Clinical trial NCT02742129
Conditions: Heart Failure
Sponsor: Adrian Hernandez
Start date: 2016-08-10; Completion date: 2017-12-27
Enrollment: 105 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
A randomized, double-blind, placebo-controlled crossover study to assess the effect of inorganic nitrite (NO2) on aerobic capacity (peak VO2) after four weeks of dosing. Approximately 100 participants will be enrolled in this 2\*2 crossover study.

Detailed description:
Screen potential HFpEF patients for eligibility criteria and interest

Study Visit 1

• Initiate consent process and obtain written informed consent.

* Confirm with the participant that HF symptoms are the primary limitation to activity. If so, they proceed to CPET screening. If not, they are considered a screen fail.
* Obtain baseline bloods \*- CBC, complete chemistry panel, biomarkers, biorepository and genetics (if agreed to participate) .
* Obtain CPET to verify patient eligibility peak VO2 ≤ 75% predicted and RER ≥ 1.0 (within 3 days prior to randomization) and establish baseline value.
* Qualifying patients perform additional baseline studies: history, assess NYHA class, physical exam, ECG, and KCCQ.
* Open label, single-dose run-in where patient receives maximal dose (80 mg) inhaled inorganic nitrite. Patients who do not tolerate the run-in are considered screen failures.
* Randomize qualifying patients.
* Dispense phase-1 study drug, nebulizers and accelerometers
* Participants take no study drug for two weeks (baseline).
* Participants take 46 mg study drug at a minimum of 4 hours apart, 3 times a day, during active part of the day for 7 days.
* Participants take 80 mg study drug at a minimum of 4 hours apart, 3 times a day, during active part of the day until returning for study visit 2 (at least 42 days but up to 49 days post-baseline visit).
* If side effects develop, participants can down-titrate to the previous dose.
* Participants are called frequently to reinforce study procedures and assess compliance.

Study Visit 2 (42-49 Days Post Study Visit 1)

• Participant holds study drug on day of visit.

* Review history, assess NYHA class, perform physical exam and KCCQ.
* Obtain blood draws \*\* - CBC, complete chemistry panel, biomarkers, biorepository (if agreed to participate).
* Obtain limited echocardiogram \*\*.
* Perform CPET with Study Drug administered immediately before starting the CPET (primary endpoint).
* Change out accelerometer and dispense phase-2 study drug.
* Participants take no study drug for two weeks (washout).
* Participants take 46 mg study drug at a minimum of 4 hours apart, 3 times a day, during active part of the day for 7 days.
* Participants take 80 mg study drug at a minimum of 4 hours apart, 3 times a day, during active part of the day until returning for study visit 3 (at least 42 but up to 49 days after study visit 2).
* If side effects develop Participants can down-titrate to the previously tolerated dose.
* Participants are called frequently to reinforce study procedures and assess compliance.

Study Visit 3 (42-49 Days Post Study Visit 2) • Participant holds study drug on day of visit.

• Review history, assess NYHA class, perform physical exam and KCCQ

* Obtain blood draws\*\* - CBC, complete chemistry panel, biomarkers, biorepository (if agreed to participate).
* Obtain limited echocardiogram\*\*.
* Perform CPET with Study Drug administered immediately before starting the CPET (primary endpoint).
* Return accelerometer and phase-2 study drug.
* End of study drug (phase out).

Phone Visit and End of Study (14 Days Post Study Visit 3)

• A final phone visit is conducted to assess for adverse events.

\*Visit 1: baseline blood draw needs to be completed prior to the CPET (if this is not feasible, then they cannot be obtained for at least 3 hours post the CPET and prior to the run-in test dose).

\*\*Visit 2 and Visit 3: blood draws and limited echo need to be obtained prior to study drug administration (if this is not feasible, then it cannot be obtained for at least 3 hours post study drug administration)

Arms: arm 1: Phase 1: Participants will wear an accelerometer device daily but take no study drug for 14 days (washout period). On day 15, participants begin phase I study drug Nebulized Sodium Nitrite (AIR001) at 46 mg, at minimum of 4 hours apart, for 3 doses per day during the active portion of the participant's day. On day 22 participants increase study drug dose to 80 mg at the same frequency. Regardless of participant's ability to tolerate study drug or if the participant requires down-titration, participants will begin Phase 2. Phase 2: Is identical to Phase 1 except subject will be taking Placebo instead of AIR001. arm 2: Phase 1: Participants will wear an accelerometer device daily but take no study drug for 14 days (washout period). On day 15, participants begin phase I study drug (Placebo) at 46 mg, at minimum of 4 hours apart, for 3 doses per day during the active portion of the participant's day. On day 22 participants increase study drug dose to 80 mg at the same frequency. Regardless of participant's ability to tolerate study drug or if the participant requires down-titration, participants will begin Phase 2. Phase 2: Is identical to Phase 1 except subject will be taking Nebulized Sodium Nitrite (AIR001) instead of Placebo.
Interventions: intervention 1: Nebulized Sodium Nitrite intervention 2: Placebo
Intervention descriptions: intervention 1: Inhaled, nebulized inorganic sodium nitrite vs. inhaled, nebulized placebo at a dose of 80 mg (or maximally tolerated dose) administered at a minimum of 4 hours apart, three times per day, during the active part of the day. intervention 2: Inhaled, nebulized placebo vs. inhaled, nebulized inorganic sodium nitrite at a dose of 80 mg (or maximally tolerated dose) administered at a minimum of 4 hours apart, three times per day, during the active part of the day.

Protocol documents:
Prot_001.pdf: 
 
INDIE-HFpEF Protocol v1.3, 22April2016 (Final) 
 
    
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Protocol for the Heart Failure Clinical Research Network 
 
Inorganic Nitrite Delivery to Improve Exercise Capacity in HFpEF 
INDIE-HFpEF 
 
 
Compiled by: 
The Heart Failure Network Research Group 
Version: April 22, 2016 
 
Distributed by: 
Heart Failure Network Coordinating Center 
Duke Clinical Research Institute 
Duke University 
P.O. Box 17969 
Durham, NC 27715 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
INDIE-HFpEF Protocol v1.3, 22April2016 (Final) 
 
    
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Study Funding: 
 
 
 
National Heart, Lung, and Blood Institute 
 
 
 
 
 
 
 
National Institutes of Health 
 
 
 
 
 
 
 
 
Aires Pharmaceuticals, Inc. 
 
 
 
 
 
 
 
San Diego, California 
 
Project Officer: 
 
 
 
Patrice Desvigne-Nickens, MD 
 
Network Chair: 
 
 
 
Eugene Braunwald, MD 
 
Protocol Principal Investigator: Barry Borlaug, MD (Mayo Clinic) 
 
Protocol Development Team:  Margaret Redfield, MD (Mayo Clinic) 
Sanjiv Shah, MD (Northwestern University) 
Greg Lewis, MD (Harvard) 
Marc Semigran, MD (Harvard) 
Julio Chirinos, MD (University of Pennsylvania) 
Martin LeWinter, MD (University of Vermont)  
 
 
 
 
 
 
 
Victor Davila, MD (Washington University) 
Lisa de las Fuentes, MD (Washington University) 
Susan Joseph, MD (Washington University) 
Ilia Halatchev, MD (Washington University) 
 
 
 
 
 
 
 
 
Sponsor/ 
 
 
 
 
Adrian Hernandez, MD  
Coordinating Center:   
 
Kevin Anstrom, PhD 
 
 
 
 
 
 
 
Duke Clinical Research Institute 
Durham, NC 
 
Biomarker Core Laboratory:  
Russell Tracy, PhD 
 
 
 
 
 
 
 
University of Vermont 
 
 
 
 
 
 
 
Burlington, VT 
 
Genetics Core Lab: 
 
 
Jean Rouleau, MD and Simon de Denus, MD 
 
 
 
 
 
 
 
University of Montreal  
 
 
 
 
 
 
 
Montreal, Quebec 
 
Echo Core Lab: 
 
 
 
Grace Lin, MD and Jae Oh, MD 
 
 
 
 
 
 
 
Mayo Clinic 
 
 
 
 
 
 
 
Rochester, MN 
 
Accelerometry Core:  
 
James Levine MD 
Mayo Clinic 
 
 
 
 
 
 
 
Rochester, MN 
 
CPET Core Lab:  
 
 
Greg Lewis, MD 
Marc Semigran, MD 


 
INDIE-HFpEF Protocol v1.3, 22April2016 (Final) 
 
    
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Massachusetts General 
Boston, MA 
 
1 
List of Abbreviations 
Abbreviation 
Definition 
6MWD 
6-Minute Walk Distance 
6MWT 
6-Minute Walk Test 
AAU 
Arbitrary Accelerometry Units 
ACC 
American College of Cardiology 
ACE 
Angiotensin-Converting Enzyme 
AE 
Adverse Event 
AHA 
American Heart Association 
ARB 
Angiotensin Receptor Blocker 
AXM 
Accelerometer 
BNP 
Brain Natriuretic Peptide 
BUN 
Blood Urea Nitrogen 
CC 
Coordinating Center 
CFR 
Code of Federal Regulations 
cGMP 
Cyclic Guanosine Monophosphate 
CPET 
Cardiopulmonary Exercise Test 
CRF 
Case Report Form 
DBP 
Diastolic Blood Pressure 
DCC 
Data Coordinating Center 
DCRI 
Duke Clinical Research Institute 
DHF 
Diastolic heart failure 
DSMB 
Data Safety Monitoring Board 
ECG 
Electrocardiogram 
eCRF 
Electronic Case Report Form 
EDC 
Electronic Data Capture 
EE 
Expedited Event 
EF 
Ejection fraction 
ER 
Emergency Room 
FDA 
Food and Drug Administration 
GFR 
Glomerular Filtration Rate 
GTN 
Glyceryl Trinitrate  
HF 
Heart Failure 
HFN 
Heart Failure Clinical Research Network 
HFN  
Heart Failure Network 
HFpEF 
heart failure with preserved ejection fraction 
HFrEF 
Heart Failure with reduced Ejection Fraction 
HFSA 
Heart Failure Society of America 
ICF 
Informed Consent Form 
 


 
INDIE-HFpEF Protocol v1.3, 22April2016 (Final) 
 
    
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ICH  
International Conference on Harmonization 
IND 
Investigational New Drug 
IRB 
Institutional Review Board 
ISDN 
Isosorbide Dinitrate 
ISMN 
Isosorbide Mononitrate 
ITT 
Intention To Treat 
IV 
Intravenous 
IVRS 
Interactive Voice Recording System 
KCCQ 
Kansas City Cardiomyopathy Questionnaire 
LV 
Left Ventricular 
NO 
Nitric Oxide 
NO2 
Inorganic Nitrite 
NO3 
Inorganic Nitrate 
NO-CGMP 
Nitric Oxide and cGMP transduction pathway 
NT-proBNP 
Pro-B-type Natriuretic Peptide 
NYHA 
New York Heart Association 
PA 
Pulmonary artery 
PCI 
Percutaneous Coronary Intervention 
PCP 
Primary Care Physician 
RCC 
Regional Clinical Center 
RCT 
Randomized Clinical Trial 
SAE 
Serious Adverse Event 
SAR 
Suspected Adverse Reaction 
SBP 
Systolic Blood Pressure 
VO2 
Oxygen consumption 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
INDIE-HFpEF Protocol v1.3, 22April2016 (Final) 
 
    
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2 
Table of Contents 
 
1 
List of Abbreviations .......................................................................................................................... 3 
2 
Table of Contents .............................................................................................................................. 5 
3 
EXECUTIVE SUMMARY ....................................................................................................................... 8 
4 
OBJECTIVES AND HYPOTHESES ........................................................................................................ 12 
4.1 
Primary Objectives ...................................................................................................................... 12 
4.2 
Secondary Objectives .................................................................................................................. 12 
4.3 
Tertiary Objectives ...................................................................................................................... 13 
5 
BACKGROUND AND SIGNIFICANCE ................................................................................................. 13 
6 
PRELIMINARY STUDIES .................................................................................................................... 15 
7 
BASIC STUDY DESIGN ....................................................................................................................... 18 
7.1 
Screening / Baseline .................................................................................................................... 18 
7.2 
Randomization ............................................................................................................................ 19 
7.3 
Study Intervention Phase ............................................................................................................ 19 
7.4 
Follow-up Phase .......................................................................................................................... 22 
8 
STUDY FLOW DIAGRAM ................................................................................................................... 22 
9 
STUDY POPULATION AND ELIGIBILITY CRITERIA ............................................................................. 23 
9.1 
Study Population ......................................................................................................................... 23 
9.2 
Inclusion Criteria ......................................................................................................................... 23 
9.3 
Exclusion Criteria ......................................................................................................................... 24 
10 
TREATMENT INTERVENTIONS.......................................................................................................... 25 
10.1 
Intervention ................................................................................................................................ 25 
10.2 
Drug Dispensing .......................................................................................................................... 26 
10.3 
Storage, accountability and destruction ..................................................................................... 27 
10.4 
Drug accountability ..................................................................................................................... 27 
10.5 
Destruction.................................................................................................................................. 27 
10.6 
Randomization, Stratification and Blinding ................................................................................ 27 
10.7 
Unblinding ................................................................................................................................... 27 
10.8 
Concomitant Medication ............................................................................................................ 28 
10.9 
Side Effect Risk Reduction Plan ................................................................................................... 28 
11 
RECRUITMENT AND SCREENING PROCEDURES ............................................................................... 28 


 
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11.1 
Common Recruitment Procedures ............................................................................................. 28 
11.2 
Estimated Enrollment Period ...................................................................................................... 28 
11.3 
Informed Consent Procedures .................................................................................................... 28 
11.3.1 Informed Consent ........................................................................................................... 28 
11.3.2 Confidentiality and HIPAA Requirements ................................................................... 28 
11.3.3 Protections of Human Subjects .................................................................................... 29 
11.3.4 Summary of the Risks and Benefits ............................................................................ 29 
12 
BASELINE EVALUATION AND RANDOMIZATION VISIT .................................................................... 29 
12.1 
Screening ..................................................................................................................................... 29 
12.2 
Baseline/Randomization Visit (Visit 1) ........................................................................................ 30 
13 
FOLLOW-UP EVALUATIONS ............................................................................................................. 31 
13.1 
Study Visits .................................................................................................................................. 31 
13.2 
Phone and Other Media Follow-up ............................................................................................. 32 
14 
OUTCOME DETERMINATIONS ......................................................................................................... 33 
14.1 
Primary Endpoint ........................................................................................................................ 33 
14.2 
Secondary Endpoints .................................................................................................................. 33 
14.3 
Tertiary Endpoints ....................................................................................................................... 33 
15 
METHODS TO PROMOTE ADHERENCE ............................................................................................ 34 
15.1 
Adherence to Study Procedures ................................................................................................. 34 
16 
PARTICIPANT SAFETY AND ADVERSE EVENTS ................................................................................. 34 
16.1 
Institutional Review Boards ........................................................................................................ 34 
16.2 
Definitions ................................................................................................................................... 34 
16.2.1 Adverse Events .............................................................................................................. 34 
16.2.2 Suspected Adverse Reaction ....................................................................................... 34 
16.2.3 Serious Adverse Events (SAE) .................................................................................... 34 
16.2.4 Laboratory Test Abnormalities ..................................................................................... 35 
16.2.5 Assessment of Causal Relationship ............................................................................ 35 
16.2.6 Assessment of Adverse Event Severity ...................................................................... 35 
16.2.7 Expectedness ................................................................................................................. 35 
16.3 
Anticipated Disease Related Events ............................................................................................ 36 
16.3.1 Recording and Reporting of Adverse Events ............................................................. 36 
16.3.2 Suspected Unexpected Serious Adverse Reaction .................................................. 37 


 
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16.3.3 Pregnancy ....................................................................................................................... 37 
17 
STATISTICAL CONSIDERATIONS ....................................................................................................... 37 
17.1 
Overview ..................................................................................................................................... 37 
17.2 
Design and Analytical Criteria ..................................................................................................... 38 
17.3 
Analysis of Secondary and Tertiary Endpoints ............................................................................ 38 
17.4 
Analysis of Safety Data and Statistical Monitoring Plan ............................................................. 38 
17.5 
Sample Size and Power Calculation ............................................................................................ 39 
18 
DATA MANAGEMENT PROCEDURES ............................................................................................... 40 
18.1 
Overview of Data Management .................................................................................................. 40 
18.2 
Data Security ............................................................................................................................... 40 
18.3 
Publication Policy ........................................................................................................................ 40 
19 
STUDY ADMINISTRATION ................................................................................................................ 41 
19.1 
Data and Safety Monitoring Board ............................................................................................. 41 
19.2 
Coordinating Center .................................................................................................................... 41 
19.3 
Core Laboratories ........................................................................................................................ 41 
19.3.1 Cardiopulmonary Exercise Testing Core Laboratory ................................................ 41 
19.3.2 Biomarker Core Laboratories ....................................................................................... 41 
19.3.3 Echocardiograph Core Laboratory .............................................................................. 41 
19.3.4 Physical Activity Measurement Core Laboratory ...................................................... 41 
20 
REGULATORY ISSUES ....................................................................................................................... 42 
20.1 
Ethics and Good Clinical Practice ................................................................................................ 42 
20.2 
Institutional Review Board/Independent Ethics Committee ...................................................... 42 
20.3 
Informed Consent ....................................................................................................................... 42 
21 
Remote Monitoring ......................................................................................................................... 42 
22 
Appendix A – Schedule of Assessments .......................................................................................... 44 
23 
REFERENCES..................................................................................................................................... 46 
 
 
 


 
INDIE-HFpEF Protocol v1.3, 22April2016 (Final) 
 
    
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3 
EXECUTIVE SUMMARY 
 
Title 
Inorganic Nitrite Delivery to Improve Exercise Capacity in HFpEF 
(INDIE-HFpEF) 
Indication 
Heart failure with preserved ejection fraction 
Location 
Approximately 20 clinical centers in the United States  
Brief Rationale 
 
 
 
 
Approximately half of Americans with heart failure (HF) have preserved 
ejection fraction (HFpEF).  There is no proven treatment that improves 
outcome.  The pathophysiology is complex and includes left ventricular 
(LV) systolic and diastolic dysfunction, pulmonary vascular disease, 
endothelial dysfunction, and peripheral abnormalities.  Multiple lines of 
evidence point to impaired NO-cGMP bioavailability as playing a central 
role in each of these abnormalities. 
 
NO-cGMP levels can be increased using direct NO donors, such as the 
organic nitrates.  The HFN NEAT trial tested whether treatment with 
organic nitrates (isosorbide mononitrate, ISMN) for 4 weeks could 
improve daily activity levels as assessed by accelerometry. 
 
However, organic nitrates have several shortcomings, including the 
development of tolerance, greater vulnerability to hypotension in patients 
with HFpEF, development of ‘pseudo-tolerance’, where chronic 
venodilation leads to renal sodium retention, and increases in oxidative 
stress and endothelial dysfunction. 
 
An alternative strategy to restore Nitric oxide-cyclic guanosine 
monophosphate (NO-cGMP) signaling is via inorganic nitrite (NO2). While 
previously considered to be an inert byproduct of NO metabolism, NO2 
has been shown to be an important in vivo reservoir for NO generation.  
Notably, reduction of NO2 to NO is enhanced under hypoxic and 
acidemic conditions, meaning that the molecule becomes most active at 
the time of greatest need (e.g., during exercise).   
 
Ancillary analyses from the CHAMPION trial have revealed that reduction 
in pulmonary artery (PA) and PA wedge pressure are associated with 
marked reduction in HF hospitalizations in HFpEF, emphasizing the 
importance of filling pressure reduction to improve morbidity in HFpEF. 
To this end, recent studies have shown that acute NO2 infusion markedly 
blunts the increase in PA and PA wedge pressure during exercise in 
HFpEF, while improving cardiac output reserve.  A similar noninvasive 
study using inorganic nitrate (NO3) (naturally occurring in beetroot juice) 
similarly improved aerobic capacity and enhanced cardiac output reserve 
and systemic vasodilation in HFpEF. 
 
Accordingly, INDIE-HFpEF will test the effects of longer term NO2 
therapy on exercise capacity and chronic daily activity levels in HFpEF. 


 
INDIE-HFpEF Protocol v1.3, 22April2016 (Final) 
 
    
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Study Design 
 
A randomized, double-blind, placebo-controlled crossover study to 
assess the effect of inorganic nitrite (NO2) on aerobic capacity (peak 
VO2) after four weeks of dosing. Approximately 100 participants will be 
enrolled in this 2*2 crossover study.  
Treatment 
Inhaled, nebulized inorganic sodium nitrite vs. inhaled, nebulized placebo 
at a dose of 80 mg (or maximally tolerated dose) administered at a 
minimum of 4 hours apart, three times per day, during the active part of 
the day.  
Primary Objective 
and Endpoint 
The primary endpoint will be the peak VO2 after 4 weeks treatment with 
inorganic nitrite as compared to the peak VO2 after 4 weeks treatment 
with placebo as assessed by cardiopulmonary exercise testing (CPET) 
performed at peak drug levels.  
Secondary 
Objectives and 
Endpoints 
1. To evaluate whether inorganic nitrite improves submaximal activity 
tolerance chronically. 
 
Average arbitrary accelerometer units (AAU) during at least 14 
days and up to 21 days of the maximally tolerated dose of study 
drug (from 28 days post Study Visit 1 until Study Visit 2 and 
from 28 days post Study Visit 2 until Study Visit 3).  
 
2. To evaluate whether inorganic nitrite improves quality of life. 
 
Quality of Life (Kansas City Cardiomyopathy Questionnaire 
score, KCCQ)  
 
NYHA class 
 
Participant preference for study phase (active vs. placebo) 
 
3. To evaluate whether inorganic nitrite improves chronic filling 
pressures as assessed by echocardiography and natriuretic 
peptide levels.  
 
E/e’ ratio on echocardiography 
 
Left atrial volume index on echocardiography 
 
Pulmonary artery systolic pressure on echocardiography 
 
N-terminal pro-B-type natriuretic peptide level 
 
4. To evaluate whether inorganic nitrite improves ventilatory efficiency 
or submaximal exercise capacity at peak drug levels. 
 
VE/VCO2 slope 
 
VO2 at ventilatory threshold 
 
5. To evaluate safety and tolerability of inhaled, nebulized sodium 
inorganic nitrite.  
 
Headache  
 
Lightheadedness/orthostasis  
 
Other AEs 
 
Abbreviated Study 
Flow 
 
Screen potential HFpEF patients for eligibility criteria and interest 
 
 


 
INDIE-HFpEF Protocol v1.3, 22April2016 (Final) 
 
    
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Study Visit 1 
 
Initiate consent process and obtain written informed consent.  
 
Confirm with the participant that HF symptoms are the primary 
limitation to activity. If so, they proceed to CPET screening.  If not, 
they are considered a screen fail.  
 
Obtain baseline bloods *- CBC, complete chemistry panel, 
biomarkers, biorepository and genetics (if agreed to participate) . 
 
Obtain CPET to verify patient eligibility peak VO2 ≤ 75% predicted 
and RER ≥ 1.0 (within 3 days prior to randomization) and establish 
baseline value. 
 
Qualifying patients perform additional baseline studies: history, 
assess NYHA class, physical exam, ECG, and KCCQ. 
 
Open label, single-dose run-in where patient receives maximal 
dose (80 mg) inhaled inorganic nitrite.  Patients who do not tolerate 
the run-in are considered screen failures. 
 
Randomize qualifying patients. 
 
Dispense phase-1 study drug, nebulizers and accelerometers 
 
Participants take no study drug for two weeks (baseline).  
 
Participants take 46 mg study drug at a minimum of 4 hours apart, 
3 times a day, during active part of the day for 7 days. 
 
Participants take 80 mg study drug at a minimum of 4 hours apart, 
3 times a day, during active part of the day until returning for study 
visit 2 (at least 42 days but up to 49 days post-baseline visit). 
 
If side effects develop, participants can down-titrate to the previous 
dose. 
 
Participants are called frequently to reinforce study procedures and 
assess compliance. 
 
Study Visit 2 (42-49 Days Post Study Visit 1) 
 
Participant holds study drug on day of visit. 
 
Review history, assess NYHA class, perform physical exam and 
KCCQ. 
 
Obtain blood draws ** - CBC, complete chemistry panel, 
biomarkers, biorepository (if agreed to participate). 
 
Obtain limited echocardiogram **. 
 
Perform CPET with Study Drug administered immediately before 
starting the CPET (primary endpoint). 
 
Change out accelerometer and dispense phase-2 study drug. 
 
Participants take no study drug for two weeks (washout). 
 
Participants take 46 mg study drug at a minimum of 4 hours apart, 
3 times a day, during active part of the day for 7 days. 
 
Participants take 80 mg study drug at a minimum of 4 hours apart, 
3 times a day, during active part of the day until returning for study 
visit 3 (at least 42 but up to 49 days after study visit 2). 
 
If side effects develop Participants can down-titrate to the 
previously tolerated dose. 
 
Participants are called frequently to reinforce study procedures and 
assess compliance. 
 


 
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Study Visit 3 (42-49 Days Post Study Visit 2) 
 
Participant holds study drug on day of visit. 
 
Review history, assess NYHA class, perform physical exam and 
KCCQ 
 
Obtain blood draws** - CBC, complete chemistry panel, 
biomarkers, biorepository (if agreed to participate).  
 
Obtain limited echocardiogram**. 
 
Perform CPET with Study Drug administered immediately before 
starting the CPET (primary endpoint). 
 
Return accelerometer and phase-2 study drug. 
 
End of study drug (phase out). 
 
Phone Visit and End of Study (14 Days Post Study Visit 3) 
 
A final phone visit is conducted to assess for adverse events.  
 
*Visit 1:  baseline blood draw needs to be completed prior to the CPET (if 
this is not feasible, then they cannot be obtained for at least 3 hours post 
the CPET and prior to the run-in test dose). 
**Visit 2 and Visit 3:  blood draws and limited echo need to be obtained 
prior to study drug administration (if this is not feasible, then it cannot be 
obtained for at least 3 hours post study drug administration) 


 
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4 
 OBJECTIVES AND HYPOTHESES 
4.1 
Primary Objectives 
To evaluate whether inhaled, nebulized inorganic sodium nitrite, as compared to placebo, 
improves maximal exercise capacity as assessed by cardiopulmonary exercise testing 
performed at peak drug levels. 
 
The primary hypothesis of the INDIE-HFpEF study is that inorganic nitrite, compared to placebo, 
will improve exercise capacity (peak oxygen consumption, VO2). 
 
The significance of this study is that it will provide evidence as to whether inorganic nitrite 
therapy improves aerobic capacity in patients with HFpEF.  This would then provide rationale to 
pursue a larger phase 3 trial of inorganic nitrate/nitrite therapy in HFpEF. 
4.2 
Secondary Objectives 
1. To evaluate whether inorganic nitrite, compared to placebo, improves submaximal exercise 
tolerance chronically throughout the course of the day, as well as at trough when plasma nitrite 
levels have dropped toward normal as assessed by:  
 
Chronic activity by averaged arbitrary accelerometry units (AAU14) during the final 
weeks of study phases (at least two and up to three weeks of peak dose of study 
drug/placebo) 
2. To evaluate whether inorganic nitrite in comparison to placebo improves quality of life as 
assessed by: 
 
KCCQ score 
 
NYHA class 
 
Patient preference for study phase (active vs. placebo) 
 
3. To test whether inorganic nitrite improves chronic filling pressures as assessed by 
echocardiography and blood natriuretic peptide levels 
 
E/e’ ratio on echocardiography 
 
Left atrial volume index on echocardiography 
 
Pulmonary artery systolic pressure on echocardiography 
 
NT-proBNP levels 
 
4. To evaluate whether inorganic nitrite improves other measures of cardiopulmonary function 
 
VE/VCO2 slope (ventilatory efficiency) 
 
VO2 at ventilatory threshold (submaximal exercise capacity) 
 
5. To evaluate the safety and tolerability of inhaled, nebulized inorganic nitrite at 80 or 46 mg 
 
Headache 
 
Lightheadedness/orthostasis 
 
Other adverse events 
 


 
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4.3 
Tertiary Objectives 
1.  Pre-specified subgroup analyses will include examination of the primary endpoint in patients 
according to baseline characteristics:  
 NT-proBNP >400pg/ml vs. ≤400 pg/ml  
 Systolic blood pressure above and below median value 
 PASP>60 mmHg vs <60 mmHg 
 Age above and below median value 
 Women vs. men 
 Estimated glomerular filtration rate by MDRD equation above and below median 
value  
 Subjects on active drug at study visit vs not on study drug at study visit 
 Subjects in atrial fibrillation/flutter vs not  
 Diabetic vs. non-diabetic patients  
 Patients with or without symptoms of exertional chest pain 
 
2. Novel accelerometry endpoints (guided by HFN NEAT ancillary paper analysis) 
 
3. Plasma nitrosothiols, cystatin-C, cGMP levels 
5 
BACKGROUND AND SIGNIFICANCE 
Therapy for HFpEF is a major unmet need: Heart failure (HF) is the leading cause of 
hospitalization among older Americans and a major public health problem. The direct and 
indirect costs of HF in 2010 were estimated at $31 billion; this is expected to increase to $70 
billion by 2030, when nearly 8 million Americans (1 in 33) will have HF.1 One-half of patients 
with HF have preserved ejection fraction (HFpEF), and the prevalence of HFpEF is growing 
relative to HF with reduced ejection fraction (HFrEF) by approximately 10% per decade.2 These 
secular trends, coupled with the current aging of the U.S. population and increasing prevalence 
of obesity and metabolic syndrome ensure that HFpEF will be the dominant form of HF by 
2020.3 Importantly, no treatment has been proven to reduce mortality in HFpEF, and few 
interventions have been observed to improve symptoms or quality of life (QOL). Thus, 
identification of effective treatments for HFpEF is an enormous unmet public health need. 
Targeting hemodynamic mechanisms of exercise intolerance in HFpEF: People with 
HFpEF suffer from dyspnea and fatigue with activity, limiting exercise tolerance and promoting 
sedentary lifestyle. Exercise capacity in HFpEF is limited by both “central” and “peripheral” 
factors. Central (cardiac) abnormalities include pathologic increases in filling pressures caused 
by diastolic dysfunction, exercise-induced pulmonary hypertension, and blunted increases in 
cardiac output (CO) with exertion.4-13 Peripheral abnormalities include changes in skeletal 
muscle capacity and endothelial dysfunction.14-17  
The fact that cardiac pressures are often normal at rest but elevated with stress has created a 
major barrier to treatment in HFpEF,10 because interventions that reduce filling pressures during 
exercise also reduce resting pressures, increasing the vulnerability to hypotension in people 
with HFpEF.18  It has recently been pointed out that HFpEF is a heterogenous syndrome and 
that this may explain the failure of previous trials testing therapies that are efficacious in HFrEF.  
However, elevation in left ventricular filling pressures and pulmonary artery pressures during 


 
INDIE-HFpEF Protocol v1.3, 22April2016 (Final) 
 
    
 14
 
 
 
 
exercise is a universal finding in HFpEF that contributes to symptoms and thus represents a 
viable target.  Other abnormalities including limited cardiac output reserve or peripheral 
limitations may apply to some patients but not others. 
Improving hemodynamics by enhancing nitric oxide signaling in HFpEF: Nitric oxide-cyclic 
guanosine monophosphate (NO-cGMP) dependent, flow-mediated vasodilation is impaired in 
people with HFpEF.9  Intriguingly, the magnitude of impaired vasodilation is directly correlated 
with severity of dyspnea and fatigue during submaximal exercise, as well as the degree of 
reduction in exercise capacity. Cardiomyocyte studies from human tissue have shown that NO-
cGMP deficiency in HFpEF is coupled to increases in diastolic ventricular stiffness.19 These 
observations have led Paulus and Tschope to recently propose that concentric ventricular 
remodeling, fibrosis and increased cardiomyocyte stiffness in HFpEF are all downstream 
consequences of impaired NO-cGMP signaling, making this a key pathway to target in trials.20   
Ongoing trials in HFpEF have or are targeting NO-cGMP through organic NO donors 
(isosorbide mononitrate, HFN NEAT trial), decreased degradation of natriuretic peptides (LCZ 
696, PARAGON trial) or direct stimulation of cGMP production (guanylate cyclase activator, 
SOCRATES-preserved).  Inorganic nitrate-nitrite represents a novel candidate molecule to 
preferentially restore NO-cGMP signaling during exercise stress, since reduction of inorganic 
nitrite to NO is enhanced in the setting of tissue hypoxia and acidosis, as develop during 
exercise.   
Exercise capacity is a clinically-meaningful, patient-centric endpoint in HF trials: Exercise 
intolerance is the ultimate symptomatic manifestation of HFpEF and can be objectively 
quantified by measurement of peak VO2.21 Cardiopulmonary exercise testing (CPET), unlike 
other measures such as 6-minute walk distance (6 MWD), permits direct assessment of the 
organ system limiting gas exchange. This is crucial in people with HFpEF who are older-aged 
and often display pulmonary, mechanical, or orthopedic limitations to exercise that might 
contribute to low 6 MWD. CPET also allows for assessment of the adequacy of exercise effort 
(respiratory exchange ratio, RER) along with other relevant gas exchange measurements that 
reflect HF severity and mirror hemodynamic abnormalities observed (VE/VCO2 slope, VO2 at 
anaerobic threshold).  Importantly, unlike changes in alternative trial end points, such as 
natriuretic peptide levels or echo parameters, there is intrinsic value to patients associated with 
improving exercise capacity, particularly if improvement of peak exercise capacity translates to 
improved functionality and quality of life. As assessment of peak VO2 is more technically 
challenging in HF trials and remains somewhat controversial, the INDIE study will provide 
unique information about the concordance of peak exercise capacity with daily activity and 
submaximal exercise capacity. 
Crossover study design: Crossover studies have been widely used in cardiovascular 
medicine, including trials testing the effects of organic nitrates in patients with coronary artery 
disease23-28 or HFpEF, as in the HFN NEAT trial.29  In a crossover design each subject is 
exposed to both interventions, which aids recruitment, and the interindividual variability is 
reduced, allowing for a much smaller sample size.  The drawbacks of a crossover design 
include the potential for a period effect and for carry-over effects.   


 
INDIE-HFpEF Protocol v1.3, 22April2016 (Final) 
 
    
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The two potential sources of bias are minimized in INDIE-HFpEF because the exposure to study 
drug is relatively short (4 weeks), decreasing the likelihood of chronic remodeling effects, and 
because the washout period is sufficiently long to minimize risk of carryover effect.  As in NEAT-
HFpEF, the concept in INDIE-HFpEF is that hemodynamic, cardiac and possibly peripheral 
benefits induced by inorganic nitrite will improve exercise capacity, as shown in two single-dose 
acute studies performed in HFpEF (see Preliminary Studies).   
6 
PRELIMINARY STUDIES  
Exercise capacity, quantified by the peak VO2 attained 
during exercise, is severely reduced in HFpEF.4, 9, 13, 14 
According to the Fick principle, VO2 is equal to the 
product of cardiac output (CO) and arterial venous O2 
content difference (A-V O2diff). These components have 
been termed ‘central’ and ‘peripheral’ determinants of 
exercise limitation.  Recent studies in HFpEF have 
identified abnormalities in both CO and A-V O2diff reserve 
in patients with HFpEF that variably correlate with 
reduced peak VO2 in different patients.13, 14, 30, 31  
However, regardless of the primacy of central vs 
peripheral mechanisms, elevation left ventricular filling pressures during exercise is universal in 
patients with HFpEF (Figure 1). 
 
Among the potential molecular pathways underlying cardiac reserve dysfunction, abnormalities 
in NO-cGMP-dependent signaling have emerged as important players.9, 19-21, 32, 33 In the RELAX 
trial we observed that sildenafil, which increases cGMP by inhibiting its breakdown, failed to 
improve exercise capacity or QOL in 
people with HFpEF.34 This suggests that 
enhancing NO-cGMP through decreased 
breakdown is not an effective approach 
in this population. Alternative strategies 
include direct provision of NO-cGMP, as 
with organic NO donors, and this 
hypothesis was tested in the HFN NEAT 
trial. However, organic nitrates are 
limited by tolerance35, increases in 
oxidative stress that may cause 
endothelial dysfunction, pseudo-
tolerance, and greater risk of 
hypotension in people with HFpEF owing 
to the steep end-systolic pressure 
volume relationship (ESPVR) observed in this HF phenotype (Figure 2).18  
 
A key factor in HFpEF that complicates treatment is that the hemodynamic perturbations 


 
INDIE-HFpEF Protocol v1.3, 22April2016 (Final) 
 
    
 16
 
 
 
 
causing morbidity (e.g., high filling pressures) are sometimes mild at rest or present only during 
stress—being absent at rest (Figure 1).10, 13 To most effectively treat these derangements, the 
ideal therapy would become more effective during stress, without untoward effects on resting 
cardiovascular homeostasis. 
 
Inorganic nitrates represent an attractive 
alternative approach to treat NO-cGMP 
deficiency in HFpEF.36-38  These molecules 
were previously considered as inert 
byproducts of endogenous NO metabolism, 
but recent studies have shown that inorganic 
nitrite functions as an alternative in vivo source 
of NO to the classical oxygen-dependent L-
arginine-NO-synthase pathway (Figure 3).  In 
the body, dietary nitrate is absorbed and then 
concentrated in the salivary glands.  Oral 
commensal bacteria then reduce nitrate to 
nitrite which is absorbed and can then be reduced to NO.  
In contrast to direct NO donors such as organic nitrates, there is no tolerance with inorganic 
nitrate/nitrite37 and importantly, reduction of inorganic nitrite to NO is enhanced under conditions 
of physiologic duress, such as tissue hypoxia and acidosis,36-38 meaning that inorganic nitrite 
becomes most effective to unload the heart precisely at the time when it would be most needed 
in patients with HFpEF, without causing hypotension at rest when filling pressures, pulmonary 
artery pressures, and CO are normal (Figure 1).  
 
In addition, inorganic nitrite has beneficial effects on skeletal muscle bioenergetics39 and 
mitochondrial respiration, improving the O2 cost of work during submaximal exercise.40-42 
Recently inorganic nitrite has been shown to improve conduit artery compliance and reduce 
pulsatile aortic load.43  Thus, inorganic nitrite represents an ideal candidate molecule to target 
both the central and peripheral limitations to exercise in HFpEF. 
 
Zamani et al. recently performed a double-blind crossover trial (n=17) showing that a single 
dose of inorganic nitrate 
(12.9 mmol) administered 
prior to exercise improves 
peak VO2 while enhancing 
CO reserve and systemic 
vasodilation in patients 
with HFpEF (Figure 4).44  
Nitrate also decreased 
arterial wave reflections 
and late systolic pressure 
augmentation.  In another 
study enrolling participants 
without HF, Omar et al. 


 
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 17
 
 
 
 
observed that inorganic nitrite vasodilates conduit arteries, an effect coupled to significant 
reductions in central blood pressure, which is typically elevated in HFpEF patients.43 
Recently, Borlaug et al. completed a randomized, double blind, placebo controlled trial testing 
the effects of acute intravenous sodium nitrite on exercise hemodynamics and ventricular 
reserve during submaximal exercise in patients with HFpEF (n=28).45   
Subjects underwent resting and 
exercise invasive hemodynamic 
assessment and were then 
treated with either intravenous 
sodium nitrite (250 μg/kg/min * 5 
min) or saline.  After 15 minutes, 
repeat rest and exercise 
hemodynamic assessment was 
performed at the same workload.  
After inorganic nitrite infusion, 
plasma levels increased to 
8.39±1.88 μM.45 
Compared to placebo, NO2- 
treated subjects displayed 
markedly attenuated increases in 
LV filling pressures during 
exercise, with a lower slope of 
the PA pressure-flow 
relationship, improved exercise CO reserve, and enhanced CO/VO2 slope (Figure 5).45   
 
Enhanced CO reserve in inorganic nitrite treated subjects was entirely due to improved stroke 
volume reserve, as there was no effect on exercise heart rate.45 Greater increases left 
ventricular stroke work with exercise were noted with inorganic nitrite, indicating an 
improvement in myocardial performance, independent of effects on cardiac loading.   
More recently, 
Borlaug et al. 
have performed 
an open label 
pilot study 
testing the 
hemodynamic 
effects of 
inhaled nitrite 
(n=4, 80 mg 
dose) at rest 
and during 
exercise, using the same protocol as the intravenous nitrite study above.45  Inhaled nitrite 
therapy produced similar increases in plasma nitrite levels as the intravenous formulation 
(Figure 6A), with similar reductions in PCWP at rest and during exercise as was observed in the 
intravenous study (Figure 6B). 


 
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In the HFpEF exercise studies of both Zamani et al.44 and Borlaug et al.45 there was improved 
vasodilation (reduction in systemic vascular resistance) with exercise but no significant 
decrease in blood pressure with nitrate/nitrite, in contrast to marked reductions in arterial 
pressure previously noted with organic nitrates in HFpEF.18  Nitrite infusion achieving plasma 
levels 30% higher than what will be tested in INDIE-HF had no effect on arterial blood pressure 
in a study of primates (95±3 vs 98±2 mmHg, p=0.8).37   
 
Inhaled nitrite has been administered at a dose of 90 mg three times daily in healthy volunteers, 
with no hypotension and no orthostatic intolerance.46  In an unpublished, open label study, 29 
patients with Group 1 pulmonary hypertension were treated with inhaled nitrite for 16 weeks, 
with open label extension out to 57 weeks.47  There were no withdrawals and no symptomatic 
hypotensive episodes.  In this trial, systolic blood pressure was unchanged during treatment, 
from 117 (95% CI: 108, 126) mmHg at study entry to 114 (107, 121) mmHg at study completion 
(p=NS).  Diastolic pressure similarly remained unchanged from 74 (68, 80) mmHg to 73 (67, 78) 
mmHg at study completion (p=NS).    
7 
BASIC STUDY DESIGN 
The INDIE-HFpEF study is a randomized, double-blind, placebo-controlled crossover study to 
assess the effect of inorganic nitrite on peak exercise capacity (peak VO2) as assessed by 
cardiopulmonary exercise testing (CPET).  
7.1 
Screening / Baseline  
Patients with a diagnosis of HFpEF are screened for entry criteria. Willing participants meeting 
entry criteria will be consented and questioned to confirm that HF symptoms are the primary 
limitation to activity.  If the participant does not meet this criteria, they will be considered screen 
failures and will not continue.  
 
All consented participants continuing to meet the screening criteria will undergo baseline blood 
draws to include complete blood count (CBC), complete chemistry panel (Sodium, Potassium, 
Chloride, Carbon Dioxide, BUN, Creatinine, Glucose, AL, AST, Alkaline Phosphatase & Total 
Bilirubin), HFN biomarkers (cystatin C, NT-proBNP, cGMP and nitrosothiols), HFN biorepository 
and genetics samples (if agreed to participate).  Afterwards, the participant will undergo a 
baseline HFN study-specific CPET to confirm Peak VO2 ≤75% with peak respiratory exchange 
ratio ≥ 1.0.  If the participant does not meet the CPET criteria, they will be considered screen 
failures and will not continue.  
 
For participants that meet the above criteria, the remaining baseline studies will be performed:   
history and physical exam, NYHA class assessment, ECG and KCCQ.  
 
Note:  The baseline bloods are to be done prior to the CPET as the biomarker values can be 
affected from the exertion of the CPET.  In the event this is not feasible, the blood draws will 
need to be obtained at least 3 hours post CPET but before the run-in test dose. 


 
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All participants will then undergo a single-dose, open label run-in to test study drug tolerance.  
Just prior to this test dose, a seated and standing BP must be obtained.  If the seated SBP is 
<115 mmHg or the standing SBP is <90 mmHg the patient is considered a screen failure and 
should not receive the test dose.   
 
Participants that pass the SBP criteria will receive one full dose (80 mg) of nebulized sodium 
nitrite with monitoring for 60 minutes following dosage for adverse effects or symptomatic 
hypotension.  Systemic blood pressures will be monitored (seated and 3 minutes after standing) 
every 15 minutes for 60 minutes.  Participants developing hypotension (SBP < 90mmHg seated 
OR standing), lightheadedness, or who otherwise do not tolerate the single-dose run-in will be 
considered run-in failures and will not be randomized.     
7.2 
Randomization 
Eligible patients successfully completing baseline and run-in activities will then be randomized 
using procedures determined by the Coordinating Center (CC) to one of 2 treatment groups 
(placebo first with crossover to inorganic nitrite or inorganic nitrite first with crossover to 
placebo). A permuted block randomization method stratified by site will be used to ensure 
relatively equal distribution of participants to each arm within each clinical site. 
 
Following randomization, participants will receive training in use of the nebulizer device and the 
accelerometry belt.  Participants will be required to demonstrate capability to administer study 
drug using the nebulizer device on their own prior to dismissal from Study Visit 1.  
7.3 
Study Intervention Phase 
Phase 1—Begins after Study Visit 1 which is considered day 0 of Phase 1 as follows:  
 
Participants will wear the accelerometer devices daily but take no study drug for 14 days 
(washout period). 
 
Study staff will call participants 4-7 days post-baseline visit to reinforce study 
procedures. 
 
Study staff will call participants 11-14 days post-baseline visit to reinforce study 
procedures. 
 
On day 15 post-baseline visit, participants will begin study drug at 46 mg, at minimum of 
4 hours apart for 3 doses per day during the active portion of the participant’s day. 
Participants will be encouraged to call study staff promptly to report any adverse 
reactions. 
 
Within 1-2 days of study drug initiation, study staff will call participants to discuss 
tolerability and potential issues with delivery and assess for adverse events. 
o In the case of study drug intolerance due to headache, participants will be 
encouraged to treat with acetaminophen and continue study drug.  
o For other intolerable side effects, study drug is discontinued but participants 
continue other study procedures.  
 
Study staff will call participants 18-21 days post-baseline visit to reinforce study 
procedures. 
 
On day 22 post-baseline visit, participants will increase study drug dose to 80 mg, at a 
minimum of 4 hours apart for 3 doses per day during the active portion of the 


 
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participant’s day. Participants will be encouraged to call study staff promptly to report 
any adverse reactions. 
 
Within 1-2 days of study drug up-titration, study staff will call participants to discuss 
tolerability and potential issues with delivery and assess for adverse events. 
o In the case of study drug intolerance due to headache, participants will be 
encouraged to treat with acetaminophen and continue study drug.  
o For other intolerable side effects, the dose of study drug is reduced to the 
previously tolerated dose (46 mg) but participants continue other study procedures.  
 
Study staff will call participants 25-28 days post-baseline visit to reinforce study 
procedures. 
 
Study staff will call participants 32-35 days post-baseline visit to reinforce study 
procedures. 
 
Study staff will call participants 3-4 days prior to Study Visit 2 to reinforce study 
procedures and important details regarding Study Visit 2. 
 
During each call, participants will be encouraged to be active within the limitations 
imposed by their HF symptoms. 
 
Regardless of participant’s ability to tolerate study drug or if the participant requires 
down-titration, study participants will begin Phase 2 as described below. 
 
Phase 2—Begins with Study Visit 2 which is considered day 0 of Phase 2:  
Study Visit 2 is performed at least 42 days but up to 49 days post-baseline visit. Participants 
should continue to wear the accelerometer and maintain study drug treatment up until Study 
Visit 2 regardless of timing of Study Visit 2.  
 
At Study Visit 2: 
 
Study staff will confirm that the participant did not take study drug on the day of 
Study Visit 2 and undergo the following: 
o Conduct interim history, physical exam, NYHA class assessment and 
KCCQ. 
o Obtain blood draws prior to the CPET (in the event this is cannot be 
completed prior to study drug administration, then it cannot be obtained 
until at least 3 hours post study drug administration) : 
 
CBC 
 
Complete chemistry panel 
 
Biomarkers 
 
Biorepository (if agreed to participate )  
o Obtain limited echocardiography prior to study drug administration and 
CPET.  (In the event, the echocardiography cannot be completed prior to 
study drug administration, then it cannot be obtained until at least 3 hours 
post study drug administration). 
 
Next, participants will undergo CPET with a dose of Phase 1 study drug 
administered immediately before the CPET under the supervision of study staff. 
 
Accelerometers are returned and changed out. 
 
Phase 1 study drug is returned. 
 
Phase 2 study drug is dispensed. 
After Study Visit 2:  
 
Participants will wear the accelerometer devices daily but take no study drug for 
14 days (washout period). 
 
Study staff will call participants 4-7 days post-Study Visit 2 to reinforce study 
procedures. 


 
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 
Study staff will call participants 11-14 days post-Study Visit 2 to reinforce study 
procedures. 
 
On day 15 post-Study Visit 2, participants will begin study drug at 46 mg, at a 
minimum of 4 hours apart, for 3 doses per day, during the active portion of the 
participant’s day. Participants will be encouraged to call study staff promptly to 
report any adverse reactions. 
 
Within 1-2 days of study drug initiation, study staff will call participants to 
discuss tolerability and potential issues with delivery and assess for adverse 
events. 
o In the case of study drug intolerance due to headache, participants will be 
encouraged to treat with acetaminophen and continue study drug.  
o For other intolerable side effects, study drug is discontinued but 
participants continue other study procedures.  
 
Study staff will call participants 18-21 days post-Study Visit 2 to reinforce study 
procedures. 
 
On day 22 post-Study Visit 2, participants will increase study drug dose to 80 
mg, at a minimum of 4 hours apart for 3 doses per day during the active portion 
of the participant’s day. Participants will be encouraged to call study staff 
promptly to report any adverse reactions. 
 
Within 1-2 days of study drug up-titration, study staff will call participants to 
discuss tolerability and potential issues with delivery and assess for adverse 
events. 
o In the case of study drug intolerance due to headache, participants will be 
encouraged to treat with acetaminophen and continue study drug.  
o For other intolerable side effects, the dose of study drug is reduced to the 
previously tolerated dose (46 mg) but participants continue other study 
procedures.  
 
Study staff will call participants 25-28 days post-Study Visit 2 to reinforce study 
procedures. 
 
Study staff will call participants 32-35 days post-Study Visit 2 to reinforce study 
procedures. 
 
Study staff will call participants 3-4 days prior to Study Visit 3 to reinforce study 
procedures and important details regarding Study Visit 3. 
 
During each call, participants will be encouraged to be active within the 
limitations imposed by their HF symptoms. 
 
Regardless of participant’s ability to tolerate study drug or if the participant 
requires down-titration, study participants will return for Study Visit 3 as 
described below. 
 
Completion—Begins with Study Visit 3:  
Study Visit 3 is performed at least 42 days but up to 49 days post-Study Visit 2. Participants 
should continue to wear the accelerometer and maintain study drug treatment up until Study 
Visit 3 regardless of timing of Study Visit 3.  
 
At Study Visit 3: 
 
Study staff will confirm that the participant did not take study drug on the day of 
Study Visit 3 and undergo the following: 
o Conduct interim history, physical exam, NYHA class assessment and 
KCCQ. 


 
INDIE-HFpEF Protocol v1.3, 22April2016 (Final) 
 
    
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o Obtain blood draws prior to the CPET(in the event this cannot be 
completed prior to study drug administration, then it cannot be obtained 
until at least 3 hours post study drug administration) : 
 
CBC 
 
Complete chemistry panel 
 
Biomarkers 
 
Biorepository (if agreed to participate )  
o Obtain limited echocardiography prior to study drug administration and 
CPET.  (In the event, the echocardiography cannot be completed prior to 
study drug administration, then it cannot be obtained until at least 3 hours 
post study drug administration). 
 
Next, participants will undergo CPET with a dose of Phase 1 study drug 
administered immediately before the CPET under the supervision of study 
staff. 
 
Accelerometers are returned. 
 
Phase 2 study drug is returned. 
 
Participants are asked to indicate the study phase during which they felt better 
(Participant preference secondary endpoint).  
 
Participants will be reminded of 2 week follow-up phone call. 
7.4 
Follow-up Phase 
 A final phone visit is conducted 2 weeks after Study Visit 3 to assess AEs and clinical stability.   
8 
STUDY FLOW DIAGRAM 
 
 


 
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9 
STUDY POPULATION AND ELIGIBILITY CRITERIA 
9.1 
Study Population 
Patients suitable for this protocol are individuals with chronic HF who have normal ejection 
fraction (LVEF ≥ 50%). 
9.2 
Inclusion Criteria 
1. Age ≥ 40 years 
2. Symptoms of dyspnea (NYHA class II-IV) without evidence of a non-cardiac or ischemic 
explanation for dyspnea 
3. EF ≥ 50% as determined on imaging study within 12 months of enrollment with no change 
in clinical status suggesting potential for deterioration in systolic function 
4. One of the following:  
 
Previous hospitalization for HF with radiographic evidence (pulmonary venous 
hypertension, vascular congestion, interstitial edema, pleural effusion) of 
pulmonary congestion or 
 
Catheterization documented elevated filling pressures at rest (PCWP ≥15 or 
LVEDP ≥18) or with exercise (PCWP ≥25) or 
 
Elevated NT-proBNP (>400 pg/ml) or BNP(>200 pg/ml) or 
 
Echo evidence of diastolic dysfunction/elevated filling pressures manifest by 
medial E/e’ ratio≥15 and/or left atrial enlargement and chronic treatment with a 
loop diuretic for signs or symptoms of heart failure 
5. Heart failure is primary factor limiting activity as indicated by answering # 2 to the 
following question: 
 
My ability to be active is most limited by: 
1. Joint, foot, leg, hip or back pain 
2. Shortness of breath and/or fatigue and/or chest pain 
3. Unsteadiness or dizziness 
4. Lifestyle, weather, or I just don’t like to be active 
 
6. Peak VO2 ≤75% predicted with peak respiratory exchange ratio≥1.0 CPET 
 
Normal 
Values for 
Peak VO2* 
Criteria (ml/kg/min) 
<75% Normal  
Value  
20-29 M 
43±7.2 
< 32.3 
20-29 F 
36±6.9 
< 27.0 
30-39 M 
42±7.0 
< 31.5 
30-39 F 
34±6.2 
< 25.5 
40-49 M 
40±7.2 
< 30.0 
40-49 F 
32±6.2 
< 24.0 
50-59 M 
36±7.1 
< 27.0 
50-59 F 
29±5.4 
< 21.8 
60-69 M 
33±7.3 
< 24.8 


 
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60-69 F 
27±4.7 
< 20.3 
> 70 M 
29±7.3 
< 21.8 
> 70 F 
27±5.8 
< 20.3 
 
*Fletcher GF et al. Circulation. 1995; 91:580-615. 
 
7. No chronic nitrate therapy or not using intermittent sublingual nitroglycerin (requirement for 
>1 SL nitroglycerin per week) within last 6 months 
8. No daily use of phosphodiesterase 5 inhibitors or soluble guanylyl cyclase activators and 
willing to withhold prn use of phosphodiesterase 5 inhibitors for duration of study 
9. Ambulatory (not wheelchair / scooter dependent) 
10. Body size allows wearing of the accelerometer belt as confirmed by ability to comfortably 
fasten the test belt provided for the screening process (belt designed to fit persons with 
BMI 20-40 kg/m2 but belt may fit some persons outside this range) 
11. Willingness to wear the accelerometer belt for the duration of the trial 
12. Willingness to provide informed consent 
9.3 
Exclusion Criteria 
1. Recent (< 1 month) hospitalization for heart failure 
2. Ongoing requirement for PDE5 inhibitor, organic nitrate or soluble guanylyl cyclase 
activators 
3. Hemoglobin (Hgb) < 8.0 g/dl within 30 days prior to randomization 
4. GFR < 20 ml/min/1.73 m2 within 30 days prior to randomization 
5. Systolic blood pressure < 115 mmHg seated or < 90 mmHg standing just prior to test dose  
6. Resting HR > 110 just prior to test dose 
7. Previous adverse reaction to the study drug which necessitated withdrawal of therapy 
8. Significant chronic obstructive pulmonary disease thought to contribute to dyspnea 
9. Ischemia thought to contribute to dyspnea 
10. Documentation of previous EF < 45% 
11. Acute coronary syndrome within 3 months defined by electrocardiographic (ECG) changes 
and biomarkers of myocardial necrosis (e.g., troponin) in an appropriate clinical setting 
(chest discomfort or anginal equivalent)   
12. PCI, coronary artery bypass grafting, or new biventricular pacing within past 3 months 
13. Primary hypertrophic cardiomyopathy 
14. Infiltrative cardiomyopathy (amyloid) 
15. Constrictive pericarditis or tamponade 
16. Active myocarditis 
17. Complex congenital heart disease  
18. Active collagen vascular disease 
19. More than mild aortic or mitral stenosis 
20. Intrinsic (prolapse, rheumatic) valve disease with moderate to severe or severe mitral, 
tricuspid or aortic regurgitation  
21. Acute or chronic severe liver disease as evidenced by any of the following: 
encephalopathy, variceal bleeding, INR > 1.7 in the absence of anticoagulation treatment 
22. Terminal illness (other than HF) with expected survival of less than 1 year 


 
INDIE-HFpEF Protocol v1.3, 22April2016 (Final) 
 
    
 25
 
 
 
 
23. Regularly (> 1x per week) swims or does water aerobics 
24. Enrollment or planned enrollment in another therapeutic clinical trial in next 3 months. 
25. Inability to comply with planned study procedures 
26. Pregnancy or breastfeeding mothers 
10 TREATMENT INTERVENTIONS 
10.1 Intervention  
Inhaled, nebulized placebo or inhaled nebulized sodium nitrite 
starting at 46 mg and titrated up to 80 mg 3 times daily, at a minimum 
of 4 hours apart, with the first dose starting at the beginning of the 
active part of the day (for example, 8:00, 12:00, and 16:00). The 
Participant should disperse the doses by at least 4 hours and deliver 
them over their normal active day time. The doses do not need to be 
given at the same time each day if the Participant’s active period 
varies by day, for example if the Participant arises later on the 
weekends.  
 
The drug or matching placebo is produced supplied in one ml 
squeezable plastic ampules. The ampules are produced in snap-
apart groups of 5 vials place in a nitrogen filled pouch. Participants 
are instructed to open and utilize one pouch at a time. The 
medication is stable at room temperature. The Participant is 
instructed to twist off the top of a vial and squeeze the content into the medication chamber of 
the nebulizer. The metered, PURPLE latch medication chamber administers the lower dose of 
46 mg. The WHITE latch medication chamber administers a full 80 mg dose of medication. 
Either chamber can be used with the nebulizer allowing two different doses to be delivered 
during the study protocol. Each Participant will be provided with a PURPLE and a WHITE study 
drug guide for use in the appropriate time in the protocol. 
  
Inhaled, nebulized sodium nitrite or placebo will be administered utilizing the Phillips I-neb 
AAD nebulizer over 10-15 minutes for each dose. The I-neb is a small, battery-powered, 
lightweight, and virtually silent drug delivery device designed to deliver a precise, reproducible 
dose of drug. (Figure 7) Throughout the treatment, the I-neb provides continuous feedback to 
the Participant through a liquid crystal display, and upon successful delivery of the treatment, 
the Participant receives audible and tactile feedback.  
 
The device has a Participant logging system that automatically records adherence and 
compliance with protocol-specified treatments. The device log will be reviewed by the research 
coordinator with study participants verbally to optimize adherence.  
 
The time to maximal concentration of NO2 in plasma is within 5 minutes, immediately after the 
end of the inhalation period (~15 minutes after start of inhalation). Maximal concentration after 
nebulization of an 80mg dose is 10-12 micromol/L, which is 2-3-fold higher than what is 


 
INDIE-HFpEF Protocol v1.3, 22April2016 (Final) 
 
    
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achieved with oral NO2 administration (PK data on file). The mean (SD) terminal half-life of 
nitrite in plasma in phase 1 testing was 0.6 (0.2) hours.46   
 
Permitted dose adjustment: If significant headache occurs, participants will be encouraged to 
treat headaches with acetaminophen.  If participants cannot tolerate the 46 mg dose, they will 
discontinue study drug but continue with all study procedures and visits. If Participants cannot 
tolerate the 80 mg dose, they will return to the previously tolerated 46 mg dose and continue all 
study procedures and visits.  If a dose is missed, the 4 hour minimum regimen should be 
resumed as prescribed with the next scheduled dose. An extra dose or increase in dose should 
not be taken to make up for the missed dose. 
 
If a participant has a temporary discontinuation for reasons other than intolerance, (e.g., such as 
hospitalization, lost device, etc), the investigator can restart at the last tolerated dose.  If the last 
tolerated dose was 80mg, and depending on duration of interruption as well as any potential 
safety concerns, the Investigator will have the flexibility to restart at the reduced dose of 46 mg 
and up titrate to the 80 mg at their discretion.  However, participants that down titrate due to 
intolerance should not be reintroduced to the higher dose. 
 
Drug interactions: Potential excess nitric oxide mediated effects (e.g., hypotension, headache) 
might be observed with co-administration of organic nitrates or phosphodiesterase-5 inhibitors 
or soluble guanylyl cyclase activators. Therefore, co-administration of nitrates, any 
phosphodiesterase-5 inhibitor formulation or soluble guanylyl cyclase activators is strictly 
contraindicated due to the risk of hypotension.  There are no other known drug interactions and 
study drug absorption is not affected by food or administration of other inhalers (inhaled steroids 
or bronchodilators). 
10.2 Drug Dispensing  
Drug dispensing will be managed by the CC who will obtain and store supplies from the 
contracted drug supply vendor. At the first study visit, participants will receive a sufficient supply 
of inorganic nitrite or placebo ampules to permit three doses a day until the second study visit, 
realizing that participants may not be able to return at the exact 42 day window due to 
scheduling conflicts (range 42-49 days). To account for potential damage or loss of ampules 
after opening the pouches containing multiple ampules and to account for unavoidable (for 
example adverse weather, family illness, etc) further delays in returning for study visits beyond 
the 42 day (+ up to 7 days) timeline, additional study drug doses will be supplied). Similarly, 
participants will receive enough inorganic nitrite or placebo ampules at the second study visit to 
last until the third (final) study visit as above. 
 
Participants will be instructed to take the medication as required by the protocol, and 
compliance will be assessed at each visit by download of the automatic Participant logging 
system contained within the nebulizer device and by phone contact (as described in the 
protocol) between visits.  Participants will be instructed to bring the nebulizer and unused drug 
supplies at each visit.   
 


 
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10.3 Storage, accountability and destruction 
Trial products (both unused and in-use) should not be exposed to moisture but can be stored at 
room temperature.  The plastic ampules come in a nitrogen filled foil pouch in snap-apart groups 
of 5 vials place.  Participants are instructed to open one pouch at a time and utilize each ampule 
before opening another pouch.  
10.4 Drug accountability 
Participants are instructed to return all used, partly used and unused trial product at each study 
visit. 
 
Returned trial product(s) (used, partly used or unused) must be stored separately from the non-
allocated trial product(s) until drug accountability has been reconciled. The investigators will 
keep track of all received, used, partly used and unused trial products. 
10.5 Destruction 
Used and unused investigational product (study drug/nebulizer) can be destroyed at the site 
according to accepted pharmacy practice, local and national guidelines, using the site’s 
destruction procedure. A copy of the current investigational product (IP) destruction SOP should 
be maintained in the pharmacy section of the Regulatory Binder and available for review in case 
of an audit. 
 
Study IP must not be destroyed until the CRA has completed IP accountability and approved 
and accounted for the study IP to be destroyed.  Study IP destruction should be documented in 
the within the Participant Specific IP Accountability Log (or equivalent tracking system).  
10.6 Randomization, Stratification and Blinding 
Randomization will occur at the first study visit.  Randomization to active drug/placebo during the 
first phase of the crossover study (1:1 allocation ratio) is stratified by site.  Blinding is ensured 
by preparation of identically appearing placebo and active drug ampules.  Participants will be 
randomized using procedures determined by the CC to one of 2 sequences. A permuted block 
randomization method stratified by site will be used to ensure relatively equal distribution of 
participants to each sequences within each clinical site.  
10.7 Unblinding 
The investigative sites will be given access to the treatment code for their Participants for 
emergency un-blinding ONLY by calling the CC. Given the safety profile of inorganic nitrite it is 
anticipated that there should be no need to un-blind the study drug for any reason.  Any 
suspected study drug-related events should be treated as though the Participant received active 
therapy.  Nevertheless, in the rare event of necessary un-blinding, the CC medical monitor must 
be contacted to discuss a given case.   
 
Randomization data are kept strictly confidential, accessible only to authorized persons, until the 
time of un-blinding.  


 
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10.8 Concomitant Medication 
Participants should be treated with standard HFpEF strategies (diuretics for congestion, blood 
pressure control and heart rate control if Participant is in atrial fibrillation) as per recommended 
guidelines48. Participants should be on stable medications and with adequate blood pressure 
control prior to entry as outlined in the entry criteria. Further adjustment of diuretics or blood 
pressure medications during the study period is discouraged and should only be performed 
according to new and clinically compelling worsening of clinical status.  As above, therapy with 
organic nitrates, phosphodiesterase-5 inhibitors or soluble guanylyl cyclase activators is contra-
indicated during the study period.  
10.9 Side Effect Risk Reduction Plan 
Confirmation of tolerance of the 80 mg dose during the run-in phase, the use of acetaminophen 
for headache and the ability to down-titrate study drug dose (from the 80 mg dose to the 46 mg 
dose) if needed are expected to enhance tolerability. 
11 RECRUITMENT AND SCREENING PROCEDURES 
11.1 Common Recruitment Procedures 
All participants admitted to the participating Heart Failure Clinical Research Network (HFN) 
centers with signs and symptoms suggestive of HFpEF will be screened by a member of the 
study staff. Patients meeting eligibility criteria will be approached regarding participation in this 
study.  
11.2 Estimated Enrollment Period 
This study will enroll approximately 100 participants at approximately 20 clinical centers in the 
U.S. The anticipated enrollment period is approximately 18 months. 
11.3 Informed Consent Procedures 
11.3.1 Informed Consent 
HFN center clinicians will explain to eligible patients the purpose of the study, study 
interventions and evaluations, and the potential risks and benefits of participation, and will 
answer any questions. If a patient agrees to participate in the INDIE-HFpEF study, they will 
review and sign the site specific IRB approved informed consent form (ICF).  
11.3.2   Confidentiality and HIPAA Requirements 
All information collected on study participants will be stored in a confidential manner using the 
procedures in place at each participating center. Only approved study personnel will have 
access to data collected as part of the study. Consented study participants will be identified by a 
participant ID number on all study documents. Data will be transmitted to the CC in a secure 
manner, and stored securely at the CC using standard Duke Clinical Research Institute (DCRI) 
operating procedures. 


 
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11.3.3 Protections of Human Subjects 
Protections for human subjects of research are required under Department of Health and 
Human Services (HHS) regulations at 21 CFR parts 50, 56, and 312. 
11.3.4 Summary of the Risks and Benefits 
Blood draws: The risks of drawing blood include bleeding at the puncture site, bruising and pain. 
These occur in a very small portion of the population. 
 
Hypotension related: A potential adverse effect of inorganic nitrite is related to venous and 
arterial vasodilatation inducing lightheadedness, presyncope or syncope and headache. In prior 
human studies nitrite-mediated blood pressure reduction typically decreases over time. Elderly 
Participants may be at increased risk of such reactions. Syncope could be associated with 
trauma.  
 
Cough: Cough and/or throat irritation may develop after the nebulizer therapy and can be 
managed symptomatically if needed (e.g., anti-tussives, lozenges).  In prior human studies 
these symptoms have not been limiting. 
 
Headache: Headache is a common and dose-related adverse effect associated with nitrate 
therapy. Both the severity and incidence of this effect appear to lessen with continued 
administration. Initiating therapy at a low dose and titrating up slowly is recommended to reduce 
the incidence of headache. Aspirin and acetaminophen have been successful in treating this 
headache.  
 
This protocol may be hazardous to an unborn child: There are no well-controlled studies of 
inorganic nitrite to determine whether there are significant risks to a mother or the fetus carried 
by a mother who is participating in this study. Therefore, female participants must be 
postmenopausal or have been surgically sterilized or have a serum negative pregnancy test.  
Women of child bearing potential must agree to use an acceptable method of birth control 
during the study period. 
 
This study involves administration of an agent (inorganic nitrite) with potential benefits in 
HFpEF.  Thus, during the phase when a participant receives active study drug (inorganic nitrite) 
rather than placebo, they could potentially experience clinical benefit.  
12 BASELINE EVALUATION AND RANDOMIZATION VISIT 
A complete schedule of assessments throughout the study is given in Appendix A. 
12.1 Screening  
Patients will be screened for entry criteria at each site using existing clinical records including 
their most recent echocardiogram (echo within 12 months) and blood work. Potential 
participants will be scheduled for Study Visit 1.   


 
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12.2 Baseline/Randomization Visit (Visit 1) 
Patients who fulfill entry criteria will undergo the consent process and written informed consent 
will be obtained prior to any study specific procedures. A screening question to assess whether 
their ability to be active is related to their heart failure symptoms (versus orthopedic, neurologic 
or behavioral factors) will be completed. If patients indicate that reasons other than heart failure 
symptoms limit their ability to be active, they will be considered a screen failure and will not 
continue. Subjects will be assessed for their ability to wear the accelerometer belt confirmed 
with a “test belt” provided to each center. Willingness to wear the accelerometer belt and to 
participate in all study procedures is confirmed.   
 
Once confirmed, the participant will undergo baseline blood draws, to include CBC, complete 
chemistry panel, biomarkers, biorepository and genetics (if agreed to participate), and then an 
HFN study-specific, baseline CPET will be conducted to ensure patient meets the entry criteria.  
Patients that do not meet the CPET criteria will be considered a screen failure and will not 
continue.  Note:  The baseline bloods are to be obtained prior to the CPET as the biomarker 
values can be affected from the exertion of the CPET.  In the event this is not feasible, the blood 
draws will need to be obtained 3 hours post CPET and prior to the run-in test dose. 
 
Once confirmed, patients will undergo the remaining baseline studies (below) and undergo 
single dose unblinded run-in receiving 80 mg nebulized sodium nitrite followed by observation 
for 60 minutes.  Patients that do not tolerate the run-in will not be randomized.   
 
Qualifying patients will be randomized using procedures determined by the CC to one of 2 
sequences (inorganic nitrite first or placebo first). Participants will be randomized in a 1:1 
allocation ratio. The Participants will be educated in study procedures including wearing of the 
accelerometer and use of the nebulizer device.  Participants will be required to demonstrate 
independent proficiency with the nebulizer prior to dismissal from Visit 1. Study drug for the first 
phase of INDIE-HFpEF will be dispensed. 
 
Remaining baseline studies and procedures include: 
 
Complete Medical History and Medication Review  
 
Physical Examination and NYHA class assessment  
 
ECG 
 
KCCQ 
 
Run-In Test Dose (post completion of baseline studies) 
 
Verify SBP > 115 mmHg seated and > 90 mmHg standing and resting HR < 110 just 
prior to test dose (if not, the participant is considered a screen failure and will not 
receive the test dose). 
 
Participants passing the SPB and HR criteria receives open label run-in with sodium 
nitrite (80 mg) given once, with monitoring for AE for 60 minutes following dosage for 
adverse effects or symptomatic hypotension.   
o Systemic blood pressures (seated and 3 minutes after standing) will be 
monitored every 15 minutes for 60 minutes. 


 
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 
Participants developing hypotension (SBP < 90 seated or standing), lightheadedness 
or who otherwise do not tolerate the single-dose run-in will be considered run-in 
failures and will not be randomized.   
Randomization 
 
Participants that pass the run-in test, will be randomized to study drug assignment.  
 
Following randomization, study staff will dispense the accelerometer, nebulizer 
device and  nebulizer supplies  and educate participant on how to use the nebulizer 
device and supplies and on how to wear the accelerometer.    
 
Compliance plan: A plan for the phone visits will be established with the participant. 
Participant-specific strategies such as sign posting, bathing-time accelerometer 
placement, and alarms may be used to enhance compliance with accelerometry and 
study drug administration. 
 
Dispense Phase 1 study drug—Participant must demonstrate proper administration 
of both the 46 mg dose and 80 mg dose.  
13 FOLLOW-UP EVALUATIONS 
13.1 Study Visits  
Study Visit 2. Participants will return no sooner than 42 days (but up to 49 days) after the first 
study visit. Participants requiring extra time beyond the 42 day window must maintain on study 
drug during this period with no interruption in therapy. 
 
Nebulizer records will be examined by the coordinator to determine compliance with study drug 
dosing and ensure proper usage.  Participants will be instructed to hold the study drug the 
morning of the visit so that it can be administered in person at the time of the CPET with study 
staff present. Participants will undergo studies and procedures as below. The Study Visit 2 
studies and procedures include: 
 
Confirm study drug held the day of the visit 
 
Interim History and Medication Review 
 
Physical Examination and NYHA class assessment 
 
KCCQ questionnaire  
 
Blood draw (prior to CPET) for CBC, Complete Chemistry Panel (Sodium, 
Potassium, Chloride, Carbon Dioxide, BUN, Creatinine, Glucose, AL, AST, 
Alkaline Phosphatase & Total Bilirubin) and HFN biomarkers (cystatin C, NT-
proBNP, cGMP and nitrosothiols), and biorepository samples (if agreed to 
participate) 
 
Limited Echocardiogram prior to study drug administration (in the event this is 
cannot be completed prior to study drug administration, then it cannot be 
obtained until at least 3 hours post study drug administration) 
 
Cardiopulmonary exercise test (with administration of a dose of Phase 1 study 
drug at the CPET and in the presence of the Study Coordinator immediately 
before starting the CPET) 
 
Compliance plan: A plan for the phone visits will be established with the 
Participant. Participant-specific strategies such as sign posting, bathing-time 
accelerometer placement, and alarms may be used to enhance compliance 
with accelerometry and study drug administration. 
 
Change out accelerometer 
 
Collect Phase 1study drug and dispense Phase 2 study drug 


 
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Study Visit 3. Participants will return no sooner than 42 days (but up to 49 days) after Study 
Visit 2. Participants requiring extra time beyond the 42 day window must maintain on study drug 
during this period with no interruption in therapy. 
 
Nebulizer records will be examined by the coordinator to determine compliance with study drug 
dosing.  Participants will be instructed to hold the study drug the morning of the visit so that it 
can be administered in person at the time of the CPET with study staff present.  Participants will 
undergo studies and procedures as below.  
 
The Study Visit 3 studies and procedures include: 
 
Confirm study drug held the day of the visit 
 
Interim History and Medication Review 
 
Physical Examination and NYHA class assessment 
 
KCCQ questionnaire  
 
Blood draws prior to study drug administration (in the event this is not feasible, 
then it cannot be obtained for at least 3 hours post study drug administration):  
CBC, Complete Chemistry Panel and HFN biomarkers (cystatin C, NT-
proBNP, cGMP and nitrosothiols), and biorepository samples (if agreed to 
participate) 
 
Limited Echocardiogram prior to study drug administration (in the event this is 
not feasible, then it cannot be obtained for at least 3 hours post study drug 
administration) 
 
Cardiopulmonary exercise test (with administration of a dose of Phase 2 study 
drug at the CPET and in the presence of the Study Coordinator immediately 
before starting the CPET) 
 
Return accelerometer 
 
Return Phase 2 study drug 
 
Participants are asked to indicate the study phase during which they felt better 
(Patient preference secondary endpoint)  
 
Final study phone visit: A final phone visit is conducted 2 weeks after Study visit 3 to assess 
clinical stability and any adverse events.  
13.2 Phone and Other Media Follow-up  
General procedures: At Study Visit 1, participants and study staff will define optimal times and 
phone numbers for the protocol specified phone contacts to encourage compliance with study 
procedures.  
 
During the follow-up phone visits, the participant will receive:  
 
Reminder of appropriate study drug dose for the stage of the protocol. 
 
Reminder regarding appropriate use of nebulizer device, dose specific study 
drug guide and compliance 
 
Encouragement of compliance with accelerometry use  
 
Encouragement of activity within the limits of their HF symptoms 
 
Confirm plans for future study visits 
 
Confirm need to bring study drug and accelerometers to future study visits 
 
Reminder to hold study drug on days of Study Visit 2 and 3.  


 
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Participants are also called 14 days ± 5 days after Study Visit 3 to assess clinical stability and 
any adverse events. 
14 OUTCOME DETERMINATIONS 
14.1 Primary Endpoint 
The primary endpoint will be the peak VO2 performed at peak drug levels after 4 weeks 
treatment with inorganic nitrite as compared to the peak VO2 after 4 weeks treatment with 
placebo. 
14.2 Secondary Endpoints 
1. Standard HF endpoints: 
 
Average arbitrary accelerometer units (AAU) during at least 14 days and up to 21 days of 
the maximally tolerated dose of study drug (from 28 days post Study Visit 1 until Study 
Visit 2 and from 28 days post Study Visit 2 until Study Visit 3).  
 
Medial E/e’ ratio at echocardiography 
 
Left atrial volume index at echocardiography 
 
Pulmonary artery systolic pressure at echocardiography 
 
KCCQ  
 
NT-proBNP 
 
NYHA class  
 
Other CPET endpoints including VE/VCO2 slope, VO2 at VAT 
 
Participant preference for study phase (active vs. placebo) 
 
Safety and tolerability 
14.3 Tertiary Endpoints 
1. Pre-specified subgroup analyses will include examination of the primary endpoint in patients 
according to baseline characteristics:  
 
NT-proBNP >400 pg/ml vs. ≤400 pg/ml  
 
Systolic blood pressure above and below median value. 
 
PASP>60 mmHg vs <60 mmHg 
 
Age above and below median value. 
 
Women vs. men 
 
Estimated glomerular filtration rate by MDRD equation above and below median value  
 
Subjects on active drug at study visit vs not on study drug at study visit 
 
Subjects in atrial fibrillation/flutter vs not  
 
Diabetic vs. non-diabetic patients  
 
Patients with or without symptoms of exertional chest pain 
2. Novel accelerometry endpoints (guided by HFN NEAT ancillary paper analysis) 
3. Plasma nitrosothiols, cystatin-C, cGMP 
 


 
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15 METHODS TO PROMOTE ADHERENCE 
15.1 Adherence to Study Procedures 
Protocol training and adherence will be a major focus of the investigator training. Based on our 
experience in prior studies, identifying and correcting non-adherence is best accomplished in a 
stepped approach. The site will be responsible for managing participant compliance with study 
drug administration, accelerometer utilization and visit compliance.  The CC will provide data 
quality reports for review and will follow-up to reemphasize the importance of adherence as 
needed. Routine data quality reports, will be provided to the sites and significant non-adherence 
issues will be discussed with the Executive Committee. 
16 PARTICIPANT SAFETY AND ADVERSE EVENTS 
16.1 Institutional Review Boards 
All HFN sites will submit the study protocol, consent form, and other study documents to their 
IRB for approval. The approval letter for each clinical center will be reviewed by the CC. Any 
amendments to the protocol, other than minor administrative changes, must be approved by 
each IRB before they are implemented. 
16.2 Definitions 
16.2.1 Adverse Events 
An adverse event (AE) is any untoward medical occurrence associated with the use of a drug in 
a participant, whether or not considered drug or biologic related. An AE can therefore be any 
undesirable sign, symptom or medical condition occurring after starting study drug, even if the 
event is not considered to be related to the pharmaceutical product. Study drug includes the 
drug under evaluation, and any reference or placebo drug given during any phase of the trial.  
Headache, dizziness and lightheadedness are AEs that will be specifically questioned about 
during phone calls and study visits. 
 
16.2.2 Suspected Adverse Reaction  
A suspected adverse reaction (SAR) is any adverse event for which there is a reasonable 
possibility that the drug caused the event. “Reasonable possibility” suggests there is a causal 
relationship between the drug and the adverse event. “Suspected adverse reaction” implies a 
lesser degree of certainty about causality than adverse reaction, which means any adverse 
event caused by a drug.  
 
16.2.3 Serious Adverse Events (SAE) 
An adverse event or suspected adverse reaction is considered serious if the investigator or 
sponsor believes any of the following outcomes occurred: 
 
Death 


 
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 
Life-threatening AE:  Places the participant at immediate risk of death at the time of the 
event as it occurred. It does not include an AE that, had it occurred in a more severe form, 
might have caused death. 
 
Persistent or significant incapacity or substantial disruption of the ability to conduct normal 
life functions. 
 
Inpatient hospitalization or prolongation of hospitalization.  
 
Congenital anomaly or birth defect.  
 
Important medical events that may not result in death, be life threatening, or require 
hospitalization may be considered a serious adverse event when, based upon appropriate 
medical judgment, they may jeopardize the participant and may require medical or surgical 
intervention to prevent one of the outcomes listed in this definition above. 
This determination is based on the opinion of either the investigator or sponsor (e.g., if either 
believes it is serious, it must be considered serious). 
16.2.4 Laboratory Test Abnormalities 
For laboratory test abnormalities that meet the definition of an SAE, that required the participant 
to have the investigational product discontinued or interrupted or required the participant to 
receive specific corrective therapy, the clinical diagnosis rather than the laboratory term will be 
used by the reporting investigator (e.g., anemia versus low hemoglobin value). 
16.2.5 Assessment of Causal Relationship 
A medically-qualified investigator must assess the relationship of any AE to the use of study 
drug, based on available information, using the following guidelines:  
 
Not related: There is not a reasonable causal relationship to the investigational product 
and the adverse event.  
 
Unlikely related: No temporal association or the cause of the event has been identified, 
or the drug or biologic cannot be implicated. 
 
Possibly related: There is reasonable evidence to suggest a causal relationship between 
the drug and adverse event. 
 
Related: There is evidence to suggest a causal relationship, and the influence of other 
factors is unlikely. 
16.2.6 Assessment of Adverse Event Severity 
The determination of adverse event severity rests on medical judgment of a medically-qualified 
Investigator. The severity of AEs will be graded using the following definitions:  
 
Mild: Awareness of sign, symptom, or event, but easily tolerated;  
 
Moderate: Discomfort enough to cause interference with usual activity and may warrant 
intervention;  
 
Severe: Incapacitating with inability to do usual activities or significantly affects clinical 
status, and warrants intervention.  
16.2.7 Expectedness 
The expectedness of an AE or SAR shall be determined according to the most current 
investigator’s brochure. Any AE that is not identified in nature, severity, or specificity in the 


 
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current investigator’s brochure is considered unexpected. Events that are mentioned in the 
investigator's brochure as occurring with a class of drugs or as anticipated from the 
pharmacological properties of the drug, but not specifically mentioned as occurring with the 
particular drug under investigation are considered unexpected. 
16.3 Anticipated Disease Related Events 
The following AEs are anticipated, disease-related events in patients with HF with preserved 
ejection fraction (HFpEF). 
 
Arrhythmias: This refers to both atrial and ventricular arrhythmias. 
 
Acute coronary syndrome: This refers to unstable angina, non ST segment 
elevation myocardial infarction (NSTEMI), and ST segment elevation myocardial 
(STEMI). 
 
Unplanned hospitalization, ER visit or clinic visit for worsening HF: This refers to 
treatment for acute heart failure such as receiving intravenous diuretics. 
 
Cerebrovascular event: This refers to cerebrovascular accidents (stroke) of any cause 
(hemorrhagic, ischemic, or embolic) and transient ischemic attack (TIA). 
 
Venous thromboembolism: This includes both deep venous thrombosis and 
pulmonary embolus. 
 
Worsening renal function: This refers to acute kidney injury, typically defined as a rise in 
creatinine > 0.3 mg/dL over 48 hours, or progressive loss of renal function over time. 
 
Anticipated disease related events will not be captured as AEs/SAEs during the study, but will 
be entered on the appropriate electronic case report form (eCRF) module (“Events of Interest” 
page). 
 
16.3.1    Recording and Reporting of Adverse Events 
The site Investigator is responsible for monitoring the safety of participants enrolled into the 
study at the study sites. Events significant enough to necessitate modification of study drug 
dosing will be captured on the AE eCRF.  
  
All AEs/SAEs, except for those anticipated AEs listed above, occurring from signed informed 
consent to 2 weeks post visit 3 will be captured on the AE/SAE eCRF.  Patients that have 
screen failed will be followed for AEs/SAEs until two weeks post last study drug dose.  Unless 
exempted as described above, all AEs/SAEs, whether or not deemed drug-related or expected, 
must be reported by the investigator or qualified designee within 24 hours of first becoming 
aware of the event.  For this study, all cause deaths will be reported on the SAE eCRF, as well 
as the Death eCRF page. The investigator or qualified designee will enter the required 
information regarding the AE/SAE into the appropriate module of the eCRF, which will 
automatically result in distribution of the information to DCRI Safety Surveillance. If the eCRF 
system is temporarily unavailable, the event, including the investigator-determined causality to 
study drug should be reported via the back-up paper SAE form to DCRI Safety Surveillance. 
Upon return of the availability of the electronic data capture (EDC) system, the SAE information 
must be entered into the eCRF. 
 


 
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Follow-up 
When additional relevant information becomes available, the Investigator will record follow-up 
information according to the same process used for reporting the initial event as described 
above. The Investigator will follow all reportable events until resolution, stabilization or the event 
is otherwise explained.  
 
DCRI Safety Surveillance will follow all SAEs until resolution, stabilization, until otherwise 
explained or until the participant completes the final follow-up, whichever occurs first.  
Investigators are also responsible for promptly reporting AEs to their reviewing IRB/EC in 
accordance with local requirements.  The DSMB will review detailed safety data approximately 
every 6 months throughout the study. 
16.3.2 Suspected Unexpected Serious Adverse Reaction 
  
AEs that meet the criteria of serious, related to study drug, and unexpected per investigator 
brochure, qualify for expedited reporting to the regulatory authorities.  DCRI will notify the FDA 
and all participating investigators in a written IND safety report of an SAR that is serious and is 
unexpected, based on the opinion of the investigator and DCRI safety medical monitor, as soon 
as possible, but not later than 15 calendar days after the event is confirmed to be a serious, 
unexpected SAR and qualifies for expedited reporting. DCRI will identify all safety reports 
previously filed with the IND concerning a similar SAR, and will analyze the significance of the 
SAR in light of the previous, similar reports. Follow-up reports will be sent to investigators to 
inform and update them about an important suspected adverse reaction if it significantly affects 
the care of the participants or conduct of the study. 
  
Day Zero - Day zero (0) is the calendar day that DCRI is first notified of an event. Day zero can 
also be the date the event qualified for expedited reporting as determined by the DCRI Safety 
Medical Monitor. 
16.3.3 Pregnancy 
Pregnancy occurring during a clinical investigation, although not considered a serious adverse 
event, must be reported to DCRI within the same timelines as a serious adverse event. The 
pregnancy will be recorded on the appropriate paper pregnancy tracking form. The pregnancy 
will be followed until final outcome. Any associated AEs or SAEs that occur to the mother or 
fetus will be recorded in the AE/SAE eCRF, within the EDC system.  
 
17 STATISTICAL CONSIDERATIONS 
17.1 Overview 
All planned analyses will be prospectively defined for this study and approved by the CC prior to 
unblinding of data. In addition, exploratory analyses will be performed to help explain and 
understand findings observed from the planned analyses. Statistical tests with a 2-sided p-value 
<0.05 will be considered statistically significant, unless otherwise stated. Analyses will be 


 
INDIE-HFpEF Protocol v1.3, 22April2016 (Final) 
 
    
 38
 
 
 
 
performed using SAS software (SAS Institute, Inc, Cary, NC). The INDIE-HFpEF statistical 
analysis plan (SAP) will contain detailed information regarding the data analysis. The SAP will 
be finalized prior to trial completion and will be approved by the coordinating center statistical 
team as well as the NHLBI program officer.  
17.2 Design and Analytical Criteria 
The primary analysis will be conducted on an intention-to-treat (ITT) basis. The ITT population 
includes all participants who are randomized. The primary endpoint is based on the within-
patient comparison of peak VO2 following 4 weeks treatment with inorganic nitrite as compared 
to following 4 weeks treatment with placebo.  It is anticipated that some data will be missing due 
to participant drop out or inability to complete both exercise tests.   
 
The primary data analysis of the primary endpoint will involve a mixed model with fixed effect 
terms for the sequence, period and treatment. When available, the baseline value of the 
variable will be included in the model.  A random effect term will be included to account for the 
correlated measurements within each participant.   The estimated treatment effect will be 
provided with a 95% confidence interval. Given the short half-life of the drug (40 minutes), the 
two-week washout period, and the assumed lack of remodeling effects with this duration of 
treatment, we anticipate no significant carry-over or residual effect from Phase 1 to Phase 2.  
 
Baseline data will be collected to characterize the study population and potentially to serve as a 
covariate in the analysis of the endpoints and to facilitate subgroup analysis, including tertiary 
endpoint analyses. As a sensitivity analysis, the data from the first period and second period will 
be presented and analyzed separately.  Exploratory analyses will be conducted using the paired 
t-test and the t-test using the crossover design structure. 
17.3 Analysis of Secondary and Tertiary Endpoints 
Analysis of continuous outcomes will be conducted using mixed models as described above. 
For endpoints such as NT-proBNP, we will consider transformations of the data to obtain more 
valid 95% confidence intervals. For nominal variables, the number and percentages in each 
category will be presented. For binary outcomes, Chi-square tests and Fisher’s exact test will be 
used for unadjusted comparisons. 
17.4    Analysis of Safety Data and Statistical Monitoring Plan 
Interim data analysis for efficacy and futility will not be conducted due to relatively small size, 
short duration, and crossover structure of this clinical trial. Safety data, summarized at the 
treatment level, will be assessed approximately every 6 months by the National Heart, Lung, 
and Blood Institute (NHLBI)-appointed DSMB. The safety analyses will be based on the entire 
ITT population. Safety will be evaluated by comparing the occurrence of AEs. Based on the 
RELAX-HFpEF study performed in patients with HFpEF within the HFN, we expect the mortality 
rate over the 12-week study period to be less than 2% (Data on file at DCRI; RELAX trial 
analysis). 
 
 


 
INDIE-HFpEF Protocol v1.3, 22April2016 (Final) 
 
    
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17.5   Sample Size and Power Calculation 
Peak VO2: From prior HFN studies, the within-patient standard deviation for peak VO2 in HFpEF 
is roughly 2 ml/min/kg.[REF 21]  A 1.2 ml/min/kg increase in peak VO2 is considered to be 
clinically meaningful for a particular patient.  Assuming a 2 ml/min/kg standard deviation, a total 
of 90 participants is adequate to provide 80% power to detect a difference of 0.6 ml/min/kg in 
peak VO2.[see Figure 8]  The assumed treatment difference allows for not all of the participants 
to have a clinically meaningful difference.  These calculations are based on a 2*2 factorial 
design and assume a 2-sided 0.05 (or 1-sided 0.025) type I error rate.  The total sample size of 
100 participants allows for approximately 10% incomplete data due to missed tests, death, and 
withdrawal of consent. The sample size estimates were calculated using nQuery and PASS 
software.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
KCCQ overall summary score: Clinically significant differences have been established for 
these endpoints. From earlier HFN studies, the within-patient standard deviation for the KCCQ 
overall summary score has ranged from 14 to 18 points. In the NEAT study, the within-patient 
standard deviation for the KCCQ overall summary score was approximately 14 points. For the 
KCCQ, a clinically-significant difference is considered 5 points and a moderately large clinical 
difference is considered to be 10 points.  Assuming a 14 point standard deviation, a total of 70 
participants is adequate to provide >80% power to detect a clinically-significant difference of 5 
points in the KCCQ overall summary score. Assuming a 16 point standard deviation, a total of 
90 participants is adequate to provide >80% power to detect a clinically-significant difference of 
5 points in the KCCQ overall summary score. Assuming an 18 point standard deviation, a total 
of 100 participants (50 per sequence) is adequate to provide approximately 79% power to 
detect a clinically-significant difference of 5 points in the KCCQ overall summary score. [See 
Figure 9] 
Figure 8. Power curve 
for the primary endpoint 
 
Power


 
INDIE-HFpEF Protocol v1.3, 22April2016 (Final) 
 
    
 40
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
18 DATA MANAGEMENT PROCEDURES 
18.1 Overview of Data Management  
The CC will have primary responsibility for data management, including the development of data 
collection systems, data monitoring processes, and data storage and back-up. State-of-the-art 
technology will be used for the management of the network’s data.  
18.2   Data Security  
Access to databases will be controlled centrally by the CC through user passwords linked to 
appropriate privileges. This protects the data from unauthorized changes and inadvertent loss or 
damage. Database and web servers will be secured by a firewall and through controlled 
physical access. Database back-up will be performed daily using standard procedures in place 
at the CC. All disk drives that provide network services, and all user computers, will be 
protected using virus-scanning software. 
18.3 Publication Policy 
Dissemination of preliminary information can adversely affect the objectivity of study data. For 
this reason, investigators will not be allowed to perform subset analyses at any point before the 
conclusion of the study, and any data, other than safety data, cannot be used for publication or 
reporting outside of this study until the study is completed or discontinued by the DSMB or HFN 
Steering Committee.  
Figure 9. Power curve for 
the KCCQ overall 
summary endpoint 
Power


 
INDIE-HFpEF Protocol v1.3, 22April2016 (Final) 
 
    
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19 STUDY ADMINISTRATION 
19.1 Data and Safety Monitoring Board 
A DSMB has been appointed by the NHLBI for the HFN, and will function as the DSMB for this 
trial. This committee consists of a group of highly experienced individuals with extensive 
pertinent expertise in HF and clinical trials. The DSMB will advise the HFN Steering Committee 
regarding the continuing safety of current participants and those yet to be recruited, as well as 
the continuing validity and scientific merit of the trial.  Safety data, summarized at the treatment 
level, will be assessed approximately every 6 months by the DSMB. The safety analyses will be 
based on the entire ITT population. Safety will be evaluated by comparing the occurrence of 
AEs and changes in laboratory values of the active arm compared to placebo. 
19.2 Coordinating Center 
The DCRI will function as the CC for this trial as specified by the National Institute of Health and 
NHLBI HFN grant. 
19.3 Core Laboratories  
19.3.1 Cardiopulmonary Exercise Testing Core Laboratory 
The Harvard DCC will serve as the core laboratory for interpretation of cardiopulmonary 
exercise tests.  
19.3.2 Biomarker Core Laboratories 
The University of Vermont will serve as the core laboratory for measurement of HFN 
biomarkers. Plasma specimens will be collected at Study Visits 1-3, processed at the clinical 
centers according to the procedures provided by the core laboratory, and shipped to the core 
laboratory on dry ice (Refer to Biomarker Core Laboratory Manual of Procedures).  Any patients 
who agree to participate in the HFN genetics biorepository will have samples collected at the 
clinical sites, stored at the University of Vermont biomarker core laboratory, and subsequently 
shipped to the HFN Genetic Core Laboratory at the University of Montreal for processing.  
19.3.3 Echocardiograph Core Laboratory 
A limited echocardiography will be performed at the clinical sites at visits 2 and 3.  All 
echocardiograms will be reviewed at the Mayo Clinic echocardiographic core laboratory using 
the techniques described in the Echocardiography Manual of Operations for the HFN.  
19.3.4 Physical Activity Measurement Core Laboratory 
The Mayo Clinic will serve as the core laboratory for the production and distribution of 
accelerometry devices, and will provide training to the sites in procedures related to 
accelerometer devices. At the completion of each phase, the devices will be returned to the 
Physical Activity Measurement Core Laboratory for downloading of accelerometry data, analysis 
and transmittal of data to the CC (refer to Physical Activity Measurement Core Laboratory 
Manual of Procedures). 


 
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20 REGULATORY ISSUES 
20.1 Ethics and Good Clinical Practice 
This study must be carried out in compliance with the protocol. These procedures are designed 
to ensure adherence to Good Clinical Practice, as described in the following documents: 
 
1. ICH Harmonized Tripartite Guidelines for Good Clinical Practice 1996. 
2. US 21 Code of Federal Regulations dealing with clinical studies (including parts 50 and 56 
concerning informed consent and IRB regulations). 
 
Participating investigators agree to adhere to the instructions and procedures described in it and 
thereby to adhere to the principles of Good Clinical Practice that it conforms to. 
20.2 Institutional Review Board/Independent Ethics Committee 
Before implementing this study, the protocol, the proposed informed consent form and other 
information to participants must be reviewed by a properly constituted Institutional Review 
Board/Independent Ethics Committee (IRB/IEC). Documentation that the protocol and informed 
consent have been approved by the IRB/IEC must be given to the Coordinating Center before 
study initiation.  Any amendments to the protocol, other than administrative ones, must be 
approved by this committee. 
20.3 Informed Consent 
The investigator or designee must explain to each participant (or legally authorized 
representative) the nature of the study, its purpose, the procedures involved, the expected 
duration, the potential risks and benefits involved and any discomfort it may entail. Each 
participant must be informed that participation in the study is voluntary and that he/she may 
withdraw from the study at any time and that withdrawal of consent will not affect his/her 
subsequent medical treatment or relationship with the treating physician. 
 
The informed consent form(s) must be submitted by the investigator for IRB/IEC approval. The 
Coordinating Center will supply template informed consent forms, which comply with regulatory 
requirements, and are appropriate for the study. Any changes to the template consent form 
suggested by the Investigator must be agreed to by the Coordinating Center before submission 
to the IRB/IEC, and a copy of the approved version must be provided to the Coordinating Center 
after IRB/IEC approval. 
21 Remote Monitoring 
The study will be monitored remotely by representatives of the sponsor or its designee 
according to the prospective clinical monitoring plan for the following purposes: 
 
 
To enable real-time monitoring of compliance with study protocol inclusion and exclusion 
criteria is enabled via triggers and range checks programmed in the EDC database.  


 
INDIE-HFpEF Protocol v1.3, 22April2016 (Final) 
 
    
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 
To assist site personnel who will verify data identified within query reports against source 
documents through frequent telephone and email contact.  
 
To verify that written informed consent was obtained before initiation of any screening 
procedures that are performed solely for the purpose of determining eligibility for the 
clinical study and/or prior to the participant’s randomization to a procedure.  
 


 
INDIE-HFpEF Protocol v1.3, 22April2016 (Final) 
 
    
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22 Appendix A – Schedule of Assessments 
 
Phase 1 
Phase 2 
 
Study Visit Window
Study Visit 1 
 (-3 to 0) 
Baseline 
Day 
4-7 
(Washout) 
Day 
11-14 
(Washout) 
Day  
15 
Day  
17 
Day 
18-21 
Day  
22  
Day  
23-24 
Day 
25-28 
Day  
32-35 
Day 
38-41 
Study 
Visit 2+ 
Day  
4-7 
(Washout) 
Day 
11-14 
(Washout) 
Day  
15 
Day  
17 
Day 
18-21 
Day 
22 
Day  
23-24 
Day  
25-28 
Day  
32-35 
Day 
38-41 
Study 
Visit 3++ 
 2 Week 
Follow-
up +++  
Days
0 
1-7 
8-14 
15-21 
15-21 
15-21 
22-28 
22-28 
22-28 
29-35 
36-42 
42-49 
1-7 
8-14 
15-21 
15-21 
15-21 
15-21 
22-28 
22-28 
29-35 
36-42 
42-49 
14 days 
Post 
Visit 3 
Study Drug Dose   
(Nitrite vs Placebo) 
 
 
46mg 
46mg 
46mg 
80mg 
80mg 
80mg 
80mg 
80mg 
80mg 
 
 
46mg 
46mg 
46mg 
80mg 
80mg 
80mg 
80mg 
80mg 
80mg 
Screening 
(Inclusion/Exclusion) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Informed Consent*
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Phone Follow-Up 
 
X 
X 
 
X 
X 
 
X 
X 
X 
X 
 
X 
X 
 
X 
X 
 
X 
X 
X 
X 
 
X 
Clinical Evaluation
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Complete Medical History 1 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Interim Medical History 
 
X 
X 
 
X 
X 
 
X 
X 
X 
X 
X 
 
 
 
X 
X 
 
X 
X 
X 
X 
X 
 
Physical Exam 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
NYHA Class 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
Medication Review 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
12-Lead ECG 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Systolic BP2 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Laboratory Evaluation
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Serum Pregnancy Test 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CBC 
X^^ 
 
 
 
 
 
 
 
 
 
 
X3 
 
 
 
 
 
 
 
 
 
 
X3 
 
Complete Chemistry Panel4 
X^^ 
 
 
 
 
 
 
 
 
 
 
X3 
 
 
 
 
 
 
 
 
 
 
X3 
 
Core-Biomarkers & 
Repository 
X^^ 
 
 
 
 
 
 
 
 
 
 
X^3 
 
 
 
 
 
 
 
 
 
 
X^3 
 
Imaging Evaluation
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Limited Echo3 
 
 
 
 
 
 
 
 
 
 
 
X3 
 
 
 
 
 
 
 
 
 
 
X3 
 
Exercise Evaluation
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CPET5  
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
QOL Evaluation
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 KCCQ 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
Study Drug and 
Accelerometer 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Test Dose (80 mg Run-in) & 
BP Observation (60 mins)6 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Randomization**
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Dispense/Change out 
Accelerometer and 
Instruction 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Dispense Study Drug, Device 
and Instructions 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Return Accelerometer (AXM), 
Study Drug and Device 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
Study Drug Initiation  
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 


 
INDIE-HFpEF Protocol v1.3, 22April2016 (Final) 
 
    
 45 
 
 
 
Study Drug Tolerance 
X 
 
 
 
X 
X 
 
X 
X 
X 
X 
X 
 
 
 
X 
X 
 
X 
X 
X 
X 
X 
 
Up-titration  (if tolerated) 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
Study Drug / AXM Adherence 
 
AXM 
only 
AXM 
only 
 
X 
X 
 
X 
X 
X 
X 
X 
AXM 
only 
AXM 
only 
 
X 
X 
 
X 
X 
X 
X 
X 
 
Adverse Events
X 
X 
X 
 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
X 
 
X 
X 
X 
X 
X 
X 
*Consented patients will be screened through IXRS  
**Only patients who have completed all inclusion/exclusion criteria, baseline studies and  tolerated the test dose  
   will be randomized through IXRS 
+Visit 2 is expected to be completed 42-49 days post Study Visit 1  
++Study Visit 3 is expected to be completed 42-49 days post Study Visit 2 
+++2 Week Follow-up visit is expected to be completed  14 days post Study Visit 3 
^ Biomarkers & Biorepository only (genetics is only required at Visit 1) 
^^Baseline bloods  must be obtained prior to CPET (in the event that is not feasible, then bloods must be at least 3 hours post  
   CPET but prior to the run-in test dose) 
1Includes etiology and duration of  HF 
2BP should be obtained just prior to test dose to confirm run-in eligibility 
3Obtain prior to study drug administration (in the event, this has to be scheduled after , then it must be at least 3 hours post 
last dose of study drug administered) 
4 Sodium, Potassium, Chloride, Carbon Dioxide, BUN, Creatinine, Glucose, ALT, AST, Alkaline Phosphatase & Total Bilirubin 
5HFN study-specific CPET:  Baseline-  within 3 days prior to randomization; Visit 2 & 3 – obtained immediately 
   post study drug administration 
6BP(seated and standing) obtained every 15 minutes for 60 minutes; patients are not to be randomized if SBP < 90 mmHg 
 


 
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23 REFERENCES  
 
1. 
Heidenreich PA, Albert NM, Allen LA, Bluemke DA, Butler J, Fonarow GC, Ikonomidis JS, 
Khavjou O, Konstam MA, Maddox TM, Nichol G, Pham M, Pina IL, Trogdon JG. 
Forecasting the impact of heart failure in the united states: A policy statement from the 
american heart association. Circ Heart Fail. 2013;6:606-619 
2. 
Owan TE, Hodge DO, Herges RM, Jacobsen SJ, Roger VL, Redfield MM. Trends in 
prevalence and outcome of heart failure with preserved ejection fraction. N Engl J Med. 
2006;355:251-259 
3. 
Borlaug BA, Paulus WJ. Heart failure with preserved ejection fraction: Pathophysiology, 
diagnosis, and treatment. Eur Heart J. 2011;32:670-679 
4. 
Borlaug BA, Melenovsky V, Russell SD, Kessler K, Pacak K, Becker LC, Kass DA. 
Impaired chronotropic and vasodilator reserves limit exercise capacity in patients with heart 
failure and a preserved ejection fraction. Circulation. 2006;114:2138-2147 
5. 
Borlaug BA, Lam CS, Roger VL, Rodeheffer RJ, Redfield MM. Contractility and ventricular 
systolic stiffening in hypertensive heart disease insights into the pathogenesis of heart 
failure with preserved ejection fraction. J Am Coll Cardiol. 2009;54:410-418 
6. 
Tan YT, Wenzelburger F, Lee E, Heatlie G, Leyva F, Patel K, Frenneaux M, Sanderson JE. 
The pathophysiology of heart failure with normal ejection fraction: Exercise 
echocardiography reveals complex abnormalities of both systolic and diastolic ventricular 
function involving torsion, untwist, and longitudinal motion. J Am Coll Cardiol. 2009;54:36-
46 
7. 
Phan TT, Abozguia K, Nallur Shivu G, Mahadevan G, Ahmed I, Williams L, Dwivedi G, 
Patel K, Steendijk P, Ashrafian H, Henning A, Frenneaux M. Heart failure with preserved 
ejection fraction is characterized by dynamic impairment of active relaxation and 
contraction of the left ventricle on exercise and associated with myocardial energy 
deficiency. J Am Coll Cardiol. 2009;54:402-409 
8. 
Maeder MT, Thompson BR, Brunner-La Rocca HP, Kaye DM. Hemodynamic basis of 
exercise limitation in patients with heart failure and normal ejection fraction. J Am Coll 
Cardiol. 2010;56:855-863 
9. 
Borlaug BA, Olson TP, Lam CS, Flood KS, Lerman A, Johnson BD, Redfield MM. Global 
cardiovascular reserve dysfunction in heart failure with preserved ejection fraction. J Am 
Coll Cardiol. 2010;56:845-854 
10. Borlaug BA, Nishimura RA, Sorajja P, Lam CS, Redfield MM. Exercise hemodynamics 
enhance diagnosis of early heart failure with preserved ejection fraction. Circ Heart Fail. 
2010;3:588-595 
11. Borlaug BA, Jaber WA, Ommen SR, Lam CS, Redfield MM, Nishimura RA. Diastolic 
relaxation and compliance reserve during dynamic exercise in heart failure with preserved 
ejection fraction. Heart. 2011;97:964-969 
12. Tartiere-Kesri L, Tartiere JM, Logeart D, Beauvais F, Cohen Solal A. Increased proximal 
arterial stiffness and cardiac response with moderate exercise in patients with heart failure 
and preserved ejection fraction. J Am Coll Cardiol. 2012;59:455-461 
13. Abudiab MM, Redfield MM, Melenovsky V, Olson TP, Kass DA, Johnson BD, Borlaug BA. 


 
INDIE-HFpEF Protocol v1.3, 22April2016 (Final) 
 
    
 47
 
 
 
 
Cardiac output response to exercise in relation to metabolic demand in heart failure with 
preserved ejection fraction. Eur J Heart Fail. 2013;15:776-785 
14. Haykowsky MJ, Brubaker PH, John JM, Stewart KP, Morgan TM, Kitzman DW. 
Determinants of exercise intolerance in elderly heart failure patients with preserved ejection 
fraction. J Am Coll Cardiol. 2011;58:265-274 
15. Bhella PS, Prasad A, Heinicke K, Hastings JL, Arbab-Zadeh A, Adams-Huet B, Pacini EL, 
Shibata S, Palmer MD, Newcomer BR, Levine BD. Abnormal haemodynamic response to 
exercise in heart failure with preserved ejection fraction. Eur J Heart Fail. 2011;13:1296-
1304 
16. Kitzman DW, Nicklas B, Kraus WE, Lyles MF, Eggebeen J, Morgan TM, Haykowsky M. 
Skeletal muscle abnormalities and exercise intolerance in older patients with heart failure 
and preserved ejection fraction. Am J Physiol Heart Circ Physiol. 2014;306:H1364-1370 
17. Haykowsky MJ, Kouba EJ, Brubaker PH, Nicklas BJ, Eggebeen J, Kitzman DW. Skeletal 
muscle composition and its relation to exercise intolerance in older patients with heart 
failure and preserved ejection fraction. Am J Cardiol. 2014;113:1211-1216 
18. Schwartzenberg S, Redfield MM, From AM, Sorajja P, Nishimura RA, Borlaug BA. Effects 
of vasodilation in heart failure with preserved or reduced ejection fraction implications of 
distinct pathophysiologies on response to therapy. J Am Coll Cardiol. 2012;59:442-451 
19. van Heerebeek L, Hamdani N, Falcao-Pires I, Leite-Moreira AF, Begieneman MP, 
Bronzwaer JG, van der Velden J, Stienen GJ, Laarman GJ, Somsen A, Verheugt FW, 
Niessen HW, Paulus WJ. Low myocardial protein kinase g activity in heart failure with 
preserved ejection fraction. Circulation. 2012;126:830-839 
20. Paulus WJ, Tschope C. A novel paradigm for heart failure with preserved ejection fraction: 
Comorbidities drive myocardial dysfunction and remodeling through coronary microvascular 
endothelial inflammation. J Am Coll Cardiol. 2013;62:263-271 
21. Redfield MM, Borlaug BA, Lewis GD, Mohammed SF, Semigran MJ, Lewinter MM, Deswal 
A, Hernandez AF, Lee KL, Braunwald E. Phosphdiesterase-5 inhibition to improve clinical 
status and exercise capacity in diastolic heart failure (relax) trial: Rationale and design. Circ 
Heart Fail. 2012;5:653-659 
22. Lundberg JO, Weitzberg E, Gladwin MT. The nitrate-nitrite-nitric oxide pathway in 
physiology and therapeutics. Nat Rev Drug Discov. 2008;7:156-167 
23. Altman DG. Practical statistics for medical research. London: Chapman and Hall; 1991. 
24. Jones B, Kenward MG. Design and analysis of cross-over trials, 2nd ed. . London: 
Chapman and Hall; 2003. 
25. Grizzle JE. The two-period change-over design an its use in clinical trials. Biometrics. 
1965;21:467-480 
26. Kahan A, Amor B, Menkes CJ, Weber S, Guerin F, Degeorges M. Nicardipine in the 
treatment of raynaud's phenomenon: A randomized double-blind trial. Angiology. 
1987;38:333-337 
27. Senn S. Cross-over trials in clinical research, 2nd ed. . Chichester: Wiley; 2002. 
28. Bland M. Cross-over trials. 2012;2012 
29. Zakeri R, Levine JA, Koepp GA, Borlaug BA, Chirinos JA, LeWinter M, VanBuren P, Davila-
Roman VG, de Las Fuentes L, Khazanie P, Hernandez A, Anstrom K, Redfield MM. 
Nitrate's effect on activity tolerance in heart failure with preserved ejection fraction trial: 
Rationale and design. Circ Heart Fail. 2015;8:221-228 


 
INDIE-HFpEF Protocol v1.3, 22April2016 (Final) 
 
    
 48
 
 
 
 
30. Santos M, Opotowsky AR, Shah AM, Tracy J, Waxman AB, Systrom DM. Central cardiac 
limit to aerobic capacity in patients with exertional pulmonary venous hypertension: 
Implications for heart failure with preserved ejection fraction. Circ Heart Fail. 2015;8:278-
285 
31. Dhakal BP, Malhotra R, Murphy RM, Pappagianopoulos PP, Baggish AL, Weiner RB, 
Houstis NE, Eisman AS, Hough SS, Lewis GD. Mechanisms of exercise intolerance in 
heart failure with preserved ejection fraction: The role of abnormal peripheral oxygen 
extraction. Circ Heart Fail. 2015;8:286-294 
32. Borbely A, Falcao-Pires I, van Heerebeek L, Hamdani N, Edes I, Gavina C, Leite-Moreira 
AF, Bronzwaer JG, Papp Z, van der Velden J, Stienen GJ, Paulus WJ. 
Hypophosphorylation of the stiff n2b titin isoform raises cardiomyocyte resting tension in 
failing human myocardium. Circ Res. 2009;104:780-786 
33. van Heerebeek L, Borbely A, Niessen HW, Bronzwaer JG, van der Velden J, Stienen GJ, 
Linke WA, Laarman GJ, Paulus WJ. Myocardial structure and function differ in systolic and 
diastolic heart failure. Circulation. 2006;113:1966-1973 
34. Redfield MM, Chen HH, Borlaug BA, Semigran MJ, Lee KL, Lewis G, Lewinter MM, 
Rouleau JL, Bull DA, Mann DL, Deswal A, Stevenson LW, Givertz MM, Ofili EO, O'Connor 
CM, Felker GM, Goldsmith SR, Bart BA, McNulty SE, Ibarra JC, Lin G, Oh JK, Patel MR, 
Kim RJ, Tracy RP, Velazquez EJ, Anstrom KJ, Hernandez AF, Mascette AM, Braunwald E. 
Effect of phosphodiesterase-5 inhibition on exercise capacity and clinical status in heart 
failure with preserved ejection fraction: A randomized clinical trial. JAMA. 2013;309:1268-
1277 
35. Munzel T, Daiber A, Mulsch A. Explaining the phenomenon of nitrate tolerance. Circ Res. 
2005;97:618-628 
36. Cosby K, Partovi KS, Crawford JH, Patel RP, Reiter CD, Martyr S, Yang BK, Waclawiw 
MA, Zalos G, Xu X, Huang KT, Shields H, Kim-Shapiro DB, Schechter AN, Cannon RO, 
3rd, Gladwin MT. Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the 
human circulation. Nat Med. 2003;9:1498-1505 
37. Dejam A, Hunter CJ, Tremonti C, Pluta RM, Hon YY, Grimes G, Partovi K, Pelletier MM, 
Oldfield EH, Cannon RO, 3rd, Schechter AN, Gladwin MT. Nitrite infusion in humans and 
nonhuman primates: Endocrine effects, pharmacokinetics, and tolerance formation. 
Circulation. 2007;116:1821-1831 
38. Maher AR, Milsom AB, Gunaruwan P, Abozguia K, Ahmed I, Weaver RA, Thomas P, 
Ashrafian H, Born GV, James PE, Frenneaux MP. Hypoxic modulation of exogenous nitrite-
induced vasodilation in humans. Circulation. 2008;117:670-677 
39. Coggan AR, Leibowitz JL, Spearie CA, Kadkhodayan A, Thomas DP, Ramamurthy S, 
Mahmood K, Park S, Waller S, Farmer M, Peterson LR. Acute dietary nitrate intake 
improves muscle contractile function in patients with heart failure: A double-blind, placebo-
controlled, randomized trial. Circ Heart Fail. 2015;8:914-920 
40. Larsen FJ, Weitzberg E, Lundberg JO, Ekblom B. Effects of dietary nitrate on oxygen cost 
during exercise. Acta Physiol (Oxf). 2007;191:59-66 
41. Bailey SJ, Winyard P, Vanhatalo A, Blackwell JR, Dimenna FJ, Wilkerson DP, Tarr J, 
Benjamin N, Jones AM. Dietary nitrate supplementation reduces the o2 cost of low-intensity 
exercise and enhances tolerance to high-intensity exercise in humans. J Appl Physiol 
(1985). 2009;107:1144-1155 


 
INDIE-HFpEF Protocol v1.3, 22April2016 (Final) 
 
    
 49
 
 
 
 
42. Larsen FJ, Schiffer TA, Borniquel S, Sahlin K, Ekblom B, Lundberg JO, Weitzberg E. 
Dietary inorganic nitrate improves mitochondrial efficiency in humans. Cell Metab. 
2011;13:149-159 
43. Omar SA, Fok H, Tilgner KD, Nair A, Hunt J, Jiang B, Taylor P, Chowienczyk P, Webb AJ. 
Paradoxical normoxia-dependent selective actions of inorganic nitrite in human muscular 
conduit arteries and related selective actions on central blood pressures. Circulation. 
2015;131:381-389; discussion 389 
44. Zamani P, Rawat D, Shiva-Kumar P, Geraci S, Bhuva R, Konda P, Doulias PT, 
Ischiropoulos H, Townsend RR, Margulies KB, Cappola TP, Poole DC, Chirinos JA. Effect 
of inorganic nitrate on exercise capacity in heart failure with preserved ejection fraction. 
Circulation. 2015;131:371-380; discussion 380 
45. Borlaug BA, Koepp KE, Melenovsky V. Sodium nitrite improves exercise hemodynamics 
and ventricular performance in heart failure with preserved ejection fraction. J Am Coll 
Card. 2015;66:1672-1682 
46. Rix PJ, Vick A, Attkins NJ, Barker GE, Bott AW, Alcorn H, Jr., Gladwin MT, Shiva S, 
Bradley S, Hussaini A, Hoye WL, Parsley EL, Masamune H. Pharmacokinetics, 
pharmacodynamics, safety, and tolerability of nebulized sodium nitrite (air001) following 
repeat-dose inhalation in healthy subjects. Clin Pharmacokinet. 2014 
47. Parsley EL. personal communication, unpublished data 
48. Hunt SA, Abraham WT, Chin MH, Feldman AM, Francis GS, Ganiats TG, Jessup M, 
Konstam MA, Mancini DM, Michl K, Oates JA, Rahko PS, Silver MA, Stevenson LW, Yancy 
CW. 2009 focused update incorporated into the acc/aha 2005 guidelines for the diagnosis 
and management of heart failure in adults a report of the american college of cardiology 
foundation/american heart association task force on practice guidelines developed in 
collaboration with the international society for heart and lung transplantation. J Am Coll 
Cardiol. 2009;53:e1-e90 
 
 

SAP_000.pdf: 
Statistical Analysis Plan Amendment 1 (INDIE)  
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INDIE – A Heart Failure Network Protocol                                                                                                      FINAL (11 April 2017) 
 
 
Statistical Analysis Plan   
 
Inorganic Nitrite Delivery to Improve Exercise Capacity in HFpEF  
 
INDIE-HFpEF 
 
 
 
 
A randomized, double-blind, placebo-controlled crossover study to assess the effect of inorganic nitrite 
(NO2) on aerobic capacity (peak VO2). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Original Protocol Date  
April 22, 2016 
 
Amendment 1 Date 
November 10, 2016 
 
Sponsor 
National Heart, Lung and Blood Institute 
 
Protocol Principal Investigators 
Barry Borlaug, MD  
 


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INDIE – A Heart Failure Network Protocol                                                                                                      FINAL (11 April 2017) 
 
Table of Contents 
1. 
Overview 
……………………………………… 
3 
 
1.1 
Synopsis 
……………………………………… 
3 
 
1.2 
Study Treatments 
……………………………………… 
3 
 
 
 
2. 
Study Design 
……………………………………… 
3 
 
2.1 
Overview 
……………………………………… 
3 
 
2.2 
Randomization 
……………………………………… 
3 
 
2.3 
Data Sources 
……………………………………… 
3 
 
 
 
3. 
Analysis Population and Missing Data 
……………………………………… 
3 
 
 
 
4. 
General Methodology 
……………………………………… 
4 
 
 
 
5. 
Primary Endpoint 
……………………………………… 
4 
 
 
 
6. 
Secondary Endpoints 
……………………………………… 
4 
 
 
 
7. 
Tertiary Endpoints 
……………………………………… 
5 
 
 
 
8. 
Primary Endpoint Description 
……………………………………… 
5 
 
8.1 
Primary Endpoint 
……………………………………… 
5 
 
 
 
9. 
Secondary Endpoint Descriptions 
……………………………………… 
6 
 
9.1 
Secondary Endpoint #1 
……………………………………… 
6 
 
9.2 
Secondary Endpoint #2 
……………………………………… 
7 
 
9.3 
Secondary Endpoint #3 
……………………………………… 
7 
 
9.4 
Secondary Endpoint #4 
……………………………………… 
8 
 
9.5 
Secondary Endpoint #5 
……………………………………… 
8 
 
9.6 
Secondary Endpoint #6 
……………………………………… 
8 
 
9.7 
Secondary Endpoint #7 
……………………………………… 
9 
 
9.8 
Secondary Endpoint #8 
……………………………………… 
9 
 
9.9 
Secondary Endpoint #9 
……………………………………… 
10 
 
9.10 
Secondary Endpoint #10 
……………………………………… 
10 
 
9.11 
Secondary Endpoint #11 
……………………………………… 
11 
 
 
 
10. 
Tertiary Endpoint Descriptions 
……………………………………… 
11 
 
10.1 
Tertiary Endpoint #1 
……………………………………… 
11 
 
10.2 
Tertiary Endpoint #2 
……………………………………… 
12 
 
10.3 
Tertiary Endpoint #3 
……………………………………… 
12 
 
 
 
11. 
Safety Analyses 
……………………………………… 
12 
 
11.1 
Safety Endpoint #1 
……………………………………… 
12 
 
 
 
12. 
Interim Analysis 
……………………………………… 
13 


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INDIE – A Heart Failure Network Protocol                                                                                                      FINAL (11 April 2017) 
 
 
1.  Overview 
 
1.1 Synopsis 
 
The Inorganic Nitrite Delivery to Improve Exercise Capacity in HFpEF (INDIE-HFpEF) trial is a placebo-
controlled 2*2 crossover trial to assess the effect of inorganic nitrite (NO2) with dose up-titration on exercise 
capacity as assessed by cardiopulmonary exercise test.    
 
1.2 Study Treatments 
 
Given the crossover nature of the study, each patient will receive both inorganic nitrite and matching 
placebo, with the treatment order determined during the randomization process. Each study phase will 
consist of a 2 week washout period, 1 week at the 46 mg dose (TID), and 3 weeks at the 80 mg dose (TID). 
Study medication is administered three times per day, at least four hours apart in a blinded fashion during 
each study phase. 
2.  Study Design 
 
2.1 Overview 
 
The INDIE-HFpEF study is a randomized, double-blind, placebo-controlled crossover study in heart failure 
patients with preserved ejection fraction. A total of 100 patients will be enrolled in the trial.     
 
The treatments in this study are blinded. Each study phase will last for 6 weeks. The first 2 weeks of each 
study phase will be the washout period with study treatment received during the last 4 weeks.  
 
The over-arching hypothesis is that, compared to placebo, treatment with inorganic nitrite will lead to 
improved exercise capacity as assessed by cardiopulmonary exercise testing.  
 
2.2 Randomization 
 
Patients are randomized in a 1:1 ratio to a specific treatment receipt order: inorganic nitrite in study phase 1 
and Placebo in study phase 2 OR Placebo in study phase 1 and inorganic nitrite in study phase 2. The 
randomization scheme consists of a permuted block design with stratification by clinical site.   
 
2.3 Data Sources 
 
A database of case report form and biomarker core lab data will be created in Inform, and the data then 
transferred to SAS for analysis.  The randomized treatment assignment will be provided through data 
provided by the Webez system, an Almac Clinical Services web-based randomization system. The 
accelerometer data was provided by the Accelerometer Core Lab located at Mayo Clinic. The core lab 
provided both cleaned and raw data for download. The data is stored on Solaris server uxctstp01 in the 
folder /hf_net/INDIE/data.  
 
3.  Analysis Population and Missing Data 


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INDIE – A Heart Failure Network Protocol                                                                                                      FINAL (11 April 2017) 
 
 
All randomized patients will be included in the analysis population for assessing the primary, secondary and 
tertiary endpoints. Given the crossover design of the study, each patient will have endpoints measured at 
two times; the first at the conclusion of Phase 1 (Visit 2) treatment and the second at the conclusion of 
Phase 2 treatment (Visit 3). All endpoints will be analyzed on an intent-to-treat basis. 
 
However, as described in subsequent sections of this document, some patients may be excluded from 
certain analyses if key data elements are missing.  With the extensive efforts being made in collaboration 
with the clinical sites to ensure data quality and completeness, it is expected that exclusion of patients for 
any endpoint analysis will be minimal.  The specific endpoint descriptions in Sections 8 through 10 describe 
the circumstances that would lead to a patient being excluded from a specific analysis. 
 
4.  General Methodology 
 
Medians, 25th and 75th percentiles will be presented for continuous variables; the number and percentage of 
patients in each category will be presented for categorical variables.  For all endpoints a p-value ≤0.05 will 
be considered statistically significant and all tests will be 2-sided.  Analyses will be performed using 
validated SAS software (SAS Institute, Inc., Cary, NC).  Appropriate statistical models will be used to 
examine the effect of treatment on both the primary, secondary, and tertiary outcomes in the study.  
 
The majority of the endpoints will be analyzed using a linear mixed model. Within the context of the mixed 
models, the period effect will be examined to determine whether both treatment effects are consistently 
higher or lower when comparing Phases 1 and 2 of the study (e.g., Peak VO2 is increased in Phase 2 for 
both treatments vs Phase 1). The sequence effect will be examined to determine if the treatment effect 
differs based on the treatment order (inorganic nitrite then Placebo or Placebo then inorganic nitrite). 
Alternative modeling may also be performed using paired t-tests or appropriately structured general linear 
models.  
 
5.  Primary Endpoint 
 
Primary Endpoint 
#1: Within patient comparison of exercise capacity as assessed by peak VO2 (ml/kg/min), comparing 
inorganic nitrite to placebo 
 
See Section 8 for a detailed description of the primary endpoint, including rules that will be followed for 
handling incomplete data. 
 
6.  Secondary Endpoints 
 
Secondary Endpoints 
#1: Within patient comparison of arbitrary accelerometer units (AAU) during the maximally tolerated dose of 
inorganic nitrite vs placebo 
#2: Within patient comparison of Kansas City Cardiomyopathy Questionnaire (KCCQ) symptom scores – 
both clinical and overall, comparing inorganic nitrite vs placebo 
#3: Within patient comparison of New York Heart Association (NYHA) functional class, comparing inorganic 
nitrite vs placebo 


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INDIE – A Heart Failure Network Protocol                                                                                                      FINAL (11 April 2017) 
 
#4: Patient preference for study phase (inorganic nitrite vs. placebo) 
#5: Within patient comparison of medial E/e’ ratio as assessed on echocardiography, comparing inorganic 
nitrite vs placebo 
#6: Within patient comparison of left atrial volume index as assessed on echocardiography, comparing 
inorganic nitrite vs placebo 
#7: Within patient comparison of pulmonary artery systolic pressure (PASP) as assessed on 
echocardiography, comparing inorganic nitrite vs placebo 
#8: Within patient comparison of core lab NT Pro BNP, comparing inorganic nitrite vs placebo 
#9: Within patient comparison of VE/VCO2 slope (ventilator efficiency) as assessed on cardiopulmonary 
exercise testing, comparing inorganic nitrite vs placebo 
#10: Within patient comparison of VO2 at ventilatory threshold (submaximal exercise capacity) as assessed 
on cardiopulmonary exercise testing, comparing inorganic nitrite vs placebo 
#11: Within patient comparison of exercise duration as assessed during cardiopulmonary exercise testing, 
comparing inorganic nitrite vs placebo 
 
See Section 9 for a detailed description of each secondary endpoint, including rules that will be followed for 
handling incomplete data. 
 
7.  Tertiary Endpoints 
 
Tertiary Endpoints 
#1: Primary endpoint comparison within the following subgroups: 
a. Baseline core lab NT Pro BNP > 400 pg/mL vs ≤ 400 pg/mL 
b. Baseline systolic blood pressure above and below the median 
c. Age above and below the median 
d. Male vs female  
e. Baseline estimated glomerular filtration rate (GFR) by MDRD above and below the median 
f. 
On vs off study drug at time of visit based on active study drug phase 
g. Atrial fibrillation/flutter vs not 
h. Diabetes vs no diabetes 
i. 
BMI above and below the median 
j. 
Baseline activity level above and below the median 
#2: Within patient comparison of core lab cystatin C, comparing inorganic nitrite vs placebo 
#3: Within patient comparison of core lab cyclic guanosine monophosphate (cGMP), comparing inorganic 
nitrite vs placebo 
 
Note: The protocol states that we will also examine novel accelerometry endpoints, guided by NEAT-HFpEF 
ancillary paper analyses (in progress). As of yet, there are no novel endpoints under consideration for this 
study. 
 
See Section 10 for a detailed description of each tertiary endpoint, including rules that will be followed for 
handling incomplete data. 
 
8.  Endpoint Descriptions 
8.1 – Primary Endpoint 


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INDIE – A Heart Failure Network Protocol                                                                                                      FINAL (11 April 2017) 
 
 
Endpoint Description: Exercise capacity as assessed by peak VO2 (ml/kg/min) measured during 
cardiopulmonary exercise testing 
 
Response Variable Definition: Visit 2 and Visit 3 peak VO2, classified as to treatment received in prior 
treatment period.  
 
Additional Covariates:  Baseline peak VO2 score, period effect, sequence effect, and random effect of 
patient 
 
Handling of Dropouts and Missing Data: No adjustment for missing data will be made.  
 
Sensitivity Analysis: Two sensitivity analyses will be performed. 
1) Due to the short washout period of the study drug, a separate analysis will be performed removing 
those patients that discontinued active study treatment prior to the next study visit where the 
cardiopulmonary exercise test was performed. 
2) To address a potential question seen in some journal reviews, the original mixed model will be run 
again, adding a new covariate for random effect of enrolling site. 
 
Statistical Tests: A mixed model (PROC MIXED in SAS) will be used to estimate and statistically compare 
the response to inorganic nitrite versus placebo on peak VO2. Secondary analyses will be performed using 
paired t-tests and appropriate general linear models. 
 
Interpretation of Results: A higher peak VO2 indicates an improved exercise capacity in the inorganic nitrite 
period vs placebo. 
 
9.  Secondary Endpoint Descriptions 
 
9.1 – Secondary Endpoint #1 
 
Endpoint Description: Arbitrary accelerometry units (AAU) during maximally tolerated dose of inorganic 
nitrite vs placebo 
 
Response Variable Definition: For each study phase, the average daily AAU will be calculated for the period 
of time that patient was taking 80 mg daily (or maximally tolerated dose if 80 mg not tolerated), approximate 
time periods are study weeks 4-6 and 10-12. Only complete days are used and there should be 21 days in 
the dosing period.  
 
Additional Covariates:  period effect, sequence effect, baseline daily average AAU, and random effect of 
patient  
 
Handling of Dropouts and Missing Data: No adjustment for missing data will be made.  
 
Sensitivity Analysis: For this sensitivity analysis, each average will be divided by the study phase 1 14 day 
baseline period average daily AAU (approximately study weeks 1-2) to create a ratio. Values greater than 1 
indicate increased activity.   


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INDIE – A Heart Failure Network Protocol                                                                                                      FINAL (11 April 2017) 
 
 
Statistical Tests: A mixed model (PROC MIXED in SAS) will be used to estimate and statistically compare 
the response to inorganic nitrite versus placebo on daily activity level. Secondary analyses will be performed 
using paired t-tests and appropriate general linear models. 
 
Interpretation of Results: Higher AAU indicates more activity in the inorganic nitrite period vs placebo.  
 
9.2 – Secondary Endpoint #2 
 
Endpoint Description: Kansas City Cardiomyopathy Questionnaire Overall and Clinical Summary Scores 
 
Response Variable Definition: Visit 2 and Visit 3 KCCQ scores, classified as to treatment received in prior 
treatment period. Each summary score will be an individual endpoint and have its own model. 
 
Additional Covariates:  Baseline KCCQ score (as appropriate), period effect, sequence effect, and random 
effect of patient 
 
Handling of Dropouts and Missing Data: No adjustment for missing data will be made.  
 
Sensitivity Analysis: Due to the short washout period of the study drug, a separate analysis will be 
performed removing those patients that discontinued active study treatment prior to the next study visit 
where the KCCQ questionnaire was completed. 
 
Statistical Tests: A mixed model (PROC MIXED in SAS) will be used to estimate and statistically compare 
the response to inorganic nitrite versus placebo on each KCCQ summary score. Secondary analyses will be 
performed using paired t-tests and appropriate general linear models. 
 
Interpretation of Results: A higher KCCQ score indicates an improved KCCQ score in the inorganic nitrite 
period vs placebo. 
 
9.3 – Secondary Endpoint #3 
 
Endpoint Description: NYHA class 
 
Response Variable Definition: Visit 2 and Visit 3 NYHA class, classified as to treatment received in prior 
treatment period.  
 
Additional Covariates:  Baseline NYHA class, period effect, sequence effect, and random effect of patient 
 
Handling of Dropouts and Missing Data: No adjustment for missing data will be made.  
 
Sensitivity Analysis: Due to the short washout period of the study drug, a separate analysis will be 
performed removing those patients that discontinued active study treatment prior to the next study visit 
where the NYHA class was assessed. 
 


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INDIE – A Heart Failure Network Protocol                                                                                                      FINAL (11 April 2017) 
 
Statistical Tests: A mixed model (PROC MIXED in SAS) will be used to estimate and statistically compare 
the response to inorganic nitrite versus placebo on NYHA class. Secondary analyses will be performed 
using paired t-tests and appropriate general linear models. 
 
Interpretation of Results: A lower NYHA class indicates an improved heart failure symptoms in the inorganic 
nitrite period vs placebo. 
 
9.4 – Secondary Endpoint #4 
 
Endpoint Description: Patient preference for study phase (inorganic nitrite vs. placebo) 
 
Response Variable Definition: Patient preferred study phase (active, placebo, no preference).  
 
Statistical Tests: No formal testing will be done.  
  
Interpretation of Results: For patients with a preference, a higher percentage of patients favoring the 
inorganic nitrite period vs placebo indicate a positive treatment experience. 
 
9.5 – Secondary Endpoint #5 
 
Endpoint Description: Medial E/e’ 
 
Response Variable Definition: Visit 2 and Visit 3 medial E/e’, classified as to treatment received in prior 
treatment period.  
 
Additional Covariates:  Period effect, sequence effect, and random effect of patient 
 
Handling of Dropouts and Missing Data: No adjustment for missing data will be made.  
 
Sensitivity Analysis: Due to the short washout period of the study drug, a separate analysis will be 
performed removing those patients that discontinued active study treatment prior to the next study visit 
where the echocardiogram was performed. 
 
Statistical Tests: A mixed model (PROC MIXED in SAS) will be used to estimate and statistically compare 
the response to inorganic nitrite versus placebo on medial E/e’. Secondary analyses will be performed using 
paired t-tests and appropriate general linear models. 
 
Interpretation of Results: A lower medial E/e’ indicates improvement in the inorganic nitrite period vs 
placebo. 
 
9.6 – Secondary Endpoint #6 
 
Endpoint Description: Left atrial (LA) volume index 
 
Response Variable Definition: Visit 2 and Visit 3 left atrial volume index, classified as to treatment received 
in prior treatment period.  


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INDIE – A Heart Failure Network Protocol                                                                                                      FINAL (11 April 2017) 
 
 
Additional Covariates:  Period effect, sequence effect, and random effect of patient 
 
Handling of Dropouts and Missing Data: No adjustment for missing data will be made.  
 
Sensitivity Analysis: Due to the short washout period of the study drug, a separate analysis will be 
performed removing those patients that discontinued active study treatment prior to the next study visit 
where the echocardiogram was performed. 
 
Statistical Tests: A mixed model (PROC MIXED in SAS) will be used to estimate and statistically compare 
the response to inorganic nitrite versus placebo on LA volume index. Secondary analyses will be performed 
using paired t-tests and appropriate general linear models. 
 
Interpretation of Results: A lower LA volume index indicates improvement in the inorganic nitrite period vs 
placebo. 
 
9.7 – Secondary Endpoint #7 
 
Endpoint Description: Pulmonary artery systolic pressure 
 
Response Variable Definition: Visit 2 and Visit 3 PASP, classified as to treatment received in prior treatment 
period.  
 
Additional Covariates:  Period effect, sequence effect, and random effect of patient 
 
Handling of Dropouts and Missing Data: No adjustment for missing data will be made.  
 
Sensitivity Analysis: Due to the short washout period of the study drug, a separate analysis will be 
performed removing those patients that discontinued active study treatment prior to the next study visit 
where the echocardiogram was performed. 
 
Statistical Tests: A mixed model (PROC MIXED in SAS) will be used to estimate and statistically compare 
the response to inorganic nitrite versus placebo on PASP. Secondary analyses will be performed using 
paired t-tests and appropriate general linear models. 
 
Interpretation of Results: A lower PASP indicates improvement in the inorganic nitrite period vs placebo. 
 
9.8 – Secondary Endpoint #8 
 
Endpoint Description: Core lab NT pro BNP 
 
Response Variable Definition: Visit 2 and Visit 3 NT pro BNP, classified as to treatment received in prior 
treatment period.  
 
Additional Covariates:  Baseline NT pro BNP, period effect, sequence effect, and random effect of patient 
 


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INDIE – A Heart Failure Network Protocol                                                                                                      FINAL (11 April 2017) 
 
Handling of Dropouts and Missing Data: No adjustment for missing data will be made.  
 
Sensitivity Analysis: Due to the short washout period of the study drug, a separate analysis will be 
performed removing those patients that discontinued active study treatment prior to the next study visit 
where the biomarkers were drawn. 
 
Statistical Tests: A mixed model (PROC MIXED in SAS) will be used to estimate and statistically compare 
the response to inorganic nitrite versus placebo on NT pro BNP. Secondary analyses will be performed 
using paired t-tests and appropriate general linear models. 
 
Interpretation of Results: A lower NT pro BNP indicates improved NT pro BNP in the inorganic nitrite period 
vs placebo. 
 
9.9 – Secondary Endpoint #9 
 
Endpoint Description: VE/VCO2 slope 
 
Response Variable Definition: Visit 2 and Visit 3 VE/VCO2 slope, classified as to treatment received in prior 
treatment period.  
 
Additional Covariates:  Baseline VE/VCO2 slope, period effect, sequence effect, and random effect of patient 
 
Handling of Dropouts and Missing Data: No adjustment for missing data will be made.  
 
Sensitivity Analysis: Due to the short washout period of the study drug, a separate analysis will be 
performed removing those patients that discontinued active study treatment prior to the next study visit 
where the cardiopulmonary exercise testing was performed. 
 
Statistical Tests: A mixed model (PROC MIXED in SAS) will be used to estimate and statistically compare 
the response to inorganic nitrite versus placebo on VE/VCO2 slope. Secondary analyses will be performed 
using paired t-tests and appropriate general linear models. 
 
Interpretation of Results: A lower VE/VCO2 slope indicates improvement in the inorganic nitrite period vs 
placebo. 
 
9.10 – Secondary Endpoint #10 
 
Endpoint Description: VO2 at ventilatory threshold 
 
Response Variable Definition: Visit 2 and Visit 3 VO2 at ventilatory threshold, classified as to treatment 
received in prior treatment period.  
 
Additional Covariates:  Baseline VO2 at ventilatory threshold, period effect, sequence effect, and random 
effect of patient 
 
Handling of Dropouts and Missing Data: No adjustment for missing data will be made.  


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INDIE – A Heart Failure Network Protocol                                                                                                      FINAL (11 April 2017) 
 
 
Sensitivity Analysis: Due to the short washout period of the study drug, a separate analysis will be 
performed removing those patients that discontinued active study treatment prior to the next study visit 
where the cardiopulmonary exercise testing was performed. 
 
Statistical Tests: A mixed model (PROC MIXED in SAS) will be used to estimate and statistically compare 
the response to inorganic nitrite versus placebo on VO2 at ventilatory threshold. Secondary analyses will be 
performed using paired t-tests and appropriate general linear models. 
 
Interpretation of Results: A higher VO2 at ventilatory threshold indicates improvement in the inorganic nitrite 
period vs placebo. 
 
9.11 – Secondary Endpoint #11 
 
Endpoint Description: Exercise duration on CPET 
 
Response Variable Definition: Visit 2 and Visit 3 exercise duration, classified as to treatment received in 
prior treatment period.  
 
Additional Covariates:  Baseline exercise duration, period effect, sequence effect, and random effect of 
patient 
 
Handling of Dropouts and Missing Data: No adjustment for missing data will be made.  
 
Sensitivity Analysis: Due to the short washout period of the study drug, a separate analysis will be 
performed removing those patients that discontinued active study treatment prior to the next study visit 
where the cardiopulmonary exercise testing was performed. 
 
Statistical Tests: A mixed model (PROC MIXED in SAS) will be used to estimate and statistically compare 
the response to inorganic nitrite versus placebo on exercise duration. Secondary analyses will be performed 
using paired t-tests and appropriate general linear models. 
 
Interpretation of Results: A longer exercise duration indicates improvement in the inorganic nitrite period vs 
placebo. 
 
10.  Tertiary Endpoint Descriptions 
 
10.1 – Tertiary Endpoint #1 
 
Endpoint Description: Exercise capacity as assessed by peak VO2 (ml/kg/min) measured during 
cardiopulmonary exercise testing within the following subgroups: 
a. Baseline core lab NT Pro BNP > 400 pg/mL vs ≤ 400 pg/mL 
b. Baseline systolic blood pressure above and below the median 
c. Age above and below the median 
d. Male vs female  
e. Baseline estimated glomerular filtration rate (GFR) by MDRD above and below the median 


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INDIE – A Heart Failure Network Protocol                                                                                                      FINAL (11 April 2017) 
 
f. 
On vs off study drug at time of visit based on active study drug phase 
g. Atrial fibrillation/flutter vs not 
h. Diabetes vs no diabetes 
i. 
BMI above and below the median 
j. 
Baseline activity level above and below the median 
 
See section 8.1 for analysis details 
 
10.2 – Tertiary Endpoint #2 
 
Endpoint Description: Core lab cystatin C 
 
Response Variable Definition: Visit 2 and Visit 3 cystatin C, classified as to treatment received in prior 
treatment period.  
 
Additional Covariates:  Baseline cystatin C, period effect, sequence effect, and random effect of patient 
 
Handling of Dropouts and Missing Data: No adjustment for missing data will be made.  
 
Statistical Tests: A mixed model (PROC MIXED in SAS) will be used to estimate and statistically compare 
the response to inorganic nitrite versus placebo on cystatin C. Secondary analyses will be performed using 
paired t-tests and appropriate general linear models. 
 
Interpretation of Results: A lower cystatin C indicates improvement in the inorganic nitrite period vs placebo. 
 
10.3 – Tertiary Endpoint #3 
 
Endpoint Description: Core lab cyclic guanosine monophosphate (cGMP) 
 
Response Variable Definition: Visit 2 and Visit 3 cGMP, classified as to treatment received in prior treatment 
period. 
 
Additional Covariates:  Baseline cGMP, period effect, sequence effect, and random effect of patient 
 
Handling of Dropouts and Missing Data: No adjustment for missing data will be made.  
 
Statistical Tests: A mixed model (PROC MIXED in SAS) will be used to estimate and statistically compare 
the response to inorganic nitrite versus placebo on cGMP. Secondary analyses will be performed using 
paired t-tests and appropriate general linear models. 
 
Interpretation of Results: A higher cGMP indicates an improvement in the inorganic nitrite period vs placebo. 
 
11.  Safety analysis 
 
11.1 – Safety Endpoint #1 
 
Safety Endpoint #1: Comparison of adverse events and serious adverse events 


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INDIE – A Heart Failure Network Protocol                                                                                                      FINAL (11 April 2017) 
 
 
Response Variable Definitions:  Each unique adverse event (AE) and serious adverse event (SAE) type 
based on preferred term within body system. The number of patients experiencing adverse events will be 
counted within each study phase, based on preferred term.  
 
Additional Covariates: None 
 
Handling of Dropouts and Missing Data: 
No adjustments for missing data will be made. 
 
Statistical Tests: 
Fisher’s mid-p will be calculated for each adverse event within each study phase.  
 
Interpretation of Results: Lower event rates indicate a better outcome for inorganic nitrite vs placebo.  
 
12.  Interim Analyses 
 
Interim data analysis for efficacy will not be conducted due to the relatively small size and short duration of 
this clinical trial. Safety data will be periodically assessed by the Data and Safety Monitoring Board (DSMB) 
based on the reporting of adverse events. There are no pre-specified guidelines for determining stopping 
rules due to a safety concern; the clinical opinion from the DSMB deliberations will be sole determinant.