Clinical trial NCT02713126
Conditions: Heart Failure
Sponsor: Barry Borlaug
Start date: 2016-09-01; Completion date: 2022-04-12
Enrollment: 92 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Participants with heart failure with preserved ejection fraction will undergo 12 weeks of cardiac rehabilitation for exercise training (ET) and be randomized to either sodium nitrite or placebo through the training period. Study drug is administered 3 times daily with one of these doses being 30 minutes prior to onset of ET sessions throughout the12 week trial. The objective is to determine if the sodium nitrite improves the clinical responses and tolerability of ET.

Detailed description:

Arms: arm 1: Subjects will undergo cardiac exercise training and receive inhaled or oral placebo three times per day for 12 weeks while wearing an accelerometer arm 2: Subjects will undergo cardiac exercise training and receive inhaled or oral sodium nitrite three times per day for 12 weeks while wearing an accelerometer
Interventions: intervention 1: Placebo intervention 2: Sodium Nitrite intervention 3: Accelerometer intervention 4: Cardiac Exercise Training
Intervention descriptions: intervention 1: administered orally or inhaled, taken every 4 hours or 3 times per day during waking hours for 12 weeks intervention 2: administered orally or inhaled, 40 mg, taken every 4 hours or 3 times per day during waking hours for 12 weeks intervention 3: External triaxial accelerometer is worn externally at the hip during exercise training sessions, and day/night to continuously measure activity levels, removed for bathing only. intervention 4: Cardiac rehabilitation exercise training consisting of supervised endurance and strength exercise training of approximately one hour, 3 times per week for 12 weeks.

Protocol documents:
Prot_SAP_000.pdf: 
 
Inorganic Nitrite to Amplify the 
Benefits and toLerability of 
Exercise TRAINING in heart 
failure with preserved ejection 
fraction (INABLE-TRAINING) 
 
NCT02713126  
 
February 10, 2021 


 
Inorganic Nitrite to Amplify the Benefits and toLerability of Exercise 
TRAINING in heart failure with preserved ejection fraction 
(INABLE-TRAINING) 
 
 
 
 
 
 
Regulatory Sponsor: 
Barry A. Borlaug, MD 
Mayo Clinic 
Internal Medicine Department 
Cardiovascular Diseases Division 
200 First Street SW 
Rochester, MN 55905 
Funding Sponsor: 
National Institutes of Health 
Aires Pharmaceuticals 
Study Product: 
Sodium Nitrite 
Protocol Number: (IRBe) 
16-001467 
IND Number: 
123333 
 
 
 
 
 
 
 
Versions: 
1 18Feb2016; Initial 
2 15Sept2016: Addition of the option to down-titrate the dose of study drug for potential intolerance (from 80 to 
46 mg). Correction of the number of subjects from 80 to 68 to reflect the target accrual number, not the target 
enrollment number 
3 11 Jan 2017: Revise inclusion criteria subject population minimum age criteria from 50 years of age to 40 
years of age. Addition of whole body DEXA scan and Cardiopulmonary Exercise Test (CPET) in the protocol 
4 22 Mar 2017: Addition of Mayo Clinic Health System in Cannon Falls as a study site. Addition of added labs 
at screening, week 4, week 8 and final visit blood draws 
5 21 Jun 2017: MRI/MRE substudy. Addition of SF- 36. AE description. 
6 20 Sept 2017: Addition of Mayo Clinic Health Systems in Eau Claire, Mankato, Red Wing, Austin, Albert Lea 
and La Crosse. 


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7 Dec 2017: Addition of whole blood collection and BAP storage. Changed inclusion/exclusion. Added thank 
you note for patients. 
 
8 10 Jun 2018: Modified protocol to change from inhaled nitrite formulation to oral formulation because of 
expiration/shutter of inhaled compound (decision made by manufacturer). 
 
9 20 Sep 2018: Modified protocol to change drug dispensing and return and allow re-enrollment 
of phase 1 INABLE subjects. 
 
10 16 Jan 2019: Modified protocol to update compounding site 
 
11 14 Feb 2019: Modified protocol to change analysis plan, randomization ratio, and treatment of 
study drug from 10 weeks to 12 weeks because of delays in provision of study drug. 
 
12  26 Jul 2019 Modified protocol to update compounding site and clerical errors. 
 
13 04 Sept 2019  Modified optional MRI substudy to include gadolinium use.  
 
14 13 Nov 2019 Modified optional MRI substudy to include hematocrit draw if not already obtained 
within the past 30 days and clarify timing of MRI. 
 
15 23 Jul 2020 Due to COVID 19 will offer home-based cardiac rehabilitation program. 
 
16   10Feb2021  Add optional muscle biopsy and add external collaborators. 
  


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Table of Contents 
STUDY SUMMARY .................................................................................................................................................. 6 
1 
INTRODUCTION ............................................................................................................................................. 8 
1.1 
BACKGROUND ................................................................................................................................................................................... 9 
1.2 
INVESTIGATIONAL AGENT .......................................................................................................................................................... 13 
PHYSICAL, CHEMICAL, PHARMACEUTICAL PROPERTIES, AND FORMULATION: ................................. 13 
DRUG SUBSTANCE: 
THE DRUG SUBSTANCE IS SODIUM NITRITE, NANO2 ........................................ 13 
1.3 
CLINICAL DATA TO DATE ........................................................................................................................................................... 14 
1.4 
DOSE RATIONALE AND RISK/BENEFITS ................................................................................................................................. 18 
2 
STUDY OBJECTIVES ................................................................................................................................... 21 
3 
STUDY DESIGN ............................................................................................................................................. 21 
3.1 
GENERAL DESIGN .......................................................................................................................................................................... 21 
3.2 
PRIMARY STUDY ENDPOINTS .................................................................................................................................................... 23 
3.3 
SECONDARY STUDY ENDPOINTS .............................................................................................................................................. 23 
3.4 
PRIMARY SAFETY ENDPOINTS................................................................................................................................................... 23 
4 
SUBJECT SELECTION ENROLLMENT AND WITHDRAWAL ........................................................... 23 
4.1 
INCLUSION CRITERIA ................................................................................................................................................................... 23 
4.2 
EXCLUSION CRITERIA .................................................................................................................................................................. 24 
4.3 
SUBJECT RECRUITMENT, ENROLLMENT AND SCREENING ................................................................................................ 25 
4.4 
EARLY WITHDRAWAL OF SUBJECTS ....................................................................................................................................... 25 
4.4.1 
When and How to Withdraw Subjects ................................................................................................. 25 
4.4.2 
Data Collection and Follow-up for Withdrawn Subjects .................................................................... 25 
5 
STUDY DRUG ................................................................................................................................................ 26 
5.1 
DESCRIPTION .................................................................................................................................................................................. 26 
5.2 
TREATMENT REGIMEN ................................................................................................................................................................. 27 
5.3 
METHOD FOR ASSIGNING SUBJECTS TO TREATMENT GROUPS ....................................................................................... 27 
5.4 
PREPARATION AND ADMINISTRATION OF STUDY DRUG ................................................................................................... 27 
5.5 
SUBJECT COMPLIANCE MONITORING ..................................................................................................................................... 28 
5.6 
PRIOR AND CONCOMITANT THERAPY ..................................................................................................................................... 28 
5.7 
PACKAGING ..................................................................................................................................................................................... 28 
5.8 
BLINDING OF STUDY ..................................................................................................................................................................... 28 
5.9 
RECEIVING, STORAGE, DISPENSING AND RETURN ............................................................................................................. 29 
5.9.1 
Receipt of Drug Supplies .................................................................................................................... 29 
5.9.2 
Storage ................................................................................................................................................ 29 
5.9.3 
Dispensing of Study Drug ................................................................................................................... 29 
5.9.4 
Return or Destruction of Study Drug .................................................................................................. 29 
6 
STUDY PROCEDURES ................................................................................................................................. 30 
6.1 
SCREENING VISIT 1 ....................................................................................................................................... 30 
6.2 
EXERCISE TRAINING (ET) VISITS ............................................................................................................................................. 30 
6.3 
ET VISITS 12 (WEEK 4) AND 24 (WEEK 8) ...................................................................................................... 31 
6.4 
FINAL VISIT ..................................................................................................................................................................................... 31 
6.5 
ANCILLARY STUDY VISITS ......................................................................................................................................................... 32 
7 
STATISTICAL PLAN .................................................................................................................................... 34 
7.1 
STATISTICAL OVERVIEW ............................................................................................................................................................. 34 
7.2 
SAMPLE SIZE DETERMINATION ................................................................................................................................................. 35 


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7.3 
PRIMARY OUTCOME ANALYSIS ................................................................................................................................................ 35 
8 
SAFETY AND ADVERSE EVENTS ............................................................................................................ 35 
8.1 
DEFINITIONS ................................................................................................................................................................................... 35 
8.2 
RECORDING OF ADVERSE EVENTS ........................................................................................................................................... 37 
8.2.1 Assessment of Causal Relationship ..................................................................................................34 
8.2.2 Assessment of Adverse Event Severity ................................................................................................ 34 
8.3 
REPORTING OF SERIOUS ADVERSE EVENTS AND UNANTICIPATED PROBLEMS ......................................................... 38 
8.3.1 
Sponsor-Investigator reporting: notifying the Mayo IRB ................................................................... 38 
8.3.2 
Sponsor-Investigator reporting: Notifying the FDA ........................................................................... 39 
8.4 
UNBLINDING PROCEDURES ........................................................................................................................................................ 39 
8.5 
STOPPING RULES ........................................................................................................................................................................... 40 
8.6 
MEDICAL MONITORING ............................................................................................................................................................... 40 
8.6.1 
Internal Data and Safety Monitoring Board ....................................................................................... 40 
9 
DATA HANDLING AND RECORD KEEPING ......................................................................................... 40 
9.1 
CONFIDENTIALITY ......................................................................................................................................................................... 40 
9.2 
SOURCE DOCUMENTS .................................................................................................................................................................. 40 
9.3 
CASE REPORT FORMS .................................................................................................................................................................. 41 
9.4 
RECORDS RETENTION .................................................................................................................................................................. 41 
10 
STUDY MONITORING, AUDITING, AND INSPECTING ...................................................................... 41 
10.1 
STUDY MONITORING PLAN .................................................................................................................................................. 41 
10.2 
AUDITING AND INSPECTING ................................................................................................................................................. 42 
11 
ETHICAL CONSIDERATIONS ................................................................................................................... 42 
12 
STUDY FINANCES ........................................................................................................................................ 42 
12.1 
FUNDING SOURCE .................................................................................................................................................................... 42 
12.2 
CONFLICT OF INTEREST ......................................................................................................................................................... 42 
13 
PUBLICATION PLAN ................................................................................................................................... 43 
14 
REFERENCES ................................................................................................................................................ 43 
15 
ATTACHMENTS ........................................................................................................................................... 50 
15.1 
INVESTIGATIONAL DRUG BROCHURE FOR AIR001 ...........................ERROR! BOOKMARK NOT DEFINED. 
15.2 
CHARTER FOR DATA SAFETY AND MONITORING BOARD ...................ERROR! BOOKMARK NOT DEFINED. 
15.3 
SCHEDULE OF ASSESSMENTS ............................................................................................................................ 50 
……………………………………………………………………….. 


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LIST OF ABBREVIATIONS 
 
AE 
Adverse Event/Adverse Experience 
CFR 
Code of Federal Regulations 
CRF 
Case Report Form 
DSMB 
Data and Safety Monitoring Board 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
HIPAA 
Health Insurance Portability and Accountability Act 
IB 
Investigator’s Brochure 
ITT 
Intent to Treat 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
IP 
Investigational Product 
PHI 
Protected Health Information 
PI 
Principal Investigator 
SAE 
Serious Adverse Event/Serious Adverse Experience 
SOP 
Standard Operating Procedure 


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Study Summary 
Title 
Inorganic Nitrite To Amplify the Benefits and Tolerability of Exercise 
Training In Heart Failure With Preserved Ejection Fraction 
Protocol Number 
16-001467 
Phase 
Phase II 
Methodology 
This is a randomized, blinded, placebo-controlled, two-stage, parallel- 
group clinical trial with all subjects receiving exercise training (ET) and 
randomized to either inorganic sodium nitrite or matching placebo. 
The trial includes 2 stages. In stage I, which has been completed, patients 
received inhaled nitrite/placebo. In stage II, patients will receive oral 
nitrite/placebo. 
Overall 
Study 
Duration 
4 years 
Subject Participation 
Duration 
14 weeks 
Single or Multi-Site 
Single site 
Objectives 
To determine if sodium nitrite can improve the clinical responses to ET 
in subjects with heart failure and preserved ejection fraction (HFpEF). 
Number of Subjects 
100 (36 received inhaled nitrite/placebo in the completed stage I, up to 
another 64 will 
receive oral nitrite/placebo in stage II of the study) 
Diagnosis and Main 
Inclusion Criteria 
Heart failure with preserved ejection fraction (HFpEF) defined by clinical 
heart failure, normal EF (≥50%) and at least 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 or with exercise or 
• Elevated NT-proBNP (>400 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 
Study Product, Dose, 
Route, Regimen 
Stage I tested Inhaled, nebulized sodium nitrite (AIR001) 80 mg at least 
4 hours apart three times daily during waking hours (i.e. 08:00, 12:00, 
16:00). 
Stage II is testing oral nitrite 40 mg tid capsules vs matching placebo 
capsules (same dosing frequency). 


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Duration 
of 
Administration 
12 weeks  
Reference therapy 
Placebo 
Statistical 
Methodology 
Data will be analyzed according to ITT principles (assigned group 
regardless of study drug administration or compliance with ET 
benchmarks achieved). Primary hypotheses will be tested using a two- 
sample t-test on the change (baseline to 12 week. Testing will be stratified 
by route of administration. 


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1 Introduction 
 
Heart failure with preserved ejection fraction (HFpEF) afflicts millions of older Americans and is 
associated with exercise intolerance, reduced quality of life (QOL), high health care costs, and 
increased mortality. People with HFpEF display increased cardiac filling pressures and inadequate 
cardiac output reserve to deliver blood to the body during exercise. Numerous lines of evidence 
have implicated abnormalities in nitric oxide-cyclic guanosine monophosphate (NO- cGMP) 
signaling as playing a key role in promoting these abnormalities. While there is currently no proven 
effective medical treatment for HFpEF, exercise training (ET) has been shown to improve aerobic 
capacity and QOL in this population. 
 
However, ET provides benefits through peripheral effects in the muscles and vasculature, without 
targeting the cardiac limitations. The specific objectives of this application are to determine 
whether treatment with sodium nitrite (which improves the cardiac abnormalities) in addition to 
ET can improve exercise capacity, chronic activity levels, and QOL in people with HFpEF as 
compared to ET alone. The guiding hypothesis is that enhancing NO-cGMP signaling in the heart 
and periphery preferentially during exercise will improve functional capacity and symptoms in 
HFpEF above and beyond what is seen with ET alone. Conversion of nitrite (NO2-) to biologically 
active NO is enhanced during tissue hypoxia and acidosis, which develop during low-level exercise 
in HFpEF, so it is expected that this intervention will preferentially target the cardiac limitations 
that develop during exercise in these patients at the time of greatest need. 
 
This document is a protocol for a human research study. This study will be carried out in 
accordance with the applicable United States government regulations and Mayo Clinic research 
policies and procedures. 
 
1.0 Summary of Protocol Changes and Rationale 
 
Significant delays in obtaining study drug supply have forced us to make another modification to 
this study. In the last modification to the protocol two main changes were made: (1) we changed 
the route of administration of sodium nitrite from inhaled to oral, and (2) we have decreased the 
duration of intervention for endpoint assessment from 12 to 10 weeks. In this modification, we are 
changing the duration of treatment back to 12 weeks to align with what was done in the first Stage 
of the study, and we are changing the randomization ratio to 2:1 for nitrite relative to placebo, for 
the reasons enumerated below. 
 
In the first Stage of the study, nitrite/placebo was administered in an inhaled formulation. However 
during the enrollment period, development of this product was discontinued by the manufacturer. 
In order to test the primary study hypotheses, we have switched from an inhaled formulation of 
nitrite to an oral formulation that achieves similar pharmacokinetics for Stage 2 of the study. 
 
 
Since that time, the research pharmacy that was compounding our study drug (Triangle Pharmacy) 
has ceased production, forcing us to identify and work with a new pharmacy (RXQ) which has 


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introduced a very large delay because of the longer start up process, and the need for additional 
batch testing.  After one shipment was received, RXQ Pharmacy failed to deliver further shipments 
of study drug and has not met production requirements, forcing us to again change the 
compounding pharmacy to The University of Iowa Pharmaceuticals (Iowa City, Iowa; 
uip.pharmacy.uiowa.edu). Potency and stability testing will be performed at University of 
Pittsburgh laboratories prior to release of study drug batches   
 
We had planned to enroll another 68 patients, 34 receiving oral nitrite and 34 receiving oral 
placebo.  However, we cannot meet this goal in two years. Our revised plan to meet our recruitment 
goal will be to use the data from the 17 patients receiving inhaled placebo in the first phase of the 
study.  We will then combine these data with that from patients receiving oral placebo drug moving 
forward. Because we do not expect the response to inhaled and oral placebo to differ, we feel that 
this is scientifically justified.   
 
Therefore, we will only require complete data from another 17 patients receiving oral placebo (for 
a combined total of 34 placebo patients) and another 34 patients receiving oral nitrite, for a total 
of 51 patients in the oral phase with complete data.  Because more data is needed for patients 
randomized to nitrite, we will randomize patients in a 2:1 ratio moving forward of nitrite to 
placebo. 
 
We have enrolled 14 patients according to the last protocol modification (on oral drug, which has 
been very well-tolerated). Therefore, at this time we will require complete data on another 47 
patients.  To allow for participant drop out and incomplete data, we will enroll up to another 60 
patients to meet our goal of reaching another 47 with complete data.  
1.1 
Background 
Heart failure (HF) is the leading cause of hospitalization among older adults in the United States. 
Half of people with HF have preserved ejection fraction (HFpEF), for which there is currently no 
proven effective treatment. People with HFpEF often have normal heart filling pressures at rest, 
but with exercise develop marked increases in heart filling pressures with blunted increases in 
cardiac output (CO). These are termed “central” (cardiac) limitations to exercise. 
 
Exercise training (ET) has been shown to improve aerobic capacity and quality of life (QOL) in 
HFpEF, but the weight of evidence indicates that ET works predominantly by improving 
“peripheral” limitations in the muscle and blood vessels, with little direct cardiac effect. This 
suggests that treatments targeting central limitations that develop with exercise may allow for 
greater benefit from ET in HFpEF. 
 
Recent studies have identified abnormal nitric oxide-cyclic guanosine monophosphate (NO- 
cGMP) signaling as a lynchpin mechanism underlying cardiac abnormalities in HFpEF. Direct NO 
donors such as organic nitrates have not improved chronic activity levels in HFpEF but are limited 
by the development of tolerance and pseudo-tolerance and by the fact that they may produce 
excessive hypotension in people with HFpEF. What is needed is an NO-cGMP enhancing agent 
that selectively targets central cardiac limitations preferentially during exercise, without untoward 
effects on resting cardiovascular homeostasis. Coupling of such an agent to ET may allow for 
synergistic benefit. Because both central and peripheral limitations will be targeted, people with 
HFpEF could potentially tolerate ET better, with fewer symptoms, increased daily activity levels, 


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and improved adherence to exercise prescription. 
 
The specific objectives of this application are to determine whether treatment with sodium nitrite 
(NO2-) in addition to ET can improve exercise capacity, activity levels, and QOL in people with 
HFpEF, as compared to ET alone. The guiding hypothesis is that enhancing NO-cGMP signaling 
in the heart and periphery during exercise will improve functional capacity and symptoms in 
HFpEF. This hypothesis is based upon work from our group showing that exercise capacity in 
HFpEF is limited by abnormalities in cardiac stress reserve that develop during exercise. 
Importantly, conversion of NO2- to biologically active NO is enhanced with tissue hypoxia and 
acidosis developing during exercise, suggesting that NO2- may preferentially target hemodynamic 
perturbations at the time of greatest need, with less effect at rest. Thus NO2- may allow for 
improved tolerance and adherence to ET in HFpEF. Successful completion of this project will 
enable understanding of the effects enhancing NO signaling through a novel, previously 
unexplored pathway, to synergize with ET to improve morbidity and QOL in people with HFpEF. 
 
Background related to Aim 1. 
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). Haykowsky et al. have observed that 
reduced peak VO2 in HFpEF is related to both low CO and impaired A-V O2diff reserve with 
exercise,14 but the improvements in peak VO2 with training are mediated exclusively by benefits 
in A-V O2diff reserve.26, 27 This observation of peripheral but not central effects of training has 
been corroborated in HFrEF populations.28, 29 Thus, coupling an agent that 


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improves cardiovascular reserve to ET would be expected to allow for the greatest benefit from 
training, which appears to work predominantly through the periphery. 
 
Among the potential molecular pathways underlying cardiac reserve dysfunction, abnormalities in 
NO-cGMP-dependent signaling have emerged as important players.9, 19, 20, 33-35 Acute 
increases in NO-cGMP activity are associated with systemic and pulmonary arterial and venous 
vaso-dilation, enabling LV and right ventricular (RV) afterload reduction, reduction in cardiac 
filling pressures and pulmonary pressures, improved flow-mediated dilation, and potentially 
enhanced LV diastolic compliance and relaxation. 
 
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.36  This suggests that enhancing NO- 
cGMP through decreased breakdown is not an effective 
approach in this population. Alternative strategies 
including organic NO donors have also failed, as 
evidenced by the recent NEAT-HFpEF trial. However, 
organic  nitrates  are  limited  by  tolerance  37,  and 
are 
associated with greater risk of hypotension in  people  
with HFpEF, especially at rest, which may predispose to 
falls and attendant morbidity in this elderly population.18 
A key factor in HFpEF that complicates treatment is that 
the  hemodynamic  perturbations  causing  morbidity (e.g., 
high filling pressures) are typically 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 such as NaNO2 represent an 
attractive approach to treat NO-cGMP deficiency 
preferentially  during   stress   in  HFpEF.38-40  These 
molecules were previously considered as inert 
byproducts of endogenous NO metabolism, but recent 
studies have shown that NO2- functions as an 
alternative in vivo source of NO to the classical 
oxygen-dependent L-arginine-NO-synthase pathway 
(Figure 2). 
 
In contrast to direct NO donors such as organic 
nitrates, there is no tolerance with NO2-.39 The NO2- reductase activity of heme-containing 
proteins peaks at O2 saturations of 40-60% but is near maximal from 20-60%, 41 meaning that 
bioconversion of NO2- to NO is enhanced in the venous blood and tissues under conditions of 
physiologic duress, such as hypoxia and acidosis 38-40. In other words, NO2- becomes most 
effective 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, NO2- may have beneficial effects on skeletal muscle bioenergetics, 
improving the O2 cost of work during submaximal exercise.42, 43 Thus; NO2- 


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represents an ideal candidate molecule to treat the central and peripheral limitations to exercise, 
with less risk of adverse toxicity, while complementing the effectiveness of ET to improve 
functional capacity in HFpEF. 
 
Participants in this trial will undergo 12 weeks of cardiac rehabilitation for exercise training (ET) 
and be randomized to either nitrite or matching placebo. In Stage 1 of the trial, participants received 
either nebulized inhaled NaNO2 (AIR001, Aires Pharmaceuticals, Inc., San Diego, CA) or 
nebulized inhaled saline (placebo) throughout the training period. Study drug (80 mg) was 
administered utilizing the Phillips I-neb AAD nebulizer over 10-15 minutes for each dose. Study 
drug was administered 3 times daily during daytime hours (e.g. 08:00, 12:00 and 16:00), with one 
of these doses being immediately prior to onset of exercise at ET sessions throughout the course 
of the 12-week trial. 
 
In Stage II of the trial, subjects will receive treatment with sodium nitrite (or matching placebo) as 
oral capsules at a dose of 40 mg tid, a lower nitrite dose than in Stage I. The oral formulation 
produces plasma nitrite levels that are similar to, but slightly less than, what is observed with 
intravenous administration in our previous study (plasma nitrite levels following 20 mg dose of 
5.5±0.7 μM with oral vs 8.4±1.9 μM in the intravenous study). 
 
While plasma nitrite levels are lower with oral nitrite than intravenous or inhaled, these levels are 
still an order of magnitude higher than what is observed with other oral formulations of 
nitrate/nitrite that have or are being tested in HFpEF (e.g. beetroot juice, potassium nitrate), and 
are similar to levels that we have observed to produce favorable hemodynamic effects in our 
preliminary invasive studies. 
 
The time to maximal concentration of NO2- in plasma with the oral formulation is 30 minutes, 
which is longer than with inhaled (5 minutes), though plasma levels return to baseline by 3 hours 
following a single dose (Hughes et al. Hypertension 2017; electronically published ahead of press, 
doi: 10.1161/HYPERTENSIONAHA.117.09016). For this reason, study drug will be administered 
30 minutes prior to training in Stage 2 of the study, as opposed to immediately prior to training as 
was performed in Stage 1. 
 
The principal investigator currently holds an Investigational New Drug application (IND 123333) 
from the FDA for use of inhaled NaNO2 to treat patients with HFpEF, and is amending this IND 
to include the oral nitrite formulation, with permission to cross reference the existing IND 
(#115926) for oral nitrite held by our collaborator, Dr. Mark Gladwin. 
 
Background related to Aim 2. 
Symptoms of exercise dyspnea and fatigue in HFpEF discourage activity and thus represent a 
barrier to succeeding with ET and maintaining an active lifestyle during and after completing  ET. 
By attenuating exercise-induced elevation in cardiac filling pressures, relaxing loading conditions, 
and improving cardiac output reserve, NO2- holds promise to reduce these symptoms and thus 
improve daily activity and tolerability of ET. The objectives of Aim 2 are to determine if treatment 
with NO2- in addition to ET improves symptoms of effort intolerance during ET and increases 
daily activity outside of ET. The working hypothesis is that administration of NO2- in the 
background of ET will improve symptoms of effort intolerance during ET and increase 


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average daily activity levels outside of ET. Our approach to test this hypothesis will be to quantify 
perceived effort and dyspnea symptoms reported during ET and evaluate whether daily activity 
assessed by 14-day averaged arbitrary accelerometer units (AAU14) increases more in subjects 
with HFpEF randomized to NO2- with ET compared to inhaled placebo with ET. The rationale for 
this aim is that treating symptoms of HF caused by hemodynamic perturbations will 
encourage/enable greater activity and tolerability of ET. After successful completion of the 
proposed studies for Aim 2, it is our expectation that we will have identified a new way to improve 
symptomatic tolerability of ET while increasing daily activity levels outside of the training 
environment, allowing for extension of the benefits of ET in the long term to maintain an active 
lifestyle and sustainable increase in long-term activity level and fitness in people with HFpEF. 
 
The duration of ET intervention is short, generally 8-12 weeks, and the long- term goal of ET is to 
achieve and maintain a durable, sustained increase in activity level beyond the original 
intervention. Data from the HF-ACTION trial showed that despite a well-organized program with 
abundant resources, only 40% of participants randomized to the intervention stage actually 
maintained ET at the target of 90 minutes per week after 3 months.24, 25 Interventions that 
improve the central cardiac limitations that contribute to symptoms of effort intolerance would be 
expected to allow for maintenance of a more active lifestyle. 
 
In patients with HFrEF, acute administration of organic nitrates reduces filling pressures and 
pulmonary artery pressures but does not increase peak exercise capacity.49-51 In contrast; long- 
term organic nitrate therapy did improve exercise capacity in this HF subtype. The discrepancy 
between acute and long-term effects with organic nitrates in HFrEF has been attributed to increases 
in physical activity and improved fitness associated with long-term improvement in HF 
symptoms—a speculation that has been corroborated by increases in self-reported physical activity 
among subjects randomized to organic nitrates.50 However, there are important fundamental 
differences between HFpEF and HFrEF, 18, 52 and between organic nitrates and NO2- . People 
with HFpEF have a much greater drop in blood pressures with organic nitrates as compared to 
HFrEF, 18 which we would expect to greatly limit compliance. Further, because NO2- conversion 
to NO is activated by venous hypoxia and acidosis with stress, 38-40 we expect that there will be 
less hypotensive effect as compared to organic nitrates in the HFpEF population at steady state. 
Finally, in contrast to organic nitrates, NO2- is not associated with tolerance or development of 
endothelial dysfunction.37, 39 
 
Externally-worn accelerometer devices provide highly quantitative, granular, high-density, 
patient-centric data which have been used to characterize activity and to assess the impact of 
interventions on activity levels in patients with chronic obstructive pulmonary disease, obesity, 
and arthritis. In HF, accelerometer data have been shown to reflect changes in clinical status in HF 
and to correlate with traditional measures of disease severity such as peak VO2, 6-minute walk 
distance, QOL, and prognosis as assessed by the Seattle Heart Failure Model.53-55 Thus, 
accelerometer data provides an ideal metric to address the hypothesis of Aim 2. 


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O 
O 
N 
1.2 
Investigational Agent 
 
Physical, Chemical, Pharmaceutical Properties, and Formulation: 
 
Drug Substance: 
The drug substance is sodium nitrite, NaNO2. 
 
Molecular Formula: 
NaNO2 
Molecular Weight: 
69.0 
Chemical Structure: 
Na    
 
Nitrite exists in the body derived from both dietary intake and as a byproduct of endogenous NO 
metabolism. Nitrite is widely used as a food preservative and is available in much higher doses for 
intravenous administration as an FDA-approved treatment for cyanide toxicity. 
 
Inhaled Nitrite (Stage 1): Inhaled, nebulized placebo or inhaled nebulized sodium nitrite was 
administered at 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 Participants 
were instructed to 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. 
 
Oral nitrite (Stage II): In Stage II of the study, nitrite will be administered using an oral 
formulation at dose of 40 mg po tid (taken using the same dosing frequency as during Stage I of 
the study, but 30 minutes prior to training rather than immediately preceding training). This 
produces peak concentrations at 30 minutes which return to baseline at 3 hours (Hughes et al. 
Hypertension 2017; published ahead of press, doi: 10.1161/HYPERTENSIONAHA.117.09016). 
Oral nitrite and matching placebo capsules will be compounded by The University of Iowa 
Pharmaceuticals, in Iowa City, IA who will ship to the University of Pittsburgh as specified in Dr. 
Mark Gladwin’s IND (#115926) for ongoing stability testing (cross reference permission letter 
provided by Dr. Gladwin to FDA to reference CMC information), and then shipped, stored and 
dispensed in the Mayo Clinic Research Pharmacy. 
 
Preliminary data administering oral nitrite 40 mg tid to (1) obese patients with metabolic syndrome 
for 12 weeks (n=20), and (2) patients with HFpEF and pulmonary hypertension for 10 weeks (n=5 
at this time) participating in separate studies at the University of Pittsburgh has revealed an 
excellent safety profile, with only 1 serious adverse event that was not attributed to study drug 
administration (Personal communication, Dr. Mark Gladwin). 
 


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1.3 
Clinical Data to Date 
 
Preliminary Studies Related to Aim 1. 
Our group has shown that exercise limitation in 
HFpEF is related to global impairments in 
cardiovascular reserve—characterized by abnormal 
systemic vasodilation (less reduction in arterial 
elastance, Ea), greater increase in pulmonary 
capillary wedge pressure (PCWP), blunted increases 
in CO relative to VO2 (lower CO/VO2 slope), and 
steeper pulmonary arterial (PA) pressure flow 
relationship 
indicating 
abnormal 
pulmonary 
vasodilation (n=74, Figure 3). Depressed CO 
reserve contributes to a net decrease in O2 delivery 
to exercising muscles, increasing the reliance in 
muscles on O2 extraction and anaerobic glycolysis 
to compensate for inadequate perfusion. 
 
We have shown that the decrease in CO during exercise in HFpEF is coupled with lower mixed 
venous O2 content and pH during submaximal exercise in patients with HFpEF compared to 
controls (n=74, Figure 4). In a different 
study, we have also observed marked 
reduction in coronary sinus O2 saturation 
during exercise in people with HFpEF 
(28±9%, n=9).44 Thus, the outgoing 
blood traveling to the left heart from the 
pulmonary arteries (mixed venous) as 
well as the venous blood returning from 
the left heart (coronary  sinus)  becomes  
hypoxic and 
acidemic during exercise in people with HFpEF, setting the stage for greater reduction of NO2- to 
NO in the central circulation. 
 
Our group has recently completed a randomized, double blind, placebo controlled trial testing 
whether acute NO2- treatment could improve hemodynamics during submaximal exercise in 
HFpEF      (n=29). 
Subjects 
underwent resting and exercise 
invasive hemodynamic assessment 
and were then treated with either 
intravenous NO2- (250  μg/kg over 
5 min) or saline. After 15 minutes, 
repeat 
rest 
and 
exercise 
hemodynamic 
assessment 
was 
performed at the same workload. 
Compared to placebo, NO2- treated subjects displayed markedly attenuated increases in PCWP 
with exertion, improved exercise CO reserve, and enhanced CO/VO2 slope (Figures 5 and 6). 


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Enhanced CO reserve in NO2- treated subjects was entirely due to improved stroke volume reserve 
(+16±19 vs +1±14 ml, p=0.02), as there was no effect on exercise heart rate (p=0.9). Acute NO2- 
infusion reduced the steepness of the PA pressure-flow relationship from 10.0 (IQR, 
4.9 to 24) mmHg*min/L to 4.1 (IQR 2.5 to 5.0) mmHg*min/L (p<0.001), revealing a significant 
reduction in right ventricular loading with stress. 
 
Notably, there was no significant effect of intravenous NO2- on blood pressure at rest or during 
exercise compared to placebo. The stroke volume increase observed, coupled with lower PCWP 
and no change in blood pressure strongly suggests a positive direct myocardial effect of acute NO2-
. In contrast to some prior studies, 41, 42 there was no reduction in the O2 cost of exercise; rather 
the proportional increase in VO2 during submaximal exercise (20 Watts) relative to the increase 
prior to study drug was 106% (IQR, 98 to 136%) with NO2- as compared to 90% with placebo 
(IQR, 84 to 111%; p<0.01 compared to NO2-). 
 
Plasma NO2- levels increased to 8.4±1.9 μM at rest in subjects receiving intravenous NO2- 
(p=0.004 vs baseline, n=4). After 5 minutes of exercise, NO2- levels decreased to 3.4±0.4 μM 
(p<0.01 compared to pre-exercise). The calculated half-life from these data is 3.9±0.6 min, which 
is 10-fold faster than previously reported kinetics for NO2- in humans at rest (30-40 min). This 
indicates active NO2- consumption during exercise, and thus greater reduction of NO2- to NO 
during stress. 
 
We have recently demonstrated equivalent hemodynamic benefits in a pilot study using inhaled 
NO2- (n=30, data from the first 4 participants receiving open label drug shown). A single inhaled 
dose of 80 mg NO2- produces similar plasma NO2- levels to the IV formulation (9.3±0.9 μM) and 
equivalent reductions in exercise PCWP (29±2 to 19±4 mmHg, p=0.01). While the effects of oral 
nitrite on rest and exercise PCWP have not yet been assessed, preliminary data from Hughan and 
Gladwin et al has revealed that plasma nitrite levels 30 minutes following a single oral dose of 40 
mg sodium nitrite (as will be used in this study) produces similar NO2- levels as we observed with 
the intravenous formulation (Figure 6). 


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As shown by Hughan et al (Hypertension 2017; 
electronically published ahead of press, doi: 
10.1161/HYPERTENSIONAHA.117.09016), 
the oral formulation of nitrite (20 mg) produces 
NO2- levels that are similar to but slightly lower 
than 
what 
is 
observed 
with 
parenteral 
administration or the 40 mg single dose (5.5±0.7 
μM, Figure 7). However, even the 20 mg oral 
nitrite dose produces plasma levels that fall 
within the range where we have observed 
favorable hemodynamic effects based upon our 
preliminary invasive studies (Figures 5, 6). 
Furthermore, the oral formulation used in Stage 
II of the study will also be easier for patients to 
administer as compared to the nebulized formulation which was cumbersome and time- consuming 
for patients to use. 
 
No episodes of hypotension were observed using directly measured pressure with the inhaled 
nitrite. As compared to inhaled placebo, AIR001 (80 mg) had no effect on mean BP (0±9 vs 1±3 
mmHg, p=0.8) and no effect on heart rate (-1±5 vs -2±4 bpm, p=0.7). 
 
With oral nitrite, which will be used in Stage II of 
this study, there is a modest vasodilator effect 
leading to reduction in mean arterial pressure 
(Figure 8) that peaks between 15 and 45 minutes 
following single dose administration (Hughan et 
al. Hypertension 2017; published ahead of press, 
doi: 10.1161/HYPERTENSIONAHA.117.09016 
and Hughan, Gladwin et al, unpublished data 
from separate trial of patients with obesity and 
metabolic syndrome, n=20). 
 
This reduction in blood pressure noted with oral 
nitrite in these studies was not associated with 
development of reflex tachycardia. In the study of Hughan et al. testing oral nitrite at a dose of 40 
mg tid, no patient developed symptomatic hypotension.  
 
In our acute invasive studies, NO2- infusion and inhalation did not affect the peripheral components 
of VO2, evidenced by no change in the increases in arterial-venous O2 difference (A-V O2diff) 
with stress (p=0.8). Because ET has been shown to improve peak VO2 through benefits on A-V 
O2diff, but not through cardiac effects, we hypothesize based upon our preliminary findings with 
both intravenous and inhaled NO2- during submaximal exercise that 


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sustained, longer-term treatment will allow for even greater synergistic benefit with ET in subjects 
with HFpEF. 
 
Our group has recently completed an invasive hemodynamic study (n=106) where 
echocardiography was performed at rest and during maximal effort exercise simultaneously with 
directly measured intracardiac and vascular pressures. PA systolic pressures (PASP) estimated by 
echocardiography were strongly correlated with directly measured pressures at rest and during 
exercise, while significant but less robust correlations were observed between PCWP and the E/e’ 
ratio during cycle ergometry. These data demonstrate the feasibility and validity of exercise 
echocardiography to assess hemodynamic reserve in our group. 
 
 
Preliminary Studies related to Aim 2. 
 
We have shown that patients with HFpEF develop 
marked increases in cardiac filling pressures, PA 
pressures, and inadequate CO reserve during 
exercise, and that each of these abnormalities can 
be improved during submaximal exercise by  acute 
infusion of NO2- (please refer to preliminary 
studies related to Aim 1, above). In addition, our 
group has shown that in subjects with HFpEF, the 
degree of NO-cGMP impairment, assessed by the 
reactive hyperemic index response to upper stage 
cuff occlusion is directly related to more severe 
symptoms of dyspnea and fatigue during matched 
submaximal exercise (Figure 9, n=31).9 Thus, 
enhancing NO-cGMP signaling with NO2- may 
improve symptoms of exercise intolerance both by 
improving central pressures and cardiac output as 
well as by improving  microvascular endothelial 
function, and in so doing, improve daily activity 
levels. 
 
Accelerometry is the primary endpoint for a recently- 
completed NHLBI-sponsored trial in which Mayo was the 
lead center, testing whether isosorbide mononitrate improves 
daily activity tolerance in people with HFpEF in a double-
blind crossover trial. Our coordinating center has been the 
leading enroller in this trial, and we have acquired high 
quality accelerometer data from all participants enrolled 
(Figure 10). Subjects have reported no difficulties 
maintaining the devices on throughout the course of the day 
and initial analysis of data quality reveals very little missing 
data. 


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1.4 Dose Rationale and Risk/Benefits 
Nitrite delivery by intravenous, inhaled and oral routes of administration has been shown to be 
safe, and the plasma nitrite levels achieved with each route have been shown by our group and 
others to cause favorable effects on cardiac hemodynamics that we expect to improve both 
symptoms and exercise capacity in patients with HFpEF, as will be tested in this study. 
 
Intravenous nitrite administration reduces pulmonary capillary wedge pressures (PCWP) at rest (-
3 mmHg, p<0.0001) and particularly during exercise (adjusted exercise values 19±5 mm Hg vs. 
28±6 mm Hg; p=0.0003). Similar effects are observed on pulmonary artery (PA) pressures, and 
there was a greater increase in cardiac output reserve (all p<0.01). Plasma nitrite levels achieved 
with the intravenous formulation were 8.39±1.88 μM (Borlaug et al. J Am Coll Cardiol 2015; 
66:1672–82). While our group has shown that nitrite reduces arterial stiffness (J Am Coll Cardiol. 
2017;70(2):136-148.), there was no symptomatic hypotension or methemoglobinemia with 
intravenous nitrite in this study, and no adverse events. 
 
Inhaled nitrite (Stage I): In Stage I of this trial, participants received a dose of inhaled nebulized 
nitrite 80 mg 3 times daily. Participants developing symptoms of intolerance could be down- 
titrated to 46 mg 3 times daily. The nebulizer route was difficult to use for many of the participants, 
and mindful of the neutral results of the INDIE trial (manuscript currently under review), the 
manufacturer of the inhaled nitrite compound discontinued manufacture. For this reason, we are 
switching to an oral route of administration of nitrite in Stage II of our study. 
 
Oral nitrite (Stage II): Like inhaled nitrite, oral nitrite has been found to be safe, without 
significant adverse effects in Phase I studies. The bioavailability of oral nitrite is excellent (95%) 
and similar to intravenous formulations. High doses of nitrite have also been provided in several 
studies by oral route in the form of beet root juice. Nutraceutical preparations are currently being 
sold in health stores with levels of nitrite (12.7 mg per tablet) that are similar to what will be used 
in Stage II of our study. 
 
As of July 31, 2017, a total of 84 subjects were enrolled in 8 clinical trials testing oral sodium 
nitrite conducted under Dr. Gladwin’s IND# 115926 at the University of Pittsburgh. Subjects 
enrolled are predominantly female (57%) and Caucasian (77%). Among the 8 clinical trials, 2 
clinical trials have been completed; one is a Phase 1 study aimed to establish the pharmacokinetics, 
metabolism and interconversion of nitrate to nitrite, nitrite to nitric oxide, and interaction with 
Conjugated Linoleic Acid (cLA) to form nitro-fatty acids in vivo in healthy adult normal 
volunteers 
(Hughan 
et 
al. 
Hypertension 
2017; 
published 
ahead 
of 
press, 
doi: 
10.1161/HYPERTENSIONAHA.117.09016). The other is a Phase 2 open labeled study designed 
to assess the safety and feasibility of oral nitrite therapy on the changes in skeletal muscle 
bioenergetics and physical capacity in older heart failure patients. 
 
Five Phase 2 studies are ongoing. Of the five active studies, 2 studies are in the patient population 
of overweight/obese adults with metabolic syndrome and hypertension, at risk for insulin 
resistance and endothelial dysfunction, one in adult obese asthmatics with metabolic syndrome, 
and another one in older HF patients, one in adult HFpEF patients aged ≥ 18 years and another one 
in older HFpEF patients aged ≥ 70 years. Dr. Gladwin’s group has also initiated 


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another Phase 1 nitrate study aimed to determine distinguishing features of the structure and 
function of the oral and gut microbiome in PH-HFpEF and controls. 
 
 
The oral nitrite for Stage II of this study will be provided under the supervision of our collaborator, 
Dr. Mark Gladwin, is an internationally recognized leader in the basic and clinical science of the 
inorganic nitrate/nitrite/NO pathway. Dr. Gladwin has held an IND for sodium nitrite (IND # 
70,411) for cardiovascular applications and currently has an approved IND for the use of sodium 
nitrite for lung transplant recipients (IND # 111,643). The cardiovascular IND involved the 
administration of sodium nitrite to 69 normal volunteers in 4 phase I-II clinical trials without 
observed adverse effects. This study will be performed under the IND held by the PI, allowing the 
FDA to cross reference the Dr. Gladwin’s current IND for the oral formulation. 
 
Dr. Gladwin has also treated 11 subjects with sickle cell disease on this IND without observed 
adverse effects. The lower doses of nitrite used in these investigational treatment regimens – 60- 
120 mg daily, or 20-40% of the intravenous dose (300mg) used in the emergency treatment of 
cyanide poisoning – do not produce methemoglobin levels greater than 3% and have not been 
associated with clinically significant hypotension. There have been no adverse events noted in the 
80 treated normal human volunteers and patients with sickle cell disease. 
 
With 20 mg oral nitrite, there are modest reductions in blood pressure with no increase in heart 
rate (Hughan et al. Hypertension 2017; electronically published ahead of press, doi: 
10.1161/HYPERTENSIONAHA.117.09016). Similar to our prior studies with inhaled and 
intravenous nitrite, oral nitrite was observed in this study to cause a very mild and clinically 
insignificant rise in plasma methemoglobin levels, with no patients developing levels >3%. 
 
The effects of oral nitrite on central hemodynamics have not yet been examined, but given the 
similar plasma nitrite levels observed with oral administration (Figure 7) and modest reduction in 
blood pressures (Figure 8), we expect reductions in pulmonary artery and pulmonary capillary 
wedge pressure at rest and during exercise of roughly similar magnitude to what we have observed 
following intravenous and inhaled preparations. 
 
Risk from Study Drug-Overview 
Sodium nitrite (NO2-) has vasodilating properties, and the most significant expected risk in this 
population would be symptomatic hypotension. NO2- is converted in vivo to nitric oxide (NO) and 
thus may cause vasorelaxation. We have observed that nitrite delivered by the intravenous and 
inhaled routes reduces arterial stiffness and vasodilates conduit vessels as well as vessels at the 
arteriolar level (J Am Coll Cardiol. 2017;70(2):136-148.), without causing excessive BP reduction 
or symptomatic hypotension. 
 
The oral formulation of nitrite has been observed to reduce blood pressure. This compound has 
been administered to patients with obesity and metabolic syndrome, as well as HFpEF and PH as 
part of Dr. Gladwin’s ongoing NIH-sponsored studies, and no severe adverse events or 
symptomatic hypotension have developed following treatment with this oral nitrite formulation 
(Dr. Mark Gladwin, personal communication 2018). We will carefully assess for any adverse 


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effects that could be related to excessive BP reduction in the study as outlined below in the 
protocol. 
 
Mechanism of Action: Inorganic sodium nitrite is an intrinsic vasodilator that is converted in 
blood and tissues to nitric oxide (NO). Endogenous NO2- represents the body’s largest reservoir of 
NO which results in vasodilation and may improve ventricular diastolic compliance. 
 
Dosage: AIR001 solution for nebulization. The dose of 80mg/ml is administered through a 
specialized nebulizer device. The oral dose of 40 mg is administered as a capsule, administered 
three times daily (same as the inhaled formulation). 
 
Metabolism: AIR001 is rapidly absorbed through the alveolar-capillary interface, achieving peak 
concentrations in less than 5 minutes. Oral nitrite is rapidly absorbed through the alimentary tract, 
achieving peak levels at 30 minutes. NO2- is elevated in plasma following both routes of 
administration and is rapidly converted to NO in areas of low oxygen tension. 
 
Interactions and Contraindications: There are no known drug interactions. NO2- is not a 
substrate or inhibitor for hepatic cytochrome p-450 enzymes. Nebulized inhaled nitrite (NO2-) 
should not be used with other NO donors (e.g. organic nitrates, nitroprusside) to minimize risk of 
hypotension, and these are listed as exclusion criteria for safety. A drug-drug interaction study 
with sildenafil dosed at 20mg three times daily did not show a pharmacodynamic interaction, but 
in this study patients treated with phosphodiesterase inhibitors will be excluded. 
 
Adverse Effects: The most frequent reported adverse effects of AIR001 used in Stage 1 are cough, 
headache, and dizziness. These effects were reported as mild to moderate intensity and rarely result 
in discontinuation of medication. 
 
In a Phase 2 study of oral nitrite therapy in older heart failure patients, Gladwin and colleagues 
found a higher propensity for hypotension in subjects aged > 70 years with heart failure compared 
to healthy controls. Two research subjects enrolled in this study were withdrawn by Investigator 
due to hypotension. In all other studies, oral nitrite and nitrate are generally safe and well tolerated, 
and all AEs thought to be related to nitrite and nitrate were mild to moderate in severity, and were 
all resolved with or without minor treatments. No drug related SAEs have been observed in all of 
the protocols. In sum, the number and nature of anticipated AEs are expected. 
 
The most frequent expected adverse effects with oral nitrite are dry mouth (infrequent), and 
nausea/abdominal pain/methemoglobinemia/flushing/headache/dizziness (rare). 
 
Abuse Potential: No risk for abuse of AIR001 has been demonstrated. 
 
Pregnancy Risk: Although nitrite exists widely in nature and human ingestion occurs daily in 
foodstuffs and through water sources, no human data exist on the safety of administration of nitrite 
during pregnancy or lactation. AIR001 and oral nitrite should not be administered to women who 
are pregnant or lactating. Effective contraceptive measures should be utilized by women of child- 
bearing potential who receive either form of nitrite. Given the ubiquitous nature 


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of nitrite in the environment and the short half-life of AIR001, prolonged contraceptive use after 
ceasing AIR001 inhalational use is unnecessary, and should  be  predicated  on  the  clinical 
judgment of the subject’s clinician. 
 
2 Study Objectives 
Aim 1: Determine whether treatment with NO2- in addition to ET for 12 weeks improves 
exercise capacity and hemodynamic reserve in HFpEF. Expired gas analysis, inert gas (C2H2) 
rebreathe, and echocardiography will be performed during rest and exercise to measure oxygen 
consumption (VO2), CO, and hemodynamics before and after completion of 12 weeks of ET with 
NO2- vs ET with inhaled placebo. 
• Our primary hypothesis is that combination therapy with ET and NO2- will be associated 
with greater improvement in peak VO2 compared to ET plus placebo. Our secondary 
hypotheses are that ET and NO2- will lead to greater improvement in CO reserve (CO/VO2 
slope), peak workload, and exercise hemodynamics (lower echocardiography estimated 
pulmonary artery pressure and E/e’ ratio). 
Aim 2: Determine whether treatment with NO2- in addition to ET for 12 weeks increases 
daily activity levels and QOL, and reduces symptoms of effort intolerance during ET. 
Subjects will use externally worn accelerometer devices to track daily physical activity. 
Tolerability of ET will be assessed by Borg perceived effort and dyspnea scores. Large and small 
vessel endothelial function (brachial and digital arteries) and quality of life (QOL) will be assessed. 
• Our primary hypothesis is that combination of NO2- and ET will be associated with greater 
improvement in daily activity levels compared to ET with placebo. Our secondary 
hypotheses are that combination of NO2- and ET will be associated with improvement in 
symptoms of dyspnea and fatigue, improved tolerability to ET, enhanced endothelial function 
and better QOL compared to ET with placebo. 
 
3 Study Design 
3.1 
General Design 
This is a Phase 2 randomized, double-blind, parallel group placebo-controlled trial testing whether 
inorganic sodium nitrite, as compared to placebo, can enhance the benefits from chronic exercise 
training (ET) in subjects with HFpEF (see flow diagram below). 
 


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The total duration of the trial is 14 weeks.  
 
Subjects will be screened from the inpatient and outpatient heart failure services and interested 
qualified subjects will be offered participation. Once consent has been obtained baseline 
assessment of cardiovascular function and exercise capacity will be performed. All subjects will 
undergo 12 weeks of ET. Participants will be randomized to receive sodium nitrite 3 times daily 
or matching placebo 3 times daily during the study period. 
 
In Stage I this was with inhaled medicine for the full 12 weeks of ET. In Stage II, study drug will 
be administered using an oral preparation for the full 12 weeks. Study subjects will wear 
accelerometry devices to track daily activity levels at home.  
 
At the Screening visit, during consent, subjects will be given the option to participate in an optional 
MRI sub-study where they will be asked to complete 2 Magnetic Resonance Imaging (MRI) scans. 
One scan will be completed prior to or as close as possible to the beginning of cardiac 
rehabilitation, the other within 2 weeks of completing week 12 of ET (for Stage II). A hematocrit 
will need to be obtained prior to the MRI if not already drawn within the past 30 days. We will 
evaluate adipose tissue content and cardiac structure and function. We will determine whether 
these variables relate to exercise capacity/intolerance in participants, and evaluate how they change 
with ET with or without nitrite. Subjects will be excluded from this optional substudy if they have 
contraindications to MRI including aneurysm clips, cardiac pacemakers, internal defibrillators, 
severe claustrophobia and pregnancy. Women of childbearing potential will undergo a urine 
pregnancy test; positive test results will yield exclusion from study participation. Patients will be 
excluded for contraindications to gadolinium-based MRI contrast material, namely glomerular 
filtration rate less than or equal to 30ml/min. 
 
The MRI scans will be performed on a clinical scanner or at the Center for Advanced Imaging Research in the 
Opus building. Patients will have ECG leads placed, as is done for cardiac MRI examinations. In some patients 
this may require shaving the chest for lead placement. Prior to scanning a peripheral intravenous (IV) line will 
be placed.  Patients may be scanned prone and/or supine. The examination will last approximately one hour. 
Intravenous contrast material (gadolinium) will be used. Subjects will receive $25/scan for participating in this 
MRI sub-study part of the main study.  
 
Also at the screening visit, during consent, subjects will be given the option to participate in an 
optional muscle biopsy where they will be asked to complete 2 muscle biopsies. One biopsy will be 
completed prior to or as close as possible to the beginning of cardiac rehabilitation, the other as close 
to the end of ET . The muscle biopsy sample will be collected from the vastus lateralis muscle (outer 
thigh).  Muscle biopsy will be performed with a percutaneous biopsy needle (modified Bergstrom 
needle).  Local subcutaneous injection of 2% lidocaine buffered with 8.4% sodium bicarbonate will be 
used for analgesia.  All biopsies will be done under sterile conditions with all necessary aseptic 
precautions.  A small incision will be made through the skin and fascia. After the biopsy, pressure will 
be held over the incision until hemostasis is achieved.  The incision is closed with sterile strips, gauze 
and an Ace wrap.  Risks of this procedure include hematoma, infection, and pain.  Hematoma 
likelihood is minimized by holding pressure after the biopsy to ensure hemostasis, followed by a 
pressure dressing.  The risk of infection is minimized by using sterile surgical techniques. Pain is 
managed by local analgesia during the procedure and Tylenol following the procedure.  Subjects will 
receive $50/biopsy for participating. 


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At the conclusion of the main trial, subjects will be offered participation in an optional ancillary 
study where they will be randomized to standard care or behavioral/motivation intervention (once 
weekly visits with co-investigator +2 phone calls per week) to evaluate durability of ET effects. 
For subjects participating in this component of the study, there will be one final visit 8-10 weeks 
following the final study visit where QOL assessment, Borg symptom severity (effort and dyspnea 
scores) will be recorded during standardized exercise training at a common objective workload, 2 
mph on treadmill at 2° grade for 5 minutes and accelerometry data will be obtained (post trial 
intervention). 
 
3.2 
Primary Study Endpoints 
 
Exercise capacity assessed by peak oxygen consumption (VO2) achieved during exercise testing 
will be the primary endpoint for Aim 1. Chronic activity levels assessed by accelerometry will be 
the primary endpoint for Aim 2. Assessments were performed after 12 weeks of intervention. 
 
3.3 
Secondary Study Endpoints 
 
Cardiac output reserve, peak exercise workload, rest and exercise hemodynamics assessed by 
echocardiography, Borg dyspnea and fatigue scores recorded during ET, endothelial function 
assessed by tonometry, QOL assessed by the Kansas City Cardiomyopathy Questionnaire and 
body composition assessed by dual energy X-ray absorptiometry (DEXA). Cardiac structure and 
function will be assessed by cardiac MRI and adipose content will be assessed by MRI in subjects 
participating in the optional MRI substudy.  The muscle biopsy will tell us about what 
improvements we see in the leg muscles, and specifically in the mitochondria of leg muscles. 
 
 
There will be ancillary trial endpoints to assess QOL, Borg symptom scores, DEXA, AXM data, 
and exercise capacity following completion of the main trial, comparing subjects randomized to 
usual care or behavioral/motivation intervention. 
 
3.4 
Primary Safety Endpoints 
 
Safety endpoints will include blood pressure, methemoglobin levels, symptoms of dizziness, 
lightheadedness, headaches, and cough. 
 
4 Subject Selection Enrollment and Withdrawal 
4.1 
Inclusion Criteria 
 
1. Age ≥ 40 years 
2. Symptoms of dyspnea (II-IV) without evidence of a non-cardiac or ischemic explanation for 
dyspnea 
3. EF of > or = 50% determined on most recent imaging study within the preceding 5 years, with no 
change in clinical status suggesting potential for deterioration in systolic function 
4. One of the following: 


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• 
Previous hospitalization for HF with radiographic evidence (pulmonary venous hypertension, 
vascular congestion, interstitial edema, pleural effusion) of pulmonary congestion or 


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• 
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 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. No chronic nitrate therapy or not using intermittent sublingual nitroglycerin (requirement for >1 SL 
nitroglycerin per week). 
7. 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 
8. Ambulatory (not wheelchair / scooter dependent) 
9. 
Body size allows wearing of the accelerometer belt as confirmed by ability to comfortably fasten 
the test belt provided for the screening process 
10. Willingness to wear the accelerometer belt for the duration of the trial 
 
4.2 
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 
6. Resting HR > 110 
7. Previous adverse reaction to the study drug which necessitated withdrawal of therapy 
8. Significant chronic obstructive pulmonary disease that is a primary contributor to symptoms in the 
opinion of the investigator 
9. Ischemia thought to contribute to dyspnea in the opinion of the investigator 
10. Documentation of previous EF < 40% 
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. Obstructive hypertrophic cardiomyopathy 
14. Known infiltrative cardiomyopathy (amyloid) 


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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 more than moderate 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 
23. Enrollment or planned enrollment in another therapeutic clinical trial in next 3 months. 
24. Inability to comply with planned study procedures 
25. Pregnancy or breastfeeding mothers 
 
4.3 
Subject Recruitment, Enrollment and Screening 
 
Eligible patients will be identified from screening of the inpatient and outpatient heart failure 
services of the Mayo Clinic in Rochester, Minnesota. As well as the cardiovascular medicine and 
general medical services of the Mayo Clinic Health Systems. Patients that participated in the first 
stage (with inhaled nitrite) will also be eligible to participate in the second stage with oral nitrite, 
provided that at least 6 months have passed since the first exercise training intervention. Data from 
the same patients in different phases will not be analyzed together, but rather separately. 
 
Eligible patients will be paid up to $500 if the entire study is completed. If they are not able to 
complete the entire study, they will be paid as follows for the study visits they do complete: $100 
each for completion of Visits 1 & 4, $50 each for Visits 2 & 3 and $5 for each cardiac rehab session 
completed. 
 
Patients will also be given a 6 hour parking pass for the screening visit, final visit and visits at 
week 4, 8 and 12. 
 
4.4 
Early Withdrawal of Subjects 
 
4.4.1 When and How to Withdraw Subjects 
 
Development of any condition that requires study withdrawal related to safety, disease 
progression, subject decision or failure to adhere to protocol requirements could qualify as reason 
to withdraw for the study. 
 
4.4.2 Data Collection and Follow-up for Withdrawn Subjects 


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Subjects withdrawing from the study will be contacted 2 weeks following last dose of study drug 
to ensure that there have been no adverse events. Participants that withdraw may be replaced to 
allow for achievement of target enrollment. 
 
5 Study Drug 
5.1 
Description 
Inhaled nitrite (Stage I): Inhaled, nebulized placebo or inhaled nebulized sodium nitrite (80 mg 3 
times daily) was given 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 dispersed the 
doses by at least 4 hours and delivers 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. 
 
Inhaled, nebulized sodium nitrite or placebo was administered utilizing the Phillips I-neb AAD 
nebulizer over 10-15 minutes for each dose. The nebulizer device has a Participant logging system 
that automatically records adherence and compliance with protocol-specified treatments. The 
device log was reviewed at the time of ET sessions by the research coordinator with study 
participants to optimize adherence. 
 
Oral Nitrite (Stage II): Because the inhaled form of nitrite is no longer available, the oral 
formulation of sodium nitrite will be used in Stage II of the study to test the study hypotheses. 
Sodium nitrite capsules and matching placebo capsules will be compounded by The University of 
Iowa Pharmaceuticals, in Iowa City, IA, then shipped to the Research pharmacy at the University 
of Pittsburgh according to specifications in the IND of Dr. Mark Gladwin for ongoing stability 
testing, and then study drug will be shipped to Mayo Clinic, where they will be stored and 
dispensed by the Mayo Clinic Research Pharmacy. 
 
Oral sodium nitrite will be administered at a dose of 40 mg capsule, 3 times daily, using the same 
dosing frequency as in Stage I of the study with inhaled nitrite. 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. 
 
Permitted dose adjustment: If significant headache occurs, participants will be encouraged to treat 
headaches with acetaminophen. If participants cannot tolerate the 80 mg dose of inhaled nitrite, 
because of headaches, despite acetaminophen, they will be able to down-titrate to the 46 mg dose. 
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. 
 
For the oral dose, headaches will be treated with acetaminophen as above. If participants cannot 
tolerate the study medication 40 mg three times daily even with acetaminophen, the dosage can be 
reduced to 20 mg three times daily. If 20 mg three times daily is not tolerated, the frequency can 
be reduced to twice daily or once daily, in consultation with study staff. It will be critically 


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important that the single day dose be administered 30 minutes prior to exercise training visits and 
patients will be instructed of this. 
 
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. 
 
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). 
 
5.2 
Treatment Regimen 
 
Inhaled nitrite (Stage I): The dose used for this part of the study was 80 mg three times per day. 
 
Oral nitrite (Stage II): In Stage II of the study, participants will receive an oral dose of 40 mg 
sodium nitrite or matching placebo three times daily. If there is intolerance, patients may (under 
the supervision of study staff) down-titrate to 20 mg tid dosing, or 20 mg at lower frequency (as 
noted above). 
 
5.3 
Method for Assigning Subjects to Treatment Groups 
 
Medidata, Rave, and Balance will be used for clinical data management and randomization. These 
tools are institutionally supported and allow for rapid database development. The implementation 
of the software within a project includes training on the system’s use, robust error checking, 
security considerations, and full audit trail. Balance, which is integrated into Rave, allows for a 
variety of randomization and minimization algorithms. 
 
5.4 
Preparation and Administration of Study Drug 
 
Drug dispensing will be managed by the research pharmacy who will obtain and store supplies 
from the contracted drug supply vendors for both the inhaled and oral nitrite/placebo formulations. 
At baseline, week 4, and week 8 study visits, participants will receive a sufficient supply of 
inorganic nitrite or placebo to permit three doses a day for 4 weeks (Stage I). At baseline, and week 
4 participants will receive a sufficient supply of inorganic nitrite or placebo to permit three doses 
a day until final visit (Stage II). To account for potential damage or loss of drug, additional study 
drug doses will be supplied. 
 
Participants will be instructed to take the medication as required by the protocol, and compliance 
will be discussed weekly with the participants and assessed every other week (at the time of QOL 
assessment). Adherence with the inhaled nitrite formulation will also be assessed by download of 
the compliance data from the nebulizer device, with oral nitrite adherence will be assessed by pill 


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counts. Participants will be instructed to bring the nebulizer (Stage I only) and unused drug 
supplies (inhaled or pills) to study visits where drug is dispensed. 


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5.5 
Subject Compliance Monitoring 
 
Inhaled nitrite (Stage I): The I-neb device has a Participant logging system that automatically 
records adherence and compliance with protocol-specified treatments. The device log was 
reviewed every other week by the research coordinator with study participants to optimize 
adherence in Stage I. 
 
Oral nitrite (Stage II): Administration of the oral capsule form of nitrite is expected to be much 
easier for participants compared with the inhaled delivery system in Stage I. Adherence will be 
encouraged through phone calls with participants and monitored by pill counts at weeks 4, 8, and 
12. 
 
5.6 
Prior and Concomitant Therapy 
 
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 
guidelines. 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. 
 
Data for baseline medication usage will be obtained for descriptive purposes at Study Visit 1. 
 
5.7 
Packaging 
 
The inhaled nitrite drug or matching placebo was supplied in one ml squeezable plastic ampules. 
The ampules are produced in snap-apart groups of 5 vials and are placed 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 oral nitrite drug or matching placebo using in Stage II will be supplied in capsules 
compounded by The University of Iowa Pharmaceuticals, Iowa City, IA, then shipped to the 
Research pharmacy at the University of Pittsburgh according to specifications in the IND of Dr. 
Mark Gladwin then stored and dispensed by the Research pharmacy at the Mayo Clinic. 
 
5.8 
Blinding of Study 
 
Only research pharmacy staff will be aware of randomization scheme and all study personnel and 
subjects will remain blinded to the identity of study drug. Placebo and nitrite ampules (Stage I) 
and capsules (Stage II) have an identical appearance assuring maintenance of the blind. 


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5.9 
Receiving, Storage, Dispensing and Return 
 
5.9.1 Receipt of Drug Supplies 
 
Drug dispensing will be managed by the research pharmacy 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 (Stage I) or capsules (Stage II) to permit three doses 
a day for 4 weeks. To account for potential damage or loss of study drug, additional study drug 
doses will be supplied. 
 
Used and unused investigational product 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 IP accountability has been documented and verified. Study 
IP destruction should be documented within the Participant Specific IP Accountability Log (or 
equivalent tracking system). 
 
5.9.2 Storage 
Trial products (both unused and in-use) should not be exposed to moisture but can be stored at 
room temperature. For inhaled nitrite, the plastic ampules come in a nitrogen filled foil pouch in 
snap-apart groups of 5. Participants are instructed to open one pouch at a time and utilize each 
ampule before opening another pouch. Oral nitrite is stored at room temperature in accordance 
with the manufacturer’s instructions. 
 
5.9.3 Dispensing of Study Drug 
 
Study drug will be provided by research pharmacy throughout the trial. Study drug may be mailed 
to participants if circumstances prevent them from collecting the drug in person, cause them to 
miss scheduled visits or any other unforeseen circumstances arise. 
 
For the inhaled nitrite formulation, the device has a Participant logging system that automatically 
records adherence and compliance with protocol-specified treatments. The device log will be 
reviewed every other week at ET sessions by the research coordinator with study participants to 
optimize adherence. For the oral formulation, adherence will be monitored by study staff using pill 
counts at follow up visits (see schedule of assessments). 
 
Regular study drug reconciliation will be performed to document drug assigned, drug dispensed, 
drug returns, and drug remaining. This reconciliation will be logged on the drug reconciliation 
form, and signed and dated by the study team. 
 
5.9.4 Return or Destruction of Study Drug 


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Participants are instructed to return all used, partly used and unused trial product at the 12 week 
visit (4, 8 and 12 week visit for Stage I, which has been completed). Returned trial product(s) 
(used, partly used or unused including empty packaging material) 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. 
 
6 Study Procedures 
 
6.1 
Screening Visit 1 
 
At Visit 1, patients will provide written informed consent followed by history and physical 
examination performed by a cardiologist. Medications will be documented. Participants will 
complete a blood draw in the CRTU where (NT-proBNP levels, blood methemoglobin, blood 
sulfhemoglobin, fasting blood glucose and insulin levels will be tested. An optional blood 
collection drawing an additional 20mL’s of whole blood will be collected, separated into serum 
and plasma by the CRTU and stored by BAP. This will be done at both the screening and final 
visits and stored for additional testing. We are storing the blood for future analysis and will be 
examining circulating inflammatory biomarkers, adipokines and how they relate to adipose tissue 
and HFpEF symptom severity. Assays to be run at a later date will include (subject to change): C-
reactive protein (C-RP), Interleukin-3 (IL-1β), Interleukin 6 (IL-6), Leptin, and Adiponectin. 
Assessment of endothelial function, baseline echocardiography, dual energy X-ray absorptiometry 
(DEXA) and a QOL assessment consisting of the KCCQ and SF 36 will be performed. Maximal 
effort cardiopulmonary exercise testing with expired gas analysis will then be performed with 
simultaneous echocardiography. Borg symptom severity (effort and dyspnea scores) will be 
recorded during standardized exercise training at a common objective workload, 2 mph on 
treadmill at 2° grade for 5 minutes. 
 
Following visit 1 assessments, subjects will be instructed on the use of accelerometer (AXM) 
devices and devices will be dispensed. Participants are to wear AXM at all times starting at Visit 
1. Participants will be randomized in a double-blind fashion at the time but will not start active 
treatment, and study drug will not be dispensed until the first ET visit (below). 
 
In contrast, Mayo health system participants (who undergo ET visits outside of Mayo Rochester) 
will receive study drug and supplies during the screening visit, as part of the nebulizer education 
the participant will be instructed on how to use the nebulizer and will self-administer a saline 
treatment (for Stage I). Mayo health system participants will be instructed to start assigned study 
drug the day prior to their initial cardiac rehab visit (ET1). 
 
6.2 
Exercise Training (ET) Visits 
 
Following the screening visit 1, subjects will enroll in standard cardiopulmonary exercise training 
(cardiac rehab) in the Mayo Cardiovascular Health Clinic (CVHC). This will involve ~1 hour 
sessions 3 times weekly wherein subjects participate in a combination of endurance and strength 
training as per current practice in the Mayo CVHC.  As a result of COVID-19, Mayo Clinic 


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(Rochester and Health System sites) cardiac rehabilitation centers remain closed or are open with 
significantly reduced capacity. Thus, we feel it prudent to offer our participants (who are all 
considered high-risk for contracting COVID-19) a home-based cardiac rehabilitation program 
(HBCR). This program will model the center-based cardiac rehab program implemented by the 
Mayo Clinic CVHC. Participants who agree to complete the HBCR program will be seen in clinic 
for their baseline assessments, 6 minute walk, and given a home exercise protocol.  A member 
from the study team will follow up with patient twice weekly to assess program compliance, 
discuss successes/barriers with the exercise program, provide modifications to the exercise 
program (if necessary) and offer support and encouragement. 
 
Once weekly, adverse events, safety and tolerability will be assessed., along with promotion of 
adherence to training regimen (for example on the third ET visit of each week). For Mayo health 
system participants this assessment will be done during a weekly telephone call. Every other week, 
study drug compliance information will be downloaded from the I-Neb device (for Stage I), 
reviewed, and discussed with the participant. For Mayo health system participants the I Neb device 
will be downloaded and reviewed during the week 4 visit, week 8 visit and final visit (for Stage I). 
For Stage II, adherence will be assessed by pill counts at these visits and review of diary. 
 
ET Visit 1: 
At the first ET visit (ET1), subjects received training on the use of I-neb device to administer study 
drug (for Stage I only). For Stage II, subjects will take their first dose of oral study drug 30 minutes 
prior to the initiation of the ET session (at ET1 and all training visits). One to two days after ET 
Visit 1, a study team member will call the participant at home to ask about compliance and 
tolerability of study drug. 
 
Prior to start of exercise training at ET1 and each subsequent ET visit, subjects will administer one 
dose of study drug (if the Visit is in the morning this will be the first dose of the day, if the Visit 
is in the afternoon, this will be the second dose of the day but must be separated from the first dose 
by ≥4 hours). For inhaled study drug (Stage I), this was administered immediately prior to ET visit. 
For the oral study drug (Stage II), the capsule is to be taken 30 minutes prior to the start of the ET 
visit. 
 
At one of the ET sessions every other week, measurement of QOL will be performed using the 
Kansas City Cardiomyopathy Questionnaire (KCCQ) and Borg symptom severity (effort and 
dyspnea scores) will be recorded during standardized exercise training at a common objective 
workload, 2 mph on treadmill at 2° grade for 5 minutes. Mayo health system participants will 
complete the KCCQ via biweekly phone visits with the study coordinator. 
 
6.3 
ET Visits 12 (week 4) and 24 (week 8) 
 
At one of the ET visits during week 4 and week 8, the following additional assessments will be 
performed: New medications over the last 4 weeks will be documented. Blood tests (NT- proBNP 
levels, blood methemoglobin, blood sulfhemoglobin, fasting blood glucose and insulin levels will 
be drawn in the CRTU. Data from the accelerometer will be downloaded from the device and the 
device will be exchanged with a fully charged device. Study drug will be administered prior to 


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start of training, as at all other ET visits. (Participants will receive additional study drug supplies 
from research pharmacy for Stage I). Mayo health system participants will be required to do the 
week 4 and week 8 visits at Mayo Clinic Rochester; they cannot be completed during an ET visit 
at their participating cardiac rehab site. 
 
6.4 
Final Visit 
 
The Final Visit of the drug interventional part of the study for Stage I occurred at week 12 
This visit will include all of the assessments performed in Screening Visit 1 (see above). 
Participants will be asked not to take their study medications the day of this visit; they will be 
asked to administer a treatment directly prior to the final visit CPET under direct investigator 
observation. For Stage I, inhaled study drug was administered immediately prior to CPET. For 
Stage II, oral study drug is administered (under direct observation by study staff) 30 minutes prior 
to CPET. AXM data will be downloaded and devices returned or changed out. Participants will 
return all other study materials at the final visit and all unused study drug. 
 
 
At the Final ET Visit (week 12), participants will be offered enrollment in an ancillary study to 
evaluate the effects of behavioral/motivational intervention on chronic activity levels and QOL. 
Subjects agreeing to this part of the trial will then be randomized to behavioral/motivational 
intervention (motivational phone calls twice weekly with one investigator visit per week) or 
standard care (no additional contact. Participants will continue to wear AXM devices following 
this visit to assess how well increases in chronic activity levels are maintained following ET 
intervention. Involvement in this part of the trial is optional. 
 
Study subjects not participating in the ancillary study will be contacted by phone 2 weeks 
following the final ET visit (week 12) to verify that there have been no adverse events. 
 
6.5 
Ancillary Study Visits 
 
6.5.1 Weekly Ancillary Study Visits 
Subjects who agree to participate in the ancillary study will be randomized to standard care or 
behavioral/motivation intervention (once weekly visits with investigator +2 phone calls per week) 
to evaluate durability of ET effects. 
 
6.5.2 Final Ancillary Study Visit 
The final ancillary study visit will occur 8-10 weeks after ET Visit 36. This visit will include a 
safety assessment, DEXA, cardiopulmonary exercise test, AXM return and final data download, 
repeat QOL assessment, Borg symptom severity (effort and dyspnea scores) will be recorded 
during standardized exercise training at a common objective workload, 2 mph on treadmill at 2° 
grade for 5 minutes. 
 
6.5.3 Optional MRI  and Muscle biopsy Study 
Subjects who agree to participate in the MRI ancillary study will undergo contrast MRI at the 
beginning of the trial (after consent but prior or as close to the start of exercise training) and again 


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at the conclusion of the drug intervention portion of the trial (within 2 weeks).  Subjects who agree 
to participate in the optional muscle biopsy study will undergo a muscle biopsy at the beginning 
of the trial (after consent but prior or as close to the start of exercise training) and again as close 
to the end of ET as possible. 
 
Study Assessments: 
Accelerometry (AXM): daily activity will be assessed using externally-worn hip accelerometers 
(GT3XP-BTLE, Actigraph; Pensacola, FL), which participants will receive along with a belt at 
Visit 1. The belt will be tailored by the site study coordinators to provide a comfortable fit over or 
under clothing. Participants will wear the belt continuously throughout the study, including while 
sleeping. The device will be removed only for bathing or swimming. 
 
Baseline (pre-intervention) daily activity will be assessed from AXM data obtained following Visit 
1 but prior to ET1 (1-2 weeks). AXM data will be downloaded from the devices and units 
exchanged (to ensure adequately charged batteries) at least every 4 weeks. Mayo health system 
participants will be sent home with an accelerometer charger and instructed to charge the device 
daily while it is off for showering or bathing. At the final visit on-trial accelerometer data will be 
downloaded.  
Subjects will be encouraged to walk for at least 30-60 minutes on non ET-days and will record 
outpatient activity levels in a log book to compare with accelerometer recorded activity levels. 
Subjects with low reported activity in log books will be encouraged to increase activity levels. 
 
Assessment of Exercise tolerance will be performed by measuring Borg perceived effort (6-20 
scale) and dyspnea (0-10 scale) scores during matched, low-level submaximal effort (2 mph on 
treadmill at 2° grade for 5 minutes). This assessment will be performed at Visit 1, on the third ET 
visit of each week (i.e. ET3, ET6, ET9…up to ET36), and at Visit 2. Submaximal exercise 
assessment of symptom severity at Visits 1 and 2 will occur after the primary endpoint assessment 
of peak exercise capacity. Patient-reported activity levels will be assessed by the IPAQ 
questionnaire, which will be completed at the screening visit and final endpoint visit (week 12). 
 
Measurement of QOL will be performed every other week at ET sessions using the Kansas City 
Cardiomyopathy Questionnaire (KCCQ).56, 57 The KCCQ score reflects clinical status in HF with 
high fidelity;56, 57 the score changes rapidly with clinical improvement or deterioration,58, 59 is 
strongly associated with outcome,60 and performs equally well in HFpEF and HFrEF.61The SF36 
will be completed at the screening visit and final endpoint visit (week 12). 
 
Endothelial function will be assessed at Visit 1 and Visit 2 using digital arterial tonometry (reactive 
hyperemia peripheral arterial tonometry, RH-PAT, EndoPAT, Itamar  Medical).9  These 
noninvasive assessments are routinely performed for both research and clinical care purposes. 
Changes in activity level, QOL and symptoms will then be correlated with changes in 
microvascular endothelial function (RH-PAT). 
 
Assessment of Exercise capacity will be performed at Visit 1 (pre drug) and Visit 2 (week 10 for 
Stage II of study) using expired gas analysis in the laboratory of Dr. Bruce Johnson (Joseph 4). 


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Subjects participating in the additional ancillary study will also undergo an additional 
cardiopulmonary exercise test at the final ancillary study visit. Participants will undergo standard, 
maximal effort cardiopulmonary testing on a semi-recumbent cycle ergometer to maximal 
subjective effort level with assessments of gas exchange as described below: 
 
• Measurement of Gas Exchange and Ventilation will be performed during exercise testing 
to measure the volumes of VO2, carbon dioxide produced (VCO2), breathing frequency 
(fb), tidal volume (VT), minute ventilation (VE), partial pressure of end-tidal oxygen and 
carbon dioxide (PETO2 and PETCO2), and derived variables (e.g., respiratory exchange 
ratio, VE/VCO2, etc.) using a low resistance, open circuit, automated metabolic system 
(Medical Graphics Co., St. Paul, MN) integrated to a mass spectrometer (Perkin Elmer, 
model 1100) as we have previously described.4,9,13,35,36 
 
• Cardiac Output (CO) will be measured at rest and during exercise by the acetylene (C2H2) 
wash-in technique first described by Stout et al., refined by Gan et al. and Nielson et al. 
and validated against direct Fick in our laboratory.45-48 
 
Echocardiography will be performed at rest and during exercise at Visit 1 and Final Visit to 
estimate central cardiac hemodynamics and ventricular function, including EF, Doppler-estimated 
pulmonary artery systolic pressure, and LV filling pressures (E/e’ ratio). 
 
Dual Energy X-ray Absorptiometry (DEXA) will be performed at the first and final visit in the 
primary study, and for subjects participating in the ancillary trial, at the final visit of that 
component, to measure changes in body composition during the trial. 
 
Magnetic Resonance Imaging (MRI) will be performed in subjects participating in the optional 
MRI substudy to measure cardiac structure and function as well as stiffness along with 
quantification of central and peripheral adipose tissue.  As part of the MRI examination we will 
measure intramyocardial fat content using multiecho Dixon water and fat separated managing with 
variable projection (VARPRO). We will also measure interstitial myocardial fibrosis by 
extracellular volume (ECV) using the MOLLI TI mapping sequence. These additional cardiac MRI 
measurements will be analyzed offline by our external collaborators, from completely de-
identified MRI scan data.  No personal health information will be included in the scans that are 
transmitted for external analysis. 
 
Muscle Biopsy  
Will be performed in subjects participating in the optional muscle biopsy substudy to tell us about   
what improvements we see in the leg muscles, and specifically in the mitochondria of leg muscles 
 
Biological Samples 
Venous blood samples will be collected at baseline, week 4, 8, and 12 to assess clinical stability 
(NT-proBNP) and safety (blood methemoglobin levels). Methemoglobin levels will be used as 
safety markers during the study and will be shared with the DSMB. All female subjects of 
childbearing potential will undergo a urine pregnancy test at study enrollment and end of study. 
 


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7 Statistical Plan 
7.1 
Statistical Overview 
 
7.2 
The full statistical details for this protocol are documented in the supplementary Statistical 
Analysis Plan (SAP). This protocol provides the key information, namely the justification 
for the sample size and the details on the primary analysis. The details regarding secondary 
endpoints, subgroup analyses, missing data and analysis sets are provided in the 
SAP.Sample Size Determination 
 
In the RELAX trial, the mean±SD change in peak VO2 with placebo in subjects with HFpEF 
treated for 6 months was 0±2.0 ml/kg/min,36 and in a previous trial of ET in HFpEF, the increase 
in peak VO in the ET arm was +2.3±2.2 ml/kg/min.22 An increase of 75% (additional 1.5 
ml/kg/min) in this change would be considered clinically relevant. Thus, we estimate that a sample 
size of N=34 participants per group will be required to provide 80% statistical power (two-tailed, 
alpha=0.05) to detect the difference of 1.5 ml/kg/min between subjects randomized to NO2- with 
ET versus placebo with ET. To account for attrition, we will increase our number of randomized 
subjects to 40/group (enroll 80 subjects total to accrue 68 total or 34 in each group). 
 
Stage 1 was discontinued early because of loss of inhaled nitrite availability, therefore we will 
accrue a total of 68 patients with complete data combining the patients with complete data 
receiving inhaled placebo in Stage I (n=17) with another 51 patients in Stage II (17 receiving 
placebo and 34 receiving nitrite) to ensure that the study is adequately powered to test the study 
hypotheses. 
 
7.3 
Primary Outcome Analysis 
 
Primary Statistical Hypotheses: 
 
H0: Change from baseline to 12 weeks in Peak VO2 in participants randomized to NO2- will be 
equal to the change in Peak VO2 in participants randomized to placebo. 
 
Ha: The change from baseline to 12 weeks in Peak VO2 will be different between the two 
randomized groups 
 
Analysis Summary: 
The primary statistical hypothesis will be tested using a regression framework. The dependent 
variable will be the change (baseline to 12 week) in Peak VO2. An indicator variable will be used 
to indicate the randomized treatment assignment (NO2- vs. placebo as the referent). This analysis 
is equivalent to a two-sample t-test. While the study design is randomized, the number of 
participants is relatively small, so imbalance between putative confounding variables may be 
observed. To address this, we will conduct an a priori sensitivity analysis that will adjust for 
baseline Peak VO2, BMI, Sex, and Age group strata through an extension of the regression model 
used for the primary analysis. The type III estimate of the treatment effect will be compared to the 
unadjusted estimate to assess the robustness of the randomization process. 
 


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Statistical testing for the primary analysis will be two-sided at the alpha=0.05 level of 
significance. 
 
8 Safety and Adverse Events 
8.1 
Definitions 
Unanticipated Problems Involving Risk to Subjects or Others (UPIRTSO) 
Any unanticipated problem or adverse event that meets the following three criteria: 


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• Serious: Serious problems or events that results in significant harm, (which may be physical, 
psychological, financial, social, economic, or legal) or increased risk for the subject or 
others (including individuals who are not research subjects). These include: (1) death; (2) 
life threatening adverse experience; (3) hospitalization - inpatient, new, or prolonged; (4) 
disability/incapacity - persistent or significant; (5) birth defect/anomaly; (6) breach of 
confidentiality and (7) other problems, events, or new information (i.e. publications, DSMB 
reports, interim findings, product labeling change) that in the opinion of the local 
investigator may adversely affect the rights, safety, or welfare of the subjects or others, or 
substantially compromise the research data, AND 
• Unanticipated: (i.e. unexpected) problems or events are those that are not already described 
as potential risks in the protocol, consent document, not listed in the Investigator’s 
Brochure, or not part of an underlying disease. A problem or event is "unanticipated" when 
it was unforeseeable at the time of its occurrence. A problem or event is "unanticipated" 
when it occurs at an increased frequency or at an increased severity than expected, AND 
• Related: A problem or event is "related" if it is possibly related to the research procedures. 
 
 
Adverse Event 
An untoward or undesirable experience associated with the use of a medical product (i.e. drug, 
device, biologic) in a patient or research subject. 
Serious Adverse Event 
Adverse events are classified as serious or non-serious. Serious problems/events can be well 
defined and include; 
• death 
• life threatening adverse experience 
• hospitalization 
• inpatient, new, or prolonged; disability/incapacity 
• persistent or significant birth defect/anomaly 
 
and/or per protocol may be problems/events that in the opinion of the sponsor-investigator may 
have adversely affected the rights, safety, or welfare of the subjects or others, or substantially 
compromised the research data. 
 
All adverse events that do not meet any of the criteria for serious, should be regarded as non- 
serious adverse events. 
 
Adverse Event Reporting Period 
 
For this study, the study treatment follow-up period is defined as 2 weeks following the last 
administration of study treatment. The study period during which adverse events must be reported 
is normally defined as the period from the initiation of any study procedures to the end of the study 
treatment follow-up. 


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Preexisting Condition 
A preexisting condition is one that is present at the start of the study. A preexisting condition 
should be recorded as an adverse event if the frequency, intensity, or the character of the condition 
worsens during the study period. 
 
General Physical Examination Findings 
At screening, any clinically significant abnormality should be recorded as a preexisting condition. 
At the end of the study, any new clinically significant findings/abnormalities that  meet the 
definition of an adverse event must also be recorded and documented as an adverse event. 
 
Post-study Adverse Event 
All unresolved adverse events should be followed by the sponsor-investigator until the events are 
resolved, the subject is lost to follow-up, or the adverse event is otherwise explained. At the last 
scheduled visit, the sponsor-investigator should instruct each subject to report, to the sponsor- 
investigator, any subsequent event(s) that the subject, or the subject’s personal physician, believes 
might reasonably be related to participation in this study. 
 
Hospitalization, Prolonged Hospitalization or Surgery 
Any adverse event that results in hospitalization or prolonged hospitalization should be 
documented and reported as a serious adverse event unless specifically instructed otherwise in this 
protocol. Any condition responsible for surgery should be documented as an adverse event  if the 
condition meets the criteria for an adverse event. 
 
Neither the condition, hospitalization, prolonged hospitalization, nor surgery are reported as an 
adverse event in the following circumstances: 
• Hospitalization or prolonged hospitalization for diagnostic or elective surgical procedures 
for a preexisting condition. Surgery should not be reported as an outcome of an adverse 
event if the purpose of the surgery was elective or diagnostic and the outcome was 
uneventful. 
• Hospitalization or prolonged hospitalization for therapy of the target disease of the study, 
unless it is a worsening or increase in frequency of hospital admissions as judged by the 
clinical investigator. 
 
8.2 
Recording of Adverse Events 
At each contact with the subject, the study team must seek information on adverse events by 
specific questioning and, as appropriate, by examination. Information on all adverse events should 
be recorded immediately in the source document, and also in the appropriate adverse event section 
of the case report form (CRF). All clearly related signs, symptoms, and abnormal diagnostic, 
laboratory or procedure results should recorded in the source document. 
 
All adverse events occurring during the study period must be recorded. The clinical course of each 
event should be followed until resolution, stabilization, or until it has been ultimately determined 
that the study treatment or participation is not the probable cause. Serious adverse events that are 
still ongoing at the end of the study period must be followed up, to determine the final outcome. 
Any serious adverse event that occurs after the study period and is considered to 


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be at least possibly related to the study treatment or study participation should be recorded and 
reported immediately. 
 
8.2.1 
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. 
 
8.2.2 
Assessment of Adverse Event Severity 
The determination of adverse event severity rests on medical judgment of a medically-qualified 
Investigator. The severity of the 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. 
 
 
8.3 
Reporting of Serious Adverse Events and Unanticipated Problems 
 
When a serious adverse event has been identified, the study team will take appropriate action 
necessary to protect the study participant and then complete the Study Adverse Event Worksheet 
and log. The sponsor-investigator will evaluate the event and determine the necessary follow-up 
and reporting required. 
 
8.3.1 Sponsor-Investigator reporting: notifying the Mayo IRB 
 
The investigator will report to the Mayo IRB any UPIRTSOs and Non-UPIRTSOs according to 
the Mayo IRB Policy and Procedures. 
 
Information collected on the adverse event worksheet 
• Subject’s name: 
• Medical record number: 
• Disease (if applicable): 


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• The date the adverse event occurred: 
• Description of the adverse event: 
• Relationship of the adverse event to the research (drug, procedure, or intervention): 
• If the adverse event was expected: 
• The severity of the adverse event: 
• If any intervention was necessary: 
• Resolution: (was the incident resolved spontaneously, or after discontinuing treatment) 
• Date of Resolution: 
 
The investigator will review all adverse event reports to determine if specific reports need to be 
made to the IRB and FDA. The investigator will sign and date the adverse event report when it  is 
reviewed. For this protocol, only directly related SAEs will be reported to the IRB. 
 
8.3.2 Sponsor-Investigator reporting: Notifying the FDA 
 
The investigator will report to the FDA all unexpected, serious suspected adverse reactions 
according to the required IND Safety Reporting timelines, formats and requirements. 
 
Unexpected fatal or life threatening suspected adverse reactions where there is evidence to suggest 
a causal relationship between the study drug/placebo and the adverse event, will be reported as a 
serious suspected adverse reaction. This will be reported to the FDA on FDA Form 3500A, no 
later than 7 calendar days after the sponsor-investigator’s initial receipt of the information about 
the event. 
 
Other unexpected serious suspected adverse reactions where there is evidence to suggest a causal 
relationship between the study drug/placebo and the adverse event, will be reported as a serious 
suspected adverse reaction. This will be reported to the FDA on FDA Form 3500A, no later than 
15 calendar days after the sponsor-investigator’s initial receipt of the information about the event. 
 
Any clinically important increase in the rate of serious suspected adverse reactions over those 
listed in the protocol or product insert will be reported as a serious suspected adverse reaction. 
This will be reported to the FDA on FDA Form 3500A no later than 15 calendar days after the 
sponsor-investigator’s initial receipt of the information about the event. 
 
Findings from other studies in human or animals that suggest a significant risk in humans exposed 
to the drug will be reported. This will be reported to the FDA on FDA Form 3500A, no later than 
15 calendar days after the sponsor-investigators initial receipt of the information about the event. 
 
8.4 
Unblinding Procedures 
 
The investigators will be given access to the treatment code for their Participants for emergency 
un-blinding. Due to the safety profile of inorganic nitrite it is anticipated that there should be no 


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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. 
 
Randomization data are kept strictly confidential, accessible only to authorized persons, until the 
time of un-blinding. 
 
8.5 
Stopping Rules 
 
The study would be stopped only if advised because of unanticipated adverse events as 
determined by the Data and Safety Monitoring Board (DSMB). 
 
8.6 
Medical Monitoring 
It is the responsibility of the Principal Investigator to oversee the conduct of the study and safety 
of subjects. This safety monitoring will include careful assessment and appropriate reporting of 
adverse events as noted above, as well as the construction and implementation of a site data and 
safety-monitoring plan (see section 10 “Study Monitoring, Auditing, and Inspecting”). Medical 
monitoring will include a regular assessment of the number and type of serious adverse events. 
 
8.6.1 Internal Data and Safety Monitoring Board 
 
An internal Data and Safety Monitoring Board (DSMB) has been formed, chaired by Dr. Bernard 
Gersh. The DSMB Charter is included as an Attachment. 
 
9 Data Handling and Record Keeping 
9.1 
Confidentiality 
Information about study subjects will be kept confidential and managed according to the 
requirements of the Health Insurance Portability and Accountability Act of 1996 (HIPAA). Those 
regulations require a signed subject authorization informing the subject of the following: 
• What protected health information (PHI) will be collected from subjects in this study 
• Who will have access to that information and why 
• Who will use or disclose that information 
• The rights of a research subject to revoke their authorization for use of their PHI. 
 
In the event that a subject revokes authorization to collect or use PHI, the investigator, by 
regulation, retains the ability to use all information collected prior to the revocation of subject 
authorization. For subjects that have revoked authorization to collect or use PHI, attempts  should 
be made to obtain permission to collect at least vital status (long term survival status that the subject 
is alive) at the end of their scheduled study period. 
 
9.2 
Source Documents 
Source data is all information, original records of clinical findings, observations, or other activities 
in a clinical trial necessary for the reconstruction and evaluation of the trial. Source data are 
contained in source documents. Examples of these original documents, and data records include: 
hospital records, clinical and office charts, laboratory notes, memoranda, subjects’ diaries or 
evaluation checklists, pharmacy dispensing records, recorded data from automated 


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instruments, copies or transcriptions certified after verification as being accurate and complete, 
microfiches, photographic negatives, microfilm or magnetic media, x-rays, subject files, and 
records kept at the pharmacy, at the laboratories, and at medico-technical departments involved in 
the clinical trial. 
 
9.3 
Case Report Forms 
 
The study case report form (CRF) is the primary data collection instrument for the study. All 
data requested on the CRF will be recorded. All missing data will be explained. 
 
Medidata Rave and Balance will be used for clinical data management and randomization (see 
Facilities and Resources for expanded description). These tools are institutionally supported and 
allow for rapid database development. The implementation of the software within a project 
includes training on the system’s use, robust error checking, security considerations, and full audit 
trail. Balance, which is integrated into Rave, allows for a variety of randomization and 
minimization algorithms. 
 
9.4 
Records Retention 
 
The investigator will maintain records and essential documents related to the conduct of the 
study. These will include subject case histories and regulatory documents. 
 
The investigator will retain the specified records and reports for; 
1. Up to 2 years after the marketing application is approved for the drug; or, if a marketing 
application is not submitted or approved for the drug, until 2 years after shipment and 
delivery of the drug for investigational use is discontinued and the FDA has been so 
notified. OR 
2. As outlined in the Mayo Clinic Research Policy Manual –“Access to and Retention of 
Research Data Policy” http://mayocontent.mayo.edu/research-policy/MSS_669717 
Whichever is longer. 
 
 
10 Study Monitoring, Auditing, and Inspecting 
10.1 
Study Monitoring Plan 
 
This study will be monitored on a routine basis during the conduct of the trial. The Mayo Clinic 
Office of Research Regulatory Support will provide periodic study monitoring as a service for the 
sponsor-investigator, to verify the validity and integrity of the data and protection of human 
research subjects. Written monitoring reports will be provided to the Sponsor-Investigator and 
should be provided to the IRB at the time of continuing review. This will  assist  with compliance 
with Food and Drug Administration regulations and GCP Guidelines. 


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10.2 
Auditing and Inspecting 
The investigator will permit study-related monitoring, audits, and inspections by the IRB, the 
sponsor, and government regulatory agencies, of all study related documents (e.g. source 
documents, regulatory documents, data collection instruments, study data etc.). The investigator 
will ensure the capability for inspections of applicable study-related facilities (e.g. pharmacy, 
diagnostic laboratory, etc.). 
 
Participation as an investigator in this study implies acceptance of potential inspection by 
government regulatory authorities and applicable compliance offices. 
 
11 Ethical Considerations 
This study is to be conducted according to United States government regulations and 
Institutional research policies and procedures. 
 
This protocol and any amendments will be submitted to a properly constituted local Institutional 
Review Board (IRB), in agreement with local legal prescriptions, for formal approval of the study. 
The decision of the IRB concerning the conduct of the study will be made in writing to  the sponsor-
investigator before commencement of this study. 
 
All subjects for this study will be provided a consent form describing this study and providing 
sufficient information for subjects to make an informed decision about their participation in this 
study. This consent form will be submitted with the protocol for review and approval by the IRB 
for the study. The formal consent of a subject, using the Approved IRB consent form, must be 
obtained before that subject undergoes any study procedure. The consent form must be signed  by 
the subject or the subject’s legally authorized representative, and the individual obtaining the 
informed consent. 
 
12 Study Finances 
12.1 
Funding Source 
 
This study is financed through a grant from the US National Institute of Health. 
Study drug for Stage 1 was provided by Aires Pharmaceuticals. 
Study drug for Stage 2 of the trial (oral nitrite/placebo) is being compounded at The University of 
Iowa Pharmaceuticals, in Iowa City, IA, shipped to the Research Pharmacy at the University of 
Pittsburgh, and then, shipped to Mayo Clinic where it will be stored and dispensed by the Mayo 
Research Pharmacy. 
 
12.2 
Conflict of Interest 
 
Any study team member who has a conflict of interest with this study (patent ownership, 
royalties, or financial gain greater than the minimum allowable by their institution, etc.) must 


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have the conflict reviewed by a properly constituted Conflict of Interest Committee with a 
Committee-sanctioned conflict management plan that has been reviewed and approved by the 
study sponsor-investigator prior to participation in this study. 
 
13 Publication Plan 
 
The primary responsibility for publication of the results lies with the principal investigator. The 
trial will be registered on ClinicalTrials.gov. 
 
14 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 
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2. Owan TE, Hodge DO, Herges RM, Jacobsen SJ, Roger VL, Redfield MM. Trends in 
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diagnosis, and treatment. Eur Heart J. 2011;32: 670-79. PMCID: PMC3056204. 
 
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8. Maeder MT, Thompson BR, Brunner-La Rocca HP, Kaye DM. Hemodynamic basis of 
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15 Attachments 
15.1 Schedule of Assessments 


 
 
 
INABLE - Training    Schedule of Assessments 
 
 
 
 
 
 
 
 
 
 
 
 
 
INABLE - Training V1 Sept 2016 
15 Attachments 
15.1 Schedule of Assessments 


Inorganic Nitrite To Amplify the Benefits and Tolerability of Exercise Training In Heart Failure With Preserved Ejection 
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