Assessing Variability of the Ventilatory Response to Duffin's Rebreathing Procedure
Phase: NA
Conditions: Ventilation, Reproducibility, Physiology
Sponsor: Food and Drug Administration (FDA)

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 1 of 46 
  
 
CLINICAL STUDY PROTOCOL 
 Assessing Variability of the Ventilatory Response to Duffin’s 
Rebreathing Procedure 
 
PROTOCOL NO. SCR-013 Lead-in 
 
Sponsor: 
U.S. Food and Drug Administration 
White Oak Building #64, Room 2072 
10903 New Hampshire Avenue 
Silver Spring, MD 20993 
 
Sponsor Study Lead: 
David Strauss, MD, PhD 
Director, Division of Applied Regulatory Science  
U.S. Food and Drug Administration 
Telephone: 301-796-6323 
Email: david.strauss@fda.hhs.gov 
 
 
Sponsor Medical 
Monitor: 
Keith Burkhart, MD 
U.S. Food and Drug Administration 
301-796-2226 
keith.burkhart@fda.hhs.gov 
 
 
Project Manager: 
Jeffry Florian, PhD  
U.S. Food and Drug Administration  
Telephone: 301-796-4847  
Email: jeffry.florian @fda.hhs.gov 
 
 
Version of Protocol: 
1.0 
 
Date of Protocol: 
27th October, 2021 
 
CONFIDENTIAL 
The concepts and information contained in this document or generated during the study 
are considered proprietary and may not be disclosed in whole or in part without the 
expressed written consent of the U.S. Food and Drug Administration. 
 
 
 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 2 of 46 
  
 
SPONSOR SIGNATURE PAGE 
This study will be conducted with the highest respect for the individual participants in 
accordance with the requirements of this clinical study protocol and also in accordance 
with the following: 
 The ethical principles that have their origin in the Declaration of Helsinki; 
 International Council for Harmonisation (ICH) harmonised tripartite guideline E6 
(R2): Good Clinical Practice; and 
 All applicable laws and regulations, including without limitation, data privacy laws 
and compliance with appropriate regulations, including human subject research 
requirements set forth by the Institutional Review Board (IRB). 
 
 
 
 
David Strauss, MD, PhD 
Director, Division of Applied 
Regulatory Science  
U.S. Food and Drug Administration 
 
Date 
 
 
 
 
10/28/2021

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 3 of 46 
  
 
INVESTIGATOR SIGNATURE PAGE 
I confirm that I have read and that I understand this protocol, the investigator brochure, 
and other product information provided by the sponsor. I agree to conduct this study in 
accordance with the requirements of this protocol and also protect the rights, safety, 
privacy, and well-being of study subjects in accordance with the following: 
 The ethical principles that have their origin in the Declaration of Helsinki; 
 ICH harmonised tripartite guideline E6 (R2): Good Clinical Practice; 
 All applicable laws and regulations, including without limitation data privacy laws 
and regulations; 
 Human subject research requirements set forth by the IRB; 
 Regulatory requirements for reporting of serious adverse events (SAEs) defined in 
Section 4.7.3.1 of this protocol; and 
 Terms outlined in the Clinical Study Site Agreement. 
I further authorize that my personal information may be processed and transferred in 
accordance with the uses contemplated in Section 6 of this protocol. 
Jan Matousek, D.O. 
Principal Investigator 
 
Date 
 
 
10/28/2021

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 4 of 46 
  
 
TABLE OF CONTENTS 
LIST OF ABBREVIATIONS .......................................................................................................................... 7 
PROTOCOL SYNOPSIS ............................................................................................................................... 8 
1. INTRODUCTION .................................................................................................................................. 15 
2. STUDY OBJECTIVES ............................................................................................................................. 16 
2.1 PRIMARY OBJECTIVE ................................................................................................................................ 16 
3. STUDY ENDPOINTS ............................................................................................................................. 16 
3.1 PRIMARY ENDPOINTS ............................................................................................................................... 16 
3.2 EXPLORATORY ENDPOINTS ........................................................................................................................ 16 
4. INVESTIGATIONAL PLAN ..................................................................................................................... 16 
4.1 STUDY DESIGN ....................................................................................................................................... 16 
4.1.1 Risk/Benefit ................................................................................................................................ 17 
4.2 SELECTION OF STUDY POPULATION ............................................................................................................. 19 
4.2.1 Inclusion Criteria ........................................................................................................................ 19 
4.2.2 Exclusion Criteria ........................................................................................................................ 20 
4.3 SCREENING FAILURES ............................................................................................................................... 21 
4.4 TERMINATION OF STUDY OR INVESTIGATIONAL SITE ....................................................................................... 21 
4.4.1 Criteria for Termination of the Study ......................................................................................... 21 
4.4.2 Criteria for Termination of the Investigational Site .................................................................... 21 
4.5 CRITERIA FOR SUBJECT WITHDRAWAL ......................................................................................................... 22 
4.5.1 Handling of Withdrawals ........................................................................................................... 23 
4.5.2 Replacement of Subjects ............................................................................................................ 23 
4.6 STUDY VISITS ......................................................................................................................................... 23 
4.6.1 Recruitment ................................................................................................................................ 23 
4.6.2 Compensation ............................................................................................................................ 23 
4.6.3 Screening .................................................................................................................................... 24 
4.6.4 Study Periods .............................................................................................................................. 25 
4.6.5 Discharge (or Early Termination) ............................................................................................... 26 
4.7 STUDY PROCEDURES ................................................................................................................................ 27 
4.7.1 Duffin’s Rebreathing Procedure ................................................................................................. 27 
4.7.2 Pupillary Assessments ................................................................................................................ 28 
4.7.3 Safety Assessments .................................................................................................................... 29 
4.7.4 Demographics and Medical History ........................................................................................... 32 
4.7.5 Prior and Concomitant Medications .......................................................................................... 32 
4.7.6 Subject Restrictions .................................................................................................................... 32 
4.8 STATISTICAL METHODS ............................................................................................................................ 34 
4.8.1 Sample Size ................................................................................................................................ 34 
4.8.2 Analysis Populations .................................................................................................................. 34 
4.8.3 General Statistical Considerations ............................................................................................. 34 
4.8.4 Subject Disposition ..................................................................................................................... 34 
4.8.5 Demographics and Baseline Characteristics .............................................................................. 34 
4.8.6 Primary Analysis ......................................................................................................................... 34 
4.8.7 Additional Analyses .................................................................................................................... 35 
4.8.8 Safety Analyses .......................................................................................................................... 35 
4.8.9 Interim Analyses ......................................................................................................................... 36 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 5 of 46 
  
 
4.8.10 Missing Data ............................................................................................................................ 36 
4.9 DATA QUALITY ASSURANCE ...................................................................................................................... 36 
4.10 DATA SHARING ..................................................................................................................................... 37 
5. ETHICAL CONSIDERATIONS................................................................................................................. 37 
5.1 ETHICAL CONDUCT OF THE STUDY .............................................................................................................. 37 
5.2 INSTITUTIONAL REVIEW BOARD (IRB) ......................................................................................................... 37 
6. ADMINISTRATIVE PROCEDURES ......................................................................................................... 38 
6.1 RESPONSIBILITIES OF THE INVESTIGATOR ...................................................................................................... 38 
6.1.1 Form FDA 1572 ........................................................................................................................... 38 
6.1.2 Adherence to Protocol ................................................................................................................ 38 
6.1.3 Reporting Requirements ............................................................................................................ 38 
6.1.4 Source Documentation ............................................................................................................... 38 
6.1.5 Retention of Records .................................................................................................................. 38 
6.1.6 Financial Disclosure and Obligations ......................................................................................... 39 
6.2 CONFIDENTIALITY AND DISCLOSURE OF DATA ............................................................................................... 39 
6.3 SUBJECT CONSENT .................................................................................................................................. 40 
6.4 DATA COLLECTION .................................................................................................................................. 40 
6.5 PUBLICATIONS ........................................................................................................................................ 40 
7. STUDY MANAGEMENT ....................................................................................................................... 41 
7.1 MONITORING ......................................................................................................................................... 41 
7.2 MANAGEMENT OF PROTOCOL AMENDMENTS AND DEVIATIONS ....................................................................... 41 
7.2.1 Modification of the Protocol ...................................................................................................... 42 
7.2.2 Protocol Violations and Deviations ............................................................................................ 42 
8. REFERENCE LIST .................................................................................................................................. 43 
9. APPENDICES ....................................................................................................................................... 45 
9.1 APPENDIX A – SCHEDULE OF EVENTS .......................................................................................................... 45 
9.2 APPENDIX B – PROTOCOL REVISION HISTORY ............................................................................................... 46 
 
 
 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 6 of 46 
  
 
LIST OF TABLES 
TABLE 4-1: STUDY SCHEDULE .............................................................................................................................. 16 
TABLE 4-3: CLINICAL LABORATORY TESTS & DIAGNOSTIC SCREENING TESTS ................................................................. 31 
TABLE 9-1: SCHEDULE OF EVENTS ........................................................................................................................ 45 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 7 of 46 
  
 
LIST OF ABBREVIATIONS 
Abbreviation 
Definition 
AE 
adverse event 
CFR 
Code of Federal Regulations 
COVID-19 
coronavirus disease of 2019 
ECG 
electrocardiogram 
eCRF 
electronic case report form 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
HBsAg 
hepatitis B surface antigen 
HepC 
hepatitis C  
HIPAA 
Health Insurance Portability and Accountability Act 
HIV 
human immunodeficiency virus 
ICH 
International Council for Harmonisation 
IRB 
Institutional Review Board 
IV 
intravenous 
MedDRA 
Medical Dictionary for Regulatory Activities 
NSAID 
nonsteroidal anti-inflammatory drug 
OTC 
over-the-counter 
QA 
Rebreathing procedure 
quality assurance 
Refers to the method consisting of three stages: relaxation, 
hyperventilation, and rebreathing assessment 
Rebreathing assessment 
Refers to a single assessment at an isoxic end tidal PO2 level 
after hyperventilation 
SAE 
serious adverse event 
SARS-CoV-2 
SD 
severe acute respiratory syndrome coronavirus 2 
standard deviation 
 
 
 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 8 of 46 
  
 
PROTOCOL SYNOPSIS 
Protocol Number: 
SCR-013 (Lead-in) 
Title: 
Assessing Variability of the Ventilatory Response to Duffin’s 
Rebreathing Procedure 
Investigators: 
Principal Investigator: Jan Matousek 
Study Physician: Jan Matousek 
Study Phase: 
Phase 1  
Study Period: 
The duration of study participation will be approximately 3 days 
(excluding the screening period). 
Study Site: 
Spaulding Clinical Research Unit, West Bend, Wisconsin 
Background and 
Motivation: 
In order to study the effects of opioids alone or in combination with 
other sedative psychotropic drugs on ventilation, we previously 
implemented the Read rebreathing procedure.1 With Read rebreathing, 
a study participant rebreathes through a circuit with a bag that initially 
contains 93% oxygen (O2) and 7% carbon dioxide (CO2). The 7% CO2 
results in relatively rapid equilibration between arterial partial pressure 
of carbon dioxide (PCO2), end tidal PCO2, and mixed venous PCO2. As 
the study participant continues to breathe, CO2 accumulates and 
triggers an increase in ventilation referred to as the "hypercapnic 
ventilatory response." Drugs can decrease the hypercapnic ventilatory 
response at drug concentrations that may have minimal-to-no 
detectable effect on ventilation when breathing room air.2-3 However, 
this method is limited in that all measurements are performed under 
hyperoxic conditions, which can have independent effects on 
ventilation, and the data cannot necessarily be used to extrapolate to the 
dynamic changes in PO2 and PCO2 that occur during real-world drug-
induced respiratory depression when a patient is breathing room air. 
Duffin modified the Read rebreathing procedure to include 1) 
hyperventilation (primarily through deep breathing) prior to rebreathing 
to achieve an end tidal PCO2 or approximately 20-25 mm Hg prior to 
rebreathing,4 2) maintaining an isoxic end tidal PO2 during 
rebreathing,5 and 3) repeating rebreathing at 2 different isoxic end tidal 
PO2 levels (i.e., at 50 mm Hg and 150 mm Hg).6 These modifications 
allow for critical physiological measurements and thresholds to be 
captured that can then be used to model the effects of drugs when there 
are dynamic changes in PO2 and PCO2, such as during a real-world 
opioid overdose. The Duffin rebreathing procedure has been used for 
multiple decades by different investigators studying different 
populations with and without drugs that can affect ventilation.1-14     

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 9 of 46 
  
 
Baseline minute ventilation is captured when end tidal PCO2 is less 
than the ventilatory recruitment threshold. The ventilatory recruitment 
threshold is the end tidal PCO2 above which minute ventilation starts to 
increase linearly with further increases in end tidal PCO2. The slope of 
the PCO2-ventilatory response curve reflects the increase in minute 
ventilation relative to the increase in end tidal PCO2. Changing the PO2 
can affect each of these variables, with hypoxia increasing baseline 
minute ventilation, decreasing the ventilatory recruitment threshold, 
and increasing the slope of the PCO2-ventilatory response curve, while 
hyperoxia has the opposite effects.  
The reproducibility of Duffin rebreathing has been assessed previously 
by Mahamed and Duffin (2001)7 performing hyperoxic and hypoxic 
rebreathing procedures measured once daily for 14 consecutive days 
and then by Jensen et al. (2010)8 performing 4 pairs of hyperoxic and 
hypoxic rebreathing procedures in 1 day followed by 1 pair on 4 
additional days separated by weeks to more than a month. The present 
study will combine Duffin’s rebreathing procedure with some 
additional study procedures, such as quantitative pupillometry, that are 
planned for subsequent clinical pharmacology studies in order to 
confirm feasibility and gather reproducibility data. 
Objectives and 
Endpoints: 
Objective: 
The objective of this study is to evaluate variability of the ventilatory 
response to Duffin’s rebreathing procedure.  
The endpoints of this study are below and will be determined for each 
rebreathing procedure: 
Primary Endpoints 
1. Baseline minute ventilation when end tidal PCO2 is less than the 
ventilatory recruitment threshold (represents non-chemoreflex 
drives to breathe) 
2. Ventilatory recruitment threshold (end tidal PCO2 above which 
minute ventilation starts to increase linearly with further 
increases in end tidal PCO2)  
3. Slope of the PCO2-ventilatory response curve that reflects the 
increase in minute ventilation relative to the increase in end 
tidal PCO2 (represents chemoreflex sensitivity) 
4. Extrapolated ventilatory recruitment threshold (intersection 
with X axis) 
Exploratory Endpoints 
1. Additional physiological assessments as specified in the 
protocol 
2. Between occasion (within a day) and between day variability for 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 10 of 46 
  
 
physiological assessments as specified in the protocol 
Study Design: 
This study is an unblinded lead-in reproducibility assessment to assess 
variability of the ventilatory response to Duffin’s rebreathing 
procedure. 
Study Schedule: 
Day -1 
Day 1 
Day 2 
Check-In 
Lead-in PD Assessments 
Check-Out 
Healthy subjects will be enrolled in this study. Subjects will report to 
the study site for screening from Days -28 to -2 and then will return to 
the site on Day -1 for baseline assessments and check-in. After check-in 
(Day -1), subjects will remain in study site for PD assessments on Day 1 
and check out on Day 2.  
 
Paired rebreathing procedures (i.e., at two different isoxic end tidal PO2 
levels) will be performed on Day 1 at 0, 2, 4, and 6 hours. An 
additional pair of rebreathing procedures will be performed on Day 2 
before checkout. Subjects will not be administered any drugs in this 
lead-in study. 
 
A summary of all procedures is described in the Schedule of Events 
(Appendix A). Subjects will be discharged from the study after 
completion of all study procedures. If a subject discontinues from the 
study prematurely, all procedures scheduled for the end of the study 
will be performed. 
Subject Population: 
The lead-in reproducibility assessment will have up to 10 healthy 
volunteer participants. Interim data analyses may be performed after 
each cohort of participants has completed. If reproducibility is 
acceptable and no procedural changes are needed, additional lead-in 
cohorts may not be enrolled. If any study procedure element changes 
are needed, they may be made and additional participants may be 
enrolled.  
Inclusion Criteria: 
Subjects who meet all the following inclusion criteria will be eligible to 
participate in the study:  
1. Subject signs an IRB-approved written informed consent and 
privacy language as per national regulations (e.g., Health 
Insurance Portability and Accountability Act authorization) 
before any study related procedures are performed.  
2. Subject is a healthy, non-smoking man or woman, 18 to 50 
years of age, inclusive, who has a body mass index of 18.5 to 32 
kg/m2, inclusive, at Screening. 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 11 of 46 
  
 
3. Subject has normal medical history findings, clinical laboratory 
results, vital sign measurements, 12 lead ECG results, and 
physical examination findings at screening or, if abnormal, the 
abnormality is not considered clinically significant (as 
determined and documented by the investigator or designee). 
4. Subject must have a negative test result for alcohol and drugs of 
abuse at screening and Check-in (Day -1).  
5. Subject must test negative for severe acute respiratory 
syndrome coronavirus (SARS-CoV-2) by a molecular 
diagnostic test at check-in (Day -1). If a subject’s test comes 
back inconclusive, it can be repeated at Check-in. 
6. Female subjects must be of non-childbearing potential or, if 
they are of childbearing potential, they must: 1) have been 
strictly abstinent for 1 month before Check in (Day -1) and 
agree to remain strictly abstinent for the duration of the study 
and for at least 1-month after the last rebreathing procedure; OR 
2) be practicing 2 highly effective methods of birth control (as 
determined by the investigator or designee; one of the methods 
must be a barrier technique) from at least 1 month before Check 
in (Day -1) until at least 1 month after the end of the study.  
7. Male subjects must agree to practice 2 highly effective methods 
of birth control (as determined by the investigator or designee) 
from at least 1 month before Check In (Day -1) until at least 1 
month after the last rebreathing procedure. 
8. Subject is highly likely (as determined by the investigator) to 
comply with the protocol defined procedures and to complete 
the study.  
Exclusion Criteria: 
Subjects who meet any of the following exclusion criteria will not be 
eligible to participate in the study: 
1. Subject has non-reactive or misshapen pupil(s) or damaged 
orbit structure or surrounding soft tissue is edematous or has an 
open lesion. 
2. Subject Duffin rebreathing data is of poor quality or subject 
does not agree to remain clean-shaven for all days when the 
Duffin rebreathing procedure is being performed. 
3. Subject has used any prescription or nonprescription drugs 
(including aspirin or NSAIDs and excluding oral contraceptives 
and acetaminophen) within 14 days or 5 half-lives (whichever is 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 12 of 46 
  
 
longer) or complementary and alternative medicines within 
28 days before check-in. 
4. Subject has used nicotine-containing products (e.g., cigarettes, 
cigars, chewing tobacco, snuff) within 6 weeks of Screening 
5. Subject has a history or evidence of a clinically significant 
disorder, condition, or disease (e.g., cancer, human 
immunodeficiency virus [HIV], hepatic or renal impairment) 
that, in the opinion of the investigator would pose a risk to 
subject safety or interfere with the study evaluation, procedures, 
or completion. This includes subjects with any underlying 
medical conditions that the Investigator believes would put 
subjects at increased risk of severe illness from COVID-19 
based on the Centers for Disease Control and Prevention (CDC) 
guidelines.  
6. Subject has any signs or symptoms that are consistent with 
COVID-19. These include subjects with fever or chills, cough, 
shortness of breath or difficulty breathing, fatigue, muscle or 
body aches, headache, new loss of taste or smell, sore throat, 
congestion or runny nose, nausea or vomiting, or diarrhea may 
have COVID-19. In addition, the subject has any other findings 
suggestive of COVID-19 risk in the opinion of the investigator. 
 
7. Subject tests positive for SARS-CoV-2 by a molecular 
diagnostic test performed prior to admission. 
 
8. Subject has clinical laboratory test results (hematology, serum 
chemistry and urinalysis) at Screening or Check-In that are 
outside the reference ranges provided by the clinical laboratory 
and considered clinically significant by the investigator.  
9. Subject has a positive test result at Screening for HIV 1 or 2 
antibody, hepatitis C virus antibodies, or hepatitis B surface 
antigen. 
10. Female subject is pregnant or lactating before enrollment in the 
study. 
11. Subject has a history of or currently has hypoventilation 
syndrome, sleep apnea, or chronic obstructive pulmonary 
disease (COPD) and is on non-invasive ventilation. 
12. Subject has a history of asthma that has required medication 
within the last five years.  
13. Subject has a history of sleep disorders, Panic Disorder, Panic 
Attacks, Generalized Anxiety Disorder, or any associated DSM 
diagnosis or condition. 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 13 of 46 
  
 
 
Assessments:  
 
The following physiologic measurements will be assessed multiple 
times throughout the study: 
 Minute ventilation 
 Tidal volume 
 Respiratory rate 
 End-tidal PCO2  
 End-tidal O2 
 Oxygen saturation (SpO2) 
 Heart rate 
 Maximum pupil diameter before constriction 
 
Safety Assessments: 
 
 
 
Safety will be evaluated in terms of adverse events (AEs), clinical 
laboratory results (hematology, serum chemistry, and urinalysis), vital 
sign measurements (blood pressure, heart rate, respiratory rate, pulse 
oximetry and oral body temperature), 12-lead ECG, and physical 
examination findings. 
Sample Size and 
Threshold 
Determination: 
Up to 10 healthy volunteer participants are planned for enrollment in 
this study. The sample size was selected empirically to acquire 
experience for the research staff with use of the rebreathing equipment 
and to assess performance over repeated assessments in a participant 
over multiple days.  
Statistical Methods: 
All data will be presented in data listings. Data from subjects excluded 
from an analysis population will be presented in the data listings but not 
included in the calculation of summary statistics. The number of subjects 
who enroll in the study and the number and percentage of subjects who 
complete each assessment will be presented. 
The frequency and percentage of subjects who withdraw or discontinue 
from the study and the reason for withdrawal or discontinuation will be 
summarized. Demographic and baseline characteristics will be 
summarized overall for all subjects. 
Descriptive statistics will be used to summarize demographic and 
baseline subject characteristics. For continuous variables, the mean, 
median, standard deviation (SD), minimum, and maximum values will 
be reported. For categorical (nominal) variables, the number and 
percentage of subjects (or observations) will be reported. 
Primary Analysis 
Baseline minute ventilation, ventilatory recruitment threshold, slope of 
the PCO2-ventilatory response curve, and the extrapolated ventilatory 
recruitment threshold will be calculated for each subject at each 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 14 of 46 
  
 
rebreathing assessment. Mean, within- and between-day variability, 
between-subject variability of the ventilatory response to modified 
Duffin’s rebreathing will be examined using mixed-effects approaches. 
Coefficients of variation (CV) will be calculated using mean within- 
and between-day values of each end point. Variability will be assessed 
separately for each isoxic gas condition (i.e. end tidal PO2 of 50 mmHg 
and 150 mmHg). Full details will be described in a separate Statistical 
Analysis Plan. 
Exploratory Analysis 
Additional physiological assessments (e.g. ventilation, respiratory rate, 
heart rate, maximum pupil diameter before constriction) as specified in 
the protocol will be reported with standard descriptive statistics. 
Safety 
The safety population will include all subjects who undergo rebreathing 
procedures starting on Day 1. All AEs will be coded using the Medical 
Dictionary for Regulatory Activities (MedDRA). The incidence of AEs, 
organized by system organ class and preferred term, will be summarized 
with a focus on treatment-emergent AEs. Vital sign measurements will 
be summarized using descriptive statistics by time point. All values will 
be evaluated for clinically notable results. Data for additional safety 
parameters (e.g., physical examination findings) will be listed. 
Date of Protocol: 
27th October 2021 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 15 of 46 
  
 
1. INTRODUCTION 
In order to study the effects of opioids alone or in combination with other sedative 
psychotropic drugs on ventilation, we previously implemented the Read rebreathing 
procedure.1 With Read rebreathing, a study participant rebreathes through a circuit with a 
bag that initially contains 93% oxygen (O2) and 7% carbon dioxide (CO2). The 7% CO2 
results in relatively rapid equilibration between arterial partial pressure of carbon dioxide 
(PCO2), end tidal PCO2, and mixed venous PCO2. As the study participant continues to 
breathe, CO2 accumulates and triggers an increase in ventilation referred to as the 
"hypercapnic ventilatory response." Drugs can decrease the hypercapnic ventilatory 
response at drug concentrations that may have minimal-to-no detectable effect on 
ventilation when breathing room air.2-3 However, this method is limited in that all 
measurements are performed under hyperoxic conditions, which can have independent 
effects on ventilation, and the data cannot necessarily be used to extrapolate to the 
dynamic changes in PO2 and PCO2 that occur during real-world drug-induced respiratory 
depression when a patient is breathing room air. 
Duffin modified the Read rebreathing method to include 1) hyperventilation (primarily 
through deep breathing) prior to rebreathing to achieve an end tidal PCO2 or 
approximately 20-25 mm Hg prior to rebreathing,4 2) maintaining an isoxic end tidal PO2 
during rebreathing,5 and 3) repeating rebreathing at 2 different isoxic end tidal PO2 levels 
(i.e., at 50 mm Hg and 150 mm Hg).6 These modifications allow for critical physiological 
measurements and thresholds to be captured that can then be used to model the effects of 
drugs when there are dynamic changes in PO2 and PCO2, such as during a real-world 
opioid overdose. The Duffin rebreathing procedure has been used for multiple decades by 
different investigators studying different populations with and without drugs that can 
affect ventilation.1-14     
Baseline minute ventilation is captured when end tidal PCO2 is less than the ventilatory 
recruitment threshold. The ventilatory recruitment threshold is the end tidal PCO2 above 
which minute ventilation starts to increase linearly with further increases in end tidal 
PCO2. The slope of the PCO2-ventilatory response curve reflects the increase in minute 
ventilation relative to the increase in end tidal PCO2. Changing the PO2 can affect each of 
these variables, with hypoxia increasing baseline minute ventilation, decreasing the 
ventilatory recruitment threshold, and increasing the slope of the PCO2-ventilatory 
response curve, while hyperoxia has the opposite effects.  
The reproducibility of Duffin rebreathing has been assessed previously by Mahamed and 
Duffin (2001)7 performing hyperoxic and hypoxic rebreathing procedures measured once 
daily for 14 consecutive days and then by Jensen et al. (2010)8 performing 4 pairs of 
hyperoxic and hypoxic rebreathing procedures in 1 day followed by 1 pair on 4 additional 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 16 of 46 
  
 
days separated by weeks to more than a month. The present study will combine Duffin’s 
rebreathing procedure with some additional study procedures, such as quantitative 
pupillometry, that are planned for subsequent clinical pharmacology studies in order to 
confirm feasibility and gather reproducibility data. 
2. STUDY OBJECTIVES 
2.1 Primary Objective 
The objective of this study is to evaluate variability of the ventilatory response to 
Duffin’s rebreathing procedure. 
3. STUDY ENDPOINTS 
The endpoints of this study are below and will be determined for each rebreathing 
procedure: 
3.1 Primary Endpoints 
1. Baseline minute ventilation when end tidal PCO2 is less than the ventilatory 
recruitment threshold (represents non-chemoreflex drives to breathe) 
2. Ventilatory recruitment threshold (end tidal PCO2 above which minute ventilation 
starts to increase linearly with further increases in end tidal PCO2)  
3. Slope of the PCO2-ventilatory response curve that reflects the increase in minute 
ventilation relative to the increase in end tidal PCO2 (represents chemoreflex 
sensitivity) 
4. Extrapolated ventilatory recruitment threshold (intersection with X axis) 
3.2 Exploratory Endpoints 
1. Additional physiological assessments as specified in the protocol 
2. Between occasion (within a day) and between day variability for physiological 
assessments as specified in the protocol 
4. INVESTIGATIONAL PLAN 
4.1 Study Design 
This study is an unblinded lead-in reproducibility assessment to assess variability of the 
ventilatory response to Duffin’s rebreathing procedure. 
Table 4-1: Study Schedule 
Day -1 
Day 1 
Day 2 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 17 of 46 
  
 
Check-In 
Lead-in PD Assessments 
Check-Out 
Healthy subjects will be enrolled in this study. Subjects will report to the study site for 
screening from Days -28 to -2 and then will return to the site on Day -1 for baseline 
assessments and check-in. After check-in (Day -1), subjects will remain in study site for 
PD assessments on Day 1 and check out on Day 2.  
Paired rebreathing procedures (i.e., at two different isoxic end tidal PO2 levels) will be 
performed on Day 1 at 0, 2, 4, and 6 hours. An additional pair of rebreathing procedures 
will be performed on Day 2 before checkout. Subjects will not be administered any drugs 
in this lead-in study. 
A summary of all procedures is described in the Schedule of Events (Appendix A). 
Subjects will be discharged from the study after completion of all study procedures. If a 
subject discontinues from the study prematurely, all procedures scheduled for the end of 
the study will be performed. 
4.1.1 Risk/Benefit 
The Duffin rebreathing procedure has been used for multiple decades by different 
investigators studying different populations with and without drugs that can affect 
ventilation.1-14 This study will utilize a lower isoxic level of 50 mmHg, which has been 
used in the majority of Duffin rebreathing studies, however some have safely used a 
lower level of 40 mmHg.9,10 When conducting studies with PO2 at 50 mmHg, SpO2 will 
be approximately 84% when end tidal pCO2 is 40 mmHg and will decrease as end-tidal 
PCO2 increases (SpO2 approximately 80% when PCO2 is 55 mmHg). Some subjects are 
likely to have SpO2 below 80%, however this is only for brief periods as the total 
rebreathing time is approximately 5 minutes. Prior studies with other methods have 
targeted an SpO2 of 80% for one hour11 (or other time limits longer than 25 minutes11, 15-
19) and thresholds for ending hypoxic challenge studies have been SpO2 of 70% in 
multiple studies.12-14 With rebreathing at isoxic level of 50 mmHg, rebreathing will end 
when PCO2 reaches 55 mmHg or if SpO2 is less than 75% for 3 minutes or if SpO2 is 
less than 70% (after confirming is not due to noise). This is well within what has been 
performed safely in prior studies. 
Subjects will be informed that participation in a human physiological measurement study 
like the present one cannot be of benefit to healthy volunteers. Nevertheless, the 
information from the physical examination, vital sign measurements, and ECG results 
may be shared with the subject’s personal physician if this is the subject’s choice. 
Subjects will be informed that it is also their choice to inform their personal physician 
that they are participating in this research study. 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 18 of 46 
  
 
Subjects will be informed that their contribution to the study is of major importance to 
agencies like the FDA to understand the ventilatory response to Duffin’s rebreathing 
procedure. However, since this is a study involving healthy volunteers, subjects will be 
informed that they have the option not to participate. 
All women must take a pregnancy test in this study. All women of childbearing potential 
enrolled on this study will be informed that they must use effective birth control methods 
(abstinence, intrauterine device, or contraceptive foam and a condom [i.e., double-barrier 
method]) during the study participation. Subjects will be informed that they must notify 
the investigator if they or their female partners become pregnant during the course of the 
study. If a subject becomes pregnant, she will be informed that neither Spaulding Clinical 
Research nor the sponsor will be responsible for the cost of any obstetric or related care, 
or for the child’s care. 
Subjects will be informed that they will be participating in the Duffin’s rebreathing 
procedure where they will alternatively breathe a hyperoxic-hypercapnic (24% O2, 6% 
CO2, N2 balanced) or hypoxic-hypercapnic (6% O2, 6% CO2, N2 balanced) gas mixture 
through a mask. They will be informed that this mixture of oxygen, carbon dioxide and 
nitrogen provided will be different than is normally found in room air (21% O2, 0.04% 
CO2, N2 balanced with minor amounts of other gasses). Subjects will be informed that the 
different gas concentrations, in particular the higher level of carbon dioxide breathed in, 
may result in a feeling of needing to breathe faster or more deeply. Subjects will be 
informed that this procedure has been used in many previous physiologic measurement 
studiesand is non-invasive. During screening, subjects will be shown the Duffin’s 
rebreathing equipment and will be trained how to use it to understand if the procedure is 
tolerable.  
Subjects will be informed that the confidentiality of their data will be respected at all 
times according to state law, and the study personnel handling their study data are bound 
by confidentiality agreements. 
Subjects will be informed that extra precautions will be put in place, including required 
screening tests, that will limit the risk of COVID-19. Precautions will be documented in a 
COVID-19 risk management plan. Subjects will be informed that despite the extra 
precautions, there is still a risk of them contracting COVID-19. Any changes to the 
COVID-19 precautions (e.g. due to updated CDC recommendations or new testing 
becoming available) will be documented in the COVID-19 risk management plan.  
Subjects will be informed that all tests, procedures, and visits required by the study are 
provided at no cost to them. If subjects become ill or physically injured because of 
participation in this study, they will be informed that costs of treatment will not be 
covered by the sponsor. 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 19 of 46 
  
 
4.2 Selection of Study Population 
Subjects will be screened, and the data collected will be reviewed by the principal 
investigator. Only those subjects who meet all the eligibility criteria will be enrolled. The 
lead-in reproducibility assessment will have up to 10 healthy volunteer participants. 
Interim data analyses may be performed after each cohort of participants have completed. 
If reproducibility is acceptable and no procedural changes are needed, additional lead-in 
cohorts may not be enrolled. If any study procedure element changes are needed, they 
may be made and additional participants may be enrolled. Every effort will be made to 
maintain an approximate 50:50 male-to-female sex distribution. 
4.2.1 Inclusion Criteria 
Subjects who meet all of the following inclusion criteria will be eligible to participate in 
the study: 
1. Subject signs an IRB approved written informed consent and privacy language as 
per national regulations (e.g., Health Insurance Portability and Accountability Act 
authorization) before any study related procedures are performed.  
2. Subject is a healthy, non-smoking man or woman, 18 to 50 years of age, 
inclusive, who has a body mass index of 18.5 to 32 kg/m2, inclusive, at Screening. 
3. Subject has normal medical history findings, clinical laboratory results, vital sign 
measurements, 12 lead ECG results, and physical examination findings at 
screening or, if abnormal, the abnormality is not considered clinically significant 
(as determined and documented by the investigator or designee). 
4. Subject must have a negative test result for alcohol and drugs of abuse at 
screening and Check-in (Day -1).  
5. Subject must test negative for severe acute respiratory syndrome coronavirus 
(SARS-CoV-2) by a molecular diagnostic test at check-in (Day -1). If a subject’s 
test comes back inconclusive, it can be repeated at Check-in. 
6. Female subjects must be of non-childbearing potential or, if they are of 
childbearing potential, they must: 1) have been strictly abstinent for 1 month 
before Check in (Day -1) and agree to remain strictly abstinent for the duration of 
the study and for at least 1-month after the last rebreathing procedure; OR 2) be 
practicing 2 highly effective methods of birth control (as determined by the 
investigator or designee; one of the methods must be a barrier technique) from at 
least 1 month before Check in (Day -1) until at least 1 month after the end of the 
study.  

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 20 of 46 
  
 
7. Male subjects must agree to practice 2 highly effective methods of birth control 
(as determined by the investigator or designee) from at least 1 month before 
Check In (Day -1) until at least 1 month after the end of the study. 
8. Subject is highly likely (as determined by the investigator) to comply with the 
protocol defined procedures and to complete the study. 
4.2.2 Exclusion Criteria 
1. Subject has non-reactive or misshapen pupil(s) or damaged orbit structure or 
surrounding soft tissue is edematous or has an open lesion. 
2. Subject Duffin rebreathing data is of poor quality or subject does not agree to 
remain clean-shaven for all days when the Duffin rebreathing procedure is being 
performed. 
3. Subject has used any prescription or nonprescription drugs (including aspirin or 
NSAIDs and excluding oral contraceptives and acetaminophen) within 14 days or 
5 half-lives (whichever is longer) or complementary and alternative medicines 
within 28 days before Check in (Day -1). 
4. Subject has used nicotine-containing products (e.g., cigarettes, cigars, chewing 
tobacco, snuff) within 6 weeks of Screening 
5. Subject has a history or evidence of a clinically significant disorder, condition, or 
disease (e.g., cancer, human immunodeficiency virus [HIV], hepatic or renal 
impairment) that, in the opinion of the investigator would pose a risk to subject 
safety or interfere with the study evaluation, procedures, or completion. This 
includes subjects with any underlying medical conditions that the Investigator 
believes would put subjects at increased risk of severe illness from COVID-19 
based on the Centers for Disease Control and Prevention (CDC) guidelines.  
6. Subject has any signs or symptoms that are consistent with COVID-19. These 
include subjects with fever or chills, cough, shortness of breath or difficulty 
breathing, fatigue, muscle or body aches, headache, new loss of taste or smell, 
sore throat, congestion or runny nose, nausea or vomiting, or diarrhea may have 
COVID-19. In addition, the subject has any other findings suggestive of COVID-
19 risk in the opinion of the investigator. 
7. Subject tests positive for SARS-CoV-2 by a molecular diagnostic test performed 
prior to admission. 
 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 21 of 46 
  
 
8. Subject has clinical laboratory test results (hematology, serum chemistry and 
urinalysis) at Screening or Check-In that are outside the reference ranges provided 
by the clinical laboratory and considered clinically significant by the investigator.  
9. Subject has a positive test result at Screening for HIV 1 or 2 antibody, hepatitis C 
virus antibodies, or hepatitis B surface antigen. 
10. Female subject is pregnant or lactating before enrollment in the study. 
11. Subject has a history of or currently has hypoventilation syndrome or sleep apnea 
and is on non-invasive ventilation. 
12. Subject has a history of sleep disorders, Panic Disorder, Panic Attacks, 
Generalized Anxiety Disorder, or any associated DSM diagnosis or condition. 
4.3 Screening Failures 
Subjects who sign and date the informed consent form but who fail to meet the inclusion 
and exclusion criteria are defined as screening failures. A screening log, which 
documents the subject initials and reason(s) for screening failure, will be maintained by 
the investigator for all screening failures. A copy of the log should be retained in the 
investigator’s study files. 
If a subject fails the screening process because of an abnormal laboratory result, they can 
receive a copy of the results upon request. The investigator will determine if follow-up 
for the abnormal laboratory result is needed and will encourage the subject to follow-up 
with his or her personal physician as appropriate. All subjects will be informed as to the 
reason(s) they are excluded from study participation, even if follow-up is not required. If 
a subject fails the screening process because of a positive test result for human 
immunodeficiency virus or hepatitis, the positive result will be reported to local health 
authorities as required by law. 
4.4 Termination of Study or Investigational Site 
4.4.1 Criteria for Termination of the Study 
The study will be completed as planned unless one of the following criteria is satisfied 
that requires early termination of the study. 
 Significant violation of Good Clinical Practice (GCP) that compromises the ability to 
achieve the primary study objective or compromises subject safety. 
4.4.2 Criteria for Termination of the Investigational Site 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 22 of 46 
  
 
The study site may be terminated if the site (including the investigator) is found in 
significant violation of GCP, the protocol, the contractual agreement, or is unable to 
ensure adequate performance of the study. 
In the event that the sponsor elects to terminate the study or the investigational site, a 
study-specific procedure for early termination will be provided by the sponsor; the 
procedure will be followed by the applicable investigational site during the course of 
termination. 
4.5 Criteria for Subject Withdrawal 
Subjects may withdraw from the study at any time at their own request, or they may be 
withdrawn by the investigator without the approval of the subject based on the 
investigator’s clinical judgment. A subject is not required to provide a written request to 
withdraw from the study; however, a written request is required if a subject withdraws 
consent for his or her personal data to be used for study-related purposes. 
A subject may be discontinued for any of the following reasons: 
 AE: The subject has experienced an AE that, in the opinion of the investigator, 
requires early termination. The appropriate electronic case report form (eCRF) must 
be completed for each AE. If a subject is discontinued from the study due to an AE, 
the investigator is required to follow-up with the subject until the event resolves or 
becomes stable. If a subject dies during the study, the cause of death must be reported 
as a serious AE (SAE), with an outcome of death noted in the eCRF. 
 Protocol Violation: The subject failed to meet protocol entry criteria or did not adhere 
to protocol requirements, and continued participation poses an unnecessary risk to the 
subject’s health. 
 Withdrawal by Subject: The subject (or other responsible individual [e.g., caregiver]) 
wishes to withdraw from the study in the absence of a medical need. 
NOTE: Withdrawal due to an AE should not be recorded in the “voluntary 
withdrawal” category. 
 Study Terminated by Sponsor: The sponsor, IRB, FDA, or other regulatory agency 
terminates the study. 
 Pregnancy: The subject is found to be pregnant. 
NOTE: If the subject is found to be pregnant, the subject must be withdrawn 
immediately. The pregnancy will be followed-up to term, and the outcome, including 
any premature termination will be recorded. All live births must be followed for a 
minimum of 30 days or until the first well-baby visit. 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 23 of 46 
  
 
 Other. 
NOTE: This category records withdrawals caused by an accidental or a medical 
emergency, and other rare cases. The specific reason should be recorded in the 
comment space of the eCRF. 
4.5.1 Handling of Withdrawals 
The investigator may terminate a subject’s study participation at any time during the 
study when the subject meets the criteria described in Section 4.5. In addition, a subject 
may discontinue his or her participation without giving a reason at any time during the 
study. Subjects will be informed that their participation in the study is voluntary, that 
refusal to participate will involve no penalty or loss of benefits to which the subject is 
otherwise entitled, and that the subject may discontinue participation at any time without 
penalty or loss of benefits to which the subject is otherwise entitled. 
Should a subject’s participation be discontinued, the primary reason for termination must 
be recorded. In addition, efforts should be made to perform all procedures scheduled for 
the early termination visit. Any data and samples collected before subject withdrawal will 
become the property of the sponsor. 
4.5.2 Replacement of Subjects 
The lead-in reproducibility assessment will have up to 10 healthy volunteer participants. 
Interim data analyses may be performed after each cohort of participants have completed. 
If reproducibility is acceptable and no procedural changes are needed, additional lead-in 
cohorts may not be enrolled and the lead-in would end with fewer than 10 participants. If 
any study procedure element changes are needed, they may be made and additional 
participants may be enrolled. While they are not expected to be needed, up to 5 
replacement participants may be enrolled. 
4.6 Study Visits 
4.6.1 Recruitment 
Recruitment materials (e.g., internet, radio, and print advertisements, social media posts) 
will be approved by the local IRB before telephone screening. The sponsor is responsible 
for registration of the study on clinicaltrials.gov. Recruitment may not occur until the 
study is fully registered on clinicaltrials.gov. 
4.6.2 Compensation 
Subjects will be offered payment for Screening; however, if the results of their alcohol 
and drug screening tests are positive, they will not be compensated. Subjects who 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 24 of 46 
  
 
complete the entire study will receive payment according to the schedule provided in the 
informed consent form. No special incentives are offered. Final payment will not be 
released until all follow-up procedures have been completed and accepted by the 
investigator. 
If a subject chooses to withdraw from the study prematurely, he or she will only be 
compensated for completed days. If subjects are withdrawn for medical reasons or if the 
study is halted temporarily or permanently, the subjects will receive compensation 
proportional to the time spent in the study. No compensation will be provided if a subject 
is dismissed from the study for noncompliance (e.g., improper conduct, ingesting alcohol 
and/or drugs [including recreational drugs], consuming any prohibited foods or 
beverages). 
If subjects are required to stay in the clinic for a longer period for safety reasons, they 
will be compensated at a rate proportional to the entire compensation for the study. If a 
subject becomes ill or physically injured because of participation in this study, the subject 
will be referred for treatment. 
4.6.3 Screening  
The following procedures and assessments will be performed at Screening (Day -28 to -
2): 
 Obtain informed consent/HIPAA authorization. The informed consent process will be 
performed by a clinical research nurse in a private room. The subject will be given 
unlimited time to ask questions regarding study participation, and each subject will be 
questioned to ensure their understanding. 
After informed consent is obtained: 
 Review inclusion/exclusion criteria to confirm subject eligibility 
 Record demographic information 
 Perform serology screening (HIV antigen/antibody [Ag/Ab] Combo 1/2, HepC 
antibody, HBsAg) 
 Record medical history 
 Perform alcohol and drug screening (amphetamines, barbiturates, benzodiazepines, 
cannabinoids, cocaine, alcohol, opiates, phencyclidine, propoxyphene, and 
methadone) 
 Perform a serum pregnancy test (female subjects only) 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 25 of 46 
  
 
 Assess follicle-stimulating hormone (FSH) for postmenopausal (i.e., without menses 
for two years) female subjects 
 Record prior medications 
 Monitor for AEs 
 Perform clinical laboratory tests (hematology, serum chemistry, and urinalysis) 
 Measure vital signs (blood pressure, heart rate, respiratory rate, oral body 
temperature, and pulse oximetry) 
 Perform a safety 12-lead ECG 
 Perform a complete physical examination 
 Duffin’s rebreathing procedure training 
4.6.4 Study Periods 
This study is an unblinded lead-in reproducibility assessment where subjects will check-
in on Day -1 and remain in the study site until checkout on Day 2.  
4.6.4.1 Check-In 
The following procedures and assessments will be performed at Check-in [Day -1]: 
 Perform/review results from SARS-CoV-2 molecular test (may be performed ~2 days 
before check-in to allow time for results) 
 Review inclusion/exclusion criteria to confirm subject eligibility 
 Review medical history 
 Perform alcohol and drug screening (amphetamines, barbiturates, benzodiazepines, 
cannabinoids, cocaine, alcohol, opiates, phencyclidine, propoxyphene, and 
methadone) 
 Perform a serum pregnancy test (female subjects only)  
 Assess follicle-stimulating hormone (FSH) for postmenopausal (i.e., without menses 
for two years) female subjects 
 Record concomitant medications 
 Monitor for AEs 
 Perform clinical laboratory tests (hematology, serum chemistry, and urinalysis) 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 26 of 46 
  
 
 Measure vital signs (blood pressure, heart rate, respiratory rate, oral body 
temperature, pulse oximetry) 
 Perform a safety 12-lead ECG  
 Perform a comprehensive physical examination 
 Admit subject to the study clinic 
4.6.4.2 Study Procedure (Day 1) 
The following procedures and assessments will be performed during the study procedure 
period (day 1) according to the Schedule of Events (Table 9-1): 
 Monitor for AEs 
 Record concomitant medications 
 Measure vital signs (blood pressure, heart rate, respiratory rate, oral body 
temperature, and pulse oximetry).  
 Collect samples for estrogen and progesterone (female subjects only) 
 Perform Duffin’s rebreathing procedure at the approximate time points 0, 2, 4, 6 and 
24 hours (last time point on morning of Day 2, Discharge day).  Preparatory steps for 
the Duffin’s rebreathing procedure (i.e., relaxation, hyperventilation) should begin 
prior to the scheduled time. At each time point, the pair of rebreathing assessments (1 
at 150 mmHg end tidal PO2 and 1 at 50 mmHg end tidal PO2) will be separated by 
approximately 30 min.  
 Quantitative pupillometry will be performed before and after each rebreathing 
procedure. 
4.6.5 Discharge (or Early Termination) 
The following procedures and assessments will be performed before the subject is 
discharged from the study or at early termination: 
 Perform a serum pregnancy test (female subjects only) 
 Record concomitant medications 
 Monitor for AEs 
 Perform clinical laboratory tests (hematology, serum chemistry, and urinalysis) 
 Measure vital signs (blood pressure, heart rate, respiratory rate, pulse oximetry and 
oral body temperature) 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 27 of 46 
  
 
 Perform Duffin’s rebreathing procedure at the 24 hour time point (last time point of 
the procedure).  Preparatory steps for the Duffin’s rebreathing procedure (i.e., 
relaxation, hyperventilation) should begin prior to the scheduled time. At each time 
point, the pair of rebreathing assessments (1 at 150 mmHg end tidal PO2 and 1 at 50 
mmHg end tidal PO2) will be separated by approximately 30 min.  
 Quantitative pupillometry will be performed before and after each rebreathing 
procedure. 
 Perform a safety 12-lead ECG  
 Perform a complete physical examination 
 Discharge subject from the study clinic after completion of all study procedures 
4.7 Study Procedures 
4.7.1 Duffin’s Rebreathing Procedure  
Training on the Duffin’s rebreathing procedure will be performed to exclude any subjects 
who cannot tolerate the procedure.   
During Duffin’s rebreathing procedure, subjects will be placed in a semi-supine position 
on a bed and connected to the respiration measurement set up. Subjects will wear a 
facemask and breathe from a pre-filled rebreathing bag containing approximately 4 L of 
either a hyperoxic-hypercapnic (24% O2, 6% CO2, N2 balanced) or hypoxic-hypercapnic 
(6% O2, 6% CO2, N2 balanced) gas mixture. During this study, subjects will be asked to 
breath room air for 5 minutes (relaxation stage) and then to hyperventilate (primarily 
through deep breathing) room air voluntarily for 5 minutes (hyperventilation stage). 
Thereafter, subjects will be switched to the rebreathing bag and perform rebreathing 
assessments at one of the 2 different isoxic end tidal PO2,  i.e., 150 mmHg and 50 mmHg. 
The isoxia at 150 mmHg or 50 mmHg end-tidal PO2 will be maintained by providing a 
computer controlled flow of 100% O2 to the rebreathing bag.   
Subjects will perform 4 pairs of rebreathing procedures (i.e., 8 procedures in total) on 
Day 1 followed by 1 pair of rebreathing procedures on Day 2 (24-hours after the first 
rebreathing procedure on Day 1). Each pair of rebreathing procedures will be separated 
by approximately 30 min. Participants can communicate to staff to discontinue an 
assessment due to discomfort at any time. Additional considerations for discontinuing a 
rebreathing procedure are described in a separate Standard Operating Procedure for the 
Rebreathing Procedure. 
Each rebreathing procedure (relaxation, hyperventilation, and rebreathing) should take 
approximately 13-15 minutes. Data collected throughout the procedure includes 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 28 of 46 
  
 
ventilatory flow and volumes as well as percent gas compositions of O2 and CO2, which 
are used to calculate multiple physiological variables.  
Data from the rebreathing procedure will be acquired using the Hans Rudolph 
SmartLabTM Data Acquisition System. Collected data will be analyzed using separate 
statistical software. Interim data analyses may be performed after each cohort of 
participants have completed. If reproducibility is acceptable and no procedural changes 
are needed, additional lead-in cohorts may not be enrolled. If any study procedure 
element changes are needed, they may be made and additional participants may be 
enrolled.  
The clock on the rebreathing recorder will be confirmed to be consistent with the study 
clock at the clinical site; this will be confirmed on each study day. Preparatory steps for 
the Duffin’s rebreathing procedure (i.e., relaxation, hyperventilation) should begin prior 
to the scheduled sample time. Timing of sample collection is planned so that the 
rebreathing assessment begins at the specified time. Additional details on the Duffin’s 
rebreathing procedure is described in a separate SOP.  
The following physiologic measurements will be assessed multiple times throughout the 
study: 
 Minute ventilation 
 Tidal volume 
 Respiratory rate 
 End-tidal PCO2  
 End-tidal O2 
 Oxygen saturation (SpO2) 
 Heart rate 
 Maximum pupil diameter before constriction 
4.7.2 Pupillary Assessments 
At Screening, subjects will be evaluated to ensure they meet requirements necessary for 
pupillary assessments (e.g., pupils are reactive and not misshapen). During the study, a 
pupillometer (a hand-held optical scanner) will be used to measure pupil size and 
dynamics in response to a light stimulus. Pupillary assessments will be performed before 
and after each rebreathing procedure while the ambient light is kept at a consistent level. 
The rubber cup of the pupillometer will be placed sequentially on the right eye followed 
by the left eye (i.e., one pupillary assessment includes measurement of both eyes). The 
actual data recording only takes seconds on each eye. If the pupillometer device reports 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 29 of 46 
  
 
the assessment was incomplete or inaccurate (based on device error codes), the 
assessment for that eye will be repeated up to three times.  
4.7.3 Safety Assessments 
Safety will be evaluated in terms of AEs, clinical laboratory results (hematology, serum 
chemistry, and urinalysis), vital sign measurements (blood pressure, heart rate, 
respiratory rate, pulse oximetry, and oral body temperature), safety 12-lead ECG results, 
and physical examination findings.  
4.7.3.1 Adverse Events 
4.7.3.1.1 Adverse Event Definitions 
An AE is defined as any untoward and/or unintended sign, including an abnormal clinical 
laboratory finding, symptom, or disease. No study drugs are being administered in this 
“lead-in” protocol to assess variability of noninvasive physiological measurements. 
A serious adverse event (SAE) is defined as any AE occurring at any assessment that 
meets the following criteria: 
 Results in death, 
 Is life threatening, 
 Requires hospitalization or prolongation of existing hospitalization, 
 Results in persistent or significant disability or incapacity, 
 Results in a congenital anomaly/birth defect due to exposure prior to conception or 
during pregnancy, or 
 Is an important medical event that may not meet the previous criteria but, based upon 
appropriate medical judgment, jeopardizes the subject or requires medical or surgical 
intervention to prevent one of the outcomes listed previously. 
4.7.3.1.2 Adverse Event Reporting 
The recording of AEs will begin after the subject signs the informed consent form and 
will continue until discharge (or early termination). All AEs, whether serious or 
nonserious, must be recorded in the eCRF.  
Any SAE must be entered into the eCRF system and reported by email to the medical 
monitor or designee using the SAE Reporting Form within 24 h of the investigator or 
study clinic staff becoming aware of the event. It is the responsibility of the investigator 
to report all SAEs to the medical monitor and to provide the most complete report 
possible. The investigator is responsible for obtaining follow-up information on all SAEs 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 30 of 46 
  
 
and submitting follow-up SAE data. Any unexpected SAEs must be reported promptly to 
the investigator’s IRB as per the IRB’s requirements. 
Adverse events that are assessed by the investigator as possibly or probably related to the 
study procedures will be followed until they resolve or stabilize. All SAEs will be 
followed until resolution. 
4.7.3.1.3 Assessment of Severity 
The investigator will assess the severity of each AE using the following scale: 
 Mild: The subject is aware of the AE but is still able to perform all activities; minimal 
or no medical intervention or therapy is required. 
 Moderate: The subject has to discontinue some activities due to the AE; minimal or 
no medical intervention or therapy is required. 
 Severe: The subject is incapacitated by the AE and is unable to perform normal 
activities; significant medical intervention or therapy is required, and hospitalization 
is possible. 
4.7.3.1.4 Pregnancy 
A serum pregnancy test will be performed for female subjects as presented in the 
Schedule of Events (Table 9-1). If a subject becomes pregnant while on the study, this 
should be reported immediately to the investigator, the subject will be withdrawn from 
the study and the medical monitor and the subject will be instructed to follow-up with his 
or her personal physician. All pregnancies are to be reported as an AE and followed for 
outcome. 
4.7.3.2 Clinical Laboratory Tests 
Clinical laboratory and diagnostic screening tests will be performed as presented in the 
Schedule of Events (Table 9-1) and will be collected in accordance with acceptable 
laboratory procedures. Clinical laboratory testing will be performed by the clinical study 
contractor(s). Tests can be repeated once in-case of isolated elevation or contamination of 
sample. The clinical laboratory tests that will be performed are presented in Table 4-2. 
Unused clinical laboratory test samples will not be stored for future use. 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 31 of 46 
  
 
 
Table 4-2: Clinical Laboratory Tests & Diagnostic Screening Tests 
4.7.3.3 Vital Sign Measurements 
Vital signs (blood pressure, heart rate, respiratory rate, pulse oximetry, and oral body 
temperature) will be measured as in the Schedule of Events (Table 9-1). The subject 
should be in a supine position, if possible, for a minimum of 5 minutes before vital signs 
are measured.  
4.7.3.4 Safety 12-lead Electrocardiograms 
12-lead ECGs will be obtained with the subjects in the supine position for a minimum of 
5 minutes before recording. ECGs will be overread by the Principal Investigator or 
designee (e.g., a medically qualified subinvestigator). If an abnormality is observed, the 
subject will be instructed to follow-up with his or her personal physician. 
Hematology 
Serum Chemistry 
Urinalysis 
Hematocrit 
Hemoglobin 
Platelet count 
Red blood cell count 
White blood cell count (with 
automated differential) 
Alanine aminotransferase 
Albumin 
Alkaline phosphatase 
Aspartate aminotransferase 
Bicarbonate 
Bilirubin (total, direct, and indirect) 
Blood urea nitrogen 
Calcium 
Chloride 
Creatinine (including calculated 
creatinine clearance by Cockcroft-
Gault) 
Glucose 
Lactate dehydrogenase 
Magnesium 
Phosphorus 
Potassium 
Sodium 
Total protein 
Uric acid 
Appearance 
Bilirubin 
Blood 
Color 
Glucose 
Ketones 
Leukocyte esterase 
Microscopic examination: red blood 
cells, white blood cells, epithelial cells, 
bacteria, crystals, and casts (if present) 
Nitrite 
pH 
Protein 
Specific gravity 
Urobilinogen 
Diagnostic Screening Tests: 
Serum 
Urine 
Other  
Serology (human 
immunodeficiency virus 
Ag/Ab Combo 1/2, hepatitis 
C virus antibody, and 
hepatitis B surface antigen) 
Female Subjects Only 
Human chorionic 
gonadotropin (for 
pregnancy) 
Estrogen 
Progesterone 
Drug screen including: 
amphetamines, barbiturates, 
benzodiazepines, cannabinoids, 
cocaine, alcohol, opiates, 
phencyclidine, propoxyphene, and 
methadone 
 SARS-CoV2 molecular test 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 32 of 46 
  
 
4.7.3.5 Physical Examinations 
A complete physical examination will be performed as presented in the Schedule of 
Events (Table 9-1). 
The complete physical examination will include, but not be limited to, assessments of the 
head, eyes, ears, nose, throat, skin, thyroid, nervous system, respiratory system, 
cardiovascular system, abdomen (liver and spleen), lymph nodes, and extremities. 
Height, weight (without shoes and wearing the lightest possible clothing), and calculation 
of body mass index will be performed at Screening and check-out. 
If a clinically significant abnormality is observed upon physical examination, the subject 
will be instructed to follow-up with his or her personal physician. 
4.7.4 Demographics and Medical History 
Demographic data (date of birth, gender, race, and ethnicity) will be collected at 
Screening. 
Each subject will provide a complete medical history at Screening that will be reviewed 
at Check-in. Specific information relating to any prior or existing medical 
conditions/surgical procedures will be recorded in the subject’s eCRF. 
4.7.5 Prior and Concomitant Medications 
Subjects are prohibited from using any prescription or nonprescription drugs (including  
aspirin or non-steroidal anti-inflammatory drugs [NSAIDs] and excluding oral 
contraceptives and acetaminophen) within 14 days or 5 half-lives (whichever is longer), 
or complementary and alternative medicines within 28 days before Day 1 of the study. 
Subjects will be asked if they have used any of these substances and their responses will 
be recorded on the eCRF. 
Subjects are also prohibited from currently participating in another clinical study of an 
investigational drug and may not have been treated with any investigational drug within 
30 days or 5 half-lives (whichever is longer) of the compound. 
Subjects must be instructed not to take any medications, including over-the-counter 
products, without first consulting with the investigator. If needed for an on-study adverse 
event, acetaminophen could be used if deemed necessary by the Principal Investigator or 
designee (e.g., a medically qualified subinvestigator). 
4.7.6 Subject Restrictions 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 33 of 46 
  
 
Subjects are not allowed to use nicotine containing products (e.g., cigarettes, cigars, 
chewing tobacco, snuff) within 6 weeks before Screening Subjects are not allowed to use 
aspirin or NSAIDs within 14 days before Day 1 of the study.  
Subjects must be able to tolerate a controlled, quiet study conduct environment, including 
avoidance of music, television, movies, games, and activities that may cause excitement, 
emotional tension, or arousal during prespecified times (e.g., before and during 
rebreathing procedures) throughout the duration of the study. 
Subjects must be willing to comply with study rules; attempting to void at specified times 
(e.g., before rebreathing procedures); remaining quiet, awake, undistracted, motionless, 
and supine during specified times; and avoiding vigorous exercise as directed throughout 
the duration of the study. Subjects will not be allowed to sleep during any rebreathing 
procedure periods. 
Standardized meals will be served at consistent times relative to the first rebreathing 
procedure, and no food or fluids will be served containing caffeine. Subjects will be 
required to fast during the intensive rebreathing procedure portions of the study. Subjects 
will be provided their first meal after the 6 h rebreathing procedure on Day 1 and an 
optional meal after the 24 h procedure on Day 2. Subjects must only eat meals and snacks 
that are provided during their stay in the study clinic, and must consume all of each meal 
that is served at a reasonable pace. Outside of meal times, the subjects will only be 
allowed to intake water, which will be available ad libitum. 
Subject must agree to remain clean-shaven for all days when the Duffin rebreathing 
procedure is being performed. 
Due to current precautions being taken for COVID-19, subjects must be willing to: 
 Always wear masks except when in a private room without anyone else present or for 
a limited time for a study procedure (e.g. rebreathing procedure or eating) when 
instructed by staff.  
 Practice social distancing. Subjects will spend most of their time in their rooms 
except for specified times for walking in the halls (with masks). 
 Practice regular handwashing with soap and water, scrubbing hands for at least 20 
seconds or with approved hand sanitizer as supplied by study staff.  
Designated isolation rooms will be set up to segregate any participant(s) that develop any 
symptoms of concern while housed in the unit and COVID-19 testing will be done when 
deemed necessary by the Investigator. If new information becomes available, there could 
be other precautions that lead to additional restrictions that will be documented in the 
COVID-19 Risk Management Plan and/or the study specific COVID-19 Procedure Plan. 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 34 of 46 
  
 
4.8 Statistical Methods 
4.8.1 Sample Size 
Up to 10 healthy volunteer participants are planned for enrollment in this study. The 
sample size was selected empirically to acquire experience for the research staff with use 
of the rebreathing equipment and to assess performance over repeated assessments in a 
participant over multiple days. 
4.8.2 Analysis Populations 
For this study the analysis population will include all subjects who complete at least one 
rebreathing procedure on Day 1. The analysis population may be defined further in the 
Statistical Analysis Plan. 
4.8.3 General Statistical Considerations 
All data will be presented in data listings. Data from subjects excluded from an analysis 
population will be presented in the data listings, but not included in the calculation of 
summary statistics. Demographic and baseline characteristics will be summarized overall 
for all subjects. 
4.8.4 Subject Disposition 
The number of subjects who enroll in the study and the number and percentage of 
subjects who complete each assessment will be presented. The frequency and percentage 
of subjects who withdraw or discontinue from the study and the reason for withdrawal or 
discontinuation will be summarized. 
4.8.5 Demographics and Baseline Characteristics 
Descriptive statistics will be used to summarize demographic and baseline subject 
characteristics. For continuous variables, the mean, median, standard deviation (SD), 
minimum, and maximum values will be reported. For categorical (nominal) variables, the 
number and percentage of subjects (or observations) will be reported. 
4.8.6 Primary Analysis  
Baseline minute ventilation, ventilatory recruitment threshold, slope of the PCO2-
ventilatory response curve, and the extrapolated ventilatory recruitment threshold will be 
calculated for each subject at each rebreathing assessment. Mean, within- and between-
day variability, between-subject variability of the ventilatory response to modified 
Duffin’s rebreathing will be examined using mixed-effects approaches. Coefficients of 
variation (CV), which will be calculated using mean within- and between-day values of 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 35 of 46 
  
 
each end point. Variability will be assessed separately for each isoxic gas condition (i.e. 
50 mmHg and 150 mmHg). Full details will be described in a separate Statistical 
Analysis Plan. 
4.8.7 Additional Analyses  
Additional physiological assessments (e.g. ventilation, respiratory rate, heart rate, 
maximum pupil diameter before constriction) as specified in the protocol will be reported 
with standard descriptive statistics. 
4.8.8 Safety Analyses 
4.8.8.1 Adverse Events 
All AEs will be coded using the latest version of the Medical Dictionary for Regulatory 
Activities. The incidence of AEs, organized by system organ class and frequency, will be 
summarized by seriousness. A detailed listing of serious AEs leading to withdrawal will 
also be provided. 
4.8.8.2 Clinical Laboratory Tests 
Clinical laboratory results will be reviewed by the investigator or designee together with 
data in the eCRF. Any values outside the reference range will be evaluated for clinical 
significance. If a value is determined to be clinically significant, the subject will be 
instructed to follow-up with his or her personal physician. The investigator or designee 
may repeat the clinical laboratory tests if deemed appropriate. The investigator will 
maintain a copy of the laboratory accreditation and the reference ranges for the laboratory 
used. 
Clinical laboratory results (hematology, serum chemistry, and urinalysis) will be 
summarized using descriptive statistics (number of subjects, mean, SD, minimum, 
median, and maximum). Clinical laboratory results will be classified as normal or 
abnormal, according to the reference ranges of the individual parameter. The number and 
percentage of subjects with abnormal laboratory results will be provided. No statistical 
testing will be performed on clinical laboratory data. 
4.8.8.3 Vital Sign Measurements 
Vital sign measurements and changes from baseline will be summarized using descriptive 
statistics (number of subjects, mean, SD, minimum, median, and maximum) by time 
point. 
4.8.8.4 Safety 12-lead Electrocardiograms  

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 36 of 46 
  
 
Safety 12-lead ECGs will be obtained with the subjects in the supine position for a 
minimum of 5 minutes before recording. ECGs will be overread by the Principal 
Investigator or designee (e.g., a medically qualified subinvestigator). If an abnormality is 
observed, the subject will be instructed to follow-up with his or her personal physician. 
4.8.8.5 Physical Examinations 
 Physical examination findings will be presented in a data listing, and abnormal physical 
examination findings will be recorded as AEs. 
4.8.8.6 Other Safety Data 
All concomitant medication usage and medications that changed in daily dose, frequency, 
or both since the subject provided informed consent will be summarized for each subject. 
4.8.9 Interim Analyses 
No formal interim analyses are planned for this study. Rebreathing results for individual 
subjects will be reviewed and analyzed as each subject completes the study. Feedback 
will be provided so that research staff can incorporate accrued experience into how the 
rebreathing equipment is used and how rebreathing procedures are conducted.  
4.8.10 Missing Data 
Missing data will not be imputed. Data that are excluded from the descriptive or 
inferential analyses will be included in the subject data listings. This will include data 
from subjects not in the particular analysis population, measurements from unscheduled 
visits, or extra measurements that may arise from 2 or more analyses of the biofluid 
sample at the same time point. Details on the handling of missing data will be further 
described in the Statistical Analysis Plan. 
4.9 Data Quality Assurance 
Completed eCRFs are required for each subject participating in study procedures. 
Electronic data entry will be accomplished through the ClinSpark® remote electronic data 
capture system, which allows for on-site data entry and data management. This system 
provides immediate, direct data transfer to the database, as well as immediate detection of 
discrepancies, enabling site coordinators to resolve and manage discrepancies in a timely 
manner. Each person involved with the study will have an individual identification code 
and password that allows for record traceability. Thus, the system, and subsequently any 
investigative reviews, can identify coordinators, investigators, and individuals who have 
entered or modified records. 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 37 of 46 
  
 
 
Furthermore, the investigator retains full responsibility for the accuracy and authenticity 
of all data entered into the electronic data capture system. The completed dataset and 
their associated files are the sole property of the sponsor and should not be made 
available in any form to third parties, except for appropriate governmental health or 
regulatory authorities, without written permission of the sponsor. 
4.10 Data Sharing 
De-identified subject-level data may be released to other researchers (including through a 
data warehouse or as a part of a publication) to enable secondary research. Additional 
secondary research may also be performed by the sponsor. 
 
5. ETHICAL CONSIDERATIONS 
5.1 Ethical Conduct of the Study 
This study will be performed in accordance with the recommendations guiding 
physicians in biomedical research involving human subjects adopted by the 18th World 
Medical Association General Assembly, Helsinki, Finland, 1964 and later revisions, as 
well as, United States Title 45 Code of Federal Regulations (CFR) Part 46 GCP, and 
International Council for Harmonisation (ICH) guidelines describing technical 
requirements for registration of pharmaceuticals for human use. 
5.2 Institutional Review Board (IRB) 
The FDA Project Lead or investigator will provide the designated IRB with all required 
documents, including the study protocol and informed consent form. The study will not 
be initiated until appropriate IRB approval is obtained from the designated IRB. The 
investigator will provide the FDA Project Lead with copies of the approval documents 
for the protocol, informed consent form, and all recruiting materials. The designated IRB 
will also receive copies of any original or amended information sheets or pamphlets 
given to the study subject in support of the informed consent process and any 
advertisements or other recruitment material. Such materials will not be employed in the 
study before approval by the designated IRB. 
 
Subjects will be informed that they have the right to contact the IRB or Office for Human 
Research Protections if they have any questions, concerns, complaints, or believe they 
have been harmed by the participation in this research study as a result of investigator 
negligence. Subjects will be given the address and phone number of the IRB. 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 38 of 46 
  
 
6. ADMINISTRATIVE PROCEDURES 
6.1 Responsibilities of the Investigator  
The following administrative items are meant to guide the investigator in the conduct of 
the study but may be subject to change based on industry and government standard 
operating procedures, working practice documents, or guidelines. Changes may be 
reported to the IRB but will not result in protocol amendments. 
6.1.1 Form FDA 1572 
The investigator will complete and sign the Form FDA 1572. 
6.1.2 Adherence to Protocol  
The investigator agrees to conduct the study as outlined in this protocol in accordance 
with the ICH E6(R2) and all applicable guidelines and regulations. 
6.1.3 Reporting Requirements 
 
By participating in this study, the investigator agrees to submit reports of SAEs according 
to the time line and method outlined in the protocol (Section 4.7.3.1.2). In addition, the 
investigator agrees to submit reports to the IRB as appropriate. The investigator also 
agrees to provide the sponsor with an adequate report shortly after completion of the 
investigator’s participation in the study. 
6.1.4 Source Documentation  
By participating in this study, the investigator agrees to maintain adequate case histories 
for the subjects treated as part of the research under this protocol. The investigator agrees 
to maintain accurate eCRFs and source documentation as part of the case histories. 
6.1.5 Retention of Records 
 
The investigator agrees to keep the records stipulated in this protocol and those 
documents that include (but are not limited to) the study-specific documents, 
identification log of all participating subjects, medical records, source worksheets, all 
original signed and dated informed consent forms, subject authorization forms regarding 
the use of personal health information (if separate from the informed consent form), 
copies of all eCRFs, query responses, and detailed records of drug disposition, to enable 
evaluations or audits from regulatory authorities, the sponsor, or its designees. 
Furthermore, ICH 4.9.5 requires the investigator to retain essential documents specified 
in Section 8 of ICH E6(R2) until at least 2 years after the last approval of a marketing 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 39 of 46 
  
 
application for a specified drug indication being investigated or, if an application is not 
approved, until at least 2 years after the investigation is discontinued and regulatory 
authorities are notified. In addition, ICH 4.9.5 states that the study records should be 
retained until an amount of time specified by applicable regulatory requirements or for a 
time specified in the clinical study site agreement between the investigator and sponsor. 
Refer to the clinical study site agreement for the sponsor’s requirements on record 
retention. The investigator should contact and receive written approval from the sponsor 
before disposing of any such documents. 
6.1.6 Financial Disclosure and Obligations 
The investigator is required to provide financial disclosure information to allow the 
sponsor to submit the complete and accurate certification or disclosure statements 
required under 45 CFR 46. In addition, the investigator must provide to the sponsor a 
commitment to update this information promptly if any relevant changes occur during the 
course of the investigation and for 1 year after the completion of the study. 
Neither the sponsor nor the study clinic is financially responsible for further testing or 
treatment of any medical condition that may be detected during the screening process. 
6.2 Confidentiality and Disclosure of Data 
All subjects will sign a HIPAA-compliant authorization form containing the mandated 
core elements and requirements before participation in this clinical study. The sponsor 
and designees affirm and uphold the principle of the subject’s right to protection against 
invasion of privacy. Throughout this study, a subject’s source data will only be linked to 
the clinical site’s electronic data capture system database or documentation via a unique 
identification number. As permitted by all applicable laws and regulations, limited 
subject attributes such as gender, age or date of birth, and subject initials are maintained 
by the clinical site and may be used to verify the subject and accuracy of the subject’s 
unique identification number. The key which allows the clinical site to link the unique 
identification number to the individual subject will be maintained by the clinical site and 
will not be released to the sponsor. The sponsor will not request or attempt to re-identify 
subjects and all personally identifiable information will be removed from the data when 
shared with the sponsor.   
To comply with ICH Guidelines for GCP and to verify compliance with this protocol, the 
sponsor requires that the investigator allow review of the subject’s original medical 
records (source data or documents) by the study monitor, representatives from any 
regulatory authority (e.g., FDA), the sponsor’s designated auditors, and the appropriate 
IRB. These medical records will include, but will not be limited to, clinical laboratory 
test result reports, ECG reports, admission and discharge summaries for hospital 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 40 of 46 
  
 
admissions occurring during a subject’s study participation, and autopsy reports. Access 
to a subject’s original medical records requires the specific authorization of the subject as 
part of the informed consent process. 
Copies of any subject source documents that are provided to the sponsor must have 
certain personally identifiable information removed (i.e., subject name, address, and other 
identifier fields not collected in the subject’s eCRF). 
Data will be maintained and backed up in the electronic data capture system. All access 
to the data is protected by username and password, and each staff member and all sponsor 
staff will have separate access that requires a separate username and password. Access is 
only given to site staff and requested sponsor staff who have completed the appropriate 
training. 
6.3 Subject Consent 
Written informed consent in compliance with 45 CFR 46 will be obtained from each 
subject before entering the study or performing any unusual or nonroutine procedure that 
involves risk to the subject. An informed consent template may be provided by the 
sponsor to the study clinic. If any institution-specific modifications to study-related 
procedures are proposed or made by the study clinic, the consent should be reviewed by 
the sponsor or its designee or both before IRB submission. Once reviewed, the consent 
will be submitted by the investigator to the IRB for review and approval before the start 
of the study. If the informed consent form is revised during the course of the study, all 
active participating subjects must sign the revised form. 
Before enrollment, each prospective subject will be given a full explanation of the study 
and be allowed to read the approved informed consent form. The informed consent 
process will be performed by a clinical research nurse in a private room. The subject will 
be given unlimited time to ask questions regarding study participation, and each subject 
will be questioned to ensure their understanding. Once the investigator is assured that the 
subject understands the implications of participating in the study, the subject will be 
asked to give consent to participate in the study by signing the informed consent form. 
The investigator will provide a copy of the signed informed consent form to the subject. 
The original form will be maintained in the subject’s medical records at the site. 
6.4 Data Collection 
Full details of procedures for data collection and handling will be documented in the data 
management plan, which is initiated with the final protocol receipt. The data management 
plan is a changing document that evolves over the course of the study. 
6.5 Publications 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 41 of 46 
  
 
No information related to or generated by this study will be released to the public until it 
has been reviewed by the sponsor. The sponsor shall own intellectual rights for the data 
and analysis resulting from this study. Authorship on publications will be determined by 
standard journal requirements. 
7. STUDY MANAGEMENT 
7.1 Monitoring 
The sponsor or its designee will monitor the study to ensure that it is being conducted 
according to the protocol, GCP standards, and applicable region-specific requirements, 
and to ensure that study initiation, conduct, and closure are adequate. The investigators 
and the study clinic staff will be expected to cooperate fully with the study monitors and 
personnel or agents of the sponsor and be available during monitoring visits to answer 
questions sufficiently and to provide any missing information. The investigators and their 
institutions will permit direct access to source data/documents for study-related 
monitoring activities, audits, IRB reviews, and regulatory inspections. 
During any on-site visits, the study monitor will:  
 Check and assess the progress of the study 
 Review all informed consent forms 
 Review study data collected 
 Conduct source document verification 
 Identify any issues and address their resolution 
 Verify that the facility remains acceptable 
These monitoring activities will be done in order to verify that the:  
 Data are authentic, accurate, and complete. 
 The safety and rights of the subjects are being protected. 
 The study is being conducted in accordance with the currently approved protocol 
(including any amendments), GCP, and all applicable regulatory requirements. 
In addition, the sponsor, designated auditors, and government inspectors must be allowed 
access to eCRFs, source documents, and other study files that may be required to evaluate 
the conduct of the study. 
7.2 Management of Protocol Amendments and Deviations 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 42 of 46 
  
 
7.2.1 Modification of the Protocol 
Any changes in this research activity, except those necessary to remove an apparent 
immediate hazard to the subject, must be submitted to the sponsor or designee and 
reviewed and approved by the local IRB before implementation. Amendments to the 
protocol must be submitted in writing to the investigator’s IRB for approval before 
subjects are enrolled into an amended protocol. 
7.2.2 Protocol Violations and Deviations 
Any significant protocol deviations that the investigator or study clinic staff believes are 
of major importance (e.g., subject enrolled but not eligible) should be reported to the 
sponsor and the investigator’s IRB as soon as possible. Significant protocol deviations 
may include the following: 
 Deviations from the inclusion/exclusion criteria that may affect subject safety 
 Deviations (omission or delay) of safety monitoring procedures 
 Deviations in obtaining informed consent 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 43 of 46 
  
 
8. REFERENCE LIST 
1. Read DJ. A clinical method for assessing the ventilatory response to carbon 
dioxide. Australas Ann Med. 1967;16(1):20-32. 
2. Cohen R, Finn H, Steen SN. Effect of diazepam and meperidine, alone and in 
combination, on respiratory response to carbon dioxide. Anesth Analg. 
1969;48(3):353-355. 
3. Rigg JR. Ventilatory effects and plasma concentration of morphine in man. Br J 
Anaesth. 1978;50(8):759-765. 
4. Casey K, Duffin J, McAvoy GV. The effect of exercise on the central-
chemoreceptor threshold in man. J Physiol. 1987;383:9-18. 
5. Duffin J, McAvoy GV. The peripheral-chemoreceptor threshold to carbon dioxide 
in man. J Physiol. 1988;406:15-26. 
6. Duffin J. Measuring the respiratory chemoreflexes in humans. Respir Physiol 
Neurobiol. 2011;177(2):71-79. 
7. Mahamed S, Duffin J. Repeated hypoxic exposures change respiratory 
chemoreflex control in humans. J Physiol. 2001;534(Pt. 2):595-603. 
8. Jensen D, Mask G, Tschakovsky ME. Variability of the ventilatory response to 
Duffin's modified hyperoxic and hypoxic rebreathing procedure in healthy awake 
humans. Respir Physiol Neurobiol. 2010;170(2):185-197. 
9. Mohan R, Duffin J. The effect of hypoxia on the ventilatory response to carbon 
dioxide in man. Respir Physiol. 1997;108(2):101-115. 
10. Duffin J, Mohan RM, Vasiliou P, Stephenson R, Mahamed S. A model of the 
chemoreflex control of breathing in humans: model parameters measurement. 
Respir Physiol. 2000;120(1):13-26. 
11. Thomson AJ, Drummond GB, Waring WS, Webb DJ, Maxwell SR. Effects of 
short-term isocapnic hyperoxia and hypoxia on cardiovascular function. J Appl 
Physiol (1985). 2006;101(3):809-816. 
12. Amin HM, Sopchak AM, Esposito BF, Henson LG, Batenhorst RL, Fox AW, 
Camporesi EM. Naloxone-induced and spontaneous reversal of depressed 
ventilatory responses to hypoxia during and after continuous infusion of 
remifentanil or alfentanil. J Pharmacol Exp Ther. 1995;274(1):34-39. 
13. Amin HM, Sopchak AM, Foss JF, Esposito BF, Roizen MF, Camporesi EM. 
Efficacy of methylnaltrexone versus naloxone for reversal of morphine-induced 
depression of hypoxic ventilatory response. Anesth Analg. 1994;78(4):701-705. 
14. Sugimori K, Amin HM, Esposito BF, Seedat MS, Camporesi EM. Effect of slow 
versus fast desaturation on the ventilatory response to hypoxia. J Med. 1996;27(5-
6):277-292. 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 44 of 46 
  
 
15. Easton PA, Slykerman LJ, Anthonisen NR. Ventilatory response to sustained 
hypoxia in normal adults. J Appl Physiol (1985). 1986;61(3):906-911. 
16. Van Mil AH, Spilt A, Van Buchem MA, Bollen EL, Teppema L, Westendorp RG, 
Blauw GJ. Nitric oxide mediates hypoxia-induced cerebral vasodilation in 
humans. J Appl Physiol (1985). 2002;92(3):962-966. 
17. Mahamed S, Cunningham DA, Duffin J. Changes in respiratory control after three 
hours of isocapnic hypoxia in humans. J Physiol. 2003;547(Pt 1):271-281. 
18. Kagawa S, Stafford MJ, Waggener TB, Severinghaus JW. No effect of naloxone 
on hypoxia-induced ventilatory depression in adults. J Appl Physiol Respir 
Environ Exerc Physiol. 1982;52(4):1030-1034. 
19. Cartwright CR, Henson LC, Ward DS. Effects of alfentanil on the ventilatory 
response to sustained hypoxia. Anesthesiology. 1998;89(3):612-619. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 45 of 46 
  
 
9. APPENDICES 
9.1 Appendix A – Schedule of Events 
Table 9-1: Schedule of Events 
 
 
 
 
 

U.S. FOOD AND DRUG ADMINISTRATION  
CDER / Office of Translational Sciences / Office of Clinical 
Pharmacology / Division Applied Regulatory Science 
Protocol Number: SCR-013 Lead-in 
(Version 1.0) 
 
CONFIDENTIAL 
Page 46 of 46 
  
 
9.2 Appendix B – Protocol Revision History 
PROTOCOL REVISION HISTORY 
 
 
Protocol 
Number 
Version 
Effective Date 
Summary of Changes 
SCR-013 
1.0 
27th October 2021 
Developed initial protocol  

Statistical Analysis Plan  
SCR-013 Lead-in: Assessing Variability of the Ventilatory 
Response to Duffin’s Rebreathing Procedure 
 
 
Page 1 of 18 
 
 
Statistical Analysis Plan  
 
 
SCR-013 Lead-in: Assessing Variability of the Ventilatory 
Response to Duffin’s Rebreathing Procedure 
 
 
Sponsor: 
U.S. Food and Drug Administration 
White Oak Building #64, Room 2072 
10903 New Hampshire Avenue 
Silver Spring, MD 20993 
 
Sponsor Study Lead: 
David Strauss, MD, PhD 
Director, Division of Applied Regulatory Science  
U.S. Food and Drug Administration 
Telephone: 301-796-6323 
Email: david.strauss@fda.hhs.gov 
 
Sponsor Medical 
Monitor: 
Keith Burkhart, MD 
U.S. Food and Drug Administration 
Telephone: 301-796-2226 
Email: keith.burkhart@fda.hhs.gov 
 
Project Manager: 
Jeffry Florian, PhD 
U.S. Food and Drug Administration 
Telephone: 301-796-4847 
Email: jeffry.florian@fda.hhs.gov 
 
Version of SAP: 
1.0 
Date of SAP: 
9 Jan 2022 
 
 
CONFIDENTIAL 
 
The concepts and information contained in this document or generated during the study are 
considered proprietary and may not be disclosed in whole or in part without the expressed 
written consent of the U.S. Food and Drug Administration. 

Statistical Analysis Plan  
SCR-013 Lead-in: Assessing Variability of the Ventilatory 
Response to Duffin’s Rebreathing Procedure 
 
 
Page 2 of 18 
 
 
Sponsor Signatures Page 
 
Prepared by 
 
 
 
 
Celine Wang, PharmD 
ORISE Fellow, Division of Applied 
Regulatory Science 
U.S. Food and Drug Administration 
 
Date 
Prepared by 
 
 
 
 
Jeffry Florian, PhD 
Associate Director, Division of Applied 
Regulatory Science 
U.S. Food and Drug Administration 
 
Date 
Approved by 
 
 
 
 
David Strauss, MD, PhD 
Director, Division of Applied Regulatory 
Science  
U.S. Food and Drug Administration 
 
Date 
 
 
SIGN
SIGN
SIGN

Statistical Analysis Plan  
SCR-013 Lead-in: Assessing Variability of the Ventilatory 
Response to Duffin’s Rebreathing Procedure 
 
 
Page 3 of 18 
 
 
Contents 
Sponsor Signatures Page .......................................................................................................... 2 
Contents .................................................................................................................................. 3 
Abbreviations and definitions................................................................................................... 5 
Change Log ......................................................................................................................... 6 
1. 
Introduction...................................................................................................................... 7 
2. 
Study Objectives .............................................................................................................. 7 
3. 
Study Overview................................................................................................................ 8 
3.1 
Study Design ............................................................................................................. 8 
4. 
Study Endpoints ............................................................................................................... 7 
4.1 
Primary Endpoints ..................................................................................................... 7 
4.2 
Exploratory Endpoints ............................................................................................... 7 
4.3 
Sample Size............................................................................................................... 8 
5. 
Analysis Populations ........................................................................................................ 9 
6. 
Data Screening and Acceptance ...................................................................................... 10 
6.1 
Handling of Missing or Incomplete Data .................................................................. 10 
7. 
General Statistical Considerations ................................................................................... 10 
7.1 
Subject Disposition .................................................................................................. 10 
7.2 
Demographic and Baseline Characteristics................................................................ 10 
7.3 
Pharmacodynamic Analyses ..................................................................................... 10 
7.3.1 
Rebreathing Analyses ........................................................................................... 10 
7.3.1.1 
Assessment of Between Occasion and Between Day Variability ......................... 14 
7.3.2 
Exploratory Ventilation Measures ......................................................................... 14 
7.3.3 
Pupillometry Analyses.......................................................................................... 15 
7.4 
Additional Exploratory Analyses .............................................................................. 15 
7.4.1 
Sex Hormone Analyses ........................................................................................ 15 
7.5 
Safety Analyses ....................................................................................................... 15 
7.5.1 
Adverse Events .................................................................................................... 15 
7.5.2 
Clinical Laboratory Tests...................................................................................... 15 
7.5.3 
Vital Sign Measurements ...................................................................................... 16 

Statistical Analysis Plan  
SCR-013 Lead-in: Assessing Variability of the Ventilatory 
Response to Duffin’s Rebreathing Procedure 
 
 
Page 4 of 18 
 
 
7.5.4 
Safety 12-lead Electrocardiograms ........................................................................ 16 
7.5.5 
Physical Examinations.......................................................................................... 16 
7.5.6 
Other Safety Data ................................................................................................. 16 
8. Data Quality Assurance ............................................................................................... 16 
9 
Appendices .................................................................................................................... 17 
9.1 
Nonlinear Regression Model .................................................................................... 17 
10 
References .................................................................................................................. 18 
 
 
 

Statistical Analysis Plan  
SCR-013 Lead-in: Assessing Variability of the Ventilatory 
Response to Duffin’s Rebreathing Procedure 
 
 
Page 5 of 18 
 
 
Abbreviations and definitions 
AE 
Adverse event 
CV 
Coefficient of variation 
ECG 
Electrocardiogram 
eCFRs 
Electronic case report forms 
FDA 
Food and Drug Administration 
mmHg 
Millimeter’s mercury 
PD 
Pharmacodynamic 
PCO2 
Partial pressure carbon dioxide 
PO2 
Partial pressure oxygen 
SAP  
Statistical analysis plan 
SD 
Standard deviation 
TEAE 
Treatment-emergent adverse event 
 
 
 

Statistical Analysis Plan  
SCR-013 Lead-in: Assessing Variability of the Ventilatory 
Response to Duffin’s Rebreathing Procedure 
 
 
Page 6 of 18 
 
 
Change Log 
Version / Date Section 
Summary of Changes 
1.0 /  
9 Jan 2022 
General  Developed initial statistical analysis plan 
 
 

Statistical Analysis Plan  
SCR-013 Lead-in: Assessing Variability of the Ventilatory 
Response to Duffin’s Rebreathing Procedure 
 
 
Page 7 of 18 
 
 
1. Introduction 
This document outlines the proposed statistical methods for data analysis of data collected from 
Protocol ‘SCR-013 Lead-in: Assessing variability of the ventilatory response to Duffin’s 
rebreathing procedure’.  
The following statistical analysis plan (SAP) provides the framework for the summarization of 
the data from this study. The SAP may change due to unforeseen circumstances. Any changes 
made to the SAP will be documented in a change log within the SAP along with a rationale for 
the changes. 
2. Study Objectives 
The objective of this study is to evaluate variability of the ventilatory response to Duffin’s 
rebreathing procedure. 
3. Study Endpoints 
3.1 Primary Endpoints 
1. Baseline minute ventilation when end tidal PCO2 is less than the ventilatory recruitment 
threshold (represents non-chemoreflex drives to breathe) 
2. Ventilatory recruitment threshold (end tidal PCO2 above which minute ventilation starts 
to increase linearly with further increases in end tidal PCO2)  
3. Slope of the PCO2-ventilatory response curve that reflects the increase in minute 
ventilation relative to the increase in end tidal PCO2 (represents chemoreflex sensitivity) 
4. Extrapolated ventilatory recruitment threshold (intersection with X axis) 
3.2 Exploratory Endpoints 
1. Between occasion (within a day) and between day variability for primary endpoints 
2. Additional physiological assessments 
a. Pharmacodynamics (respiratory and cardiovascular) (separate hyperoxic and 
hypoxic) 
i. 
Minute ventilation, tidal volume, respiratory rate, end tidal PCO2, oxygen 
saturation and heart rate during each stage of the rebreathing assessment  
ii. 
Number of apneic events lasting > 10 s 
b. Pharmacodynamics (pupillary) (before and after rebreathing) 
i. 
Maximum pupil diameter before constriction 
ii. 
Dynamic pupillary measurements after a light stimulus  
1. 
Minimum diameter at peak constriction 
2. 
Percent change between min/max diameter 
3. 
Latency of constriction  
4. 
Average constriction velocity 
5. 
Maximum constriction velocity 

Statistical Analysis Plan  
SCR-013 Lead-in: Assessing Variability of the Ventilatory 
Response to Duffin’s Rebreathing Procedure 
 
 
Page 8 of 18 
 
 
6. 
Dilation velocity after peak constriction 
7. 
Time to reach 75% recovery of maximum diameter 
4. Study Overview 
4.1 Study Design 
This study is an unblinded lead-in reproducibility assessment to assess variability of the 
ventilatory response to Duffin’s rebreathing procedure. 
Table 3--1: Study Schedule 
Day -1 
Day 1 
Day 2 
Check-In 
Lead-in PD Assessments 
Check-Out 
Healthy subjects will be enrolled in this study. Subjects will report to the study site for screening 
from Days -28 to -2 and then will return to the site on Day -1 for baseline assessments and 
check-in. After check-in (Day -1), subjects will remain in study site for PD assessments on Day 1 
and check out on Day 2.  
Paired rebreathing procedures (i.e., at two different isoxic end tidal PO2 levels) will be 
performed on Day 1 at 0, 2, 4, and 6 hours. An additional pair of rebreathing procedures will be 
performed on Day 2 before checkout. Subjects will not be administered any drugs in this lead-in 
study. 
A summary of all procedures is described in the Protocol, Schedule of Events. Subjects will be 
discharged from the study after completion of all study procedures. If a subject discontinues 
from the study prematurely, all procedures scheduled for the end of the study will be performed. 
4.2 Sample Size 
The lead-in reproducibility assessment will have up to 10 healthy volunteer participants. This 
sample size was determined empirically and was considered sufficient to gain sufficient 
experience with conducting the rebreathing procedure under different isoxic conditions and to 
characterize variability in measurements obtained using the procedure. No power calculation was 
performed with regards to variability for the study endpoints. 
A published study (Jensen et al., 2010) that also evaluated variability in the Duffin rebreathing 
procedure included 20 subjects under the same isoxic conditions. In that study, the between 
occasion (four assessments on day 1 over a period of 6 hours) and between day (five assessments 
at approximately the same time of day on day 1, 8, 15, 22, and 61) variability was evaluated for 
various rebreathing measures, including the baseline minute ventilation, ventilatory recruitment 
threshold, slope of the PCO2-ventilatory response curve, and extrapolated ventilatory recruitment 
threshold (i.e., same primary endpoints as proposed in the current study).  Between occasion and 

Statistical Analysis Plan  
SCR-013 Lead-in: Assessing Variability of the Ventilatory 
Response to Duffin’s Rebreathing Procedure 
 
 
Page 9 of 18 
 
 
between day variability across all measures proposed as primary endpoints was similar under 
hyperoxic and hypoxic conditions (Table 4-1).    
Table 4-1: Between Occasion and Between Day Variability Reported in Jensen et al for Study Primary 
Endpoints 
Variable 
Hyperoxia 
Hypoxia 
Between Occasion  
CV (%) 
Between Day  
CV (%) 
Between Occasion  
CV (%) 
Between Day  
CV (%) 
Baseline minute  
Ventilation (L/min)  
41.1 
40.0 
39.8 
33.2 
Ventilatory recruitment 
threshold (mmHg) 
4.0 
4.4 
3.8 
4.2 
Slope of PCO2-ventilatory 
response curve (L/min/mmHg) 
37.8 
44.9 
40.4 
46.4 
Extrapolated ventilatory 
recruitment threshold (mmHg) 
5.9 
5.7 
4.3 
5.1 
As the primary aim of the study is to evaluate reproducibility in Duffin rebreathing procedure, 
interim data analyses may be performed after each cohort of participants has completed. If 
reproducibility is acceptable and no procedural changes are needed, additional lead-in cohorts 
may not be enrolled. If any study procedure changes are needed, these study procedure changes 
may be made, and additional participants may be enrolled.  
5. Analysis Populations 
The safety population will include all subjects who enrolled in the study.  
The rebreathing analysis population will include all subjects who enrolled in the study and who 
completed at least one rebreathing assessment on any day or at any timepoint. A rebreathing 
assessment will be considered complete if the subject makes it through the entire procedure (e.g., 
subject makes it to the rebreathing stage with sufficient data to estimate the slope of the PCO2-
ventilatory response curve) and if there are no identifiable issues with how the procedure was 
conducted.   
Potential issues with how the procedure was conducted can include, but are not limited to, a leak 
from the system (e.g., substantially decreasing O2 during rebreathing or no evidence of an 
increase in CO2 during rebreathing), inaccurate readings from the pneumotach (e.g., non-
physiologic baseline minute ventilation readings), failure in the oxygen control loop (oxygen is 
not maintained at proper setpoint during rebreathing procedure), or a subject suddenly increasing 
minute ventilation without subsequent increases in minute ventilation as end tidal pCO2 increases 
(suggesting stress-associated or other non-CO2 mediated hyperventilation confounding the 
ventilatory response to hypercapnia through [H+] chemoreceptors). Determination of the 

Statistical Analysis Plan  
SCR-013 Lead-in: Assessing Variability of the Ventilatory 
Response to Duffin’s Rebreathing Procedure 
 
 
Page 10 of 18 
 
 
completeness of each rebreathing assessment and its individual components/measurements will 
be performed manually by a member of the data analysis team. 
6. Data Screening and Acceptance 
6.1 Handling of Missing or Incomplete Data 
The following approaches will be used for the handling of missing or incomplete data: 
• Missing PD data (e.g., subject discontinued from study; subject could not successfully 
complete rebreathing at a time point) will not be imputed.   
• Pupillometry data collected from the PLR®-3000 pupillometer device flagged as being 
anomalous based on device error codes will be excluded from analyses  
7. General Statistical Considerations 
All data will be presented in data listings. Data from subjects excluded from an analysis 
population will be presented in the data listings, but not included in the calculation of summary 
statistics. Demographic and baseline characteristics will be summarized overall and by treatment 
for all subjects. 
7.1 Subject Disposition 
The number of subjects who enroll in the study and the number and percentage of subjects who 
complete each assessment will be presented. The frequency and percentage of subjects who 
withdraw or discontinue from the study and the reason for withdrawal or discontinuation will be 
summarized. 
7.2 Demographic and Baseline Characteristics 
Continuous demographic and baseline characteristic variables (age, height, weight, and body 
mass index) will be summarized overall and by treatment using descriptive statistics (number of 
subjects, mean, standard deviation [SD], median, minimum, and maximum). The number and 
percentage of subjects in each class of categorical demographic and baseline characteristic 
variables will also be summarized. 
7.3 Pharmacodynamic Analyses 
7.3.1 Rebreathing Analyses  
Figure 7-1 below shows representative time-course results from the rebreathing procedure, with 
minute ventilation, oxygen percentage, and end-tidal PCO2 shown from top to bottom.  
Throughout the procedure subjects will undergo different steps designed to reduce noise and 
variability.   Initially, subjects breathe room air for 5 minutes and relax as much as possible. 
Around the 5-minute mark, the subject is instructed to begin hyperventilating (primarily through 
deep breathing) for 5 min to achieve an end tidal PCO2 of approximately 20-25 mmHg prior to 

Statistical Analysis Plan  
SCR-013 Lead-in: Assessing Variability of the Ventilatory 
Response to Duffin’s Rebreathing Procedure 
 
 
Page 11 of 18 
 
 
rebreathing. During this rebreathing test, subjects will be maintained at one of 2 different isoxic 
end tidal PO2. The isoxia at a hyperoxic (150 mmHg) or hypoxic (50 mmHg) end tidal PO2 will 
be maintained by providing a computer-controlled flow of 100% O2 to the rebreathing bag.   
Each of these steps can be noted on the example figure.  In this example, relaxation while 
breathing room air was performed for 5-minutes.  From 5 to 10 minutes, the test subject began 
hyperventilation and slight decreases in O2% and PCO2 with an increase in minute ventilation are 
observed.  Finally, the test subject begins rebreathing at 10 minutes.  After taking deep breaths, 
PCO2 in the rebreathing circuit and the lungs equilibrate and then PCO2 increases linearly to 
approximately 55 mm Hg at the end of rebreathing in this subject.  An increase in minute 
ventilation is triggered at a certain point during rebreathing (ventilatory recruitment threshold), 
and both measures increase until completion of the procedure based on subject tolerability or 
until other procedure stopping criteria are reached (see protocol and rebreathing SOP for full 
list). If the subject’s data does not follow these trends due to a leak in the apparatus (e.g., 
incomplete seal between the mask and subject’s face), inaccurate readings from the pneumotach, 
issues in operator recording of the data, or the subject not having a linear increase in minute 
ventilation as end tidal PCO2 increases prior to reaching PCO2 of 55 mm Hg, the run will not be 
considered completed and will be excluded from subsequent data analysis that is dependent on 
the minute ventilation / end tidal PCO2 regression line.   
 

Statistical Analysis Plan  
SCR-013 Lead-in: Assessing Variability of the Ventilatory 
Response to Duffin’s Rebreathing Procedure 
 
 
Page 12 of 18 
 
 
Figure 7-1: Sample data from a test subject during relaxation, hyperventilation, and 
rebreathing, with minute ventilation, O2 percentage, and PCO2 mm Hg over time during these 
experimental steps. A representative hyperoxic test is shown at the top (oxygen percentage 
maintained at ~21% during rebreathing) and a representative hypoxic test is shown at the 
bottom (oxygen percentage maintained at ~7.1% during rebreathing). 
 
From these results, minute ventilation and end tidal PCO2 levels over time will be matched. End 
tidal PCO2 will be calculated as % CO2 • (barometric pressure – 47 mmHg [water vapor]). 
Separate linear and nonlinear (hockey stick model) regressions will be fit to the experiment data. 
The nonlinear and linear regressions will be fit to data from the rebreathing stage of the 
rebreathing procedure (i.e., during isoxic hypercapnia).  This functional relationship for the 
nonlinear model assumes a constant relationship between minute ventilation and end tidal PCO2 
up until a threshold value is reached, after which minute ventilation and end tidal PCO2 both 
increase linearly.  Example code for the nonlinear fit can be found in the Appendix, and a sample 
result from the test case can be seen in Figure 7-2.  Because of potential inconsistencies in when 
the stage transition may have been flagged by the device operator (pressed while 
hyperventilation was still ongoing) and as the isoxic setpoint is being achieved, the first 15 
seconds of data from the rebreathing stage will be removed when calculating the linear 
regression. In addition, all other exploratory pharmacodynamic endpoints will be automatically 
calculated. 
 
Because of the potential for outlying data points in the minute ventilation and/or end-tidal PCO2 
signal at each individual breath, it is sometimes necessary to exclude outlying data points from 
the regression. Such outlying data points can be introduced by subject postural changes, sighing, 
hiccups, talking, or faulty sensors. In addition, there may be a need to remove additional data at 

Statistical Analysis Plan  
SCR-013 Lead-in: Assessing Variability of the Ventilatory 
Response to Duffin’s Rebreathing Procedure 
 
 
Page 13 of 18 
 
 
the beginning of rebreathing or to remove data at the end of rebreathing (e.g., nonlinearity as 
subject approaches or exceeds end tidal PCO2 55 mmHg).  
Outlying values for the regressions will be identified automatically through evaluation of 
standardized residuals after performing the initial regression (i.e., standardized residuals > 2).  
Then, the data analysis team will be provided with time course plots from the full assessment (all 
rebreathing procedure stages with stage transitions marked) and regression results using data 
from the rebreathing stage. The data analysis team will evaluate the completeness of the 
rebreathing assessment and visually assess the automatically calculated values in comparison to 
the underlying data. Any automatically calculated values that do not accurately capture the 
underlying data will be flagged, additional outliers will be removed, and the automated 
calculations will be re-run. A subject may have incomplete rebreathing data not supporting 
calculation of one or more primary endpoints, but other endpoint measures would still be used 
for analyses. 
If the nonlinear regression converges, the regression results will be used to derive all four 
primary endpoints for that subject at that assessment. Baseline minute ventilation when end tidal 
PCO2 is less than the ventilatory recruitment threshold and the slope of the PCO2-ventilatory 
response curve are V0 and sslope from the regression, respectively. The ventilatory recruitment 
threshold is calculated as the intersection between the two line segments. The extrapolated 
ventilatory recruitment threshold is calculated as the intersection of the PCO2-ventilatory 
response curve and the x-axis. If the nonlinear regression does not converge, results from the 
linear regression will be used to determine the slope of the PCO2-ventilatory response curve and 
data from the relaxation stage will be used to determine baseline minute ventilation. The 
ventilatory recruitment threshold and the intersection of the PCO2-ventilatory response curve 
and the x-axis will then be calculated as described above. All modeling analyses for the 
regressions will be performed in statistical software. 
 

Statistical Analysis Plan  
SCR-013 Lead-in: Assessing Variability of the Ventilatory 
Response to Duffin’s Rebreathing Procedure 
 
 
Page 14 of 18 
 
 
Figure 7-2: Sample nonlinear least squares fit between minute ventilation and end tidal PCO2 
for test subject under hyperoxic (left) or hypoxic (right) conditions. The four primary 
endpoints are annotated on each figure. 
 
7.3.1.1 Assessment of Between Occasion and Between Day Variability  
Each of the primary endpoints (denoted as endprimary) will be summarized by day and time point 
using descriptive statistics (number of subjects, mean, SD, median, minimum, and maximum).  
Time course profiles for all primary endpoints will be calculated and plotted for the overall 
group and individuals.  Linear-mixed effect modeling will be used to determine between 
occasion, between day, and between subject variability, where USUBJID, OCC, DAY are the 
subject identifier and measurements obtained at different times across the study.  The model will 
not include any fixed effects. In the event that all variabilities cannot be simultaneously 
estimated, separate analyses will be run for data from the first 6 hours (between occasion 
variability) and for data from time 0 at the start of day 1 and at the start of day 2 (i.e., 24 hours) 
(between day variability): 
endprimary ~ (1|USUBJID/OCC/DAY) 
 
7.3.2 Exploratory Ventilation Measures 
Other exploratory respiratory measures will be calculated and summarized from information 
collected during planned rebreathing procedures.  Data from the initial relaxation/resting and 
hyperventilation stages of the procedure will be used to calculate ventilation, tidal volume and 
end tidal PCO2.  All measures will be summary by day, treatment, and time using descriptive 

Statistical Analysis Plan  
SCR-013 Lead-in: Assessing Variability of the Ventilatory 
Response to Duffin’s Rebreathing Procedure 
 
 
Page 15 of 18 
 
 
statistics (number of subjects, mean, SD, median, minimum, and maximum). Time course 
profiles for all measures will be calculated and plotted.    
Apneic events lasting > 10 seconds will be determined using data collected during the 
rebreathing procedure.  An event is defined as the absence of inspiratory flow (as measured by 
the pneumotachograph) for at least 10 s during this period.  These parameters will be 
summarized using descriptive statistics (number of subjects, number of events) by study part, 
treatment, day, and time, as appropriate.  
7.3.3 Pupillometry Analyses 
Maximum pupil diameter before constriction and dynamic pupillary measurements after a light 
stimulus will be measured during relaxation and after each rebreathing assessment. This will 
include measurements before and after the rebreathing assessment and from the left and right 
eye. These exploratory parameters will be summarized using descriptive statistics (number of 
subjects, mean, SD, median, minimum, and maximum), and time courses summaries of each 
pupillary measurement will be generated.  Pupillary changes will be compared to baseline 
measurements.  In addition, time course pupillary changes will be compared to time course 
ventilatory changes across treatments to evaluate concordance between these measures. 
7.4 Additional Exploratory Analyses 
7.4.1 Sex Hormone Analyses 
Estrogen and progesterone levels will be collected at the start of the study (Day 1 prior to the 
first rebreathing assessment).  These parameters will be summarized using descriptive statistics 
(number of subjects, mean, SD, minimum, median, and maximum) and may be used to explore 
variation in ventilation across the menstrual cycle.   
7.5 Safety Analyses 
7.5.1 Adverse Events 
All adverse events (AEs) will be coded using the latest version of the Medical Dictionary for 
Regulatory Activities. The incidence of treatment-emergent adverse event (TEAEs), organized 
by system organ class and frequency, will be summarized by seriousness, severity, relationship 
to treatment, and by treatment at onset of the TEAE. A detailed listing of serious AEs and 
TEAEs leading to withdrawal will also be provided. 
7.5.2 Clinical Laboratory Tests 
Clinical laboratory results (hematology, serum chemistry, and urinalysis) will be summarized 
using descriptive statistics (number of subjects, mean, SD, minimum, median, and maximum). 
Clinical laboratory results will be classified as normal or abnormal, according to the reference 
ranges of the individual parameter. The number and percentage of subjects with abnormal 

Statistical Analysis Plan  
SCR-013 Lead-in: Assessing Variability of the Ventilatory 
Response to Duffin’s Rebreathing Procedure 
 
 
Page 16 of 18 
 
 
laboratory results will be provided. No statistical testing will be performed on clinical laboratory 
data. 
7.5.3 Vital Sign Measurements 
Vital sign measurements and changes from baseline will be summarized using descriptive 
statistics (number of subjects, mean, SD, minimum, median, and maximum) by treatment and 
time point. 
7.5.4 Safety 12-lead Electrocardiograms 
Safety 12-lead ECG data will be summarized using descriptive statistics (number of subjects, 
mean, SD, minimum, median, and maximum) by treatment and time point. 
7.5.5 Physical Examinations 
Physical examination findings will be presented in a data listing, and abnormal physical 
examination findings will be recorded as AEs. 
7.5.6 Other Safety Data 
All concomitant medication usage and medications that changed in daily dose, frequency, or 
both since the subject provided informed consent will be summarized for each subject. 
8. Data Quality Assurance 
Completed electronic case report forms (eCRFs) are required for each subject randomly assigned 
to the study drug. Electronic data entry will be accomplished through the ClinSpark® remote 
electronic data capture system, which allows for on-site data entry and data management. This 
system provides immediate, direct data transfer to the database, as well as immediate detection of 
discrepancies, enabling site coordinators to resolve and manage discrepancies in a timely 
manner. Each person involved with the study will have an individual identification code and 
password that allows for record traceability. Thus, the system, and subsequently any 
investigative reviews, can identify coordinators, investigators, and individuals who have entered 
or modified records. 
Furthermore, the investigator retains full responsibility for the accuracy and authenticity of all 
data entered into the electronic data capture system. The completed dataset and their associated 
files are the sole property of the sponsor and should not be made available in any form to third 
parties, except for appropriate governmental health or regulatory authorities, without written 
permission of the sponsor. 
 
 

Statistical Analysis Plan  
SCR-013 Lead-in: Assessing Variability of the Ventilatory 
Response to Duffin’s Rebreathing Procedure 
 
 
Page 17 of 18 
 
 
9 Appendices 
9.1 Nonlinear Regression Model 
The rebreathing procedure consists of three separate periods: i) breathing room air, termed 
‘relaxation’ period; ii) hyperventilation (primarily through deep breathing); and iii) rebreathing 
at one of 2 different isoxic end tidal PO2 (PetO2) (i.e., at 50 mm Hg and 150 mm Hg).  For the 
purposes of data analysis and determination of the primary endpoints, data from iii) will be used 
to estimate the ventilatory response to hypercapnia curve.  This relationship, which is 
characterized by a flat portion between minute ventilation and end-tidal PCO2 followed by a 
period of linear increase, can be represented using a hockey-stick function.  For purposes of 
these analyses, the following equation is pre-specified to describe data from the procedure: 
Vi = V0  (if PCO2 ≤ inflection point) 
Vi = V0 * (1 + ((PCO2,i – PCO2,inflection)*sslope /V0)g)1/g (if PCO2 > inflection point) 
Here, Vi is the minute ventilation that corresponds to an end-tidal CO2 of PCO2,i, V0 is baseline 
minute ventilation rate on the flat portion of the curve, PCO2,inflection is the end-tidal CO2 where 
increase from baseline minute ventilation begins, sslope is the slope of the curve, and g is a shape 
parameter in the hockey stick function that is set to 20 to provide the desired curvature.  Outlier 
measures, introduced by subject postural changes, sighing, talking, hiccups and other subject 
behaviors, will be identified based on evaluation of standardized residuals from the nonlinear 
regression (i.e., standardized residuals > 2 SD).  Coding for the nonlinear function will be 
performed in R (version 3.6.3 or later).  An example of the coded function is provided below. 
ff <- function(p, v0, b, s) { 
  vs <- (p-b)*s 
  vs[which(vs<0)] <- 0 
  a <- 0.95 
  g <- 1/(1-a) 
  v <- v0*(1 + (vs/v0)**g)**(1/g) 
} 
 
fits<-function (x){ 
  nls(v ~ ff(p,v0,b,s), 
      lower=list(v0=5,  b=0,  s=0), 
      start=list(v0=8,  b=30,  s=1), 
      upper=list(v0=15, b=100, s=5), 
      trace=F, algorithm="port", data=x) 
} 
 

Statistical Analysis Plan  
SCR-013 Lead-in: Assessing Variability of the Ventilatory 
Response to Duffin’s Rebreathing Procedure 
 
 
Page 18 of 18 
 
 
10 References 
Jensen D, Mask G, Tschakovsky ME. Variability of the ventilatory response to Duffin's modified 
hyperoxic and hypoxic rebreathing procedure in healthy awake humans. Respir Physiol 
Neurobiol. 2010;170(2):185-197.