive study drug during the Follow-up Period (Days 19 to 30). 
Summary of Adverse Events 
The overall incidence of treatment-emergent adverse events (TEAEs) was higher in subjects 
who received LEM (all doses except 1 mg) (50.2%), as compared with placebo (37.5%). One 
subject (2.0%) (Subject 10081004) in the LEM25 group had a severe and serious TEAE 
(generalized tonic-clonic seizure; see Appendix for further details), and one subject (1.8%) in 
the placebo group (Subject 10021003) had a serious TEAE (hyperkalemia). Both subjects 
recovered without sequelae. With LEM25, 3 subjects had TEAEs leading to study drug dose 
interruption, and 1 subject had a TEAE leading to study drug dose discontinuation (Table 3). 
There were no study drug dose interruptions or discontinuations due to TEAEs in any of the 
other dose groups. 

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Table 3  
Overview of Treatment-Emergent Adverse Events – Safety Analysis Set, Study E2006-G000-201 
Category 
Placebo 
(N=56) 
n(%) 
Lemborexant 
1 mg 
(N=32) 
n(%) 
2.5 mg 
(N=27) 
n(%) 
5 mg 
(N=38) 
n(%) 
10 mg 
(N=32) 
n(%) 
15 mg 
(N=56) 
n(%) 
25 mg 
(N=50) 
n(%) 
TEAEs 
21 (37.5) 
11 (34.4) 
11 (40.7) 
16 (42.1) 
19 (59.4) 
31 (55.4) 
30 (60.0) 
Treatment-related TEAEsa 
11 (19.6) 
8 (25.0) 
9 (33.3) 
12 (31.6) 
15 (46.9) 
24 (42.9) 
24 (48.0) 
Serious TEAEs 
1 (1.8) 
0 
0 
0 
0 
0 
1 (2.0) 
Deaths 
0 
0 
0 
0 
0 
0 
0 
Other SAEs 
1 (1.8) 
0 
0 
0 
0 
0 
1 (2.0) 
Important medical events 
1 (1.8) 
0 
0 
0 
0 
0 
1 (2.0) 
Severe TEAEs 
0 
0 
0 
0 
0 
0 
1 (2.0) 
TEAEs leading to study drug dose 
adjustment 
0 
0 
0 
0 
0 
0 
4 (8.0) 
TEAEs leading to study drug 
dose withdrawal 
0 
0 
0 
0 
0 
0 
1 (2.0) 
TEAEs leading to study drug 
dose interruption 
0 
0 
0 
0 
0 
0 
3 (6.0) 
TEAEs of Special Interest 
0 
0 
0 
1 (2.6) 
3 (9.4) 
5 (8.9) 
2 (4.0) 
Sleep paralysis 
0 
0 
0 
1 (2.6) 
3 (9.4) 
4 (7.1) 
2 (4.0) 
Cataplexy 
0 
0 
0 
0 
0 
1 (1.8) 
0 
MedDRA = Medical Dictionary for Regulatory Activities, SAE = serious adverse event, TEAE = treatment-emergent adverse event. 
Includes TEAEs considered by the investigator to be possibly or probably related to study drug or TEAEs with missing causality. 
MedDRA Version 16.1. Source:  Table 14.3.1.2 
 
 

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Adverse Events that Resulted in Discontinuation of Study Drug 
There was 1 SAE of seizure leading to discontinuation of study drug (Subject 10081004), 
and the subject was withdrawn from the study (see CIOMS form, attached). 
Adverse Events that Required Study Drug Dose Adjustment or Interruption 
Study drug treatment was interrupted in 3 subjects who received LEM25, as follows: 
Subject 10061004: On Study Day 15, the subject had a mild fever, considered not related to 
study drug by the investigator. This fever lasted less than 1 day. Study drug was interrupted 
on Study Day 15 and resumed on Study Day 16. 
Subject 10111043: A blood sample was drawn on Study Day 3 and reported on Study Day 4 
to have an incidental finding of a low white blood cell count. This TEAE was considered to 
be of mild severity and probably related to LEM by the investigator. Study drug was 
interrupted for 1 day (Study Day 5) because of this TEAE. An unscheduled blood sample 
was taken on Study Day 5 for hematology and results available on Study Day 6 indicated that 
this TEAE had resolved. 
Subject 10191018: On Study Day 3, increases in levels of ALT and AST were observed, 
considered to be of moderate severity and possibly related to LEM by the investigator. Study 
drug was interrupted for 3 nights, then was resumed after AST levels started to decline.  
Overall Safety Conclusions – Study 201 
On the basis of the reported safety data from the Phase 2 study, doses of 1 mg to LEM25 
were considered to be well tolerated. There were no clinically important differences between 
LEM treatment and placebo on blood chemistry, vital signs, weight, or ECG. Rates of AEs 
showed some evidence of dose response in the LEM groups compared to placebo subjects, 
particularly for somnolence. There were no deaths. There were 2 SAEs, 1 each in the placebo 
and LEM25 group.  
During the Phase 3 clinical development program for lemborexant, an independent 
adjudication committee was established to evaluate events potentially concerning for seizures 
and/or cataplexy, and to determine whether these potential events represented true seizure, 
cataplexy, or neither. No events of potential seizure were referred to the adjudication 
committee, although events of potential cataplexy were evaluated. None of the latter was 
adjudicated as cataplexy. 
There have been postmarketing reports of seizure with lemborexant. A review of these 
reports indicates that there is no single report where there is a clear causal relationship of the 
reported event to lemborexant therapy. Thus, evaluation of the overall available data does not 
support an association between lemborexant therapy and an increased risk of seizures.  
 
 

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Summary of All Clinical Studies with Exposure to Lemborexant 25 mg 
LEM25 has been evaluated in 4 additional studies (Table 4 below). No new safety signals at the 25 mg dose were identified through 
these studies, and the most common AEs and TEAEs were consistent with the known safety profile of lemborexant. Therefore, the 
language proposed in the consent form with respect to potential safety risks is consistent with current US prescribing information with 
additional information about rare events. 
Table 4 Clinical Studies with Exposure to LEM25 
Study Number 
NCT # 
Study Design/Population 
No. Subjects 
Exposed to 
LEM25 
E2006-A001-001 
Randomized, Double-blind, Placebo- and Active- controlled, Single Dose Study to Assess 
the Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of E2006 in Healthy 
Subjects and Otherwise Healthy Subjects with Primary Insomnia 
18 
NCT01463098 
E2006-A001-002 
NCT01673451 
 
Double-blind, PBO-controlled safety, tolerability, and PK study of MAD of LEM in healthy 
nonelderly and elderly subjects 
11 
E2006-A001-003 
NCT02039089 
 
Double-blind, PBO-controlled safety, tolerability, PK, and PD MAD study of LEM in 
healthy Japanese and White subjects 
6 
E2006-A001-005 
NCT01673880 
 
Open-label, crossover, bioavailability study of LEM capsule versus tablet formulations in 
healthy subjects 
16 
E2006-A001-102 
NCT03471871 
Double-blind, PBO-controlled, crossover study of respiratory safety of LEM10 and LEM25 
in adult and elderly healthy subjects 
49 
E2006-G000-201 
NCT01995838 
Multicenter, randomized, double-blind, PBO-controlled, parallel group, Bayesian adaptive 
randomization design, dose- response study in subjects with insomnia disorder 
50 
LEM: lemborexant, LEM10: lemborexant 10 mg, LEM25: lemborexant 25 mg, MAD: multiple ascending dose, NCT: National 
Clinical Trial, PBO: placebo, PD: pharmacodynamic, PK: pharmacokineticL 

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18.2 Appendix 2: Schedule of Events 
Visits 
Pre-
Screen 
Visit 1 
Visit 2 
Visit 3 
Visit 4 
Admission 
48-Hour 
Sampling 
8-12 Hour 
Observation 
Discharge 
Informed Consent 
 
X 
 
 
 
 
 
 
Demographics 
 
X 
 
 
 
 
 
 
Medical History 
 
X 
 
 
 
 
 
 
MMSE  
 
X 
 
 
 
 
 
 
C-SSRS  
 
X 
 
 
 
 
 
 
MEQ  
 
X 
 
 
 
 
 
 
PSQIa 
X 
X 
 
 
 
 
 
 
STOP-BANG  
X 
 
 
 
 
 
 
 
Amyloid-Beta Blood Testb 
 
X 
 
 
 
 
 
 
Physical Exam 
 
X 
 
 
 
 
 
X 
Neurological Exam 
 
X 
 
 
 
 
 
 
Concomitant Medication 
 
X 
 
X 
X 
 
 
X 
Vital Signs  
 
X 
X 
 
X 
X 
X 
 
Height 
 
X 
 
 
 
 
 
 
Weight 
 
X 
 
X 
X 
 
 
 
12-Lead ECG 
 
X 
 
 
 
 
 
 
Blood Collection (CBC w Diff 
& Comprehensive Metabolic 
Panel)  
 
X 
 
 
 
 
 
 
Blood Collection (Coagulation 
Panel) 
 
 
 
X 
 
 
 
 
Blood Draw (APOE) 
 
X 
 
 
 
 
 
Attended PSG 
 
 
X 
 
 
 
 
 
Unattended PSG 
 
 
 
 
X 
X 
X 
 
Lumbar Catheter Placement  
 
 
 
 
X 
X 
 
 
Leucine Infusion  
 
 
 
 
X 
 
 
 

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Visits 
Pre-
Screen 
Visit 1 
Visit 2 
Visit 3 
Visit 4 
Admission 
48-Hour 
Sampling 
8-12 Hour 
Observation 
Discharge 
Study Drug Administration (2 
nights) 
 
 
 
 
 
X 
 
 
CSF/Blood Catheter Sampling 
 
 
 
 
 
X 
 
 
PVT  
 
 
 
 
X 
 
X 
 
SSS  
 
 
 
 
X 
 
X 
 
CSF Safety Labs 
 
 
 
 
 
X 
 
 
Assess for AE’s 
 
X 
X 
X 
X 
X 
X 
X 
Blood Patch  
 
 
 
 
 
 
 
PRN 
24-Hour Phone Follow-Up  
 
 
 
 
 
 
 
X 
48-Hour Phone Follow-Up 
 
 
 
 
 
 
 
X 
a May be obtained during phone screen or Visit 1 
b Not required if referred by the SEABIRD study 

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