Clinical trial NCT05439603
Conditions: Major Depressive Disorder
Sponsor: Ancora Bio, Inc. d/b/a EmbarkNeuro, Inc.
Start date: 2022-09-19; Completion date: 2023-10-18
Enrollment: 15 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
A Phase 2 Study of ANC-501 in the treatment of adults with Major Depressive Disorder

Detailed description:
This is a single-arm, open-label Phase 2 study to assess the safety, tolerability, pharmacokinetics (PK), and activity of ANC-501 oral capsules as adjunctive treatment in subjects diagnosed with major depressive disorder (MDD)

Arms: arm 1: 50 mg/day
Interventions: intervention 1: ANC-501
Intervention descriptions: intervention 1: Five 10 mg capsules per day

Protocol documents:
Prot_000.pdf: 
EmbarkNeuro 
Clinical Study Protocol 
ANC-501 
ANC501D0005 (Version 6.0) 
03 March 2023 Version 6.0 
Confidential 
Page 1 of 63 
CLINICAL STUDY PROTOCOL 
Protocol Title: 
A Phase 2 Study of ANC-501 in the Treatment of Adults with 
Major Depressive Disorder 
Protocol Number: 
ANC501D0005 
Study Drug: 
ANC-501 
Brief Title: 
ANC-501 in Adults with Major Depressive Disorder 
Phase of Development: 
2 
Sponsor Name and Address: 
Ancora Bio d/b/a EmbarkNeuro, Inc. 
1111 Broadway 
Suite 1300 
Oakland, CA  95607 
Sponsor Contact: 
Philip Perera, MD, MBA 
Chief Medical Officer 
philip.perera@embarkneuro.com 
Regulatory Agency 
Identifier Number(s): 
IND 119048 
Version Number: 
Date 
Protocol Version 6.0, Amendment 05 
03 March 2023 
Protocol Version 5.0, Amendment 04 
17 January 2023 
Protocol Version 4.0, Amendment 03 
21 November 2022 
Protocol Version 3.0, Amendment 02 
22 August 2022 
Protocol Version 2.0, Amendment 01 
04 May 2022 
Original Protocol 1.0 
09 March 2022 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 (Version 6.0) 
03 March 2023 Version 6.0 
Confidential 
Page 2 of 63 
This clinical study will be conducted in accordance with the International Council for Harmonisation of Technical 
Requirements for Pharmaceuticals for Human Use (ICH), Good Clinical Practice (GCP) Guidelines, and applicable 
regulatory requirements. 
CONFIDENTIALITY STATEMENT 
The confidential information in this document is provided to you as an Investigator or consultant for review by you, 
your staff, and the applicable Institutional Review Board/Independent Ethics Committee. Your acceptance of this 
document constitutes agreement that you will not disclose the information contained herein to others without written 
authorization from the Sponsor. 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 (Version 6.0) 
03 March 2023 Version 6.0 
Confidential 
Page 3 of 63 
PROTOCOL SIGNATURE PAGE 
 
Protocol: ANC501D0005 Version 6.0, Amendment 05 
 
I, the undersigned, have read this protocol and agree that it contains all necessary information 
required to conduct the study. 
 
 
 
 
Stephen J. Kanes, MD, PhD 
Chief Executive Officer 
EmbarkNeuro 
 
Date 
 
DocuSign Envelope ID: 45242E7D-47EB-4423-B6F3-40A97577BF93
07-Mar-2023


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 (Version 6.0) 
03 March 2023 Version 6.0 
Confidential 
Page 4 of 63 
INVESTIGATOR SIGNATURE 
By signing below, I agree that: 
I have read this protocol. I agree that it contains all necessary details for carrying out the study as 
described. I will conduct this study in accordance with the design and specific provisions of this 
protocol. I will not initiate the study until it has received approval from the Institutional Review 
Board/Independent Ethics Committee. I will provide copies of this protocol and access to all 
information furnished by EmbarkNeuro to study personnel under my supervision. I will discuss 
this material with them to ensure they are fully informed about the study drug and study 
procedures. I will inform them that this information is confidential and proprietary to 
EmbarkNeuro and that may not be further disclosed to third parties. I understand that the study 
may be terminated or enrollment suspended at any time by EmbarkNeuro with or without cause, 
or by me if it becomes necessary to protect the best interests of the study patients. 
I agree to conduct this study in accordance with the ethical principles that have their origin in the 
Declaration of Helsinki, the International Council for Harmonisation guidelines for Good 
Clinical Practice, and applicable regional regulations. 
INVESTIGATOR SIGNATURE 
 
 
 
Investigator Signature 
 
Date 
 
 
 
Investigator Printed Name 
 
 
 
 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 (Version 6.0) 
03 March 2023 Version 6.0 
Confidential 
Page 5 of 63 
SUMMARY OF MAJOR PROTOCOL CHANGES 
 
Protocol 
Version 
Summary of change 
Rationale for change 
6.0, A05 
Change inclusion # 8 
Allows less restrictive MADRS cutoff 
score from minimum of 28 to 
minimum of 26  
6.0, A05 
Change inclusion # 9 
Allows screening of subjects with 
fewer weeks on continuous 
antidepressant therapy  
Broadens the types of concomitant 
antidepressants allowed 
6.0, A05 
Change inclusion # 11 
Allows less restrictive MADRS cutoff 
score from minimum of 28 to 
minimum of 26 
6.0, A05 
Added clarification language for the screening period 
Clarifies the restriction around 
medication changes during the 
screening period 
Clarifies the screening period does not 
need to be a full 30 days 
6.0, A05 
Updated exclusion #22 to be consistent with change in 
Inclusion #9  
Clarifies the length of treatment on a 
stable dose prior to day1 
5.0, A04 
Removed exclusion #5 
Conforms with FDA IND advice  
5.0, A04 
Updated exclusion #18 (previously #19) to indicate 
positive alcohol breath test 
New exclusion #19 covers the urine 
drug screen 
5.0, A04 
Added new exclusion #19 to clarify the positive urine 
drug test criteria 
Offers specific guidance to 
investigators and no longer eliminates 
subjects with recreational cannabis use  
5.0, A04 
Updated abbreviation for CTQ 
Reflects the appropriate 28-Item CTQ 
used in this study 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 (Version 6.0) 
03 March 2023 Version 6.0 
Confidential 
Page 6 of 63 
5.0, A04 
Removed the word “cannabis” from footnote ‘g’ in the 
Schedule of Events 
Consistent with exclusion #19 
5.0, A04 
Clarified footnote ‘i' in the Schedule of Events 
Offers greater latitude for blood draw 
times 
4.0, A03 
Updated Sponsor name throughout document from 
Ancora Bio to EmbarkNeuro 
Ancora Bio is now doing business as 
EmbarkNeuro 
4.0, A03 
Clarified the conversion from nmol/L to mcg/L for 12-
hour urine cortisol (>22.7 nmol/L = greater than or equal 
to 8.3 mcg/L). 
 
Clarification  
4.0, A03 
Clarification to that exclusion #16 history of seizures does 
not include isolated febrile seizures of childhood 
Clarification 
4.0, A03 
Clarification that the eye exam will be referred to as an 
ophthalmic exam instead of an ophthalmological exam 
since optometrists are now permitted into the study 
Clarification which conforms to the 
current standard of practice 
4.0, A03 
The addition of an optometrist has been included along 
with an ophthalmologist  
Conforms to the current standard of 
practice 
3.0, A02 
 
LOCS III scale changes from baseline in the LOCS III 
grade of ≥0.5 (Nuclear Opalescence), ≥0.5 (Cortical). ≥0.5 
(Posterior Subcapsular) in either eye or any changes 
considered to be clinically significant (e.g., incidence of 
cataract) will be reported as an adverse event and as an 
event of special interest. 
Response to FDA comments 
3.0, A02 
Clarified C-SSRS exclusion criterion #2 
Clarification to the critical questions 
and the timeframe. 
3.0 A02 
Clarification of trained personnel qualified to administer 
Ophthalmic exam and slit-lamp imaging  
Conforms to the current standard of 
care 
3.0 A02 
Addition of Dosing Diary 
Improves ability to monitor study drug 
accountability and compliance 
2.0, A01 
Updated list of abbreviations 
To reflect the updates deleted and 
added, as applicable 
2.0, A01 
Updated follow-up visits to include Day 112 for all 
subjects 
Response to FDA comments 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 (Version 6.0) 
03 March 2023 Version 6.0 
Confidential 
Page 7 of 63 
2.0, A01 
Updated method of alcohol test at screening 
Clarification 
2.0, A01 
Updated list of scales patients will be completing 
Clarification 
2.0, A01 
Change from AREDS to LOCS III grading scale to detect 
changes in lens opacities 
Response to FDA comments 
2.0, A01 
Added word “emergent” to the C-SSRS endpoint 
Clarification 
2.0, A01 
Updated inclusion #4 to include BCVA and clarify what is 
meant by significant findings 
Clarification 
2.0, A01 
Updated Exclusion #1 to include current MDD episode 
Clarification 
2.0, A01 
Added new Exclusion (now #5) for History of 
implantation of an artificial lens or missing lens in one or 
both eyes 
Improves ability to identify risk of 
lens opacity 
2.0, A01 
Added new Exclusion (now #6) to read as follows 
Based on an ophthalmic examination performed at 
Screening, patient must not have any of the following: 
• 
Nuclear opalescence (NO) with a LOCSIII 
classification > 3.0 in either eye 
• 
Nuclear color (NC) with a LOCSIII 
classification > 3.5 in either eye 
• 
Cortical lens opacities (C) with a LOCSIII 
classification > 2.0 in either eye 
• 
Posterior subcapsular lens opacities (P) with a 
LOCSIII classification > 0.3 in either 
Response to FDA comments 
2.0, A01 
Updated Exclusion #18 to remove benzodiazepines from 
exclusion 
Clarification 
2.0, A01 
Updated Exclusion #19 to clarify method and type drug 
tests at screening 
Clarified breath test or urine drug 
screen 
2.0, A01 
Updated Schedule of Events to reflect other updates in 
this Summary of Changes 
Clarification 
2.0, A01 
Include HAMD-6 and GAD-7 as exploratory endpoints 
Would like to assess patient reported 
changes in depression and anxiety 
symptoms 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 (Version 6.0) 
03 March 2023 Version 6.0 
Confidential 
Page 8 of 63 
2.0, A01 
Included Section 8.4.9 to include Events of Special 
Interest – Lens Opacity 
Response to FDA comments 
2.0, A01 
Removed mention of Local laboratories 
Clarification 
2.0, A01 
Removed use of AiCure platform throughout the 
document 
AiCure no longer being used for drug 
adherence 
2.0, A01 
Administration changes/corrections throughout 
Clarifications/corrections 
 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 (Version 6.0) 
03 March 2023 Version 6.0 
Confidential 
Page 9 of 63 
TABLE OF CONTENTS 
CLINICAL STUDY PROTOCOL ..................................................................................................1 
CONFIDENTIALITY STATEMENT .............................................................................................2 
PROTOCOL SIGNATURE PAGE .................................................................................................3 
INVESTIGATOR SIGNATURE.....................................................................................................4 
SUMMARY OF MAJOR PROTOCOL CHANGES ......................................................................5 
LIST OF ABBREVIATIONS ........................................................................................................13 
1. 
PROTOCOL SUMMARY ..........................................................................................16 
1.1. 
Synopsis ......................................................................................................................16 
1.2. 
Study Schema .............................................................................................................23 
1.3. 
Schedule of Events .....................................................................................................24 
2. 
INTRODUCTION ......................................................................................................28 
2.1. 
Major Depressive Disorder .........................................................................................28 
2.2. 
Mechanism of Action of Vasopressin Receptor 1b (V1b Receptor) ..........................29 
2.3. 
Rationale for Targeting V1b Receptor in Treatment of Depression ...........................29 
2.3.1. 
Evaluation in Humans .................................................................................................30 
2.4. 
Risk/Benefit ................................................................................................................31 
3. 
OBJECTIVES AND ENDPOINTS ............................................................................33 
4. 
STUDY DESIGN .......................................................................................................34 
4.1. 
Overall Design ............................................................................................................34 
4.2. 
Justification for Dose ..................................................................................................36 
4.3. 
End of Study Definition ..............................................................................................36 
5. 
STUDY POPULATION .............................................................................................37 
5.1. 
Inclusion Criteria ........................................................................................................37 
5.2. 
Exclusion Criteria .......................................................................................................38 
5.3. 
Subject Enrollment .....................................................................................................39 
5.4. 
Screen Failures ............................................................................................................40 
6. 
STUDY TREATMENT ..............................................................................................41 
6.1. 
Study Treatment Administered ...................................................................................41 
6.2. 
Handling, Storage, and Accountability .......................................................................41 
6.3. 
Measures to Minimize Bias: Randomization and Blinding ........................................41 
6.4. 
Treatment Compliance ................................................................................................41 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 (Version 6.0) 
03 March 2023 Version 6.0 
Confidential 
Page 10 of 63 
6.5. 
Dose Modifications .....................................................................................................41 
6.6. 
Continued Access to Study Drug after the End of Study ...........................................42 
6.7. 
Treatment of Overdose ...............................................................................................42 
6.8. 
Concomitant Medications and Therapies ...................................................................42 
7. 
DISCONTINUATION OF STUDY TREATMENT AND SUBJECT 
WITHDRAWAL ........................................................................................................43 
7.1. 
Discontinuation of Study Treatment ...........................................................................43 
7.2. 
Subject Withdrawal from Study .................................................................................43 
8. 
STUDY ASSESSMENTS AND PROCEDURES ......................................................44 
8.1. 
General Study Assessments ........................................................................................44 
8.2. 
Efficacy Assessments .................................................................................................44 
8.3. 
Safety Assessments .....................................................................................................45 
8.3.1. 
Laboratory Assessments .............................................................................................46 
8.3.1.1. 
Reporting and Evaluation of Laboratory Test Results ...............................................47 
8.3.1.2. 
Repeat Testing ............................................................................................................47 
8.3.2. 
COVID-19 Pandemic Accommodations ....................................................................48 
8.4. 
Adverse Events, Serious Adverse Events, and Other Safety Reporting .....................48 
8.4.1. 
Definition of Adverse Event .......................................................................................48 
8.4.1.1. 
Events Meeting the Adverse Event Definition ...........................................................49 
8.4.1.2. 
Events NOT Meeting the Adverse Event Definition ..................................................49 
8.4.2. 
Definition of Serious Adverse Event (SAE) ...............................................................50 
8.4.3. 
Time Period for Collecting AE and SAE Information ...............................................51 
8.4.4. 
Recording of Adverse Events and/or Serious Adverse Events ...................................51 
8.4.4.1. 
Terms of Reported Adverse Events ............................................................................51 
8.4.4.2. 
Detection of Adverse Events and Serious Adverse Events ........................................51 
8.4.4.3. 
Severity of Adverse Events ........................................................................................51 
8.4.4.4. 
Causal Relationship with Study Treatment ................................................................51 
8.4.4.5. 
Follow-up of Adverse Events .....................................................................................52 
8.4.5. 
Reporting of Serious Adverse Events (SAE)..............................................................52 
8.4.6. 
Reporting of Deaths ....................................................................................................53 
8.4.7. 
Pregnancy ...................................................................................................................53 
8.4.8. 
Overdose .....................................................................................................................53 
8.4.9. 
Events of Special Interest- Lens Opacity ....................................................................53 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 (Version 6.0) 
03 March 2023 Version 6.0 
Confidential 
Page 11 of 63 
8.5. 
Pharmacokinetics ........................................................................................................53 
8.6. 
Biomarker Evaluations ...............................................................................................54 
9. 
STATISTICAL CONSIDERATIONS .......................................................................55 
9.1. 
Statistical Hypotheses .................................................................................................55 
9.2. 
Sample Size Determination ........................................................................................55 
9.3. 
Analysis Populations ..................................................................................................55 
9.4. 
Statistical Analyses .....................................................................................................55 
9.4.1. 
General Considerations ...............................................................................................55 
9.4.2. 
Analysis of Safety .......................................................................................................55 
9.4.3. 
Analysis of Efficacy ...................................................................................................56 
9.4.4. 
Analysis of Pharmacokinetics .....................................................................................56 
9.5. 
Interim Analysis ..........................................................................................................56 
10. 
ADMINISTRATIVE CONSIDERATIONS ..............................................................57 
10.1. 
Protocol Compliance/Deviations ................................................................................57 
10.2. 
Study Termination ......................................................................................................57 
10.3. 
Case Report Forms .....................................................................................................57 
10.4. 
Source Documents ......................................................................................................57 
10.5. 
Data Protection ...........................................................................................................58 
10.6. 
Recordkeeping ............................................................................................................58 
10.7. 
Quality Control and Quality Assurance ......................................................................58 
10.8. 
Financial Disclosure ...................................................................................................58 
11. 
ETHICS ......................................................................................................................60 
11.1. 
Ethical Conduct of the Study ......................................................................................60 
11.2. 
Written Informed Consent ..........................................................................................60 
11.3. 
Institutional Review Board/Independent Ethics Committee ......................................60 
12. 
PUBLICATION POLICY ..........................................................................................61 
13. 
REFERENCES ...........................................................................................................62 
 
LIST OF TABLES 
Table 1: 
General Study Assessments ........................................................................................44 
Table 2: 
Efficacy Assessments .................................................................................................45 
Table 3: 
Safety Assessments .....................................................................................................45 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 (Version 6.0) 
03 March 2023 Version 6.0 
Confidential 
Page 12 of 63 
Table 4: 
Laboratory Assessments .............................................................................................46 
Table 5: 
Definitions of Analysis Populations ...........................................................................55 
 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 (Version 6.0) 
03 March 2023 Version 6.0 
Confidential 
Page 13 of 63 
LIST OF ABBREVIATIONS 
Abbreviation 
Term 
ACTH 
adrenocorticotropic hormone 
ADT 
antidepressant therapy 
AE 
adverse event 
ALT 
alanine aminotransferase 
aPTT 
activated partial thromboplastin time 
AST 
aspartate aminotransferase 
AUC 
area under the concentration-time curve 
AVP 
arginine vasopressin 
BCVA 
best corrected visual acuity 
BMI 
body mass index 
BUN 
blood urea nitrogen 
CGI-I 
Clinical Global Impression-Improvement 
CGI-S 
Clinical Global Impression-Severity 
CLr 
renal clearance 
Cmax  
maximum plasma concentration 
CONSORT 
Consolidated Standards of Reporting Trials 
COVID-19 
coronavirus disease 2019 
CRF 
corticotropin-releasing factor 
CRH 
corticotropin-releasing hormone 
CRO 
clinical research organization 
C-SSRS 
Columbia-Suicide Severity Rating Scale 
CTQ 
Childhood Trauma Questionnaire 
CYP 
cytochrome P450 
DSM-IV 
Diagnostic and Statistical Manual of Mental Disorders, fourth edition 
ECG 
electrocardiogram 
eCRF 
electronic case report form 
ECT 
electroconvulsive therapy 
EMA 
Ecological Momentary Assessment 
ENT 
ears, nose, and throat 
ET 
early termination 
FDA 
Food and Drug Administration 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 (Version 6.0) 
03 March 2023 Version 6.0 
Confidential 
Page 14 of 63 
Abbreviation 
Term 
GAD-7 
General Anxiety Disorder (7-item) 
GCP 
Good Clinical Practice 
HAM-A 
Hamilton Anxiety Scale 
HAMD-6 
Hamilton Depression Rating Scale (6-item) 
HBsAg 
hepatitis B surface antigen 
HCV Ab 
hepatitis C virus antibody 
HIPAA 
Health Insurance Portability and Accountability Act 
HIV 
human immunodeficiency virus 
HPA 
hypothalamus-pituitary-adrenal 
IB 
Investigator’s Brochure 
ICF 
informed consent form 
ICH 
International Council for Harmonisation 
IND 
Investigational New Drug 
INR 
International Normalized Ratio 
IRB 
Institutional Review Board 
IRT 
Interactive Response Technology 
LOCS III 
Lens Opacities Classification System III 
MAD 
multiple ascending dose 
MADRS 
Montgomery-Åsberg Depression Rating Scale 
MDD 
major depressive disorder 
MedDRA 
Medical Dictionary for Regulatory Activities 
MGH 
Massachusetts General Hospital 
MGH ATRQ 
Massachusetts General Hospital Antidepressant Treatment Response 
Questionnaire 
mRNA 
messenger RNA 
NO 
Nuclear Opalescence 
PET 
positron emission tomography 
PGI-I 
Patient Global Impression of Improvement 
PK 
pharmacokinetic(s) 
PT 
Preferred Term; prothrombin time 
RBC 
Red blood cell(s) 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 (Version 6.0) 
03 March 2023 Version 6.0 
Confidential 
Page 15 of 63 
Abbreviation 
Term 
rTMS 
repetitive transcranial magnetic stimulation 
SAD 
single ascending dose 
SAE 
serious adverse event 
SAFER 
State vs trait, Assessability, Face and Ecological validity, Rule of 3Ps [pervasive, 
persistent, and pathological] 
SAP 
Statistical Analysis Plan 
SCID-5-CT 
Structured Clinical Interview for DSM-5 – Clinical Trials Version 
SF-36 
Short Form-Health Survey (36-item) 
SNRI 
serotonin norepinephrine reuptake inhibitor 
SOC 
System Organ Class 
SSRI 
selective serotonin reuptake inhibitor 
STAR*D 
Sequenced Treatment Alternatives to Relieve Depression 
t½ 
apparent half-life 
TRD 
treatment-resistant depression 
US 
United States 
V1b 
vasopressin receptor 1b 
WBC 
white blood cell(s) 
 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 (Version 6.0) 
03 March 2023 Version 6.0 
Confidential 
Page 16 of 63 
1. 
PROTOCOL SUMMARY 
1.1. 
Synopsis 
Sponsor: 
EmbarkNeuro 
Study Title: 
A Phase 2 Study of ANC-501 in the Treatment of Adults with Major Depressive Disorder 
Protocol Number: 
ANC501D0005 
Phase of Development: 
2 
Investigational 
Product: 
ANC-501 Capsules 
Study Design and 
Duration: 
This is a single-arm, open-label Phase 2 study to assess the safety, tolerability, 
pharmacokinetics (PK), and efficacy of ANC-501 oral capsules as adjunctive treatment in 
subjects diagnosed with major depressive disorder (MDD).  
With the exception of subjects permitted to use current stable antidepressant treatment, 
initiation of psychotropic medications and other medications that may potentially have an 
impact on efficacy or safety endpoints will not be allowed between screening and 
completion of the Day 56 assessments. Psychotropic medications, which must have been 
initiated at least 8 weeks prior to screening, must remain at a stable dose until completion 
of the Day 56 assessments. During the study, efficacy and safety assessments will be 
performed and blood samples collected for PK analysis of ANC-501and future exploratory 
biomarker and genetic analysis. A wearable watch-like device will collect data on 
biometrics, which may be used for exploratory analyses. 
Subjects will receive 50 mg ANC-501 Capsules (5 × 10 mg capsules) orally once daily at 
least 1 hour prior to the morning meal for 8 weeks beginning with the first dose 
administered in the clinic on Day 1 and will return to the clinic for scheduled visits on 
Days 8, 15, 29, 43, and 56. Follow-up visits will occur on Day 70 and 112. All visits after 
Day 1 allow a visit window of ±3 days. 
Total duration of participation is 20 weeks (up to 30 days screening, 8 weeks dosing, 8 
weeks follow-up). 
The following is an overview of study procedures/assessments (refer to the Schedule of 
Events [Section 1.3] for specific procedures/assessments and their timing, with further 
details located in Section 8): 
Screening Period (Day -30 to Day -1) 
After providing informed consent, eligibility assessments will include review of 
demographics, medical history, prior/concomitant medications, full physical examination, 
vital sign assessments, body weight/height/body mass index (BMI) measurements, urine 
pregnancy test (for female subjects of childbearing potential), urine drug and alcohol breath 
test screen, safety laboratory tests (i.e., hematology, serum chemistry, coagulation, and 
urinalysis), hepatitis and human immunodeficiency virus (HIV) screen, and plasma, urine 
and saliva cortisol collection. 
The Structured Clinical Interview for DSM-5 – Clinical Trials Version (SCID-5-CT) will 
be employed for the determination of MDD and will be performed by a qualified, trained, 
validated healthcare professional. 
Qualified staff will also complete the Montgomery-Åsberg Depression Rating Scale 
(MADRS), Hamilton Anxiety Rating Scale (HAM-A), Clinical Global Impression-Severity 
(CGI-S), Columbia-Suicide Severity Rating Scale (C-SSRS), and Childhood Trauma 
Questionnaire (CTQ). 
Ophthalmic exams, including slit-lamp imaging, may be performed by an appropriately 
trained and qualified individual as delegated by the PI and documented in the Study 
Delegation Log. However, a licensed ophthalmologist or optometrist must oversee this 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 (Version 6.0) 
03 March 2023 Version 6.0 
Confidential 
Page 17 of 63 
work and will make the determination on subject eligibility as per inclusion criteria #4.  Slit 
lamp imaging of the lens of both eyes will be taken and sent to a central reader for 
assessment using the LOCS III classification. 
 
Study site personnel will dispense equipment for 12-hour urine collection to measure 
cortisol levels. 
A call between the subject and the qualified central rater will be scheduled to allow the 
rater to administer the Massachusetts General Hospital Antidepressant Treatment Response 
Questionnaire (MGH ATRQ) for assessment of current antidepressant therapy (ADT), 
adequacy of duration and dose of prior and current ADT, as well as degree of 
improvement, to ensure only patients with inadequate response (<50% improvement) are 
included. The central rater will also complete the MADRS assessment and SAFER 
inventory (State vs trait, Assessability, Face and Ecological validity, Rule of 3Ps 
[pervasive, persistent, and pathological]) for confirmation of eligibility (diagnosis, severity, 
and treatment history). Results will be communicated to the site prior to Day 1. 
No changes to medications are allowed during the screening period, and screening can be 
less than 30 days if all other criteria are met. 
 
Treatment Period (Days 1, 8, 15, 29, 43, 56) 
At the Day 1 visit only, subjects confirmed as eligible will receive a 50 mg dose of study 
drug prior to eating. Subjects who cannot tolerate 50 mg will receive 40 mg for the 
remainder of the study. Subjects who cannot tolerate 40 mg will be terminated from the 
study. Before releasing subjects, study site personnel will dispense and train subjects on a 
dosing diary, the wearable watch-like device and mobile application. ANC-501 will be 
dispensed to subjects with instructions to take the medication every morning at least 1 hour 
prior to eating breakfast. 
During visits, safety procedures/assessments and administration of assessments/scales 
(including from Day 8 onward, the Clinical Global Impression-Improvement [CGI-I] and 
the Patient Global Impression of Improvement [PGI-I]) will be conducted. During specific 
visits, PK blood samples will be collected, as well as blood samples for future exploratory 
biomarker and genetic analysis. Concomitant medications and dosing diary will be 
reviewed, the dosing diary will be returned to the subject, and adverse events (AEs) 
recorded. At all visits after Day 1, recording/accounting for any returned/unused ANC-501 
will be performed and ANC-501 will be dispensed to subjects with a reminder to take the 
medication every morning at least 1 hour prior to eating breakfast. 
At home activities 
Subjects will take study medication every morning (at least 1 hour prior to eating 
breakfast), make entries into the dosing diary, and wear their watch-like device 
throughout the day and night except when charging. Subjects will also complete 
EMA orienting questions, the Hamilton Depression Rating Scale (6-item) 
(HAMD-6) and the General Anxiety Disorder (7-item) (GAD-7) twice a day for 
three days per week, and the Short Form-Health Survey (36-item) (SF-36) weekly 
using the EMA Wellness smartphone/tablet application. 
At the Day 43 visit, study site personnel will dispense equipment and retrain subjects on 
the 12-hour urine collection to measure cortisol levels (the collection will be returned to the 
site at the Day 56 visit). On Day 54, the site will contact the subject to remind them to 
begin collection on Day 55. 
At the Day 56 visit, subjects will take their last study medication and visit the site to return 
their 12-hour urine collection, all remaining medication, the dosing diary, and wearable 
watch-like devices. Study procedures/assessments will be conducted, including an 
ophthalmic exam and slit lamp imaging of the lenses of both eyes. Images will be sent to a 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 (Version 6.0) 
03 March 2023 Version 6.0 
Confidential 
Page 18 of 63 
central reader for assessment using the LOCS III. Collection of plasma and saliva cortisol 
samples will also be conducted. 
The same rater should perform the CGI-S, CGI-I, and MADRS assessments throughout the 
study on a given subject when possible. 
 
Follow-up Period (Day 70 and Day 112) 
Subjects will return to the site for a follow-up visit on Day 70 and Day 112. Study 
procedures/assessments will be conducted, including an ophthalmic exam and slit lamp 
imaging of the lenses of both eyes. Images will be sent to a central reader for assessment 
using the LOCS III. Concomitant medications will be reviewed, and AEs recorded. 
Objectives and 
Endpoints: 
 
Objectives 
Endpoints 
Primary 
• To evaluate the effect of 
treatment with ANC-501 
capsules on depressive 
symptoms in subjects with 
major depressive disorder 
(MDD) 
• Change from baseline (Day 1) to Day 56 in 
Montgomery-Åsberg Depression Rating Scale 
(MADRS) total score 
• To evaluate the safety and 
tolerability of ANC-501 
capsules in subjects with 
MDD 
• Safety and tolerability as assessed by: 
o Frequency and severity of adverse events 
(AEs) 
o Vital signs 
o Body weight 
o Electrocardiograms (ECGs) 
o Clinical laboratory tests 
o Physical examination 
o Lens Opacities Classification System III 
(LOCS III) 
o Emergent behavior using Columbia-Suicide 
Severity Rating Scale (C-SSRS) 
Secondary 
• To evaluate effect of 
ANC-501 at all time points 
and percent reduction in total 
symptom scores compared 
with baseline. 
• Change from baseline (Day 1) in the MADRS 
total score at all time points 
• Percentage of MADRS responders (≥50% 
reduction in total score) 
• Percentage of MADRS remitters (total 
score ≤10) 
• Change in Hamilton Anxiety Scale (HAM-A) 
total score from Baseline to Day 56 
• Change in Clinical Global Impression-Severity 
(CGI-S) score from Baseline to Day 56 
• Short Form-Health Survey (36-item) (SF-36) 
total score and subscale scores from Baseline to 
Day 56 and at all time points 
• Percentage of Clinical Global Impression-
Improvement (CGI-I) improvers (“Very much 
improved” or “Much improved”) 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 (Version 6.0) 
03 March 2023 Version 6.0 
Confidential 
Page 19 of 63 
• Patient Global Impression of Improvement 
(PGI-I) score at Day 56 
• To assess the PK profile of 
ANC-501 Capsules in 
plasma using a sparse 
sampling model 
• Plasma concentrations of ANC-501 
 
 
 
Objectives 
Endpoints 
Exploratory 
• To evaluate changes in 
12-hour urine, plasma, and 
saliva cortisol 
• Changes in 12-hour urine, plasma, and saliva 
cortisol from baseline to Day 56 
• To evaluate changes in 
biometrics 
• Changes in biometrics using the wearable 
watch-like device 
• To evaluate biological 
samples as potential 
biomarkers and metabolites 
• Patient reported changes in 
Depression and Anxiety 
symptoms 
• Baseline levels and/or changes in exploratory 
biomarkers through Day 56 
• Changes in Hamilton Depression Rating Scale 
(6-item) (HAMD-6) scores from baseline across 
all time points using ePRO. 
• Changes in General Anxiety Disorder (7-item) 
(GAD-7) from baseline across all time points 
using ePRO. 
 
 
Population: 
Inclusion Criteria 
Patients who meet ALL the following inclusion criteria will be eligible to participate in the 
study: 
1. Demonstrate understanding of the study procedures, restrictions, and willingness to 
participate as evidenced by voluntarily signing and dating the written informed 
consent form. 
2. Adult male or female between 18 and 65 years of age, inclusive. 
3. Good physical health and has no clinically significant findings, as determined by the 
Investigator, on physical examination, 12-lead electrocardiogram (ECG), vitals, 
or clinical laboratory tests. 
4. Has no clinically significant findings on the ophthalmic examination including, Best 
Corrected Visual Acuity (BCVA) worse than 20/30 or, in the opinion of the 
ophthalmologist or optometrist, any cataract that may become clinically significant 
and/or need surgical intervention during the course of the trial. 
5. Able and willing to complete all study requirements. 
6. Diagnosis of current episode of major depressive disorder (MDD) at least 8 weeks 
prior to screening, confirmed by Structured Clinical Interview for DSM-5 – Clinical 
Trials Version (SCID-5-CT). 
7. Have not responded to their current antidepressant therapy or to dose 
adjustment/treatment changes following a loss of response to their current 
antidepressant therapy. 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 (Version 6.0) 
03 March 2023 Version 6.0 
Confidential 
Page 20 of 63 
8. Complete the SAFER (State vs trait, Assessability, Face and Ecological validity, 
Rule of 3Ps [pervasive, persistent, and pathological]) interview, administered via 
teleconference by a Massachusetts General Hospital (MGH) psychiatrist or 
psychologist, and receive a passing score which confirms a diagnosis of MDD, and 
a minimum Montgomery-Åsberg Depression Rating Scale (MADRS) total score of 
26. The SAFER interview will also confirm the subject’s treatment history and 
response to antidepressant therapies (ADTs) through the administration of the MGH 
Antidepressant Treatment Response Questionnaire (ATRQ). 
9. Receiving a stable dose of the same antidepressant (selective serotonin reuptake 
inhibitor [SSRI], serotonin and norepinephrine reuptake inhibitor [SNRI], 
bupropion or trazodone monotherapy) for the current episode for at least 6 weeks of 
continuous treatment, which can include some or all of the screening period, with 4 
weeks on a stable dose prior to day 1 and has an inadequate response (<50% 
improvement) using the MGH ATRQ. 
10. Willing to maintain stable dose and delay start of other antidepressant or antianxiety 
medications and any new pharmacotherapy regimens, including benzodiazepine 
anxiolytics, during the screening and treatment periods. 
11. Has a MADRS total score of ≥26 at screening and Day 1 (prior to dosing). 
12. Has a 12-hour urine cortisol level >22.7 nmol/L (greater than or equal to 8.3 
mcg/L). 
13. Body mass index (BMI) ≥18 and ≤38 kg/m2 at screening using the formula: 
𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤ℎ𝑡𝑡 (𝑘𝑘𝑘𝑘) ℎ𝑒𝑒𝑒𝑒𝑒𝑒ℎ𝑡𝑡 (𝑚𝑚)2
⁄
. 
14. Agrees to practice an acceptable method of highly effective birth control at 
screening, throughout study participation, and for 90 days after the last dose of 
study drug. Highly effective methods of birth control include sexual abstinence 
(for males and females); vasectomy; or a condom with spermicide in combination 
with a highly effective female partner’s method (for males); and hormonal methods 
of contraception (i.e., established use of oral, implantable, injectable, or transdermal 
hormones); placement of an intrauterine device; placement of an intrauterine 
system; and mechanical/barrier method of contraception (i.e., condom or occlusive 
cap [diaphragm or cervical/vault cap] in conjunction with spermicide [foam, gel, 
film, cream, or suppository]) (for females). 
Exclusion Criteria 
Patients who meet any of the following exclusion criteria will be excluded from 
participation in the study: 
1. History of suicide attempt in the past 2 years or within current MDD episode 
2. Active suicidal ideation with plan or a “yes” response to items 4 or 5 on the C-SSRS 
in the past 24 months is exclusionary as assessed at Screening and Baseline (Day 1).  
3. Inadequate response to >2 prior ADTs (not including current antidepressant) of at 
least 6 weeks duration each for the episode current at screening. 
4. Recent history or active clinically significant manifestations of metabolic, hepatic, 
renal, hematological, pulmonary, cardiovascular, gastrointestinal, musculoskeletal, 
dermatological, urogenital, neurological, ophthalmic (especially cataracts), or ears, 
nose, and throat (ENT) disorders, or any other acute or chronic condition that, in the 
Investigator's opinion, would limit the subject's ability to complete or participate in 
this clinical study. 
5. Based on an ophthalmic examination performed at Screening, patient must not have 
any of the following: 
• 
Nuclear opalescence (NO) with a LOCSIII classification > 3.0 in either eye 
• 
Nuclear color (NC) with a LOCSIII classification > 3.5 in either eye 
• 
Cortical lens opacities (C) with a LOCSIII classification > 2.0 in either eye 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 (Version 6.0) 
03 March 2023 Version 6.0 
Confidential 
Page 21 of 63 
• 
Posterior subcapsular lens opacities (P) with a LOCSIII classification > 0.3 in 
either 
6. History or presence of intellectual disability, pervasive developmental disorder, 
cognitive disorder, neurodegenerative disorder (e.g., Alzheimer’s disease, 
Parkinson’s disease, multiple sclerosis, Huntington’s disease), or brain injury. 
7. Receipt of electroconvulsive therapy (ECT) within 12 months of screening, 
lifetime receipt of more than one course of ECT, or plans to receive ECT during 
the study. 
8. Receipt of repetitive transcranial magnetic stimulation (rTMS) within 12 months 
of screening or planned to receive rTMS during the study. 
9. Plans to initiate or terminate cognitive or behavioral psychotherapy or alter the 
frequency of ongoing therapy during this study. 
10. Works night shifts or needs to work night shifts during the study. 
11. Females who are pregnant, intend to become pregnant (within 90 days of the last 
dose of ANC-501), are breastfeeding, or have a positive pregnancy test at 
screening or on Day 1 prior to study drug administration. 
12. Known allergy to ANC-501, or related compounds. 
13. Detectable hepatitis B surface antigen (HBsAg), hepatitis C virus antibody 
(HCV Ab), or human immunodeficiency virus (HIV) antibody at screening. 
14. Active psychosis per Investigator assessment. 
15. Medical history of seizures (does not include isolated febrile seizures of 
childhood). 
16. Medical history of bipolar disorder, schizophrenia, and/or schizoaffective 
disorder. 
17. History of alcohol or substance use disorder in the 12 months prior to screening. 
18. Positive alcohol breath test. 
19. Positive urine drug screen for hallucinogens, psychoactive inhalants, stimulants, 
opioids, barbiturates, non-prescribed anxiolytics or other non-prescribed sedatives. 
Does not include positive test for cannabinoids, tobacco or caffeine, unless the 
subject meets criteria for substance use disorder for these substances. 
20. Exposure to another investigational medication or device within 30 days or 5 half-
lives (whichever is longer) prior to screening. 
21. Has been previously treated in this study or randomized or treated in any other 
study employing ANC-501 (i.e., subject may not have received study drug and 
then re-enrolled). 
22. Administration of drugs to treat  psychiatric or neurologic conditions that have not 
been taken at a stable dose for at least 4 weeks prior to day 1. 
23. Need/plan to take moderate to strong inhibitors and/or inducers of 
cytochrome P450 (CYP)3A4 (food, beverages, medications, and 
supplements). 
24. Any condition not identified in the protocol that in the opinion of the 
Investigator would confound the evaluation and interpretation of the study 
data or may put the subject at risk. 
Dosage Forms 
and Route of 
Administration: 
50 mg ANC-501 Capsules (5 × 10 mg capsules), administered orally. 
Dose Adjustment 
for Safety or 
Tolerability 
Reasons: 
During the Open-Label Treatment Period, subjects may receive study drug as long as there 
are no dose-limiting safety/tolerability concerns as determined by the Investigator. Subjects 
who experience moderate or severe AEs while receiving the 50 mg dose of study drug 
which, according to the clinical judgement of the Investigator, are related to study drug, 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 (Version 6.0) 
03 March 2023 Version 6.0 
Confidential 
Page 22 of 63 
may receive 40 mg for the remainder of the Open-Label Treatment Period if the AEs are 
intolerable. Subjects who experience moderate or severe related AEs while receiving the 
40 mg dose of study drug might not be able to continue receiving study drug, based on the 
evaluation and clinical judgment of the Investigator, and may be discontinued from further 
treatment. 
Statistical 
Methods: 
General 
Continuous endpoints will be summarized with n, mean, standard deviation, median, 
minimum, and maximum. In addition, change from baseline values will be calculated at 
each time point and summarized descriptively. For categorical endpoints, descriptive 
summaries will include counts and percentages. 
 
Analysis Populations and Methods 
The Safety Population (defined as all subjects administered study drug) will be used to 
provide descriptive summaries of safety data. Treatment-emergent adverse events (TEAEs) 
will be classified by type, incidence, severity, and causality. The overall incidence of AEs 
will be summarized by Medical Dictionary for Regulatory Activities (MedDRA) System 
Organ Class (SOC) and Preferred Term (PT). Data for vital signs, clinical laboratory tests, 
ECG, physical examinations, and concomitant medication usage will also be summarized. 
Out-of-range safety endpoints may be categorized as low or high, where applicable. 
 
Suicidality data collected using the C-SSRS at baseline and at each visit during the active 
Open-Label Treatment Period will be listed for all subjects. The C-SSRS listings will 
include behavior type and/or category for suicidal ideation and suicidal behavior of the 
C-SSRS. 
 
Slit lamp imaging performed during the study showing a change of greater than or equal to 
0.5 on the LOCS III scale in either eye or any new incidence of cataract will be considered 
adverse events and events of special interest. 
 
The Efficacy Population (defined as all subjects in the Safety Population who complete at 
least one day of dosing of study drug and have at least one post-baseline efficacy 
evaluation) will be used to analyze efficacy data. Efficacy data will be analyzed using 
appropriate descriptive statistics and pre-specified statistical methods, as well as other data 
presentation methods where applicable; subject listings will be provided for all efficacy 
data. Efficacy data will be summarized descriptively. 
Sample Size 
Determination: 
Up to 20 subjects will be enrolled. The sample size was selected based on clinical and not 
statistical considerations. 
 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 (Version 6.0) 
03 March 2023 Version 6.0 
Confidential 
 
Page 23 of 63 
1.2. 
Study Schema 
 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
 
Page 24 of 63 
1.3. 
Schedule of Events 
Study Procedure 
Screening 
Period 
Open-Label Treatment Period a 
Follow-up Period b 
Visit Days 
Window 
-30 to -1 
Day 1 
c 
Day 8 
(±3 
days) 
Day 15 
(±3 
days) 
Day 29 
(±3 
days) 
Day 43 
(±3 
days) 
Day 
56/ET 
(±3 days) 
Day 70 
(±3 days) 
Day 112 
(±3 days) 
Informed consent 
X 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
 
 
Full physical examination 
X 
 
 
 
 
 
X 
 
 
Body weight/height/BMI d 
X 
X 
 
 
 
 
 
 
 
Vitals e 
X 
X 
X 
 
 
 
X 
 
 
12-lead ECG 
 
X 
X 
 
 
 
X 
 
 
Safety laboratory tests f 
X 
X 
X 
 
 
 
X 
X 
 
Urine drug and alcohol breath test 
screen g 
X 
X 
 
 
 
 
 
 
 
Urine pregnancy test h 
X 
X 
 
 
 
 
 
 
 
Hepatitis and HIV serology  
X 
 
 
 
 
 
 
 
 
Sparse PK blood sampling i 
 
X 
X 
X 
X 
X 
X 
 
 
Exploratory biomarker sampling j 
 
X 
X 
 
 
 
X 
 
 
Genetic sampling k 
 
X 
 
 
 
 
 
 
 
Ophthalmic exam and LOCS III l 
X 
 
 
 
 
 
X 
X 
X 
SCID-5-CT diagnostic interview 
X 
 
 
 
 
 
 
 
 
MADRS 
X 
X 
X 
X 
X 
X 
X 
X 
 
HAM-A 
X 
X 
X 
X 
X 
X 
X 
X 
 
CGI-S 
X 
X 
X 
X 
X 
X 
X 
X 
 
C-SSRS m 
X 
X 
X 
X 
X 
X 
X 
X 
 
HAMD-6 n 
 
 
 
 
GAD-7 n 
 
 
 
 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
 
Page 25 of 63 
Study Procedure 
Screening 
Period 
Open-Label Treatment Period a 
Follow-up Period b 
Visit Days 
Window 
-30 to -1 
Day 1 
c 
Day 8 
(±3 
days) 
Day 15 
(±3 
days) 
Day 29 
(±3 
days) 
Day 43 
(±3 
days) 
Day 
56/ET 
(±3 days) 
Day 70 
(±3 days) 
Day 112 
(±3 days) 
SF-36 n 
 
 
 
 
EMAW Orienting Questions n 
 
 
 
 
CTQ 
X 
 
 
 
 
 
 
 
 
CGI-I 
 
 
X 
X 
X 
X 
X 
X 
 
PGI-I 
 
 
X 
X 
X 
X 
X 
X 
 
SAFER Interview (central rater) o 
X 
 
 
 
 
 
 
 
 
Inclusion/Exclusion Criteria 
Review 
X 
X 
 
 
 
 
 
 
 
12-hour urine collection for 
cortisol instructions p 
X 
 
 
 
 
 
X 
 
 
Plasma and saliva cortisol sample 
collection 
X 
 
 
 
 
 
X 
 
 
Wearable monitoring instructions 
and Mobile application training 
 
X 
 
 
 
 
 
 
 
Continuous Wearable monitoring q 
 
 
 
 
Site download of Wearable data 
 
 
X 
X 
X 
X 
X 
 
 
ANC-501 dosing r 
 
 
 
 
ANC-501 Dosing Diary dispensed 
and reviewed by site 
 
 
 
 
Dispense study medication 
 
X 
X 
X 
X 
X 
 
 
 
Adverse Events s 
X 
 
Prior/Concomitant Medications t 
X 
 
Abbreviations: AE = Adverse Event; BMI = body mass index; CGI-I = Clinical Global Impression-Improvement; CGI-S = Clinical Global Impression-Severity; 
C-SSRS = Columbia-Suicide Severity Rating Scale; CTQ = Childhood Trauma Questionnaire, 28-item; ECG = electrocardiogram; ET = early termination; HAM-A = Hamilton 
Anxiety Rating Scale; HIV = human immunodeficiency virus; LOCS III= Lens Opacities Classification System III; MADRS = Montgomery-Åsberg Depression Rating Scale; 
MDD = major depressive disorder; MGH ATRQ = Massachusetts General Hospital Antidepressant Treatment Response Questionnaire; PGI-I = Patient Global Impression of 
Improvement; PK = pharmacokinetic; SAE = serious adverse event; SAFER = State vs trait, Assessability, Face and Ecological validity, Rule of 3Ps (pervasive, persistent, and 
pathological); SCID-5-CT = Structured Clinical Interview for DSM-5 – Clinical Trials Version; HAMD-6= Hamilton Depression Rating Scale (6-item); GAD-7= General 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
 
Page 26 of 63 
Study Procedure 
Screening 
Period 
Open-Label Treatment Period a 
Follow-up Period b 
Visit Days 
Window 
-30 to -1 
Day 1 
c 
Day 8 
(±3 
days) 
Day 15 
(±3 
days) 
Day 29 
(±3 
days) 
Day 43 
(±3 
days) 
Day 
56/ET 
(±3 days) 
Day 70 
(±3 days) 
Day 112 
(±3 days) 
Anxiety Disorder (7-item); SF-36 = Short Form-Health Survey (36-item). 
 
Schedule of Events (continued)  
Refer to Section 8 for additional details regarding assessments/procedures. 
a. 
Subjects who discontinue study treatment before completing the study and those who prematurely withdraw from the study for any reason should undergo 
Day 56 visit procedures in-clinic as soon as possible and schedule the Day 70 safety assessments (at +2 weeks post ET visit) and Day 112 follow-up 
ophthalmic visit (at +8 weeks post ET visit). 
b. Subjects will return to the site on Day 70 and 112 for a follow-up visit. 
c. 
Day 1 procedures are to be completed prior to dosing, except for sparse PK sampling which will be performed prior to dosing and between 0.75 and 2 hours 
post-dose. 
d. Body weight and height will be measured at screening and BMI will be calculated. Only body weight and BMI calculation will be performed at subsequent 
visits. 
e. 
Vital signs include oral temperature (°C), respiratory rate, heart rate, and blood pressure (supine and standing). 
f. 
Safety laboratory tests will include hematology, serum chemistry, coagulation, and urinalysis. Refer to Table 4 for details. 
g. Urine toxicology for selected drugs of abuse (barbiturates, amphetamines, cocaine, opioids,) and breath test for alcohol. 
h. Urine pregnancy test will be conducted only in female subjects of childbearing potential. Female subjects who prematurely discontinue will have a 
pregnancy test performed at the ET visit. 
i. 
A blood sample for sparce PK sampling will be collected pre-dose and between 0.75 and 2 hours post-dose at the Day 1 visit and between 0.75 and 2 hours 
post-dose, whenever possible, at other indicated visits (with the date and time of the previous dose provided by the subject). 
j. 
A blood sample for exploratory biomarker analysis will be collected in conjunction with PK sampling on Days 1 (pre- post-dose), 8, and 56. 
k. A genetic sample (blood) for exploratory biomarker testing will be collected before the first dose of drug. 
l. 
Ophthalmic exam, including slit-lamp imaging, may be performed by an appropriately trained and qualified individual as delegated by the PI and 
documented in the Study Delegation Log. However, a licensed ophthalmologist or optometrist must oversee this work and will make the determination on 
subject eligibility as per inclusion criteria #4.  Slit lamp imaging of the lens of both eyes will be taken and sent to a central reader for assessment using the 
LOCS III. Ideally the exam would be performed after cortisol results are known and the subject is otherwise deemed eligible to continue with screening 
assessments. Additional slit lamp imaging of the lens of both eyes may be performed during the treatment period and sent to a central reader for assessment 
using the LOCS III, per Investigator’s discretion. 
m. The “Baseline/Screening” C-SSRS form will be completed during screening. The “Since Last Visit” C-SSRS form will be completed at any time of day at 
all subsequent time points. 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
 
Page 27 of 63 
n. The Short Form-Health Survey (36-item) (SF-36) weekly.  The Hamilton Depression Rating Scale (6-item) (HAMD-6) and the General Anxiety Disorder (7-
item) (GAD-7) will be completed up to twice a day for a minimum of three days per week (to achieve 6 collections per week).  Subjects will answer EMA 
orienting questions up to three times per day in conjunction with the other assessments being collected via the EMA application on their cell phones or the 
provided device. 
o. Conducted by central reader after Screening visit, but prior to Day 1 visit. The MADRS, MGH ATRQ and confirmation of MDD will be conducted by 
central rater for determination of eligibility. 
p. Study site personnel will dispense equipment and train subjects on 12-hour urine collection for measuring cortisol level. At the Day 43 visit, study site 
personnel will dispense equipment and retrain subjects on the 12-hour urine collection to measure cortisol levels (the collection will be returned to the site at 
the Day 56 visit). On Day 54, the site will contact the subject to remind them to begin collection on Day 55. 
q. Subjects will wear their wearable watch-like device throughout the day and night except when charging. 
r. 
At the Day 1 visit, subjects confirmed as eligible will receive a 50 mg dose of study drug at the study site prior to eating. All other doses will be taken by the 
subjects at home. 
s. 
All AEs will be recorded from study drug administration on Day 1 until Day 112 visit. Serious adverse events that occur subsequent to the signing of the 
informed consent (i.e., during the Screening Period) as a result of study participation will be recorded, as well as all SAEs occurring from study drug 
administration on Day 1 until Day 112 visit. 
t. 
Includes medications taken within 30 days prior to informed consent and throughout the study. 
 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 28 of 63 
2. 
INTRODUCTION 
2.1. 
Major Depressive Disorder 
Depression is ranked as the leading cause of disability worldwide by the World Health 
Organization.1 Major depressive disorder (MDD) is a psychiatric disorder that is characterized 
by the occurrence of one or more major depressive episodes, along with an absence of any 
history of manic, mixed, or hypomanic episodes. It is a serious, often recurrent medical 
condition, which is associated with a 15.9% lifetime risk of suicide attempt.2 Results of the 
World Mental Health Survey Initiative (2011) found that the average lifetime incidence of 
Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) major 
depressive episodes was 14.6%, with a 12-month prevalence of 5.5% in higher income counties.3 
Similarly, the National Comorbidity Survey-Replication4 found the incidence of depression to be 
6.6% over 12-months, with a 16.2% lifetime incidence. In addition to the high mortality rate due 
to suicide, depressed patients are more likely to develop coronary artery disease and type 2 
diabetes.5 
Current treatments available for depression include psychotherapy (for mild depression), 
antidepressant medications, a combination of medication and psychotherapy (for mild-to-
moderate depression), and electroconvulsive therapy (moderate-to-severe depression). Despite 
the availability of numerous antidepressant drugs (e.g., monoamine oxidase inhibitors, tricyclic 
antidepressants, selective serotonin reuptake inhibitors [SSRIs], serotonin norepinephrine 
reuptake inhibitors [SNRIs]), there remains an unmet need for the pharmacological treatment of 
MDD. With currently available medications, alleviation of depressive symptoms is often not 
observed until several weeks of treatment have been received. Even after an adequate duration of 
treatment, the most commonly used treatments today (e.g., SSRIs and SNRIs) only result in 
approximately 35% to 45% of patients achieving clinical remission.6 Approximately 30% of 
patients are resistant to a series of treatments according to STAR*D (Sequenced Treatment 
Alternatives to Relieve Depression) study.7 Moreover, side effects are still a concern, even with 
newer medications. Thus, there is a clear need for efficacious and well-tolerated agents that can 
effectively treat depression and prevent recurrent episodes of depression. 
Stress has also been hypothesized to be a pivotal factor in the pathophysiology of depression, 
specifically by way of the hypothalamus-pituitary-adrenal (HPA) axis, which appears to be 
a prominent mechanism by which the brain reacts to both acute and chronic stress through 
feedback loops involving several neuroendocrine hormones, including adrenocorticotropic 
hormone (ACTH) and cortisol. In addition, both corticotropin-releasing factor (CRF) 
and arginine vasopressin (AVP), both of which are produced in the paraventricular nucleus of the 
hypothalamus8,9 are considered primary factors in the regulation of HPA axis activity. Receptor 
subtypes for these neuropeptides, which may be involved in the regulation of HPA axis activity, 
have attracted much attention as potential targets for the treatment of depression and anxiety. 
ANC-501 is an investigational new drug with antagonistic activity of the vasopressin receptor 1b 
(V1b receptor), which plays a role in the modulation of stress and mood. Based on nonclinical 
studies conducted, ANC-501 appears to be a promising candidate for clinical development with 
a novel mode of action that may benefit MDD patients. 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 29 of 63 
2.2. 
Mechanism of Action of Vasopressin Receptor 1b (V1b Receptor) 
Arginine vasopressin, a cyclic nonapeptide, together with CRF, are principal factors in the 
regulation of ACTH release from the anterior pituitary,8 and have been reported to play an 
important role in mood regulation. Arginine vasopressin is synthesized in the parvocellular 
division of the paraventricular nucleus of the hypothalamus, which is linked to activation of the 
HPA axis, and upon stress exposure, AVP is released from the median eminence into the 
pituitary portal circulation, where it strongly potentiates the effects of CRF on the release of 
ACTH. Clinically, the plasma AVP levels are elevated in patients with major depression, 
compared with healthy controls.10,11 In previous studies, subjects with melancholic-type or 
anxious-retarded depression exhibited elevated plasma AVP levels. Plasma AVP levels are also 
reportedly positively correlated with cortisol levels during depression, particularly in suicide 
victims.12 Moreover, the quantities of AVP-immunoreactive neurons and AVP messenger RNA 
(mRNA) in the paraventricular nucleus are increased during depression,13,14 particularly in 
subjects in the melancholic subgroup,13 and pituitary AVP responsibility in the regulation of the 
HPA axis is increased.15 The involvement of the AVP system in anxiety disorders has also been 
suggested. 
AVP exerts its effects via three receptor subtypes: V1a, V1b, and V2. These subtypes are G-protein 
coupled receptors.16 Among them, the V1b receptor is mainly expressed in the anterior pituitary 
and is thought to be involved in the regulation of HPA axis activity and emotional processes.17 
Indeed, SSR149415, a non-peptide V1b receptor antagonist with high affinity and potent 
antagonistic activity for the V1b receptor,18 has been reported to attenuate increases in 
plasma ACTH induced by corticotropin-releasing hormone (CRH)+AVP,19 and to exhibit 
antidepressant- and anxiolytic-like activities in a variety of rodent models of depression 
and anxiety, with more pronounced effects in models involving stressful situations.18,19,20 
Therefore, blockade of V1b receptor may provide a novel approach to treating stress-related 
disorders such as depression and anxiety disorders. 
ANC-501 is a potent and selective antagonist of V1b receptor in both in vitro and in vivo studies 
and has been shown to possess antidepressant activity in several animal models of depression, 
including one which mimics severely impaired HPA axis function. 
2.3. 
Rationale for Targeting V1b Receptor in Treatment of Depression 
A series of in vitro studies was conducted to define the affinity of ANC-501 to the V1b receptor, 
as well as a battery of in vivo studies to characterize the efficacy profile in MDD and its 
anxiolytic-like potential in animal models. 
During in vivo rat studies, the immobility time in the forced swimming test and the 
hyperemotionality in a rat olfactory bulbectomy model, both of which are indicative of 
depressive states, were each significantly reduced by treatment with ANC-501. In the olfactory 
bulbectomy model, ANC-501 exhibited efficacy following chronic treatment for 14 days, as did 
fluvoxamine. In addition, ANC-501 significantly improved depressive-like behavior induced by 
repeated corticosterone injection, a depression model which has been reported to be resistant to 
fluvoxamine and imipramine,21 indicating that ANC-501 may be effective for patients with 
severely impaired HPA axis function. In an anxiety model (social interaction behavior in rats), 
ANC-501 demonstrated anxiolytic activity as well. This preclinical evidence further suggests 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 30 of 63 
that a V1b receptor antagonist may be useful in the treatment of MDD with co-morbid anxiety 
disorders. 
2.3.1. 
Evaluation in Humans 
Three Phase 1 studies (a single ascending dose [SAD] study [TS121-US101], a multiple 
ascending dose [MAD] study [TS121-US102], and a PET receptor occupancy study 
[TS121-US103]) and one Phase 2 study (TS121-US201) have been conducted in the United 
States.  
Pharmacokinetic (PK) studies indicate that following single and multiple oral administration of 
ANC-501, increases in plasma ANC-501 peak and total exposures (maximum plasma 
concentration [Cmax] and area under the concentration-time curves [AUCs]) were observed with 
increases in dose. ANC-501 reached Cmax within 4 hours after dosing and declined with apparent 
half-life (t½) of approximately 20 hours. No food effect was observed on AUCs of ANC-501, 
while Cmax was reduced under fed conditions. In the MAD study, the plasma trough 
concentrations of ANC-501 appeared to reach steady state by 5 days after dosing and the 
exposures accumulated to approximately 2 times. Low renal clearance (CLr) suggested minimal 
contribution of the urinary excretion of ANC-501. Though two metabolites (M5 and M9) were 
found in plasma, the unchanged form was proposed as a predominant component. In addition, 
PET study revealed the relationship between plasma exposure of ANC-501 and V1b receptor 
occupancy in the pituitary. 
Overall, 78 healthy and 32 adult subjects with MDD were administered ANC-501 to date. In the 
Phase 2 study, there was no statistically significant difference observed in the primary endpoint 
(Montgomery-Åsberg Depression Rating Scale [MADRS] Change from Baseline to Week 
6positive trends were observed for the scores of depression scales in ANC-501 treated groups 
(10 mg and 50 mg) versus placebo. The lack of statistical significance may be attributable to the 
small number of subjects enrolled. 
Safety Findings in Clinical Studies 
A top dose of 50 mg for 6 weeks was deemed safe and well tolerated. There were no deaths or 
serious adverse events (SAEs) reported in any of the studies. No safety trends were observed in 
clinical laboratory, electrocardiogram (ECG), vital signs, physical examinations, or cardiac 
telemetry. In consideration of the nonclinical findings at the time of the MAD study, 
comprehensive eye examinations were performed. In Phase 2a study, eye examinations with a 
standardized lens opacity grading scale (Lens Opacities Classification System III [LOCS III]), 
were conducted to evaluate this risk in the study. Increase from baseline in the LOCS III grade of 
≥0.5 (Nuclear Opalescence [NO]), ≥0.8 (Cortical) or ≥0.5 (Posterior Subcapsular), or changes 
considered to be clinically significant as per the ophthalmologist or optometrist, were defined in 
the protocol as events of clinical significance. Two events of lenticular opacities were reported in 
Phase 2a study TS121-US201 in patients dosed with ANC-501 in the 50 mg dose group. One of 
2 patients had an increase of NO score from baseline at Visit 6 in the left and right eye of 0.9 and 
0.8 points, respectively, which was reportable as an event of clinical significance per protocol. 
Per the Investigator, the difference from the initial LOCS III assessment did not reflect actual 
change in lens opacity but rather intra-observer variability. The Investigator rated the event as 
mild in intensity and considered it to be not related to the study drug. The second patient had an 
increase of NO score from baseline at visit 6 in the unilateral (right) eye of 0.7 points, which was 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 31 of 63 
reportable as an event of clinical significance per protocol. The Investigator rated this event as 
mild in intensity and considered it to be possibly related to the study drug. The nonclinical 
studies demonstrated changes in the lens in a select number of study animals that were noted 
either in the posterior pole of the lens or affecting cortical lens fibers. In the clinical program, 
neither of the 2 patients with reports of progression of NO that met the level of clinical 
significance according to the study protocol were noted to have posterior polar or cortical 
changes. Further, the LOCS III scoring has a standardized range for NO from 1–6, with 1 being 
the lowest standard image available for comparison. The scores reported for one patient were 0.3 
at baseline and increasing to 1.0 at the time of the event. The initial score is below the level of 
the first comparative image on the LOCS scale template, and thus inter-visit variability could 
account for the noted difference. The second patient had changes noted in both eyes, with 0.7 
and 0.6 noted at baseline in the right and left eye, respectively and 1.5 noted at the time of the 
reported event in both eyes. Given the relative subjective nature of lens grading, and the fact that 
at initial examination the scoring was below the lowest standard image standard, intra-grader 
variability likely accounted for these changes, as noted by the Investigator for the second patient. 
In the Phase 2 study, there was no statistically significant difference observed in the primary 
endpoint (Montgomery-Åsberg Depression Rating Scale [MADRS] Change from Baseline to 
Week 6), although positive trends were observed for the scores of depression scales in ANC-501 
treated groups (10 mg and 50 mg) versus placebo. The lack of statistical significance may be 
attributable to the small number of subjects enrolled. 
In conclusion, these results suggest that ANC-501 (ANC-501) may be useful for the treatment of 
patients with MDD. 
2.4. 
Risk/Benefit 
Three Phase 1 studies, a SAD study (TS121-US101), a MAD study (TS121-US102) and a PET 
receptor occupancy study (TS121-US103), and one Phase 2 study in MDD (TS121-US201) have 
been conducted in the US. Overall, 78 healthy male and female adult subjects and 32 adult 
subjects with MDD were administered ANC-501 to date. A top dose of 50 mg for 6 weeks was 
deemed safe and well-tolerated. There were no deaths or SAEs reported in any of the studies. All 
events across trials were either mild or moderate in intensity. Moreover, no safety trends were 
observed in clinical laboratory, ECG, vital signs, physical examinations, or cardiac telemetry. 
In consideration of the nonclinical findings (rat) at the time of the MAD study, comprehensive 
prospective slit lamp examinations were performed in the Phase 2 trial. Two events of lenticular 
opacity were reported as clinically significant by prespecified criteria on the LOCS III rating 
scale in 2 subjects (1 event each) in the 50 mg group. These events were rated as mild in 
intensity, were not accompanied by any changes in vision, and did not require treatment. One 
was judged as “possibly related” and the other was judged as “not related” to ANC 501. While 
Study TS121-US201 was not enrolled to completion, patients treated with ANC-501 in 
combination with their underlying antidepressant showed numerical improvement compared to 
placebo. Consistent with the hypothesized mechanism of action of ANC-501, those patients with 
elevated baseline 12-hour urine cortisol concentration showed substantially larger separation 
from placebo at the end of 6 weeks in both dose groups tested. In the case of the high cortisol 
50 mg sub-group, these differences continued to increase throughout the follow-up period. 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 32 of 63 
Approximately 30% of patients are resistant to a series of treatments according to the STAR*D 
study. There is little data-driven guidance on next steps to treat MDD patients in the event of a 
first-line failure. Importantly, side effects are a major concern for patients particularly the use of 
antipsychotic agents as adjunctive therapies and those used to treat treatment-resistant depression 
(TRD). Thus, there is a clear need for new, efficacious, well-tolerated agents to both treat 
depression episodes and prevent recurrent episodes of depression. 
The safety profile of ANC-501, based on the previously conducted trials and preliminary clinical 
data, suggest that ANC-501 may also be well tolerated in patients and efficacious as an 
adjunctive treatment in select MDD patients. The current significant unmet need in the treatment 
of depression, remaining as a number one cause of disability worldwide, justifies a favorable 
risk-benefit ratio, and investigation of ANC-501 in MDD. 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 33 of 63 
3. 
OBJECTIVES AND ENDPOINTS 
Objectives 
Endpoints 
Primary 
• To evaluate the effect of treatment with 
ANC-501 capsules on depressive 
symptoms in subjects with major 
depressive disorder (MDD) 
• Change from baseline (Day 1) to Day 56 in Montgomery-Åsberg 
Depression Rating Scale (MADRS) total score 
• To evaluate the safety and tolerability 
of ANC-501 capsules in subjects with 
MDD 
• Safety and tolerability as assessed by: 
o Frequency and severity of adverse events (AEs) 
o Vital signs 
o Body weight 
o Electrocardiograms (ECGs) 
o Clinical laboratory tests 
o Physical examination 
o Lens Opacities Classification System III (LOCS III) 
o Emergent Suicidal behavior using Columbia-Suicide Severity 
Rating Scale (C-SSRS) 
Secondary 
• To evaluate effect of ANC-501 at all 
time points and percent reduction in 
total symptom scores compared with 
baseline. 
• Change from baseline (Day 1) in the MADRS total score at all 
time points 
• Percentage of MADRS responders (≥50% reduction in total 
score) 
• Percentage of MADRS remitters (total score ≤10) 
• Change in Hamilton Anxiety Scale (HAM-A) total score from 
Baseline to Day 56 
• Change in Clinical Global Impression-Severity (CGI-S) score 
from Baseline to Day 56 
• Short Form-Health Survey (36-item) (SF-36) total score and 
subscale scores from Baseline to Day 56 and at all time points 
• Percentage of Clinical Global Impression-Improvement (CGI-I) 
improvers (“Very much improved” or “Much improved”) 
• Patient Global Impression of Improvement (PGI-I) score at 
Day 56 
• To assess the pharmacokinetic (PK) 
profile of ANC-501 Capsules in plasma 
using a sparse sampling model 
• Plasma concentrations of ANC-501  
Exploratory 
• To evaluate changes in 12-hour urine, 
plasma, and saliva cortisol 
• Changes in 12-hour urine, plasma, and saliva cortisol from 
baseline to Day 56 
• To evaluate changes in biometrics 
• Changes in biometrics using the wearable watch-like device 
• To evaluate biological samples as 
potential biomarkers and metabolites 
• Patient reported changes in Depression 
and Anxiety symptoms 
• Baseline levels and/or changes in exploratory biomarkers through 
Day 56 
• Changes in Hamilton Depression Rating Scale (6-item) (HAMD-
6) scores from baseline across all time points using ePRO 
• Changes in General Anxiety Disorder (7-item) (GAD-7) from 
baseline across all time points using ePRO 
 
 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 34 of 63 
4. 
STUDY DESIGN 
4.1. 
Overall Design 
This is a single-arm, open-label Phase 2 study to assess the safety, tolerability, pharmacokinetics 
(PK), and efficacy of ANC-501 oral capsules as adjunctive treatment in subjects diagnosed with 
major depressive disorder (MDD). 
With the exception of subjects permitted to use current stable antidepressant treatment, initiation 
of psychotropic medications and other medications that may potentially have an impact on 
efficacy or safety endpoints will not be allowed between screening and completion of the Day 56 
assessments. Psychotropic medications, which must have been initiated at least 8 weeks prior to 
screening, must remain at a stable dose until completion of the Day 56 assessments. During the 
study, efficacy and safety assessments will be performed and blood samples collected for PK 
analysis of ANC-501and future exploratory biomarker and genetic analysis. A wearable 
watch-like device will collect data on biometrics, which may be used for exploratory analyses. 
Subjects will receive 50 mg ANC-501 Capsules (5 × 10 mg capsules) orally once daily at least 
1 hour prior to the morning meal for 8 weeks beginning with the first dose administered in the 
clinic on Day 1, and will return to the clinic for scheduled visits on Days 8, 15, 29, 43, and 56. 
Follow-up visits will occur on Day 70 and 112. All visits after Day 1 allow a visit window of ±3 
days. 
Total duration of participation is 20 weeks (up to 30 days screening, 8 weeks dosing, 8 weeks 
follow-up). 
The following is an overview of study procedures/assessments (refer to the Schedule of Events 
[Section 1.3] for specific procedures/assessments and their timing, with further details located in 
Section 8): 
Screening Period (Day -30 to Day -1) 
After providing informed consent, eligibility assessments will include review of demographics, 
medical history, prior/concomitant medications, full physical examination, vital sign 
assessments, body weight/height/body mass index (BMI) measurements, urine pregnancy test 
(for female subjects of childbearing potential), urine drug and alcohol breath test screen, safety 
laboratory tests (i.e., hematology, serum chemistry, coagulation, and urinalysis), hepatitis and 
human immunodeficiency virus (HIV) screen, and plasma and saliva cortisol sample collection. 
The Structured Clinical Interview for DSM-5 – Clinical Trials Version (SCID-5-CT) will be 
employed for the determination of MDD and will be performed by a qualified, trained, validated 
healthcare professional. 
Qualified staff will also complete the Montgomery-Åsberg Depression Rating Scale (MADRS), 
Hamilton Anxiety Rating Scale (HAM-A), Clinical Global Impression-Severity (CGI-S), 
Columbia-Suicide Severity Rating Scale (C-SSRS), and Childhood Trauma Questionnaire 
(CTQ). 
Ophthalmic exams, including slit-lamp imaging, may be performed by an appropriately trained 
and qualified individual as delegated by the PI and documented in the Study Delegation Log. 
However, a licensed ophthalmologist or optometrist must oversee this work and will make the 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 35 of 63 
determination on subject eligibility as per inclusion criteria #4.  Slit lamp imaging of the lens of 
both eyes will be taken and sent to a central reader for assessment using the LOCS III 
classification. 
Study site personnel will dispense equipment for 12-hour urine collection to measure cortisol 
levels. 
A call between the subject and the qualified central rater will be scheduled to allow the rater to 
administer the Massachusetts General Hospital Antidepressant Treatment Response 
Questionnaire (MGH ATRQ) for assessment of current antidepressant therapy (ADT), adequacy 
of duration and dose of prior and current ADT, as well as degree of improvement, to ensure only 
patients with inadequate response (<50% improvement) are included. The central rater will also 
complete the MADRS assessment and SAFER inventory (State vs trait, Assessability, Face 
and Ecological validity, Rule of 3Ps [pervasive, persistent, and pathological]) for confirmation of 
eligibility (diagnosis, severity, and treatment history). Results will be communicated to the site 
prior to Day 1. 
No changes to medications are allowed during the screening period, and screening can be less 
than 30 days if all other criteria are met. 
Open-Label Treatment Period (Days 1, 8, 15, 29, 43, 56) 
At the Day 1 visit only, subjects confirmed as eligible will receive a 50 mg dose of study drug 
prior to eating. Subjects who cannot tolerate 50 mg will receive 40 mg for the remainder of the 
study. Subjects who cannot tolerate 40 mg will be terminated from the study. Before releasing 
subjects, study site personnel will dispense and train subjects on a dosing diary, the wearable 
watch-like device and mobile application. ANC-501 will be dispensed to subjects with 
instructions to take the medication every morning at least 1 hour prior to eating breakfast. 
During visits, safety procedures/assessments and administration of assessments/scales (including 
from Day 8 onward, the Clinical Global Impression-Improvement [CGI-I] and the Patient Global 
Impression of Improvement [PGI-I]) will be conducted. During specific visits, PK blood samples 
will be collected, as well as blood samples for future exploratory biomarker and genetic analysis. 
Concomitant medications and dosing diary will be reviewed, the dosing diary will be returned to 
the subject, and adverse events (AEs) recorded. At all visits after Day 1, recording/accounting 
for any returned/unused ANC-501 will be performed and ANC-501 will be dispensed to subjects 
with a reminder to take the medication every morning at least 1 hour prior to eating breakfast. 
At home activities 
Subjects will take study medication every morning (at least 1 hour prior to eating breakfast), 
make entries into the dosing diary, and wear their wearable watch-like device throughout the 
day and night except when charging. Subjects will also complete EMA orienting questions, 
the Hamilton Depression Rating Scale (6-item) (HAMD-6) and the General Anxiety Disorder 
(7-item) (GAD-7) twice a day for three days per week, and the Short Form-Health Survey 
(36-item) (SF-36) weekly in the EMA Wellness smartphone/tablet application. 
At the Day 43 visit, study site personnel will dispense equipment and retrain subjects on the 
12-hour urine collection to measure cortisol levels (the collection will be returned to the site at 
the Day 56 visit). On Day 54, the site will contact the subject to remind them to begin collection 
on Day 55. 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 36 of 63 
At the Day 56 visit, subjects will take their last study medication and visit the site to return their 
12-hour urine collection, all remaining medication, the dosing diary, and wearable watches. 
Study procedures/assessments will be conducted, including an ophthalmic exam and slit lamp 
imaging of the lenses of both eyes. Images will be sent to a central reader for assessment using 
the LOCS III. Collection of plasma and saliva cortisol samples will also be conducted.  
The same rater should perform the CGI-S, CGI-I, and MADRS assessments throughout the study 
on a given subject when possible. 
Follow-up Period (Day 70 and Day 112) 
Subjects will return to the site for a follow-up visit on Day 70 and Day 112. Study 
procedures/assessments will be conducted, including an ophthalmic exam and slit lamp imaging 
of the lenses of both eyes. Images will be sent to a central reader for assessment using the LOCS 
III. Concomitant medications will be reviewed, and Aes recorded. 
4.2. 
Justification for Dose 
ANC-501 dose selection was based on accumulated experience from preclinical studies 
and previous clinical trials. The SAD study (TS121-US101) and MAD study (TS121-US102) 
provide safety and tolerability support for the current doses. A PET receptor occupancy study 
(TS121-US103) provides rationale for doses of 10 mg and 50 mg orally once daily. From results 
of the PET receptor occupancy study, 10 mg and 50 mg doses were modeled to provide 
approximately 55% and 75% receptor occupancy, respectively. In the Phase 2 study 
(TS121-US201), the 50 mg dose suggested efficacy in the sub-population of subjects with 
higher cortisol levels. 
4.3. 
End of Study Definition 
End of study is defined as the date when the last subject has completed the final scheduled 
procedure shown in the Schedule of Events (Section 1.3). 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 37 of 63 
5. 
STUDY POPULATION 
Subject eligibility must be verified prior to enrollment. Prospective approval of protocol 
deviations to recruitment and enrollment criteria (also known as protocol waivers or exemptions) 
is not permitted. 
5.1. 
Inclusion Criteria  
Patients who meet ALL the following inclusion criteria will be eligible to participate in the 
study: 
1. Demonstrate understanding of the study procedures, restrictions, and willingness to 
participate as evidenced by voluntarily signing and dating the written informed consent 
form. 
2. Adult male or female between 18 and 65 years of age, inclusive. 
3. Good physical health and has no clinically significant findings, as determined by the 
Investigator, on physical examination, 12-lead electrocardiogram (ECG), vitals, 
or clinical laboratory tests. 
4. Has no clinically significant findings on the ophthalmic examination, including Best 
Corrected Visual Acuity (BCVA) worse than 20/30 or, in the opinion of the 
ophthalmologist or optometrist, any cataract that may become clinically significant 
and/or need surgical intervention during the course of the trial. 
5. Able and willing to complete all study requirements. 
6. Diagnosis of current episode of major depressive disorder (MDD) at least 8 weeks prior 
to screening confirmed by Structured Clinical Interview for DSM-5 – Clinical Trials 
Version (SCID-5-CT). 
7. Have not responded to their current antidepressant therapy or to dose 
adjustment/treatment changes following a loss of response to their current antidepressant 
therapy. 
8. Complete the SAFER interview, administered via teleconference by a Massachusetts 
General Hospital (MGH) psychiatrist or psychologist, and receive a passing score which 
confirms a diagnosis of MDD, and a minimum Montgomery-Åsberg Depression Rating 
Scale (MADRS) total score of 26. The SAFER interview will also confirm the subject’s 
treatment history and response to antidepressant therapies (ADTs) through the 
administration of the MGH Antidepressant Treatment Response Questionnaire (ATRQ). 
9.  Receiving a stable dose of the same antidepressant (selective serotonin reuptake inhibitor 
[SSRI], serotonin and norepinephrine reuptake inhibitor [SNRI], bupropion or trazodone 
monotherapy) for the current episode for at least 6 weeks of continuous treatment, which 
can include some or all of the screening period, with 4 weeks on a stable dose prior to 
day 1 and has an inadequate response (<50% improvement) using the MGH ATRQ. 
10. Willing to maintain stable dose and delay start of other antidepressant or antianxiety 
medications and any new pharmacotherapy regimens, including benzodiazepine 
anxiolytics, during the screening and treatment periods. 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 38 of 63 
11. Has a MADRS total score of ≥26 at screening and Day 1 (prior to dosing). 
12. Has a 12-hour urine cortisol level >22.7 nmol/L (greater than or equal to 8.3 mcg/L). 
13. Body mass index (BMI) ≥18 and ≤38 kg/m2 at screening using the formula: 
𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤ℎ𝑡𝑡 (𝑘𝑘𝑘𝑘) ℎ𝑒𝑒𝑒𝑒𝑒𝑒ℎ𝑡𝑡 (𝑚𝑚)2
⁄
. 
14. Agrees to practice an acceptable method of highly effective birth control at screening, 
throughout study participation, and for 90 days after the last dose of study drug. Highly 
effective methods of birth control include sexual abstinence (for males and females); 
vasectomy; or a condom with spermicide in combination with a highly effective female 
partner’s method (for males); and hormonal methods of contraception (i.e., established 
use of oral, implantable, injectable, or transdermal hormones); placement of an 
intrauterine device; placement of an intrauterine system; and mechanical/barrier method 
of contraception (i.e., condom or occlusive cap [diaphragm or cervical/vault cap] in 
conjunction with spermicide [foam, gel, film, cream, or suppository]) (for females). 
5.2. 
Exclusion Criteria 
Patients who meet any of the following exclusion criteria will be excluded from participation in 
the study: 
1. History of suicide attempt in the past 2 years or within current MDD episode. 
2. Active suicidal ideation with plan or a “yes” response to items 4 or 5 on the C-SSRS in 
the past 24 months is exclusionary as assessed at Screening and Baseline (Day 1). 
3. Inadequate response to >2 prior ADTs (not including current antidepressant) of at least 
6 weeks duration each for the episode current at screening. 
4. Recent history or active clinically significant manifestations of metabolic, hepatic, renal, 
hematological, pulmonary, cardiovascular, gastrointestinal, musculoskeletal, 
dermatological, urogenital, neurological, ophthalmic (especially cataracts), or ears, nose, 
and throat (ENT) disorders, or any other acute or chronic condition that, in the 
Investigator's opinion, would limit the subject's ability to complete or participate in this 
clinical study. 
5. Based on an ophthalmic examination performed at Screening, patient must not have any 
of the following: 
• Nuclear opalescence (NO) with a LOCSIII classification > 3.0 in either eye 
• Nuclear color (NC) with a LOCSIII classification > 3.5 in either eye 
• Cortical lens opacities (C) with a LOCSIII classification > 2.0 in either eye 
• Posterior subcapsular lens opacities (P) with a LOCSIII classification > 0.3 in 
either 
6. History or presence of intellectual disability, pervasive developmental disorder, cognitive 
disorder, neurodegenerative disorder (e.g., Alzheimer’s disease, Parkinson’s disease, 
multiple sclerosis, Huntington’s disease), or brain injury. 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 39 of 63 
7. Receipt of electroconvulsive therapy (ECT) within 12 months of screening, lifetime 
receipt of more than one course of ECT, or plans to receive ECT during the study. 
8. Receipt of repetitive transcranial magnetic stimulation (rTMS) within 12 months 
of screening or planned to receive rTMS during the study. 
9. Plans to initiate or terminate cognitive or behavioral psychotherapy or alter the frequency 
of ongoing therapy during this study. 
10. Works night shifts or needs to work night shifts during the study. 
11. Females who are pregnant, intend to become pregnant (within 90 days of the last dose of 
ANC-501), are breastfeeding, or have a positive pregnancy test at screening or on Day 1 
prior to study drug administration. 
12. Known allergy to ANC-501, or related compounds. 
13. Detectable hepatitis B surface antigen (HBsAg), hepatitis C virus antibody (HCV Ab), 
or human immunodeficiency virus (HIV) antibody at screening. 
14. Active psychosis per Investigator assessment. 
15. Medical history of seizures (does not include isolated febrile seizures of childhood). 
16. Medical history of bipolar disorder, schizophrenia, and/or schizoaffective disorder. 
17. History of alcohol or substance use disorder in the 12 months prior to screening. 
18. Positive alcohol breath test. 
19. Positive urine drug screen for hallucinogens, psychoactive inhalants, stimulants, opioids, 
barbiturates, non-prescribed anxiolytics or other non-prescribed sedatives. Does not 
include positive test for cannabinoids, tobacco or caffeine, unless the subject meets 
criteria for substance use disorder for these substances. 
20. Exposure to another investigational medication or device within 30 days or 5 half-lives 
(whichever is longer) prior to screening. 
21. Has been previously treated in this study or randomized or treated in any other study 
employing ANC501 (i.e., subject may not have received study drug and then reenrolled). 
22. Administration of drugs to treat  psychiatric or neurologic conditions that have not been 
taken at a stable dose for at least 4 weeks prior to day 1. 
23. Need/plan to take moderate to strong inhibitors and/or inducers of cytochrome P450 
(CYP)3A4 (food, beverages, medications, and supplements). 
24. Any condition not identified in the protocol that in the opinion of the Investigator would 
confound the evaluation and interpretation of the study data or may put the subject at risk. 
5.3. 
Subject Enrollment 
Subject eligibility must be verified prior to enrollment. Upon eligibility verification, subject 
information will be entered into an Interactive Response Technology (IRT) system. The first 
dose of study drug should be administered within 72 hours (all efforts should be made to initiate 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 40 of 63 
treatment as soon as possible after registration) following entry into the IRT. The date the first 
dose of study drug is administered is the date the subject is considered enrolled into the study. 
5.4. 
Screen Failures 
Screen failures are defined as subjects who consent to participate in the clinical study but not 
subsequently enrolled in the study. A minimal set of screen failure information is required to 
ensure transparent reporting of screen failure subjects to meet the Consolidated Standards of 
Reporting Trials (CONSORT) publishing requirements and to respond to queries from regulatory 
authorities. Minimal information includes demography, screen failure details, eligibility criteria, 
and any serious adverse event (SAE) that occurs after completion of the informed consent form 
(ICF). 
Subjects who do not meet the criteria for participation in this study (i.e., screen failures) may be 
rescreened after discussion with the Medical Monitor. Previously completed study assessments 
remaining within the protocol-defined window will be accepted for rescreening. 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 41 of 63 
6. 
STUDY TREATMENT 
6.1. 
Study Treatment Administered 
Subjects will receive 50 mg ANC-501 Capsules (5 × 10 mg capsules) orally once daily at least 
1 hour prior to the morning meal for 8 weeks beginning with the first dose administered in the 
clinic on Day 1. 
6.2. 
Handling, Storage, and Accountability 
ANC-501 Capsules are stored in child-resistant 35-count bottles at controlled room temperature 
(15–25°C/59–77°F). 
The Investigator or designee must confirm appropriate temperature conditions have been 
maintained during transit for all study drug received, and any discrepancies are reported and 
resolved before use of the study drug. 
Only subjects enrolled in the study may receive study drug, which may only be supplied or 
administered by authorized site staff. All study drug must be stored in a secure, environmentally 
controlled, and monitored (manual or automated) area in accordance with the labeled storage 
conditions, with access limited to the Investigator and authorized site staff. 
The Investigator is responsible for study drug accountability, reconciliation, and record 
maintenance (i.e., receipt, reconciliation, and final disposition records). The Investigator or 
designee will maintain a record of all study drug received and dispensed. 
Further guidance and information for the final disposition of unused study drug are provided in 
the Pharmacy Manual. 
6.3. 
Measures to Minimize Bias: Randomization and Blinding 
Not applicable; this is an open-label study. 
6.4. 
Treatment Compliance 
Drug accountability will be performed by the study site using appropriate source documentation 
and recorded in the eCRF. 
 
6.5. 
Dose Modifications 
During the Open-Label Treatment Period, subjects will be able to receive study drug as long as 
there are no dose-limiting safety/tolerability concerns as determined by the Investigator. Subjects 
who experience moderate or severe AEs while receiving the 50 mg dose of study drug which, 
according to the clinical judgement of the Investigator, are related to study drug, may receive 
40 mg for the remainder of the Open-Label Treatment Period if the AEs are intolerable. Subjects 
who experience moderate or severe related AEs while receiving the 40 mg dose of study drug 
might not be able to continue receiving study drug based on the evaluation and clinical judgment 
of the Investigator and may be discontinued from further treatment. 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 42 of 63 
6.6. 
Continued Access to Study Drug after the End of Study 
There are currently no plans to provide ANC-501 to subjects after the study ends. 
6.7. 
Treatment of Overdose 
No information regarding overdose of ANC-501 in humans is available. The subject should be 
closely monitored for signs of toxicity, and the Medical Monitor notified immediately. There is 
no specific antidote for overdose of ANC-501, and general supportive care should be provided. 
6.8. 
Concomitant Medications and Therapies 
Any medication or vaccine(s) (including over-the-counter or prescription medicines, recreational 
drugs, vitamins, and/or herbal supplements) that the subject received within 30 days prior to 
informed consent or receives during the study must be recorded along with the reason for use, 
start and end dates, dose, and frequency. The Medical Monitor should be contacted if there are 
any questions regarding prior or concomitant therapy. 
Subjects may not have received another investigational medication or device within 30 days 
prior to screening. Psychotropic medications, which must have been initiated at least 8 weeks 
prior to screening, must remain at a stable dose until completion of the Day 56 assessments. 
Strong inhibitors and/or inducers of CYP3A4 (food, beverages, medications, and supplements) 
are not permitted. 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 43 of 63 
7. 
DISCONTINUATION OF STUDY TREATMENT AND SUBJECT 
WITHDRAWAL 
7.1. 
Discontinuation of Study Treatment 
A subject must be discontinued from study treatment for any of the following: 
1. Unacceptable toxicity, despite maximal supportive care and/or dose reduction. 
2. Subjects exhibiting a change in the LOCS III greater than or equal to 0.5 in Nuclear 
Opalescence, Cortical Opalescence or Posterior Subcapsular Opalescence in either eye or 
clinically significant eye changes (e.g., incident of cataract). 
3. Investigator decision (may include the need for antidepressant therapy or other therapy 
not otherwise permitted in the study, including ECT or changes to cognitive/behavioral 
psychotherapy) or noncompliance with study requirements. 
4. Subject becomes or intends to become pregnant. 
5. Subject withdrawal of consent for further study treatment. 
6. Study termination by the Sponsor. 
Subjects discontinuing treatment should complete the Early Termination visit procedures and 
enter the Follow-up Period. 
7.2. 
Subject Withdrawal from Study 
A subject will be withdrawn from all study participation (i.e., discontinued without further 
follow-up): 
1. Withdrawal of consent to further follow-up assessments, irrespective of the reason 
2. Lost to follow-up 
a. A subject will be considered lost to follow-up after repeated failure to attend 
scheduled study visits, and who is unable to be contacted by the study site. Only 
after 3 phone calls (if possible) are attempted and no response received from a 
certified letter (or equivalent local method) to the subject’s last known mailing 
address should a subject be considered lost to follow-up. All contact attempts 
should be documented in the subject’s source records. 
3. Death 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 44 of 63 
8. 
STUDY ASSESSMENTS AND PROCEDURES 
Written informed consent must be provided before any study-related procedures/assessments are 
performed. Refer to the Schedule of Events (Section 1.3) for procedures/assessments and timing. 
8.1. 
General Study Assessments 
General study assessments are outlined in Table 1. 
 Table 1: 
General Study Assessments 
Assessment 
Details 
Review of inclusion/exclusion criteria 
Eligibility is to be confirmed prior to the first dose of study treatment 
(see Section 5.1 and Section 5.2 for eligibility criteria).  
Medical history, demographics 
Demographics (age, gender, race, ethnicity), relevant medical history 
(past and concurrent). 
Height and body weight 
Height is measured at screening only (for body mass index [BMI] 
calculation). 
Prior and concomitant 
medications/concomitant therapy 
Record all medications that are taken within 30 days prior to informed 
consent and throughout the study. 
Structured Clinical Interview for 
DSM-5 – Clinical Trials Version 
(SCID-5-CT) Diagnostic Interview 
Subjects will be screened to determine whether they fulfill the criteria 
for diagnosis of MDD according to the SCID-5-CT, which will be 
performed by a qualified, trained, validated healthcare professional. 
SAFER Interview 
The interview will be conducted by a central rater for determination of 
eligibility. The SAFER inventory for confirmation of eligibility 
(diagnosis, severity, and treatment history) includes confirmation of 
MDD using the MADRS, and use of the Massachusetts General 
Hospital Antidepressant Treatment History Questionnaire (MGH 
ATRQ) for assessment of current antidepressant therapy (ADT), 
adequacy of duration and dose of prior and current ADT, as well as 
degree of improvement, so that only patients with inadequate response 
(<50% improvement) are included. 
Site staff will schedule the interview for the central rater, who will 
contact the patient as scheduled and conduct the interview. Results will 
be communicated to the site prior to Day 1. 
Childhood Trauma Questionnaire 
(CTQ) 
Retrospective measure of childhood trauma containing 5 subscales 
(emotional abuse, physical abuse, sexual abuse, emotional neglect, 
and physical neglect). Assessed at screening only. 
8.2. 
Efficacy Assessments 
Efficacy assessments are outlined in Table 2. 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 45 of 63 
Table 2: 
Efficacy Assessments 
Assessment 
Details 
Montgomery-Åsberg Depression 
Rating Scale (MADRS) 
10 questions, each rated on a 7-point scale, to stratify severity of 
depressive episodes in adults. 
Includes change from baseline in the MADRS total score at all time 
points, percentage of MADRS responders (≥50% reduction in total 
score), percentage of MADRS remitters (total score ≤10). 
The same rater should perform MADRS assessments throughout the 
study on a given patient when possible. 
Hamilton Anxiety Scale (HAM-A) 
14 questions, each rated on a 5-point scale, to rate the level of anxiety. 
Includes change in HAM-A total score from baseline to Day 56, 
and change in total score at all time points. 
Clinical Global Impression-Severity 
(CGI-S) 
7-point scale for clinician to rate the severity of the patient’s illness at 
the time of assessment, relative to past experience with patients having 
the same diagnosis. 
Includes change in CGI-S total score from baseline to Day 56, 
and change in total score at all time points. 
The same rater should perform CGI assessments throughout the study 
on a given patient when possible. 
Clinical Global Impression-
Improvement (CGI-I) 
7-point scale for clinician to assess how much the patient’s illness has 
improved or worsened relative to a baseline state. 
Includes percentage of CGI-I improvers (“Very much improved” or 
“Much improved”) at all time points. 
The same rater should perform CGI assessments throughout the study 
on a given patient when possible. 
Patient Global Impression of 
Improvement (PGI-I) 
7-point scale used to evaluate patient satisfaction with treatment. 
Includes PGI-I score at Day 56, and score at all time points. 
Hamilton Depression Rating Scale (6-
item) (HAMD-6) 
6-point scale derived from the 17-item Hamilton Rating Scale for 
Depression to evaluate severity of depression. Subjects will complete 
the HADM-6 twice a day for three days per week. 
General Anxiety Disorder (7-item) 
(GAD-7) 
7-point scale to measure the level of anxiety. Subjects will complete 
the GAD-7 twice a day for three days per week. 
Short Form-Health Survey (36-item) 
(SF-36) 
36-question survey self-reported measure of physical and mental 
health. 
Includes total score from baseline to Day 56, and change from baseline 
in total score at all time points. 
8.3. 
Safety Assessments 
Safety assessments are outlined in Table 3. 
Table 3: 
Safety Assessments 
Assessment 
Details 
AE monitoring 
Serious adverse events (SAEs) that occur subsequent to the signing of the 
informed consent (i.e., during the Screening Period) as a result of study 
participation will be recorded. All adverse events (AEs) and SAEs occurring 
subsequent to the first dose of study drug on Day 1 will be recorded. 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 46 of 63 
Assessment 
Details 
Physical examination 
Physical examination and review of relevant systems at screening, including 
body weight and height. 
Vital signs 
Oral temperature (°C), respiratory rate, heart rate, and blood pressure (supine 
and standing). 
12-lead electrocardiograms 
A standard 12-lead ECG (ventricular rate, RR, PR, QRS, QT, QTcB, and QTcF) 
will be performed by the Investigator or designee after the subject has been in the 
supine position for at least 5 minutes at specified visits. The subject must be in 
a supine position while the ECG is obtained. Electrocardiogram tracings will be 
reviewed and interpreted by a qualified clinician. Electrocardiogram tracings and 
results with Investigator’s interpretation are to be included and summarized in 
the subject’s study records. 
The date, time, subject demographics (e.g., age, and sex) and subject number 
should be recorded on the ECG. 
Ophthalmic examination 
Ophthalmic exam, including slit-lamp imaging, may be performed by an 
appropriately trained and qualified individual as delegated by the PI and 
documented in the Study Delegation Log. However, a licensed ophthalmologist 
or optometrist must oversee this work and will make the determination on 
subject eligibility as per inclusion criteria #4. Additional slit lamp imaging of the 
lenses of both eyes may be performed during the treatment period and sent to a 
central reader for assessment using the LOCS III as per investigator’s discretion. 
Slit lamp imaging performed during the study showing a change of greater than 
or equal to 0.5 on the LOCS III scale and any changes determined by the 
investigator to be clinically significant will be recorded and considered an 
adverse event of special interest. 
Clinical significance is defined as a class I lens event (minimal change) [increase 
from baseline in the LOCS III grade of ≥0.5 (Nuclear Opalescence), ≥0.5 
(Cortical) or ≥0.5 (Posterior Subcapsular)] and any changes considered to be 
clinically significant (e.g., incidence of cataract). 
Columbia-Suicide Severity 
Rating Scale (C-SSRS) 
Rating scale used to assess suicidal ideation and behavior. 
The “Baseline/Screening” C-SSRS form will be completed at screening. The 
“Since Last Visit” C-SSRS form will be completed at any time of day at all 
subsequent time points. 
Safety laboratory tests 
See Table 4. 
Abbreviations: AE = adverse event; ECG = electrocardiogram; ET = Early Termination; PK = pharmacokinetic; 
QTcF = QT interval corrected for heart rate using Fridericia’s formula. 
8.3.1. 
Laboratory Assessments 
See Table 4 for the list of clinical laboratory tests to be performed and the Schedule of Events 
(Section 1.3) for timing and frequency. 
Table 4: 
Laboratory Assessments 
Laboratory Test 
Parameters 
Hematology 
Hemoglobin, hematocrit, RBC (including mean corpuscular hemoglobin, mean 
corpuscular volume, mean corpuscular hemoglobin concentration), WBC with 
differential (neutrophils [absolute count] and lymphocytes, monocytes, 
eosinophils, basophils), and platelet count. 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 47 of 63 
Laboratory Test 
Parameters 
Coagulation 
PT, INR, aPTT. 
Serum chemistry  
Non-fasting: albumin, alanine aminotransferase (ALT), aspartate 
aminotransferase (AST), alkaline phosphatase, amylase/lipase, bicarbonate, 
bilirubin (total and direct), BUN (urea where BUN not tested), calcium, chloride, 
creatinine, glucose, magnesium, phosphorus, potassium, sodium, and total 
protein. Subjects found to have treatment-emergent laboratory toxicity of 
Grades 3 or 4 will be monitored at least weekly until resolution. 
Urinalysis 
pH, protein, ketones, bilirubin, nitrite, blood, leukocytes, specific gravity, 
urobilinogen. Reflex to microscopic exam when positive for protein, blood, 
nitrite, or leukocytes. 
Pregnancy test 
Urine pregnancy test will be conducted in only female subjects of childbearing 
potential. Female subjects who prematurely discontinue will have a pregnancy 
test performed at the ET visit. 
Serology 
HBsAg, HCV Ab, HIV antibody 
Urine drug and alcohol 
screen 
Urine toxicology for selected drugs of abuse (barbiturates, amphetamines, 
cocaine, opioids) and breath test for alcohol. 
Plasma, saliva, and 12-hour 
urine collection for 
measurement of cortisol 
level,  
Study site personnel will dispense the equipment and train subjects on how to 
collect a 12-hour urine collection for measuring cortisol level. 
Saliva collection at screening should be a morning sample. 
Abbreviations: ALT = alanine aminotransferase; aPTT = activated partial thromboplastin time; AST = aspartate 
aminotransferase; BUN = blood urea nitrogen; ET = early termination; HBsAg = hepatitis B surface antigen; 
HCV Ab = hepatitis C virus antibody; HIV = human immunodeficiency virus; INR = International Normalized Ratio; 
PT = prothrombin time; RBC = red blood cell; WBC = white blood cell. 
 
8.3.1.1. 
 Reporting and Evaluation of Laboratory Test Results 
Adverse events will be collected from the first dose of ANC-501, therefore the screening 
laboratory results are considered baseline. All laboratory test results (except for those at 
screening, which are considered baseline) must be reviewed for clinical significance by the 
Investigator. Laboratory test results outside the normal range (hematology, coagulation, 
chemistry, urinalysis) that worsen in severity from baseline and are deemed clinically significant 
by the Investigator should be reported as AEs. A new laboratory abnormality resulting in study 
drug interruption, dose reduction, treatment discontinuation, intervention, or otherwise having an 
impact on the subject should be reported as an AE, unless it is part of a clinical diagnosis already 
reported as an AE. 
All laboratory values outside the normal range are to be evaluated. 
8.3.1.2. 
Repeat Testing 
Repeat testing of any clinically significant laboratory test will be performed until the value 
returns to within the normal range, returns to the baseline level, or clinically stabilizes. 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 48 of 63 
8.3.2. 
COVID-19 Pandemic Accommodations 
The US Food and Drug Administration (FDA) has issued guidance to provide general 
considerations to assist sponsors in assuring the safety of study participants, maintaining 
compliance with Good Clinical Practice (GCP), and minimizing risks to trial integrity during the 
coronavirus disease 2019 (COVID-19) public health emergency. It is recognized that the 
COVID-19 public health emergency may impact the conduct of clinical trials. Challenges may 
arise, for example, from quarantines, site closures, travel limitations, interruptions to the supply 
chain for the investigational product, or other considerations if site personnel or study subjects 
become infected with COVID-19. These challenges may lead to difficulties in meeting 
protocol-specified procedures, including administering or using the investigational product or 
adhering to protocol-mandated visits and laboratory/diagnostic testing. Protocol modifications 
may be required, and there may be unavoidable protocol deviations due to COVID-19 illness 
and/or COVID-19 public health control measures. EmbarkNeuro, or its affiliated clinical 
research organizations (CROs), representatives, and clinical sites may need to make decisions 
regarding continuing study recruitment, continuing use of the investigational product for subjects 
already participating in the study, and the need to change subject monitoring during the study. 
EmbarkNeuro acknowledges that it is critical that study subjects are kept informed of changes to 
the study and monitoring plans that could impact them. EmbarkNeuro, following consultation 
with clinical investigators and Institutional Review Boards (IRBs), may determine that the 
protection of a subject’s safety, welfare, and rights is best served by continuing a subject in the 
study as per the protocol or by discontinuing the administration or use of the investigational 
product or even participation in the study. In addition, home health services may be utilized in 
this study per FDA guidance to enable subjects to remain on study and have testing performed 
per the protocol. It is generally the practice of EmbarkNeuro to implement protocol changes only 
after review and approval by the IRB, and in some cases, by the FDA. EmbarkNeuro or its 
affiliated CROs and clinical investigators will engage with IRBs as early as possible when urgent 
or emergent changes to the protocol or informed consent are anticipated as a result of 
COVID-19. Such changes to the protocol or investigational plan to minimize or eliminate 
immediate hazards or to protect the life and well-being of research subjects (e.g., to limit 
exposure to COVID-19) may be implemented without IRB approval or before filing an 
amendment to the Investigational New Drug (IND) application, but will be reported afterwards. 
These considerations are intended to remain in effect only for the duration of the public health 
emergency related to COVID-19. 
8.4. 
Adverse Events, Serious Adverse Events, and Other Safety 
Reporting 
8.4.1. 
Definition of Adverse Event 
AE Definition 
An AE is defined as any untoward medical occurrence in a clinical study subject administered 
a pharmaceutical product and does not necessarily have a causal relationship with this 
treatment. An AE can therefore be any unfavorable and unintended sign (including an 
abnormal laboratory finding), symptom, or disease temporally associated with the use of 
a treatment, whether or not related to the treatment. 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 49 of 63 
8.4.1.1. 
Events Meeting the Adverse Event Definition 
Events Meeting the AE Definition 
• Laboratory test results outside the normal range (hematology, coagulation, clinical 
chemistry, or urinalysis) or other safety assessments (e.g., ECG, vital signs measurements, 
questionnaires), including any that worsen in severity from baseline, which are considered 
clinically significant in the medical and scientific judgement of the Investigator. 
• Exacerbation of a chronic or intermittent pre-existing condition resulting in an increase 
in severity. 
• Condition detected or diagnosed that is new in onset or increased in severity from the 
baseline condition. 
• Signs, symptoms, or the clinical sequelae of a suspected intervention-intervention 
interaction. 
• Signs, symptoms, or the clinical sequelae of a suspected overdose of either study treatment 
or a concomitant medication. An overdose or incorrect administration of study treatment is 
not itself an AE, but it may result in an AE. 
8.4.1.2. 
Events NOT Meeting the Adverse Event Definition 
Events NOT Meeting the AE Definition 
• Any clinically significant abnormal laboratory findings or other abnormal safety 
assessments which are associated with the underlying disease, unless judged by the 
Investigator to be more severe than expected for the patient’s condition. 
• The disease/disorder being studied or expected progression, signs, or symptoms of the 
disease/disorder being studied, unless more severe than expected for the patient’s 
condition. 
• Medical or surgical procedure (e.g., endoscopy, appendectomy). The condition that leads 
to the procedure is the AE. 
• Pre-planned medical or surgical procedures, or pre-planned treatments or hospital 
admissions used to facilitate study visit arrival or procedures. 
• Situations in which an untoward medical occurrence did not occur (social and/or 
convenience admission to a hospital). 
• Anticipated day-to-day fluctuations of pre-existing disease(s) or condition(s) present 
or detected at the start of the study that do not worsen in severity. 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 50 of 63 
8.4.2. 
Definition of Serious Adverse Event (SAE) 
An SAE is defined as any untoward medical occurrence that at any dose: 
1. Results in death 
2. Is life-threatening 
• The term “life-threatening” refers to an event in which the patient was at risk of death 
at the time of the event. It does not refer to an event which hypothetically might have 
caused death if it were more severe. 
3. Requires inpatient hospitalization or prolongation of existing hospitalization 
• In general, hospitalization signifies that the participant has been detained (usually 
involving at least an overnight stay) at the hospital or emergency ward for observation 
and/or treatment that would not have been appropriate in the physician’s office 
or outpatient setting. Complications that occur during hospitalization are AEs. 
If a complication prolongs hospitalization or fulfills any other serious criteria, the event 
is serious. When in doubt as to whether “hospitalization” occurred or was necessary, 
the AE should be considered serious. 
• Hospitalization for preplanned procedure and/or study-related treatment or procedure 
that did not worsen from baseline is not considered an AE. 
• Hospitalization for elective treatment of a pre-existing condition that did not worsen 
from baseline is not considered an AE. 
4. Results in persistent disability/incapacity 
• Disability is defined as a substantial disruption of a person’s ability to conduct normal 
life functions, either reported or per clinical judgment. 
5. Is a congenital anomaly/birth defect (if exposure to product just before conception 
or during pregnancy resulted in an adverse outcome in the child) 
6. Other situations (Important medical events): 
• Medical or scientific judgment should be exercised in deciding whether SAE reporting 
is appropriate in other situations such as important medical events that may not be 
immediately life-threatening or result in death or hospitalization but may jeopardize the 
participant or may require medical or surgical intervention to prevent one of the other 
outcomes listed in the above definition. These events should usually be considered 
serious (e.g., invasive or malignant cancers, intensive treatment in an emergency room 
or at home for allergic bronchospasm, blood dyscrasias or convulsions that do not 
result in hospitalization, development of drug dependency or drug abuse). 
• Is any other important medical event that based upon appropriate medical judgment 
may jeopardize the patient or may require medical or surgical intervention to prevent 
one of the other outcomes listed in the definitions above (e.g., may not result in death, 
be life threatening, or require hospitalization). 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 51 of 63 
8.4.3. 
Time Period for Collecting AE and SAE Information 
All AEs will be recorded from study drug administration on Day 1 until Day 112 (see 
Section 8.3.1.1 regarding laboratory-associated AE assessments). 
Serious adverse events that occur subsequent to the signing of the informed consent (i.e., during 
the Screening Period) as a result of study participation will be recorded, as well as all SAEs 
occurring from study drug administration on Day 1 until Day 112. If the Investigator becomes 
aware of an SAE that is possibly related to the investigational drug treatment at any time after 
the final follow-up visit, the Sponsor should be notified immediately (i.e., within 24 hours). 
Report the SAE using the method described below. 
8.4.4. 
Recording of Adverse Events and/or Serious Adverse Events 
8.4.4.1. 
Terms of Reported Adverse Events 
All AEs will be recorded in the electronic case report form (eCRF), including start and stop 
dates, severity/grade, study drug relationship, and event outcome. 
The Investigator should attempt to establish a diagnosis of the event based on signs, symptoms, 
and/or other clinical information. Whenever possible, the diagnosis (not the individual 
signs/symptoms) should be documented as the AE. 
8.4.4.2. 
Detection of Adverse Events and Serious Adverse Events 
The Investigator will record all observed and reported AEs. In addition, each subject will be 
questioned with regard to AEs, but not occurrence of specific AEs. 
8.4.4.3. 
Severity of Adverse Events 
Severity of AEs will be graded as: 
• Mild: event may be noticeable to the subject; does not influence daily activities; usually 
does not require intervention 
• Moderate: event may be of sufficient severity to make the subject uncomfortable; 
performance of daily activities may be influenced; intervention may be needed 
• Severe: event may cause severe discomfort; usually interferes with daily activities; 
subject may not be able to continue in the study; treatment or other intervention usually 
needed 
If the severity of an AE changes more than once a day, the maximum severity for the event 
should be listed. If the severity changes over a number of days, these mini-events or changes 
should be recorded separately (i.e., having distinct onset dates). 
8.4.4.4. 
Causal Relationship with Study Treatment 
Assessment of Causality 
• The Investigator is obligated to assess the relationship between study drug and each 
occurrence of each AE/SAE. A “reasonable possibility” of a relationship conveys that 
there are facts, evidence, and/or arguments to suggest a causal relationship. Investigators 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 52 of 63 
Assessment of Causality 
should use their knowledge of the subject’s medical/disease history, the specific 
circumstances of the event, and potential alternative causes to determine if an AE is 
considered related to the study drug: 
o Definitely: The event has a plausible time relationship to drug intake, cannot be 
 
explained by the disease under study or other drugs, or has a definitive 
 
pharmacologic relationship to the study drug. 
o Probably: 
The event has a reasonable time relationship to drug intake, and is 
 
unlikely to be attributed to the disease under study or other drugs. 
o Possibly: 
The event has a reasonable time relationship to drug intake, and could also 
 
be attributed to the disease under study or other drugs. 
o Not related: The event has a time relationship to drug intake that makes a relationship 
 
impossible, or is clearly attributable to the disease under study, to other 
 
drugs, or to other factors or events. 
• The Investigator will consult the Investigator’s Brochure (IB) in the assessment and will 
use clinical judgment in the determination. 
• For each AE/SAE, the Investigator must document in the medical notes that the event has 
been reviewed and an assessment of causality provided. 
• There may be situations in which an SAE has occurred, and the Investigator has minimal 
information to include in the initial report to the Sponsor or designee. However, it is very 
important that the Investigator always assessment causality for every event before the 
initial transmission of the SAE data. The Investigator may change causality determination 
in light of follow-up information, and send an SAE follow-up report with the updated 
causality assessment. 
8.4.4.5. 
Follow-up of Adverse Events 
Adverse events should be followed until the AE has resolved, returned to baseline, or stabilized; 
withdrawal of consent; or death. 
8.4.5. 
Reporting of Serious Adverse Events (SAE) 
The Sponsor or designee must be notified using an SAE Report form within 24 hours of the 
Investigator becoming aware of an SAE. This timeframe is also applicable to follow-up 
information for previous reported SAEs. 
The Investigator is obligated to obtain supplemental measurements and/or evaluations as 
medically indicated or as requested by the Sponsor or designee to elucidate the nature and/or 
causality of the event as fully as possible. This may include additional laboratory tests or 
investigations, histopathological examinations, radiographic imaging, or consultation with other 
health care professionals. 
The SAE Report form and redacted medical records should be submitted to 
safety@safeharborpv.com or via fax to (919) 591-0004. 
Follow-up information will be reported in the same method as above. 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 53 of 63 
8.4.6. 
Reporting of Deaths 
Death must be reported if it occurs during the SAE reporting period as noted in Section 8.4.3. 
8.4.7. 
Pregnancy 
Details of all pregnancies in female subjects or in female partners of male subjects (after 
appropriate consent for use of medical information has been obtained) will be reported to the 
Sponsor or designee at the email noted in Section 8.4.5 within 24 hours of the Investigator’s 
awareness, using a Pregnancy Report form. 
While pregnancy itself is not considered to be an AE or SAE, any pregnancy complication or 
elective termination of a pregnancy for medical reasons will be reported as an AE or SAE. 
Abnormal pregnancy outcomes (e.g., spontaneous abortion, fetal death, stillbirth, congenital 
anomalies, ectopic pregnancy) are considered SAEs and will be reported as such. 
The subject/pregnant female partner will be followed to determine the outcome of the pregnancy. 
The Investigator will collect follow-up information on the subject/pregnant female partner 
and the neonate, and forward to the Sponsor or designee. 
Any female subject who becomes pregnant while participating in the study will discontinue 
study drug. 
8.4.8. 
Overdose 
Refer to Section 6.7 for information regarding ANC-501 overdose. 
8.4.9. 
Events of Special Interest- Lens Opacity 
Lens opacity is an event of special interest in this study. Slit lamp imaging of the lenses of both 
eyes will be taken at Screening, Day 56, follow-up Day 70 and follow-up Day 112 and sent to a 
central reader for assessment using the LOCS III. Additional slit lamp imaging may be 
performed during the treatment period and sent to a central reader for assessment using the 
LOCS III as per the investigator’s discretion. 
 LOCS III scale changes from baseline of ≥0.5 (Nuclear Opalescence), ≥0.5 (Cortical). ≥0.5 
(Posterior Subcapsular) and any changes considered to be clinically significant (e.g., incidence of 
cataract) are considered adverse events and events of special interest. Any changes considered to 
be clinically significant  will be immediately reported to the Sponsor and/or designee within 24 
hours of becoming aware of the event whether or not the event is deemed to be related to 
investigational product. 
8.5. 
Pharmacokinetics 
The serum concentrations and calculated PK parameters of ANC-501 will be assessed based on 
blood samples collected from subjects during the study. Collection time points for the assessment 
of PK are detailed in the Schedule of Events (Section 1.3). Additional details regarding sample 
collection timing will be included in a separate Laboratory Manual. 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 54 of 63 
8.6. 
Biomarker Evaluations 
Blood samples will be collected for future exploratory biomarker and genetic analysis. 
Collection time points are detailed in the Schedule of Events (Section 1.3). 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 55 of 63 
9. 
STATISTICAL CONSIDERATIONS 
9.1. 
Statistical Hypotheses 
No formal statistical hypotheses testing will be performed. 
9.2. 
Sample Size Determination 
Up to 20 subjects will be enrolled. The sample size was selected based on clinical and not 
statistical considerations. 
9.3. 
Analysis Populations 
For the purposes of analysis, the following populations are defined: 
Table 5: 
Definitions of Analysis Populations 
Analysis Population 
Definition 
Safety 
Includes all subjects who received at least one dose of ANC-501. 
This is the primary population for exposure and safety analyses. 
Efficacy 
Includes all subjects who received at least one dose of ANC-501 
and have at least one post-baseline efficacy evaluation. This is the 
primary population for efficacy endpoints. 
9.4. 
Statistical Analyses 
9.4.1. 
General Considerations 
A Statistical Analysis Plan (SAP) will be prepared as a separate document. The SAP will include 
a more technical and detailed description of the planned statistical summaries and will be 
finalized prior to closing of database. 
Continuous endpoints will be summarized with n, mean, standard deviation, median, minimum, 
and maximum. In addition, change from baseline values will be calculated at each time point and 
summarized descriptively. For categorical endpoints, descriptive summaries will include counts 
and percentages. 
Methodology and analyses for missing data will be defined in the SAP. 
9.4.2. 
Analysis of Safety 
The Safety Population will be used to provide descriptive summaries of safety data. Treatment-
emergent adverse events will be classified by type, incidence, severity, and causality. The overall 
incidence of adverse events will be summarized by Medical Dictionary for Regulatory Activities 
(MedDRA) System Organ Class (SOC) and Preferred Term (PT). Data for vital signs, clinical 
laboratory tests, ECG, physical examinations, and concomitant medication usage will also be 
summarized. 
Changes in LOCS III will be assessed from Screening to Day 56, 70 and 112 visits.  An increase 
from baseline in the LOCS III grade of ≥0.5 (Nuclear Opalescence), ≥0.5 (Cortical) or ≥0.5 
(Posterior Subcapsular)] in either eye and any changes considered to be clinically significant 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 56 of 63 
(e.g., incidence of cataract) as per the ophthalmologist or optometrist will be reported as an 
adverse event and an event of special interest. 
Suicidality data collected using the C-SSRS at baseline and at each visit during the active 
Open-Label Treatment Period will be listed for all subjects. The C-SSRS listings will include 
behavior type and/or category for suicidal ideation and suicidal behavior of the C-SSRS. 
9.4.3. 
Analysis of Efficacy 
The Efficacy Population will be used to analyze efficacy data. Efficacy data will be analyzed 
using appropriate descriptive statistics and pre-specified statistical methods, as well as other data 
presentation methods where applicable; subject listings will be provided for all efficacy data. 
Efficacy data will be summarized descriptively. 
9.4.4. 
Analysis of Pharmacokinetics 
Plasma concentrations of ANC-501 will be listed by individual and summarized by treatment in 
tabular format using descriptive statistics (number, arithmetic mean, standard deviation, median, 
minimum, maximum, geometric mean, and geometric coefficient of variation). ANC-501 plasma 
concentrations will be plotted against time points by dose level/cohort (mean and individual). 
ANC-501 concentration data may be combined with concentration data from other studies to 
build a PK model. 
9.5. 
Interim Analysis 
No formal interim analysis is planned. 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 57 of 63 
10. 
ADMINISTRATIVE CONSIDERATIONS 
10.1. 
Protocol Compliance/Deviations 
The Investigator agrees to conduct the study in compliance with the protocol and all 
amendments. Any protocol deviation(s) should be recorded in the source documents along with 
a clear description of the deviation(s) and cause. 
10.2. 
Study Termination 
Premature termination of this study may occur because of a regulatory authority decision,  change 
in opinion of the Institutional Review Board (IRB), issues with study drug, or at the discretion of 
the Sponsor. In addition, the Sponsor retains the right to suspend or discontinue ANC-501 
development at any time. 
The Sponsor will notify the Investigator if the study is prematurely terminated. The Investigator 
must promptly contact all subjects to arrange final study visit(s) and procedures, as directed by 
the Sponsor. 
10.3. 
Case Report Forms 
An eCRF is required for each enrolled subject. For screen failure subjects, a minimal set of data 
will be collected as noted in Section 5.4. Data entered into the eCRF must be consistent with the 
source documents. 
The Investigator has ultimate responsibility for the collection and reporting of all data entered 
in the eCRF and any other data collection forms, and will be electronically signed by the 
Investigator to attest that the data contained in the eCRF are accurate. 
10.4. 
Source Documents 
Source documents provide evidence for the existence of the subject and substantiate the integrity 
of the data collected. Source documents (e.g., hospital records, patient hard-copy/electronic files, 
clinical/office charts, pharmacy dispensing records, X-rays) are filed at the Investigator’s site. 
Data entered in the eCRF that are transcribed from source documents must be consistent with the 
source documents or the discrepancies must be explained. The Investigator may need to request 
previous medical records or transfer records, depending on the study. Also, current medical 
records must be available. 
Study monitors will perform ongoing source data verification to confirm that data entered into 
the eCRF by authorized site personnel are accurate, complete, and verifiable from source 
documents; that the safety and rights of participants are being protected; and that the study is 
being conducted in accordance with the currently approved protocol and any other study 
agreements, International Council for Harmonisation Good Clinical Practice guideline E6 
(ICH GCP), and all applicable regulatory requirements. 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 58 of 63 
10.5. 
Data Protection 
Subjects will be assigned a unique identifier by the Sponsor. Any subject records or datasets that 
are transferred to the Sponsor will contain the identifier only; names or any information which 
would make the subject identifiable will not be transferred. 
The subject must be informed that his/her personal study-related data will be used by the 
Sponsor in accordance with local data protection law. The level of disclosure must also be 
explained to the subject, who will be required to give consent for the data to be used as described 
in the informed consent form (ICF). 
The subject must also be informed that medical records may be examined by Clinical Quality 
Assurance auditors or other authorized personnel appointed by the Sponsor, by appropriate IRB 
members, and by inspectors from regulatory authorities. 
10.6. 
Recordkeeping 
The Investigator agrees to keep and maintain records that would permit the Sponsor 
and regulatory authorities to evaluate/audit the study. These include the identity of subjects, IRB 
correspondence, original signed ICFs, source documents, the eCRFs, SAE/Pregnancy Report 
forms, drug storage/accountability/disposition records, and correspondence (e.g., letters, email, 
meeting minutes, telephone call records) with the Sponsor or designee. Records should be 
retained by the Investigator for a period of 2 years after the last marketing application approval 
(or if not approved, for 2 years following the discontinuation of the investigational use), 
according to the ICH guidelines, local regulations, or as specified in the clinical study agreement, 
whichever is longer. 
If the Investigator becomes unable to retain study records for any reason (e.g., retirement, 
relocation), they must be transferred to the Sponsor or to a designee acceptable to the Sponsor 
(e.g., another investigator, another institution, or independent third party). 
10.7. 
Quality Control and Quality Assurance 
The Sponsor or designee will conduct periodic visits or audits during or after the study to ensure 
that the protocol and GCP are being followed. The monitors may review the drug storage area, 
study drug stocks, drug storage/accountability/disposition records, source documents, and 
regulatory documentation. The Investigator and institution will permit direct access to the 
physical spaces, records, and source documents to perform this verification to the Sponsor or 
designee monitors/auditors, IRB members, and appropriate regulatory authority inspectors. 
The Investigator must immediately notify the Sponsor or its designee of any regulatory 
inspection notification, will cooperate with the Sponsor or designee to prepare the study site 
for the inspection, will be present during the inspection along with relevant study staff, will 
allow the Sponsor or designee to be present during the inspection, and will provide copies of any 
inspection findings. 
10.8. 
Financial Disclosure  
Investigators and sub-investigators will provide the Sponsor with sufficient, accurate financial 
information as requested to allow the Sponsor to submit complete and accurate financial 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 59 of 63 
certification or disclosure statements to the appropriate regulatory authorities. Investigators are 
responsible for providing information on financial interests during the course of the study and for 
1 year after completion of the study. 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 60 of 63 
11. 
ETHICS 
11.1. 
Ethical Conduct of the Study 
The study will be conducted in accordance with legal and regulatory requirements, as well as the 
general principles set forth in the International Ethical Guidelines for Biomedical Research 
Involving Human Subjects (Council for International Organizations of Medical Sciences 2002), 
ICH GCP Guidelines, and the Declaration of Helsinki (World Medical Association 1996 and 
2008). 
In addition, the study will be conducted in accordance with the protocol, ICH GCP, 
and applicable local regulatory requirements and laws. 
11.2. 
Written Informed Consent 
An informed consent form (ICF) meeting the requirements of 21 CFR §50, local regulations, 
ICH guidelines, Health Insurance Portability and Accountability Act (HIPAA) requirements 
(where applicable), must be reviewed and approved by the Sponsor, approved by the IRB before 
use, and available for inspection. 
The Investigator or authorized representative will explain the nature of the study to the subject 
and answer all questions regarding the study. Subjects must be informed that their participation 
is voluntary, and if they choose to participate, will be required to sign the approved ICF. The 
medical record must include a statement that written informed consent was obtained before any 
study procedures were performed, along with the date written consent was obtained. The 
authorized person obtaining the informed consent must also sign the ICF. The original signed 
ICF will be retained in the Investigator’s records, with a copy provided to the subject. 
Subjects must be re-consented to the most current version of the ICF during their participation in 
the study. 
11.3. 
Institutional Review Board/Independent Ethics Committee 
The protocol, protocol amendments, ICF, Investigator’s Brochure (IB), and other relevant 
documents (e.g., advertisements) must be submitted to an IRB by the Investigator, and reviewed 
and approved by the IRB before the study is initiated. 
Protocols and any substantial amendments to the protocol will require health authority approval 
prior to initiation except for changes necessary to eliminate an immediate hazard to study 
subjects. Any amendments to the protocol will require IRB approval before implementation of 
changes made to the study design, except for changes necessary to eliminate an immediate 
hazard to study subjects. 
The Investigator will provide written summaries of the status of the study and any other 
significant safety findings to the IRB annually or more frequently in accordance with its 
requirements, policies, and procedures. At the end of the study, the Investigator will notify the 
IRB of the conclusion of the study and its outcome. 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 61 of 63 
12. 
PUBLICATION POLICY 
The Sponsor will comply with the requirements for publication of study results. In accordance 
with standard editorial and ethical practice, the Sponsor will generally support publication 
of multicenter studies only in their entirety and not as individual site data. 
Authorship will be determined by mutual agreement and in line with International Committee of 
Medical Journal Editors authorship requirements. 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 62 of 63 
13. 
REFERENCES 
1. World Health Organization, Fact Sheet Number 369. 2012. 
2. Chen YW, Dilsaver SC. Lifetime rates of suicide attempts among subjects with bipolar 
and unipolar disorders relative to subjects with other Axis I disorders. Biol Psychiatry. 
1996; 39(10): 896–899. 
3. Bromet E, Andrade LH, Hwang I, Sampson NA, Alonso J, de Giralamo G, et al. 
Crossnational epidemiology of DSM-IV major depressive episode. BMC Med. 2011; 9: 
90. 
4. Kessler RC, Berglund P, Demler O, Jin R, Koretz D, Merikangas K, et al. The 
epidemiology of major depressive disorder: results from the National Comorbidity 
Survey Replication (NCS-R). JAMA. 2003; 289(23): 3095–3105. 
5. Knol MJ, Twisk JWR, Beekman ATF, Heine RJ, Snoek FJ, Pouwer F. Depression as a 
risk factor for the onset of type 2 diabetes mellitus. A meta-analysis. Diabetologia. 2006; 
49(5): 837–845. 
6. Thase ME, Entsuah AR, Rudolph RL. Remission rates during treatment with venlafaxine 
or selective serotonin reuptake inhibitors. Br J Psychiatry. 2001. 178: 234–241. 
7. Rush AJ, Trivedi MH, Wisniewski SR, Nierenberg AA, Stewart JW, Warden D, et al. 
Acute and longer-term outcomes in depressed outpatients requiring one or several 
treatment steps: a STAR*D report. Am J Psychiatry. 2006; 163(11): 1905–1917. 
8. Aguilera G, Rabadan-Diehl C. Vasopressinergic regulation of the hypothalamic-pituitary-
adrenal axis: implications for stress adaptation. Regul Pept. 2000; 96(1-2): 23–29. 
9. Vale W, Spiess J, Rivier J. Characterization of a 41-residue ovine hypothalamic peptide 
that stimulates secretion of corticotropin and beta-endorphin. Science. 1981; 213(4514): 
1394–1397. 
10. de Winter RFP, van Hemart AM, DeRijk RH, Zwinderman KH, Frankhuijzen-Serievogel 
AC, Wiegant VM, et al. Anxious-retarded depression: relation with plasma vasopressin 
and cortisol. Neuropsychopharmacology. 2003; 28(1): 140–147. 
11. van Londen L, Goekoop JG, van Kempen GM, Frankhuijzen-Sierevogel AC, Wiegant 
VM, van der Velde EA, et al. Plasma levels of arginine vasopressin elevated in patients 
with major depression. Neuropsychopharmacology. 1997; 17(4): 284–292. 
12. Inder WJ, Donald RA, Prickett TC, Frampton CM, Sullivan PF, Mulder RT, et al. 
Arginine vasopressin is associated with hypercortisolemia and suicide attempts in 
depression. Biol Psychiatry. 1997; 42(8): 744–747. 
13. Meynen G, Unmehopa UA, van Heerikhuize JJ, Hofman MA, Swaab DF, Hoogendijk 
WJG. Increased arginine vasopressin mRNA expression in the human hypothalamus in 
depression: A preliminary report. Biol Psychiatry. 2006; 60(8): 892–895. 
14. Purba JS, Hoogendijk WJ, Horman MA, Swaab DF. Increased number of vasopressin- 
and oxytocin-expressing neurons in the paraventricular nucleus of the hypothalamus in 
depression. Arch Gen Psychiatry. 1996; 53(2): 137–143. 


EmbarkNeuro 
 
Clinical Study Protocol 
ANC-501 
 
ANC501D0005 
03 March 2023 Version 6.0 
Confidential 
Page 63 of 63 
15. Dinan TG, Lavelle E, Scott LV, Newell-Price J, Medbak S, Grossman AB. Desmopressin 
normalizes the blunted adrenocorticotropin response to corticotropin-releasing hormone 
in melancholic depression: evidence of enhanced vasopressinergic responsivity. J Clin 
Endocrinol Metab. 1999; 84(6): 2238–2240. 
16. Serradeil-Le Gal C, Wagnon J, Valette G, Pascal M, Maffrand JP, Le Fur G. Nonpeptide 
vasopressin receptor antagonists: development of selective and orally active V1a, V2 and 
V1b receptor ligands. Prog Brain Res. 2002; 139: 197–210. 
17. Lolait SJ, O’Carroll AM, Mahan LC, Felder CC, Button DC, Young 3rd WS, et al. 
Extrapituitary expression of the rat V1b vasopressin receptor gene. Proc Natl Acad Sci 
USA. 1995; 92(15): 6783–6787. 
18. Griebel G,Stemmelin J, Serradeil-Le Gal C, Soubrié P. Non-peptide vasopressin V1b 
receptor antagonists as potential drugs for the treatment of stress-related disorders. Curr 
Pharm Des. 2005; 11(12): 1549–1559. 
19. Serradeil-Le Gal C, et al. Characterization of (2S,4R)-1-[5-chloro-1-[(2,4-
dimethoxyphenyl)sulfonyl]-3-(2-methoxy-phenyl)-2-oxo- 2,3-dihydro-1H-indol-3-yl]-4-
hydroxy-N,N-dimethyl-2-pyrrolidine carboxamide (SSR149415), a selective and orally 
active vasopressin V1b receptor antagonist. J Pharmacol Exp Ther. 2002; 300(3): 
1122–1130. 
20. Iijima M, Chaki S. An arginine vasopressin V1b antagonist, SSR149415 elicits 
antidepressant-like effects in an olfactory bulbectomy model. Prog 
Neuropsychopharmacol Biol Psychiatry. 2007; 31(3): 622–627. 
21. Iijima M, Ito A, Kurosu S, Chaki S. Pharmacological characterization of repeated 
corticosterone injection-induced depression model in rats. Brain Res. 2010; 1359: 75–80. 

SAP_001.pdf: 
1 
 
Statistical Analysis Plan 
 
 
 
 
Protocol Title 
A Phase 2 Study of ANC-501 in the Treatment of Adults 
with Major Depressive Disorder 
Protocol Number: 
ANC501D0005 
Protocol Version: 
6.0 Amendment 05, 03MAR2023 
SAP Version: 
1.1 
Date: 
29SEP2023 
Sponsor: 
Ancora Bio d/b/a EmbarkNeuro, Inc. 
1111 Broadway 
Suite 1300 
Oakland, CA 95607 
 
 
Author: 
Carrie Anderson, PhD 
Instat Clinical Research 
 
 
 
 
 
 
 
 
 
This study will be conducted in compliance with the protocol, Good Clinical Practice 
and all other applicable regulatory requirements, including the archiving of 
essential documents. 
 
Confidential Information 
No use or disclosure outside EmbarkNeuro is permitted without prior written 
authorization from EmbarkNeuro. 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
2 
 
 
 
 
Signatures/Approvals 
 
 
 
 
 Philip Perera 
 
Philip Perera (Sep 30, 2023 07:34 PDT) 
Signing Reason: I approve this document. 
 
Philip Perera, MD, MBA 
Chief Medical Officer 
EmbarkNeuro, Inc. 
 
 
 
 
 
 Carrie Anderson 
 
Carrie Anderson (Sep 30, 2023 19:13 PDT) 
Signing Reason: I am the author of this document. 
 
Carrie Anderson, PhD 
Senior Biostatistician 
Instat Clinical Research 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
3 
 
 
Table of Contents 
Signatures/Approvals........................................................................................................... 2 
Table of Contents ................................................................................................................ 3 
List of Abbreviations ........................................................................................................... 6 
1. 
Introduction .................................................................................................................. 8 
2. 
Study Objectives .......................................................................................................... 8 
2.1. Primary Objectives ............................................................................................. 8 
2.2. Secondary Objectives ..........................................................................................8 
2.3. Exploratory Objectives ....................................................................................... 8 
3. 
Study Design ................................................................................................................ 9 
4. 
Analysis Populations .................................................................................................. 15 
5. 
Statistical Methods ..................................................................................................... 15 
5.1. Handling Missing Data ..................................................................................... 16 
5.2. Definitions ........................................................................................................ 16 
6. 
Analysis Considerations ............................................................................................. 18 
6.1. Determination of Sample Size .......................................................................... 18 
6.2. Adjustments for Covariates .............................................................................. 18 
6.3. Interim Analysis and Data Monitoring ............................................................. 18 
6.4. Examination of Subgroups ............................................................................... 18 
6.5. Multicenter Studies ............................................................................................ 18 
6.6. Multiplicity Comparison/Multiplicity ............................................................... 18 
7. 
Population Analysis ................................................................................................... 19 
7.1. Subject Disposition ........................................................................................... 19 
7.2. Protocol Deviations .......................................................................................... 19 
7.3. Demographic and Baseline Characteristics....................................................... 19 
7.4. Baseline Major Depressive Disorder Characteristics........................................ 20 
7.5. Screening Mental Health Assessments ............................................................. 20 
7.5.1. 
Structured Clinical Interview for DSM-5 – Clinical Trials Version ... 20 
7.5.2. 
SAFER Interview ................................................................................ 20 
7.5.3. 
Childhood Trauma Questionnaire ....................................................... 20 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
4 
 
 
7.6. Medical History ................................................................................................. 21 
7.7. Prior and Concomitant Medications ................................................................. 21 
7.8. Duration and Compliance ................................................................................. 22 
8. 
Efficacy Analysis ....................................................................................................... 22 
8.1. Efficacy Parameters ........................................................................................... 22 
8.2. Primary Efficacy Analysis ................................................................................ 23 
8.2.1. 
MADRS Total Score – Day 56 Only .................................................. 23 
8.3. Secondary Efficacy Analysis ............................................................................ 24 
8.3.1. 
MADRS Total Score – Scheduled Visits ............................................ 24 
8.3.2. 
MADRS Responders ........................................................................... 24 
8.3.3. 
MADRS Remitters .............................................................................. 25 
8.3.4. 
Hamilton Anxiety Scale (HAM-A) ..................................................... 25 
8.3.5. 
Clinical Global Impression-Severity (CGI-S) ..................................... 25 
8.3.6. 
Short Form-Health Survey (36-item) (SF-36) ..................................... 26 
8.3.7. 
Clinical Global Impression-Improvement (CGI-I) ............................. 27 
8.3.8. 
Patient Global Impression-Improvement (PGI-I) ............................... 27 
8.4. Exploratory Efficacy Analysis .......................................................................... 28 
8.4.1. 
Hamilton Depression Rating Scale (6-item) (HAMD-6) .................... 28 
8.4.2. 
General Anxiety Disorder (7-item) (GAD-7)...................................... 28 
9. 
Safety Analysis .......................................................................................................... 28 
9.1. Safety Parameters ............................................................................................. 29 
9.2. Primary Safety Analysis ................................................................................... 29 
9.2.1. 
Adverse Events .................................................................................... 29 
9.2.2. 
Vital Signs, Height, and Weight ......................................................... 30 
9.2.3. 
Electrocardiogram ............................................................................... 30 
9.2.4. 
Clinical Laboratory Tests .................................................................... 31 
9.2.5. 
Physical Examination .......................................................................... 31 
9.2.6. 
Lens Opacities Classification System III (LOCS III) ......................... 32 
9.2.7. 
Columbia-Suicide Severity Rating Scale (C-SSRS) ........................... 33 
9.3. Secondary Safety Analysis ............................................................................... 34 
9.3.1. 
Pharmacokinetics ................................................................................. 34 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
5 
 
 
9.4. Exploratory Safety Analysis ............................................................................. 35 
9.4.1. 
Cortisol ................................................................................................ 35 
9.4.2. 
Wearable Device Biometrics ............................................................... 35 
9.4.3. 
Biomarkers .......................................................................................... 36 
10. Tables, Listings, and Figures ..................................................................................... 36 
11. Protocol and SAP Differences ................................................................................... 36 
12. References .................................................................................................................. 36 
 
 
 
 
 
 
 
SAP Version 
Changes from Previous Version 
1.0 
Not applicable. 
1.1 
Clarify reason for change in definition of efficacy population and 
remove language pertaining to a treatment policy estimand. 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
6 
 
 
List of Abbreviations 
 
ADaM 
Analysis Data Model 
AE 
Adverse Event 
ATC 
Anatomical Therapeutic Chemical 
C-SSRS 
Columbia-Suicide Severity Rating Scale 
CGI-I 
Clinical Global Impression – Improvement 
CGI-S 
Clinical Global Impression – Severity 
CRF 
Case Report Form 
CRO 
Contract Research Organization 
CS 
Clinically Significant 
CTQ 
Childhood Trauma Questionnaire (CTQ) 
ECG 
Electrocardiogram 
EDC 
Electronic Data Capture 
EFF 
Efficacy Population 
EOT 
End of Treatment 
ET 
Early Termination 
GAD-7 
General Anxiety Disorder (7-item) 
HAM-A 
Hamilton Anxiety Scale 
HAMD-6 
Hamilton Depression Rating Scale (6-item) 
IB 
Investigator’s Brochure 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonization 
ITT 
Intent to Treat 
LOCS III 
Lens Opacities Classification System III 
MADRS 
Montgomery-Åsberg Depression Rating Scale 
MDD 
Major Depressive Disorder 
MedDRA 
Medical Dictionary of Regulatory Affairs 
NCS 
Not Clinically Significant 
PGI-I 
Patient Global Impression of Improvement (PGI-I) 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
7 
 
 
 
PK 
Pharmacokinetics 
SAE 
Serious Adverse Event 
SAF 
Safety Population 
SAP 
Statistical Analysis Plan 
SCID-5-CT 
Structured Clinical Interview for DSM-5 – Clinical Trials Version 
SD 
Standard Deviation 
SDTM 
Study Data Tabulation Model 
SF-36 
Short Form-Health Survey (36-item) 
SOC 
System Organ Class 
TEAE 
Treatment-Emergent Adverse Event 
TLF 
Tables, Listings, and Figures 
WHO 
World Health Organization 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
8 
 
 
1. 
Introduction 
EmbarkNeuro is conducting a study to investigate a new treatment for Major Depressive 
Disorder (MDD). The study background, design and subject assessments for the study 
are described in the study specific protocol. 
The statistical methods to be implemented during the analyses of data collected within 
the scope of this study will be outlined in this document. The purpose of this plan is to 
provide specific guidelines from which the statistical analysis will proceed. Any 
deviations from this plan will be documented in the clinical study report. 
2. 
Study Objectives 
2.1. Primary Objectives 
The primary objectives of this study are: 
 
• To evaluate the effect of treatment with ANC-501 capsules on depressive 
symptoms in subjects with MDD 
• To evaluate the safety and tolerability of ANC-501 capsules in subjects with 
MDD 
2.2. Secondary Objectives 
The secondary objectives of this study are: 
 
• To evaluate effect of ANC-501 at all time points and percent reduction in total 
symptom scores compared with baseline 
• To assess the pharmacokinetic (PK) profile of ANC-501 capsules in plasma using 
a sparse sampling model 
2.3. Exploratory Objectives 
The exploratory objectives of this study are: 
 
• To evaluate changes in 12-hour urine, plasma, and saliva cortisol 
• To evaluate changes in biometrics 
• To evaluate biological samples as potential biomarkers and metabolites 
• Patient reported changes in depression and anxiety symptoms 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
9 
 
 
3. 
Study Design 
This is a single-arm, open-label Phase 2 study to assess the safety, tolerability, PK, and 
efficacy of ANC-501 oral capsules as adjunctive treatment in subjects diagnosed with 
MDD. 
With the exception of subjects permitted to use current stable antidepressant treatment, 
initiation of psychotropic medications and other medications that may potentially have an 
impact on efficacy or safety endpoints will not be allowed between screening and 
completion of the Day 56 assessments. Psychotropic medications, which must have been 
initiated at least 8 weeks prior to screening, must remain at a stable dose until completion 
of the Day 56 assessments. During the study, efficacy and safety assessments will be 
performed. Blood samples will also be collected for PK analysis of ANC-501 and future 
exploratory biomarker and genetic analysis. A wearable watch-like device will collect 
data on biometrics, which may be used for exploratory analyses. 
Subjects will receive 50 mg ANC-501 Capsules (5  10 mg capsules) orally once daily at 
least 1 hour prior to the morning meal whenever possible for 8 weeks beginning with the 
first dose administered in the clinic on Day 1 and will return to the clinic for scheduled 
visits on Days 8, 15, 29, 43, and 56. Follow-up visits will occur on Day 70 and 112. All 
scheduled visits after Day 1 allow a visit window of  3 days. 
Total duration of participation is 20 weeks (up to 30 days screening, 8 weeks dosing, 8 
weeks follow-up). 
A summary of the schedule of events is provided in Table 1 below. 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
10 
 
 
 
Table 1: Schedule of Events 
 
Study Procedure 
Screening 
Period 
Open-Label Treatment Period a 
Follow-up Period b 
Visit Days 
Window 
-30 to -1 
Day 1 c 
Day 8 
(3 days) 
Day 15 
(3 days) 
Day 29 
(3 days) 
Day 43 
(3 days) 
Day 56/ET 
(3 days) 
Day 70 
(3 days) 
Day 112 
(3 days) 
Informed Consent 
X 
Demographics 
X 
Medical history 
X 
Full physical examination 
X 
X 
Body weight/height/BMI 
d 
X 
X 
Vitals e 
X 
X 
X 
X 
12-lead ECG 
X 
X 
X 
Safety laboratory tests f 
X 
X 
X 
X 
X 
Urine drug and alcohol 
breath test screen g 
X 
X 
Urine pregnancy test h 
X 
X 
Hepatitis and HIV 
serology 
X 
Sparse PK blood 
sampling i 
X 
X 
X 
X 
X 
X 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
11 
 
 
 
Exploratory biomarker 
sampling j 
X 
X 
X 
Genetic sampling k 
X 
Ophthalmic exam and 
LOCS III l 
X 
X 
X 
X 
SCID-5-CT diagnostic 
interview 
X 
MADRS 
X 
X 
X 
X 
X 
X 
X 
X 
HAM-A 
X 
X 
X 
X 
X 
X 
X 
X 
CGI-S 
X 
X 
X 
X 
X 
X 
X 
X 
C-SSRS m 
X 
X 
X 
X 
X 
X 
X 
X 
HAMD-6 n 
X 
GAD-7 n 
X 
SF-36 n 
X 
EMAW Orienting 
Questions n 
X 
CTQ 
X 
CGI-I 
X 
X 
X 
X 
X 
X 
PGI-I 
X 
X 
X 
X 
X 
X 
SAFER Interview (central 
rater) o 
X 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
12 
 
 
 
Inclusion/Exclusion 
Criteria Review 
X 
X 
12-hour urine collection 
for cortisol instructions p 
X 
X 
Plasma and saliva cortisol 
sample collection 
X 
X 
Wearable monitoring 
instructions and Mobile 
application training 
X 
Continuous Wearable 
monitoring q 
X 
Site download of 
Wearable data 
X 
X 
X 
X 
X 
ANC-501 dosing r 
X 
ANC-501 Dosing Diary 
dispensed and reviewed 
by site 
X 
Dispense study 
medication 
X 
X 
X 
X 
X 
Adverse events s 
X 
X 
Prior/Concomitant 
Medications t 
X 
X 
 
Abbreviations: AE = Adverse Event; BMI = body mass index; CGI-I = Clinical Global Impression-Improvement; CGI-S = Clinical Global Impression-Severity; C-SSRS = Columbia-Suicide Severity 
Rating Scale; CTQ = Childhood Trauma Questionnaire, 28-item; ECG = electrocardiogram; ET = early termination; HAM-A = Hamilton Anxiety Rating Scale; HIV = human immunodeficiency virus; 
LOCS III= Lens Opacities Classification System III; MADRS = Montgomery-Åsberg Depression Rating Scale; MDD = major depressive disorder; MGH ATRQ = Massachusetts General Hospital 
Antidepressant Treatment Response Questionnaire; PGI-I = Patient Global Impression of Improvement; PK = pharmacokinetic; SAE = serious adverse event; SAFER = State vs trait, Assessability, Face 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
13 
 
 
 
and Ecological validity, Rule of 3Ps (pervasive, persistent, and pathological); SCID-5-CT = Structured Clinical Interview for DSM-5 – Clinical Trials Version; HAMD-6= Hamilton Depression Rating 
Scale (6-item); GAD-7= General Anxiety Disorder (7-item); SF-36 = Short Form-Health Survey (36-item). 
a. 
Subjects who discontinue study treatment before completing the study and those who prematurely withdraw from the study for any reason should undergo Day 56 visit 
procedures in-clinic as soon as possible and schedule the Day 70 safety assessments (at +2 weeks post ET visit) and Day 112 follow-up ophthalmic visit (at +8 weeks post ET 
visit). 
b. 
Subjects will return to the site on Day 70 and 112 for a follow-up visit. 
c. 
Day 1 procedures are to be completed prior to dosing, except for sparse PK sampling which will be performed prior to dosing and between 0.75 and 2 hours post-dose. 
d. 
Body weight and height will be measured at screening and BMI will be calculated. Only body weight and BMI calculation will be performed at subsequent visits. 
e. 
Vital signs include oral temperature (°C), respiratory rate, heart rate, and blood pressure (supine and standing). 
f. 
Safety laboratory tests will include hematology, serum chemistry, coagulation, and urinalysis. 
g. 
Urine toxicology for selected drugs of abuse (barbiturates, amphetamines, cocaine, opioids, cannabis) and breath test for alcohol. 
h. 
Urine pregnancy test will be conducted only in female subjects of childbearing potential. Female subjects who prematurely discontinue will have a pregnancy test performed 
at the ET visit. 
i. 
A blood sample for sparse PK sampling will be collected pre-dose and between 0.75 and 2.0 hours post-dose at the Day 1 visit, and between 0.75 and 2.0 hours post-dose at 
all other indicated visits whenever possible (with date/time of previous dose provided by the subject). 
j. 
A blood sample for exploratory biomarker analysis will be collected in conjunction with PK sampling on Days 1 (pre- post-dose), 8, and 56. 
k. 
A genetic sample (blood) for exploratory biomarker testing will be collected before the first dose of drug. 
l. 
Ophthalmic exam, including slit-lamp imaging, may be performed by an appropriately trained and qualified individual as delegated by the PI and documented in the Study 
Delegation Log. However, a licensed ophthalmologist or optometrist must oversee this work and will make the determination on subject eligibility as per inclusion criteria #4. 
Slit lamp imaging of the lens of both eyes will be taken and sent to a central reader for assessment using the LOCS III. Ideally the exam would be performed after cortisol 
results are known and the subject is otherwise deemed eligible to continue with screening assessments. Additional slit lamp imaging of the lens of both eyes may be 
performed during the treatment period and sent to a central reader for assessment using the LOCS III, per Investigator’s discretion. 
m. The “Baseline/Screening” C-SSRS form will be completed during screening. The “Since Last Visit” C-SSRS form will be completed at any time of day at all subsequent time 
points. 
n. 
The Short Form-Health Survey (36-item) (SF-36) weekly. The Hamilton Depression Rating Scale (6-item) (HAMD-6) and the General Anxiety Disorder (7-item) (GAD-7) 
will be completed up to twice a day for a minimum of three days per week (to achieve 6 collections per week). Subjects will answer EMA orienting questions up to three 
times per day in conjunction with the other assessments being collected via the EMA application on their cell phones or the provided device. 
o. 
Conducted by central reader after Screening visit, but prior to Day 1 visit. The MADRS, MGH ATRQ and confirmation of MDD will be conducted by central rater for 
determination of eligibility. 
p. 
Study site personnel will dispense equipment and train subjects on 12-hour urine collection for measuring cortisol level. At the Day 43 visit, study site personnel will dispense 
equipment and retrain subjects on the 12hour urine collection to measure cortisol levels (the collection will be returned to the site at the Day 56 visit). On Day 54, the site will 
contact the subject to remind them to begin collection on Day 55. 
q. 
Subjects will wear their wearable watch-like device throughout the day and night except when charging. 
r. 
At the Day 1 visit, subjects confirmed as eligible will receive a 50 mg dose of study drug at the study site prior to eating. All other doses will be taken by the subjects at home 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
14 
 
 
 
s. 
All AEs will be recorded from study drug administration on Day 1 until Day 112visit. Serious adverse events that occur subsequent to the signing of the informed consent 
(i.e., during the Screening Period) as a result of study participation will be recorded, as well as all SAEs occurring from study drug administration on Day 1 until Day 112 
visit. 
t. 
Includes medications taken within 30 days prior to informed consent and throughout the study 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
15 
 
 
 
4. 
Analysis Populations 
Safety Population (SAF): The Safety population will consist of all subjects who 
received at least one dose of ANC-501. This population will be used for all Safety 
analyses. 
Enrolled Subjects: The date the first dose of ANC-501 is administered is the date 
a subject is considered enrolled into the study. Enrolled subjects and the safety 
population are equivalent. 
Efficacy Population (EFF): The Efficacy population will consist of all subjects who 
received at least one dose of ANC-501 and have completed treatment. This population 
will be used for all Efficacy analyses. 
EFF differs from the protocol due to two subjects who were discontinued near the 
beginning of the study due to a failed Day 1 drug screen. 
5. 
Statistical Methods 
The statistical analyses will be reported using tables, listings, and figures (TLFs). 
Numbering for TLFs will be based on the recommended numbering convention provided 
by the International Conference on Harmonization (ICH). Continuous variables will be 
summarized with means, standard deviations, medians, minimums, and maximums unless 
mentioned otherwise in this statistical analysis plan (SAP). Categorical variables will be 
summarized by counts and percentage of subjects in corresponding categories. Footnotes 
will be used to specify the percent calculation when applicable. 
Tables will be summarized for scheduled visits, and individual subject data obtained via 
the electronic data capture (EDC) system and from external vendors may be presented in 
listings, as indicated in this SAP. If there are multiple readings taken per visit, the most 
recent reading from that visit will be using in summaries, unless there are pre-dose and 
post-dose readings taken at the same visit, as mentioned elsewhere in this SAP. 
The analyses described in this plan are considered a priori, in that they have been defined 
prior to database lock. Any analysis added after the database lock will be considered post 
hoc and exploratory. Post hoc analyses will be labeled as such on the output and 
identified in the CSR. All analyses and tabulations will be performed using SAS version 
9.4 or higher. TLFs will be presented in .pdf format. SDTM datasets will be produced 
using Version 3.3 of the Implementation Guide. ADaM datasets will be produced using 
Version 1.0 of the Implementation Guide. Upon completion, all SAS programs used to 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
16 
 
 
produce datasets (SDTM, ADaM) and tables will be validated by an independent 
programmer. The validation process will be used to confirm that statistically valid 
methods have been implemented and that all data manipulations and calculations are 
accurate. Checks will be made to ensure accuracy, consistency with this SAP, 
consistency within tables, and consistency between tables and corresponding listings. 
5.1. Handling Missing Data 
For incomplete dates related to adverse events and concomitant medications, the dates 
will be imputed as follows: 
• Incomplete start dates missing day with known month will be imputed as the later 
of (a) day of first dose started in that month or (b) first of the month. 
• Incomplete start dates missing month and day will be imputed as the later of (a) 
month and day of first dose started in that year or (b) January 1. 
• Dates missing year regardless of month or day will be considered as missing. 
• An unknown start time with known or imputed date will be imputed with a time 
of 23:59 for events or medications on Day 1 or as 00:00 on other days. 
• An unknown end time with known or imputed date will be imputed the same as a 
missing start time unless the event or medication is ongoing. 
• Events with completely unknown start dates will be considered as treatment- 
emergent adverse events unless otherwise indicated by a known end date. 
• Medications with completely unknown start dates will be considered as 
concomitant medications unless otherwise indicated by a known end date. 
• Any imputed start date or time must come before any known end date or time. 
• Missing dates, date parts, and time will be shown as missing in the listings. 
 
For missing values not described in this section of the SAP, imputation will not be 
performed. 
5.2. Definitions 
• The baseline measurement for a variable is defined as the most recent non- 
missing value prior to study treatment. 
• The change from baseline measurement for a variable at a post-baseline visit will 
be derived as the measurement taken at the specified visit minus the baseline 
measurement. 
• Treatment Duration will be derived as the total number of days on treatment as 
follows: Date of Last Dose – Date of First Dose + 1. 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
17 
 
 
• Study Duration will be defined as the total number of days in the study as follows: 
Date of Last Visit – Date of Informed Consent + 1. 
• Treatment Period is defined to be the period that the subject is active on 
treatment: Date of First Dose through Date of Last Dose. 
• Treatment Compliance over the Treatment Period will be calculated as follows 
based on drug accountability: Treatment Compliance (%) = Number of capsules 
taken / Number of capsules expected to be taken  100. 
• Morning Meal Compliance over the Treatment Period will be calculated as 
follows based on Day 1 administration and the dosing diary: Morning Meal 
Compliance is Yes if all responses to the following questions are yes; otherwise, 
Morning Meal Compliance is No: 
o Was subject dosed at least 1 hour prior to morning meal? 
o Since last visit, has subject been compliant with dosing at least 1 hour 
prior to the morning meal? 
• MADRS responders are defined as subjects who experienced a >= 50% reduction 
in MADRS total score compared to baseline. 
• MADRS remitters are defined as subjects with a MADRS total score <= 10. 
 
• CGI-I improvers are defined as subjects who reported “Very much improved” or 
“Much improved” on the CGI-I scale. 
• An adverse event is defined as Related if the event is reported as Definitely 
Related, Probably Related, or Possibly Related. 
• Adverse Events of Special Interest are defined as a change from baseline of  0.5 
in Nuclear Opalescence, Cortical Lens Opacity, or Posterior Subcapsular Lens 
Opacity on the LOCS III scale, or any other ophthalmic results deemed clinically 
significant by the investigator. 
• Age at First MDD Diagnosis will be calculated as follows: Year of the First MDD 
Diagnosis – Year of Birth, where the Year of the First MDD Diagnosis is defined 
as the year from the earliest start date of the major depressive episodes 
determined in Section 7.4 of this SAP. 
• Number of Prior MDD Episodes will be calculated as follows: The number of 
MDD Episodes with a Start Year that is prior to the Year of the Day 1 Visit, 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
18 
 
 
where MDD Episodes are defined as the major depressive episodes determined in 
Section 7.4 of this SAP. 
• Duration of Current MDD Episode will be calculated as follows: Year of the Day 
1 Visit – Start Year of the Current MDD Episode, where the Start Year of the 
Current MDD Episode is defined as the year from the most recent start date prior 
to the Day 1 Visit of the major depressive episodes determined in Section 7.4 of 
this SAP. 
• Duration from First MDD Diagnosis will be calculated as follows: Year of Day 1 
Visit – Year of the First MDD Diagnosis, where the Year of the First MDD 
Diagnosis is defined as the year from the earliest start date of the major depressive 
episodes determined in Section 7.4 of this SAP. 
6. 
Analysis Considerations 
The objectives of this study are to evaluate the safety and tolerability, and to evaluate the 
efficacy, of ANC-501 capsules in subjects with MDD. 
6.1. Determination of Sample Size 
Up to 20 subjects will be enrolled. The sample size was selected based on clinical and 
not statistical considerations. 
6.2. Adjustments for Covariates 
No adjustment for covariates is planned. 
 
6.3. Interim Analysis and Data Monitoring 
No interim analyses are planned. 
 
6.4. Examination of Subgroups 
No subgroup analyses are planned. 
 
6.5. Multicenter Studies 
All sites will be pooled for this analysis. 
 
6.6. Multiplicity Comparison/Multiplicity 
Not Applicable. 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
19 
 
 
7. 
Population Analysis 
The subjects will be summarized using descriptive statistics. All population summaries 
will be based on the set of subjects mentioned in the relevant section of this SAP. 
Results for population parameters will also be listed by subject according to the relevant 
section in this SAP. 
7.1. Subject Disposition 
Information regarding subject disposition will be summarized using all subjects. 
Summaries will include the number of screen failures, the number of enrolled subjects, 
the number of subjects in the safety and efficacy populations, number of subjects 
completing the study treatment, number of subjects who discontinue the study treatment 
early, number of subjects completing the study, and number of subjects who discontinue 
the study early. For those who discontinue the study treatment early or discontinue the 
study early, the primary reason for discontinuation will be summarized. 
A listing of subjects showing screen failures, analysis populations, study treatment status, 
study status, reason for discontinuing study treatment early, if applicable, and reason for 
discontinuing the study early, if applicable, will also be provided. Additionally, a listing 
of inclusion/exclusion criteria not met will be provided. 
7.2. Protocol Deviations 
Protocol deviations will be collected throughout the duration of the study. Protocol 
deviations will be assigned a sponsor-defined category and subcategory and classified as 
major or minor. A categorical summary of all major subject level deviations will be 
provided by deviation category and subcategory. 
A listing of all subject level deviations will also be provided. 
 
7.3. Demographic and Baseline Characteristics 
Demographics variables will include age, sex and childbearing potential if females, race, 
ethnicity, height at baseline, weight at baseline, and BMI at baseline. Baseline 
characteristics will include the Montgomery-Åsberg Depression Rating Scale (MADRS) 
total score obtained at the SAFER screening interview and the Childhood Trauma 
Questionnaire (CTQ) subscale scores (Emotional Abuse, Physical Abuse, Sexual Abuse, 
Emotional Neglect, Physical Neglect) and CTQ Minimization/Denial scale score. 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
20 
 
 
Demographics and baseline characteristics will be summarized for the safety and efficacy 
analysis populations, and a listing of demographics will also be provided. 
7.4. Baseline Major Depressive Disorder Characteristics 
Major depressive episodes will be determined by using the Medical Dictionary for 
Regulatory Activities (MedDRA) coded terms of “Depression” and “Major depression” 
from medical history. The Age at First MDD Diagnosis (years), Number of Prior MDD 
Episodes, Duration of Current MDD Episode (years), and Duration from First MDD 
Diagnosis (years) will be calculated as defined in Section 5.2 of this SAP. 
A summary will be provided for the Age at First MDD Diagnosis, Number of Prior MDD 
Episodes, Duration of Current MDD Episode, Duration from First MDD Diagnosis, 
baseline MADRS total score, and baseline HAM-A total score using the safety 
population. 
A listing of major depressive episodes from medical history with their start and end dates, 
identified by the aforementioned MedDRA coded terms, and the Age at First MDD 
Diagnosis, Number of Prior MDD Episodes, Duration of Current MDD Episode, and 
Duration from First MDD Diagnosis will also be provided. 
7.5. Screening Mental Health Assessments 
7.5.1. Structured Clinical Interview for DSM-5 – Clinical Trials Version 
 
Subjects will be screened to determine whether they fulfill the criteria for diagnosis of 
MDD according to the Structured Clinical Interview for DSM-5 – Clinical Trials Version 
(SCID-5-CT). No listing or summary table will be provided. 
7.5.2. SAFER Interview 
 
The SAFER interview is conducted for confirmation of MDD. The interview uses the 
MADRS and also makes use of the Massachusetts General Hospital Antidepressant 
Treatment History Questionnaire (MGH ATRQ) for assessment of current antidepressant 
therapy (ADT), adequacy of duration, and dose of prior and current ADT, as well as 
degree of improvement. No listing or summary table will be provided. 
7.5.3. Childhood Trauma Questionnaire 
 
CTQ is assessed at screening only. It is a retrospective measure of childhood trauma and 
contains five subscales (Emotional Abuse, Physical Abuse, Sexual Abuse, Emotional 
Neglect, Physical Neglect) and a Minimalization/Denial scale. The results will be 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
21 
 
 
presented in a listing. No summary table will be provided. The CTQ scores are 
addressed in Section 7.3 of this SAP. 
7.6. Medical History 
Medical history verbatim terms captured via the EDC system will be mapped to System 
Organ Class (SOC) and Preferred Term (PT) using MedDRA version 25.0. Any new, 
untoward, or worsening medical or psychiatric condition reported after first 
administration of the investigational product is to be captured as an adverse event. 
Medical history events will be summarized by SOC and PT using the safety population. 
The summary will present the number and percent of subjects having each event. 
Subjects may have more than one event per SOC and PT. However, a subject is counted 
once if one or more events at the same level of SOC and PT is reported for the subject. 
The summary will be displayed in descending order of incidence of SOC and then 
descending order of PT within SOC. 
A listing of medical history events will also be provided. 
 
7.7. Prior and Concomitant Medications 
Prior and concomitant medication verbatim terms captured via the EDC system will be 
mapped to Anatomical Therapeutic Chemical (ATC) classification and preferred name 
using the World Health Organization Drug (WHODrug) Dictionary Global March 2022 
release. 
Prior and concomitant medications (defined below) will be summarized separately by 
WHO ATC classification (using ATC4 and then using the closest available parental ATC 
classification if ATC4 is not available) and preferred name using the safety population. 
• Prior Medication: Medications with a start date prior to first dose date will be 
considered as prior medications regardless of when the medications ended or 
whether the medications are ongoing. 
• Concomitant Medication: Prior medications that continue to be taken on or after 
the first dose date or new medications with a start date on or after first dose date 
will be considered concomitant medications. 
Note that these definitions are not mutually exclusive in that it is possible 
for a medication to be both prior and concomitant if that medication 
started prior to first dose and continued after first dose, such as with for 
example a high blood pressure medication. 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
22 
 
 
These summaries will present the number and percent of subjects using each medication. 
Subjects may have more than one medication per ATC classification and preferred name. 
However, a subject is counted once if one or more medications at the same level of ATC 
classification and preferred name is reported for the subject. Each summary will be 
displayed in descending order of incidence of ATC classification and then descending 
order of preferred name within ATC classifications. 
A listing of prior and concomitant medications will also be provided. In the listing, each 
medication will be marked as prior, concomitant, or prior and concomitant. 
7.8. Duration and Compliance 
Treatment Duration, Study Duration, Treatment Compliance, and Morning Meal 
Compliance will be derived as stated in Section 5.2 of this SAP. A summary will be 
provided for Treatment Duration, Study Duration, and Compliance as continuous, and for 
Morning Meal Compliance as categorical, using the safety population. 
A listing of Treatment Duration, Study Duration, Treatment Compliance, and Morning 
Meal Compliance, and a listing of dosing diary information, will also be provided. 
Additionally, a listing of study drug administration (in clinic) and a listing of drug 
accountability will be provided. 
8. 
Efficacy Analysis 
The efficacy objectives of this study are to evaluate the efficacy of ANC-501 capsules in 
subjects with MDD. There will be no imputations for missing data except as outlined in 
Section 5.1 of this SAP. 
The efficacy endpoints will be summarized using descriptive statistics. All efficacy 
analyses will be based on the efficacy population. 
Results for efficacy parameters will also be listed by subject according to the relevant 
section in this SAP. 
8.1. Efficacy Parameters 
The primary efficacy parameter is the change from baseline to Day 56 in the 
Montgomery-Åsberg Depression Rating Scale (MADRS) total score. 
Secondary parameters for efficacy to be considered include: 
 
- 
Change from baseline in MADRS total score at all scheduled post-baseline visits 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
23 
 
 
- 
Percentage of MADRS responders (>= 50% reduction in total score) at all 
scheduled post-baseline visits 
- 
Percentage of MADRS remitters (total score <= 10) at all scheduled post-baseline 
visits 
- 
Change from baseline in Hamilton Anxiety Scale (HAM-A) total score at all 
scheduled post-baseline visits 
- 
Change from baseline in Clinical Global Impression-Severity (CGI-S) score at all 
scheduled post-baseline visits 
- 
Short Form-Health Survey (36-item) (SF-36) total score and subscale scores from 
Baseline to Day 56 and at all time points 
- 
Percentage of Clinical Global Impression-Improvement (CGI-I) improvers (“Very 
much improved” or “Much improved”) at all scheduled post-baseline visits 
- 
Patient Global Impression of Improvement (PGI-I) score at Day 56 
Exploratory parameters for efficacy to be considered include: 
- 
Change in Hamilton Depression Rating Scale (6-item) (HAMD-6) scores from 
baseline across all time points 
- 
Change in General Anxiety Disorder (7-item) (GAD-7) from baseline across all 
time points 
8.2. Primary Efficacy Analysis 
The primary efficacy objective of this study is to evaluate the effect of treatment with 
ANC-501 capsules on depressive symptoms in subjects with MDD. 
8.2.1. MADRS Total Score – Day 56 Only 
 
The primary efficacy endpoint is the change from baseline to Day 56 in Montgomery- 
Åsberg Depression Rating Scale (MADRS) total score. 
The MADRS contains 10 questions, each rated on a 7-point scale. The total score is 
calculated by summing the 10 item scores and will range from 0 to 60. A higher score 
indicates more severe depression. 
Summary statistics on the observed and change from baseline MADRS total score will be 
displayed at baseline and Day 56. In addition, a one-sample two-sided t-test will be 
performed and the 95% confidence interval and p-value for the mean change from 
baseline (i.e., mean difference) will be included. 
MADRS total score will be shown on the listing described in Section 8.3.1 of this SAP. 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
24 
 
 
One figure will be provided showing a line plot of baseline MADRS total score and Day 
56 MADRS total score for all subjects, one line per subject. 
8.3. Secondary Efficacy Analysis 
The secondary efficacy objective of this study is to evaluate the effect of ANC-501 at 
scheduled visits and percent reduction in total symptom scores compared with baseline. 
8.3.1. MADRS Total Score – Scheduled Visits 
 
MADRS scores will be collected at Screening, Day 1, Day 8, Day 15, Day 29, Day 43, 
Day 56, and Day 70. The total score will be calculated as described in Section 8.2.1. 
Summary statistics on the observed and change from baseline MADRS total score will be 
displayed at baseline and all scheduled visits. Additionally, summary statistics on the 
observed and change from baseline MADRS item scores will be displayed at baseline and 
all scheduled visits. 
A listing of MADRS total score, MADRS responder (Yes, No) as described in Section 
8.3.2 of this SAP, MADRS remitter (Yes, No) as described in Section 8.3.3 of this SAP, 
and the ten MADRS item scores will also be provided. MADRS responder and MADRS 
remitter will appear in the listing starting on Day 8. 
Two figures will be provided, one showing the mean MADRS total score at baseline and 
at each post-baseline scheduled visit, and the other showing the mean change from 
baseline MADRS total score at baseline (i.e., zero) and each post-baseline scheduled 
visit. Both figures will including standard error bars at baseline and each post-baseline 
visit, where standard error = standard deviation / √n, except for mean change from 
baseline in MADRS total score at baseline. 
8.3.2. MADRS Responders 
 
MADRS responders are defined as subjects who experienced a >= 50% reduction in total 
score from baseline. The total score will be calculated as described in Section 8.2.1. 
For all subjects with a MADRS measurement at the corresponding visit, the frequency 
and percentage of MADRS responders (Yes, No) will be displayed at all scheduled post- 
baseline visits. MADRS responders will be shown on the listing described in Section 
8.3.1 of this SAP. 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
25 
 
 
8.3.3. MADRS Remitters 
 
MADRS remitters are defined as subjects with a total score <= 10. The total score will 
be calculated as described in Section 8.2.1. 
For all subjects with a MADRS measurement at the corresponding visit, the frequency 
and percentages of MADRS remitters (Yes, No) will be displayed at all scheduled post- 
baseline visits. MADRS remitters will be shown on the listing described in Section 8.3.1 
of this SAP. 
8.3.4. Hamilton Anxiety Scale (HAM-A) 
 
Hamilton Anxiety Scale (HAM-A) scores will be collected at Screening, Day 1, Day 8, 
Day 15, Day 29, Day 43, Day 56, and Day 70. HAM-A consists of 14 questions, each 
rated on a 5-point scale. The total score is calculated by summing the responses to all 14 
questions and will range from 0 to 56. A higher score indicates more severe anxiety. 
Summary statistics on the observed and change from baseline in HAM-A total score will 
be displayed at baseline and all scheduled visits. 
A listing of the fourteen HAM-A items as well as the HAM-A total score will also be 
provided. 
One figure will be provided showing the mean HAM-A total score at baseline and at each 
post-baseline scheduled visit. The figure will including standard error bars at baseline 
and each post-baseline visit, where standard error = standard deviation / √n. 
8.3.5. Clinical Global Impression-Severity (CGI-S) 
 
The Clinical Global Impression-Severity (CGI-S) score will be collected at Screening, 
Day 1, Day 8, Day 15, Day 29, Day 43, Day 56, and Day 70. CGI-S consists of one 
question, where the clinician is asked, “Considering your total clinical experience with 
this particular population, how mentally ill is the patient at this time?” This question is 
rated on a scale from 0 to 7, where a higher score indicates greater severity of mental 
illness, as follows: 
0 = Not assessed 
1 = Normal, not at all ill 
2 = Borderline mentally ill 
3 = Mildly ill 
4 = Moderately ill 
5 = Markedly ill 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
26 
 
 
6 = Severely ill 
7 = Among the most extremely ill patients 
 
Summary statistics on the observed and change from baseline in the CGI-S score will be 
displayed at baseline and all scheduled visits. A value of zero will be treated as missing 
and not included in the summary statistics. 
CGI-S score will be shown on the listing described in Section 8.3.7 of this SAP. 
 
One figure will be provided showing the mean CGI-S score at baseline and at each post- 
baseline scheduled visit. The figure will including standard error bars at baseline and 
each post-baseline visit, where standard error = standard deviation / √n. 
8.3.6. Short Form-Health Survey (36-item) (SF-36) 
 
Short Form-Health Survey (36-item) (SF-36) consists of 36 questions. Each item’s score 
will be recoded to a value on a scale from 0 to 100. For items 1, 2, 20, 22, 34, and 36, the 
scores are recoded as 1=100, 2=75, 3=50, 4=25, 5=0. For items 3, 4, 5, 6, 7, 8, 9, 10, 11, 
and 12, scores are recoded as 1=0, 2=50, 3=100. For items 13, 14, 15, 16, 17, 18, and 19, 
scores are recoded as 1=0 and 2=100. For items 21, 23, 26, 27, and 30, scores are 
recoded as 1=100, 2=80, 3=60, 4=40, 5=20, 6=0. For items 24, 25, 28, 29, and 31, scores 
are recoded as 1=0, 2=20, 3=40, 4=60, 5=80, 6=100. For items 32, 33, and 35, scores are 
recoded as 1=0, 2=25, 3=50, 4=75, 5=100. 
 
For each of the 8 scales, the corresponding items are averaged for a scale score. Items 
that are left blank (missing data) are not considered when calculating the scale scores. 
The scales and corresponding items are as follows: physical functioning (PF) (items 3- 
12), role limitations due to physical health (RP) (items 13-16), role limitations due to 
emotional problems (RE) (items 17-19), energy/fatigue (ENGY) (items 23, 27, 29, 31), 
emotional well-being (EMWB) (items 24-26, 28, 30), social functioning (SF) (items 20, 
32), Pain (PAIN) (items 21-22), and general health (GH) (items 1, 33-36). 
 
The physical health summary measure includes four scales: PF (10 items), RP (4 items), 
PAIN (2 items), and GH (5 items). The mental health summary measure includes four 
scales: RE (3 items), ENGY (4 items), EMWB (5 items), and SF (2 items). The total 
score is an average of all 36 items. A higher score indicates a more favorable physical 
and mental health state. 
All SF-36 summaries, listings, figures, and analyses will be described in a separate 
analysis plan and performed separately by an external vendor, including any analysis of 
orienting questions. 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
27 
 
 
8.3.7. Clinical Global Impression-Improvement (CGI-I) 
 
The Clinical Global Impression-Improvement (CGI-I) score will be collected on Day 8, 
Day 15, Day 29, Day 43, Day 56, and Day 70. CGI-I consists of one question, where the 
clinician is asked, “Compared to his condition at admission to the project, how much has 
he changed?” This question is rated on a scale from 0 to 7, where a higher score 
indicates greater lack of improvement, as follows: 
0 = Not assessed 
1 = Very much improved 
2 = Much improved 
3 = Minimally improved 
4 = No change 
5 = Minimally worse 
6 = Much worse 
7 = Very much worse 
 
A CGI-I improver is defined as a subject with a CGI-I score of “Very much improved” or 
“Much improved”. 
A categorical summary of CGI-I improvers (Yes, No) will be displayed at all scheduled 
post-baseline visits. A value of “Not assessed” will be treated as missing and not 
included in the categorical summary. 
A listing of the CGI-S and CGI-I scores will also be provided. 
 
8.3.8. Patient Global Impression-Improvement (PGI-I) 
 
The Patient Global Impression of Improvement (PGI-I) score will be collected on Day 8, 
Day 15, Day 29, Day 43, Day 56, and Day 70. PCGI-I consists of one question where the 
subject is asked to describe his or her current condition with how it was before treatment. 
This question is rated on a scale from 0 to 7, where a higher score indicates greater 
dissatisfaction with treatment, as follows: 
0 = Not assessed 
1 = Very much better 
2 = Much better 
3 = A little better 
4 = No change 
5 = A little worse 
6 = Much worse 
7 = Very much worse 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
28 
 
 
Summary statistics on the observed and change from baseline in the PGI-I score will be 
displayed at baseline and Day 56. A value of zero will be treated as missing and not 
included in the summary statistics. 
A listing of the PGI-I score will also be provided. 
 
8.4. Exploratory Efficacy Analysis 
The exploratory efficacy objective of this study is to evaluate subject-reported changes in 
depression and anxiety symptoms. 
8.4.1. Hamilton Depression Rating Scale (6-item) (HAMD-6) 
 
The Hamilton Depression Rating Scale (6-item) (HAMD-6) measures six core symptoms 
of depression on a 0-4-point scale for items 1 to 5 and 0-2 points for item 6. The total 
score is calculated by summing the 6 item scores and will range from 0 to 22. A higher 
score indicates more severe depression. 
All HAMD-6 summaries, listings, figures, and analyses will be described in a separate 
analysis plan and performed separately by an external vendor. 
8.4.2. General Anxiety Disorder (7-item) (GAD-7) 
 
The General Anxiety Disorder (7-item) (GAD-7) Scale consists of 7 items scored on a 4- 
point scale. The total score is calculated as the sum of all item scores and ranges from 0 
to 21. A higher score indicates more severe anxiety. 
All GAD-7 summaries, listings, figures, and analyses will be described in a separate 
analysis plan and performed separately by an external vendor. 
9. 
Safety Analysis 
The primary safety objectives of this study are to evaluate the overall safety of ANC-501 
capsules in subjects with MDD. There will be no imputations for missing data except as 
outlined in Section 5.1 of this SAP. 
The safety endpoints will be summarized using descriptive statistics. All safety analyses 
will be based on the safety population. 
Results for safety parameters will also be listed by subject according to the relevant 
section in this SAP. 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
29 
 
 
9.1. Safety Parameters 
Primary parameters for safety to be considered include: 
 
- 
Frequency and severity of adverse events (AEs) 
- 
Vital signs 
- 
Body weight 
- 
Electrocardiograms (ECGs) 
- 
Clinical laboratory tests 
- 
Physical examination 
- 
Lens Opacities Classification System III (LOCS III) 
- 
Emergent behavior using Columbia-Suicide Severity Rating Scale (C-SSRS) 
The secondary safety parameter is the plasma concentrations of ANC-501. 
Exploratory parameters for safety to be considered include: 
 
- 
Changes in 12-hour urine, plasma, and saliva cortisol from baseline to Day 56 
- 
Changes in biometrics using the wearable watch-like device 
- 
Baseline levels and/or changes in exploratory biomarkers through Day 56 
 
9.2. Primary Safety Analysis 
The primary safety objective of this study is to evaluate the safety and tolerability of 
ANC-501 capsules in subjects with MDD. 
9.2.1. Adverse Events 
 
Adverse events (AEs) summaries will only consider treatment-emergent adverse events 
(TEAEs), using the safety population. TEAEs are defined as those AEs that occurred 
after dosing and those existing AEs that worsened during the study. If it cannot be 
determined whether the AE is treatment-emergent due to an incomplete or missing onset 
date or time, the AE will be considered a treatment-emergent AE. AEs with an onset day 
after the last day study medication was applied will not be considered TEAEs. 
An overall categorical summary of TEAEs will be provided where the total number of 
TEAEs, the number and percent of subjects with at least one mild, moderate, or severe 
TEAE, and the number and percent of each of the following items will be displayed: 
• Subjects with at Least One TEAE 
• Subjects with at Least One TEAE Related to Study Treatment 
• Subjects with at Least One TEAE Leading to Discontinuation of Study Treatment 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
30 
 
 
• Subjects with at Least One TEAE of Special Interest 
• Subjects with at Least One Serious TEAE 
• Subjects with at Least One TEAE Leading to Death 
 
AEs of Special Interest and Related AEs are defined as stated in Section 5.2 of this SAP. 
If a subject reports one or more events within the same SOC and PT, the subject is 
classified (Related/Not Related) according to the most related relationship within the 
same SOC and PT. 
In addition, for each bulleted item in the above list, categorical summaries for TEAEs by 
system organ class (SOC) and preferred term (PT) via MedDRA version 25.0 will be 
provided, and subjects having more than one TEAE may appear in more than one SOC or 
PT but will be counted at most once per each SOC and PT. Furthermore, a summary of 
TEAEs by SOC, PT, and maximum relatedness (Related, Not Related) will be provided 
where the maximum relatedness will be used for subjects with multiple TEAEs within the 
same SOC and PT, and a summary of TEAEs by SOC, PT, and maximum severity (Mild, 
Moderate, Severe) will be provided where the maximum severity will be used for 
subjects with multiple TEAEs within the same SOC and PT. Summaries that are 
displayed by SOC and PT will be ordered by descending incidence of SOC and then PT 
within each SOC, and if applicable ascending severity within SOC and PT. 
A listing of adverse events and a listing of serious adverse events will also be provided. 
 
9.2.2. Vital Signs, Height, and Weight 
 
Blood pressure (standing and supine), heart rate, respiration rate, temperature, weight, 
and BMI will be collected at screening, Day 1, Day 8, and Day 56. Height will be 
collected at screening only. A summary of change from baseline in the vital signs at 
baseline and all scheduled post-baseline visits will be provided. 
A listing of vital signs will also be provided. 
 
9.2.3. Electrocardiogram 
 
A standard 12-lead electrocardiogram (ECG) will be performed on Day 1 prior to dosing, 
Day 8, and Day 56. A summary for change from baseline in the ECG parameters 
(Ventricular Rate, PR Interval, RR Interval, QRS Interval, QT Interval, QTcB Interval, 
QTcF Interval) at baseline and all scheduled post-baseline visits will be provided. 
The ECG results are to be interpreted as follows: Normal; Abnormal, NCS (not clinically 
significant); Abnormal, CS (clinically significant). A shift table of the interpretation will 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
31 
 
 
be presented for all scheduled post-baseline visits. Categories for the shift table will be 
as follows: Missing; Normal; Abnormal, NCS; Abnormal, CS; Total. 
Additionally, a categorical summary of maximum post-baseline QTcB Interval (> 0 to <= 
500 msec, > 500 msec), maximum post-baseline QTcF Interval (> 0 to <= 500 msec, > 
500 msec), and maximum change from baseline (> 0 to < 30 msec, >= 30 to <= 60 msec, 
> 60 msec) for both QTcB and QTcF will be provided. 
A listing of ECG data will also be provided. 
9.2.4. Clinical Laboratory Tests 
 
Laboratory evaluations (hematology, serum chemistry, coagulation, urinalysis) will be 
conducted at Screening, Day 1, Day 8, Day 56, and Day 70. Any clinically significant 
laboratory result after baseline is to be captured as an adverse event and thus included in 
the summary of adverse events. 
Summary statistics for the observed and change from baseline continuous laboratory 
results will be displayed at baseline and all scheduled post-baseline visits via the 
following tables: 
• Hematology 
• Serum Chemistry 
• Coagulation 
• Urinalysis 
 
For categorical urinalysis results, frequency counts and percentages will be presented. 
 
For laboratory parameters (hematology, serum chemistry, coagulation) where normal 
ranges are available, shift tables will be presented for all scheduled post-baseline visits. 
Categories for the shift tables will be Missing, Low, Normal, High, and Total. 
A listing for each category of laboratory results shown in the above bulleted list as well 
as a listing for serology will also be provided, and values will be flagged as Low, 
Normal, or High relative to normal ranges, if available, and included in the listings. In 
addition, a listing will be provided for urine drug screen results and a listing will be 
provided for alcohol breath test screen results. 
9.2.5. Physical Examination 
 
A physical examination (PE) will be conducted during the screening period and at end of 
treatment. Any abnormalities noted prior to first administration of the investigational 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
32 
 
 
product are to be captured as medical history. Findings after first administration of the 
investigational product that meet the criteria of an AE are to be captured as an AE. 
A listing of PE results will be provided. No summary tables will be provided. 
 
9.2.6. Lens Opacities Classification System III (LOCS III) 
 
An ophthalmic exam including slit-lamp imaging may be performed and would be 
assessed using LOCS III. Any result that is an AE of Special Interest as defined in 
Section 5.2 of this SAP is to be recorded. 
LOCS III data are collected at Screening, Day 56, Day 70, and Day 112. A summary for 
change from baseline in four LOCS III parameters (Cortical Lens Opacity, Nuclear 
Color, Nuclear Opalescence, Posterior Subcapsular Lens Opacity) by eye (OD, OS) at all 
scheduled post-baseline visits will be provided. 
In addition, a shift table for each of the LOCS III parameters by eye will be provided. 
The possible values for Cortical Lens Opacity (C) range from 0.1 to 5.9, for Nuclear 
Color (NC) range from 0.1 to 6.9, for Nuclear Opalescence (NO) range from 0.1 to 6.9, 
and for Posterior Subcapsular Lens Opacity (P) range from 0.1 to 5.9. The exclusion 
criteria based on LOCS III are as follows: 
• C with a LOCS III classification > 2.0 in either eye 
• NC with a LOCS III classification > 3.5 in either eye 
• NO with a LOCS III classification > 3.0 in either eye 
• P with a LOCS III classification > 0.3 in either eye 
 
Given the above exclusion criteria and the definition of AEs of Special Interest as 
provided in Section 5.2 of this SAP, the categories for the shift tables will be defined as 
follows, where Nuclear Color will follow the same shift table categorization strategy as 
Nuclear Opalescence, Cortical Lens Opacity, and Posterior Subcapsular Lens Opacity: 
Baseline categorization: 
 
• C: Missing, 0.1 to <= 1.0, > 1.0 to <= 2.0, > 2.0, Total 
• NC: Missing, 0.1 to <= 1.5, > 1.5 to <= 2.5, > 2.5 to <= 3.5, > 3.5, Total 
• NO: Missing, 0.1 to <= 1.0, > 1.0 to <= 2.0, > 2.0 to <= 3.0, > 3.0, Total 
• P: Missing, 0.1 to <= 0.2, > 0.2 to <= 0.3, > 0.3, Total 
Post-baseline categorization: 
• Change: Missing, <= -0.5, > -0.5 to < 0, No Change, > 0 to < 0.5, >= 0.5, Total 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
33 
 
 
Primary ophthalmic data (assessment of first reading) will be used for summaries, and a 
listing of primary ophthalmic data will also be provided. Secondary ophthalmic data 
(secondary readings) will not be summarized or listed. 
9.2.7. Columbia-Suicide Severity Rating Scale (C-SSRS) 
 
The Columbia-Suicide Severity Rating Scale (C-SSRS) will be used to assess suicidal 
ideation and behavior. The “Baseline/Screening” C-SSRS form will be completed at 
screening. The “Since Last Visit” C-SSRS form will be completed at all other visits (Day 
1, Day 8, Day 15, Day 29, Day 43, Day 56, Day 70). 
The “Baseline/Screening” C-SSRS form contains the following possible types of 
responses per each item: response based on Past Month, response based on Life, and/or 
Describe event. The “Since Last Visit” C-SSRS form contains the following possible 
types of responses per each item: response based on Since Last Visit and/or Describe 
event. Not all types of responses are present for all items. 
C-SSRS Suicidal Ideation contains the following five ordered Yes/No response items: 
 
1. Wish to be Dead 
2. Non-Specific Active Suicidal Thoughts 
3. Active Suicidal Ideation with Any Methods (Not Plan) without Intent to Act 
4. Active Suicidal Ideation with Some Intent to Act, without Specific Plan 
5. Active Suicidal Ideation with Specific Plan and Intent 
 
C-SSRS Intensity of Ideation contains the following six numeric response items: 
 
• Most Sever Ideation (1-5) 
• Frequency – How many times have you had these thoughts (1-5)? 
• Duration – When you have the thoughts how long do they last (1-5)? 
• Controllability – Could/can you stop thinking about killing yourself or wanting to 
die if you want to (1-6)? 
• Deterrents – Are there things - anyone or anything (e.g., family, religion, pain of 
death) - that stopped you from wanting to die or acting on thoughts of committing 
suicide (1-6)? 
• Reasons for Ideation – What sort of reasons did you have for thinking about 
wanting to die or killing yourself? Was it to end the pain or stop the way you 
were feeling (in other words you couldn’t go on living with this pain or how you 
were feeling) or was it to get attention, revenge or a reaction from others? Or 
both (1-6)? 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
34 
 
 
The Suicidal Ideation Subscale Score is determined as follows. For the five C-SSRS 
Suicidal Ideation items, Yes/No responses are recorded. The maximum item number 
where Yes is the response is the Suicidal Ideation Subscale Score, i.e., the most severe 
ideation endorsed is the score. Zero is to be assigned (a) if all responses to Categories 1- 
5 are No or (b) if responses to Categories 1-2 are both No and responses to Categories 3- 
5 are missing. If a score is not able to be determined, them it will be left as missing. 
The Intensity of Ideation Subscale Score is determined as follows. The sum of responses 
on five C-SSRS Intensity of Ideation items (Frequency, Duration, Controllability, 
Deterrents, Reasons for Ideation) is the Intensity of Ideation Subscale Score. If the 
Suicidal Ideation Subscale Score is zero or missing, the Intensity of Ideation Subscale 
Score is also zero or missing, respectively. Higher values for the Intensity of Ideation 
Subscale Score indicate more intense ideation. 
Summary statistics for the Suicidal Ideation Subscale Score and Intensity of Ideation 
Subscale Score from the “Baseline/Screening” C-SSRS form and “Since Last Visit” C- 
SSRS form will be presented by scheduled visit, where the screening visit will contain 
Past Month and Life summaries and other scheduled visits will contain the Since Last 
Visit summary. 
A shift table of Suicidal Ideation Items 1 and 2 (Wish to be Dead, Non-Specific Active 
Suicidal Thoughts) will be presented using “Since Last Visit” for all scheduled post- 
baseline visits. Categories for the shift table will be Missing, No, Yes, and Total. 
A listing of C-SSRS Suicidal Ideation and C-SSRS Intensity of Ideation will be provided. 
The Suicidal Ideation Subscale Score and Intensity of Ideation Subscale Score will be 
included in the listing. 
9.3. Secondary Safety Analysis 
The secondary safety objective of this study is to assess the pharmacokinetic profile of 
ANC-501 capsules in plasma using a sparse sampling model. 
9.3.1. Pharmacokinetics 
 
The secondary endpoint is the plasma concentrations of ANC-501. 
 
Sparse pharmacokinetic (PK) blood sampling will be collected pre-dose and between 
0.75 and 2 hours post-dose on Day 1 and between 0.75 and 2 hours post-dose, whenever 
possible, on Day 8, Day 15, Day 29, Day 43, and Day 56. 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
35 
 
 
Summary statistics (number, arithmetic mean, standard deviation, median, minimum, 
maximum, geometric mean, geometric coefficient of variation) for the observed PK 
concentration data will be displayed by scheduled visit for the ANC-501 analyte using 
the safety population. Reported values marked as Below Limit of Quantitation (BLQ) 
will be set to 0.5  Lower Limit of Quantitation (LLOQ), and reported values marked as 
Above Limit of Quantitation (ALQ) will be set to 1.0  Upper Limit of Quantitation 
(ULOQ), for summarization purposes. LLOQ is 2.50 ng/mL, and ULOQ is 2,500 ng/mL. 
A listing of PK concentration results will also be provided. 
 
Any additional PK summaries, listings, figures, and analyses will be described in a 
separate analysis plan and performed separately by an external vendor. 
9.4. Exploratory Safety Analysis 
The exploratory safety objectives of this study are to evaluate changes in 12-hour urine, 
plasma, and saliva cortisol, to evaluate changes in biometrics, and to evaluate biological 
samples as potential biomarkers and metabolites. 
9.4.1. Cortisol 
 
Cortisol sampling (12-hour urine cortisol, plasma and/or saliva cortisol) will be 
performed at screening and Day 56. 
Summary statistics for the observed and change from baseline cortisol level will be 
displayed at baseline and all scheduled post-baseline visits. 
A listing for cortisol level will also be provided. The type of sampling will be included in 
the listing. 
9.4.2. Wearable Device Biometrics 
 
Subjects are to wear their watch-like device throughout the day and night between Day 1 
to Day 56 except when charging. The wearable device collects actigraphy data (such as 
total daily steps, hour with the most steps, waking hour with the least steps, total minutes 
of sleep divided into daytime sleep and nighttime sleep, resting heart rate, average daily 
heart rate, and/or heart rate variation) on a daily basis. As the first round of daily data 
comes after Day 1 dosing, there is no baseline measure. 
All wearable device biometric summaries, listings, figures, and analyses will be described 
in a separate analysis plan and performed separately by an external vendor. 


EmbarkNeuro 
Protocol: ANC501D0005 
Statistical Analysis Plan 
29SEP2023 
36 
 
 
9.4.3. Biomarkers 
 
Biomarker samples will be collected on Day 1 (pre-dose, post-dose), Day 8, and Day 56. 
 
All biomarker summaries, listings, figures, and analyses will be described in a separate 
analysis plan and performed separately. 
10. 
Tables, Listings, and Figures 
Tables will generally be presented with parameter and visits as applicable in the first 
column followed by the summary column(s). Descriptive statistics such as mean, 
median, minimum, and maximum will typically be presented to one decimal place and 
standard deviation will typically be presented to two decimal places. Numeric laboratory 
tables may vary for each test depending on the precision of the test. 
Listings will be sorted by subject ID and visit within subject ID as appropriate. Data 
reported in EDC, external vendor data, and/or derived values may be included in the 
listings, as indicated in this SAP. 
Tables, listings, and figures will be maintained outside of this document and may be 
amended as needed. The spacing, overall presentation, or layout of the displays may 
need to be altered once data are available. 
11. 
Protocol and SAP Differences 
If there are any differences between the study protocol and this SAP, this SAP takes 
precedent. 
12. 
References 
EmbarkNeuro, Inc. ANC501D0005: A Phase 2 Study of ANC-501 in the Treatment of 
Adults with Major Depressive Disorder, Protocol Version 6.0, Amendment 05. 03 March 
2023.