Conditions: Major Depressive Disorder Treatment-Resistant Depression Depression
Keywords: Biomarker Neurobiology Glutamate Magnetoencephalography Magnetic Resonance Spectroscopy
Allocation: RANDOMIZED | Intervention model: CROSSOVER
Enrollment: 11
Healthy volunteers: no

Brief summary:
Background:

Major depressive disorder (MDD) is a common, chronic mental illness. It can take weeks to months for antidepressants to work. Researchers want to test a new drug that might act more rapidly.

Objective:

To see if TS-161 will improve symptoms of depression in people with MDD.

Eligibility:

Adults ages 18-65 with MDD without psychotic features.

Design:

Participants will be screened under a separate protocol. They will have blood tests. They will complete surveys about their symptoms.

Participants will have an inpatient visit at NIH. Participation may last 12-16 weeks.

During the first phase of the study, participants will be tapered off their psychiatric medicines. For 2 weeks they will have a drug-free period.

During Phase II participants will take TS-161 or placebo. They will take TS-161 for 3 weeks and placebo for 3 weeks. In between the 3-week time period, they will have 2-3 weeks where they will be drug free. Participants will also have the following tests during this time:

* Interviews
* Physical exams
* Psychological tests and surveys about their symptoms
* Blood draws and urine samples
* They may complete tests of mood and thinking
* Magnetic resonance imaging (MRI): Participants will lie in a machine that takes pictures of their brain.
* Functional MRIs: They will perform tasks displayed on a computer screen inside the MRI scanner
* Magnetoencephalography (MEG): Participants will lie down and do tasks of memory, attention, and thinking. A cone lowered on their head will record brain activity.
* Electrocardiograms to record the heart s electrical activity. Electrodes will be placed on the skin....

Detailed description:
OBJECTIVE

Modulation of glutamatergic signaling is implicated in improvement of depressive symptoms and related constructs/dimensions of observable behavior and neurobiological measures with treatment. Current standard monoaminergic pharmacological approaches for major depressive disorder (MDD) have proven to be only modestly effective during acute major depressive episodes (MDEs). We have systematically tested different glutamatergic modulators in subjects with mood disorders in order to develop improved therapeutics. We found that the N-methyl-D-aspartate receptor (NMDAR) antagonist, ketamine, produces rapid antidepressant effects in patients with treatment-resistant depression (TRD in MDD, Bipolar Disorder) and in suicidal ideation. However, despite being highly efficacious, ketamine produces psychotomimetic effects and has the risk of abuse. The antidepressant effects of mGlu2/3 receptor antagonists are worthy of pursuit, since the antidepressant profile in preclinical assays as well as the synaptic/neural cellular and molecular mechanisms involved in their actions are comparable to those of ketamine, but without the side effects and abuse potential of ketamine.

In the present protocol, we aim to evaluate a new glutamate-mediated mechanism associated with antidepressant efficacy by targeting the mGlu2/3 receptor with a potent and selective antagonist. Targeting the mGlu2/3 receptor with an antagonist is anticipated to, and similar to ketamine, result via pre-synaptic mechanisms in a "glutamate surge" with subsequent alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) activation and gamma power increases but without potential adverse effects that occur with ketamine.

The present Phase 2 proof-of-concept (POC) study is designed to evaluate in subjects with MDD, the antidepressant effects of TS-161, the prodrug of a potent and selective mGlu2/3 receptor antagonist TP0178894 that crosses the blood brain barrier (BBB). In animal model assays of antidepressant efficacy, TS-161 induced acute and prolonged antidepressant-like effects without exhibiting ketamine-like side effects as determined by the lack of increase in locomotor activity or abuse potential.

We will also evaluate the putative neurobiological mechanisms involved in the antidepressant response to TS-161. We expect that this effect may modulate glutamate transmission and reverse the clinical symptoms of depression. The demonstration that an mGlu2/3 receptor antagonist produces antidepressant effects without psychotomimetic side effects would support the therapeutic relevance of the mGlu2/3 receptor and could direct the development of novel drug targets for the treatment of depression.

STUDY POPULATION

Twenty-five individuals with treatment-resistant major depressive disorder (MDD) will be consented.

DESIGN

Male and female subjects, ages 18 to 65 years, with a diagnosis of MDD, currently in an episode of major depression, will be recruited for this study. This study will consist of a randomized, double-blind crossover administration of either the mGlu2/3 receptor antagonist prodrug TS-161 (50 to 100 mg/day given orally) or placebo for 3 weeks. The study will assess the efficacy in improving overall depressive symptomatology and tolerability of TS-161 in treatment-resistant MDD. Other aims of the study include determining whether changes in gamma power obtained via magnetoencephalography (MEG), brain neurochemicals (e.g. glutamate) obtained via magnetic resonance spectroscopy (MRS), and peripheral measures correlate with drug effects and/or antidepressant response to TS-161 in subjects with treatment-resistant MDD.

OUTCOME MEASURES

Primary: Montgomery-Asberg Depression Rating Scale (MADRS) total score.

Secondary: Proportion of subjects achieving remission (MADRS\<=10) and response (\>=50% reduction from baseline in MADRS total score); change from baseline on the Hamilton Rating Scale (HDRS), change from baseline in Hamilton Anxiety Rating Scale (HAM-A), and the Columbia Suicide Severity Rating Scale (C-SSRS) total scores. Surrogate biomarkers of drug effect/response include: changes in gamma power measured with MEG, changes in prefrontal glutamate levels measured with 7T 1H-MRS, resting and task based functional connectivity with fMRI, neurocognitive functioning, and changes in peripheral biological indices (neurotrophic factors, cell cycle/signal transduction regulators, neuroinflammatory, neuroendocrinological measures, and metabolomic and proteomic measures).

Arms:
arm 1: Participants with major depressive disorder (MDD) were randomized to receive TS-161 100mg capsule orally once per day for three weeks (with option to decrease to 50 mg due to drug intolerance), followed by Placebo capsule orally once per day for three weeks. arm 2: Participants with major depressive disorder (MDD) were randomized to receive Placebo capsule orally once per day for three weeks, followed by TS-161 100mg capsule orally once per day for three weeks (with option to decrease to 50 mg due to drug intolerance).

Interventions: intervention 1: Placebo intervention 2: TS-161
Intervention descriptions: intervention 1: Participants received Placebo capsule orally once daily for three weeks. intervention 2: Participants received TS-161 50-100 mg capsule orally once daily for three weeks. TS-161 is a mGlu2/3 receptor antagonist prodrug.

Protocol document:
Prot_SAP_000.pdf: 
Abbreviated Title: Antidepressant effect of TS-161 
 
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Abbreviated Title:  Antidepressant Effect of TS-161 
Protocol #:  
000101 
Version Date:  
06 November 2023 
Title:  
An Investigation of the Antidepressant Effects of the mGlu2/3 receptor 
antagonist TS-161 in Treatment-Resistant Depression 
 
NIH Principal Investigator: Carlos A. Zarate, MD 
Experimental Therapeutics and Pathophysiology Branch 
National Institute of Mental Health 
Building 10, Rm 7-5342 
9000 Rockville Pike 
Bethesda, MD 20892 
Phone: 301-451-0861 
E-mail: zaratec@mail.nih.gov 
Investigational Agents: 
DRUG NAME: 
TP0473292 (TS-161) 
IND NUMBER: 
153429 
SPONSOR: 
NIMH 
MANUFACTURER: 
Taisho Pharmaceutical Co., Ltd 
 
Responsible Data Safety Monitoring Board (DSMB):  
NIMH-IRP Data and Safety Monitoring Board 
 
 


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STATEMENT OF COMPLIANCE 
The trial will be carried out in accordance with International Conference on Harmonisation 
Good Clinical Practice (ICH GCP) and the following:  
• United States (US) Code of Federal Regulations (CFR) applicable to clinical studies (45 
CFR Part 46, 21 CFR Part 50, 21 CFR Part 56, 21 CFR Part 312, and/or 21 CFR Part 812)  
National Institutes of Health (NIH)-funded investigators and clinical trial site staff who are 
responsible for the conduct, management, or oversight of NIH-funded clinical trials have 
completed Human Subjects Protection and ICH GCP Training. 
The protocol, informed consent form(s), recruitment materials, and all participant materials 
will be submitted to the Institutional Review Board (IRB) for review and approval.  Approval of 
both the protocol and the consent form must be obtained before any participant is enrolled.  Any 
amendment to the protocol will require review and approval by the IRB before the changes are 
implemented to the study.  In addition, all changes to the consent form will be IRB-approved; a 
determination will be made regarding whether a new consent needs to be obtained from 
participants who provided consent, using a previously approved consent form. 
 
 
 


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TABLE OF CONTENTS 
1 
Protocol Summary ................................................................................................................................ 5 
1.1 Synopsis ......................................................................................................................................... 5 
1.2 Schema ........................................................................................................................................... 8 
1.3 Schedule of Events ......................................................................................................................... 9 
1.4 Schedule of Clinical Rating Scales .............................................................................................. 17 
2 
Introduction ........................................................................................................................................ 18 
2.1 Study Rationale ............................................................................................................................ 18 
2.2 Background .................................................................................................................................. 18 
2.3 Risk/Benefit Assessment .............................................................................................................. 35 
3 
Objectives and Endpoints .................................................................................................................. 38 
4 
Study Design ....................................................................................................................................... 44 
4.1 Overall Design ............................................................................................................................. 44 
4.2 Scientific Rationale for Study Design .......................................................................................... 44 
4.3 Justification for Dose ................................................................................................................... 45 
5 
Study Population ................................................................................................................................ 45 
5.1 Inclusion Criteria .......................................................................................................................... 45 
5.2 Exclusion Criteria ......................................................................................................................... 46 
5.3 Inclusion of Vulnerable Participants ............................................................................................ 47 
5.4 Persons over 65 Years of Age ...................................................................................................... 48 
5.5 Non-English Speakers .................................................................................................................. 48 
5.6 Lifestyle Considerations ............................................................................................................... 48 
5.7 Screen Failures ............................................................................................................................. 49 
5.8 Strategies for Recruitment and Retention .................................................................................... 49 
6 
Study Intervention .............................................................................................................................. 52 
6.1 Study Interventions(s) Administration ......................................................................................... 52 
6.2 Preparation/Handling/Storage/Accountability ............................................................................. 55 
6.3 Acquisition and Accountability .................................................................................................... 55 
6.4 Formulation, Appearance, Packaging, and Labeling ................................................................... 56 
6.5 Measures to Minimize Bias: Randomization and Blinding .......................................................... 56 
6.6 Study Intervention Compliance .................................................................................................... 56 
6.7 Concomitant Therapy ................................................................................................................... 56 
7 
Study Intervention Discontinuation and Participant Discontinuation/Withdrawal .................... 56 
7.1 Discontinuation of Study Intervention ......................................................................................... 56 
7.2 Participant Discontinuation/Withdrawal from the Study ............................................................. 57 
7.3 Lost to Follow-up ......................................................................................................................... 57 
8 
Study Assessments and Procedures .................................................................................................. 58 
8.1 Screening Procedures ................................................................................................................... 58 


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8.2 Medication Tapering .................................................................................................................... 58 
8.3 TS-161 and Placebo Administration ............................................................................................ 58 
8.4 Efficacy Assessments ................................................................................................................... 59 
8.5 Biospecimen Evaluations ............................................................................................................. 64 
8.6 Safety and Other Assessments ..................................................................................................... 66 
8.7 Adverse Events and Serious Adverse Events ............................................................................... 68 
8.8 Unanticipated Problems ............................................................................................................... 71 
9 
Statistical Considerations .................................................................................................................. 72 
9.1 Statistical Hypothesis ................................................................................................................... 72 
9.2 Sample Size Determination .......................................................................................................... 73 
9.3 Populations for Analyses .............................................................................................................. 74 
9.4 Statistical Analyses ...................................................................................................................... 74 
10 Regulatory and Operational Considerations ................................................................................... 76 
10.1 Informed Consent Process ............................................................................................................ 76 
10.2 Study Discontinuation and Closure .............................................................................................. 77 
10.3 Confidentiality and Privacy .......................................................................................................... 77 
10.4 Future Use of Stored Specimens and Data ................................................................................... 78 
10.5 Safety Oversight ........................................................................................................................... 78 
10.6 Clinical Monitoring ...................................................................................................................... 79 
10.7 Quality Assurance and Quality Control ....................................................................................... 79 
10.8 Data Handling and Record Keeping ............................................................................................. 80 
10.9 Protocol Deviations ...................................................................................................................... 80 
10.10 Publication and Data Sharing Policy .......................................................................................... 81 
10.11 Collaborative Agreements .......................................................................................................... 81 
10.12 Conflict of Interest Policy ........................................................................................................... 81 
11 Abbreviations ...................................................................................................................................... 82 
12 References ........................................................................................................................................... 86 
13 Appendices .......................................................................................................................................... 99 
13.1 Appendix A. Five Question Consent Quiz ................................................................................... 99 
13.2 Appendix B. Drugs Allowed and Not Allowed ......................................................................... 100 
13.3 Appendix C. Peripheral (Plasma/Serum) Measures of Interest as Biomarkers in Major 
Depressive Disorders.................................................................................................................. 106 
13.4 Appendix D: Contraceptive Guidance and Collection of Pregnancy Information ..................... 110 
 
 
 


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1  PROTOCOL SUMMARY 
1.1  SYNOPSIS 
TITLE: 
An Investigation of the Antidepressant Effects of the mGlu2/3 receptor 
antagonist TS-161 in Treatment-Resistant Depression 
STUDY 
DESCRIPTION: 
This is a randomized, double blind, placebo-controlled, crossover, 
single–site study. 
This experimental study will assess the efficacy and safety of three 
weeks of 50 to 100 mg/day of TS-161, an mGlu2/3 receptor antagonist 
prodrug. 
OBJECTIVES: 
Primary Objective:   
The primary objective is to evaluate the ability of the mGlu2/3 receptor 
antagonist prodrug, TS-161, to improve overall depressive 
symptomatology in subjects with Major Depressive Disorder (MDD). 
The efficacy of a three-week course of TS-161 will be compared to three 
weeks of placebo in a crossover study. The Montgomery-Asberg 
Depression Rating Scale (MADRS) will serve as the main outcome 
measure.  
 
Secondary Objectives:  
1. To evaluate the antidepressant efficacy of TS-161 at 230 min, 1, 2, 3, 
7, 14, and 21 days post-initial dosing compared to placebo in a 
crossover study, as assessed by change from baseline on MADRS 
total scores. 
2. To determine whether TS-161 will demonstrate a superior 
antidepressant efficacy compared to placebo in a crossover study, as 
assessed by the proportion of subjects in remission (defined as 
MADRS total score ≤10). 
3. To determine whether TS-161 will demonstrate a superior 
antidepressant response compared to placebo in a crossover study, as 
assessed by the proportion of subjects achieving response (defined as 
a ≥50% reduction from baseline in MADRS total score). 
4. To evaluate the antisuicidal ideation effects of TS-161 at 230 min, 1, 
2, 3, 7, 14, and 21 days post-initial dosing compared to placebo in a 
cross over study, as assessed by change from baseline on item 10 
(suicidality) of the MADRS, the Columbia Suicide Severity Rating 
Scale (C-SSRS), and the Scale for Suicidal Ideation (SSI). 
5. To investigate the effects of TS-161 on mood, anxiety, and anhedonia 
symptoms at 230 min, 1, 2, 3, 7, 14, and 21 days post-initial dosing 
compared to placebo in a cross over study, as assessed by change 


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from baseline on the Hamilton Depression Rating Scale (HDRS), 
Hamilton Anxiety Rating Scale (HAM-A), Positive and Negative 
Affect Schedule (PANAS), Snaith–Hamilton Pleasure Scale 
(SHAPS), and the Temporal Experience of Pleasure Scale (TEPS) 
scales.  
6. To assess the safety and tolerability of a three-week course of TS-161 
compared to placebo in a crossover study, by incidence of adverse 
events (AEs) and total scores using the Clinician Administered 
Dissociative States Scale (CADSS), Young Mania Rating Scale 
(YMRS), the Brief Psychiatric Rating Scale (BPRS), vital signs, 
changes in clinical laboratory evaluations, and electrocardiograms 
(ECGs). 
ENDPOINTS: 
Primary Endpoint: 
• Change from baseline in the MADRS total score 
Secondary Endpoints: 
• Proportion of subjects in remission (defined as MADRS total score 
≤10). 
• Proportion of subjects with response (defined as ≥50% reduction 
from baseline in MADRS total score). 
• Change from baseline in HDRS, BPRS, CADSS, C-SSRS, HAM-A, 
PANAS, SHAPS, SSI, TEPS, YMRS total scores.  
• Incidence and nature of adverse events; vital signs; weight and body 
mass index (BMI) changes; physical examination changes; clinical 
laboratory evaluations; ECG. 
Surrogate Markers of Drug Effect, Target Engagement, and 
Antidepressant Response  
• Change in magnetoencephalography (MEG) spectral power (gamma 
power). 
• Change in glutamate levels in magnetic resonance spectroscopy 
(MRS). 
• Change in resting and task based functional connectivity in fMRI. 
• Change in peripheral biomarkers. 
STUDY 
POPULATION: 
Up to 25 individuals with Treatment-Resistant Depression (TRD) 
between the ages of 18 and 65 will be enrolled in the study.  
Participation will not be limited by gender identity or geographic 
location. 
PHASE: 
Phase 2 Proof-of-Concept (POC) Clinical Trial 
DESCRIPTION OF 
SITES/FACILITIES 
Research will be conducted at the NIH Clinical Center in Bethesda, 
Maryland, United States. 


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ENROLLING 
PARTICIPANTS: 
DESCRIPTION OF 
STUDY 
INTERVENTION: 
This study will consist of a randomized, double-blind crossover 
administration of the mGlu2/3 receptor antagonist prodrug TS-161 (50 to 
100 mg/day given orally) and placebo. 
STUDY DURATION: The estimated duration of this protocol is 36 months. 
PARTICIPANT 
DURATION: 
The total duration of the study, which consists of two phases, is 12 to 16 
weeks. 


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1.2  SCHEMA 
 


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1.3  SCHEDULE OF EVENTS 
 
Timepointi 
Procedures 
Phase I 
Medication Taper, Drug-Free Period, and Baseline Assessments 
Day -28 to Day -
15 
Medication Taper 
Day -14 to Day -1 
Drug-Free Period 
Day -8 to Day -2 
Clinical Rating Scalesii 
Neurocognitive Battery 
Imaging : Structural neuroimaging studies (if clinically indicated, unless completed within the past 
12 months), fMRI/MRS, MEG 
Day -1 
Clinical Rating Scale 
Sample Collection: Safety Labs iii,iv  
AE Assessments 
Phase II 
Research Conditions 1 & 2 
Research 
Condition 1 
 
Day 0 
 
-60 to 0 min 
Clinical Rating Scalesii 
AE Assessments 
Sample Collection: Research Blood, PK Blood 
Clinical Evaluations: Orthostatic Vital Signs, Height and Weight 
0 min 
Study Drug Administration: TS-161/ Placebo (100mg) 
+60 min (1 hrs.) 
Sample Collection: PK Blood 
+120 min (2 
hrs.) 
Sample Collection: PK Blood 
Imaging: MEG 


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Timepointi 
Procedures 
Clinical Evaluations: Orthostatic Vital Signs  
+180 min (3 
hrs.) 
Sample Collection: PK Blood 
+230 min 
Clinical Rating Scalesii 
AE Assessments 
Sample Collection: Research Blood 
+240 min (4 
hrs.) 
Sample Collection: PK Blood 
Imaging: fMRI/MRS 
Clinical Evaluations: Orthostatic Vital Signs  
+6 hrs. 
Sample Collection: PK Blood 
Clinical Evaluations: Orthostatic Vital Signs  
+8 hrs. 
Sample Collection: PK Blood 
+10 hrs. 
Sample Collection: PK Blood 
+12 hrs. 
Sample Collection: PK Blood 
Day 1 
Clinical Rating Scalesii 
AE Assessments 
Sample Collection: Research Blood, PK Blood 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg)v  
Clinical Evaluations: Orthostatic Vital Signs 
Day 2 
Clinical Rating Scalesii 
AE Assessments 
Neurocognitive Battery 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg)v 
Clinical Evaluations: Orthostatic Vital Signs  


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Timepointi 
Procedures 
Day 3 
Clinical Rating Scalesii 
AE Assessments 
Sample Collection: Research Blood 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) v  
Clinical Evaluations: Orthostatic Vital Signs  
Day 4 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) v  
Clinical Evaluations: Orthostatic Vital Signs  
Day 5 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) v  
Clinical Evaluations: Orthostatic Vital Signs  
Day 6 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) v  
Clinical Evaluations: Orthostatic Vital Signs  
Day 7 
Clinical Rating Scalesii 
AE Assessments 
Sample Collection: Research Blood, PK Blood, Safety Labsiii, エラー! ブックマークが定義されていません。 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) v  
Clinical Evaluations: Orthostatic Vital Signs, Physical Exam 
Day 8 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) v  
Clinical Evaluations: Orthostatic Vital Signs  
Day 9 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) v  
Clinical Evaluations: Orthostatic Vital Signs  
Day 10 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) v 
Clinical Evaluations: Orthostatic Vital Signs  
Day 11 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) v 
Clinical Evaluations: Orthostatic Vital Signs  
Day 12 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) v 
Clinical Evaluations: Orthostatic Vital Signs  
Day 13 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) v 


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Timepointi 
Procedures 
Clinical Evaluations: Orthostatic Vital Signs  
Day 14 
Clinical Rating Scalesii 
AE Assessments 
Sample Collection: Research Blood, PK Blood, Safety Labs エラー! ブックマークが定義されていません。,エラー! ブック
マークが定義されていません。 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg)v 
Clinical Evaluations: Orthostatic Vital Signs 
Day 15 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg)  
Clinical Evaluations: Orthostatic Vital Signs  
Day 16 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg)  
Clinical Evaluations: Orthostatic Vital Signs  
Day 17 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg)  
Clinical Evaluations: Orthostatic Vital Signs  
Day 18 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg)  
Clinical Evaluations: Orthostatic Vital Signs  
Day 19 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg)  
Sample Collection: Urine pregnancy test 
Clinical Evaluations: Orthostatic Vital Signs  
Day 20 
Sample Collection: Research Blood, PK Blood, Safety Labsiii 
Neurocognitive Battery 
Imaging: fMRI/MRS, MEG 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg)  
Clinical Evaluations: Orthostatic Vital Signs, Weight & Height, ECG 
Day 21 to Day 34 
Washout Period 
Day 21 
Clinical Rating Scalesii 
AE Assessments 


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Timepointi 
Procedures 
Day 28 to Day 33 
Neurocognitive Battery 
Imaging: Clinical MRI, fMRI/MRS, MEG 
Day 34 エラー! ブック
マークが定義されていませ
ん。 
Clinical Rating Scalesii 
Sample Collection: Safety Labs iii, エラー! ブックマークが定義されていません。 
AE Assessments 
Research 
Condition 2 
 
Day 35 
 
-60 to 0 min 
Clinical Rating Scalesii 
AE Assessments 
Sample Collection: Research Blood, PK Blood 
Clinical Evaluations: Orthostatic Vital Signs, Height and Weight 
0 min 
Study Drug Administration: TS-161/ Placebo (100mg) 
+60 min (1 hrs.) 
Sample Collection: PK Blood 
+120 min (2 
hrs.) 
Sample Collection: PK Blood 
Imaging: MEG 
Clinical Evaluations: Orthostatic Vital Signs  
+180 min (3 
hrs.) 
Sample Collection: PK Blood 
+230 min 
Clinical Rating Scalesii 
AE Assessments 
Sample Collection: Research Blood 
+240 min (4 
hrs.) 
Sample Collection: PK Blood 
Imaging: fMRI/MRS 
Clinical Evaluations: Orthostatic Vital Signs  
+6 hrs. 
Sample Collection: PK Blood 
Clinical Evaluations: Orthostatic Vital Signs  


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Timepointi 
Procedures 
+8 hrs. 
Sample Collection: PK Blood 
+10 hrs. 
Sample Collection: PK Blood 
+12 hrs. 
Sample Collection: PK Blood 
Day 36 
Clinical Rating Scalesii 
AE Assessments 
Sample Collection: Research Blood, PK Blood 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) v  
Clinical Evaluations: Orthostatic Vital Signs 
Day 37 
Clinical Rating Scalesii 
AE Assessments 
Neurocognitive Battery 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) v  
Clinical Evaluations: Orthostatic Vital Signs  
Day 38 
Clinical Rating Scalesii 
AE Assessments 
Sample Collection: Research Blood 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) v 
Clinical Evaluations: Orthostatic Vital Signs  
Day 39 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) v  
Clinical Evaluations: Orthostatic Vital Signs  
Day 40 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) v 
Clinical Evaluations: Orthostatic Vital Signs  
Day 41 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) v  
Clinical Evaluations: Orthostatic Vital Signs  


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Timepointi 
Procedures 
Day 42 
Clinical Rating Scalesii 
AE Assessments 
Sample Collection: Research Blood, PK Blood, Safety Labsiii,エラー! ブックマークが定義されていません。 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) v  
Clinical Evaluations: Orthostatic Vital Signs, Physical Exam 
Day 43 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) v  
Clinical Evaluations: Orthostatic Vital Signs  
Day 44 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) v  
Clinical Evaluations: Orthostatic Vital Signs  
Day 45 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) v  
Clinical Evaluations: Orthostatic Vital Signs  
Day 46 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) v  
Clinical Evaluations: Orthostatic Vital Signs  
Day 47 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) v  
Clinical Evaluations: Orthostatic Vital Signs  
Day 48 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) v 
Clinical Evaluations: Orthostatic Vital Signs  
Day 49 
Clinical Rating Scalesii 
AE Assessments 
Sample Collection: Research Blood, PK Blood, Safety Labsiii,エラー! ブックマークが定義されていません。 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) v  
Clinical Evaluations: Orthostatic Vital Signs 
Day 50 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) 
Clinical Evaluations: Orthostatic Vital Signs  
Day 51 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) 
Clinical Evaluations: Orthostatic Vital Signs  
Day 52 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) 


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Timepointi 
Procedures 
Clinical Evaluations: Orthostatic Vital Signs  
Day 53 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) 
Clinical Evaluations: Orthostatic Vital Signs  
Day 54 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) 
Sample Collection: Urine pregnancy test 
Clinical Evaluations: Orthostatic Vital Signs  
Day 55 
Sample Collection: Research Blood, PK Blood  
Neurocognitive Battery 
Imaging: fMRI/MRS, MEG 
Study Drug Administration: TS-161/ Placebo (100mg or 50mg) 
Clinical Evaluations: Orthostatic Vital Signs  
Day 56 (or End 
of Study) 
Clinical Rating Scalesii 
AE Assessments 
Sample Collection: Safety Labsiii,エラー! ブックマークが定義されていません。 
Clinical Evaluations: Urine Toxicology, Orthostatic Vital Signs, Weight, Height, ECG, Physical 
Exam 
Standard 
Treatment 
 
Up to 2 months 
Standard Treatment 
 
 
i Allow up to 3 hours for procedures scheduled on the same day. Allow up to 3 days on procedures scheduled on different days to accommodate changes to the 
schedule. Some procedures may not be performed due to unforeseen technical or scheduling problems. 
ii See the Schedule of Clinical Rating Scales for specific scales used.   
iii See the Safety Labs section for a list of labs conducted. 
iv A urine pregnancy test (HCG) will be obtained as indicated and no more than 24 hours prior to start of each research condition and imaging procedure.  A urine 
toxicology screen will be obtained within 24 hrs of administration of study drug. 
v See Dosing and Administration for more details on dose adjustments. 


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1.4  SCHEDULE OF CLINICAL RATING SCALES 
Timepointi 
C-SSRS- 
lifetime, FHS BPRS, YMRS 
CADSS 
C-SSRS, HDRS, 
HAM-A, PANAS, 
SHAPS, SSI, TEPS 
MADRS 
Study Phase I 
Days -8 to -2 
Xii 
 
 
 
 
Day -1 
Xii 
X 
X 
X 
X 
Study Phase II 
Research Condition 1 
Day 0 
 
 
 
 
 
-60 min 
 
 
 
 
X 
+230 min  
 
X 
X 
X 
X 
Day 1 
 
X 
X 
X 
X 
Day 2 
 
 
X 
X 
X 
Day 3 
 
 
X 
X 
X 
Day 7  
 
 
X 
X 
X 
Day 14 
 
 
X 
X 
X 
Day 21 
 
X 
X 
X 
X 
Research Condition 2 
Day 34 
 
X 
X 
X 
X 
Day 35 
 
 
 
 
 
-60 min 
 
 
 
 
X 
+230 min  
 
X 
X 
X 
X 
Day 36 
 
X 
X 
X 
X 
Day 37 
 
 
X 
X 
X 
Day 38 
 
 
X 
X 
X 
Day 42 
 
 
X 
X 
X 
Day 49 
 
 
X 
X 
X 
Day 56 (or End 
of Study) 
 
X 
X 
X 
X 
 
i Allow up to 3 hours on same day for procedures scheduled on the same day. 
ii These will be completed once during Phase I, if not previously done under protocol 01-M-0254. 
 


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2  INTRODUCTION 
2.1  STUDY RATIONALE 
Modulation of glutamatergic signaling is implicated in improvement of depressive symptoms 
and related constructs/dimensions of observable behavior and neurobiological measures with 
treatment. Current standard monoaminergic pharmacological approaches for major depressive 
disorder (MDD) have proven to be only modestly effective during acute major depressive episodes 
(MDEs). We have systematically tested different glutamatergic modulators in subjects with mood 
disorders in order to develop improved therapeutics. We found that the N-methyl-D-aspartate 
receptor (NMDAR) antagonist, ketamine, produces rapid antidepressant effects in patients with 
treatment-resistant depression (TRD in MDD, Bipolar Disorder) and in suicidal ideation. However, 
despite being highly efficacious, ketamine produces psychotomimetic effects and has the risk of 
abuse. The antidepressant effects of mGlu2/3 receptor antagonists are worthy of pursuit, since the 
antidepressant profile in preclinical assays as well as the synaptic/neural cellular and molecular 
mechanisms involved in their actions are comparable to those of ketamine, but without the side 
effects and abuse potential of ketamine.  
In the present protocol, we aim to evaluate a new glutamate-mediated mechanism associated 
with antidepressant efficacy by targeting the mGlu2/3 receptor with a potent and selective 
antagonist. Targeting the mGlu2/3 receptor with an antagonist is anticipated to, and similar to 
ketamine, result via pre-synaptic mechanisms in a “glutamate surge”  with subsequent α-amino-3-
hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) activation and gamma power increases but 
without potential adverse effects that occur with ketamine.  
The present Phase 2 proof-of-concept (POC) clinical trial is designed to evaluate in subjects 
with MDD, the antidepressant effects of TS-161, the prodrug of a potent and selective mGlu2/3 
receptor antagonist TP0178894 that readily crosses the blood brain barrier (BBB). In animal model 
assays of antidepressant efficacy, TS-161 induced acute and prolonged antidepressant-like effects 
without exhibiting ketamine-like side effects as determined by the lack of increase in locomotor 
activity or abuse potential.  
We will also evaluate the putative neurobiological mechanisms involved in the antidepressant 
response to TS-161. We expect that this effect may modulate glutamate transmission and reverse 
the clinical symptoms of depression. The demonstration that an mGlu2/3 receptor antagonist 
produces antidepressant effects without psychotomimetic side effects would support the 
therapeutic relevance of the mGlu2/3 receptor and could direct the development of novel drug 
targets for the treatment of depression.  
2.2  BACKGROUND  
2.2.1  Ketamine Induces Rapid Antidepressant Effects, but Produces Dissociative Side 
Effects and has the Potential for Abuse 
Mood disorders (MDD and bipolar disorder [BD]) are common, chronic, recurring, serious 
mental illnesses associated with significant morbidity, and often become life-threatening. A variety 
of antidepressants have been developed; unfortunately, these medications take weeks to months to 
achieve their full effects and in the meantime, patients continue to suffer from their symptoms and 
continue to be at risk of self-harm as well as harm to their personal and professional lives. Although 
a significant proportion of patients usually respond to currently available medications within three 


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to four weeks, remission rates are usually low at this point and may take several more weeks to 
become manifest (Entsuah, Huang et al. 2001). Moreover, up to one-third of patients still do not 
respond with four consecutive trials of antidepressants. A more rapidly acting antidepressant 
medication in TRD would significantly impact the treatment of depression and have major health 
care implications.  
In subjects with TRD, ketamine is the only pharmacological treatment that has antidepressant 
effects that both occur within hours and are sustained for one week or more with a single-
administration (Zarate, Singh et al. 2006, Kishimoto, Chawla et al. 2016). In the 2006 study, of 17 
subjects, 71% of participants met criteria for antidepressant response (a 50% decrease in HDRS 
scores), and 29% achieved remission (≤7 HDRS) at 24 hours following infusion of ketamine. Six 
(35%) subjects maintained response to ketamine for at least one week; in contrast, no subject on 
placebo responded at Day 1 or Day 7. In a recent study, we found that the antidepressant efficacy 
of ketamine lasted at least 11 days with single administration in subjects with TRD (Nugent, 
Ballard et al. 2019). Within six hours to one day, a single ketamine infusion led to response rates 
comparable to those seen following eight weeks of treatment with monoaminergic based 
antidepressants. Furthermore, ketamine induced remission in approximately one-third of subjects 
within one day, which is in stark contrast to that of monoaminergic-based approaches, which 
usually require 10-14 weeks of chronic daily usage to produce similar remission rates (Trivedi, 
Rush et al. 2006).  
A recent meta-analysis that included seven clinical trials examining the antidepressant 
response to ketamine in subjects with depression (several of these studies were from our group) 
found that ketamine produced an antidepressant effect at 24 hours post-infusion with odds ratios 
for response of 9.87 (4.37-22.29) and transient remission of 14.47 (2.67-78.49) (Newport, 
Carpenter et al. 2015). In addition, considerable evidence demonstrates that ketamine also has 
rapid antisuicidal ideation, anxiolytic, and anti-anhedonic effects in individuals with treatment-
resistant MDD (Zarate, Singh et al. 2006, DiazGranados, Ibrahim et al. 2010, Murrough, Iosifescu 
et al. 2013, Lally, Nugent et al. 2014). Furthermore, preliminary evidence suggests that acute 
ketamine administration has salutary effects on a number of immune and inflammatory and bone 
biological indices associated with heart disease and osteopenia, which are potentially modifiable 
risk factors and have often been found to be altered in depression (Kadriu, Gold et al. 2017). 
Whether acutely rectifying these indices in addition to improving depressive symptoms could have 
long-term benefits on brain and body health, particularly as one ages, remains unknown. However, 
it is quite clear that treatments capable of producing a rapid (within hours), robust, and sustained 
antidepressant effect would be of major importance to individuals with depression. Such an 
intervention could also potentially minimize the cumulative morbid effects of depression and 
altered immune and inflammatory states that place MDD subjects at increased risk of premature 
death from either medical conditions or suicide (Zivin, Ilgen et al. 2012, Choi, DiNitto et al. 2017). 
Taken together, the evidence gleaned from these studies have introduced a new paradigm for the 
research into the mechanisms implicated in antidepressants with a rapid onset of action. 
Synaptic plasticity changes involving AMPA receptors (AMPARs) may underlie the acute 
and sustained antidepressant actions of ketamine (Koike and Chaki 2014, Duman, Aghajanian et 
al. 2016, Zanos, Moaddel et al. 2016). Ketamine’s NMDAR blockade is hypothesized to be the 
first step in a cascade of events (“disinhibition hypothesis”) that includes rapid increases in 
presynaptic glutamate release (“glutamate burst”), enhanced regional activity in excitatory 
networks and, ultimately, changes in synaptic plasticity and connectivity, thus rapidly restoring 


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the impaired synaptic and circuit homeostasis associated with depression (Krystal, Karper et al. 
1994). We first demonstrated in antidepressant assays (Maeng, Zarate et al. 2008) that the 
administration of AMPAR antagonist (NBQX) blocks the antidepressant-like effects of ketamine, 
which indicates that the neurotransmission at AMPARs is involved in these effects. This finding 
has now been replicated by multiple labs (Li, Lee et al. 2010, Autry, Adachi et al. 2011, Zanos, 
Moaddel et al. 2016). 
Unfortunately, the widespread clinical use of ketamine for the treatment of major depression 
is restricted to certain subgroups (e.g., TRD, severe suicidal ideation) as ketamine requires close 
monitoring when administered, due to its side effects, including dissociation, psychotomimetic 
properties, and abuse potential(Acevedo-Diaz, Cavanaugh et al. 2019). Consequently, alternative 
medications that share the robust antidepressant actions of ketamine, but lack its side effects and 
abuse potential, are urgently needed. mGlu2/3 receptor antagonists have a similar antidepressant 
profile and share similar mechanisms implicated in antidepressant response to ketamine, including 
AMPAR stimulation (Maeng, Zarate et al. 2008, Dong, Zhang et al. 2017), but without the 
limitations of ketamine (Witkin, Monn et al. 2016). 
2.2.2  Modulating mGlu2/3 Receptor Results in Antidepressant-like Properties in 
Antidepressant Assays   
The metabotropic glutamate receptor family is comprised of eight different receptor subtypes 
(mGlu1–mGlu8 receptors), which are divided into three main groups (group I: mGlu1 and mGlu5 
receptors; group II: mGlu2 and mGlu3 receptors; group III: mGlu4, mGlu6, mGlu7, and mGlu8 
receptors). Of these, group II mGlu receptors (mGlu2 and mGlu3 receptors) are coupled to Gi/Go 
to negatively regulate adenylyl cyclase activity, and are highly expressed within the cortical and 
limbic regions of the brain, the brain areas associated with emotion and cognition.  
Preclinical studies have reported the efficacy of group II metabotropic glutamate receptor 
(mGlu2/3 receptor) antagonists in reducing behavioral despair in the acute forced-swim test at 30–
60 min following drug administration (Chaki, Yoshikawa et al. 2004, Bespalov, van Gaalen et al. 
2008, Witkin, Monn et al. 2016) and decreasing escape failures in the learned helplessness 
paradigm (Yoshimizu, Shimazaki et al. 2006). mGlu2/3 receptors are expressed in hippocampal, 
synaptic, mossy fiber-CA3 pyramidal cells and at excitatory synapses in the prefrontal cortex 
(PFC) (Ohishi, Ogawa-Meguro et al. 1994). mGlu2 receptors are primarily localized peri-
synaptically in close proximity to the pre-synaptic terminals (Petralia, Wang et al. 1996, 
Shigemoto, Kinoshita et al. 1997), where they act as auto-receptors to decrease synaptic glutamate 
transmission when activated, presumably serving as a homeostatic mechanism to prevent 
excessive glutamate release (Tzounopoulos, Janz et al. 1998, Chen, Huang et al. 2001). In contrast, 
mGlu3 receptors are primarily localized to glial cells (Ohishi, Shigemoto et al. 1993) and their 
activation inhibits astrocyte growth (Ciccarelli, Sureda et al. 1997) and increases levels of 
glutamate transporter proteins (Aronica, Gorter et al. 2003), thus indirectly decreasing 
extracellular glutamate levels. 
mGlu2/3 receptor inhibition elicits rapid antidepressant actions in preclinical studies, similar 
to ketamine. In particular, a single administration of an mGlu2/3 receptor antagonist reduced 
immobility time in the 24-h forced-swim test (Dwyer, Lepack et al. 2012), decreased time delay 
until food consumption in the novelty-suppressed feeding test (Koike, Kasamatsu et al. 2013, 
Fukumoto, Iijima et al. 2014), rapidly reversed chronic stress-induced decreases in sucrose 
preference, which was sustained for at least 10 days (Dwyer, Lepack et al. 2013), and reversed 


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chronic corticosterone-induced behavioral deficits(Ago, Yano et al. 2013, Koike, Iijima et al. 
2013) in rodents. In addition, mGlu2/3 receptor blockade reversed the decrease in sucrose 
preference and increased behavioral despair produced by chronic social defeat stress in mice within 
24 hours after a single administration (Dong, Zhang et al. 2017). While a large (n = 310 subjects) 
clinical trial of a negative allosteric modulator of mGlu2/3 receptor (RG1578; decoglurant) failed 
to demonstrate antidepressant responses compared with placebo, no measure of target engagement 
was included in this trial and therefore we cannot conclude that the target was engaged. Additional 
studies are needed to determine the potential of mGlu2/3 receptor antagonists in the treatment of 
TRD (Quiroz, Tamburri et al. 2016). Both orthosteric and allosteric modulator for mGlu2/3 
receptor have been shown to exert antidepressant effects, however the potency of allosteric 
modulators was demonstrated to be weaker than that of the orthosteric antagonists (Campo, 
Kalinichev et al. 2011). For this reason, we are pursuing investigation with a potent and selective 
mGlu2/3 receptor orthosteric antagonist.  
Our work challenges the existing hypothesis that N-methyl-D-aspartate receptor (NMDAR) 
inhibition is ketamine’s primary mechanism of antidepressant action; rather, we found that 
ketamine’s antidepressant effects may be due to its metabolite (2R,6R)-hydroxynorketamine 
(HNK), whose mechanism may be NMDAR-independent (Zanos, Moaddel et al. 2016, Lumsden, 
Troppoli et al. 2019). We recently found that (2R,6R)-HNK induces synaptic plasticity as shown 
by increases in AMPAR-mediated EPSPs (Zanos, Moaddel et al. 2016). We subsequently found 
that this effect appears to be due to pre-synaptic mechanisms, as (2R,6R)-HNK induced paired-
pulse depression (Zanos, Highland et al. 2019). In addition, whole-cell patch clamp data showed 
increased mEPSC frequencies, but not amplitude, in the presence of tetradotoxin (TTX), indicating 
pre-synaptic regulation of synaptic strengthening. mGlu2 receptor specifically acts as an 
autoreceptor and, when inhibited, increases the probability of glutamate release, similar to 
(2R,6R)-HNK. Our preclinical data indicate that administration of the mGlu2/3 receptor agonist 
LY379268 to mice blocked ketamine and (2R,6R)-HNK’s antidepressant actions and that, together, 
sub-effective doses of an mGlu2/3 receptor antagonist and sub-effective doses of ketamine exerted 
antidepressant effects, suggesting synergistic mechanisms. Furthermore, the antidepressant actions 
of ketamine and (2R,6R)-HNK were also blocked in mGlu2 receptor, but not mGlu3 receptor, 
knockout mice (Zanos, Highland et al. 2019). Notably, recent studies have also indicated important 
roles of mGlu3 receptor in the antidepressant effects, presumably through postsynaptic 
mechanisms (Joffe, Santiago et al. 2020). Studies suggest that mGlu2/3 receptor antagonists exert 
antidepressant effects in multiple antidepressant assays in rodents (Yoshimizu, Shimazaki et al. 
2006, Palucha-Poniewiera, Wieronska et al. 2010, Dong, Zhang et al. 2017); these appeared within 
24 hours after a single administration in the chronic unpredictable stress (Dwyer, Lepack et al. 
2013) and chronic social defeat stress models (Dong, Zhang et al. 2017). These compounds have 
also been associated with sustained antidepressant effects lasting ~ one week after a single 
administration (Dwyer, Lepack et al. 2013, Dong, Zhang et al. 2017). It is notable that mGlu2/3 
receptor antagonists share mechanisms implicated in the antidepressant response to ketamine, 
including AMPAR stimulation (Maeng, Zarate et al. 2008), brain derived neurotrophic factor 
(BDNF)/TrkB signaling, mechanistic target of rapamycin complex 1 (mTORC1) signaling, and 
synthesis of synaptic proteins (such as GluA1) in the PFC (Dong, Zhang et al. 2017, Chaki 2019).  
In addition to synaptic mechanisms, serotonergic and dopaminergic systems have also been 
suggested to have important roles in the antidepressant actions of the mGlu2/3 receptor antagonists. 
The mGlu2/3 receptor antagonists increased the firing of the 5-HT neurons in the dorsal raphe 
nucleus and 5-HT release in the mPFC, indicating activation of the serotonergic system. 


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Furthermore, the antidepressant actions of mGlu2/3 receptor antagonists were blocked by 
depletion of 5-HT and a 5-HT1A receptor antagonist, suggesting the serotonergic system is 
necessary for mGlu2/3 receptor antagonists to exert their antidepressant actions (Fukumoto, Iijima 
et al. 2014, Fukumoto, Iijima et al. 2016, Fukumoto, Iijima et al. 2018). Likewise, an mGlu2/3 
receptor antagonist has been demonstrated to increase number of spontaneously active dopamine 
neurons in the ventral tegmental area and to increase extracellular dopamine level in the nucleus 
accumbens and medial prefrontal cortex (Witkin, Monn et al. 2016), suggesting the dopaminergic 
system is also implicated in the antidepressant effects of mGlu2/3 receptor antagonists. These 
serotonergic and dopaminergic mechanisms are also shared with ketamine (Dong, Zhang et al. 
2017, Chaki 2019, Chaki and Fukumoto 2019). In terms of safety, mGlu2/3 receptor antagonists 
did not induce side effects similar to those of ketamine in mouse tests, nor do they appear to have 
abuse potential (Witkin, Monn et al. 2017). Furthermore, mGlu2/3 receptor antagonists increase 
gamma oscillations (Ahnaou, Ver Donck et al. 2014) which is linked to gamma activation (see 
below) thus, this gives us a putative biomarker for our POC studies. The candidate drug we selected, 
TS-161, is a prodrug of TP0178894, an orthosteric mGlu2/3 receptor antagonist. The Phase 2 POC 
study will be conducted on our research unit. 
2.2.3  Surrogate Neurobiological Markers of mGlu2/3 receptor antagonist TS-161’s 
Mechanism of Action and/or Antidepressant Therapeutic Response 
An impediment to progress in the discovery and testing of novel medications for serious 
psychiatric disorders is the lack of established surrogate neurobiological markers capable of 
predicting therapeutic response or identifying mechanisms of action. Identifying such markers may 
greatly facilitate drug discovery. The proposed project examines the utility of several potential 
neurobiological surrogate markers. A surrogate biomarker is most useful in that the number of 
subjects required to demonstrate an effect is less than that needed to prove clinical efficacy. 
Therefore, by assessing the same number of subjects with the neurobiological markers as those 
being evaluated for clinical efficacy, the proposed study should have sufficient power to identify 
surrogate neurobiological markers of practical significance. For example, previous studies from 
our laboratory found that gamma power changes, as determined by MEG, may be a putative 
biomarker to identify a subgroup of subjects with TRD who will respond favorably to ketamine’s 
antidepressant effects (Cornwell, Salvadore et al. 2012, Nugent, Ballard et al. 2019). 
In the proposed project, we will evaluate the following approaches as potential surrogate 
markers of drug effects and/or treatment response:  
1.  Gamma oscillatory power as well as other spectral analyses with MEG 
2.  Resting and task based functional connectivity with fMRI. Resting and task based 
functional 
3.  Prefrontal glutamate levels (and other brain metabolites) measured with 7T 1H-MRS 
4.  Plasma/serum/Lymphocytes/mRNA for neurotrophic biomarkers 
2.2.4  Convergent Evidence that Enhancing AMPAR Activity is Critical to the Mechanism 
of Action of Rapid Acting Antidepressants 
Understanding the mechanism of action by which ketamine exerts its antidepressant effects 
could lead to advances in the development of the next generation of antidepressants. As noted 
above, preclinical data from our laboratory indicates that ketamine’s antidepressant effects are 
mediated through AMPARs. We demonstrated blockage of  the antidepressant-like properties of 


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ketamine in the forced swim test (FST) with a relatively selective AMPAR antagonist (NBQX) 
(Maeng, Zarate et al. 2008). Other investigators subsequently replicated this finding (Li, Lee et al. 
2010, Autry, Adachi et al. 2011, Andreasen, Gynther et al. 2013, Beurel, Grieco et al. 2016, Zanos, 
Moaddel et al. 2016). 
The present study proposes to expand our preclinical findings by evaluating whether an 
mGlu2/3 receptor antagonist also produces the same effects of ketamine in subjects with TRD (i.e., 
antidepressant effects, increases in glutamate release and gamma power).  
2.2.5  Gamma Oscillations as a Putative Cross-Species Biomarker of Synaptic Plasticity 
and AMPAR Throughput 
One theory posits that the rapid antidepressant effects of ketamine are probably initiated by 
its antagonistic effects on NMDARs; these effects regulate glutamate/GABA and/or 
AMPAR/NMDAR activity. Preclinical studies have shown that ketamine rapidly induces 
synaptogenesis and reverses synaptic deficits caused by chronic stress, thus restoring network 
connectivity (Duman and Aghajanian 2012), while clinical studies support enhanced plasticity in 
response to ketamine (Cornwell, Salvadore et al. 2012, Nugent, Wills et al. 2019). Animal studies 
have begun to elucidate ketamine’s downstream effects that might underlie its beneficial effects in 
depressed subjects. Ketamine’s NMDAR blockade is hypothesized to be the first step in a cascade 
of events that includes rapid increases in presynaptic glutamate release, enhanced regional activity 
in excitatory networks and, ultimately, changes in synaptic plasticity and connectivity. In rodents, 
low-dose ketamine administration rapidly triggers three successive events: first, presynaptic 
disinhibition of glutamatergic neurons, which leads to a glutamate burst; second, increased 
activation of the AMPAR, combined with the blockade of extrasynaptic NMDARs; and third, 
postsynaptic activation of neuroplasticity-related signaling pathways involving BDNF and 
mTORC1, which results in overall synaptogenesis and synaptic potentiation (Li, Lee et al. 2010, 
Autry, Adachi et al. 2011, Liu, Lee et al. 2012). Both animal (Pinault 2008, Zhang, Yoshida et al. 
2012, Anderson, Pinault et al. 2014, Jones, Anderson et al. 2014) and human (Rivolta, Heidegger 
et al. 2015, Shaw, Saxena et al. 2015) studies indicate that acute, subanesthetic ketamine infusion 
is associated with robust increases in gamma power. Multiple synaptic mechanisms play a role in 
regulating gamma oscillations, including AMPAR-mediated depolarization and GABAA receptor-
mediated inhibition. Ketamine may influence both of these systems, both by silencing GABAergic 
inhibitory synapses and by increasing glutamate release, thereby activating AMPARs (Ren, 
Pribiag et al. 2016). The decreased activity in GABAergic interneurons and the disinhibition of 
excitatory pyramidal neurons (Homayoun and Moghaddam 2007) presumably provide the 
mechanism for increased gamma oscillations (Carlen, Meletis et al. 2012) and corresponding 
measured power. The antidepressant mechanism is likely more complex, however, given that 
blockade of NMDARs on interneurons and the subsequent disinhibition of pyramidal neurons does 
not consistently produce an antidepressant effect (Miller, Moran et al. 2016).  
Notably, acute administration of the ketamine metabolite (2R,6R)-HNK also increases gamma 
oscillations likely via mGlu2 receptor signaling, despite the fact that the metabolite does not inhibit 
NMDARs at concentrations that increase gamma power (Zanos, Moaddel et al. 2016, Zanos, 
Highland et al. 2019). Because AMPAR blockade can attenuate (2R,6R)-HNK-induced gamma 
oscillations, enhanced AMPAR activity is likely to be the mechanism by which (2R,6R)-HNK and 
mGlu2/3 receptor antagonists increase gamma power, although the mechanism of increased 
AMPAR activity is not known (Bartos, Vida et al. 2007). Taken together, these findings 


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underscore the close relationship between gamma oscillations and inhibition/excitation balance 
(Kehrer, Maziashvili et al. 2008, Gandal, Sisti et al. 2012).  
Previous studies from our laboratory also used cortical excitability as a plasticity measure of 
antidepressant response to ketamine. Twenty unmedicated subjects with treatment-resistant MDD 
received a single, open-label intravenous infusion of ketamine hydrochloride (0.5 mg/kg) 
(Cornwell, Salvadore et al. 2012). MEG recordings were performed at baseline (prior to) and 
approximately seven hours after the ketamine infusion. At these two timepoints, subjects passively 
received tactile stimulation (somatosensory evoked potentials) to the right and left index fingers 
while they rested with eyes closed. Subjects exhibiting robust improvements in depressive 
symptoms 230 minutes post-ketamine infusion (responders) had increased cortical excitability 
within this antidepressant response window. Specifically, we found that stimulus-evoked 
somatosensory cortical responses (i.e., gamma-band power) increased after infusion relative to 
pretreatment responses in treatment responders but not in nonresponders. Spontaneous 
somatosensory cortical gamma-band activity during rest did not change within the same time frame 
after ketamine in either responders or nonresponders. These findings suggest that NMDAR 
antagonism does not directly lead to increased cortical excitability hours later and thus might not 
be sufficient for the therapeutic effects of ketamine to take hold. Rather, increased cortical 
excitability as depressive symptoms improve is consistent with the hypothesis that enhanced non-
NMDAR-mediated glutamatergic neurotransmission via synaptic potentiation is central to the 
antidepressant effects of ketamine.  


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These results support the hypothesis that ketamine’s rapid antidepressant effects are due to 
enhanced AMPAR throughput in cortex.  
In a follow-up study, we used the same task (passive tactile stimulation) but with a more 
rigorous design (Nugent, Wills et al. 2019), again finding increased gamma power in responders 
compared with nonresponders. In a double-blind, placebo-controlled study of 35 unmedicated, 
TRD- MDD subjects and 25 healthy controls, we also assessed ketamine’s mechanism of action 
using gamma power with MEG as a proxy measure for homeostatic balance (Nugent, Ballard et 
al. 2019). MDD subjects showed significant improvements in depressive symptoms, and 
unexpectedly, healthy subjects exhibited modest but significant increases in depressive symptoms 
for up to one day after ketamine administration. Both groups showed increased resting gamma 
power six to nine hours following ketamine. In controls, increased gamma power was associated 
with increased depressive symptoms. In MDD subjects, baseline gamma power moderated the 
relationship between post-ketamine gamma power and antidepressant response. While higher post-
ketamine gamma power was associated with better response in MDD subjects with lower baseline 
gamma, the relationship was inverted in MDD subjects with higher baseline gamma (Nugent, 
Ballard et al. 2019). We next sought to replicate previous findings of increased gamma response 
Figure 1A shows the contrasts for Ketamine-Day 0 versus Placebo-Day 0 for the MDD group. Figure 1B shows 
the same contrast in the healthy control group. Regions include those involved in the central executive (CEN), 
salience (SN), and default mode (DMN) networks. Figure 1C illustrates the estimated marginal means from mixed
models of RMS gamma power extracted from selected regions of interest defined on the Ketamine-Day 0 vs. 
Placebo-Day 0 contrast across both groups. MDD subjects exhibited increases post-ketamine to levels 
commensurate with those seen in healthy control subjects following placebo infusion. 
A. MDD: Ketamine > Placebo 
C. 
B. HC: Ketamine > Placebo 


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to a somatosensory stimulus at 230 min and Day 1 in ketamine responders versus non-responders 
in 31 depressed subjects and 25 healthy controls (Nugent, Wills et al. 2019). A significant 
difference in peak gamma power was seen in the depressed ketamine responders versus non-
responders. These results implicate AMPAR throughput in ketamine's mechanism of 
antidepressant action. While potentially promising, it is important to note that MEG gamma power 
as a biomarker is still in the exploratory stages. No dose-dependent gamma power changes have 
been yet demonstrated with ketamine.
 
 
It is important to emphasize that the SAD TS-161 study was conducted in healthy 
volunteers and as such the results might be different in depressed subjects. For this reason, we will 
examine all MEG frequency band changes with TS-161.   
The blood oxygen level dependent (BOLD) signal measured using functional MRI (fMRI) 
has been shown to be modulated by other mGluR2 agonists in the striatal, dorsolateral prefrontal 
cortex and ACC (Krystal 2013, Wolf, Ruparel et al. 2013).  In this study we plan to use resting 
state fMRI to probe disruption of neural circuits connected to these regions.  Additionally, we plan 
to use simple cognitive tasks, such as working memory, continuous performance and emotion 
identification to localize any effects of TS-161. 
2.2.6  Prefrontal Glutamate Levels Measured with 7T 1H-MRS and fMRI 
Several recent preclinical studies have demonstrated the need of a “glutamate surge” and the 
accompanying AMPA activation in generating the antidepressant-like response of ketamine and 
other NMDA antagonists. However, blocking NMDAR receptors likely results in its 
psychomimetic properties and contributes to its abuse potential. We hypothesized that TS-161 will 
also rapidly increase glutamate release by selectively inhibiting mGlu2 receptors thus activating 
AMPA receptors and intracellular neuroprotective cascades including AKT and mTORC1.  
In a recent study (Evans, Lally et al. 2018), we found only a trend-level increase in glutamate 
levels in the pgACC using 1H-MRS 7T in MDD subjects 24 hours post-ketamine, suggesting that 
the changes (i.e., glutamate burst) could have been more immediate to the infusion of ketamine. 
In this study, we plan to measure glutamate levels in the pgACC using 1H-MRS 7T in MDD 
subjects at plasma Cmax of active metabolite of the initial dose of TS-161 (100 mg) at a timeframe 
that would presumably coincide with the glutamate surge.  
2.2.7  Neurochemical Indices as Putative Biomarkers of Synaptic Plasticity, Drug Effects, 
and Antidepressant Response of Ketamine 
BDNF is the most abundant neurotrophin in the brain, and its activation of tropomyosin-
related kinase B (TrkB) receptors stimulates neural growth, survival, and plasticity. In preclinical 
models of despair, ketamine activates mTORC1, which is a critical hub of cellular growth and 
proliferation (Hay and Sonenberg 2004). Ketamine was found to increase mTORC1 
phosphorylation and other downstream molecular targets critical for transcriptional activation 
within one hour of administration (Li, Lee et al. 2010). Twenty-four hours following ketamine 
exposure, greater numbers of mature dendritic spines were observed. These molecular and cellular 
effects were lost if the rodent was pretreated with rapamycin, an mTORC1 antagonist. These 
findings suggest that mTORC1 activation is necessary for ketamine’s rapid-acting antidepressant 
effects in a rodent model of despair, which may occur via BDNF. After initial studies from our 
group suggested that peripheral BDNF levels do not correlate with antidepressant response to 


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ketamine without genotypic stratification (Machado-Vieira, Yuan et al. 2009, Laje, Lally et al. 
2012), several subsequent studies with larger samples (including ours) reported a positive 
association between baseline serum BDNF and ketamine’s antidepressant effects. One study found 
that in a sample of 22 TRD subjects, plasma BDNF levels at 4 hours were associated with greater 
antidepressant improvement at the same timepoint 24 hours and 72 hours post-ketamine infusion 
(Haile, Murrough et al. 2014). In a subsequent study, we found that changes in plasma BDNF were 
proportional to changes in EEG slow wave activity (SWA), and that this link was present only in 
subjects who responded to ketamine treatment, suggesting enhanced synaptic plasticity (Duncan, 
Sarasso et al. 2013). In order to identify pathways that are responsible for ketamine's effect, we 
carried out a targeted metabolomic approach using a double-blind, placebo-controlled crossover 
design, with infusion order randomized with medication-free patients with TRD- MDD (29 
subjects) and healthy controls (25 subjects). Ketamine treatment resulted in a general increase in 
circulating sphingomyelins, levels which were not correlated with response. Ketamine response 
resulted in more pronounced effects in the kynurenine pathway and the arginine pathway at 4 h 
post-infusion, where a larger decrease in circulating kynurenine levels and a larger increase in the 
bioavailability of arginine were observed in responders to ketamine treatment, suggesting possible 
mechanisms for response to ketamine treatment (Moaddel, Shardell et al. 2018). 
We will thus be collecting whole blood for the purpose of transcriptional profiling, 
metabolomics, proteomics, and the measurement of inflammatory measures, oxidative stress 
markers and neurotrophic factors, as well as glycine/serine levels, all directly implicated in the 
pathophysiology and treatment response in mood disorders (reviewed in (Machado-Vieira, Soeiro-
De-Souza et al. 2014)). We will also explore inflammatory markers such as cytokines and how 
they may be related to drug effects or treatment response. 
2.2.8  RDoC, Synaptic Plasticity, and Antidepressant Response to Ketamine 
Depression is a multidimensional disorder characterized by a variety of symptoms that include 
depressed mood, anhedonia, negative cognitive biases, and altered activity levels. The focus on 
diagnostic entities rather than the component constructs has likely limited attempts to understand 
the biology of mental illness. A more dimensional approach, like that described by the Research 
Domain Criteria (RDoC) initiative, is a potential solution to this issue. Indeed, in an exploratory 
factor analysis of a variety of depression ratings collected in 119 subjects with either MDD or 
bipolar depression, we found that the best solution comprised eight unidimensional factors: 
Depressed Mood, Tension, Negative Cognition, Impaired Sleep, Suicidal Thoughts, Reduced 
Appetite, Anhedonia, and Amotivation. Importantly, a differential pattern of response, both to 
ketamine and placebo, was observed across these unidimensional constructs (factors) (Ballard, 
Yarrington et al. 2018). In other words, the use of these unidimensional constructs may reveal 
patterns not observed with traditional scoring of individual instruments. The empirical 
identification of unidimensional constructs creates more refined scores that may elucidate the 
connection between specific symptoms and underlying pathophysiology. For this reason, we plan 
to conduct an exploratory analysis to measure the different constructs derived from our exploratory 
factor analysis (i.e., Depressed Mood, Tension, Negative Cognition, Impaired Sleep, Suicidal 
Thoughts, Reduced Appetite, Anhedonia, and Amotivation).  


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2.2.9  Neurocognition and Glutamatergic Modulation 
The acute cognitive effects of ketamine treatment for depression have been minimally studied. 
In healthy participants, acute infusions using larger ketamine doses have been associated with 
significant short-term deficits particularly in working memory, source memory and episodic 
memory, as well as subjective cognitive effects (e.g., impaired memory, confusion) (Morgan, 
Mofeez et al. 2004).  
Further, few studies have examined possible cumulative cognitive effects of repeated 
ketamine treatment in depressed subjects (Blier, Zigman et al. 2012, Irwin, Iglewicz et al. 2013, 
Diamond, Farmery et al. 2014, Shiroma, Albott et al. 2014, George, Galvez et al. 2017, Galvez, Li 
et al. 2018). Cognitive deficits following repeated use of NMDAR antagonists have been reported 
in animal studies (Jentsch, Tran et al. 1997, Mandillo, Rinaldi et al. 2003) and in recreational users 
of ketamine (Morgan, Muetzelfeldt et al. 2010). In humans, these deficits have been observed 
primarily on measures of verbal fluency, episodic memory and attention/working memory 
(Morgan, Mofeez et al. 2004, Morgan, Muetzelfeldt et al. 2010). A prior study showed that 
cognitive functioning was associated with subsequent antidepressant response to ketamine at 24 
hours (Murrough, Wan et al. 2013). Individuals who demonstrated a positive antidepressant 
response to ketamine had slower processing speed at baseline, compared with individuals who did 
not respond. Furthermore, the authors did not find adverse cognitive effects of ketamine 7 days 
post treatment.  
The precise mechanism for the cognitive side effects of ketamine is unclear but could possibly 
be linked to enhanced glutamate release. Aspects of the disinhibition hypothesis was originally 
proposed by Moghaddam and colleagues where they suggested that  hyperactivation of non-
NMDA receptors, as opposed to a “glutamatergic deficiency” from NMDAR blockade may 
account for some of the cognitive deficits and schizophrenia-like symptoms of NMDA receptor 
antagonists (Moghaddam, Adams et al. 1997). Thus, it stands that the side effect of ketamine, 
dissociation, could be related to the mechanism of antidepressant action as they appear to share 
important mechanistic processes implicated in ketamine’s therapeutic properties. In support, as 
was previously discussed, there is increasing evidence to suggest that the primary mechanism of 
ketamine for its antidepressant effects is NMDAR-independent (Zanos, Moaddel et al. 2016). 
Since mGlu2/3 receptor antagonists would also be linked to an increase in glutamate release, it is 
imperative that we monitor cognition in subjects exposed to such drugs. Preclinical evidence 
however, does not suggest that cognitive deficits will occur with mGlu2/3 receptor antagonists 
(Witkin, Monn et al. 2017). In fact, mGlu2/3 receptor antagonists do appear to have pro-cognitive 
effects as demonstrated in the social recognition test (Higgins, Ballard et al. 2004, Shimazaki, 
Kaku et al. 2007). We will obtain a detailed neurocognitive battery during the study following 
dose administration. 
2.2.10  Current Proposal 
In Study Phase I, subjects will be 2-weeks drug-free prior to their Study Phase II 
randomization except if subjects were taking a medication with a long half-life such as fluoxetine, 
aripiprazole, or brexpiprazole in which case the washout will be five weeks. The MADRS will be 
the primary outcome measure for depression severity. Other measures obtained will include 
behavioral measures, peripheral biomarkers, MEG, fMRI, and 1H-MRS. Male and female subjects, 
ages 18 to 65 years, with a diagnosis of MDD, currently in an episode of major depression, will be 
recruited for this study, which will take place at the NIH Clinical Center in Bethesda, MD. 


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2.2.10.1  The Candidate Drug: TS-161, an mGlu2/3 Receptor Antagonist Prodrug 
TP0473292 (TS-161), an orally bioavailable prodrug of TP0178894 (a potent and selective 
mGlu2/3 receptor antagonist), is being developed as a novel antidepressant by Taisho 
Pharmaceutical Co., Ltd. Preclinical evaluation of TP0473292 (TS-161)  showed rapid conversion 
into the pharmacologically active metabolite TP0178894 on oral administration in rats and 
monkeys. The pharmacokinetic profile of TP0178894 was consistent with once-a-day oral dosing 
in humans. In animal models of depression and behavior, including those refractory to current 
medications, TP0178894 or its prodrug TP0473292 (TS-161) demonstrated potent antidepressant-
like effects. 
 
 
 
 
  
2.2.10.2  Nonclinical Pharmacological Profiles of TP0178894 and TP0473292  
TP0178894 showed high affinity for human mGlu2 receptor (Ki=4.27 nM), and human 
mGlu3 receptor (Ki=2.83 nM). TP0178894 also had potent antagonist activity for human mGlu2 
receptor (IC50=23.3 nM), and human mGlu3 receptor (IC50=20.9 nM). In contrast, TP0178894 
had a negligible antagonistic effect for mGlu4, 6 and 8 receptors. Moreover, TP0178894 at 10 μM 
showed no significant affinity for other 66 molecules including receptors, transporters and ion 
channels, indicating that TP0178894 is a selective mGlu2/3 receptor antagonist. In addition, 
TP0178894 also showed potent antagonist activity for rat mGlu2 receptor (IC50=23.2 nM), and 
rat mGlu3 receptor (IC50=27.8 nM), indicating that TP0178894 has no species difference in 
antagonist activity for mGlu2/3 receptor.  
Oral administration of TP0473292 was converted into the active metabolite TP0178894 in 
rodents and monkeys. TP0473292 per se had a negligible affinity for human mGlu2 receptor and 
mGlu3 receptor. Moreover, TP0473292 at 10 μM did not show significant affinity for other 65 
molecules including receptors, transporters and ion channels. Oral administration of TP0473292 
exerted antidepressant effects in a rat FST (the lowest effective dose: 1 mg/kg) at concentration of 
TP0178894 in rat cerebrospinal fluid (CSF) of 1.87 ng/mL. In addition, TP0473292 also exhibited 
the antidepressant effect in a repeated corticosterone treatment model (the lowest effective dose: 
1 mg/kg, p.o.), which is refractory to current medications such as fluvoxamine and imipramine 
(Iijima, Ito et al. 2010). In contrast, TP0473292 did not affect spontaneous locomotor activity in 
rats up to 10 mg/kg, p.o., indicating that TP0473292 may not cause sedation or excess excitation. 
In addition, TP0473292 counteracted an mGlu2/3 receptor agonist, LY379268-induced 
hypolocomotion, indicating that TP0473292 has antagonist activity for mGlu2/3 receptor in vivo. 
These results show that TP0473292 is an orally active mGlu2/3 receptor antagonist with 
antidepressant potential.  
2.2.10.3  Preclinical Pharmacokinetic Profiles of TP0178894 and TP0473292 
TP0473292, a prodrug of TP0178894, an mGlu2/3 receptor antagonist, is orally bioavailable, 
and presystemically converts into TP0178894 in rats and monkeys.  
Absorption, distribution, metabolism, excretion, and pharmacokinetic drug-drug interaction 
of TP0178894 and TP0473292 were investigated in in vivo in rats and monkeys, and in in vitro 
studies. The summary of results was as follows: After single oral administration of TP0473292 to 


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rats (1 mg/kg) and monkeys (1.63 mg/kg), plasma levels of the active form TP0178894 reached a 
maximum concentration (Cmax) within 3 hours post-dose, and decreased with an elimination half-
life (T1/2) of 1 to 5 hours. The oral bioavailability as TP0178894 was 58.4% (rats) and 56.6% 
(monkeys). The prodrug TP0473292 was not measurable in plasma at all the sampling time points, 
indicating complete presystemic hydrolysis of TP0473292 to TP0178894 after gastrointestinal 
absorption. 
TP0178894 was found to cross the BBB into rat 
 CSF after a single oral 
administration of TP0473292 (1 mg/kg). The TP0178894 levels in 
 CSF reached Cmax 
of 
 2.78 ng/mL at 3 hours post-dose, respectively. 
 
The 
 CSF-
to-plasma exposure (area under the concentration-time curve: AUC) ratios of TP0178894 were 
 0.0149, 
 
TP0473292 was almost completely hydrolyzed to TP0178894 in cryopreserved hepatocytes 
of rats, monkeys, and humans, and no metabolites related to TP0178894 were detected. A 
metabolite derived from an ester side-chain moiety 
 was also released from the 
prodrug TP0473292 via hydrolysis, which was further metabolized to glucuronide 
 
via glucuronidation. No human specific metabolites were found in in vitro studies. In the human 
recombinant carboxylesterase (CES) system, TP0473292 was hydrolyzed by both CES1 and CES2 
which express predominantly in liver and intestine, respectively.  
Following single intravenous administration of TP0178894 to rats (1 mg/kg) and monkeys (1 
mg/kg), 24-hour-urinary excretion rates of TP0178894 were 76.6% (rats) and 97.4% (monkeys) 
of the dose, indicating renal excretion as the main route of clearance of TP0178894. 
TP0473292 and its metabolites (TP0178894, 
 showed no 
inhibitory potential on seven major CYP isoforms. TP0473292 and TP0178894 had no induction 
potential on the CYP1A2, CYP2B6, and CYP3A4 messenger ribonucleic acids (mRNA) 
expression in human hepatocytes. Under the conditions of the CYP induction study, TP0473292 
would likely undergo degradation to yield TP0178894, 
 therefore, 
the metabolites derived from modifications of the ester side-chain moiety were also considered to 
have no induction potential for CYP1A2, CYP2B6, and CYP3A4 mRNA expression. 
 
  
 
 
 
 
2.2.10.4  Nonclinical Toxicology of TP0473292 and TP0178894 
  
 
 
 
 
 
  


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2.2.10.5  Prior Human Experiences - Single and Multiple Ascending Dose Studies (TS161-
US101) 
A first-in-human, randomized, double-blinded, placebo-controlled, single and multiple 
ascending dose study was conducted to evaluate the safety, tolerability, pharmacokinetics, and 
pharmacodynamics of TS-161 administered orally to healthy male and female participants.  


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Methods 
The study was composed of three parts; Part A (SAD, Single Ascending Dose), Part B (CSF), 
and Part C (MAD, Multiple Ascending Dose).  
A total of 70 healthy participants received TS-161 (15 mg to 400 mg; n=54) or placebo (n=16) 
in the study.  
In Part A, 40 participants were enrolled to receive TS-161 (15 mg to 400 mg; n=30) or placebo 
(n=10). Eight participants were randomized (6 active and 2 placebo) in each cohort to receive a 
single dose of TS-161 (15, 50, 100, 200, or 400 mg) or placebo. All participants were dosed under 
a fasted condition except for participants in Cohort 2, who were first dosed under a fasted condition 
(Cohort 2 [Fasted]), and then dosed again under a fed condition (Cohort 2 [Fed]).  
In Part B, six participants were enrolled to receive a single dose of 100 mg TS-161 under a 
fasted condition to evaluate CSF concentrations of TP0178894 (active metabolite).  
In Part C, 24 participants were enrolled to receive TS-161 (50 mg to 150 mg; n=18) or placebo 
(n=6). Eight participants were randomized (6 active and 2 placebo) in each cohort to receive a 10-
day multiple dose of TS-161 (50, 100, or 150 mg) or placebo. 
Safety  
There were no deaths or serious adverse events (SAEs) during the study. Thirty-nine (72.2%) 
out of 54 participants in the total TS-161 group reported 85 adverse events (AEs) and 7 (43.8%) 
out of 16 participants in the total placebo group reported 20 AEs. In Part C (MAD), 2 participants 
in the 150 mg TS-161 group and 1 participant in the placebo group discontinued from the study 
by participants’ request due to AEs. Overall, the frequently experienced AEs with exposure-related 
increases in their incidence were nausea, vomiting, and dizziness (including dizziness postural).  
Single Ascending Dose  
In Part A (SAD), 16 (53.3%) out of 30 participants in the fasted TS-161 group reported 25 
AEs and 4 (40.0%) out of 10 participants in the fasted placebo group reported 7 AEs. In Cohort 2, 
one (16.7%) out of 6 participants in the fasted TS-161 group reported 4 AEs, whereas 3 (50.0%) 
out of 6 participants in the fed TS-161 group reported 7 AEs. In Part B (CSF), 6 (100.0%) of 6 
participants reported 8 AEs.  
In Part A, the most frequent AEs included orthostatic heart rate response increased (13.3%), 
orthostatic hypotension (13.3%), and somnolence (10.0%) during fasted and fed combined TS-161 
treatment. The incidences of these AEs were not dose-related. All AEs were mild in intensity. The 
total number of AEs and participants with AEs increased with increase in dose levels; however, 
there were no other apparent dose-related or meal condition-related trends in the incidences of 
individual AEs. In Part B, the most frequent AE was post lumbar puncture syndrome (83.3%).  
In Part A, 15 (50.0%) out of 30 participants in the fasted TS-161 group reported 24 AEs that 
were considered possibly or probably related to the study drug and 1 participant reported 1 AE that 
was considered not related to the study drug (skin laceration). In the fed TS-161 group, 3 (50.0%) 
out of 6 participants reported 6 AEs that were considered possibly or probably related to the study 
drug and 1 participant reported 1 AE that was considered not related to the study drug (blood 
creatine phosphokinase increased). In Part B, 2 (33.3%) out of 6 participants reported 2 AEs that 


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were considered possibly related to the study drug and 5 participants reported 6 AEs that were 
considered not related to the study drug (post lumbar puncture syndrome and anxiety).  
There was no apparent treatment- or dose-related trends in clinical laboratory results, vital 
sign measurements, 12-lead ECG measurements or physical/neurological examination findings.  
The results from Parts A and B indicate that single oral doses of up to 400 mg TS-161 under fasted 
conditions were safe and well-tolerated in healthy participants.  
Multiple Ascending Dose  
In Part C (MAD), 15 (83.3%) out of 18 participants in the TS-161 group reported 45 AEs and 
3 (50.0%) out of 6 participants in the placebo group reported 9 AEs. Two subjects who received 
TS-161 had discontinued early from the study by subjects’ request due to AEs. One subject 
experienced AEs of dissociation, nausea, vomiting and anxiety, which were moderate in intensity, 
as well as mild headache, constipation, panic attack, abnormal dreams and dizziness. The second 
subject experienced nausea, headache and dizziness postural, which were moderate in intensity, 
and mild constipation.  
The system organ classes (SOCs) with high incidences of AEs in TS-161 treatment included 
Nervous System Disorders (10 participants [55.6%]) and Gastrointestinal Disorders (8 participants 
[44.4%]).  
The total number of AEs increased with increase in dose levels. The most frequent AEs in 
TS-161 treatment included nausea (44.4%), headache (22.2%), somnolence (22.2%), vomiting 
(16.7%), and abnormal dreams (16.7%). All AEs were mild in intensity, except for the moderate 
AEs observed in participants who discontinued early from the study and one moderate AE of 
vomiting which was observed in a participant who completed the study. The AEs with dose-related 
increases in incidences were nausea, vomiting, dizziness (including dizziness postural), headache, 
and abnormal dreams.  
Fifteen (83.3%) out of 18 participants in the TS-161 group reported 45 AEs that were 
considered possibly or probably related to study drug.  
There were no apparent treatment- or dose-related trends in clinical laboratory results, vital 
sign measurements, 12-lead ECG measurements or physical/neurological examination findings.  
The results from Part C indicate that TS-161 was safe and well-tolerated up to 100 mg when 
administered daily for 10 days under fed conditions to healthy participants. 
In summary, TP0473292 an orally bioactive prodrug of TP0178894, a potent and selective 
mGlu2/3 receptor antagonist, was well-tolerated with a favorable pharmacokinetic profile. 
TP0473292 (TS-161) exerts ketamine-like antidepressant effects in relevant animal models of 
depression. Based on these results TP0473292 (TS-161) was selected as a candidate drug for 
clinical development. The daily clinically efficacious dose of TP0473292 was estimated to be 50–
100 mg/day by predicting the human dose required to give an efficacious CSF exposure of 
TP0178894.  


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2.3  RISK/BENEFIT ASSESSMENT 
2.3.1  Known Potential Risks 
This protocol involves more than minimal risks to subject. By agreeing to participate in this 
study, subjects will be temporarily forgoing the opportunity to receive routine clinical care in the 
community. This will be clearly explained to all patients, along with the treatment strategies that 
are generally used in patients with MDD. Including the two-week medication-free period before 
Phase II, participants with MDD may not receive traditional standard treatment for MDD during 
the study. If subjects discontinue the study, we will provide short-term treatment at the NIMH as 
long as they remain here on a voluntary basis. However, if subjects do not remain on a voluntary 
status, then they may have to be transferred to a local hospital for further treatment. If subjects are 
hospitalized elsewhere, the NIH will not cover the costs associated with that hospitalization. 
2.3.1.1  TS-161 Administration 
None of the adverse events reported for TS-161 in the Phase 1 study were considered serious. 
The most common side effects associated with the doses used in this study include: nausea, 
increased heart rate, sleepiness, and decreased blood pressure.  Overall, the clinical laboratory 
assessments indicated that there were no meaningful changes in laboratory parameters as a result 
of TS-161 treatment, and the number of abnormal results did not present a safety concern during 
the study. In addition, there were no apparent trends in the vital signs, physical examinations, and 
12-lead ECGs. 
Clinical studies on drug interactions between TS-161 and other products have not been 
conducted. Results from in vitro cytochrome P-450 (CYP) inhibition and induction studies showed 
that the prodrug (TP0473292) and its metabolites (TP0178894, 
 were 
not potent inhibitors or inducers of the major human CYP isoforms evaluated. Thus, it is unlikely 
that TS-161 will demonstrate drug-drug interaction through CYP inhibition or induction in humans.  
 
Therefore, concomitant use of TS-
161 with substrates of these transporters should be avoided. Appendix B provides a list of 
allowable and not allowable concomitant medications. 
Pulse, blood pressure, adverse events, and laboratory values will be monitored throughout the 
study.  
2.3.1.2  Medication Taper/Drug-Free Period 
Hospitalization may be somewhat upsetting to patients. It is essential to ensure patient safety 
while they are being withdrawn from concomitant medication. There may be a worsening of 
depressive symptoms including suicidal ideation as the patient’s concomitant medications are 
being discontinued. Subjects with MDD may not receive standard treatment for 12-16 weeks.  
All patients will be admitted to the 7SE Mood and Anxiety Disorders Research Unit at the 
NIMH for supervision. In order to assess if a patient should be withdrawn from/remain off 
medications to participate, the following criteria must be met: a) subject is experiencing a MDE of 
sufficient severity (see Study Population for inclusion/exclusion criteria); and, b) the subject does 
not appear to have significantly benefited from their current medication regimen. We will attempt 
to verify the latter with the treating clinician(s). Subjects will be told to immediately inform 


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research staff if they develop suicidal ideation, intent, a suicidal plan, or arrangements for suicide 
during withdrawal and the medication-free period. Risk management procedures performed during 
the medication-taper period will continue during this and future periods of the study. With subjects 
off medication, there may be a worsening of symptoms if it is necessary that a patient discontinue 
medication/s and during the medication-free period. Such worsening could include increased 
depressive symptoms, sleep disorder, and/or suicidal ideation. The inpatient nursing staff has 
extensive experience handling voluntary adult patients with severe mental illness and is adept at 
maintaining patient safety and comfort during inpatient stays. Furthermore, patients will be 
assigned a Clinical Research Advocate (CRA) from the Human Subjects Protection Unit (HSPU). 
The CRA will monitor subjects during research participation from initial consent until study 
completion. All participants will be hospitalized for the duration of the protocol. 
2.3.1.3  Worsening of Symptoms/Delay in Treatment 
There could be a delay in treatment for as long as 16 weeks from the initial screening visit; 
this period of time includes the taper off medications, the drug free period, and the double-blind 
study phase. There may be a worsening of depressive and anxiety symptoms, including suicidal 
ideation, as the patient’s psychotropic medications are discontinued.  Subjects will be told to 
immediately inform their research physician if they develop suicidal ideation or symptoms of 
mania, or a worsening of depressive symptoms. We have included safety measures such as 
discontinuation of the study drug and transition to standard clinical treatment should subjects 
worsen to a sufficient degree. These precautions are likely to be effective in minimizing risks. 
2.3.1.4  Blood Draw 
Venipuncture or intravenous lines may be associated with the momentary discomfort of the 
needle stick, as well as a small potential risk of hematoma (bruise) formation. 
The total amount of research blood drawn during the whole study will not be more than 
370mL during the 14-16 week study.   
2.3.1.5  Clinical Rating Scales 
The risks and discomforts of clinical ratings or psychological assessments are minimal. No 
discomfort is expected to occur during clinical interviews or psychological assessments, other than 
the potential emotional stress caused by discussing difficult psychiatric topics. 
2.3.1.6  Neurocognitive Battery 
There are also minimal risks associated with the neurocognitive tasks, beyond possible stress 
or frustration.   Participants will be informed that they can stop participation in these tasks at any 
time. 
2.3.1.7  MEG 
MEG is associated with minimal medical risk or discomfort. Remaining still for the duration 
of the scan may result in mild muscle stiffness or fatigue. 
The potential risks associated with MEG will be minimized as follows:  
• In addition to a Basic Life Support (BLS)-certified staff member, ancillary staff will 
be present throughout in case of emergency.  


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2.3.1.8  MRI 
There is minimal risk associated with exposure to magnetic waves during MR imaging, 
though the potential risk of heart rhythm disturbances exists for patients with a history of heart 
rhythm abnormalities or those who have certain types of pacemakers. A substantial risk to persons 
who have metallic objects inside their bodies exists, as the magnet in the scanner can cause these 
to move. Pregnant women should not undergo MRI because of the possible harmful effects to the 
fetus. People with claustrophobia may find this procedure uncomfortable as it involves having 
one's head confined to a relatively small space. A small amount of discomfort may be associated 
with having to be still for the 90 minutes required for the MR scan, possibly resulting in mild 
muscle stiffness and fatigue. In addition, very high field magnets (above 3 Tesla) may cause 
additional side-effects of peripheral nerve stimulation. This is experienced as twitching in the 
muscles, or a tingling sensation. Additionally, some subjects report dizziness, mild nausea, 
headache, a metallic taste in their mouth, or a sensation of flashing lights. These sensations are not 
considered harmful and desist upon cessation of scanning. On very rare occasions, subjects may 
experience some eye discomfort. No biological hazards to humans have been associated with high 
static magnetic fields. Finally, the MRI scanning procedure is loud, and may affect hearing; we 
will place earplugs to wear during the scan to minimize discomfort and prevent adverse effects on 
hearing.  
The potential risks associated with MRI will be minimized as follows: 
• The potential for claustrophobia will be mitigated by explaining the nature of the 
procedure in detail prior to entering the MR scanner. 
• In addition to a BLS-certified staff member, ancillary staff will be present 
throughout in case of emergency.  
• A member of the research team will place hearing protection, such as earplugs, in 
each subject to be worn during the scan to minimize discomfort and prevent any 
adverse effects on hearing. 
• A history of any intraocular, intra-aural, intracranial, or intra-thoracic ferromagnetic 
implants or devices is an exclusion criterion. Ferromagnetic objects can be heated 
and/or moved in a strong magnetic field (3T and 7T), which may result in bodily 
harm. 
• Women of child-bearing potential will undergo urine pregnancy testing to rule out 
pregnancy no more than 24 hours prior to each imaging session. 
• A power meter will be used to ensure that experimental sequences fall within the 
accepted guidelines for radio frequency (RF) energy exposure. Applied RF power 
will be limited so that the specific absorption averaged over the head does not exceed 
3W/kg for 10 minutes.  
• While subjects are in the scanner, it is standard procedure to provide them with a 
squeeze bulb. Subjects are instructed to squeeze it in case of emergency (e.g. eye 
discomfort, nausea, emesis), after which the scan will stop immediately. If subjects 
experience eye discomfort or desire to stop for any reason, the scan will stop 
immediately. 


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2.3.1.9  Standard Treatment Phase 
During the optional traditional standard treatment phase, participants may have worsening of 
symptoms, including suicidal ideation, as medications and dosages are adjusted. In addition, 
pharmacologic treatment may result in side effects.  
At the end of study participation, patients with MDD will be offered short-term traditional 
standard treatment (up to two months). During this period, patients will be assessed and discussed 
at daily morning rounds by nursing staff and licensed independent practitioners (MD, APRN, part 
of the research team) for changes in psychiatric symptomatology and medication side effects. 
Additional interventions will be made depending on their status/progress. Additionally, our branch 
holds several weekly meetings to discuss our inpatients in the standard treatment phase, including 
an interdisciplinary treatment team meeting. During this meeting, ancillary team members not 
typically present at morning rounds (e.g., physical therapy, occupational therapy, pharmacy) will 
also have the opportunity to present their observations and weigh in on patient progress. If issues 
arise - for instance, psychiatric decompensation or intolerable side effects- medications may be 
adjusted/discontinued and/or additional medications or therapeutic interventions initiated based on 
a risk-benefit discussion between the inpatient treatment team and the patient. Additionally, if the 
patient expresses suicidal or homicidal thinking in the treatment phase, is judged to be an imminent 
risk of harm to self and/or others, and is not amenable to additional voluntary treatment on 7SE, 
he or she may be involuntarily psychiatrically hospitalized for his or her safety at an outside 
hospital. 
2.3.2  Known Potential Benefits 
This study offers no direct benefits to participants but is likely to yield important and 
generalizable knowledge about the development of new treatments for MDD.   
2.3.3  Assessment of Potential Risks and Benefits 
The primary risks of study participation include possible adverse events associated with TS-
161, discomfort during the medication taper and drug-free periods, and mild discomfort resulting 
from blood draws, imaging procedures. This study is likely to yield generalizable knowledge about 
the antidepressant effects of TS-161. Overall, the risks are reasonable in relation to anticipated 
benefit. 
 
3  OBJECTIVES AND ENDPOINTS 
 
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
Primary 
 
 
To evaluate the ability of the 
mGlu2/3 receptor antagonist 
prodrug, TS-161, to improve 
overall depressive 
Montgomery-Asberg 
Depression Rating Scale 
(MADRS) change from 
baseline 
The MADRS is a tool used 
for the evaluation of 
depressive symptoms in 
adults and for the assessment 


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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
symptomatology in subjects 
with MDD. The efficacy of a 
three-week course of TS-161 
will be compared to three 
weeks of placebo in a 
crossover study. The 
Montgomery-Asberg 
Depression Rating Scale 
(MADRS) will serve as the 
main outcome measure. 
Hypothesis:  
Among TRD-MDD subjects, 
the active drug (TS-161; 50-
100 mg/day) will result in less 
severe depressive symptoms 
than placebo after three weeks 
of treatment.  
of any changes to those 
symptoms. 
Secondary 
 
 
1. To evaluate the 
antidepressant efficacy of 
TS-161 230 min and 1, 2, 
3, 7, and 14 days post-
initial dosing compared to 
placebo in a crossover 
study.  
Hypothesis:  
Among TRD-MDD subjects, 
the active drug (TS-161; 50-
100 mg/day) compared to 
placebo will result in less 
severe depressive symptoms 
than placebo 230 min, 1, 2, 3, 
7, and 14 days post-initial 
dosing. 
1. Change from baseline on 
MADRS total scores 
1. The MADRS is a tool 
used for the evaluation of 
depressive symptoms in 
adults and for the 
assessment of any changes 
to those symptoms. 
2. To determine whether TS-
161 will demonstrate a 
superior antidepressant 
efficacy compared to 
2. Proportion of subjects in 
remission (defined as 
MADRS total score ≤10) 
2. The MADRS is a tool 
used for the evaluation of 
depressive symptoms in 
adults and for the 


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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
placebo in a crossover 
study 
Hypothesis:  
Among TRD-MDD subjects, 
the active drug compared to 
placebo will result in a greater 
proportion of subjects in 
remission at 230 min, 1, 2, 3, 
7, and 14 days post-initial 
dosing. 
assessment of any changes 
to those symptoms. 
3. To determine whether TS-
161 will demonstrate a 
superior antidepressant 
response compared to 
placebo in a crossover 
study. 
Hypothesis:  
Among TRD-MDD subjects, 
the active drug compared to 
placebo will result in a greater 
proportion of responders at 
230 min, 1, 2, 3, 7, 14, and 21 
days post-initial dosing. 
3. Proportion of subjects 
achieving response (defined 
as a ≥50% reduction from 
baseline in MADRS total 
score). 
3. The MADRS is a tool 
used for the evaluation of 
depressive symptoms in 
adults and for the 
assessment of any changes 
to those symptoms. 
4. To evaluate the antisuicidal 
ideation effects of TS-161 
at 230 min, 1, 2, 3, 7, 14, 
and 21 days post-initial 
dosing compared to 
placebo in a cross over 
study. 
Hypothesis:  
Among TRD-MDD subjects, 
the active drug will result in 
less severe suicidal ideation 
than placebo at 230 min, 1, 2, 
3, 7, 14, and 21 days post-
initial dosing. 
4. Change from baseline on 
item 10 (suicidality) of the 
MADRS and total score on 
the C-SSRS, and the Scale 
for Suicidal Ideation (SSI). 
4. Item-10 of the MADRS 
used to assess suicidal 
ideation. 
5. To investigate the effects 
of TS-161 on mood, 
5. Change from baseline on the 
HDRS, HAM-A, the 
5. These rating scales 
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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
anxiety, and anhedonia 
symptoms at 230 min, 1, 2, 
3, 7, 14, and 21 days post-
initial dosing compared to 
placebo in a cross over 
study.  
Hypothesis:  
Among TRD-MDD subjects, 
TS-161 will result in less 
severe mood, anxiety, and 
anhedonia symptoms than 
placebo at 230 min, 1, 2, 3, 7, 
14, and 21 days post-initial 
dosing. 
PANAS, Snaith–Hamilton 
Pleasure Scale (SHAPS), 
and the Temporal 
Experience of Pleasure 
Scale (TEPS) scales. 
mood, anxiety, and 
anhedonia. 
6. To assess the safety and 
tolerability of a three-week 
course of TS-161 
compared to placebo in a 
crossover study. 
Hypothesis:  
Among TRD-MDD subjects, 
the active drug will result in 
comparable safety and 
tolerability to placebo at the 
different timepoints measured 
during the 3 weeks of 
treatment. 
6. Incidence of AEs and total 
scores using the Clinician 
Administered Dissociative 
States Scale (CADSS), 
Young Mania Rating Scale 
(YMRS), the Brief 
Psychiatric Rating Scale 
(BPRS), vital signs, changes 
in clinical laboratory 
evaluations, and ECGs. 
6. These measures and 
evaluations assess various 
aspects of clinical 
condition, adverse events, 
and mood and anxiety 
symptomology. 


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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
Surrogate Markers of Drug 
Effect, Target Engagement, and 
Antidepressant Response  
1. To determine whether the 
glutamate peak in the 
medial PFC (mPFC) 
correlates with changes in 
depression scores to TS-
161. 
Hypothesis 1:  
Among TRD-MDD subjects, 
pre-treatment mPFC glutamate 
levels will predict the 
magnitude of clinical 
improvement to TS-161 in 
subjects with MDD, but not to 
placebo. 
Hypothesis 2:  
Among TRD-MDD subjects, 
post-treatment increases in 
glutamate levels will predict 
the magnitude of clinical 
improvement to TS-161 (but 
not placebo).  
 
 
 
 
1. 1H-MRS correlates with 
changes in MADRS score 
 
 
 
1. The MADRS is a tool 
used for the evaluation of 
depressive symptoms in 
adults and for the 
assessment of any changes 
to those symptoms.  MRS 
is used to measure 
glutamate levels. 
2. To determine whether 
gamma power increases 
with TS-161are associated 
with treatment response to 
TS-161. 
Hypothesis 1:  
Among TRD-MDD subjects, 
pre-treatment gamma power 
levels will predict the 
magnitude of clinical 
improvement to TS-161, but 
not to placebo. 
2. Gamma power measured via 
MEG 
2. MEG scanning is used to 
measure gamma power. 
 


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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
Hypothesis 2:  
Among TRD-MDD subjects, 
post-treatment increases in 
gamma power will be 
positively associated with 
antidepressant response to TS-
161 (but not placebo). 
3. To determine whether TS-
161 modulates task-
dependent and resting-state 
activity in the frontolimbic 
circuitry and whether 
changes in activity in the 
frontolimbic circuitry are 
correlated with 
antidepressant response to 
TS-161 
Hypothesis 1: 
Among TRD-MDD subjects, 
TS-161 administration will 
increase pgACC activity and 
decrease amygdala activity to 
emotional faces more than 
placebo. Similar effects will 
be observed in activity 
associated with the resting-
state networks. TS-161 will 
also decrease pgACC activity 
during the tasks more than 
placebo. 
3. Changes in activity in the 
frontolimbic circuitry 
3. fMRI is used to measure 
frontolimbic circuitry 
Tertiary/Exploratory  
 
 
1. To determine whether TS-
161 will increase 
plasma/serum BDNF and 
vascular endothelial growth 
factor (VEGF) and 
decrease glycine/serine and 
alter oxidative 
stress/inflammatory 
parameters in peripheral 
1. Plasma/serum BDNF, 
vascular endothelial growth 
factor (VEGF), 
glycine/serine, and 
oxidative 
stress/inflammatory 
parameters in peripheral 
leukocytes 
1. To identify potential 
biomarkers of TS-161 
antidepressant activity.  


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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
leukocytes compared to 
placebo and/or baseline 
levels.  
1. To collect blood samples 
for optional exploratory 
studies focusing on 
identification of genes, 
proteins, and metabolic 
responses that may 
influence the disposition, 
efficacy, safety and 
tolerability of TS-161. 
2. Blood samples for the 
identification of genes, 
proteins, and metabolic 
responses 
2. To identify potential 
biomarkers of TS-161 
antidepressant activity. 
3. To evaluate neurocognitive 
functioning on TS-161 
compared to placebo 
and/or baseline levels. 
3. Neurocognitive functioning 
3. To evaluate any 
neurocognitive effects of 
TS-161 
 
4  STUDY DESIGN 
4.1  OVERALL DESIGN 
This is a Phase 2 randomized, double blind, placebo-controlled, crossover, single–site, 
approximately 16-week inpatient experimental study that will assess the efficacy and safety of 
three weeks of 50 to 100 mg/day of TS-161, an mGlu2/3 receptor antagonist prodrug. Subjects 
will meet diagnostic criteria for MDD without psychotic features, according to DSM-IV or DSM-
5 guidelines and confirmed by the SCID-P. Approximately 25 subjects with MDD will be recruited 
for this study to allow 20 subjects to crossover and complete the study. TS-161 and placebo will 
be taken orally in capsule form as a single daily dose for 3 weeks in each of two crossover Research 
Conditions.  
This single site, inpatient study will be conducted at the NIH Clinical Center in Bethesda, MD. 
The protocol will not include interim analyses, substudies, or stratifications. 
4.2  SCIENTIFIC RATIONALE FOR STUDY DESIGN 
This study will use a double-dummy blind dosing design, all subjects will receive two 
capsules in the AM approximately 30 minutes after start of breakfast, and dose adjustment, if 
required due to intolerance, will be done blindly. A flexible dose design (50 or 100 mg/day) will 
be used in this study since this type of dosing permits better assessment of safety and efficacy than 
does a fixed dose, especially when this compound has not been previously tested in subjects with 
major depression. The maximum daily dose of TS-161 administered in this study will be 100 
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will cross over. To avoid carry-over effects between the different test sessions, there will be an 
interval of 14-21 days, pending response to Research Condition 1. Subjects will then be blindly 
crossed over to the second treatment condition (either TS-161 or placebo) for another three weeks. 
Subjects maintaining response to Research Condition 1 after two weeks will receive an additional 
one-week washout before being crossed over to Research Condition 2. Subjects maintaining 
response (50% improved from baseline on the MADRS) after this additional week will be 
withdrawn from the study and will receive standard clinical treatment. 
4.3  JUSTIFICATION FOR DOSE 
The dose of TS-161 was arrived at based on toxicology studies in animals, as well as from 
Single Ascending and Multiple Ascending Dose Studies conducted in humans. The daily clinically 
efficacious dose of TS-161 was estimated to be 50–100 mg/day by predicting human dose required 
for giving an efficacious CSF exposure of TP0178894 based on preclinical studies. 
 
5  STUDY POPULATION 
5.1  INCLUSION CRITERIA 
In order to be eligible to participate in this study, an individual must meet all of the following 
criteria: 
1.  Ability of subject to understand and the willingness to sign a written informed consent 
document. To verify this, subjects must score ≥ 80% on the consent quiz (Appendix A). 
2.  Stated willingness to comply with all study procedures and availability for the duration of 
the study.  
3.  Aged 18 to 65. 
4.  Subjects must fulfill DSM-IV or DSM-5 criteria for MDD, single episode or recurrent 
without psychotic features, based on clinical assessment and confirmed by a structured 
diagnostic interview (SCID-P). Subjects must be experiencing a current major depressive 
episode of at least 4 weeks duration.  
5.  Subjects must have an initial score of ≥ 20 on the MADRS and a YMRS score of <12 
within one week of the start of Phase I and upon entry into Phase II. 
6.  Ability to take oral medication and be willing to adhere to the TS-161 regimen. 
7.  Subjects must have a current or past history of lack of response to at least one adequate 
antidepressant trial (may be from the same chemical class) in the current major 
depressive episode, operationally defined using the modified-Antidepressant Treatment 
History Form (ATHF)); a failed adequate trial of ECT [or TMS] would count as an 
adequate antidepressant trial. 
8.  For females of reproductive potential: use of contraception starting at the time of 
enrollment and agreement to use such a method during study participation and for an 
additional 4 weeks after the end of Phase II (see Contraception Guidance, Appendix D).  
9.  For males of reproductive potential: use of condoms or other methods from the time of 
enrollment to ensure effective contraception with partner, and for an additional 90 days 
after the end of Phase II (see Contraception Guidance, Appendix D).  
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5.2  EXCLUSION CRITERIA 
An individual who meets any of the following criteria will be excluded from participation in 
this study: 
1.  Current use of disallowed concomitant medications (see Appendix B: Drugs Allowed and 
Not Allowed) or transcranial magnetic stimulation (TMS) less than 2 weeks prior to the 
start of Phase II. 
2.  Treatment with a reversible monoamine oxidase inhibitor (MAOI) less than 4 weeks prior 
to the start of Phase II. 
3.  Treatment with fluoxetine, aripiprazole, or brexpiprazole less than 5 weeks prior to the 
start of Phase II. 
4.  Treatment with clozapine or electroconvulsive therapy (ECT) less than 4 weeks prior to 
the start of Phase II. 
5.  Lifetime history of deep brain stimulation. 
6.  Previous antidepressant non-response to ketamine or esketamine. 
7.  No structured psychotherapy will be permitted during the total duration of the study. 
Subjects unable or unwilling to stop psychotherapy will be unable to participate in the 
study.  
8.  Pregnancy or lactation 
9.  Current psychotic features or a diagnosis of schizophrenia or any other psychotic disorder 
as defined in the DSM-IV or DSM-5. 
10. Subjects with a history of DSM-IV drug or alcohol dependency or abuse (or “alcohol use 
disorder” per DSM-5), except for caffeine or nicotine dependence, within the preceding 3 
months. 
11. Subjects with a DSM-IV or DSM-5 Axis II diagnosis of borderline or antisocial 
personality disorder. 
12. Subjects with a history of a head injury that resulted in loss of consciousness exceeding 5 
minutes (for the imaging component of the study).  
13. Serious, unstable illnesses including hepatic, renal, gastroenterologic, respiratory, 
cardiovascular (including ischemic heart disease), endocrinologic, neurologic, 
immunologic, or hematologic disease. 
14. Subjects with any current or chronic history of liver disease, hepatic, or other biliary 
abnormalities. 
15. Subjects with a history of persistent orthostatic hypotension or other cardiac conditions 
where orthostatic hypotension would be a safety concern 
16. Subjects with unstable clinical hyperthyroidism or hypothyroidism. 
17. Subjects with one or more seizures without a clear and resolved etiology. 
18. Clinically significant abnormal laboratory tests. In particular, participants with the 
following based on baseline liver safety values will be excluded: 
• Alkaline phosphatase (Alk Phos) > 150 U/L 
• Alanine aminotransferase (ALT) > 55 U/L 
• Aspartate aminotransferase (AST) > 34 U/L 


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• Total bilirubin (TB)> 1.2 mg/dL 
• Direct bilirubin (DB) > 0.5 mg/dL 
19. Subjects who, in the Principal Investigator’s judgment, pose a current serious suicidal or 
homicidal risk. 
20. Positive HIV test. 
21. For those participating in imaging procedures, contraindications to imaging (metal in 
body, claustrophobia, etc.)  
22. Participants with COVID-19, or suspected COVID-19 
23. A current NIMH employee/staff or their immediate family member. 
5.3  INCLUSION OF VULNERABLE PARTICIPANTS 
5.3.1  Children 
We are excluding minors as we feel preliminary safety and efficacy data should be obtained 
in adults before proceeding to vulnerable populations.   
5.3.2  Pregnant Women, Fetuses, or Neonates 
The safety of TS-161 during pregnancy and breast-feeding is unknown. In addition, the safety profiles 
for MRI in pregnant women are unknown.  
5.3.3  Prisoners 
Prisoners will be excluded from participation. 
5.3.4  Decisionally Impaired Adults 
We are excluding cognitively impaired persons as we feel that preliminary safety and efficacy 
data should be obtained in non-impaired adults before proceeding to vulnerable populations. Given 
that this study presents no direct clinical benefit for the participant and the research involves more 
than a minor increase in minimal risk, the risks will outweigh the benefits for this population. We 
also want to ensure that all participants understand the consenting process as well as all study 
procedures, including the study tests and measures. Therefore, cognitively impaired persons, who 
may not be able to provide their own consent, will not be enrolled. 
5.3.5  Participation of NIH Staff or family members of study team members 
NIH staff and family members of study team members may be enrolled in this study as this 
population meets the study entry criteria.  Neither participation nor refusal to participate as a 
subject in the research will have an effect, either beneficial or adverse, on the participant’s 
employment or position at NIH. 
Every effort will be made to protect participant information, but such information may be 
available in medical records and may be available to authorized users outside of the study team in 
both an identifiable and an unidentifiable manner. 
The NIH Information Sheet on NIH Staff Research Participation will be made available.  
Please see Informed Consent Process for consent of NIH Staff. 


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5.3.6  Patients with Comorbid Anxiety or Other Psychiatric Disorders, Besides Those 
Listed Under Inclusion/Exclusion Criteria 
Exclusion of patients with comorbid anxiety or other psychiatric disorders would affect the 
generalizability of our findings, given that a substantial percentage of patients with MDD may 
have additional diagnoses. Allowing the participation of patients with a history of comorbid 
disorders broadens the inclusion criteria to more closely approximate patients seen in real world 
settings. Thus, we will exclude patients with comorbid diagnoses only if it has been the primary 
focus of treatment in the past six months. 
5.3.7  HIV/AIDS Positive Subjects 
The drug used in this study is experimental and may have unknown interactions with 
medications used to control HIV. In addition, the effects of HIV on the brain may be a confound 
to our examination of brain biomarkers. For experimental therapeutic depression studies, we have 
historically excluded HIV-positive individuals. 
5.4  PERSONS OVER 65 YEARS OF AGE 
Subjects older than 65 will be excluded to prevent age-related changes in brain structure and 
function from confounding our analyses. In addition, experimental TS-161 use in older and/or 
medically compromised patients has not been explored due to greater theoretical risk of serious 
adverse events.  
5.5  NON-ENGLISH SPEAKERS 
The majority of the required monitoring and rating instruments are not validated in languages 
other than English, and we do not have the ability to provide clear written and verbal 
communication with those individuals. 
5.6  LIFESTYLE CONSIDERATIONS 
During this study, participants are asked to: 
1.  Abstain from alcohol and drug use (except for caffeine and nicotine) while in the study. 
• For individuals of reproductive potential: use of highly effective contraception 
starting at the time of enrollment and agreement to use such a method during study 
participation and for an additional 4 weeks after the end of Phase II.  
• For males of reproductive potential: use of condoms or other methods from the time 
of enrollment to ensure effective contraception with partner, and for an additional 
90 days after the end of Phase II.  
• Agree to remain inpatient, unless authorized by the Principal or Lead Associate 
Investigator.  Passes will be permitted for inpatients if the subject is clinically 
assessed and deemed psychiatrically and medically stable, including not an 
imminent danger to self or others and the pass does not interfere with the study or 
unit procedures. 
• The taper off of medications and drug-free period of the study may be done as an 
outpatient if clinically appropriate. 


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5.7  SCREEN FAILURES 
Screening for inclusion into the study will be done under protocol 01-M-0254, “The 
Evaluation of Patients with Mood and Anxiety Disorders and Healthy Volunteers.” 
5.8  STRATEGIES FOR RECRUITMENT AND RETENTION 
Our recruitment program seeks to increase awareness of our studies among a broad 
demographic (e.g. patients and families, scientific community, mental health clinicians) through a 
range of recruitment materials and venues. Participants will be recruited under Screening Protocol 
01-M-0254: “The Evaluation of Patients with Mood and Anxiety Disorders and Healthy 
Volunteers” or under this protocol using IRB approved recruitment materials. 
NIH employees/staff will not be directly recruited by or through their supervisors to 
participate in this study but are eligible for participation. NIMH employees/staff and their 
immediate family members cannot participate in this protocol. 
We will use the following recruitment methods: 
1.  Non-Paid Advertisements 
a.  Internal NIH media (e.g. NIH Record) 
b.  NIH Internet (e.g. http://patientinfo.nimh.nih.gov/) 
c.  Listservs:  
With the permission of listserv administrators, we will distribute information 
regarding our studies.  We will not post directly on listservs. We will distribute 
information to the administrators of listservs such as the: NIMH Outreach 
Partnerships Updates, NIH research assistant listserv, Community Services 
Announcements- City of Gaithersburg, Montgomery County Providers, Howard 
County Providers, and Frederick Providers Council listservs. We will request that 
the following disclaimer be included with any distributed information: 
“You are receiving this message because your email address is included in the 
above listserv.  The purpose of this message is to inform you of our NIMH 
research studies.  The moderator of the listserv has permitted its use for this 
distribution.” 
d.  Short Text Descriptions: 
Short Text Descriptions of this study will appear on social networking sites as 
Facebook, Instagram, and Twitter.  Only official NIH accounts will be used.  
These may also be publicized using Community Resource Listings (e.g. 
Community Calendars, Newsletters, electronic and print- twenty-five word text 
summary). 
2.  Paid Advertisements: 
a.  Local print publications (e.g. Washington Post Express, The Gazette) 
b.  National print publications (e.g. Washington Post, NAMI Advocate Magazine) 
c.  Public transit advertisements (e.g. posters on Washington Metropolitan Area 
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d.  Radio advertisements (e.g. WMZQ, WASH, WLZL/CBS Radio) 
3.  Brochures:  
a.  Mental Health Clinics, (e.g. Threshold Services, Adventist Potomac Ridge 
Behavioral Health, Northern VA Mental Health Institute), Hospitals (e.g. 
Suburban Hospital, Walter Reed National Military Medical Center), and local 
Clinicians 
b.  Professional Conferences (e.g. American Psychiatric Association and American 
College of Neuropsychopharmacology annual conferences), Symposiums (e.g. 
Challenging Depression: New Insights into Research and Treatment (Suburban 
Hospital)), and other professional settings (e.g. Latino Behavioral Health Institute, 
Frederick Providers Council, DC Department of Mental Health) 
c.  Mental Health Advocacy Conferences (e.g. NIMH Outreach Partnership Annual 
Meeting, Maryland’s Annual Suicide Prevention Conference) 
d.  Self-Help and Advocacy Groups (e.g. local NAMI and DBSA chapters, Jewish 
Social Service Agency, Active Minds) 
4.  NIH Referrals: 
a.  Investigators from other NIH inpatient units and outpatient clinics, including 
those from the NIMH, may refer patients to the screening protocol, as they may 
be eligible for additional studies. 
b.  CenExcelCBH, a recruitment and screening database developed by the Centers 
for Behavioral Health (CBH), will be added as an additional referral/recruitment 
source for this protocol. CBH is a local research facility that recruits and enrolls 
participants in psychiatric studies. Subjects that present at CBH sign an IRB-
approved CBH consent form to be enrolled in their database and collect screening 
information. The medical and psychiatric history information collected will assist 
in determining if an individual is a potential candidate for this protocol. If the 
individual meets the initial eligibility for this study, a CBH team member will 
give them the contact information for the NIMH staff along with IRB approved 
recruitment materials for the study. If the potential referral is interested in 
participating in the study, it will be up to them to contact the NIMH research 
team.  None of the individuals’ information will be given to the NIMH for 
recruitment purposes. If they are interested in learning more about the NIH study, 
they will call and complete a brief telephone screening with NIMH staff. If they 
meet study criteria, we will invite them for an in-person screening visit under 
protocol 01-M-0254. We may ask the individual to sign a CBH release of 
information form to share existing diagnostic information from their CBH records 
with NIH. 
Brochures and other IRB-approved recruitment materials or study information may be given 
directly to individuals interested in our research in both hard copy and electronic formats, 
depending on request. Associate Investigators and members of the NIMH Marketing & 
Community Relations Unit will distribute recruitment materials to individuals/groups, such as 
mental health clinics, hospitals, self-help and advocacy groups, and local clinicians.  Clinicians 
who are contacted will be provided with information to disseminate to patients as they see fit.  We 


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will explain to them that individuals interested in participating in our studies will need to initiate 
contact with our group and that we will not make this initial contact. 
5.8.1  Costs 
There are no costs to participants enrolled in this study. 
5.8.2  Compensation 
All participants will be compensated for time and research-related inconveniences. 
Compensation will be in the form of a check payment and will be given at the end of each study 
phase.  Payment will be prorated for parts completed if subjects do not complete the study. Escorts 
will not be compensated. 
The Designation of Reimbursement for Travel and Subsistence (DRTS) form for this protocol, 
which details the compensation for hotel and transportation, is attached in iRIS. 
NIH employees or staff who participate during work hours must have permission from their 
supervisor. NIH employees or staff must either participate outside of work hours or take leave in 
order to receive compensation. 
 
PHASE I 
  
  
  
Procedure 
Number 
Pay per Procedure 
Total Pay 
Clinical Rating Scales 
2 
20 
40 
Blood Draw 
1 
10 
10 
Neurocognitive Testing 
1 
80 
80 
Clinical MRI 
1 
40 
40 
fMRI/MRS 
1 
80 
80 
MEG 
1 
100 
100 
Phase I Total 
 
 
$ 350 
 


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PHASE II 
  
  
  
Procedure 
Number 
Pay per Procedure 
Total Pay 
Clinical Rating Scales 
17 
20 
340 
Blood Draws 
26 
10 
260 
Neurocognitive Testing 
5 
80 
400 
Additional Safety Monitoring (study drug days) 
42 
48 
2,016 
fMRI/MRS 
5 
80 
400 
MEG 
5 
100 
500 
Maximum Phase II Total 
 
 
$3,916 
The maximum compensation, including Phases I and II is $4,266.  
 
6  STUDY INTERVENTION 
6.1  STUDY INTERVENTIONS(S) ADMINISTRATION 
6.1.1  Study Intervention: TP0473292 (IND) 
The use of TP0473292 (TS-161) in this study has been approved by the FDA under IND 
#153429.  The study drug will be supplied by Taisho Pharmaceutical Co., Ltd. The sponsor of the 
IND is the National Institute of Mental Health.  The IB is attached in iRIS. 
TS-161 is not available for commercial use. 
6.1.1.1  Study Intervention: MRI Machines (NSR Device) 
MRI are non-significant risk (NSR) devices that will be used for investigational research 
under this protocol. Under this protocol, we are using a 7T Magnetom MRI Prototype Siemens 
System manufactured by Siemens Medical Solutions USA, Inc. Images generated by the MRI 
differ from what are created in a routine clinical scan. However, all studies done under this protocol 
will be performed within FDA safety guidelines. The use of research coils in the MRI has not been 
approved by the FDA and is considered investigational. Additionally, some of the MR machines 
used in this study are considered investigational (not yet approved by the FDA for this use) but are 
used within the FDA safety guidelines. 
 
Criteria establishing MRI scanners as NSR devices: 
a.  is intended as an implant and presents a potential for serious risk to the health, safety, 
or welfare of a subject 
An MRI scanner is not an implantable device. 
b.  is purported or represented to be for a use in supporting or sustaining human life and 
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An MRI scanner is not for use in supporting or sustaining human life. It does not 
present a potential for serious risk to the health, safety, or welfare of participants when 
used as described in this protocol. 
c.  is for a use of substantial importance in diagnosing, curing, mitigating, or treating 
disease, or otherwise preventing impairment of human health and presents a potential 
for serious risk to the health, safety, or welfare of a subject 
An MRI scanner, as used under this protocol, is not of substantial importance in 
diagnosing, curing, mitigating, or treating disease, or otherwise preventing impairment 
of human health and does not present a potential for serious risk to the health, safety or 
welfare of a subject. 
d.  otherwise presents a potential for serious risk to the health, safety or welfare of a subject 
MRI scanners have been in use for over three decades. Safety guidelines have been 
developed and updated allowing its dissemination to a wide range of clinical and non- 
clinical settings. As long as subjects are properly screened and sessions are run 
according to these safety guidelines, MRI scanners are generally considered NSR. 
While operated in research mode, the MRI will be under the International 
Electrotechnical Commission (IEC) 60601-2-33 First Level Controlled Operating 
Mode, which allows for research pulse sequences to be used within the FDA/IEC safety 
limits for MRI devices. 
6.1.1.2  Research Pulse Sequences for 3T and 7T MRI 
Research pulse sequences used during imaging procedures under this protocol are considered 
non-significant risk (NSR) devices. 
Criteria establishing research pulse sequences as NSR devices: 
a.  is intended as an implant and presents a potential for serious risk to the health, safety, 
or welfare of a subject 
Research pulse sequences are not an implantable device. 
b.  is purported or represented to be for a use in supporting or sustaining human life and 
presents a potential for serious risk to the health, safety, or welfare of a subject 
Research pulse sequences are not for use in supporting or sustaining human life. 
They do not present a potential for serious risk to the health, safety, or welfare of 
participants when used as described in this protocol. 
c.  is for a use of substantial importance in diagnosing, curing, mitigating, or treating 
disease, or otherwise preventing impairment of human health and presents a potential 
for serious risk to the health, safety, or welfare of a subject 
Research pulse sequences, as used under this protocol, are not of substantial 
importance in diagnosing, curing, mitigating, or treating disease, or otherwise 
preventing impairment of human health and does not present a potential for serious risk 
to the health, safety or welfare of a subject. 
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Research pulse sequences, as used under this protocol, do not present a potential 
for serious risk to the health, safety, or welfare of a subject.  While operated in an 
investigational manner, research pulse sequences are used within the FDA/IEC safety 
limits for MRI devices. 
6.1.1.3  Radiofrequency Research Coils for 3T and 7T MRI 
Research coils used during MRI scans under this protocol are considered non-significant risk 
(NSR) devices. 
Criteria establishing research coils as NSR devices: 
a.  is intended as an implant and presents a potential for serious risk to the health, safety, 
or welfare of a subject 
Research coils are not an implantable device. 
b.  is purported or represented to be for a use in supporting or sustaining human life and 
presents a potential for serious risk to the health, safety, or welfare of a subject 
Research coils are not for use in supporting or sustaining human life. They do not 
present a potential for serious risk to the health, safety, or welfare of participants when 
used as described in this protocol. 
c.  is for a use of substantial importance in diagnosing, curing, mitigating, or treating 
disease, or otherwise preventing impairment of human health and presents a potential 
for serious risk to the health, safety, or welfare of a subject 
Research coils, as used under this protocol, are not of substantial importance in 
diagnosing, curing, mitigating, or treating disease, or otherwise preventing impairment 
of human health and does not present a potential for serious risk to the health, safety or 
welfare of a subject. 
d.  otherwise presents a potential for serious risk to the health, safety or welfare of a subject 
Research coils, as used under this protocol, do not present a potential for serious 
risk to the health, safety, or welfare of a subject.  The research coils are used within the 
FDA/IEC safety limits for MRI devices. 
6.1.1.4  Image Reconstruction and Analysis Software for 3T and 7T MRI 
Image reconstruction and analysis software used during MRI scans under this protocol are 
considered non-significant risk (NSR) devices. 
Criteria establishing image reconstruction and analysis software as NSR devices: 
a.  is intended as an implant and presents a potential for serious risk to the health, safety, 
or welfare of a subject 
Image reconstruction and analysis software are not implantable devices. 
b.  is purported or represented to be for a use in supporting or sustaining human life and 
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Image reconstruction and analysis software are not for use in supporting or 
sustaining human life. They do not present a potential for serious risk to the health, 
safety, or welfare of participants when used as described in this protocol. 
c.  is for a use of substantial importance in diagnosing, curing, mitigating, or treating 
disease, or otherwise preventing impairment of human health and presents a potential 
for serious risk to the health, safety, or welfare of a subject 
Image reconstruction and analysis software, as used under this protocol, are not of 
substantial importance in diagnosing, curing, mitigating, or treating disease, or 
otherwise preventing impairment of human health and does not present a potential for 
serious risk to the health, safety or welfare of a subject. 
d.  otherwise presents a potential for serious risk to the health, safety or welfare of a subject 
Image reconstruction and analysis software, as used under this protocol, do not 
present a potential for serious risk to the health, safety, or welfare of a subject.  The 
software are used within the FDA/IEC safety limits for MRI devices. 
6.1.2  Dosing and Administration 
 
 
 
  
The starting dose will be 100 mg/day (given as a single AM dose approximately 30 minutes 
after start of breakfast) and the treatment target for all participants will be 100 mg/day unless 
tolerability issues ensue. The dose of TS-161/placebo will remain at 100 mg/day in subjects until 
the end of the study. If subjects are unable to tolerate the current dose, then the dose may be 
decreased down to 50 mg/day for one week at which time the dose may be increased back to 100 
mg/day if response criteria are not met and side effects are tolerable. In case of continued side 
effects at 100 mg/day, the dose may be lowered to 50 mg/day until the end of the study. Subjects 
who are unable to tolerate the lowest allowable dose of study medication (50 mg/day) will be 
discontinued from the study. No dose increases will be allowed two weeks after the start of each 
research condition (Days 14 and 49) to allow ample time to evaluate the effect of the drug. Only 
licensed independent practitioners who are investigators on this protocol will be able to make 
dosing decisions. The maximum daily dose of TS-161 administered in this study will be 100 
mg/day. 
6.2  PREPARATION/HANDLING/STORAGE/ACCOUNTABILITY  
Preparation, handling, storage, and accountability will be handled by the Clinical Center’s 
Investigational Drug Management and Research Section (IDMRS) in accordance with the 
Investigational Brochure (IB). 
6.3  ACQUISITION AND ACCOUNTABILITY 
The study drug and placebo will be provided by the manufacturer, Taisho Pharmaceutical Co., 
Ltd, in accordance with a CRADA (see Collaborative Agreements).  The study site pharmacist(s) 
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Study personnel will pick up the study intervention or the control product from the NIH 
pharmacy the day of the study visit. Any unused study intervention or control product will be 
returned to the NIH pharmacy for disposal. 
6.4  FORMULATION, APPEARANCE, PACKAGING, AND LABELING 
The name of the participant, name of the study, participant number, directions for 
administration, day of study visit, and the signature of the physician should appear on the study 
intervention and the control product. 
6.4.1  Product Storage and Stability 
 
 
  
6.4.2  Preparation 
 Instructions for the preparation of study drugs 
are provided in a separate manual. 
6.5  MEASURES TO MINIMIZE BIAS: RANDOMIZATION AND BLINDING 
A randomization code will be generated by the unblinded statistician. The study site 
pharmacist(s) responsible for study drug dispensation will also be unblinded. 
6.6  STUDY INTERVENTION COMPLIANCE 
The study drug will be administered to participants on the inpatient unit to ensure adherence 
to the protocol. 
6.7  CONCOMITANT THERAPY 
For this protocol, a prescription medication is defined as a medication that can be prescribed 
only by a properly authorized/licensed clinician. Medications to be reported in the Case Report 
Form (CRF) are concomitant prescription medications, over-the-counter medications and 
supplements.  See Appendix B: Drugs Allowed and Not Allowed. 
 
7  STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL  
7.1  DISCONTINUATION OF STUDY INTERVENTION 
Discontinuation from TS-161 does not mean discontinuation from the study, and remaining 
study procedures should be completed as indicated by the study protocol.  If a clinically significant 
finding is identified after enrollment (including, but not limited to changes from baseline), the 
investigator or qualified designee will determine if any change in participant management is 
needed. Any new clinically relevant finding will be reported as an AE. 
To the extent possible, data to be collected at the time of study intervention discontinuation 
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Participants are free to withdraw from participation in the study at any time upon request. 
An investigator may discontinue or withdraw a participant from the study for the following 
reasons: 
• Completion of study intervention 
• Disease progression which requires discontinuation of the study intervention 
• If any clinical AEs, laboratory abnormality, or other medical condition or situation occurs 
such that continued participation in the study would not be in the best interest of the 
participant.  Examples include: 
• Extreme changes in vital signs 
• Persistent systolic blood pressure (SBP) > 165 mm Hg, diastolic blood pressure 
(DBP) > 100 mm Hg, SBP <  80 (symptomatic), pulse >130, pulse < 45 
(symptomatic), SaO2 <90%, and T > 39 degrees C 
• Investigator discretion 
• Positive pregnancy test 
• Participant unable to receive TS-161 for 3 weeks. 
7.2  PARTICIPANT DISCONTINUATION/WITHDRAWAL FROM THE STUDY 
Prior to removal from study, effort must be made to have all subjects complete all end of study 
procedures listed on the Schedule of Events. 
Participants are free to withdraw from participation in the study at any time upon request.  
An investigator may discontinue or withdraw a participant from the study for the following 
reasons: 
• Significant study intervention non-compliance  
• If the participant meets an exclusion criterion (either newly developed or not previously 
recognized) that precludes further study participation 
• Subject has completed the study follow-up period 
• Death 
• Screen Failure 
The reason for participant discontinuation or withdrawal from the study will be recorded on 
the Enrollment log. Subjects who sign the informed consent form and are randomized but do not 
receive the study intervention may be replaced.  Subjects who sign the informed consent form, and 
are randomized and receive the study intervention, and subsequently withdraw, or are withdrawn 
or discontinued from the study, will not be replaced. 
7.3  LOST TO FOLLOW-UP 
This section is not applicable as this is an inpatient protocol. 
 


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8  STUDY ASSESSMENTS AND PROCEDURES 
8.1  SCREENING PROCEDURES 
Prior to consenting to this study, subjects will have undergone screening under protocol 01-
M-0254, “The Evaluation of Patients with Mood and Anxiety Disorders and Healthy Volunteers”. 
Psychiatric history and diagnosis of MDE will be made using the SCID-P (First, Spitzer et al. 
2001) as well as DSM-IV or DSM-5 diagnostic criteria. Subjects who meet DSM-IV or DSM-5 
criteria for major depressive episode (without psychotic features) must have a score of ≥20 on the 
MADRS within one week of the start of Phase I and upon entry into Study Phase II. Subjects who 
previously enrolled in and completed studies at the ETPB may be enrolled in this protocol. 
We will require that all subjects have physical examinations and specific laboratory tests (e.g. 
Acute Care Panel, CBC+Diff, Hepatic Panel, LFT, TFT, urinalysis, urine pregnancy, urine 
toxicology, HIV). Results of these tests will identify patients who should be excluded because of 
active medical problems or substance abuse that might affect clinical phenomenology or make 
participation in the protocol unsafe. Subjects are expected to meet all other eligibility criteria to be 
enrolled in the study. Protocol 01-M-0254 allows for the sharing of collected data, including 
history and physical exams, structural neuroimaging studies, and lab testing. 
8.2  MEDICATION TAPERING 
If participants with MDD are medicated at the time of consent, they will undergo tapering of 
all prohibited medications (see Appendix B, Drugs Allowed and Not Allowed for details). The 
medication taper will be followed with a subsequent washout of two weeks during which time 
patients will be medication-free. Subjects will be reminded to report all medications, OTC products, 
and other agents to the investigators so that they can screen to avoid interactions that might make 
participation unsafe or might confound the research results. The taper off of medications and drug-
free period of the study may be done as an outpatient if clinically appropriate. Medications that 
are required for non-psychiatric medical reasons (e.g., antihistamines and topical antifungals) will 
not be discontinued for the purpose of the study. 
8.3  TS-161 AND PLACEBO ADMINISTRATION 
 
 
 All participants will use this dose range unless 
tolerability issues ensue. A randomization code will be generated by the unblinded statistician. The 
study site pharmacist(s) responsible for study drug dispensation will also be unblinded. 
 
This study will use a double-dummy blind dosing design, all 
subjects will receive two capsules in the AM after start of breakfast, and dose adjustment, if 
required due to intolerance, will be done blindly. Instructions for the preparation of study drugs 
are provided in a separate manual. A flexible dose design (50 or 100 mg/day) will be used in this 
study since this type of dosing permits better assessment of safety and efficacy than does a fixed 
dose, especially when this compound has not been previously tested in subjects with major 
depression. All subjects except those who are responders at the end of the first Research Condition 
will cross over. To avoid carry-over effects between the different test sessions, there will be an 
interval of 14-21 days, pending response to Research Condition 1. Subjects will then be blindly 
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No subject will receive more than 3 weeks of TS-161. All subjects who discontinue the study or 
who complete Study Phase II will then receive either short-term standard clinical treatment (for up 
to 2 months) or the opportunity to participate in another research protocol. Subjects maintaining 
response to Research Condition 1 after two weeks will receive an additional one-week washout 
before being crossed over to Research Condition 2. Subjects maintaining response (50% improved 
from baseline on the MADRS) after this additional week will be withdrawn from the study and 
will receive standard clinical treatment. 
The starting dose will be 100 mg/day (given as a single AM dose approximately 30 minutes 
after start of breakfast) and the treatment target for all participants will be 100 mg/day unless 
tolerability issues ensue. The dose of TS-161/placebo will remain at 100 mg/day in subjects until 
the end of the study. If subjects are unable to tolerate the current dose, then the dose may be 
decreased down to 50 mg/day for one week at which time the dose may be increased back to 100 
mg/day if response criteria are not met and side effects are tolerable. In case of continued side 
effects at 100 mg/day, the dose may be lowered to 50 mg/day until the end of the study. Subjects 
who are unable to tolerate the lowest allowable dose of study medication (50 mg/day) will be 
discontinued from the study. No dose increases will be allowed after Day 14 of each treatment 
period to allow ample time to evaluate the effect of the drug. Only licensed independent 
practitioners who are investigators on this protocol will be able to make dosing decisions. 
Concurrent with the first dose administration of study medication, participants will undergo 
MEG and MRS procedures (described below), unless technical or availability issues prevent their 
acquisition. Participants will also undergo MRI/MRS, MEG procedures on the final day of each 
Research Condition, unless technical or availability issues prevent acquisition.  
8.4  EFFICACY ASSESSMENTS 
8.4.1  Clinical Rating Scales 
The following rating scales will be administered to participants at scheduled intervals during 
their participation (see the Schedule of Clinical Rating Scales). 
Brief Psychiatric Rating Scale (BPRS, (Overall and Gorham 1962))  
Symptoms and behaviors that are characteristic of schizophrenia will be assessed by using the 
BPRS. Four key BPRS items will be used as an index of positive symptoms of schizophrenia based 
on previous reports that indicated their utility and validity (Bowers, Heninger et al. 1980, Kane, 
Honigfeld et al. 1988) and inclusion within the empirically derived thought disorder factor of the 
BPRS. These four key positive symptoms are conceptual disorganization, hallucinatory behavior, 
suspiciousness, and unusual thought content. Three key BPRS items, blunted affect, emotional 
withdrawal, and motor retardation are selected as a measure of negative symptoms of 
schizophrenia based on a report of their reliability and validity and their inclusion within the 
empirically derived withdrawal-retardation factor of the BPRS. 
Clinician Administered Dissociative States Scale (CADSS, (Bremner, Krystal et al. 1998))  
The CADSSS is a 28-item scale for the assessment of dissociative states at discrete points in 
time. The instrument contains both subjective and objective items. This is intended to capture the 
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observed by an outside observer. The CADSS can be used as a change measure to assess, for 
example, dissociative states before and after a course of treatment. 
Columbia Suicide Severity Rating Scale (C-SSRS, (Posner, Brown et al. 2011))  
The C-SSRS is a suicidal ideation rating scale created by researchers at Columbia University. 
It rates an individual's degree of suicidal ideation on a scale, ranging from "wish to be dead" to 
"active suicidal ideation with specific plan and intent. The scale identifies behaviors, which may 
be indicative of an individual's intent to commit suicide. An individual exhibiting even a single 
behavior identified by the scale was 8 to 10 times more likely to commit suicide. 
Family History Screen (FHS, (Weissman, Wickramaratne et al. 2000))  
The FHS is a clinician-administered structured interview designed to collect the mental health 
history of an informant and their first-degree relatives on 15 psychiatric disorders and suicidal 
behavior. A series of 31 questions are used to detect any evidence of psychiatric symptomatology 
in the participant’s relatives. Its validity is best demonstrated for major depression, anxiety 
disorders, substance abuse, and suicide attempts. If the FHS was not conducted under 01-M-0254 
it will be done under the current protocol. 
Hamilton Depression Rating Scale (HDRS, (Hamilton 1960))  
The HDRS is a widely used observational rating measure of depression severity. The 
estimated time to administer this scale is 30 minutes. It assesses both the presence and severity of 
individual signs and symptoms characterizing depression without psychotic features.   
Hamilton Psychiatric Rating Scale for Anxiety (HAM-A, (Hamilton 1959))  
The HAM-A is a widely used observational rating measure of anxiety severity. The scale 
consists of 14 items. Each item is rated on a scale of 0 to 4. This scale will be administered to 
assess the severity of anxiety and its improvement during the course of therapy. The HAM-A total 
score is the sum of the 14 items and the score ranges from 0 to 56. 
Montgomery and Asberg Depression Rating Scale (MADRS; (Montgomery and Asberg 1979))  
The MADRS is a 10-item instrument used for the evaluation of depressive symptoms in adults 
and for the assessment of any changes to those symptoms. The estimated time to administer this 
scale is 20 minutes. Inter-rater reliability of the scale is high and scores correlate significantly with 
those of the HAMD. Each of the 10 items are rated on a scale of 0 to 6, with differing descriptors 
for each item. These individual item scores are added together to form a total score, which can 
range between 0 and 60 points. 
Positive and Negative Affect Schedule (PANAS, (Watson, Clark et al. 1988))  
The PANAS is a questionnaire that consists of a number of words that describe different 
feelings and emotions. 
Snaith–Hamilton Pleasure Scale (SHAPS, (Snaith, Hamilton et al. 1995))  
The SHAPS is a self-reported scale evaluating anhedonia for neuropsychiatric disorders. This 
is a 14-item checklist covering four domains of hedonic experience, namely interest/pastimes, 
social interaction, sensory experience, and food/drink. 


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Scale for Suicidal Ideation (SSI, (Beck, Kovacs et al. 1979))  
The SSI is a 19-item observer scale designed to quantify the intensity of current conscious 
suicidal ideation in various dimensions of self-destructive thoughts or wishes: the extent of the 
wish to die, the desire to make an actual suicide attempt, and details of any plans; also, internal 
deterrents to an active attempt, and subjective feelings of control and/or courage regarding a 
proposed attempt. 
Temporal Experience of Pleasure Scale (TEPS, (Gard, Gard et al. 2006))  
The TEPS is a self-report measure designed to distinguish between consummatory and 
anticipatory anhedonia. 
Young Mania Rating Scale (YMRS; (Young, Biggs et al. 1978))  
The YMRS consists of 11 items. Items 5, 6, 8, and 9 are rated on a scale from 0 (symptom 
not present) to 8 (symptom extremely severe). The remaining items are rated on a scale from 0 
(symptom not present) to 4 (symptom extremely severe). Items 5, 6, 8, and 9 (irritability, speech, 
content and disruptive-aggressive behavior) are given twice the weight of the remaining 7 in order 
to compensate for the poor condition of severely ill patients. The YMRS total score ranges from 0 
to 60. The time to administer this scale is 15-30 minutes. The YMRS scale is obtained should 
hypomanic/manic symptoms develop during the study protocol. 
8.4.2  Magnetoencephalography (MEG) (Optional) 
All subjects will have a total of six MEG scans: baselines preceding each treatment phase, 2 
hours after the first dose administrations and at the end of each Research Condition (see the 
Schedule of Events for timing of procedures). 
 During the MEG scanning, participants will be in the shielded recording room with their 
heads inside the helmet. Brain magnetic fields will be recorded with the 275-channel OMEGA 
system. The 275 SQUID sensors are uniformly distributed, in a grid, over the inner surface of the 
helmet that covers the entire head with provisions for the eyes and ears. Visual and two-way audio 
communication with the participant will be maintained throughout the session. Head position 
within the magnetometer will be determined using three indicator coils that are attached to the 
preauricular and the nasion fiducial points. The positions define the coordinate system for the 
signals and allow for post-hoc registration. Digital photographs or 3d laser scans of the fiducial 
points on the participant's head may be used to facilitate registration with the anatomical MRI. A 
10-minute resting state MEG scan will consist of continuous acquisition without presentation of a 
task, during which participants will be instructed to relax with their eyes closed. Tasks which 
measure visual, auditory, and somatosensory evoked potentials as well as simple cognitive tasks 
will also be performed (see below for a description of tasks).  Physiological activity will be 
recorded via pulse oximetry and electrooculogram. 
During MEG scanning, the following protocol of rest/task scans will be collected: 
Auditory, Somatosensory, and Visual Evoked Fields Task 
Subjects will be instructed to attend to a fixation while binaural auditory tones, left and right 
visual field static checkerboards, and left and right somatosensory “airpuff” stimulations are 
randomly presented during a 10-minute scan session. Subjects will be asked to maintain fixation 
on a centrally-presented dot during the scan session. 


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Monetary Incentive Delay Task 
Subjects will be instructed to attend to stimuli (three different shapes: triangle, circle, square) 
that are presented briefly, followed by a variable delay, then a response window. During the 
response window, subjects will be instructed to button-press as quickly as possible. Some of the 
objects will allow participants to win money, some objects will allow participants to avoid losing 
money, and some will not be associated with reward/loss. This task will be collected during a 10-
minute scan session.  
Hariri Hamer Task 
Subjects will be instructed to match faces and shapes during a 10-minute scan session. 
Subjects will first be presented with a target object (either face or shape), then after a brief delay, 
two simultaneous images (either 2 faces or 2 shapes). They will indicate via left or right button-
press which of the two simultaneous images matches the target. For faces, subjects will be 
instructed to match the emotion displayed (either happy or sad). For shapes, subjects will be 
instructed to match the shape, even if the orientation changes.  
8.4.3  MRI and MRS (Optional) 
All subjects will have a total of six MRI/MRS scans: baselines preceding each treatment phase, 
3 hours, and 4 hours after the first dose administrations and at the end of each Research Condition 
(see the Schedule of Events for the timing of procedures). Participants who have not received MRI 
scans at the NIH within the past 12 months will be scheduled for a battery of clinical scans. These 
scans take approximately 15 minutes to acquire. These clinical scans may be performed on either 
a 1.5, 3, or 7 Tesla system and may be acquired either during a separate scanning session before 
the baseline scanning session, or during the baseline scanning session. A structural MRI scan will 
be acquired for MEG data coregistration if one has not already been obtained.  
MRI scans will be acquired using a multi-channel coil for increased signal-to-noise. The 
whole brain structural scan will be acquired with a gradient echo based T1 weighted whole brain 
sequence with a minimum of 1.2mm isotropic voxels. 
 
Besides the clinical MRI, all subjects will have a total of six MRI/MRS scans: at baseline for 
both Research Conditions and at the cmax of the first and last dose administration for each treatment 
phase (placebo or active drug).  
The MRS scan will consist of the following elements and will last approximately 60-90 
minutes. It is anticipated that these scans may not be collected in some subjects due to logistical 
issues (e.g., scheduling, or scanner not functioning). 
• Whole brain structural image (15 minutes) 
• Resting state functional MRI (10 min) 
• Glutamate MRS (3 voxels, 10 minutes/voxel), pending sequence development  
• Task MRI: motor/sensory/cognitive/emotional/working memory/ attention tasks (10 
minutes each, time permitting) 
MRIs are accomplished by having the subject lie down on a bed that is slid into the hollow, 
cylindrical “bore” of a magnet. Two persons with experience conducting MRIs will perform the 
scan from an adjacent room. Audible communication will occur by intercom. The NIH code team 
with full medical coverage will be available in case of emergency. Individuals will be removed 


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from the scanner immediately upon request or in case of emergency. During the scan, the subjects 
will hear a thumping or buzzing sound, but earplugs or MRI compatible headphones will be placed 
to minimize noise.  
A customized, 3-D printed pillow may also be created to help stabilize the participant’s head 
if movement is of particular concern in the study.  If motion artifacts (e.g. ghosting, ringing) are 
present in the screening MRI, the optional custom pillow will be considered for use in subsequent 
research scans. In advance of imaging procedures, we will use a camera to take a series of pictures 
of the subject’s head. These images will be used to map the participant’s head shape and generate 
a pillow that is uniquely fitted to them. 
Video monitoring may be used for motion correction and eye gaze tracking. These cameras 
are MR compatible and have been approved by the NMR safety committee. Video monitoring is 
optional. 
8.4.4  Neurocognitive Battery (Optional) 
The following optional neurocognitive tests will be administered as time/resources permit at 
each of the timepoints listed in the Schedule of Events. At each timepoint, a minimum of one test 
will be administered for each domain- verbal episodic memory, working memory/attention, and 
verbal fluency. 
8.4.4.1  Verbal Episodic Memory 
California Verbal Learning Test (CVLT; (Delis, Freeland et al. 1988))  
The CVLT is a comprehensive battery used in memory research.  For the purpose of this study, 
only one component, measuring immediate recall, will be employed.  There are four standardized 
lists of 16 items to remember, allowing for repeated measurement. The test takes approximately 
25 minutes to complete. 
Rey Auditory Verbal Learning Test (RAVLT; (Ryan and Geisser 1986, Peaker and Stewart 
1989)) 
The RAVLT is designed to assess verbal memory, including proactive inhibition, retroactive 
inhibition, retention, encoding versus retrieval, and subjective organization. The test also evaluates 
the severity of memory disjunction and changes that occur in memory function over a period of 
time. The test takes 10-15 minutes to complete. 
Hopkins Verbal Learning Test – Revised (HVLT-R; (Benedict, Schretlen et al. 1998)) 
The HVLT-R is a test of verbal learning and delayed memory. The test includes a series trials. 
Each trial consists of a list of 12 nouns organized into semantic categories (e.g. precious gems, 
types of structures, animals).  Participants are read the list and told to memorize the words. They 
are then asked to free-recall the items on the list. This same procedure is repeated during two 
additional trials.  On the final trial, they are read 24 words and asked to respond with a “yes” if the 
word was included in the list or “no” if it was not. The test takes approximately 10 minutes to 
complete. There may be a 20- to 25-minute delay between hearing the list and recalling the words. 


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8.4.4.2  Working Memory/Attention 
Digit Symbol Substitution Test (DSST; (Wechsler 1939)) 
The DSST, part of the original Wechsler Adult Intelligence Scale (WAIS), measures the 
presence of and change in cognitive dysfunction. The procedure involves matching symbols to 
corresponding Arabic number (e.g. ↔ is 1, ↕ is 2, ≡ is 3).  The test is a series of boxes in two rows.  
The top row contains a number and the bottom row boxes are empty.  Using the key, participants 
are asked to put the symbols in the boxes below their corresponding numbers.  The score is the 
number of correct matches made within the specified length of time.  The test takes approximately 
5 minutes to complete. 
Conners Continuous Performance Test 3rd Ed. (CPT 3; (Keith Conners, Sitarenios et al. 2018)) 
The CPT-3 is a revision of its predecessor, the CPT-II, and was designed to assess attention-
related problems in four domains of attention.  The test integrates the same key elements as its 
predecessor with a number of new features, including Validity scales, Screener items, Critical 
items, Impairment items, an assessment of Executive Functioning, and strengthened linkage to the 
Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5).  The test takes 
approximately 15 minutes to administer. 
8.4.4.3  Verbal Fluency 
Controlled Oral Word Association Test (COWAT; (Benton and Hamsher 1983)) 
The COWAT is a measure used to assess verbal fluency. Patients are asked to produce words 
beginning with a letter as well as words that belong to a specific category. Patients have exactly 
one minute to produce as many words as possible per letter and category. The task takes 
approximately 5 minutes to complete. 
Delis-Kaplan Executive Function System (D-KEFS; (Delis, Kaplan et al. 2001)), Verbal Fluency 
subtest 
The D-KEFS evaluates the main components of executive function that are believed to be 
mediated by the frontal lobe.  The assessment includes nine separate, stand-alone tests. The Verb 
al Fluency Test, one such component, measures letter fluency, category fluency, and category 
switching. The test takes approximately 10 minutes to complete. 
8.5  BIOSPECIMEN EVALUATIONS 
Blood will be collected according to site procedures through either an i.v. saline lock or 
directly by a needle. During the time that blood is obtained on multiple occasions, a saline lock 
will be used, thus the saline lock will be used via an antecubital iv line. If a single blood draw is 
done the sample will be obtained using a butterfly needle. Samples for safety and research purposes 
(samples for PK purposes are exclusionary) will be collected between the hours of 10 am and 2pm, 
to minimize potential diurnal variability (Begliuomini, Lenzi et al. 2008) at baseline prior to 
randomization and at the timepoints listed in the Schedule of Events. This will afford us the 
opportunity to explore the temporal relationship between changes in serum biomarkers and clinical 
improvement and changes in plasticity measures. 
Blood samples will be drawn for safety, research, and PK purposes. No more than 370mL (25 
tablespoons) of research blood will be drawn during the study. 


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8.5.1.1  Safety Labs 
Samples will be collected for safety labs, including Acute Care Panel (sodium, potassium, 
chloride, total CO2, creatinine, eGFR, glucose, urea nitrogen anion gap), CBC+Diff (complete 
blood count with differential, including WBC, RBC, HGB, HCT, MCV, MCH, RDW, platelet 
count, MPY, nucleated RBC, absolute neutrophils, bands, immature granulocytes, lymphocytes, 
monocytes, eosinophils, basophils, neutrophil absolute, immature granulocytes absolute, 
lymphocyte absolute, monocyte absolute, eosinophil absolute, basophil absolute), hepatic panel 
(alkaline phosphatase, alanine aminotransferase, aspartame aminotransferase, bilirubin total, 
bilirubin direct), thyroid function tests (screening; thyroid stimulating hormone, free thyroxine). 
Safety labs will be collected at the timepoints listed in the table below, as well as in the Schedule 
of Events. Additional safety draws will be conducted as clinically indicated. 
8.5.2  Correlative Studies for Research/Pharmacokinetic Studies 
These draws will be conducted at the timepoints listed in the Schedule of Events.  See 
Appendix C, for a detailed description of research-related blood draws. 
We will not send PK samples for outside analysis taken from participants with COVID-19 or 
suspected COVID-19 (e.g., have respiratory symptoms or chest imaging findings). 
8.5.3  Samples for Genetic/Genomic Analysis 
8.5.3.1  Description of the scope of genetic/genomic analysis 
See Appendix C for a description of genetic/genomic analyses.  
Samples and data (including genomic data) will be shared with NIMH Data Archive, dbGaP, 
and the NIMH Repository and Genomics Resource.  
8.5.3.2  Description of how privacy and confidentiality of medical information/biological 
specimens will be maximized 
All research activities will be conducted in as private a setting as possible. Clinical data will 
be 
managed 
in 
accordance 
with 
the 
NIH 
Clinical 
Center’s 
privacy 
policy 
(http://www.cc.nih.gov/participate/patientinfo/legal.shtml). Research data and records will be 
coded, no individual will be identified by name, and the data will be stored in a locked filing 
cabinet stored in the principal investigator’s locked offices to protect subject anonymity. Electronic 
data with identifiers (including neuroimaging) will be saved password-protected NIH-issued 
computers on secured servers.  Neuroimaging data will be maintained on a secure internet-based 
server.  Only study investigators will have access to the data.  De-identified results from clinical 
trials will be posted on ClinicalTrials.gov.  Clinical data will be managed according to NIH 
Clinical Center’s standard policies. 
Biological samples (i.e. blood) will be kept in freezers by the NIMH/ETPB with no participant 
identifiers. Information on processing and storage of samples is on record with the Clinical and 
Scientific Director’s offices at the NIMH.  Plasma samples will be collected, handled, stored, and 
shipped as outlined in the Data Handling and Record Keeping section. Results will be published 
as group data without the use of characteristics that would identify individual subjects. Samples 
and date will be stored using codes that we assign. Only study investigators will have access to the 
stored samples. 


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This study collects sensitive information, including personal medical and treatment history. 
The PI will train study staff regarding obtaining and handling potentially sensitive and private 
information about NIH employees and staff through staff discussions and written branch/section 
procedures.   
Samples and data will be stored using codes that we assign. Data will be kept in password-
protected computers.  Samples will be kept in locked storage. Only study investigators will have 
access to the samples and data.  De-identified results from clinical trials will be posted on 
cctrials.gov. 
8.5.3.3  Management of results 
Results from genetic testing will be obtained in a non-CLIA laboratory; therefore, results will 
not be provided back to the participant or health care provider for purposes of providing clinical 
diagnostic information.  
8.5.3.4  Genetic counseling 
Genetic counseling will not be provided under this protocol. 
8.6   SAFETY AND OTHER ASSESSMENTS 
8.6.1  Blood draw and laboratory screen 
A blood draw and laboratory screen (e.g. Acute Care Panel, CBC+Diff, Hepatic Panel, LFT, 
TFT, HIV) will be conducted to screen for any medical condition that would increase risk for 
adverse events. Blood draw and laboratory screens will be conducted for screening and evaluation 
purposes. 
8.6.2  Blood pressure 
An assessment of blood pressure will be conducted as cardiovascular illness is an exclusion 
criterion for this study. Blood pressure will be collected for screening and evaluation purposes.  
8.6.3  Structural neuroimaging studies  
Structural neuroimaging studies will be conducted within one year prior to study participation. 
In rare cases when an abnormality is discovered, participants are offered further assessment at the 
NIH via a neurology consult. Minor abnormalities with no clinical significance to participants may 
not be shared. Structural neuroimaging studies will be conducted for screening purposes. 
8.6.4  Electrocardiogram 
The assessment of cardiovascular function via electrocardiogram (ECG) will be conducted as 
cardiovascular illness is an exclusion criterion for this study. ECG will be collected for screening 
purposes. 
8.6.5  Heart rate 
The assessment of heart rate will be conducted as cardiovascular illness is an exclusion 
criterion for this study. Heart rate will be collected for screening and evaluation purposes. 


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Additionally, heart rate will be collected during the study visit to monitor for potential adverse 
events. 
8.6.6  Height 
The assessment of height will be conducted for screening and evaluation purposes. 
8.6.7  Medical history 
A review of the participants medical history will be conducted. The review will involve the 
assessment of current medications as the use of psychoactive medications is exclusion criteria for 
this study. The review of medical history will also involve screening for medical conditions that 
would increase risk for MRI or any component of study participation. A review of medical history 
will be conducted for screening purposes. 
8.6.8  Monitoring of clinical symptoms 
Clinical Rating Scales will be used to monitor mood and anxiety symptoms. Additionally, 
clinical personnel with monitor participants for adverse events and other clinical symptoms (e.g. 
headache, nausea, vomiting, abdominal pain, chest pain, light-headedness, dizziness, and blurry 
vision). 
8.6.9  Physical exam 
Participants will undergo a physical exam to screen for medical conditions that would increase 
risk for MRI, TS-161, or any component of study participation. Physical exams will be conducted 
for screening purposes and safety assessment at the end of study participation. 
8.6.10  Respiration 
The assessment of respiration rate will be conducted to monitor responses to the 
administration of the study intervention. 
8.6.11  Review of changes to Medical Report Form and Medication Review 
Changes to participant’s medical report forms and medication histories will be conducted to 
screen for medical conditions that would increase risk for the study intervention or other 
components of study participation. Review of changes to their medical report form and medication 
history will be performed in the 24 hours prior to administration of the study intervention.  
8.6.12  Temperature 
The assessment of body temperature will be conducted to monitor responses to the 
administration of the study intervention. 
8.6.13  Urine pregnancy test 
Women of child-bearing potential will undergo urine pregnancy testing to rule out pregnancy. 
Urine pregnancy tests will be completed at the time of screening and no more than 24 hours prior 
to start of each research condition and imaging procedure.  


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8.6.14  Urine toxicology 
Participants will undergo a urine toxicology screen as drugs that act on the central nervous 
system is exclusion criteria for this study. Urine toxicology will be conducted for screening 
purposes. 
8.6.15  Weight 
Participants will undergo the measurement of their weight for screening purposes and at time 
points indicated in the Schedule of Events. 
8.7  ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS 
8.7.1  Definition of Adverse Event 
Adverse event means any untoward medical occurrence associated with the use of an 
intervention in humans, whether or not considered intervention-related (21 CFR 312.32 (a)). 
8.7.2  Definition of Serious Adverse Events (SAE) 
An  AE or suspected AE is considered "serious" if, in the view of either the investigator or 
sponsor, it results in any of the following outcomes: death, a life-threatening adverse event, 
inpatient hospitalization or prolongation of existing hospitalization, a persistent or significant 
incapacity or substantial disruption of the ability to conduct normal life functions, or a congenital 
anomaly/birth defect. Important medical events that may not result in death, be life-threatening, or 
require hospitalization may be considered serious when, based upon appropriate medical judgment, 
they may jeopardize the participant and may require medical or surgical intervention to prevent 
one of the outcomes listed in this definition. Examples of such medical events include allergic 
bronchospasm requiring intensive treatment in an emergency room or at home, blood dyscrasias 
or convulsions that do not result in inpatient hospitalization, or the development of drug 
dependency or drug abuse. 
8.7.3  Classification of an Adverse Event 
8.7.3.1  Severity of Event 
For AEs not included in the protocol defined grading system, the following guidelines will be 
used to describe severity.  
Mild 
Events require minimal or no treatment and do not interfere with the participant’s daily 
activities.  
Moderate 
Events result in a low level of inconvenience or concern with the therapeutic measures. 
Moderate events may cause some interference with functioning. 


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Severe 
Events interrupt a participant’s usual daily activity and may require systemic drug therapy or 
other treatment. Severe events are usually potentially life-threatening or incapacitating.  Of note, 
the term “severe” does not necessarily equate to “serious”.] 
8.7.3.2  Relationship to Study Intervention 
All AEs must have their relationship to study intervention assessed by the investigator who 
examines and evaluates the participant based on temporal relationship and their clinical judgment. 
The degree of certainty about causality will be graded using the categories below. In a clinical trial, 
the study product must always be suspect.  
Definitely Related 
There is clear evidence to suggest a causal relationship, and other possible contributing factors 
can be ruled out. The clinical event, including an abnormal laboratory test result, occurs in a 
plausible time relationship to study intervention administration and cannot be explained by 
concurrent disease or other drugs or chemicals. The response to withdrawal of the study 
intervention (dechallenge) should be clinically plausible. The event must be pharmacologically or 
phenomenologically definitive, with use of a satisfactory rechallenge procedure if necessary. 
Probably Related 
There is evidence to suggest a causal relationship, and the influence of other factors is unlikely. 
The clinical event, including an abnormal laboratory test result, occurs within a reasonable time 
after administration of the study intervention, is unlikely to be attributed to concurrent disease or 
other drugs or chemicals, and follows a clinically reasonable response on withdrawal (dechallenge). 
Rechallenge information is not required to fulfill this definition. 
Potentially Related 
There is some evidence to suggest a causal relationship (e.g., the event occurred within a 
reasonable time after administration of the trial medication). However, other factors may have 
contributed to the event (e.g., the participant’s clinical condition, other concomitant events). 
Although an AE may rate only as “possibly related” soon after discovery, it can be flagged as 
requiring more information and later be upgraded to “probably related” or “definitely related”, as 
appropriate. 
Unlikely to be related 
A clinical event, including an abnormal laboratory test result, whose temporal relationship to 
study intervention administration makes a causal relationship improbable (e.g., the event did not 
occur within a reasonable time after administration of the study intervention) and in which other 
drugs or chemicals or underlying disease provides plausible explanations (e.g., the participant’s 
clinical condition, other concomitant treatments). 
Not Related 
The AE is completely independent of study intervention administration, and/or evidence 
exists that the event is definitely related to another etiology. There must be an alternative, definitive 
etiology documented by the clinician. 


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8.7.3.3  Expectedness  
The PI and LIPs on the study will be responsible for determining whether an AE is expected 
or unexpected.  An AE will be considered unexpected if the nature, severity, or frequency of the 
event is not consistent with the risk information previously described for the study intervention. 
8.7.4  Time Period and Frequency for Event Assessment and Follow-Up 
The occurrence of an AE or SAE may come to the attention of study personnel during study 
visits and interviews of a study participant presenting for medical care, or upon review by a study 
monitor. 
All AEs including local and systemic reactions not meeting the criteria for SAEs will be 
captured on the appropriate CRF. Information to be collected includes event description, time of 
onset, clinician’s assessment of severity, relationship to study product (assessed only by those with 
the training and authority to make a diagnosis), and time of resolution/stabilization of the event. 
All AEs occurring while on study must be documented appropriately regardless of relationship. 
All AEs will be followed to adequate resolution. 
Any medical condition that is present at the time that the participant is screened will be 
considered as baseline and not reported as an AE. However, if the study participant’s condition 
deteriorates at any time during the study, it will be recorded as an AE.  
Changes in the severity of an AE will be documented to allow an assessment of the duration 
of the event at each level of severity to be performed. AEs characterized as intermittent require 
documentation of onset and duration of each episode. 
LIPs on the study will record all reportable events with start dates occurring any time after 
informed consent is obtained until 7 (for non-serious AEs) or 30 days (for SAEs) after the last day 
of study participation.  At each study visit, the investigator will inquire about the occurrence of 
AE/SAEs since the last visit.  Events will be followed for outcome information until resolution or 
stabilization. 
8.7.5  Adverse Event Reporting  
Reportable events for this protocol will be tracked and reported in compliance with Policy 
801. 
8.7.6  Serious Adverse Event Reporting  
All serious adverse event (SAE) reporting will adhere to the U.S. Code of Federal Regulations 
(21 CFR Part 312.32) for IND drugs. The PI will immediately report any SAEs to the sponsor, 
whether or not these are considered study intervention-related, including those listed in the 
protocol or investigator brochure; any such report must include an assessment of whether there is 
a reasonable possibility that the study intervention caused the event.  The PI or designee must send 
all SAEs on a MedWatch form to the Sponsor, copying the REGSupportORSC@nih.gov inbox at 
the time of awareness (within 24 hours for deaths, within 48 hours for all other SAEs). Additional 
information to include in the MedWatch Form 3500A is as follows: 
• IND Number 
• Protocol Number 
• Principal Investigator’s Name 


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• Event Attribution 
The sponsor or designee will be responsible for determining if an SAE is reportable to the 
FDA as an IND Safety Report, i.e. the event is considered a serious unexpected suspected adverse 
reaction (SUSAR). The study sponsor will be responsible for notifying the Food and Drug 
Administration (FDA) or other applicable regulatory agency, of any unexpected fatal or life-
threatening suspected AE as soon as possible, but in no case later than seven calendar days after 
the sponsor's initial receipt of the information. In addition, the sponsor must notify the FDA and 
all participating investigators of an Investigational New Drug (IND) safety report of potential 
serious risks, from clinical trials or any other source, as soon as possible, but in no case later than 
15 calendar days after the sponsor determines that the information qualifies for reporting.  
All SAEs will be followed until satisfactory resolution. All AEs will be documented on 
appropriate study records, including case report forms and AE tracking logs. All AEs will be 
provided in aggregate to the sponsor upon request and no less than once a year.   
8.7.7  Events of Special Interest  
Events of Special Interest are not applicable to this study.  
8.7.8  Reporting of Pregnancy  
Pregnant or nursing women or women who plan to become pregnant are excluded from this 
study. Participants who tested positive for pregnancy or become pregnant while enrolled in the 
study will be discontinued from the protocol and followed through pregnancy outcome. 
8.8  UNANTICIPATED PROBLEMS 
8.8.1  Definition of Unanticipated Problems (UP) 
Any incident, experience, or outcome that meets all of the following criteria: 
• Unexpected in terms of nature, severity, or frequency given (a) the research procedures 
that are described in the protocol-related documents, such as the Institutional Review Board 
(IRB)-approved research protocol and informed consent document; and (b) the 
characteristics of the participant population being studied; and 
• Related or possibly related to participation in the research (“possibly related” means there 
is a reasonable possibility that the incident, experience, or outcome may have been caused 
by the procedures involved in the research); and 
• Suggests that the research places participants or others (which many include research staff, 
family members or other individuals not directly participating in the research) at a greater 
risk of harm (including physical, psychological, economic, or social harm) than was 
previously known or expected. 
8.8.2   Unanticipated Problem Reporting  
The investigator will report unanticipated problems (UPs) to the NIH Institutional Review 
Board (IRB) as per Policy 801. 


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8.8.3  NIH Intramural IRB Reporting of IND Safety Reports 
Only IND Safety Reports that meet the definition of an unanticipated problem will need to be 
reported to the NIH Intramural IRB. 
 
9  STATISTICAL CONSIDERATIONS 
9.1  STATISTICAL HYPOTHESIS 
9.1.1  Primary Endpoint 
• Change from baseline in the MADRS total score 
• H0: The 95% confidence interval for the effect of drug (TS-161 minus placebo) on 
MADRS total at 3 weeks, after controlling for baseline scores, will include zero. 
• HA: The 95% confidence interval for the effect of drug (TS-161 minus placebo) on 
MADRS total at 3 weeks, after controlling for baseline scores, will be less than and 
exclude zero. 
9.1.2  Secondary Endpoints 
• Proportion of subjects in remission (defined as MADRS total score ≤10). 
• H0: The 95% confidence interval for the odds ratio (TS-161 versus placebo) 
predicting MADRS total score ≤10 at 3 weeks will include one. 
• HA: The 95% confidence interval for the odds ratio (TS-161 versus placebo) 
predicting MADRS total score ≤10 at 3 weeks will be greater than and exclude one. 
• Proportion of subjects with response (defined as ≥50% reduction from baseline in MADRS 
total score). 
• H0: The 95% confidence interval for the odds ratio (TS-161 versus placebo) 
predicting 50% change from baseline in MADRS total score at 3 weeks will include 
one. 
• HA: The 95% confidence interval for the odds ratio (TS-161 versus placebo) 
predicting 50% change from baseline in MADRS total score at 3 weeks will be 
greater than and exclude one. 
• Change from baseline in HDRS, BPRS, CADSS, C-SSRS, HAM-A, MADRS, PANAS, 
SHAPS, SSI, TEPS, YMRS total scores.  
• H0: The 95% confidence interval for the effect of drug (TS-161 minus placebo) on 
[all listed outcomes] at the timepoints listed in the Schedule of Events after 
controlling for baseline scores, will include zero. 
• HA: The 95% confidence interval for the effect of drug (TS-161 minus placebo) on 
[all listed outcomes] at the timepoints listed in the Schedule of Events after 
controlling for baseline scores, will be less than and exclude zero. 


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9.1.3  Surrogate Markers of Drug Effect, Target Engagement, and Antidepressant 
Response  
• Change in MEG spectral power (gamma power). 
• H0: Among TRD-MDD subjects, pre-treatment gamma power levels will predict the 
magnitude of clinical improvement to TS-161, but not to placebo. 
• Change in resting and task based functional connectivity in fMRI. 
• H0: Among TRD-MDD subjects, TS-161 administration will increase pgACC 
activity and decrease amygdala activity to emotional faces more than placebo. 
Similar effects will be observed in activity associated with the resting-state networks. 
TS-161 will also decrease pgACC activity during the tasks more than placebo. 
• Change in glutamate levels in MRS. 
• H0: Among TRD-MDD subjects, pre-treatment mPFC glutamate levels will predict 
the magnitude of clinical improvement to TS-161 in subjects with MDD, but not to 
placebo. 
• HA: Among TRD-MDD subjects, post-treatment increases in glutamate levels will 
predict the magnitude of clinical improvement to TS-161 (but not placebo).  
• Change in peripheral biomarkers (exploratory). 
9.2  SAMPLE SIZE DETERMINATION 
The estimated duration of this protocol is 36 months and we anticipate recruiting 1-2 subjects 
a month over this period.  
The primary outcome measure is the MADRS total score. The null hypothesis is that the 95% 
confidence interval for the effect of drug (TS-161 minus placebo) on MADRS total at 3 weeks, 
after controlling for baseline scores, will include zero; the alternative hypothesis is that this 95% 
CI will be less than and exclude zero. We have observed in previous crossover studies of the rapid 
acting antidepressant ketamine an estimated difference of -6.7 (SE = 1.5) [95%CI: -9.6, -3.76]. 
This study will be powered to detect an effect within that confidence interval that translates to 
moderate effect size (difference = -7.5; d = 0.70) on the MADRS total score at end point. While 
we are primarily interested in identifying drugs with a large effect size, this effect size was selected 
to balance the risk of an underpowered study if the true effect size were not large and the cost of 
powering the study to detect a small effect size. Assuming α=.05, two-tailed, 20 patients with 
MDD would provide 80% power to detect a drug effect of this magnitude at end point. As 
discussed, we are interested only in a moderate effect size and have powered the study accordingly. 
However, if we were to power the study based on the reasonable range of treatment effect observed 
in the previous studies of rapid acting antidepressants, the sample size requirements would range 
from 13 subjects for the largest effect in the 95% CI to 67 subjects for the smallest effect. To 
account for a 25% rate of attrition, either from participants who do not complete the drug-free 
period or who drop out during the crossover double-blind portion of the study, we will recruit 25 
participants. No interim analyses are planned. Secondary analyses with continuous outcomes will 
likely be sufficiently powered, as we anticipate that the effects on most secondary outcomes will 
be similar to the primary outcome. However, the power of the study to detect effects on the 
categorical secondary outcomes (response and remission) will be reduced as categorical outcomes 
de facto require a larger sample size. SAS 9.4 PROC POWER for paired mean designs was used 
for sample size calculations. 


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9.3  POPULATIONS FOR ANALYSES 
There will be no interim analysis; the blind will be broken after the accrual goal has been met 
and all analyses will be based on the full dataset. Analyses will be based on the intent-to-treat 
(ITT) principle where all randomized subjects who receive baseline and at least one post-baseline 
assessment during period 1 will be included. The ITT dataset will be used for the primary and 
secondary analyses. 
Safety and tolerability assessments will be based on the safety analysis set which will include 
all randomized subjects who are given study treatment. 
9.3.1  Evaluable for Toxicity  
All patients will be evaluable for toxicity from the time of their first treatment with TS-161. 
9.4  STATISTICAL ANALYSES 
9.4.1  General Approach 
The assumptions of all models will be checked. Specifically, given that the primary model of 
analysis is the generalized linear model, we will graphically assess the distribution and spread of 
residuals. We will perform transformation if exploratory data analysis suggests that it will improve 
adherence to model assumptions; we do not expect that this will be necessary for behavioral 
outcomes but a log transformation is commonly applied to biological outcomes. For the primary 
analysis no a priori covariates are specified, except for the baseline values of the outcome as 
described in this section. Significance will be evaluated at p<.05, two-tailed. The primary 
hypothesis has only one outcome, the treatment effect at week 3, and therefore no correction to 
alpha (or to p-values) will be made. Following the current guidelines of the American Statistical 
Association (Wasserstein, Schirm et al. 2019), no threshold will be applied to secondary outcomes 
of this trial. Instead, parameter estimates will be presented alongside both their 95%  
9.4.2  Analysis of the Primary Endpoints 
The primary aim will be accomplished by assessing the efficacy of three weeks of oral TS-
161 (50 to 100 mg/day) compared with placebo in improving overall depressive symptomatology. 
The primary assessment of efficacy is a comparison of MADRS scores between treatment groups.  
The MADRS total score is measured at the interval level, and although repeated measurement of 
the outcome is obtained the primary hypothesis concerns the value at 3 weeks.  
The difference between TS-161 and placebo will be examined using a mixed model for 
repeated measures with restricted maximum likelihood estimation where drug and time are within 
subjects factors. Fixed effects for drug, time period, and their interaction will be specified. Period-
specific baseline scores and the subject-specific average baseline score will be entered as a 
covariate (Kenward and Roger 2010); no other covariates will be included a priori. The degrees 
of freedom will be corrected using the Kenward-Roger approach. The residual covariance matrix 
will account for nesting of observations within period for each participant; during the model fitting 
stage, fit indices (e.g., Schwarz’s Bayesian criteria) will be used to determine the most appropriate 
structure for the residual covariance matrix, including the possibility that it will be specified by 
drug to allow for heteroscedasticity. Also, during the model fitting stage, a subject-specific 
(random) intercept will be tested and retained if indicated by model fit indices. During the model 


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fitting stage, the residuals will be graphically assessed for distribution and spread; if they violate 
the assumptions of the model then appropriate transformations will be applied before selecting the 
final model. The analysis will be performed in the ITT sample, and missing data may occur. 
Maximum likelihood estimation will be implemented, which allows for missingness without 
excluding the participant; no imputation will be performed. The effect of interest in this model is 
the effect of drug at the 3-week timepoint (with 95% CI), which will be obtained from the drug-
by-time interaction using a contrast statement. Cohen’s d, calculated using the model-estimated 
difference between groups, the standard error of that difference, and the degrees of freedom 
associated with that comparison, will be used to describe the effect size.   
9.4.3  Analysis of the Secondary Endpoints 
Secondary objectives are to evaluate the remission and response rates on the primary outcome 
measure (MADRS). A similar model to the one described above will be employed, with the 
difference that the outcome distribution will be specified as binary logistic. Because 
remission/response is determined based on change from baseline within a period, no baseline 
covariate will be included in the model. The parameter estimate for the effect of drug on 
remission/response rate at a given time point will be expressed as an odds ratio with a 95% CI. 
The remaining secondary analyses of clinical outcomes measured on the interval level (HAMD, 
BPRS, CADSS, C-SSRS, HAM-A, PANAS, SHAPS, SSI, TEPS, and YMRS) will be conducted 
in the manner described above for MADRS score. Following the current guidelines of the 
American Statistical Association (Wasserstein, Schirm et al. 2019), no threshold will be applied 
to secondary outcomes of this trial. Instead, parameter estimates will be presented alongside both 
their 95% confidence intervals and the raw p-value.     
9.4.4  Safety Analyses 
Safety will be evaluated throughout study participation in order to assess symptomology and 
the development of adverse events.  AEs will be tracked in CRFs and reported in compliance with 
Policy 801. 
9.4.5  Baseline Descriptive Statistics 
The demographic composition of the sample, including sex assigned at birth, gender identity, 
race, ethnicity, age, markers of socioeconomic status, and BMI will be tabulated for the full sample 
in order to aid interpretation of the generalizability of the sample. Because the sequence groups 
are randomized, inferential tests of baseline differences are inappropriate.  
9.4.6  Tabulation of individual Participant Data 
Individual participant data will not be listed; only summary statistics will be presented. 
9.4.7  Exploratory Analyses 
We will conduct primary and secondary analyses as specified in Sections 9.4.2 and 9.4.3. We 
will not conduct exploratory analysis for confirmatory proof for registration trials. 
 


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10  REGULATORY AND OPERATIONAL CONSIDERATIONS 
10.1  INFORMED CONSENT PROCESS 
10.1.1  Consent/Assent Procedures and Documentation 
All participants will receive a verbal explanation in terms suited to their comprehension of 
the purposes, procedures and potential risks of the study and of their rights as research participants. 
In-person consent will be obtained at the NIH CC (see below for telephone and telehealth consent 
processes).  Participants will be provided as much time as is required to carefully review the written 
consent form and ask questions of consenting investigators regarding this study prior to signing. 
Consent will be obtained by authorized licensed independent practitioners with patient care 
and clinical research privileges in the NIH CC.  All those study investigators obtaining informed 
consent have completed the NIMH HSPU ‘Elements of Successful Informed Consent’ training. 
An HSPU team member will monitor the informed consent process for patients, which may 
then, in certain circumstances, trigger the need for a formal independent capacity assessment.  
Subjects without consent capacity excluded from participation. 
10.1.2  For assent of children, if applicable 
This section is not applicable. 
10.1.3  Consent for minors when they reach the age of majority  
This section is not applicable. 
10.1.4  Telephone and Telehealth Consent  
The informed consent document will be sent to the subject. An explanation of the study will 
be provided over the telephone after the subject has had the opportunity to read the consent form. 
The subject will sign and date the informed consent. The original informed consent document will 
be sent back to the consenting investigator who will sign and date the consent form with the date 
the consent was returned. A fully executed copy will be returned via mail for the subject’s records. 
The informed consent process will be documented on a progress note by the consenting 
investigator. The investigator will confirm that, when required, written legally effective consent 
has been obtained prior to initiating any study interventions. 
Sometimes it is necessary to obtain information about symptoms by electronic media, obtain 
measures of symptom severity, or conduct other eligible procedures (e.g. interviews, 
questionnaires, and psychological testing) by telehealth.  Under such circumstances, this process 
will be utilized to obtain telehealth consent for this study using NIH-approved platforms. 
10.1.5  Telephone Assent 
This section is not applicable. 
10.1.6  Consent of subjects who are or become, decisionally impaired 
Adults unable to provide consent are excluded from enrolling in the protocol. However, it is 
possible that subjects enrolled in the protocol may permanently lose the capacity to consent for 


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themselves during the course of this study.  In the event this occurs, the subjects will be withdrawn 
from the study. 
10.1.7  Considerations for Consent of NIH staff, or family members of study team members 
Consent for NIH staff will be obtained as detailed above with following additional 
protections: 
Consent from staff members will be obtained by an individual independent of the staff 
member’s team whenever possible.  Otherwise, the consent procedure will be independently 
monitored by the CC Department of Bioethics Consultation Service in order to minimize the risk 
of undue pressure on the staff member. 
10.2  STUDY DISCONTINUATION AND CLOSURE 
This study may be temporarily suspended or prematurely terminated if there is sufficient 
reasonable cause.  Written notification, documenting the reason for study suspension or 
termination, will be provided by the suspending or terminating party to study participants, 
investigator, funding agency, and regulatory authorities.  If the study is prematurely terminated or 
suspended, the PI will promptly inform study participants, the IRB, and sponsor and will provide 
the reason(s) for the termination or suspension.  Study participants will be contacted, as applicable, 
and be informed of changes to study visit schedule. 
Circumstances that may warrant termination or suspension include, but are not limited to: 
• Determination of unexpected, significant, or unacceptable risk to participants 
• Demonstration of efficacy that would warrant stopping    
• Insufficient compliance to protocol requirements 
• Data that are not sufficiently complete and/or evaluable 
• Determination that the primary endpoint has been met 
• Determination of futility 
Study may resume once concerns about safety, protocol compliance, and data quality are 
addressed, and satisfy the sponsor, IRB and, as applicable, the FDA. 
10.3  CONFIDENTIALITY AND PRIVACY 
Participant confidentiality and privacy are strictly held in trust by the participating 
investigators, their staff, and the sponsor. This confidentiality is extended to cover testing of 
biological samples and genetic tests in addition to the clinical information relating to participants. 
Therefore, the study protocol, documentation, data, and all other information generated will be 
held in strict confidence. No information concerning the study or the data will be released to any 
unauthorized third party without prior written approval of the sponsor.  
This study collects sensitive information, including personal and family medical and 
treatment history. The PI will train study staff regarding obtaining and handling potentially 
sensitive and private information about NIH employees and staff through staff discussions and 
written branch/section procedures.   
Hard copy research data/records will be coded, no individual will be identified by name, and 
the data will be stored in a locked filing cabinet stored in locked offices maintained by the PI to 


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protect subject anonymity. Electronic data with identifiers will be saved on NIH secured servers. 
Data will be kept in password-protected computers.  Samples will be kept in locked storage.  
Samples and data will be stored using codes that we assign. Only study investigators and 
clinical monitors will have access to the samples and data with patient identifiable information 
(PII).  Offsite associate investigators will not have access to PII. 
The study monitor, other authorized representatives of the sponsor, representatives of the IRB, 
and/or regulatory agencies may inspect all documents and records required to be maintained by 
the investigator, including but not limited to, medical records (office, clinic, or hospital) and 
pharmacy records for the participants in this study. The clinical study site will permit access to 
such records. 
The study participant’s contact information will be securely stored for internal use during the 
study. At the end of the study, all records will continue to be kept in a secure location for as long 
a period as dictated by the reviewing IRB, Institutional policies, or sponsor requirements. 
Study participant research data, which is for purposes of statistical analysis and scientific 
reporting, will be transmitted to and stored at the Clinical Trials Database (CTDB) at NIH. This 
will not include the participant’s contact or identifying information. Rather, individual participants 
and their research data will be identified by a unique study identification number. The study data 
entry and study management systems used by clinical sites and by ETPB/NIMH research staff will 
be secured and password protected. At the end of the study, all study databases will be de-identified 
and archived at the CTDB. 
To further protect the privacy of study participants, a Certificate of Confidentiality has been 
issued by the NIH.  This certificate protects identifiable research information from forced 
disclosure. It allows the investigator and others who have access to research records to refuse to 
disclose identifying information on research participation in any civil, criminal, administrative, 
legislative, or other proceeding, whether at the federal, state, or local level. By protecting 
researchers and institutions from being compelled to disclose information that would identify 
research participants, Certificates of Confidentiality help achieve the research objectives and 
promote participation in studies by helping assure confidentiality and privacy to participants. 
10.4  FUTURE USE OF STORED SPECIMENS AND DATA 
Biological samples will be kept in freezers by the NIMH/ETPB with no patient identifiers. 
Information on processing and storage of samples is on record with the Clinical and Scientific 
Director’s offices at the NIMH. Samples will be collected, handled, stored, and shipped as outlined 
in the Data Handling and Record Keeping section. Results will be published as group data without 
the use of characteristics that would identify individual subjects. Samples and date will be stored 
using codes that we assign. Only study investigators will have access to the stored samples. 
10.5  SAFETY OVERSIGHT 
This protocol will be reviewed by the NIMH-IRP Data and Safety Monitoring Board.  The 
DSMB meets two times per year.  The board will review study accrual and progress, adverse events 
related to the study, and safety and outcome data.  Specific data elements required by the DSMB 
will be established at the first meeting the protocol is reviewed.  The DSMB will have the authority 
to require changes in the study design, or to stop all or part of any study based on accumulating 
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The medically responsible investigator has the authority to break the blind in the event of an 
emergency.  Breaking of the blind, life threatening injury or death all require immediate reporting 
to the DSMB and the NIMH Clinical Director. A written notification to the DSMB chair must 
follow within 7 days. 
10.6  CLINICAL MONITORING 
Details of clinical site monitoring are documented in a Clinical Monitoring Plan (CMP). The 
CMP describes in detail who will conduct the monitoring, at what frequency monitoring will be 
done, at what level of detail monitoring will be performed, and the distribution of monitoring 
reports. 
As per ICH-GCP 5.18 and FDA 21 CFR 312.50 clinical protocols are required to be 
adequately monitored. Monitoring for the NIH site will be conducted according to the “NIMH 
Intramural Program Guidelines for Monitoring of Clinical Trials”. Monitors under contract to the 
NIMH OCD ORO will visit the NIH site to monitor aspects of the study in accordance with the 
appropriate regulations and the approved protocol. The objectives of a monitoring visit will be: 1) 
to verify the existence of signed informed consent documents and documentation of the ICF 
process for each monitored subject; 2) to verify the prompt and accurate recording of all monitored 
data points, and prompt reporting of all SAEs; 3) to compare abstracted information from clinical 
databases (e.g. CTDB) with individual subjects’ records and source documents (subjects’ charts, 
laboratory analyses and test results, physicians’ progress notes, nurses’ notes, and any other 
relevant original subject information); and 4) to help ensure investigators are in compliance with 
the protocol. The monitors also will inspect the clinical site regulatory files to ensure that 
regulatory requirements (Office for Human Research Protections-OHRP), FDA, and applicable 
guidelines (ICH-GCP) are being followed. During the monitoring visits, the investigator (and/or 
designee) and other study personnel will be available to discuss the study progress and monitoring 
visit. 
The investigator (and/or designee) will make study documents (e.g., consent forms, clinical 
database records and pertinent hospital/sources or clinical records) readily available for inspection 
by the local IRB, FDA, the site monitors, and the NIMH staff for confirmation of the study data. 
A specific protocol monitoring plan will be discussed with the Principal Investigator and study 
staff. The plan will outline the frequency of monitoring visits based on such factors as study 
enrollment, data collection status and regulatory obligations. 
10.7  QUALITY ASSURANCE AND QUALITY CONTROL 
Each clinical site will perform internal quality management of study conduct, data and 
biological specimen collection, documentation and completion.  An individualized quality 
management plan will be developed to describe a site’s quality management. 
Quality control (QC) procedures will be implemented beginning with the data entry system 
and data QC checks that will be run on the database will be generated. Any missing data or data 
anomalies will be communicated to the site(s) for clarification/resolution. 
Following written Standard Operating Procedures (SOPs), the monitors will verify that the 
clinical trial is conducted and data are generated and biological specimens are collected, 
documented (recorded), and reported in compliance with the protocol, International Conference 


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on Harmonisation Good Clinical Practice (ICH GCP), and applicable regulatory requirements (e.g., 
Good Laboratory Practices (GLP), Good Manufacturing Practices (GMP)).  
The investigational site will provide direct access to all trial related sites, source 
data/documents, and reports for the purpose of monitoring and auditing by the sponsor, and 
inspection by local and regulatory authorities. 
10.8  DATA HANDLING AND RECORD KEEPING 
10.8.1  Data Collection and Management Responsibilities 
Data collection is the responsibility of the clinical trial staff at the site under the supervision 
of the site investigator. The investigator is responsible for ensuring the accuracy, completeness, 
legibility, and timeliness of the data reported. 
All source documents should be completed in a neat, legible manner to ensure accurate 
interpretation of data.   
Hardcopies of the study visit worksheets will be provided for use as source document 
worksheets for recording data for each participant enrolled in the study.  Data recorded in the 
electronic case report form (eCRF) derived from source documents should be consistent with the 
data recorded on the source documents.  
Clinical data (including demographics information, clinical rating scales, and AEs) and 
clinical laboratory data will be recorded in paper form or entered into CTDB, a 21 CFR Part 11-
compliant data capture system provided by the NICHD.  The data system includes password 
protection and internal quality checks, such as automatic range checks, to identify data that appear 
inconsistent, incomplete, or inaccurate. Clinical data will be entered directly or from the source 
documents.  
Electronic data (including imaging data) and electronic personally-identifiable health 
information (ePHI) will be stored on secure servers within the NIH firewall with access for study 
personnel only. 
10.8.2  Study Records Retention 
Study documents should be retained for a minimum of 2 years after the last approval of a 
marketing application in an International Conference on Harmonisation (ICH) region and until 
there are no pending or contemplated marketing applications in an ICH region or until at least 2 
years have elapsed since the formal discontinuation of clinical development of the study 
intervention, and as per the NIH Intramural Records Retention Schedule. No records will be 
destroyed without the written consent of the sponsor, if applicable. It is the responsibility of the 
sponsor to inform the investigator when these documents no longer need to be retained. 
10.9  PROTOCOL DEVIATIONS 
It is the responsibility of the investigator to use continuous vigilance to identify and report 
deviations to the NIH Institutional Review Board as per Policy 801.  All deviations must be 
addressed in study source documents, reported to the NIMH Program Official and Office of the 
Clinical Director.  The investigator is responsible for knowing and adhering to the reviewing IRB 
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10.9.1  NIH Definition of Protocol Deviation 
A protocol deviation is any changed, divergence, or departure from the IRB-approved 
research protocol.  
• Major deviations: Deviations from the IRB approved protocol that have, or may have the 
potential to, negatively impact the rights, welfare or safety of the subject, or to substantially 
negatively impact the scientific integrity or validity of the study. 
• Minor deviations: Deviations that do not have the potential to negatively impact the rights, 
safety or welfare of subjects or others, or the scientific integrity or validity of the study. 
10.10  PUBLICATION AND DATA SHARING POLICY 
10.10.1  Human Data Sharing Plan 
This study will be conducted in accordance with the following publication and data sharing 
policies and regulations: 
National Institutes of Health (NIH) Public Access Policy, which ensures that the public has 
access to the published results of NIH funded research. It requires scientists to submit final peer-
reviewed journal manuscripts that arise from NIH funds to the digital archive PubMed Central 
upon acceptance for publication. 
This study will comply with the NIH Data Sharing Policy and Policy on the Dissemination of 
NIH-Funded Clinical Trial Information and the Clinical Trials Registration and Results 
Information Submission rule. As such, this trial will be registered at ClinicalTrials.gov, and results 
information from this trial will be submitted to ClinicalTrials.gov. In addition, every attempt will 
be made to publish results in peer-reviewed journals.  Data from this study may be requested from 
other researchers 6 years after the completion of the primary endpoint by contacting the ETPB. 
10.10.2  Genomic Data Sharing Plan 
This study will comply with the NIH Genomic Data Sharing Policy, which applies to all NIH-
funded research that generates large-scale human or non-human genomic data, as well as the use 
of these data for subsequent research. Large-scale data include genome-wide association studies 
(GWAS), single nucleotide polymorphisms (SNP) arrays, and genome sequence, transcriptomic, 
epigenomic, and gene expression data. 
10.11  COLLABORATIVE AGREEMENTS  
10.11.1  Agreement Type 
A CRADA was established between the NIMH and Taisho Pharmaceutical Co., Ltd.  Taisho 
Pharmaceutical Co., Ltd. will manufacture the study drug and placebo for this protocol. NIMH 
will be responsible for obtaining the IND, subject recruitment, clinical care, conduct of the research 
study and procedures, study monitoring, analyzing and writing, and publishing study results. 
10.12  CONFLICT OF INTEREST POLICY 
The independence of this study from any actual or perceived influence, such as by the 
pharmaceutical industry, is critical.  Therefore, any actual conflict of interest of persons who have 
a role in the design, conduct, analysis, publication, or any aspect of this trial will be disclosed and 


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managed. Furthermore, persons who have a perceived conflict of interest will be required to have 
such conflicts managed in a way that is appropriate to their participation in the design and conduct 
of this trial.  The study leadership in conjunction with the NIMH has established policies and 
procedures for all study group members to disclose all conflicts of interest and will establish a 
mechanism for the management of all reported dualities of interest. 
 
11  ABBREVIATIONS 
 
AMPA 
α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid 
ANCOVA 
Analysis of Covariance 
ATHF 
Antidepressant Treatment History Form 
BBB 
Blood Brain Barrier  
BCRP 
Breast Cancer Resistance Protein 
BDNF 
Brain Derived Neurotrophic Factor 
BLS 
Basic Life Support 
BMI 
Body Mass Index 
BOLD 
Blood Oxygenation Level Dependent 
BPRS 
Brief Psychiatric Rating Scale  
CADSS 
Clinician-Administered Dissociative States Scale 
CAMKIII 
Calcium-Calmodulin Dependent Protein Kinase III 
CBC 
Complete Blood Count 
CES 
Carboxylesterase  
CLIA 
Clinical Laboratory Improvements Amendments  
Cmax 
Maximum plasma concentration 
CMP 
Clinical Monitoring Plan 
COC 
Certificate of Confidentiality 
COVID-19 
Coronavirus Disease 2019 
COWAT 
Controlled Oral Word Association Test 
CPT 3 
Conners Continuous Performance Test 3rd Edition 
CRA 
Clinical Research Advocate 
CRP 
C-Reactive Protein  
CSF 
Cerebrospinal Fluid 
C-SSRS 
Columbia-Suicide Severity Rating Scale 
CTDB 
Clinical Trials Database 
CVLT-II 
California Verbal Learning Test - Second Edition 
CYP 
Cytochrome P-450 


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dbGap 
database of Genotypes and Phenotypes 
DSST 
Digit Symbol Substitution Test 
D-KEFS 
Delis-Kaplan Executive Function System 
DRTS 
Designation of Reimbursement for Travel and Subsistence 
ECG 
Electrocardiogram 
ECT 
Electroconvulsive therapy 
eEF2K 
eukaryotic Elongation Factor-2 Kinase 
ePHI 
electronic Personally-Identifiable Health Information  
FDA 
Food and Drug Administration 
fMRI 
functional Magnetic Resonance Imaging 
FST 
Forced Swim Test 
GDNF 
Glial-Derived Neurotrophic Factor  
HAM-A 
Hamilton Anxiety Rating Scale 
HCG 
Human Chorionic Gonadotropin 
HDRS 
Hamilton Depression Rating Scale 
HIV 
Human Immunodeficiency Virus 
HNK 
Hydroxynorketamine 
HSPU 
Human Subjects Protection Unit 
HVLT-R 
Hopkins Verbal Learning Test - Revised 
IDMRS 
Investigational Drug Management and Research Section 
IEC 
International Electrotechnical Commission 
IFN 
Interferon 
IGF-1 
Insulin-like Growth Factor-1 
IL 
Interleukin 
ISM 
Independent Safety Monitor 
ISO 
International Organization for Standardization 
ITT 
Intent-To-Treat 
LPS 
Lipopolysaccharide  
MAD 
Multiple Ascending Dose 
MADRS 
Montgomery-Asberg Depression Rating Scale  
MAOI 
Monoamine Oxidase Inhibitor  
MDD 
Major Depressive Disorder 
MDEs 
Major Depressive Episodes 
MEG 
Magnetoencephalography  
mGlu 
metabotropic Glutamate 


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mPFC 
medial Prefrontal Cortex  
mRNA 
Messenger Ribonucleic Acid 
MRS 
Magnetic Resonance Spectroscopy 
mTORC1 
mammalian Target of Rapamycin  
NIH 
National Institutes of Health  
NMDAR 
N-Methyl-D-Aspartate Receptor 
NO 
Nitric Oxide  
NOAEL 
No Observed Adverse Effect Level 
NSR 
Non-Significant Risk 
OAT3 
Organic Anion Transporter 3 
OATP1B1 
Organic Anion Transporting Polypeptide 1B1 
OHRP 
Office for Human Research Protections 
PANAS 
Positive and Negative Affect Schedule 
PBMC 
Peripheral Blood Mononuclear Cell 
PCR 
Polymerase Chain Reaction 
PFC 
Prefrontal Cortex 
PI 
Principal Investigator 
PII 
Personally Identifiable Information 
POC 
Proof-of-Concept 
PRN 
Pro Re Nata 
RAVLT 
Rey Auditory Verbal Learning Test  
RDoC 
Research Domain Criteria  
RF 
Radio Frequency 
RH 
Relative Humidity 
SAD 
Single-Ascending Dose 
SCID-P 
Structured Diagnostic Interview-Patient Version 
SHAPS 
Snaith Hamilton Pleasure Scale 
SNP 
Single Nucleotide Polymorphism 
SOC 
System Organ Class 
SOP 
Standard Operating Procedure 
SSI 
Scale for Suicidal Ideation 
SWA 
Slow-Wave sleep Activity 
TEPS 
Temporal Experience of Pleasure Scale 
TMS 
Transcranial Magnetic Stimulation  
TRD 
Treatment Resistant Depression 


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TrkB 
Tropomyosin-Related Kinase B 
TS-161 
TP0473292 
TSH 
Thyroid Stimulating Hormone 
TTX 
Tetradotoxin 
VEGF 
Vascular Endothelial Growth Factor 
YMRS 
Young Mania Rating Scale 
  
 


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13  APPENDICES 
13.1  APPENDIX A. FIVE QUESTION CONSENT QUIZ 
 
TS-161 is not an FDA-approved for major depressive disorder (MDD). 
True 
False 
You will receive a placebo during this study.  
True 
False 
If you are able to get pregnant, you will have a pregnancy test before taking 
TS-161.  There may be birth defects with TS-161.  
True 
False 
We will look at chemicals in the brain and how they respond to TS-161.  
True 
False 
You may not get any benefit from participating in this study.  
True 
False 
Note: Correct responses are highlighted for this document 
 
 
 


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13.2  APPENDIX B. DRUGS ALLOWED AND NOT ALLOWED 
13.2.1  Medications Allowed (Y) and Not Allowed (N) as Concomitant Medications 
Drug Name or Class 
Episodic 
Use 
(PRN) 
Chronic 
Use 
Exclusion: 
Safety or 
Study 
Confound 
Restrictions/Comments 
Analgesics 
Y 
N 
— 
Narcotic analgesics or long-acting 
nonsteroidal anti-inflammatory 
drugs are not allowed  
Anesthetics, general 
N 
N 
Safety 
Anorexics 
N 
N 
Confound — 
Antacids 
Y 
N 
— 
— 
Antiacne 
N 
N 
— 
Antianginal agents 
N 
N 
Safety 
— 
Antiarrhythmics 
N 
N 
Safety 
— 
Antiasthma agents 
N 
N 
— 
— 
Antibiotics 
Y 
N 
Confound —  
Anticoagulants 
N 
N 
Safety 
Warfarin (Coumadin) and 
antiplatelet agents are not allowed  
Anticholinergics 
N 
N 
Confound 
Anticonvulsants 
N 
N 
Confound — 
Antidepressants 
N 
N 
Confound — 
Antidiarrheal preparations 
Y 
N 
Safety 
Only loperamide HCl (Imodium), 
bismuth subsalicylate (Pepto-
Bismol), and kaolin preparations 
are allowed 
Antifungals, systemic 
N 
N 
Safety 
— 
Antifungals, topical 
Y 
Y 
— 
— 


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Drug Name or Class 
Episodic 
Use 
(PRN) 
Chronic 
Use 
Exclusion: 
Safety or 
Study 
Confound 
Restrictions/Comments 
Antihistamines 
Y 
Y 
Confound 
Sedating antihistamines are not 
allowed. Only, loratadine (Claritin), 
desloratadine (Clarinex), and 
cetirizine (Zyrtec) are allowed for 
episodic or chronic use. The use of 
combinations containing 
pseudoephedrine or phenylephrine 
is not allowed. Combination 
products containing the word 
nighttime or some synonym 
routinely include a sedating 
antihistamine are not allowed.  
Antihypertensives 
N 
N 
Both 
Anti-impotence 
medications 
N 
N 
Safety 
— 
Anti-inflammatory drugs 
Y 
N 
Confound Long-acting nonsteroidal anti-
inflammatory drugs are not allowed 
Antimigraine 
N 
N 
Both 
Antinauseants/ Antiemetics 
Y 
N 
Confound 
Only phosphoric acid preparations 
(Emetrol, Emecheck), bismuth 
subsalicylate (Pepto-Bismol), and 
cola syrup are allowed. 
Antineoplastics/ 
Immunosuppressant agents 
N 
N 
Safety 
Use of chemotherapy agents or 
history of cancer, other than 
resolved skin cancer, within 5 years 
before the screening visit. 
Antiobesity/Appetite 
suppressants 
N 
N 
Confound 
OTC Alli (Xenical), sibutramine 
(Meridia), and phentermine; 
(Adipex-P and others) are not 
allowed. 
Antiplatelet agents 
N 
N 
Safety 
Antipsoriatic treatments 
N 
N 
— 
Only topical agents and acitretin; 
(Soriatane) is not allowed. 


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Drug Name or Class 
Episodic 
Use 
(PRN) 
Chronic 
Use 
Exclusion: 
Safety or 
Study 
Confound 
Restrictions/Comments 
Antipsychotics 
N 
N 
Confound — 
Antismoking medications 
N 
N 
Confound Varenicline (Chantix) is not 
allowed.  
Antiviral agents 
N 
N 
— 
Anxiolytics 
N 
N 
Confound — 
Benign prostatic 
hyperplasia treatments 
N 
N 
Safety 
Buspirone 
N 
N 
Confound — 
Cough/cold preparations 
Y 
N 
Confound 
Use of cough and cold preparations 
containing pseudoephedrine or 
phenylephrine are not allowed. Any 
sedative OTC (night) products are 
not allowed. Combination products 
containing the word nighttime or 
some synonym routinely include a 
sedating antihistamine and are not 
allowed.  
Diuretics 
N 
N 
— 
Dopaminergics 
N 
N 
Confound Dopamine agonists for restless leg 
syndrome are not allowed.  
Gastrointestinal: 
H2-blockers/ 
proton pump inhibitors/ 
prokinetic agents 
N 
N 
— 
Hormonal 
(noncontraceptive) 
therapies 
N 
Y 
— 
see below 
Hormone suppressants 
N 
N 
Safety 


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Drug Name or Class 
Episodic 
Use 
(PRN) 
Chronic 
Use 
Exclusion: 
Safety or 
Study 
Confound 
Restrictions/Comments 
Hormones: reproductive 
N 
Y 
Safety 
Systemic hormonal contraceptives 
(oral contraceptives of estrogen and 
progestin combinations, depot 
injections such as Depo-Provera, 
the contraceptive implant Implanon, 
or transdermally delivered 
contraceptives such as Ortho Evra) 
are allowed. 
Hormones: thyroid 
N 
Y 
Confound 
Thyroid hormone replacement is 
allowed (dosage of thyroid 
medication should be stable for 2 
months before screening). 
Therapeutic use for psychiatric 
disorders (e.g., T3 augmentation) is 
not allowed  
Hypoglycemic agents 
N 
N 
Safety 
Oral hypoglycemic agents or 
insulin are not allowed.  
Hypolipidemics 
N 
N 
Safety 
Hypolipidemics: 
bile acid sequestrants 
N 
N 
Confound — 
Hypolipidemics: fibrates 
N 
N 
Confound 
Hypolipidemics: niacin 
N 
N 
Confound 
Hypolipidemics: statins 
N 
N 
Safety 
Investigational drug 
N 
N 
Safety 
No investigation drug within 30 
days of starting the study. 
Laxatives 
Y 
N 
— 
Only fiber-based products and 
docusate sodium (Colace) are 
allowed. 
Lithium 
N 
N 
Confound — 
Muscle relaxants 
N 
N 
Confound — 


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Drug Name or Class 
Episodic 
Use 
(PRN) 
Chronic 
Use 
Exclusion: 
Safety or 
Study 
Confound 
Restrictions/Comments 
Psychotropic drugs not 
otherwise specified 
(including herbal products) 
N 
N 
Confound 
No drugs with psychomotor effects 
or with anxiolytic, stimulant, 
antipsychotic, or sedative properties 
are allowed. Herbal/dietary 
products and supplements with 
potential psychoactive actions 
including St. John’s wort, kava 
kava, SAMe, valerian root, DHEA, 
tyrosine, methylfolate. and 5-HTP 
are not allowed. 
Sedatives/hypnotics 
N 
N 
Confound — 
Steroids/systemic 
N 
N 
Confound 
Steroids/topical and 
inhalant 
N 
N 
— 
— 
Steroids/intra-articular 
Y 
NA 
— 
— 
Stimulants 
N 
N 
Confound 
Oral or transdermal 
methylphenidate, amphetamine 
products or prodrugs, 
pseudoephedrine, modafinil 
(Provigil), and other medications of 
same category are not allowed. 
Vaccines 
Y 
NA 
— 
— 
Elagolix 
N 
N 
Safety 
Elagolix, for endometriosis 
treatment, is not allowed. This 
compound is a substrates of a drug 
transporter (OATP1B1) which 
might be inhibited by TP0473292 
(prodrug).   
Eluxadoline 
N 
N 
Safety 
Eluxadoline, for irritable bowel 
syndrome with diarrhea treatment, 
is not allowed. This compound is a 
substrates of a drug transporter 
(OATP1B1) which might be 
inhibited by TP0473292 (prodrug).   


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Drug Name or Class 
Episodic 
Use 
(PRN) 
Chronic 
Use 
Exclusion: 
Safety or 
Study 
Confound 
Restrictions/Comments 
Pulmonary arterial 
hypertension treatment 
N 
N 
Safety 
Ambrisentan, bosentan, and 
selexipag are not allowed. These 
compounds are substrates of drug 
transporter (OATP1B1 and/or 
OATP1B3) which might be 
inhibited by TP0473292 (prodrug).   
 
5-HTP = 5-hydroxytryptophan; DHEA = dehydroepiandrosterone; N = no; NA = not 
applicable; OATP = organic anion transporting polypeptide; OTC = over the counter; PRN = as 
needed (pro re nata); SAMe = S-adenosylmethionine; T3 = triodothyronine; Y = yes. 
 
 
13.2.2  List of Psychiatric Medications Allowed and Not Allowed During the Study* 
Drug Class 
Episodic Use 
(as needed) 
Chronic 
Use 
Restrictions 
Antidepressants 
No 
No 
Treatment with fluoxetine or 
aripiprazole is an exclusion because 
of their long half-life. 
Antipsychotics 
No 
No 
  
Anxiolytics 
No 
No 
  
Mood Stabilizers 
No 
No 
 
Psychotropic drugs not 
otherwise specified 
(including herbal 
products) 
No 
No 
No drugs with psychomotor effects or 
with anxiolytic, stimulant, 
antipsychotic, or sedative properties 
are allowed. 
Sedatives/Hypnotics 
No 
No 
  
* Some medications in this table may be indicated for exclusionary conditions; therefore, it 
would be unlikely that participants meeting inclusion will be taking them. 
 
 
 


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13.3  APPENDIX C. PERIPHERAL (PLASMA/SERUM) MEASURES OF INTEREST AS BIOMARKERS 
IN MAJOR DEPRESSIVE DISORDERS 
13.3.1  General Background 
Our conceptualization of the pathophysiology of major depressive disorders has evolved 
significantly over the past decade from a simple chemical imbalance model to a model of impaired 
neural plasticity and resilience (Zarate, Singh et al. 2006). This new conceptual framework affords 
us the opportunity to specifically examine the clinical utility of novel drugs affecting 
neuroplasticity (Manji, Quiroz et al. 2003, Zarate, Du et al. 2003). Moreover, this conceptual 
approach 
facilitates 
exploration 
of 
drug 
effects 
and 
several 
potential 
predictors, 
moderators/mediators and the surrogate endpoint response to treatment. Here, we provide a brief 
discussion of the rationale and goals of our group’s concerted effort to identify biomarkers of TS-
161 antidepressant effects.  
13.3.2  Plasma/Serum Biomarkers: Neurotrophic Factors, Cell Cycle/Signal Transduction 
Regulators, Neuroinflammatory, Neuroendocrinological Measures, PBMC, 
Metabolomic and Proteomic Measures 
Neurotrophic Factors 
Animal studies clearly demonstrate that neurotrophins like brain-derived neurotrophic factor 
(BDNF), basic fibroblast growth factor (bFGF/FGF-2), vascular endothelial growth factor (VEGF), 
glial-derived neurotrophic factor (GDNF) and insulin-like growth factor (IGF-1) mediate some of 
the beneficial neuromodulatory effects of antidepressant medications.  
S-100B protein is highly specific for neural tissue, where it is predominantly synthesized and 
secreted by glial cells; yet, its role is not yet fully understood. It may have intracellular and 
extracellular neurotrophic as well as neurotoxic effects. Structural damage to glial cells causes 
leakage of S-100B protein into the extracellular milieu and CSF, which then enters the blood. 
Recently, serum S-100B protein has emerged as an attractive surrogate marker of CNS injury. It 
can be measured in arterial and venous serum, is not affected by hemolysis and remains stable for 
several hours (Beaudeux, Dequen et al. 1999, Beaudeux 2009). Several studies have demonstrated 
elevated levels of S-100B in the CSF (Grabe, Ahrens et al. 2001) and serum in MDD, especially 
melancholia (Rothermundt, Arolt et al. 2001). A recent meta-analysis of nine studies with 198 
depressed and 209 healthy subjects revealed very strong evidence of elevated S-100B levels in 
MDD (Cohen’s d=2.57, 95% CI: 1.87 to 3.27) (Schroeter, Abdul-Khaliq et al. 2008). S-100B 
levels are also positively correlated with the numbers of depressive episodes, family history of 
major depression and cognitive disruption (Yang, Xie et al. 2008), which can be normalized by 
effective antidepressant treatment (Schroeter, Abdul-Khaliq et al. 2002, Schroeter, Abdul-Khaliq 
et al. 2008). An association between S-100B levels and memory processes in patients with 
recurrent depression has been hypothesized, which suggests a neuroprotective role for moderately-
increased S-100B serum levels in the course of affective disorders (Zhang, Rothermundt et al. 
2009). In sum, although still preliminary, there is now mounting evidence to suggest serum S-
100B levels may serve as a useful biomarker in MDD. Considering its proposed function in 
regulating cell survival and its putative origin from damaged glial cells, it is a likely candidate 
surrogate endpoint of ketamine’s antidepressant response and thus a promising area of study with 
TS-161. 


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Samples will be collected at the timepoints listed in the Schedule of Events at the timepoints 
listed in the to minimize potential diurnal variability (Begliuomini, Lenzi et al. 2008) at baseline 
prior to randomization and at the timepoints listed in the Schedule of Events. This will afford us 
the opportunity to explore the temporal relationship between levels, drug effects, and clinical 
improvement. 
Objective 
To determine if there are baseline differences in peripheral neurotrophins in MDD/current 
MDE and whether changes in serum concentrations are correlated with neurophysiological 
parameters and the antidepressant effects and response to TS-161. 
13.3.3  Cell Cycle/Signal Transduction Regulators 
The importance of intracellular signal transduction cascades in mood disorders may be 
inferred from the activation of synaptic membrane receptors leading to antidepressant effects. 
Preclinical models of despair have demonstrated that the modulation of signal transduction 
pathways, e.g. activation of mammalian target of rapamycin (mTORC1) (Li, Lee et al. 2010) and 
release of inhibition on eukaryotic elongation factor-2 kinase (eEF2K)/calcium-calmodulin 
dependent protein kinase III (CAMKIII) (Autry, Adachi et al. 2011), is necessary (as inhibition 
abrogates antidepressant-like effects) and sufficient (as activation induces antidepressant-like 
effects) for ketamine’s rapid antidepressant effects. To date, there have been few clinical studies 
to analyze signal transduction mediators and their response to antidepressants. A significant 
reduction in mTORC1 and other downstream signal transduction mediators was observed in 
postmortem MDD PFC relative healthy control brains (Jernigan, Goswami et al. 2011). In a single 
treatment-resistant depressed subject, ketamine’s antidepressant time course correlated with 
increasing mTORC1 expression in peripheral leukocytes (Denk, Rewerts et al. 2011), but, to date, 
this response has yet to be replicated in a larger sample. 
Other cell cycle regulators have been implicated in the pathogenesis of depression, i.e. p11 
(Snyder 2011). p11 is a member of the S-100 family of proteins (discussed above) that interacts 
with both the 5-HT1B (Svenningsson, Chergui et al. 2006) and 5-HT4 (Warner-Schmidt, Flajolet et 
al. 2009) receptors. p11 mutant mice display despair-like phenotypes, and overexpressing p11 
mice are more resilient to stress than their wild-type littermates ((Svenningsson, Chergui et al. 
2006)). In clinical studies, p11 levels are decreased in depressed patients (including suicide 
completers) (Zhang, Su et al. 2011).    
13.3.4  Inflammatory Mediators 
Proinflammatory cytokines have also been implicated in the underlying etiopathogenesis of 
mood disorders.  TNFα,  IL-1, IL-6 administration (either centrally or peripherally) has been linked 
to “sickness behavior”, which mimics several characteristic signs and symptoms of major 
depression including anhedonia, decreased social exploration, psychomotor disturbances and 
decreased libido (Dantzer 2001, Dantzer 2001). A recent PET study in non-human primates with 
a ligand binding to activated microglia revealed lipopolysaccharide (LPS)-induced activation 
within hours, which is likely mediated by alterations in cytokine release (Hannestad, Gallezot et 
al. 2012). Likewise, peripheral cytokine levels are elevated in isolated and co-occurring depressive 
disorders (Penninx, Kritchevsky et al. 2003, Fitzgerald, O'Brien et al. 2006, Kahl, Bens et al. 2006, 
O'Brien, Scully et al. 2007), and proinflammatory cytokines decrease in response to effective 


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antidepressant therapy (Lanquillon, Krieg et al. 2000, Narita, Murata et al. 2006, O'Brien, Scully 
et al. 2007). Microglial NMDAR upregulation (as detected in vitro with the agonist quinolinic 
acid) was detected in several brain areas in MDD but not BD (Steiner, Walter et al. 2011). 
Similar neuroinflammatory effects have also been observed in preclinical models of 
depression. Repeated restraint stress in rodents induced microglial activation in the medial PFC, 
which could be reversed by tetracycline antibiotic and microglial inhibitor minocycline (Hinwood, 
Morandini et al. 2012). TNF receptor (TNFR1-/- and TNFR2-/-) knockout mice display increased 
resilience in the FST relative to their wild-type littermates (Simen, Duman et al. 2006). 
Additionally, TNFR1 knockout mice exhibited reduced fear conditioning while TNFR2 nulls 
displayed increased sucrose drinking after water deprivation stress; however, no difference was 
observed between wild-type and knockout littermates in anxiety-related tests, e.g. open field test 
and elevated plus maze (Simen, Duman et al. 2006). Treatment with kinin B1 receptor antagonists 
(an important modulator of TNFα secretion from activated microglia) reduced depressive-like 
behaviors in rodents (Viana, Maciel et al. 2010).   
Several studies have reported physiological effects of antidepressants on microglial cells. 
SSRIs and the SNRI venlafaxine are anti-inflammatory in vitro as demonstrated by decreased 
production of several pro-inflammatory cytokines from microglia (Liu, Wang et al. 2011, Tynan, 
Weidenhofer et al. 2012). Additionally, paroxetine and sertraline significantly inhibited the 
generation of nitric oxide (NO) and TNFα from interferon (IFN)-γ-activated microglia in an 
intracellular Ca2+-dependent manner. However, the monoamine oxidase inhibitor (MAOI) 
phenelzine increased the release of TNFα and IL-6 in a NF-kB-dependent manner in cultured 
microglia, which suggests differential effects among different antidepressant classes. It should be 
noted that the mGlu3 receptor on astrocytes mediates IL-6 secretion in the presence of IL-1β 
(Aronica, Gorter et al. 2005), while the mGlu2 receptor on microglia mediates TNFα secretion 
(Taylor, Jones et al. 2005). 
Several groups have investigated immunomodulatory therapy as treatment alternatives in 
MDD due to the retrospective antidepressant effects of novel immunomodulators in autoimmune 
disease. For example, 618 subjects with moderate-to-severe psoriasis were randomized to receive 
the TNFα monoclonal antibody etanercept or placebo. Although not the primary outcome of the 
study, etanercept treatment resulted in a ~50% reduction in depression scores; additionally, unlike 
fatigue, which was highly correlated with psoriatic symptomatology, the antidepressant effects did 
not strongly correlate with improvement in the dermatological and/or musculoskeletal 
signs/symptoms of psoriasis (Tyring, Gottlieb et al. 2006). Although a recent placebo controlled 
trial of the TNFα neutralizing antibody infliximab did not reveal global efficacy in TRD, subjects 
with a baseline level of the acute phase reactant C-reactive protein (CRP) >5 mg/L had a positive 
antidepressant response (Raison, Rutherford et al. 2012). Targets of other novel 
immunomodulators, e.g. IL-1 and IFN inhibitors, are also rational drug approaches in depressive 
disorders. 
Objective 
To assess baseline differences in cell cycle/signal transduction and inflammatory mediators 
in MDD/current MDE and whether changes in serum concentrations are correlated with 
neurophysiological responses of drug effects, and the antidepressant response to TS-161. 


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13.3.5  Neuroendocrinological Mediators 
There has been extensive research to date on putative neuroendocrinological measures in 
depressive disorders.  These findings, e.g. impaired dexamethasone suppression and aberrant 
thyroid functioning, have been observed in some but not all depressed patients, which may assist 
in identifying biologically relevant subtypes among the clinical heterogeneity of MDD. Many 
preclinical studies (with rodents and non-human primates) have implicated the hypothalamic-
pituitary-adrenal axis in stress-induced relapse and craving, and these findings are presently being 
translated into humans. Neuropeptides and stress-related hormones may be altered acutely and/or 
chronically in currently depressed subjects. As such, we will investigate stress-related 
neuropeptide and hormone levels at baseline and after TS-161 treatment.   
Objective 
To determine if there are baseline differences in neuroendocrinological markers including but 
not limited to cortisol, ACTH, corticotropin-releasing hormone in MDD/current MDE and whether 
changes in these markers are correlated with neurophysiological parameters during TS-161 and 
the antidepressant effects of TS-161. 
13.3.6  Peripheral Blood Mononuclear Cells (PBMCs)  
Objective 
To determine if there are baseline differences in PBMC (lymphocyte) NMDARs gene 
expression levels in MDD/current MDE and whether changes in PBMC are correlated with 
neurophysiological parameters during TS-161 administration and the antidepressant response to 
TS-161. Real-time PCR will be used to investigate the mRNA expression level of 
NMDAR/AMPA subunits in peripheral blood lymphocytes. Metabolomic and Proteomic 
Measures 
Objective 
The objective of this part of study is to determine whether there are specific metabolomic 
signatures in patients with MDD receiving TS-161 and whether these signatures or changes in 
these signatures with treatment are correlated with antidepressant change scores following 
treatment within different subgroups. 
13.3.7  Transcriptional Profiling 
Objective 
Gene expression changes in neuropsychiatric and neurodegenerative disorders, and gene 
responses to therapeutic drugs, provide new ways to identify CNS targets for drug discovery. In 
the absence of a deeper understanding of disease pathology and mechanisms of side effects, CNS 
drug discovery would remain dominated by the redesign of drugs for familiar targets and reduced 
approval rates for ‘me too’ drugs. The ability to evaluate changes in the expression of the entire 
genome in brain areas affected by CNS disease and drug effects on multiple pathways is an 
alternative, ‘bottom–up’ approach to drug discovery (Altar, Vawter et al. 2009). 
 
 
 


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13.4  APPENDIX D: CONTRACEPTIVE GUIDANCE AND COLLECTION OF PREGNANCY 
INFORMATION 
Contraceptive use by men or women should be consistent with local regulations regarding the 
methods of contraception for those participating in clinical studies. In this section, the terms 
“male”, “female”, or “woman” refer to biological sex. 
13.4.1  Male participants 
Male participants are eligible to participate if they agree to the following during the 
intervention period and for at least 90 days after the last dose of study intervention: 
• Refrain from donating sperm 
• PLUS either a) OR b): 
a.  Be abstinent from heterosexual intercourse as their preferred and usual lifestyle 
(abstinent on a long term and persistent basis) and agree to remain abstinent; 
b.  Must agree to use contraception/barrier as detailed below 
• Agree to use a male condom and female partner use of an additional highly 
effective contraceptive method with a failure rate of <1% per year as described 
in this appendix, when having sexual intercourse with a woman of childbearing 
potential (WOCBP) who is not currently pregnant 
• Agree to use male condom when engaging in any activity that allows for passage 
of ejaculate to another person  
13.4.2  Female participants 
A female participant is eligible to participate if they are not pregnant or breastfeeding, and at 
least 
• One of the following conditions, c) or d) applies: 
a.  Is not a WOCBP  
b.  Is a WOCBP and using a contraceptive method that is highly effective (with a failure 
rate of <1% per year), preferably with low user dependency, as described in this 
appendix, during the trial and for at least 30 days after the last dose of study intervention. 
The investigator should evaluate the effectiveness of the contraceptive method in 
relationship to the first dose of study intervention. 
• A WOCBP must have a negative serum pregnancy test on Screening Visit and Day -1. 
• The investigator is responsible for review of medical history, menstrual history, and 
recent sexual activity to decrease the risk for inclusion of a woman with an early 
undetected pregnancy 
WOCBP 
A woman is considered fertile following menarche and until becoming post-menopausal 
unless permanently sterile (see below). 
If fertility is unclear (e.g., amenorrhea in adolescents or athletes) and a menstrual cycle cannot 
be confirmed before first dose of study intervention, additional evaluation should be considered. 


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Women in the following categories are not considered WOCBP 
• Premenarchal 
• Premenopausal female with one of the following: 
• Documented hysterectomy 
• Documented bilateral salpingectomy 
• Documented bilateral oophorectomy 
For individuals with permanent infertility due to an alternate medical cause other than the 
above, (e.g., Mullerian agenesis, androgen insensitivity), investigator discretion should be applied 
to determining study entry. 
Note: Documentation can come from the site personnel’s: review of the participant’s medical 
records, medical examination, or medical history interview. 
 
• Postmenopausal female 
• A postmenopausal state is defined as no menses for 12 months without an alternative 
medical cause (A high follicle stimulating hormone [FSH] level in the 
postmenopausal range may be used to confirm a postmenopausal state in women not, 
using hormonal contraception or hormonal replacement therapy [HRT]). 
• Females on HRT and whose menopausal status is in doubt will be required to use 
one of the non-estrogen hormonal highly effective contraception methods if they 
wish to continue their HRT during the study. Otherwise, they must discontinue HRT 
to allow confirmation of postmenopausal status before study enrollment. 
13.4.3  Contraception Guidance 
Contraception for Female (WOCBP) Participants 
Female participants of child-bearing potential must agree to use a contraceptive method that 
is highly effective, preferably with low user dependency, as listed below during the trial and at 
least 30 days following the last dose of TS-161. 
Contraceptives1 Allowed During the Study Include: 
Highly Effective Methods2 That Have Low User Dependency 
Failure rate of <1% per year when used consistently and correctly. 
• Implantable progestogen-only hormone contraception associated with inhibition of 
ovulation3 
• Intrauterine device (IUD) 
 
1 Contraceptive use by men or women should be consistent with local regulations regarding the use of contraceptive 
methods for those participating in clinical studies. 
2 Failure rate of <1% per year when used consistently and correctly. Typical use failure rates differ from those when 
used consistently and correctly. 
3 If locally required, in accordance with Clinical Trial Facilitation Group (CTFG) guidelines, acceptable contraceptive 
methods are limited to those which inhibit ovulation as the primary mode of action. 


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• Intrauterine hormone-releasing system (IUS)3 
• Bilateral tubal occlusion 
• Vasectomized partner 
• Vasectomized partner is a highly effective contraceptive method provided that 
the partner is the sole sexual partner of the WOCBP and the absence of sperm has 
been confirmed. If not, an additional highly effective method of contraception 
should be used. Spermatogenesis cycle is approximately 90 days. 
Highly Effective Methods3 That Are User Dependent Failure rate of <1% per year when 
used consistently and correctly.  
• Combined (estrogen- and progestogen-containing) hormonal contraception associated with 
inhibition of ovulation3 
• Oral 
• Intravaginal 
• Transdermal 
• injectable 
• Progestogen-only hormone contraception associated with inhibition of ovulation3 
• Oral 
• Injectable 
• Sexual abstinence 
Sexual abstinence is considered a highly effective method only if defined as refraining 
from heterosexual intercourse during the entire period of risk associated with the study 
intervention. The reliability of sexual abstinence needs to be evaluated in relation to the 
duration of the study and the preferred and usual lifestyle of the participant. 
Note: Periodic abstinence (calendar, symptothermal, post-ovulation methods), 
withdrawal (coitus interruptus), spermicides only, and lactational amenorrhoea method 
(LAM) are not acceptable methods of contraception. Male condom and female condom 
should not be used together (due to risk of failure with friction) 
Contraception for Male Participants 
Male participants with a partner of child-bearing potential must agree to use following 
methods of contraception listed below from the initial administration of study intervention until at 
least 90 days following the last dose of TS-161. 
Contraceptives Allowed During the Study Include: 
• Condom for male participant AND his partner of child-bearing potential must use one of 
the methods of contraception for female (WOCBP) participants (listed above) 
• Sexual abstinence 
Sexual abstinence is considered a highly effective method only if defined as refraining 
from heterosexual intercourse during the entire period of risk associated with the study 


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intervention. The reliability of sexual abstinence needs to be evaluated in relation to the 
duration of the study and the preferred and usual lifestyle of the participant. 
 
Note: Male participants should refrain from fathering a child or donating sperm from the 
initial administration of study intervention until at least 90 days following the last dose of TS-161. 
13.4.4  Collection of Pregnancy Information 
Male participants with partners who become pregnant 
• The investigator will attempt to collect pregnancy information on any male participant’s 
female partner who becomes pregnant while the male participant is in this study or prior to 
90 days following his last dose of study intervention. This applies only to male participants 
who receive TS-161 (excluding placebo). 
• After obtaining the necessary signed informed consent from the pregnant female partner 
directly, the investigator will record pregnancy information on the appropriate form and 
submit it to the sponsor within 24 hours of learning of the partner’s pregnancy. Pregnancy 
of the participant’s partner is not considered to be an AE, however, the female partner will 
be followed to determine the outcome of the pregnancy. Information on the status of the 
mother and child will be forwarded to the sponsor. Generally, the follow-up will be no 
longer than 6 to 8 weeks following the estimated delivery date. Any termination of the 
pregnancy will be reported regardless of fetal status (presence or absence of anomalies) or 
indication for the procedure. Any congenital anomaly must be reported as an SAE. 
Female participants who become pregnant 
• The investigator will collect pregnancy information on any female participant who 
becomes pregnant while participating in this study. Information will be recorded on the 
appropriate form and submitted to the sponsor within 24 hours of learning of a participant's 
pregnancy. 
• The participant will be followed to determine the outcome of the pregnancy. The 
investigator will collect follow-up information on the participant and the neonate and the 
information will be forwarded to the sponsor. Generally, follow-up will not be required for 
longer than 6 to 8 weeks beyond the estimated delivery date. Any termination of pregnancy 
will be reported, regardless of fetal status (presence or absence of anomalies) or indication 
for the procedure. 
• While pregnancy itself is not considered to be an AE or SAE, any pregnancy complication 
or elective termination of a pregnancy will be reported as an AE or SAE. A spontaneous 
abortion or congenital anomaly is always considered to be an SAE and will be reported as 
such. Any post-study pregnancy related SAE considered reasonably related to the study 
intervention by the investigator will be reported to the sponsor as described in this appendix. 
While the investigator is not obligated to actively seek this information in former study 
participants, they may learn of an SAE through spontaneous reporting. 
• Any female participant who becomes pregnant while participating in the study will 
discontinue study intervention or be withdrawn from the study.