Clinical trial NCT03015402
Conditions: Pulmonary Hypertension Secondary Heart Failure
Sponsor: University of Pittsburgh
Start date: 2017-10-30; Completion date: 2023-01-25
Enrollment: 33 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
The main objective of this study is to determine the clinical efficacy of oral inorganic nitrite verses placebo and the therapeutic response with regards to exercise tolerance in patients with pulmonary hypertension and heart failure with preserved ejection fraction (PH-HFpEF).

Detailed description:
This is a single-center, 22-week double-blind, randomized placebo-controlled cross-over trial of oral nitrite on exercise capacity and hemodynamic measurements in subjects with PH-HFpEF.

Arms: arm 1: Study arm 2: Control
Interventions: intervention 1: Sodium Nitrite intervention 2: Placebo Oral Capsule
Intervention descriptions: intervention 1: 40 mg PO (by mouth) TID (three times each day) for 10 weeks intervention 2: Placebo capsule that is of identical size, shape, and color to experimental drug capsule PO (by mouth) TID (three times each day) for 10 weeks

Protocol documents:
Prot_SAP_000.pdf: 
 
 
 
 
 
Oral Nitrite in Patients with Pulmonary Hypertension and Heart Failure with Preserved 
Ejection Fraction (PH-HFpEF) 
 
 
 
 
 
 
 
Michael Risbano, MD 
Principal Investigator 
 
 
 
NCT03015402 
 
 
 
 
 
 
 
University of Pittsburgh 
School of Medicine 
Division of Pulmonary, Allergy and Critical Care 
 
 
 
 


Study Short Title: Oral Nitrite in Patients with PH-HFpEF 
 
Sponsor: Mark T. Gladwin, MD 
    
PI: Michael Risbano, MD 
STUDY20010014 
Page 1 of 47 
V14.0  02/14/2022  
 
 
Table of Contents 
PROTOCOL SYNOPSIS .........................................................................................................................4 
1.   OBJECTIVE, SPECIFIC AIMS, BACKGROUND, AND SIGNIFICANCE .....................................................8 
1.1 OBJECTIVE ........................................................................................................................................... 8 
1.2 SPECIFIC AIMS ..................................................................................................................................... 8 
1.3 BACKGROUND ..................................................................................................................................... 8 
1.3.1 Harnessing the nitrate-nitrite-NO pathway for PH therapeutics ................................................. 8 
1.4 SIGNIFICANCE .................................................................................................................................... 10 
2.   RESEARCH DESIGN AND METHODS .............................................................................................. 11 
2.1 CLASSIFICATION AND METHODOLOGICAL DESIGNS ......................................................................... 11 
2.2 DETAILED DESCRIPTION OF STUDY DESIGN ....................................................................................... 11 
2.2.1. Study Drug Preparation and Distribution .................................................................................. 11 
2.2.2. Treatment Period ...................................................................................................................... 12 
2.2.3. Safety Monitoring: .................................................................................................................... 12 
2.2.4. Medication Compliance ............................................................................................................ 12 
2.2.5. Medication Accountability ........................................................................................................ 13 
2.3 STUDY ASSESSMENT .......................................................................................................................... 13 
2.3.1. Screening Procedures ................................................................................................................ 13 
2.3.2. Baseline Procedures .................................................................................................................. 14 
2.3.3. Treatment Period and Monitoring ............................................................................................ 14 
2.3.4. End of Trial and Follow-Up Period ............................................................................................. 17 
2.3.5.   Open Label Period ................................................................................................................... 17 
2.3.6. Schedule of Assessments ........................................................................................................... 18 
2.4 DESCRIPTION OF STUDY PROCEDURES .............................................................................................. 19 
2.4.1 Echocardiogram ......................................................................................................................... 19 
2.4.2 Six Minute Walk Test (6MWT and Borg Scale) ........................................................................... 19 
2.4.3 Accelerometer ............................................................................................................................ 19 
2.4.4 Spirometry .................................................................................................................................. 19 
2.4.5 Cardiopulmonary Exercise Testing (CPET) and Gas Exchange Measurements: ......................... 20 
2.4.6 Laboratory Testing ..................................................................................................................... 20 
2.4.7 Methemoglobin Levels ............................................................................................................... 21 
2.4.8 Right Heart Catheterization, Simultaneous Pressure and Doppler Flow Velocity Measurement, 
and Exercise RHC ................................................................................................................................. 21 
2.4.9 Intravenous Glucose Tolerance Test (IVGTT) ............................................................................. 22 
2.5 SPECIMEN COLLECTION AND MANAGEMENT ................................................................................... 22 
2.5.1 Specimen Handling and Labeling (De-Identification) ................................................................. 23 
2.5.2 Specimen Management and Storage ......................................................................................... 23 
2.5.3 Restrictions to Direct Access of Specimens ................................................................................ 23 


Study Short Title: Oral Nitrite in Patients with PH-HFpEF 
 
Sponsor: Mark T. Gladwin, MD 
    
PI: Michael Risbano, MD 
STUDY20010014 
Page 2 of 47 
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2.6 ENDPOINTS ........................................................................................................................................ 23 
2.6.1 Primary Endpoints ...................................................................................................................... 23 
2.6.2 Secondary Endpoints .................................................................................................................. 23 
2.7 SUBJECT WITHDRAWAL ..................................................................................................................... 24 
2.7.1. Dropouts and withdrawals ........................................................................................................ 24 
2.8 STATISTICAL ANALYSIS....................................................................................................................... 24 
2.8.1 Sample Size and Power .............................................................................................................. 24 
3.   HUMAN SUBJECTS ...................................................................................................................... 25 
3.1 SUBJECT POPULATION ....................................................................................................................... 25 
3.1.1 Inclusion of Women and Minorities ........................................................................................... 25 
3.1.2 Inclusion of Children ................................................................................................................... 25 
3.2 INCLUSION CRITERIA ......................................................................................................................... 25 
3.3 EXCLUSION CRITERIA ......................................................................................................................... 26 
4.   RECRUITMENT AND INFORMED CONSENT PROCEDURES .............................................................. 26 
4.1 RECRUITMENT METHODS ................................................................................................................. 26 
4.2 INFORMED CONSENT PROCEDURES .................................................................................................. 28 
5.   POTENTIAL RISKS AND BENEFITS ................................................................................................. 29 
5.1. RISK OF EXPERIMENTAL DRUG INTERVENTION ........................................................................................... 29 
5.2 RISK OF STUDY PROCEDURES ................................................................................................................... 31 
5.3 ALTERNATIVE TREATMENTS .............................................................................................................. 34 
5.4 POTENTIAL BENEFITS ......................................................................................................................... 34 
5.5 DATA SAFETY MONITORING PLAN ..................................................................................................... 34 
5.5.1 Data Safety Monitoring Board ................................................................................................... 34 
5.5.2 Data Safety Monitoring Plan ...................................................................................................... 34 
5.5.3 Parameters to be Monitored ...................................................................................................... 36 
5.5.4 Frequency of Monitoring ............................................................................................................ 37 
5.5.5 Reportable Adverse Events......................................................................................................... 37 
5.5.6 Adverse Events Reporting Timeline ............................................................................................ 37 
5.6 RISKS MANAGEMENT PROCEDURES ................................................................................................. 38 
5.6.1 Protection Against Risks ............................................................................................................. 38 
5.6.2 Protection Against Potential Risks of Experimental Intervention .............................................. 39 
6. STUDY ADMINSTRATION .............................................................................................................. 39 
6.1 QUALITY CONTROL AND QUALITY ASSURANCE ................................................................................. 39 
6.2 DATA HANDLING AND RECORD-KEEPING .......................................................................................... 39 
6.2.1 Data recording ........................................................................................................................... 39 
6.2.2 Record maintenance and retention ........................................................................................... 39 
6.3 ETHICS ............................................................................................................................................... 40 
7.  COSTS AND PAYMENTS ................................................................................................................ 41 
7.1 COSTS ................................................................................................................................................ 41 


Study Short Title: Oral Nitrite in Patients with PH-HFpEF 
 
Sponsor: Mark T. Gladwin, MD 
    
PI: Michael Risbano, MD 
STUDY20010014 
Page 3 of 47 
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7.2 PAYMENTS ......................................................................................................................................... 41 
8.  QUALIFICATIONS AND SOURCE OF SUPPORT ................................................................................ 42 
8.1 QUALIFICATIONS OF THE INVESTIGATORS ........................................................................................ 42 
8.2   SOURCE OF SUPPORT ....................................................................................................................... 43 
REFERENCES .................................................................................................................................... 43 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


Study Short Title: Oral Nitrite in Patients with PH-HFpEF 
 
Sponsor: Mark T. Gladwin, MD 
    
PI: Michael Risbano, MD 
STUDY20010014 
Page 4 of 47 
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PROTOCOL SYNOPSIS 
 
Protocol Title: 
Oral Nitrite in Patients with Pulmonary Hypertension and Heart 
Failure with Preserved Ejection Fraction  
(PH-HFpEF) 
Protocol Number: 
PRO16110594 
NCT Number: 
03015402 
Version # and Date: 
Version 14.0/February 14, 2022 
Clinical Phase: 
Phase II clinical investigation 
Investigational Drug: 
Oral Nitrite (NO2
−) 
Trial Site: 
Single-Center Trial 
Sponsor: 
Mark Gladwin, MD 
Professor of Medicine 
Chair, Department of Medicine 
University of Pittsburgh 
 
Investigator: 
 
Michael Risbano, MD, 
Assistant Professor of Medicine 
University of Pittsburgh 
UPMC PACCM 
 
Sub-Investigators: 
Mark T. Gladwin, MD   
Marc Simon, MD 
Adil Yunis, MD 
Vinaya Mulkareddy, MD 
Michael Bashline, MD 
Frederico Toledo, MD 
Carl Koch, MD 
Jessica Huston, MD 
Study Monitor: 
Mark T. Gladwin, MD 
Chairman, Department of Medicine 
University of Pittsburgh School of Medicine 
3550 Terrace St. - 1218 Scaife Hall 
Pittsburgh, PA  15261 
 
Research Facilities: 
UPMC Presbyterian Hospital 
200 Lothrop Street 
Pittsburgh, PA 15213 
 
Clinical Laboratories: 
University of Pittsburgh Medical Center 
3477 Euler Way 
Pittsburgh, PA  15213 


Study Short Title: Oral Nitrite in Patients with PH-HFpEF 
 
Sponsor: Mark T. Gladwin, MD 
    
PI: Michael Risbano, MD 
STUDY20010014 
Page 5 of 47 
V14.0  02/14/2022  
 
 
Manufacturer: 
Triangle Compounding Pharmacy, Inc. 
3700 Regency Parkway, Suite 140 
Cary, NC 27518 
University of Iowa Pharmaceuticals 
Iowa City, Iowa 52242 
Study Rationale: 
A strong foundation of knowledge supports that nitrite, as a 
source of nitric oxide (NO), can prevent and reverse 
experimental pulmonary hypertension. 
 
Study Hypothesis: 
We hypothesize that: 
𝑵𝑶𝟐
− 40mg given orally three times a day for 10 weeks will 
improve hemodynamics, with a reduction in peak mean 
pulmonary arterial pressure (mPAP) during submaximal 
exercise as compared to placebo (primary endpoint), as 
well as resting mPAP, pulmonary capillary wedge pressure 
(PCWP) at rest and with submaximal exercise, right atrial 
pressure at rest and with submaximal exercise, and an 
increase in pulmonary artery compliance at rest and with 
submaximal exercise (all secondary endpoints).  
 
𝑵𝑶𝟐
− 40mg given orally three times a day for 10 weeks will 
improve exercise performance, including exercise time and 
maximal oxygen consumption during maximal and 
submaximal exercise as compared to placebo (secondary 
endpoint).  
 
𝑵𝑶𝟐
− 40mg given orally three times a day for 10 weeks will 
improve insulin sensitivity, fasting blood glucose, and 
HgbA1c, compared to placebo (exploratory endpoint).  
 
Study Aims: 
Specific Aims: 
1. To evaluate the clinical efficacy and signaling 
mechanisms of a new FDA-IND approved oral 
formulation of nitrite (IND# 119526) on exercise 
tolerance and hemodynamics in PH-HFpEF. 
 
2. To monitor in vivo formation and metabolism of this 
new class of signaling electrophile using new FDA IND 
approved oral nitrite. 
 
3. To leverage the methodological tools and insights 
gleaned over this time to explore fundamental signaling 
mechanisms of the reactive nitrogen species, nitrite 
(𝑵𝑶𝟐
−), in the context of pulmonary hypertension (PH) in 
the setting of the metabolic syndrome. 
 


Study Short Title: Oral Nitrite in Patients with PH-HFpEF 
 
Sponsor: Mark T. Gladwin, MD 
    
PI: Michael Risbano, MD 
STUDY20010014 
Page 6 of 47 
V14.0  02/14/2022  
 
    
The study aims to assess the use of oral nitrite vs. placebo on 
exercise capacity and hemodynamic measurements in 
subjects with PH-HFpEF. 
Subjects will undergo screening procedures to determine that 
eligibility requirements are met. Prior to receiving the study 
drug or placebo, subjects will complete baseline assessments. 
Subjects (n=26) who meet the inclusion criteria and none of 
the exclusion criteria will be randomized to study drug (oral 
nitrite or placebo) for 10 weeks. Subjects will then cross-over 
to the alternative therapy for an additional 10 weeks and 
complete all the same testing procedures.  
Drug: oral formulation of sodium nitrite dose strength in 20 
or 40 mg capsules or matching placebo three times 
daily or TID (once in the morning, in the early 
afternoon and again in the evening) for the duration of 
the study.  
 
Subjects will be evaluated as outpatients with the first dose of 
study drug. Subjects will return at weeks 4 and 8 for safety visits 
after the start of each study arm. The safety visit will occur to 
assess adverse events (AE), methemoglobin level, interval 
histories, brief physical exams, medication compliance review, 
and dispensing of study drug.  
Subjects will also be contacted by the study investigator and/or 
the study coordinator for a follow-up phone assessment 
approximately 30 days following the completion of the study. 
Planned Sample Size: 
26 subjects 
Duration of Treatment: 
22 weeks 
Inclusion Criteria: 
Age 18 years and older 
 
PH-HFpEF confirmed diagnosis by RHC: 
• 
Mean Pulmonary Arterial Pressure (mPAP) ≥ 25    
mmHg  
AND 
 
• 
Pulmonary capillary wedge pressure (PCWP)  ≥ 15 
mmHg 
AND 
• 
Transpulmonary Gradient (TPG) ≥ 12 mmHg 
 
Patient has one or more of the following heart failure associated 
signs or symptoms: shortness of breath with activity, overweight, 
atrial fibrillation and/or systemic hypertension. 
 
Exclusion Criteria: 
• 
Age less than 18 years;  
• 
SBP > 170 or < 110 mmHg 
• 
DBP >95 or < 60 mmHg 


Study Short Title: Oral Nitrite in Patients with PH-HFpEF 
 
Sponsor: Mark T. Gladwin, MD 
    
PI: Michael Risbano, MD 
STUDY20010014 
Page 7 of 47 
V14.0  02/14/2022  
 
• 
Hemoglobin A1C > 10 
• 
Positive urine pregnancy test or breastfeeding 
• 
Ejection Fraction (EF) < 40% 
• 
Dementia 
• 
End-stage malignancy 
• 
Major cardiovascular event or procedure within 6 weeks 
prior to enrollment 
• 
Severe valvular disease 
• 
Known chronic psychiatric or medical conditions that may 
increase the risk associated with study participation in the 
judgment of the investigator, would make the subject 
inappropriate for entry into this study 
• 
Smoker  
• 
Hemoglobin <9 g/dL 
• 
Serum creatinine > 3.0 mg/dL 
• 
Recent (<3 months) addition or change in dosing of 
hormonal 
contraceptive 
medications 
(OCP, 
IUD, 
DepoProvera) 
• 
Receipt of an investigational product or device, or 
participation in a drug research study within a period of 15 
days 
• 
RHC < 2 weeks from study screening RHC unless clinically 
indicated 
Study Endpoints: 
Primary endpoint: 
The primary endpoint of the trial will be mean pulmonary artery 
pressure (mPAP) during submaximal exercise as compared 
between placebo and nitrite at 10 weeks (i.e. the week 10 RHC 
of placebo vs week 10 RHC of nitrite). 
 
 
Secondary endpoints:  
1.The differences in 6MWT with Borg dyspnea scale 
assessments and pulse oxygen saturation measurements  
2. RHC hemodynamics (right atrial pressure, pulmonary 
capillary wedge pressure, pulmonary artery pressures and 
resistance, pulmonary artery compliance, cardiac output, 
diastolic pressure gradients, and transpulmonary gradient)  
3. Doppler-echocardiography 
4. NT-proBNP plasma levels  
5. NYHA functional classification changes 
6. NO2
− metabolites 
7. Endurance exercise time (ET), changes in CPET 
measurements taken at isotime (oxygen consumption [VO2, 
L/min], minute ventilation [VE, L/min], ventilatory equivalent 
[VE/VO2], dead space to tidal volume ratio [Vd/Vt], Borg 
dyspnea and leg score, and changes in time constants for 
increases of Vt, respiratory rate [RR], VE, and heartrate) 
8. Changes in fasting glucose and insulin sensitivity 
 
 


Study Short Title: Oral Nitrite in Patients with PH-HFpEF 
 
Sponsor: Mark T. Gladwin, MD 
    
PI: Michael Risbano, MD 
STUDY20010014 
Page 8 of 47 
V14.0  02/14/2022  
 
1.   OBJECTIVE, SPECIFIC AIMS, BACKGROUND, AND SIGNIFICANCE 
1.1 OBJECTIVE 
The main objective of this study is to determine the clinical efficacy of oral inorganic nitrite 
verses placebo and the therapeutic response with regards to exercise tolerance in patients 
with Group II PH-HFpEF. 
1.2 SPECIFIC AIMS 
Hypothesis: 
1. Acute open-label dosing of 40 mg of oral nitrite in the cardiac catheterization laboratory 
will safely vasodilate the pulmonary circulation and reduce left ventricular filling 
pressures of patients with Group II PH-HFpEF.  
2. Blinded chronic oral nitrite supplementation (40 mg PO TID for 10 weeks) will improve 
exercise capacity, hemodynamics, and insulin resistance compared with placebo.  
 
Specific Aims: 
 
Aim 1: To evaluate the clinical efficacy and signaling mechanisms of a new FDA-IND approved 
oral formulation of nitrite (IND# 119526) on exercise tolerance and hemodynamics in PH-
HFpEF. 
 
Aim 2: To monitor in vivo formation and metabolism of this new class of signaling electrophile 
using new FDA IND approved oral nitrite. 
 
Aim 3: To leverage the methodological tools and insights gleaned over this time to explore 
fundamental signaling mechanisms of the reactive nitrogen species, nitrite (NO2
−), in the 
context of pulmonary hypertension (PH) in the setting of the metabolic syndrome. 
 
 
1.3 BACKGROUND  
 
1.3.1 Harnessing the nitrate-nitrite-NO 
pathway for PH therapeutics  
Reduced 
bioavailability 
and 
impaired 
production of nitric oxide (NO) are thought to 
contribute to the development of both 
metabolic 
syndrome 
and 
PH.[1-6] 
An 
increasing number of therapeutic approaches, 
which enhance NO generation and bioactivity, 
have been proposed over the past decade and 
include administration of nitrite and nitrate.[7] 
Nitrite, the active metabolite of dietary nitrate, 
has been recognized as a natural reservoir of 
NO.[7],[8] This is now referred to as the 
nitrate-nitrite-NO pathway and involves a 
series 
of 
oxygen-independent 
and 
NO 
synthase-independent single electron transfer reactions.[7] Nitrate accumulates in the plasma 
from oral intake of foods rich in nitrate, such as green leafy vegetables and root plants, or from 
the intravascular oxidation of NO to nitrate by oxyhemoglobin (Figure 1). Nitrate is then 
concentrated in the saliva and reacts with oral commensal bacteria, which contain nitrate 


Study Short Title: Oral Nitrite in Patients with PH-HFpEF 
 
Sponsor: Mark T. Gladwin, MD 
    
PI: Michael Risbano, MD 
STUDY20010014 
Page 9 of 47 
V14.0  02/14/2022  
 
reductase enzymes.[7, 9] Humans do not possess nitrate reductase enzymes so require these 
bacteria for conversion of nitrate to nitrite. Nitrite is then swallowed and systemically absorbed 
where it can be further reduced via one-electron transfer reactions with hemoglobin, 
myoglobin, neuroglobin, and molybopterrin-containing enzymes (such as xanthine oxidase, 
aldehyde oxidase and mARC).[10-13] Nitrite has now been shown to regulate blood pressure, 
hypoxic vasodilation, mitochondrial efficiency, and exercise performance. From a therapeutic 
standpoint, nitrite has been shown to exhibit therapeutic efficacy in mouse, rat, and sheep 
pre-clinical models of PH.[14-16] Additionally, oral supplementation of nitrate and nitrite 
prevents the development of hypoxia- and monocrotaline-induced PH in mice.[17]  
Furthermore, a recent study suggests that dietary supplementation of nitrate reverses the 
features of metabolic syndrome in eNOS-deficient mice, yet the mechanism behind this 
observation and the role of nitrite on metabolic syndrome remain uncertain.[18]  
 
Nitrate-nitrite-NO signaling improves exercise capacity and efficiency  
In addition to effects on improving pulmonary hypertension, nitrate and nitrate-containing 
foods such as beet root juice, and purified nitrate solutions, improve exercise efficiency (3-6% 
increase VO2/watts), exercise time to exhaustion (≈16%), increase tolerance to high intensity 
exercise (≈25%), and improve mitochondrial efficiency (P/O ratios).[19-21] Studies in highly 
trained athletes show that higher plasma baseline levels of nitrite are associated with superior 
exercise capacity and nitrate therapy increases time trial performance (the time required to 
achieve a pre-specified work level) for several sports.[22-33] A recent meta-analysis of 17 
studies investigating the effect of inorganic nitrate supplementation on exercise performance 
for several types of exercise, including cycling, walking, running, and knee extension exercise, 
showed a significant benefit on performance for time to exhaustion tests, performance for time 
trials, and graded exercise tests.[26] Studies in patients with cardiopulmonary disease are 
now being published including a recently completed trial of a high nitrate containing beet root 
juice in 15 subjects with COPD (placebo-controlled cross over trial) that increased plasma 
nitrite levels by 379% and increased median submaximal constant work rate exercise time by 
28.8 seconds.[34] A recently completed study evaluated a single dose of 12.9 mmoles of 
nitrate in beetroot juice compared with nitrate depleted juice in a double-blinded placebo-
controlled cross over trial of HFpEF patients without PH and found significant increases in 
exercise duration, VO2 max, and total work performed.[35, 36] These studies provide relevant 
background to test oral nitrite in this longer term trial and we expect additional benefit in the 
targeted enrollment population of patients with HFpEF that also have an increased pulmonary 
vascular resistance and metabolic syndrome.  
 
Sirtuins and AMPK as regulators of metabolism, activated by nitrite therapy 
AMPK is a cellular energy sensor that participates in many 
metabolic processes, and controls liver and skeletal muscle 
insulin receptor-independent glucose uptake.[37] Dysregulation 
of AMPK and its signaling network have been associated both 
with metabolic syndrome and idiopathic PAH.[38-41] Several 
studies have shown that NO donors increase glucose uptake 
and GLUT4 expression via AMPK activation,[42, 43] and our 
group has shown that nitrite increases AMPK activation in 
cardiomyocytes, identifying AMPK as a candidate signaling 
pathway for the effects of nitrate and nitrite on glucose 
homeostasis.[44] As shown in Figure 2, activation of AMPK can 
be mediated by upstream kinases, including liver kinase B1 
(LKB1), 
calcium-sensitive 
calmodulin-dependent 
protein 
kinase 
II 
(CaMKII), 
calcium/calmodulin-dependent protein kinase-beta (CaMKKβ), and TGFβ-activated kinase-1 
(TAK1).[45-47] Recently, sirtuin-3 (SIRT3), a major mitochondrial deacetylase that is 


Study Short Title: Oral Nitrite in Patients with PH-HFpEF 
 
Sponsor: Mark T. Gladwin, MD 
    
PI: Michael Risbano, MD 
STUDY20010014 
Page 10 of 47 
V14.0  02/14/2022  
 
upregulated in skeletal muscle with exercise, has been shown to activate AMPK in skeletal 
muscle and neurons.[48-50] On the basis of such observations, we hypothesize that nitrite, 
similar to metformin, might improve metabolic syndrome via the activation of AMPK signaling 
pathways. In the skeletal muscle, we propose that nitrite and metformin activate mitochondrial 
SIRT3, which in turn promotes the phosphorylation and activation of AMPK and membrane 
translocation of the glucose transporter 4 (GLUT4). Nitrite also directly activates pulmonary 
vascular smooth muscle AMPK via alternative SIRT3-independent pathways that will be 
carefully explored. Systemic improvements in metabolic syndrome and localized effects on 
the pulmonary vasculature suggest that nitrite may be an ideal therapy for PH-HFpEF in the 
setting of metabolic syndrome.  
 
Development of sodium nitrite administration for human use  
A phase 1a and 1b dose escalation study designed to define the maximum tolerated and safe 
doses of inhaled sodium nitrite delivered to normal volunteers following electronic nebulization 
has shown an increase in heart rate (+24 ± 5 bpm) without clinically significant changes in 
systolic (-3 ± 2 mmHg) or diastolic (-3 ± 2 mmHg) blood pressure at the maximal tolerated 
dose (MTD of 125 mg).[51] At the highest delivered dose (176 mg), there was a significant 
decrease in systemic blood pressure and increase in heart rate. Reduction of nitrite to NO 
was demonstrated by an increase in exhaled NO. Methemoglobin levels increased at the 
highest doses administered, but remained less than 3.5% in all subjects. Pharmacokinetic 
modeling demonstrated dose-proportional increases in peak and AUC plasma nitrite. There 
is an ongoing phase 2 study (clinicaltrials.gov NCT01431313) of inhaled nitrite therapy in 
patients with group II PH (PH-HFpEF), Group I PAH, and Group III PH (PH due to lung 
disease), with data suggesting likely efficacy in patients with PH-HFpEF at doses of both 45 
and 90 mg.[52-54] We have also now completed phase 1 safety and PK analyses of a newly 
developed FDA-IND approved oral nitrite preparation in normal volunteers, with and without 
15-nitrogen labeling (both with matching placebos).[55, 56] The oral bioavailability of nitrite is 
excellent (95-98%) and similar to intravenous delivery.[57] An ongoing safety and efficacy trial 
of 3 months of treatment with oral nitrite (20 mg, twice daily) in patients with metabolic 
syndrome (clinical trials.gov NCT01681810) continues to accrue. 
 
1.4 SIGNIFICANCE 
Group II pulmonary hypertension (PH) or post-capillary pulmonary venous hypertension is 
known to occur secondary to left ventricular (LV) systolic or diastolic dysfunction. The latter is 
more commonly referred to as heart failure with preserved ejection fraction (HFpEF) or non-
systolic heart failure, and is the most common cause of Group II PH. With chronic elevations 
in the diastolic filling pressure of the left heart, the pulmonary vasculature can vasoconstrict 
and undergo pathological remodeling and secondary right ventricular (RV) hypertrophy and 
dilation, leading to an elevated pulmonary vascular resistance (PVR) and high transpulmonary 
pressure gradients.[58-61] It is now appreciated that features of the metabolic syndrome, 
including systemic hypertension, obesity, diabetes, and hyperlipidemia, represent risk factors 
for the development of pulmonary vascular disease.  In fact, patients with pulmonary arterial 
hypertension (Group I PAH) exhibit an increased prevalence of glucose intolerance (increased 
hemoglobin A1c levels).[60-64]  Patients with Group II PH-HFpEF commonly present with two 
or more features of the metabolic syndrome.[63]  Mouse models of metabolic syndrome, such 
as the apo E knock-out mice fed a high fat diet, have been shown to develop pulmonary 
hypertension.[65] There are no currently approved therapies for PH-HFpEF and most 
medications approved for Group I PAH have been shown to be ineffective in patients with left 
heart disease.  New therapies that target both the metabolic syndrome and the pulmonary 
vasculature are being tested in pre-clinical models and in patients, including peroxisome 
proliferator-activated 
receptor-γ 
activators, 
and, 
in 
our 
studies, 
metformin 
and 


Study Short Title: Oral Nitrite in Patients with PH-HFpEF 
 
Sponsor: Mark T. Gladwin, MD 
    
PI: Michael Risbano, MD 
STUDY20010014 
Page 11 of 47 
V14.0  02/14/2022  
 
nitrate/nitrite.[18, 65-68]  To date, there are no established animal models of PH-HFpEF 
associated with metabolic syndrome and no specific therapy for patients with PH-HFpEF.   
 
 2.   RESEARCH DESIGN AND METHODS 
2.1 CLASSIFICATION AND METHODOLOGICAL DESIGNS 
This is a single-center, 22-week, double-blind, randomized, placebo-controlled cross-over 
trial of oral nitrite on exercise capacity and hemodynamic measurements in subjects with 
PH-HFpEF. 
 
2.2 DETAILED DESCRIPTION OF STUDY DESIGN 
Potential subjects will undergo screening visit(s) to determine that eligibility requirements are 
met. An echocardiogram, bloodwork, and screening right heart catheterization hemodynamic 
measurements will determine subject’s eligibility.  Subjects who meet all the inclusion criteria, 
and none of the exclusion criteria, will be included in the study. Once eligibility is confirmed, 
subjects will complete a series of baseline procedures prior to receiving study drug. The study 
population (n=26) will receive either oral sodium nitrite or matching placebo as follows: 
 
Drug:  The study drugs are oral formulation of sodium nitrite and matching placebo. Sodium 
nitrite is available in dose strengths of 20 mg and 40 mg. Sodium nitrite of both dose 
strengths and matching placebo will be supplied as capsules for oral administration. All 
capsule formulations will be identical in appearance (size, shape, color) and smell. The 
packaging and labeling will be designed to maintain blinding to the Investigator’s team and 
to subjects. Oral formulation of sodium nitrite, total dose strength of 40 mg or placebo three 
times daily or TID (once in the morning and again in the early afternoon and again in the 
evening). 
 
During the treatment period, subjects will undergo periodic clinical evaluations and weekly 
follow-up phone calls in between clinic visits.  Throughout the study period, subjects will be 
asked to remain diligent in recording in a diary the date and time of study drug dosing and any 
associated symptoms. 
 
2.2.1. Study Drug Preparation and Distribution 
Sodium nitrite are prepared at Triangle Compounding Pharmacy, Inc. (Cary, NC 27518) and 
matching placebos were prepared at NIH pharmacy. The labeling and packaging will be 
conducted according to Good Clinical Practice and regulatory requirements. The University of 
Pittsburgh Medical Center Investigational Drug Pharmacy Service (UPMC-IDS) will be utilized 
for dispensing of the study drugs (nitrite and placebo). The study drug is administered only to 
subjects enrolled in the study and in accordance with the protocol. 
 
Sodium nitrite  
Standard sodium nitrite will be supplied as capsules for three times daily oral administration 
with a capsule dose strength of 20 mg or 40 mg. Total dose strength will be 40mg TID. 
 
Matching placebo 
Placebo will be supplied as matching capsules for three times daily oral administration. 
 


Study Short Title: Oral Nitrite in Patients with PH-HFpEF 
 
Sponsor: Mark T. Gladwin, MD 
    
PI: Michael Risbano, MD 
STUDY20010014 
Page 12 of 47 
V14.0  02/14/2022  
 
2.2.2. Treatment Period 
Subjects will undergo study treatment duration of 10 weeks of sodium nitrite three times daily 
or matching placebo. This is considered appropriate to demonstrate the potential effects of 
nitrite in adults with PH-HFpEF.  Upon completion of the 1st 10 weeks plus a 2-week wash-
out period, the subjects will cross-over to receive the alternative therapy (nitrite or matching 
placebo) and undergo the same study procedures as noted for the 1st 10 weeks. Subjects will 
be given a diary to monitor study drug administration and will be asked to return the diary at 
each clinic visit to monitor compliance.  Upon completion of the cross-over portion of the trial, 
subjects will be given the option to receive open-label drug for 6 months.   
 
2.2.3. Safety Monitoring: 
Weekly phone follow-up (+/- 7 days) assessments will occur while on active study treatment 
(when subjects are not followed up in clinic) that will include symptom review, assessment of 
adverse events, review of concomitant medications, and interval histories. Subjects may 
return to clinic for outpatient assessments at weeks 4 and 8 (both Arm 1 and Arm 2) if the PI 
deems it necessary to bring the patient in for an evaluation. No weekly phone follow up may 
be necessary if subjects are evaluated in clinic.  Clinical visits are completed in the outpatient 
clinics at UPMC Presbyterian outpatient cardiology and/or Falk Clinic, Comprehensive Lung 
Center, or Translational Research Center (TRC).  The in-person clinical visits include a brief 
physical exam, NYHA functional class assessment, vital signs including oxygen and 
methemoglobin levels via co-oximetry, NTproBNP lab, 6MWT and Borg scale, interval 
histories, medication compliance review, AE assessment/symptom review, and dispensing of 
study drug.   
 
In addition to these formal evaluations, subjects will be encouraged to immediately contact 
the study investigator and/or the study coordinator with questions, concerns, or to report new 
symptoms that occur during their study participation. If there are particular concerns that need 
to be addressed sooner than the in-person visits, the subject will be asked to return to the 
clinic as soon as possible for evaluation. If appropriate, based upon the evaluation, medical 
treatments will be provided to subjects, including appropriate referral to physicians or other 
services at the UPMC.  
 
2.2.4. Medication Compliance 
Randomization to study drug verses placebo will occur after eligibility has been confirmed by 
screening RHC and baseline procedures are completed. Randomization of sequence of 
nitrite-placebo versus placebo-nitrite followed by cross-over to the alternative treatment in a 
1:1 ratio according to a computer generated pseudo-random code. 
 
Subjects will self-administer their first dose of study medication or placebo under supervision 
of the physician investigator or the primary study coordinator after eligibility has been 
confirmed.   
 
Subjects will also be dispensed their study medication for home use and a diary to track drug 
administration. Subjects will return the previously dispensed study medication and diary at 
every study visit thereafter, and medication compliance will be assessed 
 
The study investigator and/or the study coordinator will review the daily diary. In the event that 
the compliance rate is < 80%, subjects will be re-educated on medication compliance. If 
medication compliance falls outside of the acceptable range more than 2 times, the study 
investigators will discuss subject eligibility for continued participation in the study.  
 


Study Short Title: Oral Nitrite in Patients with PH-HFpEF 
 
Sponsor: Mark T. Gladwin, MD 
    
PI: Michael Risbano, MD 
STUDY20010014 
Page 13 of 47 
V14.0  02/14/2022  
 
2.2.5. Medication Accountability 
The study investigators or the study coordinator will document the amount of study drug 
dispensed from UPMC-IDS. The study drug accountability records will be maintained at 
UPMC-IDS throughout the course of the clinical trial. 
 
2.3 STUDY ASSESSMENT 
      The complete study assessment and procedures are outlined in Table 1.  
Subjects will undergo study procedures and screening RHC to confirm that eligibility criteria 
have been met.  The RHC procedure may be performed on a separate day.  If a RHC has 
been completed within 12 months from enrollment, test results may be considered for baseline 
measurements. 
 
2.3.1. Screening Procedures  
The outpatient visits will take place in various outpatient clinics and/or procedural areas 
throughout the University of Pittsburgh Medical Center (UPMC) Presbyterian and UPMC 
Montefiore Hospitals.  The screening procedures will take place over 2 days if screening RHC 
is needed. Subjects will be asked to fast a minimum of 8 hours prior to each visit. These 
screening procedures will determine eligibility for the study.  
 
• 
Obtain written informed consent 
• 
Complete history and physical examination to include body weight, height  
• 
Vital signs that include: blood pressure (BP), pulse, respirations, and pulse oximetry 
• 
Obtain waist measurement 
• 
Review of concomitant medications 
• 
NYHA functional class assessment 
• 
6 minute walk test (6MWT) and Borg scale 
• 
MetHb level via co-oximetry (pre single dose and 30 minutes post single dose) . Due 
to unknown reasons the co-oximeter device may not be able to read metHb levels. In 
that event, a blood sample will be collected to test metHb levels at approximately 30-
45 minutes after the 40mg dose. 
• 
Blood samples: fasting glucose, comprehensive metabolic panel, hemoglobin A1C, 
fasting lipid profile, PT/PTT, complete blood count, platelet and differential, NT-
proBNP. The total volume of blood drawn is approximately 1 ½ tablespoons (20 mL). 
If any of the above tests were done within 30 days before the screening visit, they 
may or may not be repeated for research purposes.  
• 
Urine pregnancy test for women of childbearing potential 
• 
Echocardiogram (if subject has had an echocardiogram within the past year we may 
not obtain during screening) 
• 
Right heart catheterization (with optional micromanometry), research blood samples 
for plasma nitrite concentrations, red cell iron-nitrosyl-hemoglobin and platelet studies, 
and open label 40mg oral nitrite dosing with methemoglobin level. Research blood 
samples will be collected prior to taking the 40mg oral nitrite and approximately 
30-45 minutes post-dose. 
• 
A RHC may be performed to determine the PH-HFpEF eligibility criteria. The RHC will 
be conducted at UPMC Presbyterian Hospital, 3rd FL Catheterization Laboratory. The 
RHC hemodynamic measurement parameters for PH-HFpEF eligibility are as follows: 
mPAP ≥ 25 mm Hg; PCWP ≥15 and TPG ≥ 12. If a RHC has been completed within 
12 months from enrollment, test results may be considered for baseline 
measurements. The total volume of blood drawn is approximately 1 ¾ tablespoons 


Study Short Title: Oral Nitrite in Patients with PH-HFpEF 
 
Sponsor: Mark T. Gladwin, MD 
    
PI: Michael Risbano, MD 
STUDY20010014 
Page 14 of 47 
V14.0  02/14/2022  
 
(26mL). Subjects who report using PDE-5 inhibitor medications will be asked to hold 
the medication 5 days before the screening RHC and throughout the study. 
 
2.3.2. Baseline Procedures  
After eligibility has been confirmed, the following procedures will take place in various   
outpatient clinics and/or procedural areas throughout the University of Pittsburgh Medical 
Center (UPMC) Presbyterian Hospital, UPMC Montefiore Hospital, Kaufmann Building, 
and/or Falk Clinic.  Subjects will need to fast a minimum of 2 hours prior to these visits. 
These procedures will be completed over 2-3 days. 
• 
Cardiopulmonary exercise test (CPET): Incremental exercise testing (IET), a practice 
constant work rate (CWRP) test, and baseline constant workrate (CWRB) test, 
Spirometry- Forced vital capacity (FVC) maneuver only 
• 
Accelerometer – activity monitor to be worn for about 7-10 days 
• 
Completion of The Kansas City Cardiomyopathy Questionnaire (KCCQ), 36-Item 
Short Form Health Survey (SF-36), and Pulmonary Arterial Hypertension – Symptoms 
and Impact Questionnaire (PAH-SYMPACT).  
 
A physical exam with NYHA functional class assessment may be performed and this will be 
completed on a case by case basis at the discretion of the study physician. 
Subjects who report using PDE-5 inhibitor medications and do not need a screening RHC will 
be asked to hold the medication 5 days before the initial study drug or placebo dose and 
throughout the study. Patients who are fatigued or short of breath after stopping PDE5i, if 
specifically done for the study, will be evaluated and if it is felt to be related to stopping PDE5i 
will be restarted and they will continue in the trial with monitoring as per the trial. 
 
Randomization 
Randomization to study drug verses placebo will occur upon completion of all screening and 
baseline procedures and confirmation by a study physician that subjects have met all 
eligibility criteria.  Randomization of sequence of nitrite-placebo versus placebo-nitrite 
followed by cross-over to the alternative treatment in a 1:1 ratio according to a computer 
generated pseudo-random code.   
Subjects will self-administer their first dose of study medication or placebo under supervision 
of the physician investigator or the primary study coordinator after eligibility has been 
confirmed. Each patient will have their vital signs and methemoglobin measured before taking 
the study medication and approximately 30-45 minutes after taking their first dose.   
 
Subjects will also be dispensed their study medication for home use including a medication 
administration diary. Subjects will return the previously dispensed study medication and diary 
at every study visit thereafter, and medication compliance will be assessed.   
 
2.3.3. Treatment Period and Monitoring  
The follow up visits will take place in various outpatient clinics and/or procedural areas 
throughout the University of Pittsburgh Medical Center (UPMC) Presbyterian Hospital, UPMC 
Montefiore Hospital, Kaufmann Building, and/or Falk Clinic. The intravenous glucose 
tolerance test (IVGTT) will be performed at the Montefiore Clinical and Translational Research 
Center (CTRC).  
 
Phone Visits: Weeks 4 and 8 (+/- 7 Days) following the start of each treatment arm 


Study Short Title: Oral Nitrite in Patients with PH-HFpEF 
 
Sponsor: Mark T. Gladwin, MD 
    
PI: Michael Risbano, MD 
STUDY20010014 
Page 15 of 47 
V14.0  02/14/2022  
 
 
• Interval history (ER visits/hospitalizations) 
• Review of concomitant medications 
• Review for compliance and symptoms 
• AE/SAE assessment 
 
***If the PI deems it necessary to bring a participant to clinic, the following will be 
performed in addition to the items listed under the Phone visits: 
These visits may take up to 2 hours to complete.  Subjects will be instructed to hold the 
morning or afternoon dose of study drug depending on the appointment time. 
• 
Self-administration of drug by subject under direct supervision of study team 
• 
Interval history  
• 
Physical examination including vital signs, pulse co-oximetry, and body weight 
• 
Methemoglobin percentage level via co-oximetry (approximately 30-45 minutes post study 
medication) 
• 
Review of concomitant medications 
• 
Collection of used study drug and diary; review for compliancy and symptoms 
• 
Functional Class assessment 
• 
NT-proBNP blood sample. The total volume of blood drawn for this sample is 
approximately 1 teaspoon. 
• 
6MWT and Borg scale 
• 
Dispense study drug and new diary cards (if needed)  
• 
AE/SAE assessment 
 
Clinic Visit Week 10 (+/- 14  and End of Treatment (+/- 14) to begin testing visits) 
 
These visits may take  up to 4 days to complete all the assessments. Subjects e instructed to 
arrive in a fasted state and hold study medication the morning of these scheduled visits. 
Participants will take their morning or afternoon dose under direct supervision of the study 
team and will continue to take the study medication TID until all testing is completed. 
 
• 
Self-administration of drug by subject under direct supervision of study team at the start 
of each visit 
• 
Interval history  
• 
Physical examination including vital signs, pulse co-oximetry, and body weight 
• 
Waist measurement 
• 
Methemoglobin percentage level via co-oximetry (approximately 30-45 minutes post study 
medication) 
• 
Review of concomitant medications 
• 
Collection of used study drug and diary; review for compliancy and symptoms 
• 
Functional Class assessment 
• 
6MWT and Borg scale 
• 
Accelerometer 
• 
Urine pregnancy if applicable 
• 
Echocardiogram 


Study Short Title: Oral Nitrite in Patients with PH-HFpEF 
 
Sponsor: Mark T. Gladwin, MD 
    
PI: Michael Risbano, MD 
STUDY20010014 
Page 16 of 47 
V14.0  02/14/2022  
 
• 
Blood samples: fasting glucose, comprehensive metabolic panel, hemoglobin A1C, fasting 
lipid profile, PT/PTT, complete blood count, platelet and differential, and NT-proBNP. The 
total volume of blood drawn for these samples is approximately 1 ½  tablespoons (20 mL). 
• 
Cardiopulmonary exercise test (CPET): Constant Work Rate Test 
• 
RHC: Hemodynamic measurements will be completed at rest. Once the resting 
measurements are completed, the patient will take a single dose of their randomly 
assigned study medication. Another set of hemodynamic measurements will be taken 
approximately 30-45 minutes after the study medication is taken. Following these 
measurements, the patient will complete a constant workrate supine exercise test lasting 
about 4-12 minutes.  Patients will pedal until exhaustion. The information collected from 
the baseline constant workrate exercise test during the baseline procedures will determine 
the watts used for the constant workrate supine exercise tests in the catheterization lab 
during weeks 10 and EOT. Hemodynamic measurements will be taken about every 2 
minutes during the exercise test.   
• 
Micromanometry (optional) during RHC 
• 
Research blood samples (plasma nitrite concentrations, red cell iron-nitrosyl-hemoglobin, 
and platelet studies) during RHC: pre-dose and approximately 30-45 minutes post dosing 
of study drug.  The total volume of blood drawn is approximately 1 ¾ tablespoons (26 mL). 
• 
AE/SAE assessment 
• 
IVGTT (optional) 
• 
Completion of the KCCQ, SF-36 and PAH-SYMPACT Questionnaires 
• 
Once all of the testing is completed, subjects will be given a diary card to keep track of 
any symptoms during the 2-week washout period and again after the EOT testing for the 
30 day follow up phone call. 
 
2-Week Washout Period (+ 7 days) 
Upon completion of the first 10 weeks of study treatment, a 2-week washout period will 
occur.  Subjects will not receive the study drug or placebo for two (2) weeks.  Subjects will 
be given a diary card to keep track of any symptoms, doctor appointments, etc. during this 
time period. Subjects will return to clinic after the washout to cross over to alternative 
therapy and undergo the same study procedures as the first 10 weeks. 
Cross-Over Visit  
This visit may be completed over 2 days dependent on patient and staff availability.  Subjects 
will be instructed to fast for a minimum of 8 hours on the day of the bloodwork. Patients will 
only need to fast for 2 hours prior to the constant work rate exercise test.  
 
• 
Self-administration of drug by subject under direct supervision of study team 
• 
Interval history  
• 
Collection of diary; review for compliancy and symptoms 
• 
Physical examination including vital signs, pulse co-oximetry, and body weight 
• 
Urine pregnancy if applicable 
• 
Methemoglobin percentage level via co-oximetry (approximately 30-45 minutes post study 
medication) 
• 
Review of concomitant medications 
• 
Functional Class assessment 
• 
6MWT and Borg scale 
• 
Constant work rate exercise test 
• 
Accelerometer 


Study Short Title: Oral Nitrite in Patients with PH-HFpEF 
 
Sponsor: Mark T. Gladwin, MD 
    
PI: Michael Risbano, MD 
STUDY20010014 
Page 17 of 47 
V14.0  02/14/2022  
 
• 
Blood samples: fasting glucose, comprehensive metabolic panel, hemoglobin A1C, fasting 
lipid profile, PT/PTT, complete blood count, platelet and differential, NT-proBNP, research 
blood samples: plasma nitrite concentration, red cell iron-nitrosyl-hemoglobin, and platelet 
studies. The total volume of blood drawn for these samples is approximately 2 ½  
tablespoons (35 mL). 
• 
Dispense study drug and diary cards  
• 
AE/SAE assessment 
• 
Completion of the KCCQ, SF-36 and PAH-SYMPACT Questionnaires 
 
Weekly phone follow-up (+/- 7 days) assessments will occur while on active study treatment 
(when subjects are not followed up in clinic) that will include symptom review, assessment of 
adverse events, review of concomitmant medications, and interval histories.  Subjects will return 
to clinic at week 10 and undergo study procedures as noted in the first 10 weeks. 
2.3.4. End of Trial and Follow-Up Period  
Post Study Drug Telephone Assessment (approximately 30 days after the final day of testing) 
• 
Telephone assessment for interval histories and AEs. 
 
2.3.5.   Open Label Period 
Upon completion of the end of treatment visit, subjects will be given the option to receive open-
label drug for 6 months.  Subjects will receive open-label of Oral Nitrite (NO2
−). Subjects may be 
asked to return monthly for blood pressure, methemoglobin checks (optional). Oral nitrite will be 
dispensed at each of these visits. Patients will be asked to return in-person for month 3 and 
month 6 of the open label period, and will be asked to complete 3 questionnaires (KCCQ, SF-
36, and PAH-SYMPACT), perform a six-minute walk test, and have a blood collection to 
measure NT-proBNP level. The total amount of blood drawn is 4ml.  
 
Currently HFpEF is a disease with no approved treatments. The main purpose of the open label 
period is to provide specific treatment to those patients that have a response during the 
crossover trial. This can also be a benefit to the patients if they are getting relief from their 
symptoms from the oral nitrite. It will also provide longer term safety data. Patient reported 
outcomes such as QOL are collected. 
 
 
 


Study Short Title: Oral Nitrite in Patients with PH-HFpEF 
 
Sponsor: Mark T. Gladwin, MD 
    
PI: Michael Risbano, MD 
STUDY20010014 
Page 18 of 47 
V14.0  02/14/2022  
 
2.3.6. Schedule of Assessments  
The table below summarizes the protocol procedures that will be performed throughout the study trial 
TABLE 1 
Study Phase 
Screening 
Baseline 
Weekly  
Phone 
Call 
+/-  7 
days 
Weeks 
4 and 8 
(per PI 
only) 
+/- 7 
days 
Week 10 
 
+/-14 
days  
Cross-
over 
Visit 
Weekly  
Phone 
Call 
+/-  7 
days 
Weeks  
4 and 8 
(per 
PI 
only) 
+/- 7 
days 
End of 
Treatment 
(EOT) 
Follow-up 
Period Phone 
Call 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
I/E Criteria 
X 
 
 
 
 
 
 
 
 
 
Medical History & 
Demographics 
X 
 
 
 
 
 
 
 
 
 
Physical Exam 
X 
 
 
X 
X 
X 
 
X 
X 
 
Vital Signs & Body 
Weight 
X 
X 
 
X 
X 
X 
 
X 
X 
 
Oxygen Saturation 
X 
 
 
X 
X 
X 
 
X 
X 
 
Blood Samples: fasting 
glucose,CMP,hemoglo
bin A1C, fasting lipid 
profile, PT/PTT, 
complete blood count, 
platelet and differential, 
research labs 
X 
 
 
 
X 
X 
 
 
X 
 
α NTproBNP blood test  
X 
 
 
X 
X 
X 
 
X 
X 
 
Urine Pregnancy test (if 
applicable) 
X 
 
 
 
X 
X 
 
 
X 
 
NYHA functional class 
assessment 
X 
 
 
X 
X 
X 
 
X 
X 
 
α 6 minute walk test 
(6MWT)  
X 
 
 
X 
X 
X 
 
X 
X 
 
Echocardiogram 
X∞ 
 
 
 
X 
 
 
 
X 
 
▪ Spirometry lung 
function test  
 
X 
 
 
 
 
 
 
 
 
Incremental 
exercise 
testing (IET) 
 
X 
 
 
 
 
 
 
 
 
Practice Constant Work 
Rate (CWRP) 
 
X 
 
 
 
 
 
 
 
 
Constant Work Rate 
(CWR) test 
 
X 
 
 
X 
X 
 
 
X 
 
(RHC): RA, RV, PA, 
PCWP, CO/CI 
Resting hemodynamics 
and (sub-maximal 
exercise at Week 10 
and EOT only) 
Xꜛ 
 
 
 
X 
 
 
 
X 
 
נּ Micromanometry 
(optional) 
X 
 
 
 
X 
 
 
 
X 
 
Accelerometer 
 
X 
 
 
X 
X 
 
 
X 
 
תּ IVGTT 
 
 
 
 
X 
 
 
 
X 
 
MetHgb levels via co-
oximetry 
X 
X 
 
X 
X 
X 
 
X 
X 
 
Randomization 
 
X 
 
 
 
 
 
 
 
 
Dispense and study 
drug administration 
 
X 
 
X 
X 
X 
 
X 
If patient wants to 
continue open 
label 
 
Study drug compliancy 
assessment 
 
 
 
X 
X 
 
 
X 
X 
 
Dispense subject diary 
 
X 
 
X 
X 
X 
 
X 
X 
 
α KCCQ, SF-36 and 
PAH-SYMPACT 
questionnaires  
 
X 
 
 
X 
X 
 
 
X 
 
AE Assessment 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
Review of concomitant 
medications 
X 
 
X 
X 
X 
X 
X 
X 
X 
 
Interval History 
Assessment 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
 


Study Short Title: Oral Nitrite in Patients with PH-HFpEF 
 
Sponsor: Mark T. Gladwin, MD 
    
PI: Michael Risbano, MD 
STUDY20010014 
Page 19 of 47 
V14.0  02/14/2022  
 
∞ If subject has had an echocardiogram within the past year, we may not obtain during screening. 
▪ Spirometry lung function test may not be needed at baseline visit if results are available within 6 months from enrollment. 
ꜛ A screening RHC may not be performed if a RHC was completed within 12 months from enrollment per PI discretion. 
נּ Micromanometry is optional and completed during RHC. 
תּ Insulin-dependent diabetics will not receive the IVGTT. 
α At month 3 and month 6 of open label period, subjects will complete three questionnaires, six-minute walk test and NT-proBNP blood 
draw. 
 
2.4 DESCRIPTION OF STUDY PROCEDURES 
 
2.4.1 Echocardiogram 
Transthoracic echocardiograms will be completed to assess RV function by measures such 
as TAPSE as well as to assess the Doppler waveform of the pulmonary artery.  This is a 
noninvasive test. 
2.4.2 Six Minute Walk Test (6MWT and Borg Scale) 
The six-minute walk test (6MWT) will be conducted in accordance with local standard 
operating procedures. The 6MWT measures the distance an individual is able to walk over a 
total of six minutes on a hard, flat surface.  The subject is allowed to self-pace and rest as 
needed as they traverse back and forth along a marked walkway.   The test is used as a 
performance-based measure of functional exercise capacity.  It evaluates the global and 
integrated responses of all the systems involved during exercise, including the pulmonary and 
cardiovascular systems, systemic circulation, peripheral circulation, blood, neuromuscular 
units, and muscle metabolism. The self-paced 6MWT assesses the submaximal level of 
functional capacity. Most subjects do not achieve maximal exercise capacity during the 
6MWT; instead, they choose their own intensity of exercise and are allowed to stop and rest 
during the test. However, because most activities of daily living are performed at submaximal 
levels of exertion, the 6MWT may best reflect the functional exercise level for daily physical 
activities. 
 
The Borg Scale is a method of rating perceived exertion during exercise and will be used while 
the subject performs a 6MWT.  
 
2.4.3 Accelerometer 
Physical activity: The SenseWear Pro Armband Accelerometer (BodyMedia, Pittsburgh, PA 
and/or Actigraph, Pensacola, FL) multi-sensor device will be used to provide an objective 
measure of physical activity for about 7-10 days. 
 
2.4.4 Spirometry 
Spirometry testing at baseline will consist of the Forced Vital Capacity (FVC) maneuver. A 
trained pulmonary function technician will administer the pulmonary function testing. The FVC 
is the maximal volume of air exhaled with a maximally forced effort from a maximal inspiration. 
The FVC maneuver will be performed using the Easy on-PC (NDD) with the subject in a seated 
position. Within the FVC maneuver, the maximal volume of air exhaled in the first second of 
a forced expiration (FEV1) will be obtained as well. The best of three efforts will be defined as 
the highest FEV1 and the highest FVC, each obtained on any of three efforts that meet the 
American Thoracic Society (ATS) guidelines for acceptability and repeatability criteria (with a 
maximum of eight attempts). The highest FEV1 and FVC will be selected regardless of 
whether they come from different spirometric maneuvers or from the same maneuver.  
 
If the ratio of FEV1/FVC is less than 0.70, then salbutamol (albuterol) will be administered. 
After a gentle and incomplete expiration, a dose of 100 μg of salbutamol (albuterol) is inhaled 


Study Short Title: Oral Nitrite in Patients with PH-HFpEF 
 
Sponsor: Mark T. Gladwin, MD 
    
PI: Michael Risbano, MD 
STUDY20010014 
Page 20 of 47 
V14.0  02/14/2022  
 
in one breath to total lung capacity. The breath is inhaled for 10 seconds before the subject 
exhales. Four separate doses (total dose 400μg) are delivered at approximately one minute 
intervals. Three additional, acceptable post-bronchodilator FVC manoeuver tests will be 
recorded 15 to 30 minutes after the last dose of salbutamol (albuterol) is inhaled. 
  
2.4.5 Cardiopulmonary Exercise Testing (CPET) and Gas Exchange Measurements: 
CPET will be performed using an electromagnetically braked cycle ergometer (Ergoline 800, 
SensorMedics) and the gas exchange measurements will be analyzed using the Viasys Vmax 
Encore metabolic cart system. 
CPET at baseline will consist of an incremental exercise test (IET) and a constant work rate 
test for practice (CWRP). The CWRP test will determine the constant workrate (CWR) that will 
be used at baseline, Week 10, crossover, and EOT. 
 
Determining Workload (WL) and CWR at Baseline 
• 
IET:The work rate will increase in a stepwise manner, using an individualized incremental 
WL (based on age, height, weight, and gender) to achieve a targeted duration of 4 to 12 
minutes to volitional exhaustion. The incremental WL will be determined to the nearest 
multiple of 5 watts [69].  
 
• 
CWRP: A resting period of at least 2 hours will be scheduled between exercise tests. 
The CWR is a set work rate that remains unchanged throughout the duration of the test.  
The CWRP will be performed around 70-75% of the maximal WL from the IET [70, 71]. 
Further adjustment in CWR will be based on the exercise endurance time (ET) of CWRP 
to optimize CWR at baseline, Week 10, crossover, and EOT to achieve a target duration 
of 4 to 12 minutes. ET will be measured from the start of loaded pedaling to volitional 
exhaustion and the WL has returned to unloaded pedaling. If a patient is unable to reach 
4 minutes, the patient may be asked to return on a separate day to repeat the CWRP to 
adjust to a workload the patient can endure for at least 4 minutes. The second test will be 
optional.  
 
CPET for baseline, Week 10, crossover, and EOT 
The CWR determined from CWRP will be used for baseline, Week 10, crossover, and EOT. 
ET will be measured from the start of loaded pedaling to volitional exhaustion and the WL has 
returned to unloaded pedaling. The endurance exercise challenge will be used to compare 
oral nitrite to placebo.  
 
CPET will measure the integrated response of the pulmonary mechanics, gas exchange, 
cardio/pulmonary vascular, peripheral muscle systems, and the subject’s perceived exertion. 
For each subject, isotime measurements of cardiopulmonary metabolic responses will be 
compared over the minimum ET of Week 10 and EOT.  Data will be reported on an exercise 
test form. 
A detailed description of the CPET procedures is provided by the CPET lab. 
Emergency equipment including a defibrillator will be readily available. 
2.4.6 Laboratory Testing 
The following peripheral blood samples will be evaluated at the screening/baseline, week 10, 
cross-over, and end of treatment visits for follow-up comparison purposes: fasting glucose, 
comprehensive metabolic panel, hemoglobin A1C, fasting lipid profile, PT/PTT, complete 
blood count, platelet and differential, NT-proBNP, and research blood samples for plasma 


Study Short Title: Oral Nitrite in Patients with PH-HFpEF 
 
Sponsor: Mark T. Gladwin, MD 
    
PI: Michael Risbano, MD 
STUDY20010014 
Page 21 of 47 
V14.0  02/14/2022  
 
nitrite concentrations, red cell iron-nitrosyl-hemoglobin, and platelet studies.  Urine pregnancy 
test will be performed on females of child-bearing potential at baseline, week 10, cross-over 
visit, and at the end of study treatment.  The samples will be collected and analyzed at the 
UPMC Presbyterian laboratories. The NT-proBNP blood sample will be sent to the 
Translational Research Core Laboratory (TRCL) for storage and analysis. Research samples 
will be sent to Shiva/Gladwin labs in Vascular Medicine Institute (VMI) for storage and 
analysis. 
2.4.6.1 Research Laboratory Profiles 
Blood samples for platelet mitochondrial analysis, plasma nitrite concentrations and red cell 
iron-nitrosyl-hemoglobin may be collected at screening/baseline, Week 10, crossover, and 
EOT visits. These de-identified blood samples will be sent to the Shiva/Gladwin labs in the 
Vascular Medicine Institute for storage and analysis under the supervision of Drs. Gladwin, 
Risbano,and Shiva.  
2.4.7 Methemoglobin Levels 
The Masimo Rainbow SET® CO-Oximeter device that will be used in this clinical trial has been 
validated to provide accurate determinations of methemoglobin levels under conditions of 
hypoxia and methemoglobinemia. Measurements will be obtained before initial dose and 
approximately 30-45 minutes post initial dose and approximately 30-45 minutes after the dose 
is given at the clinical visits. Due to unknown reasons the co-oximeter device may not be able 
to read metHb levels. In that event, a blood sample will be collected to test metHb levels. 
  
2.4.8 Right Heart Catheterization, Simultaneous Pressure and Doppler Flow Velocity 
Measurement, and Exercise RHC 
Right heart catheterization (RHC) is performed routinely as a clinical standard of care 
procedure for diagnostic purposes in this disease population. The standard right heart 
catheterization is performed with a balloon tipped, flow-directed pulmonary artery catheter 
(Swan-Ganz catheter), inserted through a sheath in the internal jugular vein. As per standard 
clinical protocol, hemodynamic recordings of right atrial, right ventricular, pulmonary artery 
pressures, pulmonary capillary wedge pressure, in addition to cardiac output, are made at 
baseline.    
RHC may be completed at screening for eligibility purposes, week 10, and end of treatment 
visits.  Resting and supine constant workrate exercise RHC measurements will be performed 
at week 10 and end of treatment. Hemodynamic measurements may be obtained every 2 
minutes during the exercise test.   
Subjects may also undergo additional hemodynamic measurements using a micromanometer 
pressure catheter that may be inserted via the Swann-Ganz catheter distal port.  
Measurements with micromanometer and/or Swann-Ganz catheter may be made with 
simultaneous flow velocity from transthoracic echocardiographic doppler ultrasound at resting 
and after the exercise portion of RHC. This portion of the study that involves the 
micromanometer instrumentation is expected to add an additional 5-10 minutes onto the 
procedure at each time point. 
Cardiac output (CO) is the amount of blood pumped through the left ventricle of the heart in 
a time interval of one minute.  There are a number of ways to calculate CO.  Thermodilution 
and/or Fick cardiac index are two means that will be used for measuring CO for this 
protocol.  The CO will be the average of the recorded CO collected.    


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Cardiac index (CI) is a measurement that relates the CO from the left ventricle to body 
surface area (BSA), therefore determining cardiac function to size of the individual.  CI is 
calculated by CO/BSA. 
Pulmonary capillary wedge pressure (PCWP) provides an indirect estimate of left atrial 
pressure (LAP). PCWP is measured by the balloon-tipped, multi-lumen catheter (Swan-
Ganz catheter) while it is in a branch of the pulmonary artery. Just behind the tip of the 
catheter is a small balloon that can be inflated with air (~1 cc). The catheter has one 
opening (port) at the tip (distal to the balloon) and a second port several centimeters 
proximal to the balloon. These ports are connected to pressure transducers. When properly 
positioned in a branch of the pulmonary artery, the distal port measures pulmonary artery 
pressure and the proximal port measures right atrial pressure. The balloon is then inflated, 
which occludes the branch of the pulmonary artery. When this occurs, the pressure in the 
distal port rapidly falls, and after several seconds, reaches a stable lower value that is very 
similar to left atrial pressure (normally about 8-10 mmHg). The balloon is then deflated and 
pulmonary artery pressure can again be recorded. The same catheter can be used to 
measure cardiac output. Anticoagulants would need to be held prior to the right heart 
catheterization as this is standard clinically for that procedure. Antiplatelets and beta 
blockers are continued, also clinical standard of care. We will be holding only the morning 
dose of oral hypoglycemics for all non-insulin dependent diabetics until they complete the 
study later that morning - this is also standard clinically for this procedure. Holding diuretics 
along with potassium may also occur as this is often done prior to RHC but will be 
addressed on a case by case basis by study investigators. 
 
2.4.9 Intravenous Glucose Tolerance Test (IVGTT) 
This visit will take place in the Montefiore CTRC.  This test will occur at least 48 hours after 
the last exercise bout to account for the effects of acute exercise on insulin resistance. 
Subjects will be asked not to drink or eat anything other than plain water for 8 hours prior to 
their arrival for this visit.  
Both glucose and insulin will be injected intravenously into the arm. Blood samples will be 
taken over the course of 3.5 hours, totaling about 15 1/2 tablespoons. 
 
The IVGTT is considered a gold-standard test to measure insulin secretion and sensitivity. It 
has been standardized and used worldwide for more than 20 years. In our study, the 
expected duration is 180 mins after the test is started with an IV glucose bolus of 0.3g/kg. 
After the glucose bolus, a small dose of human regular insulin is given IV to moderate the 
plasma glucose rise. The insulin dose is 25 mUnits/Kg body weight. Patients taking oral 
hypoglycemics may be asked to hold those medications the morning of the IVGTT but can 
resume taking them following the completion of the IVGTT the same day.  
 
2.4.10 Self Administered Health Questionnaires 
The Kansas City Cardiomyopathy Questionnaire (KCCQ) helps quantify physical function, 
symptoms, social function, self-efficacy and knowledge and quality of life.  The 36-Item 
Short Form Health Survey (SF-36) measures physical, mental and general health.  The 
Pulmonary Arterial Hypertension – Symptoms and Impact Questionnaire aims to assess 
pulmonary hypertension symptoms and their impact on quality of life.  
 
2.5 SPECIMEN COLLECTION AND MANAGEMENT 
 


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2.5.1 Specimen Handling and Labeling (De-Identification) 
Specimens collected will be properly labeled. All research biological specimens and all 
records associated with the samples will be labeled only with a unique code that contains no 
personal identifiers. The information linking these code numbers to the corresponding 
subject’s identity will be kept in a secure location in the investigator’s office, and will not be 
available to staff managing samples at the research laboratories.  
Immediately upon receipt of the biological specimens, all attempts will be made to process, 
isolate, collect, and store the specimens. The code number and date on which the specimen 
is frozen, all other information about the specimen, and subsequent processing will be entered 
on the specimen processing worksheet. 
2.5.2 Specimen Management and Storage 
Specimens in excess of immediate assay requirements may be stored indefinitely in a locked 
freezer under the control of the principal investigator. Subject’s de-identified biological 
samples and data will be stored for future research analyses and may be shared with other 
researchers’, their laboratories or federal repositories. Samples will be sent to the Lai lab at 
Indiana University, Indianapolis for further analysis. Future sharing outside the institution will 
be done under an approved data or specimen sharing agreement. 
The blood samples will be stored after appropriate coding to remove patient identifiers. The 
coding information linking patient identifiers to the stored samples will be maintained in a 
locked, secure area that will be accessible only to the study investigator. Subjects may request 
to have their samples destroyed at any time. These samples will be destroyed immediately 
upon receipt of the subjects’ written request to do so. Identification of which samples to destroy 
will be available from the coding information linking patient identifiers to the stored samples 
as described earlier in this paragraph.  
 
2.5.3 Restrictions to Direct Access of Specimens 
Specimens will be kept in the responsible study investigators’ laboratories indefinitely and will 
be under the control of the principal investigator. Investigators or other personnel not involved 
with the management or operations of the study are not permitted direct access to the 
specimens.  
 
2.6 ENDPOINTS 
 
2.6.1 Primary Endpoints 
The primary endpoint of the trial will be mean pulmonary artery pressure (mPAP) during 
submaximal exercise as compared between placebo and nitrite at 10 weeks (i.e. the week 
10 RHC of placebo vs week 10 RHC of nitrite). 
2.6.2 Secondary Endpoints 
Secondary endpoints:  
1.The differences in 6MWT with Borg dyspnea scale assessments and pulse oxygen 
saturation measurements  
2. RHC hemodynamics (right atrial pressure, pulmonary capillary wedge pressure, 
pulmonary artery pressures and resistance, pulmonary artery compliance, cardiac 
output, diastolic pressure gradients, and transpulmonary gradient)  
 
3. Doppler-echocardiography 
 
4. NT-proBNP plasma levels  


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PI: Michael Risbano, MD 
STUDY20010014 
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5. NYHA functional classification changes 
 
6. NO2
− metabolites 
7. Endurance exercise time (ET), changes in CPET measurements taken at isotime 
(oxygen consumption [VO2, L/min], minute ventilation [VE, L/min], ventilatory equivalent 
[VE/VO2], dead space to tidal volume ratio [Vd/Vt], Borg dyspnea and leg score, and 
changes in time constants for increases of Vt, respiratory rate [RR], VE, and heartrate). 
8. Changes in fasting glucose and insulin sensitivity 
 
2.7 SUBJECT WITHDRAWAL  
 
A subject may voluntarily discontinue participation in this study at any time. The investigator 
may also, at his or her discretion, discontinue a subject from this study at any time. Every 
effort should be made by the investigator to keep the subject in the study.   
 
Subjects may be withdrawn from the study prior to completion if any of the following criteria 
are observed: 
 
• 
Intercurrent illness or an unexpected fatal or life-threatening adverse event, which 
requires discontinuation of study treatment 
• 
Pregnancy 
• 
Subject reached protocol-defined stopping criteria 
• 
Request by the subject to withdraw from the study 
• 
Protocol violations 
• 
Persistent non-compliance 
• 
Lost to follow-up 
• 
Investigator discretion 
• 
Study closed/terminated 
 
2.7.1. Dropouts and withdrawals 
To be considered complete, a subject must complete all study visits as specified in the 
protocol without violations of the protocol so significant as to obscure the response to study 
treatment. 
 
Subjects who fail to complete all study required visits will not be considered complete and 
may not enroll at a later date and will not be replaced. A record will be kept of all subjects who 
fail to complete all study visits and their primary reasons for discontinuation. 
 
In the event of subject withdrawal, subjects will be encouraged to continue all follow-up visits 
for safety monitoring or to continue follow up as directed by their personal primary physicians, 
unless the subject withdraws consent at any time (without having to justify the decision). All 
available data from subjects who discontinued during the study, for whatever reason, will be 
included in the safety analysis. 
 
2.8 STATISTICAL ANALYSIS 
 
2.8.1 Sample Size and Power 
Sample size: Prior study has indicated that oral nitrate reduces baseline mean PAP -7 (SD = 
4) mm Hg vs. -3 (4) mmHg in placebo group. A sample size of 12 in each group provides 88% 


Study Short Title: Oral Nitrite in Patients with PH-HFpEF 
 
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PI: Michael Risbano, MD 
STUDY20010014 
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power to detect this difference. This sample size will provide 80% power to detect -10 (6) vs. 
-5 (6) decrease in post-exercise mPAP in treatment and placebo group, respectively.  
Statistical analysis: Pre-randomization characteristics of two groups will be presented 
using the median (interquartile range) or frequency tables at each study sequence. The 
difference of mPAP during submaximal exercise as compared between placebo and nitrite 
at 10 weeks (i.e. the week 10 RHC of placebo vs week 10 RHC of nitrite) using the 
parametric PK-cross analysis (i.e. an ANOVA which determines the sequence, period, 
carryover, and treatment effects). Post-exercise values of before and after crossover will be 
compared between two groups using similar approach. For secondary outcomes, we will 
compare the difference of baseline and follow-up value and compare these differences with 
a parametric test. Alternatively a nonparametric test will be used for these comparisons. For 
both primary and secondary outcomes, treatment effect in each sequence as well as mean 
(SD) total treatment effect and corresponding P values will be reported.  
 
We will also test the effect of treatment on pre-exercise mPAP and secondary outcomes using 
mixed effect models. In all analysis, missing data will be imputed and sensitivity analysis with 
imputed data will be performed. All analyses will be performed by ITT approach. 
 
3.   HUMAN SUBJECTS 
3.1 SUBJECT POPULATION 
 
The racial, gender and ethnic characteristics of the proposed subject population in this 
research protocol shall reflect the demographics of the population of Pittsburgh and the 
surrounding area. We shall attempt to recruit subjects in proportion to these demographics. 
No exclusion criteria shall be based on race, ethnicity, or gender. 
 
Every effort will be made to keep subjects in the study until they complete all study 
procedures. 
 
3.1.1 Inclusion of Women and Minorities 
Both men and women of all races and ethnic groups are eligible for this trial. Women who 
meet the inclusion criteria, and have none of the exclusion criteria, will be enrolled without 
restriction as dictated by the study protocol.  Because of the use of a study medication, woman 
of child bearing potential must meet specialized inclusion/exclusion criteria to minimize this 
risk.   
 
 
3.1.2 Inclusion of Children 
Children under the age of 18 will not be recruited for this protocol because of the need to 
explore potential adverse effects of the study treatment more fully in adults. 
3.2 INCLUSION CRITERIA 
Potential study subjects must satisfy the following criteria to be enrolled in the study: 
1. Age 18 years and older 
2. PH-HFpEF 
Confirmed diagnosis by RHC: 
• 
Mean Pulmonary Arterial Pressure (mPAP) ≥ 25 mm Hg  
AND 
• 
Pulmonary capillary wedge pressure (PCWP)  ≥ 15 mm Hg 


Study Short Title: Oral Nitrite in Patients with PH-HFpEF 
 
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PI: Michael Risbano, MD 
STUDY20010014 
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AND 
• 
Transpulmonary Gradient (TPG) ≥ 12 mm Hg 
3. Patient has one or more of the following heart failure associated signs or symptoms: 
shortness of breath with activity, overweight, atrial fibrillation and/or systemic 
hypertension.  
 
3.3 EXCLUSION CRITERIA 
Subjects meeting any of the exclusion criteria at baseline will be excluded from participating 
in study. 
• 
Age less than 18 years  
• 
SBP > 170 or < 110 mmHg 
• 
DBP > 95 or < 60 mmHg 
• 
Hemoglobin A1C > 10 
• 
Positive urine pregnancy test or breastfeeding 
• 
Ejection Fraction < 40% 
• 
Dementia 
• 
End-stage malignancy 
• 
Major cardiovascular event or procedure within 6 weeks prior to enrollment 
• 
Severe valvular disease 
• 
Known chronic psychiatric or medical conditions that may increase the risk associated 
with study participation in the judgement of the investigator would make the subject 
inappropriate for entry into this study 
• 
Smoker  
• 
Hemoglobin < 9 g/dL 
• 
Serum creatinine > 3.0 mg/dL 
• 
Recent (<3 months) addition or change in dosing of hormonal contraceptive 
medications (OCP, IUD, DepoProvera) 
• 
Receipt of an investigational product or device, or participation in a drug research 
study within a period of 15 days; 
• 
RHC < 2 weeks from study screening RHC unless clinically indicated 
 
4.   RECRUITMENT AND INFORMED CONSENT PROCEDURES 
4.1 RECRUITMENT METHODS 
 
• 
A variety of mechanisms will be used to recruit subjects for this protocol.  
 
a. UPMC 
Heart failure patients can be referred by their physician and/or cardiologist from the UPMC 
Advanced Heart Failure Center, General Cardiology clinic, Benedum Geriatric Center, the 
Comprehensive Pulmonary Hypertension (PH) Program, internal medicine clinics, or other 
UPMC or community facilities. For UPMC Presbyterian/Montefiore-based clinics, we will 
obtain physician and HRPO approval for screening of subject medical records for 
identification of eligible candidates. He will engage with additional cardiologists, internal 
medicine physicians, and geriatricians for permission to screen from those clinics. Once 
identified as preliminarily eligible, the study coordinator/staff will reaffirm with the subject's 
physician on the clinic visit day that recruitment of the subject is medically appropriate. 


Study Short Title: Oral Nitrite in Patients with PH-HFpEF 
 
Sponsor: Mark T. Gladwin, MD 
    
PI: Michael Risbano, MD 
STUDY20010014 
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Staff will also request direction from the attending physician as to the timing of speaking 
to the subject, i.e., before or after the subject has seen the physician for their visit. This 
will be done on a case by case basis. Study staff will speak with candidates at clinic visits 
or by telephone call to assess interest and review medical history to assess eligibility.  
 
Subjects referred from facilities outside the UPMC Epic medical record system will be 
provided a medical record release for their cardiologist/physician to release needed 
documents to confirm eligibility. 
 
Subjects can also be identified from the inpatient Cardiology service. The study 
coordinator will follow the same procedure as for outpatients by screening the inpatient 
service for subjects admitted with heart failure symptomatology, check medical history for 
heart failure diagnoses, and engage the attending cardiologist as to appropriateness for 
the study. As noted, permission to speak with subject will be obtained from the Cardiology 
attending. 
 
 
b. Research Registry 
The HRPO submission will include the request to use the Clinical Translational Science 
Institute’s Pitt+Me Research Registry, which includes a database of over 90,000 
individuals who have indicated their interest in participating in research studies. The 
Pitt+Me initiative includes the use of social media to engage subjects.  We will also request 
to use the University of Pittsburgh’s Pepper Center Research Registry which includes a 
database of older individuals interested in research, fostering inclusion of older adults and 
covers individuals in the community, assisted living and skilled nursing facilities.  
 
 
c. Advertisements 
An approved study flier and recruitment brochure will be placed in key places and/or be 
distributed to physician offices, related clinics, or on other occasions/venues that present 
as an opportunity to recruit (e.g., a PI speaking engagement or a community outreach 
event to reach minority subjects). Potential subjects can self-refer by contacting the study 
staff via a telephone number/email address that is provided on these advertisements. 
Study staff will utilize the approved phone screening script when responding to interested 
candidates. With subject permission, they will be screened on the phone to make a 
preliminary assessment of eligibility. We will obtain permission to access their medical 
records in the UPMC database or request records from their provider as needed to further 
document eligibility.  
 
Advertisements, such as on radio, television, or print copy in newspapers, or bus signs, 
may also be utilized depending on recruitment rates.  
 
• 
Once subjects have been determined to be eligible from medical record review and 
attending approval, recruitment and enrollment procedures will then follow, including: 
 
a) Confirmation of eligibility by study physician.  
 
b) Setting dates for patient visits will also occur prior to screening visit to maintain timeliness 
of visits per protocol. 
 


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PI: Michael Risbano, MD 
STUDY20010014 
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c) Scheduling outpatient screening visit (Visit 1) where study risks and potential benefits and 
rights as a research subject will be described in detail, informed consent will be obtained, 
and final eligibility will be confirmed. 
 
d) To minimize the possibility that subjects will feel obligated to participate, investigators will 
reinforce with their subjects that participation is voluntary, that they do not have to 
participate, and that the decision not to participate will not affect their care, now or in the 
future. The investigator will also allow subjects to make further inquiries if they are 
interested. 
 
4.2 INFORMED CONSENT PROCEDURES 
 
Informed consent is a process that is initiated prior to the individual’s agreeing to participate 
in the study and continues throughout the individual’s study participation. Extensive 
discussion of risks and possible benefits of participation in this study will be provided to the 
subjects and their families.  Consent forms describing in detail the study procedures and risks 
are given to the subject and written documentation of informed consent is required prior to 
enrolling in the study.  Consent forms will be IRB approved and the subject will be asked to 
read and review the document. Upon reviewing the document, the investigator will explain the 
research study to the subject and answer any questions that may arise.  The subjects will sign 
the informed consent document prior to being enrolled in the study.  The subjects should have 
the opportunity to discuss the study with their surrogates or think about it prior to agreeing to 
participate. The subjects may withdraw consent at any time throughout the course of the 
study. A copy of the informed consent document will be given to the subjects for their records. 
The rights and welfare of the subjects will be protected by emphasizing to them that the quality 
of their medical care will not be adversely affected if they decline to participate in this study. 
Prior to performing any of the research study procedures or interventions, subjects must 
provide informed consent. The investigator will verbally explain the study to the potential 
subject in a language understandable to subjects, providing all pertinent information (purpose, 
procedures, risks, benefits, alternatives to participation, etc.), and will allow potential subjects 
ample opportunity to ask questions to elicit a better understanding of the study. Following this 
verbal explanation, potential subjects will be provided with a local IRB approved consent form 
and will be asked to read and review the document. Upon reviewing the document, the 
investigator will provide adequate opportunity for the subject to consider all options, and 
answer any additional questions the potential subject may have. Every effort will be made to 
ensure that subjects have comprehended the study information prior to obtaining subject's 
voluntary agreement to participate. 
Remote Consenting 
Remote consenting may be used as opposed to in person consenting where possible to 
reduce unnecessary in person encounters. In the event remote consenting is utilized, subjects 
will be provided with a copy of the informed consent prior to the teleconsent meeting either 
via email, fax, mail or previously provided during an in person visit. After the physician 
investigator and subject review the consent form via video conferencing (e.g., telephone, 
conference call, videoconference, telemedicine, etc.), the subject will be offered the 
opportunity to ask any questions and have those questions answered. The subject will sign 
and date the informed consent. 


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PI: Michael Risbano, MD 
STUDY20010014 
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The physician investigator will verify the subject physically signed the consent document 
either by viewing via video conference or obtaining a photo of the complete signed consent 
signature page or obtaining verbal confirmation from the subject that he/she signed the 
consent form or agreed to participate electronically. The signed document is then mailed, 
emailed, photo/scanned to text or faxed to the study team. 
If the signed consent is provided as an electronic copy (emailed, photo/scanned, or faxed), 
the subject will return the original signed document on their first in person visit or by mail. If 
the signed consent form is mailed to the study team, the physician investigator will sign their 
copy which they possess after the subject has acknowledged signature on their copy. Once 
the subject’s original copy is received the study team’s copy will be attached to make a single 
document. 
The study team will document the remote consent process, including the date and time the 
conversation occurred, how it occurred (e.g., telephone, video conference), the names of the 
participants, confirmation that the subject received the informed consent, and confirmation of 
verbal informed consent to participate. 
In addition, older potential participants whose competency to consent is in question will be 
tested for sufficient comprehension and recall of the information presented. Prospective 
subjects who do not remember the important facts about participation in the research study 
after repeated testing will not be included in the study.  
The subjects may withdraw consent at any time throughout the course of the study. A copy of 
the informed consent document will be given to the subjects for their records. The rights and 
welfare of the subjects will be protected by emphasizing to them that the quality of their 
medical care will not be adversely affected if they decline to participate in this study. 
5.   POTENTIAL RISKS AND BENEFITS 
5.1. Risk of Experimental Drug Intervention 
 
Numerous studies have evaluated acute, subacute, and chronic drinking water exposures of 
nitrite in laboratory animals, and drinking water and dietary exposures in humans. Recent 
studies are available using a high dose of nitrite by oral route in the form of beet root juice. 
Recent studies have evaluated acute exposures of oral preparations of nitrite on PK and blood 
pressure and are characterized below. More extensive human data is available on parenteral 
sodium nitrite as it is currently available and approved by the FDA for use in the emergency 
treatment of cyanide poisoning. [72, 73] It is also notable that neutrapharmaceutical 
preparations are currently being sold with levels of nitrite (12.7 mg per tablet). 
   
Nitrite:  
Sodium nitrite has been used commercially as a food preservative, an anti-corrosive agent, a 
coloring agent, and an anti-anginal agent, with additional uses in laxatives, burn ointments, 
and liniments. Amyl nitrite has been inhaled or ingested as an euphoric stimulant.  Nitrite has 
also been found as a contaminant in well water. Literature searches generated case reports 
of nausea, vomiting, abdominal pain, dizziness, headache, flushing, cyanosis, tachypnea, 
dyspnea, hypotension, and death attributed to excess nitrite (high-dose) exposure from these 
sources as a consequence of methemoglobinemia due to oxidation of heme-iron in 
oxyhemoglobin. Normal background methemoglobin production is 1-3%. If levels of 
methemoglobin rise above approximately 30% of total hemoglobin, a subject may appear 
cyanotic and experience dyspnea, due to the reduced oxygen carrying capacity of hemoglobin 


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PI: Michael Risbano, MD 
STUDY20010014 
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(methemoglobin cannot bind oxygen). Levels above 50% can cause seizures, hypotension, 
coma, and death. Sodium nitrite administration for cyanide poisoning at the labeled dosage of 
300 mg causes methemoglobinemia, a desirable effect, as methemoglobin binds to cyanide, 
thus protecting cellular mitochondria. A standard dose of nitrite used for cyanide poisoning is 
300 mg up to 600 mg. Note that methemoglobin levels have never risen higher than 3% at the 
currently used therapeutic doses (< 75 mg) in 80 volunteers in phase I studies at the NIH. 
   
The Sponsor of this study proposal, Dr. Gladwin, has previously 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. He 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 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 disease33-35. In another study by Gladwin et al., 18 healthy adults received an 
infusion of sodium nitrite totaling 75 mg (15 minutes each x 2 infusions). This was associated 
with a 7 mmHg decrease in mean arterial pressure, a peak methemoglobin of less 3%, and 
no other significant effects34. Note this single dose is 1.9 times the single dose per time of day 
we plan to use in this trial.  
 
In an open-label three-way crossover study, 9 healthy adult subjects received two single high 
dose oral sodium nitrite aqueous solutions (0.12 and 0.06 mmol NaNo2/mmol Hb, equivalent 
to 290-380 mg and 140-190 mg sodium nitrite, respectively, depending on the total body 
hemoglobin level of the person) and one intravenous sodium nitrite dose (0.12 mmol 
NaNo2/mmol Hb)18. Note that this is 1.2-3.2 times the daily dose we plan to use in this trial. 
There was a washout period of at least 7 days between each of the treatments. Mild headache 
occurred in 44-55% of subjects and was the most frequent complaint during each treatment 
session, which the authors ascribed to the sodium nitrite, not methemoglobinemia, as the 
percentage of methemoglobinemia stayed below clinically toxic levels (<15%). By report, up 
to 22% experienced nausea, which subsided within half an hour18. The pharmacokinetic 
analysis of this study indicated similar bioavailability of oral and IV delivery of nitrite, as well 
as similar side effect and safety profiles. 
 
A recent study determined the safety and feasibility of prolonged intravenous nitrite infusion. 
Twelve adult volunteers received increasing starting doses of sodium nitrite, 4.2 to 533.8 
μg/kg/hr for 48 hours. Dose limiting toxicity occurred at 445.7 μg/kg/hr (10.6 mg/kg/day) and 
was limited to asymptomatic transient decreases of arterial blood pressure of up to 20 mmHg 
and asymptomatic increases of methemoglobin levels above 5%. No tolerance or clinically 
significant rebound was observed36. Note, this is 8.2 times the daily dose we plan to use in 
this trial (based on an adult body weight of 95 kg). 
 
For nitrite, two retrospective case-control studies have shown that high maternal dietary nitrite 
intake from cured meat or drinking water during pregnancy might be associated with risk of 
childhood brain tumors and possibly gastric and esophageal cancer. This evidence is only 
based on retrospective case-control studies; cohort studies have found no significantly 
increased risks24. 
 
In the 2001 National Toxicology Program (NTP) Report summarizing 2-year rodent drinking 
water studies, there was no evidence of carcinogenic activity of sodium nitrite in male or 


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STUDY20010014 
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female F344/N rats exposed to up to 130 mg/kg/day in males and 150 mg/kg/day in females, 
or in male B6C3F1 mice exposed to up to 220 mg/kg/day. There was equivocal evidence of 
carcinogenic activity of sodium nitrite in the highest dose of 165 mg/kg/day in female B6C3F1 
mice based on the positive trend in the incidences of squamous cell papilloma or carcinoma 
(combined) of the forestomach. Exposure to sodium nitrite in drinking water resulted in 
increased incidences of epithelial hyperplasia in the forestomach. However, no chromosomal 
damage (genetic toxicity) was observed in three studies conducted in rats and mice in vivo37. 
Taken together, these findings suggest minimal carcinogenic nitrite-mediated risk. 
 
In the current study, we will use lower doses of nitrite than used safely in any of these studies. 
Our nitrite dose of 40 mg three times daily is lower than the dose used on the cardiovascular 
IND and < 40% of the dose for cyanide poisoning.  
 
To summarize, we anticipate the following symptoms by organ system and likely frequency of 
risk: 
Gastrointestinal 
• 
Common: none 
• 
Frequent: none 
• 
Infrequent: dry mouth 
• 
Rare: nausea, abdominal pain, and vomiting 
 
Hematologic 
• 
Common: none 
• 
Frequent: none 
• 
Rare: methemoglobinemia 
 
Cardiovascular 
• 
Common: none 
• 
Frequent: none 
• 
Rare: flushing, tachycardia, hypotension 
 
Neurologic 
• 
Common: none 
• 
Frequent: none 
• 
Rare: headache, dizziness, seizure, coma 
 
Respiratory 
• 
Common: none 
• 
Frequent: none 
• 
Rare: tachypnea, dyspnea, cyanosis 
 
5.2 Risk of Study Procedures 
 
Risks of Blood Drawing 
The amount of blood to be drawn over the course of this research study could be a maximum 
of 40 tablespoons. To minimize the risks of blood tests, all blood draws are performed by 
trained RN, MD, technician or phlebotomist.  
Common risks include temporary discomfort, bruising which may last for several days, 
redness, swelling, lower hemoglobin level. Infrequent risks include a subject may feel 


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STUDY20010014 
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lightheaded or faint when blood is drawn. This is usually due to nervousness and is not usually 
serious. Rare risks include infection and bleeding.  
Risks of Echocardiography 
This is a noninvasive procedure. There is no known risk associated with this procedure. Gel 
will be applied during the test which may cause coldness and/or irritation. 
Risks of Accelerometer Use 
Common risks associated with wearing the accelerometer are redness, irritation, and chaffing.  
 
Risks of Right heart Catheterization 
Common risks: pain at the needle entry site and slight risk of bleeding around the site, bruising 
at site, lightheadedness or dizziness during the needle stick. 
Infrequent risks: puncturing the lung which would require a chest tube insertion, irregular 
heartbeats which usually stop when the long tube is removed from the heart. 
Very rare complications include cardiac arrhythmias, cardiac tamponade, low blood 
pressure, infection, or embolism caused by blood clots at the tip of the catheter. 
 
Risks of the Use of Micromanometer Catheter during Catheterization 
The micromanometer pressure and flow catheters are FDA approved, and have been used 
for clinical/investigative indications for over 25 years. Moreover, these catheters do not pose 
any inherent risk to patients. The catheter will be placed through the clinical Swann-Ganz 
catheter that is within the preexisting jugular venous sheath. This obviates the need for an 
additional venipuncture. This catheter has successfully been used without complications by 
the study Principal Investigator for the past 5+ years. Risks associated with this procedure are 
estimated to be rare, which include abnormal heart rhythm, bleeding, or perforation of the 
heart or pulmonary artery. 
Risks of Medications used during Catheterization 
The medications used for sedation are relatively brief in duration and should wear off within 
several hours. The side effects are listed below for each drug. 
• 
1% Lidocaine: will be used to numb the area prior to the insertion of the cardiac catheter 
into either the arm, neck or groin vein. A common side effect is slight burning at the site 
which dissipates quickly.  
• 
Fentanyl: Common side effects include temporary light-headedness, dizziness, and 
nausea, vomiting or sweating.  
• 
Midazolam: Common side effect includes drowsiness. Infrequent side effects are nausea, 
vomiting. Breathing problems are rare.  Allergic reactions (e.g., hives, itching, etc.) to 
lidocaine, fentanyl, or midazolam are rare. 
 
Risks of Fluoroscopy during the Catheterization 
An x-ray may be done, if indicated, on participants whose neck vein is used for the 
catheterization. An X-ray performed for the purpose of this research study involves exposure 
to radiation. Each x-ray will result in a radiation dose of approximately 3 rems to the neck with 
minimal exposure of other body areas. A total of 9 rems if all three RHC’s are performed. For 
comparison, radiation workers are permitted, by Federal regulation, to receive a maximum 
annual radiation dose of 20 rems to the most sensitive organs of their body. There is no 
minimum amount of radiation exposure that is recognized as being totally free of the risk of 
causing genetic mutations (abnormal cells) or cancer. However, the risk associated with the 


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amount of radiation exposure received from participation in this study is considered to be low 
and comparable to everyday risks.  
Risks of Cardiopulmonary Exercise Test (CPET) 
CPET is a well-established clinical tool. The risk of serious or fatal complications is rare. The 
risks include but are not limited to the following: 
• 
Common risks include shortness of breath, mild angina, musculoskeletal discomfort. 
• 
Infrequent risk includes hypotension, chest pain. 
• 
Rarely, exercise may cause moderate to extreme pain which could be due to muscle 
sprains, muscle strains, broken bones, or chest pain, changes include shortness of breath, 
abnormal blood pressure, or fainting, myocardial infarction, prolonged cardiac arrhythmia, 
stroke, or sudden death are rare (occur in <1% of people).  
 
Risk of Spirometry  
Spirometry infrequently can make the chest tight or lead to shortness of breath. Albuterol will 
be available if this occurs. Rarely, a subject may have coughing or lightheadedness with 
spirometry. If Albuterol is given, the subject will get up to 4 puffs of albuterol with these tests. 
Albuterol may make a subject feel slightly jittery or nervous, but the feelings are temporary. 
 
Risks of Intravenous Glucose Tolerance Test (IVGTT) 
• 
IV Catheter Insertion Common Risks: bruising, soreness, phlebitis. Infrequent Risks: 
infiltration (a leakage of anything that has been given through the vein (such as the 
saline, dextrose, deuterated glucose, insulin) into the arm tissue that surrounds the vein 
and holds the IV). Other Risks: infection, dizziness/fainting, bleeding. 
 
• 
IVGTT Common Risks: (occurring in less than 25% of people) include: phlebitis 
(inflammation of a vein), transient hyperglycemia (high blood sugar not requiring 
treatment). Rare side effects (occurring in less than 1% of people) include: 
Hypoglycemia (low blood sugar) which may cause symptoms such as shakiness, 
nervousness, sweatiness, hunger, dizziness, or fast heart rate. If severe, hypoglycemia 
can cause coma, seizure, or even death. 
 
Reproductive Risks   
It is not known if the study drug can affect an unborn baby. Therefore, subjects should not 
become pregnant or father a baby while on this study.  If subjects are physically able to father 
a baby, subjects must use an effective method of birth control while on this study. If subjects 
become aware that they or their sexual partner is pregnant during the course of their 
participation in this research study, subjects must contact, as soon as possible, the study 
investigator.  
Risk of Collection and Storage of Private Health Information and Internet Usage 
Although we are taking many steps to protect the participants' information, there is always a 
chance that their information or identity could be disclosed. We will continue to review and 
improve the ways we keep their information private. To protect their information, paper-based 
records will be kept in a secure location and only be accessible to personnel involved in the 
study. Paper charts will contain subject identifiers but will be in locked cabinets within a locked 
office on a unit that has restricted access. 
 


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5.3 ALTERNATIVE TREATMENTS 
 
The alternative treatment for the subjects participating in this investigation is to continue 
their medical care under the direction of their primary physicians. 
 
5.4 POTENTIAL BENEFITS 
 
Participation in the proposed research may or may not provide a direct benefit to participants 
in this research. This research involves interventions and/or procedures that present greater 
than minimal risk to the involved adults, but which holds out the potential for direct individual 
benefit. The risk is justified by the extent of potential benefit to the involved adults, which 
includes possibly revealing previously-undiagnosed morbidities commonly seen in obesity 
such as hyperlipidemia, impaired fasting glucose, or type 2 diabetes mellitus. Information 
obtained from the proposed research will provide information about the relationship between 
metabolic syndrome and PH-HFpEF and patient outcome. 
Based on the preceding assessment of risks and potential benefits, the risks to subjects are 
reasonable in relation to anticipated benefits. The research presents a balance of risks and 
expected direct benefits similar to that available in the clinical setting. 
 
5.5 DATA SAFETY MONITORING PLAN 
      
5.5.1 Data Safety Monitoring Board 
A Data and Safety Monitoring Board (iDSMB) independent of the study investigators will 
monitor this clinical trial for additional measure of subject protection. The iDSMB consists of 
clinicians completely independent of the investigators who have no financial, scientific, or 
other conflict of interest with the trial. 
The iDSMB will conduct interim monitoring of accumulating data from research activities to 
assure the continued safety of human subjects, relevance and appropriateness of the study, 
and the integrity of research data. 
5.5.2 Data Safety Monitoring Plan 
Assuring patient safety is an essential component of this protocol. The study Principal 
Investigator has primary responsibility for the oversight of the data and safety monitoring.  The 
study investigators will evaluate all adverse events. All subjects who have AEs, whether 
considered associated with the use of the study medication or not, must be monitored to 
determine the outcome. The clinical course of the AE will be followed up according to accepted 
standards of medical practice, even after the end of the period of observation, until a 
satisfactory explanation is found or the Principal Investigator considers it medically justifiable 
to terminate follow-up. 
All untoward medical occurrences observed in subjects receiving the study drug will be 
recorded on the participants’ adverse event worksheets by the study coordinator under the 
supervision of the principal investigator. The worksheets will then be reviewed for 
completeness and internal consistency. In addition to internal safeguards built into a 
computerized system, external safeguards will be put in place to ensure that access to the 
computerized system and to the data is restricted to authorized personnel. Training conducted 
by qualified individuals on a continuing basis will be provided to individuals in the specific 


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operations with regard to computerized systems that they are to utilitze during the course of 
the study.  
The Sponsor and Investigator will prepare a detailed written summary of serious, unexpected, 
and treatment-related adverse events, and will compare and contrast each event with prior 
events. The written summary will be provided to the DSMB and the IRB.  
In addition, the DSMB Report addressing the following information will be submitted to the 
IRB at the time of continuing review, annually or more often as required: 
 
• 
A list of the research personnel who participated in the data and safety monitoring. 
• 
The frequency of monitoring that took place during the renewal intervals and/or the dates 
that data and safety monitoring was conducted. 
• 
A summary of cumulative data related to unanticipated problems (including adverse 
events) including a determination of causality and whether the risk to benefit assessment 
has changed. 
• 
If appropriate, a summary of pertinent scientific literature reports, therapeutic 
developments, or results of related studies that may have an impact on the safety of 
study participants or the ethics of the research study. 
• 
A summary of the outcome of reviews conducted to ensure subject privacy and research 
data confidentiality. 
• 
Final conclusions regarding changes to the anticipated benefit-to-risk assessment of the 
study participation and final recommendations related to continuing, changing, or 
terminating the study. 
 
Stopping Rule 
For safety reasons, we propose to temporarily suspend enrollment and review with our DSMB 
if any two subjects experience the following despite dose reduction:  
 
1. A single occurrence of venous methemoglobin level >5% 
If subjects report change in skin tone and dizziness or other signs of methemoglobinemia 
for >24 hours, an interim visit will be scheduled (if not already being seen for a research 
visit) and the following steps will occur. The visit can be conducted in UPMC Presbyterian 
or the TRC.  
The physician investigator will factor relevant subject symptoms and hemodynamics and: 
• 
Determine if unblinding is needed (if methemoglobin % >5) and, 
• 
If subject is willing to do an interim visit with repeat drug dosing of 20 mg with follow 
up methemoglobin and BP pressure monitoring or 
• 
Subject should be withdrawn 
 
Interim visit: If methemoglobin is greater than 5%, the PI will be unblinded to drug or 
placebo assignment. If subjects are on nitrite, subjects will be asked to repeat drug dosing 
of 20 mg sodium nitrite with follow up methemoglobin and hemodynamic monitoring. A 
study physician or PI will evaluate data and make a determination regarding discharge on 
20 mg tid. If discharged on 20 mg tid, subjects will be contacted via phone for the next 2 
days for symptom re-assessment. If symptoms remain resolved, subjects will continue on 
20 mg tid. If symptoms persist, subjects will be requested to come in for repeat 


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methemoglobin testing after 3 days.  If level persists above 5% on lower dose, 
participation in the study for the subject will be discontinued.  
2. Blood pressure (BP) and related symptoms: If subjects experience lightheadedness or 
dizziness, we request that they call the study coordinator who will assess their 
symptoms, compare to baseline levels, and discuss need for interim visit with study 
physician.  If a visit is indicated, BP, RR, heart rate, pulse co-oximetry and 
methemoglobin level via co-oximetry will be measured.   
• 
If systolic BP ≤ 90mm Hg, the study drug will be reduced from 40 TID to 20 TID.  
Subjects will be reassessed via telephone to ensure that the patient is stable on 
the new dose. If stable and asymptomatic, subjects will continue with the half dose.  
• 
If subjects are symptomatic with dizziness, lightheadedness for ≥ 24 hours, and 
require fluid replacement or other therapy for hypotension, subjects will be 
requested to return to clinic for an interim visit for assessment of vitals, 
methemoglobin percent, and evaluation by an investigator-physician to reevaluate 
the subject’s suitability for continued participation based on symptoms. 
• 
If SBP remains ≤ 90mm Hg and symptomatic and at the lowest dosage of 20mg 
TID, subject will be withdrawn. 
 
If an unexpected fatal event or any of the above life-threatening events occur despite 
dose reduction, the study will be halted, until data review by investigators and the Data 
Safety Monitoring Board has rendered a final recommendation about study 
continuation. 
Other potential reasons for stopping participation in study activities 
1. Subject participation will be discontinued if a participant has a positive pregnancy test 
at any point during study participation.  
2. In the event that the medication compliance rate is < 80%, subjects will be re-
educated on medication compliance. If medication compliance falls outside of the 
acceptable range more than 2 times, the study investigators will discuss subject 
eligibility for continued participation in the study. 
 
5.5.3 Parameters to be Monitored 
The following progress will be monitored throughout the course of the research to ensure the 
safety of subjects as well as the integrity and confidentiality of their data. 
 
• 
An evaluation of the progress of the research study, including subject recruitment 
and retention, and an assessment of the timeliness and quality of the data. 
 
• 
A review of collected data (including adverse events, unanticipated problems, and 
subject withdrawals) to determine whether there is a change to the anticipated 
benefit-to-risk assessment of study participation and whether the study should 
continue as originally designed, should be changed, or should be terminated. 
 
• 
An assessment of external factors or relevant information (eg. pertinent scientific 
literature reports or therapeutic development, results of related studies) that may 
have an impact on the safety and study participants or the ethics of the research 
study. 
 


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• 
A review of study procedures designed to protect the privacy of the research subjects 
and the confidentiality of their research data. 
 
The severity of adverse changes in physical signs or symptoms will be classified as follows: 
• 
Grade 1 (Mild): asymptomatic or mild symptoms; clinical or diagnostic observation only; 
intervention not indicated. 
 
• 
Grade 2 (Moderate): minimal, local, or noninvasive intervention indicated; limiting age-
appropriate ADL (Activities of Daily Living). 
 
• 
Grade 3 (Severe): medically significant but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated; disabling; limiting self-care 
ADL. 
 
• 
Grade 4 (Life-threatening): consequences; urgent intervention indicated. 
 
• 
Grade 5 (Death): event is a direct cause of death. 
 
5.5.4 Frequency of Monitoring 
The Investigator will review subject safety data as it is generated. The Investigator and the 
research staff will meet at approximately monthly intervals to re-evaluate study goals, subject 
recruitment, data coding and retention, documentation and identification of adverse events, 
complaints, and confidentiality of subjects. There will be an evaluation of the progress of the 
research study, including assessments of data quality, time lines, participant recruitment, 
accrual, and retention. The Investigator will also review the outcome and adverse event data 
to determine whether there is any change to the anticipated benefit-to-risk ratio of study 
participation and whether the study should continue as originally designed or should be re-
evaluated and changed. 
  
The DSMB will also be expected to meet as needed, but not less than every six months to 
provide an overall summary status report to the regulatory agencies. 
 
5.5.5 Reportable Adverse Events 
For this study, a serious adverse event is any untoward clinical event that is thought by the 
investigator to be study-related, that is also: 
1. Fatal or immediately life threatening 
2. Permanently disabling, or severely incapacitating 
3. Requires or prolongs inpatient hospitalization   
4. Important medical events that may not result in death, be life threatening, or require 
hospitalization may be considered serious adverse events when, based upon 
appropriate medical judgment, they may jeopardize the patient, or subject, and may 
require medical or surgical intervention to prevent one of the serious outcomes listed 
above. 
 
If clinically important and unexpected adverse experiences, or clinically important study-
related adverse experiences occur, they will be recorded on the adverse event case report 
form.    
 
5.5.6 Adverse Events Reporting Timeline 
Life-threatening or fatal unexpected adverse events associated with the use of the study drug 


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or procedures must be reported to the DSMB and the IRB within 24 hours of discovery of the 
incident with subsequent follow-up submission of a detailed written report. 
 
The FDA must be notified by telephone or facsimile transmission of a human adverse event 
that is fatal or life-threatening no later than 7 calendar days after receiving the respective 
human adverse event information, followed by the subsequent submission of a written IND 
Safety Report. 
 
Serious and unexpected adverse events associated with the use of the study drug or 
procedures must be reported to the DSMB and the IRB with subsequent follow-up submission 
of a detailed written report in accordance with the respective policies and procedures of the 
IRB. Written IND Safety Reports will be submitted to the FDA as soon as possible and, in no 
event, later than 15 calendar days following the investigator-sponsor’s receipt of the 
respective adverse event information. 
 
A summary report of the DSMB’s findings will be prepared and submitted to the regulatory 
agencies. 
 
5.6 RISKS MANAGEMENT PROCEDURES 
 
5.6.1 Protection Against Risks  
General Risks of Study Protocol and Procedures 
All research interventions/activities will be conducted in private patient care areas. The 
collection of sensitive information about subjects is limited to the amount necessary to achieve 
the aims of the research, so that no unneeded sensitive information is being collected. 
 
All demographic and clinical information about the subject will be stored on an electronic 
password-guarded study database under the supervision of the Investigator for this protocol. 
The electronic database that is being used for the purpose of this study has not been fully 
validated to be in compliance with the FDA regulations at 21 CFR Part 11; i.e. taking into 
account the limited scope of this clinical investigation. The data will be stripped of individual 
identifiers and stored anonymously with a subject number. Information linking subject 
identifiers with the coded subject number will be stored under password protection on 
computers in locked areas, with access only to the database manager. Maintaining records in 
locked files in locked offices will protect confidentiality of subjects. All staff will sign 
confidentiality statements. Access to the database will be limited to the data manager and 
staff under the supervision of the Investigator. 
 
Specimens will be stripped of subject identifiers and stored according to a similar coding 
protocol as described above. These specimens will be stored safely in the custody of the 
Investigator responsible for the individual assays. The Investigators will limit future access to 
any remaining sample to only those investigators with prior IRB approval for their studies. 
 
All staff involved in this study are properly credentialed and instructed in the areas of testing, 
confidentiality, and safety.  
 
The Investigator will retain the data for the entire period of this study. The Investigator may 
continue to use and disclose subjects’ de-identified information for the purpose of this study 
for a minimum of seven years after final reporting or publication of the study. If the subject  


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STUDY20010014 
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decides to withdraw or be withdrawn from study participation, they may request that the study 
data and samples be destroyed. 
 
5.6.2 Protection Against Potential Risks of Experimental Intervention 
The study will include involvement by trained staff / investigators with experience in the 
administration of the study drug. CPET will be conducted by experienced exercise 
physiologists along with a physician to continuously observe a 12 lead ECG monitor (GE 
Cardiosoft software) that will utilize electrodes and detection electronics which minimize 
motion artifact during the IET. A 3 lead ECG will be used during the CWR tests. If ECG 
changes develop or the participant becomes distressed the test will be stopped. Subjects’ vital 
signs are monitored closely throughout the procedure. 
 
Continuous monitoring by the Data and Safety Monitoring Board. 
 
Required Education in the Protection of Human Research Participants 
• 
The Investigator and all sub-investigators listed on University of Pittsburgh Institutional 
Review Board approved protocol are required to complete the CITI modules.  This web-
based tutorial is a requirement of the IRB for protocol submission.  
 
6. STUDY ADMINSTRATION 
6.1 QUALITY CONTROL AND QUALITY ASSURANCE 
 
Independent monitoring of the clinical study for protocol and GCP compliance will be 
conducted periodically (i.e., at a minimum of annually) by qualified staff of the Education and 
Compliance Office – Human Subject Research, Research Conduct and Compliance Office, 
University of Pittsburgh. 
The Investigator and Sponsor and the University of Pittsburgh and University of Pittsburgh 
Medical Center will permit direct access of the study monitors and appropriate regulatory 
authorities to the study data and to the corresponding source data and documents to verify 
the accuracy of this data. 
 
6.2 DATA HANDLING AND RECORD-KEEPING 
 
6.2.1 Data recording 
Study worksheets will be completed for each subject enrolled into the clinical study.  The 
Investigator will review, sign and date completed worksheets; the Investigator’s signature 
serving as attestation of the Investigator’s responsibility for ensuring that all clinical and 
laboratory data are complete, accurate, and authentic.   
Appropriate coded identifications (i.e. Subject ID number) will be used. Every effort will be 
made to collect complete data for each study visit. Causes of missing data will be fully 
documented. With respect to safety evaluation, it is not planned to impute missing data. 
6.2.2 Record maintenance and retention   
The Sponsor and Investigator will maintain records in accordance with Good Clinical Practice 
guidelines, to include: 


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• 
FDA correspondence related to the IND and clinical protocol, including copies of 
submitted Safety Reports and Annual Reports  
• 
IRB correspondence (including approval notifications) related to the clinical protocol; 
including copies of adverse event reports and annual or interim reports 
• 
Current and past versions of the IRB-approved clinical protocol and corresponding 
IRB-approved consent form(s) and, if applicable, subject recruitment advertisements 
• 
Signed FDA Form 1572 Statements of Investigator (i.e., for the Sponsor and all 
identified sub-investigators) 
• 
Financial disclosure information (Investigator-sponsor and clinical protocol sub-
investigators) 
• 
Curriculum vitae (Sponsor and clinical protocol sub-investigators) 
• 
Certificates of required training (e.g., human subject protections, Good Clinical 
Practice, etc.) for Sponsor and listed sub-investigators 
• 
Listing of printed names/signatures of Investigator-sponsor and listed sub-
investigators 
• 
Normal value(s)/range(s) for medical/laboratory/technical procedures or tests included 
in the clinical protocol 
• 
Laboratory certification information 
• 
Instructions for on-site preparation and handling of the investigational drug(s), study 
treatment(s), and other study-related materials (i.e., if not addressed in the clinical 
protocol) 
• 
Decoding procedures for blinded trials 
• 
Master randomization list  
• 
Signed informed consent forms 
• 
Completed worksheets; signed and dated by Investigator 
• 
Source Documents or certified copies of Source Documents 
• 
Monitoring visit reports  
• 
Copies of Sponsor communications to the Investigator and copies of Investigator 
communications to sub-investigators  
• 
Subject screening and enrollment logs 
• 
Subject identification code list 
• 
Investigational drug accountability records, including documentation of drug disposal 
• 
Retained biological specimen log 
• 
Interim data analysis report(s)  
• 
Final clinical study report 
 
Subject-specific data and will be coded and the subject identification code list will be stored 
so as to protect the subjects’ confidentiality.  Subject names or other directly identifiable 
information will not appear on any reports, publications, or other disclosures of clinical study 
outcomes. 
The Investigator and Sponsor and will retain the specified records and reports for up to 2 
years after the marketing application is approved for the investigational drug; or, if a 
marketing application is not submitted or approved for the investigational drug, until 2 years 
after investigations under the IND have been discontinued and the FDA so notified. 
 
6.3 ETHICS  
 
Institutional Review Board (IRB) Approval 


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The Investigator will obtain, from the University of Pittsburgh Human Research Protection 
Office (HRPO), prospective approval of the clinical protocol and corresponding informed 
consent form(s), modifications to the clinical protocol and corresponding informed consent 
forms, and brochures (i.e., directed at potential research subjects and clinical faculty/staff) for 
study recruitment.    
The only circumstance in which a deviation from the current IRB-approved clinical 
protocol/consent form(s) may be initiated in the absence of prospective IRB approval is to 
eliminate an apparent immediate hazard to the research subject(s). In such circumstances, 
the Investigator will promptly notify the University of Pittsburgh IRB of the deviation.  
The University of Pittsburgh IRB operates in compliance with FDA regulations at 21 CFR Parts 
50 and 21 CFR 56, and in conformance with applicable International Conference on 
Harmonization (ICH) Guidelines on Good Clinical Practice (CGP).  
In the event that the University of Pittsburgh IRB requires, as a condition of approval, 
substantial changes to a clinical protocol submitted under an FDA-accepted IND application, 
or in the event of the Investigator’s decision to modify the previously accepted clinical protocol 
the Investigator will submit (i.e., in advance of implementing the change) a Protocol 
Amendment to the IND describing any change to this Phase 2 clinical protocol that significantly 
affects the safety of the subjects.  For changes that do not affect critical safety assessments, 
the revisions to the clinical protocol will be addressed in the Annual Report to the IND.  
 
Ethical and scientific conduct of the clinical study  
The clinical study will be conducted in accordance with the current IRB-approved clinical 
protocol; ICH Guidelines on GCP; and relevant policies, requirements, and regulations of the 
University of Pittsburgh HRPO, University of Pittsburgh and UPMC, Commonwealth of 
Pennsylvania, and applicable federal agencies. 
The Investigator will make certain that appropriate processes and procedures are in place to 
ensure that ongoing questions and concerns of enrolled subjects are adequately addressed 
and that the subjects are informed of any new information that may affect their decision to 
continue participation in the clinical study.  In the event of substantial changes to the clinical 
study or the risk-to-benefit ratio of study participation, the Investigator will obtain the informed 
consent of enrolled subjects for continued participation in the clinical study. 
 
7.  COSTS AND PAYMENTS 
7.1 COSTS 
 
Study drug and all costs associated with this research protocol will be supported by ongoing 
research grants.  
 
7.2 PAYMENTS 
Subjects may be paid a total of $1,400 on a ‘WePay’ debit card if all study procedures are 
completed. This is a breakdown of payments: 
• 
Screening – patients will be compensated $200 if the screen fail following the screening 
RHC 


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• 
Baseline visits with screening RHC - $200 
• 
Baseline visits with no screening RHC - $100 
 
• 
Completion of Week 10 procedures - $400 
o Day 1 - $100 
o Day 2 - $200 - RHC 
o Day 3 - $100 – IVGTT 
 
• 
Cross-over visit - $50 
 
• 
Completion of end of treatment procedures - $750 
o Day 1 - $200 
o Day 2 - $450 - RHC 
o Day 3 - $100 - IVGTT 
 
Subjects will receive the first payment following the completion of week 10 and will receive 
the final payment at the final visit for EOT. Subjects will also be provided a ticket for 
outpatient parking costs at all visits. In the event that a subject must be withdrawn due to a 
study related adverse event, they will be reimbursed for the individual study visits that were 
completed to date per the above reimbursement schedule. Travel and hotel may be 
provided in certain circumstances. Subjects may also receive meals or meal tickets for visits 
greater than 4 hours in length. 
 
Subjects who require an interim visit will be compensated $25 and provided parking. 
 
8.  QUALIFICATIONS AND SOURCE OF SUPPORT 
8.1 QUALIFICATIONS OF THE INVESTIGATORS 
Sponsor: 
 
Mark Gladwin, M.D., is Professor of Medicine, University of Pittsburgh. Dr Gladwin is the 
Chair of the Department of Medicine and Director of the Heart, Lung, Blood, and Vascular 
Medicine Institute at the University of Pittsburgh. He is an internationally recognized authority 
in the field of sodium nitrite including both the basic science and a broad range of clinical 
applications in cardiovascular disease. He is a current IND holder for the investigation of 
sodium nitrite in lung transplant. 
   
Investigator: 
Michael Risbano, MD, MA is an Assistant Professor in the Department of Medicine, Division 
of Pulmonary, Allergy, and Critical Care Medicine. Dr. Risbano’s clinical interests are primarily 
focused on the diagnosis, management, and treatment of pulmonary hypertension. He is an 
attending physician in the Comprehensive Pulmonary Hypertension Program. Dr. Risbano’s 
past research has focused on the discovery and implementation of biomarkers for the early 
diagnosis of pulmonary hypertension in patients with scleroderma (SSc-PAH). He is interested 
in the hemodynamic evaluation of subjects with pulmonary hypertension and correlation of 
hemodynamic values with biomarker levels. Dr. Risbano also has a research interest in the 


Study Short Title: Oral Nitrite in Patients with PH-HFpEF 
 
Sponsor: Mark T. Gladwin, MD 
    
PI: Michael Risbano, MD 
STUDY20010014 
Page 43 of 47 
V14.0  02/14/2022  
 
hemodynamic responses to vasoactive medications. He will be responsible for the overall 
accrual of patients who are seen at the UPMC Comprehensive Pulmonary Hypertension 
Program on a regular basis as well as for performance of the cardiac catheterizations, analysis 
and reporting of all data. 
 
Sub-investigators: 
 
Marc A. Simon, M.D., is an Adjunct Professor in the Heart Failure and Cardiac 
Transplantation Section at the University of Pittsburgh. Dr. Simon has been involved in many 
research projects. He will assist with confirming eligibility and interpreting cardiac 
catheterizations, and assist with analysis  of thedata. 
 
Adil Yunis, MD, Vinaya Mulkareddy, MD, and Michael Bashline, MD are cardiology 
fellows in the Heart and Vascular Institute workingin pulmonary hypertension research.  
 
Frederico Toledo, MD: Dr. Toledo is an Associate Professor of Medicine. Dr. Toledo is board-
certified in endocrinology and has been a clinical researcher for one and a half decades. He 
has performed more than 500 euglycemic clamps/IVGTTs as part of his studies and as a 
collaborator in others. 
 
Carl Koch, MD, is a Clinical Instructor of Medicine in the Division of Pulmonary, Allergy and 
Critical Care division of the University of Pittsburgh. Since beginning his postdoctoral 
fellowship at the University of Pittsburgh, Dr. Koch has continued to foster his research into 
the role of nitric oxide and its metabolites in pulmonary and vascular physiology. He has further 
developed interest in the role of the microbiome in nitric oxide metabolism as it pertains to the 
development of pulmonary hypertension and cardiovascular disease. Dr. Koch will assist with 
with subject enrollment and implementation of the study. 
 
Jessica Huston, MD, is an Assistant Professor, Advanced Heart Failure, Pulmonary 
Hypertension, and Cardiac Transplant In the Division of Cariology at the University of 
Pittsburgh. Dr. Huston will assist with subject enrollment and implementation of the study. 
 
 
8.2   SOURCE OF SUPPORT 
 
National Institute of Health- tPPG Grant- Mark Gladwin, MD 
 
 
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PI: Michael Risbano, MD 
STUDY20010014 
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