Clinical trial NCT02457260
Conditions: Heart Failure
Sponsor: Gladwin, Mark, MD
Start date: 2016-01-08; Completion date: 2017-03-06
Enrollment: 21 participants; Allocation: NON_RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Studies have demonstrated nitrite therapy increases adenosine triphosphate (ATP) synthesis in skeletal muscle mitochondria concomitant with reduced whole-body oxygen cost during steady state exercise. Our own work has demonstrated safety and efficacy of an FDA investigational new drug (IND) approved sodium nitrite (10 milligram \[mg\]) capsule, and its utility to upregulate the sirtuin 3 adenosine monophosphate protein kinase (SIRT3-AMP) pathway of skeletal muscle of younger heart failure (HF) patients. It now seems exceptionally logical and opportune to apply these insights to older HF patients and to delineate mechanisms of disease and aging that respond to nitrite therapy.

Detailed description:
Heart failure (HF) is epidemic with aging and prevalence of HF is steadily increasing as the population of older adults expands. Despite the fact that age always stands out as a leading risk factor for HF incidence as well as for poor HF prognosis, few HF trials focus specifically on aging physiology as a key determinant of the disease, and/or on the utility of targeting mechanisms associated with aging as beneficial therapeutic targets. Consistently, HF trials have tended to focus primarily on central mechanisms of cardiac pumping dysfunction despite the fact that HF-outcomes are strongly related to functional decrements that are largely mediated by peripheral manifestations of the disease, and which are particularly interrelated with aging physiology. HF-related skeletal muscle myopathy is a manifestation of HF that diminishes physical function, and which is likely exacerbated by sarcopenia, vascular stiffening, and other aspects of aging such that exercise intolerance is disproportionate among older HF populations as well as its insidious clinical implications. In a pilot investigation, the investigators will study older (age ≥70 years) adults, including patients with HF with reduced ejection fraction (HFrEF) and HF with preserved ejection fraction (HFpEF) and age-matched healthy controls, to study benefits of nitrite therapy (in addition to established standards of HF care) to improve physical function. In this pilot analysis the investigators will focus on the utility of daily nitrite supplements to moderate aerobic (maximal and submaximal) and strength (maximal, endurance, and power) indices as well as underlying skeletal muscle mechanisms (skeletal muscle mitochondrial performance, gene expression, and capillarity).

Atrophy of type 1 skeletal muscle myocytes is associated with HFrEF and HFpEF. Multiple studies of normal aging have also demonstrated typical atrophy of type 2 skeletal muscle fibers. Consistently, older adults are compromised by cumulative atrophy risks, with studies showing losses of lean body mass as well as intrinsic skeletal muscle weakening, increased interstitial fat, and increased inflammation, with associated functional decrements and fatigue. While aerobic and strength exercise training may be used to modify such HF-related muscle patterns, deconditioning remains pervasive among older HF patients, and efforts to promote exercise interventions are typically confounded by comorbidity (e.g. arthritis, peripheral arterial disease, diabetes, depression), geriatric syndromes (e.g., falls, frailty, incontinence, dementia, poor sleep, malnutrition, auditory and vision impairments), as well as pain, anxiety, and logistic limitations. Even major exercise-training trials that provided strong reinforcements to ensure requisite behavioral changes yielded only poor exercise adherence. There is high conceptual rationale for a therapy that intrinsically improves skeletal muscle performance in HF as a vital means to improve physical function and moderate effects of disease itself as well as to frailties and enfeeblement associated with the disease. This will potentially improve efficacy and quality of care, and also potentially mitigate the skyrocketing costs associated with aggregate HF management.

Studies have demonstrated nitrate therapy increases adenosine triphospate (ATP) synthesis in skeletal muscle mitochondria concomitant with reduced whole-body oxygen cost during steady state exercise. Our own work has demonstrated safety and efficacy of an FDA-IND approved sodium nitrite (10 mg) capsule, and its utility to upregulate the SIRT3-AMP pathway of skeletal muscle of younger HF patients. It now seems exceptionally logical and opportune to apply these insights to older HF patients and to delineate mechanisms of disease and aging that respond to nitrite therapy.

Overall aims:

1. To demonstrate that oral nitrite pills provide skeletal muscle physiological benefit in old HFrEF and HFpEF patients:

   * To show that oral supplements are manifest as increased plasma nitrite.
   * To show that increased plasma nitrite is associated with improved skeletal muscle (mitochondrial respirometry) and platelet (Seahorse XF) metabolism.
   * To demonstrate that improved metabolism is associated with shifts in skeletal muscle anabolic gene expression (Fibronectin type III domain-containing protein 5 \[FNDC5\], peroxisome proliferator-activated receptor-γ coactivator \[PGC1α\], Sirtuin 3) as well as reduced catabolic gene expression (ubiquitin, muscle RING-Finger Protein \[MuRF\], Atrogin1\]) and inflammation (Tumor necrosis factor alpha \[TNFα\], Interleukin 1beta (IL-1β), Interleukin six (IL-6).
2. To demonstrate that improved skeletal physiology achieved using oral nitrate pills is associated with improved clinical indices in old HFrEF and HFpEF patients:

   * To show that oral nitrite supplements increase efficiency of work, i.e., reduced oxygen uptake (VO2) required for the same work intensity.

Arms: arm 1: 10 healthy adults, age 70 or older to receive 14 Nitrogen (14N) sodium nitrite, 40 mg tid arm 2: 10 adults with heart failure and preserved ejection fraction age 70 or older to receive 14N sodium nitrite, 20 or 40 mg tid depending on dose stratification for safety arm 3: 10 adults with heart failure and reduced ejection fraction aged 70 or older to receive 14N sodium nitrite, 20 or 40 mg tid depending on dose stratification for safety
Interventions: intervention 1: 14 N Sodium Nitrite
Intervention descriptions: intervention 1: oral formulation of sodium nitrite 40 mg three times daily for 4 weeks

Protocol documents:
Prot_SAP_000.pdf: 
Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
1 
 
 
Study of the Utility of Oral Nitrite Therapy to Improve Skeletal Muscle 
Bioenergetics and Physical Capacity in Older Heart Failure Patients. 
 
 
 
 
 
 
 
 
 
Daniel Forman, MD 
Principal Investigator 
 
 
 
 
 
 
Professor of Medicine 
University of Pittsburgh Medical Center 
Chair, Geriatric Cardiology Section  
 
 
 
 
 
Version 7  
September 2, 2016 
 


Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
2 
 
TABLE OF CONTENTS 
PROTOCOL SYNOPSIS………………………………………………………………………………3 
 
1. 
STUDY OBJECTIVE, SPECIFIC AIMS, BACKGROUND AND SIGNIFICANCE…......8 
 
1.1 OBJECTIVE………………………………………………………………………………..8 
 
1.2 SPECIFIC AIMS…………………………………………………………………………...9 
 
1.3 BACKGROUND……………………………………………………………………………9 
 
1.4 SIGNIFICANCE……………………………………………………………………………10 
 
2. 
RESEARCH DESIGN AND METHODS…………………………………………………….10 
 
 
2.1 CLASSIFICATION AND METHODOLOGICAL DESIGN……………………………..10 
 
2.2 DETAILED DESCRIPTION OF STUDY DESIGN……………………………………..11 
 
2.3 STUDY ASSESSMENT………………………………………………………………...  14 
 
2.4 STUDY ENDPOINTS……………………………………………………………………..21 
 
2.5 STATISTICAL ANALYSIS………………………………………………………………..21 
 
3. 
HUMAN SUBJECTS…………………………………………………………………………..22 
 
 
3.1 SUBJECT POPULATION………………………………………………………………...22 
 
3.2 INCLUSION CRITERIA…………………………………………………………………...22 
 
3.3 EXCLUSION CRITERIA…………………………………………………………………..23 
 
4. 
RECRUITMENT AND INFORMED CONSENT PROCEDURES…………………………23 
 
 
4.1 RECRUITMENT METHODS……………………………………………………………..23 
 
4.2 INFORMED CONSENT PROCEDURES……………………………………………….24 
 
5. 
POTENTIAL RISKS AND BENEFITS………………………………………………………24 
 
 
5.1 POTENTIAL RISKS……………………………………………………………………….24 
 
5.2 ALTERNATIVE TREATMENTS………………………………………………………….28 
 
5.3 POTENTIAL BENEFITS………………………………………………………………….28 
 
5.4 DATA SAFETY MONITORING PLAN…………………………………………………..28 
 
5.5 RISK MANAGEMENT PROCEDURES…………………………………………………32 
 
6. 
COSTS AND PAYMENTS……………………………………………………………………33 
 
 
6.1 COSTS……………………………………………………………………………………..33 
 
6.2 PAYMENTS………………………………………………………………………………..33 
 
7. 
QUALIFICATIONS AND SOURCE OF SUPPORT………………………………………..34 
 
 
7.1 QUALIFICATIONS OF THE INVESTIGATORS………………………………………..34 
 
7.2 SOURCE OF SUPPORT…………………………………………………………………35 
 
8. 
REFERENCES…………………………………………………………………………………36 
 
 
 


Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
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PROTOCOL SYNOPSIS 
 
Protocol Title: 
Study of the Utility of Oral Nitrite therapy to improve Skeletal 
Muscle Bioenergetics and physical capacity in older heart 
failure patients. 
 
Protocol Number: 
PRO15020481 
NCT Number: 
 
Version # and Date: 
Version 7.0 / September 2, 2016 
 
Clinical Phase: 
 
Phase II clinical investigation 
 
Investigational Drugs: 
Nitrogen 14 Sodium Nitrite (14 N Sodium Nitrite) 
Trial Site: 
Single-Center Trial 
 
IND Sponsor: 
Mark T. Gladwin, MD 
Professor of Medicine 
Chairman, Department of Medicine 
University of Pittsburgh School of Medicine 
 
Investigator: 
 
Daniel Forman, MD 
Professor of Medicine 
Chair, Geriatric Cardiology Section, University of Pittsburgh   
Medical Center 
University of Pittsburgh 
3471 Fifth Avenue, Suite 500 
Pittsburgh, PA 15213 
 
Sub-Investigators: 
Mark T. Gladwin, MD  
Sruti Shiva, PhD  
John Gorcsan, MD 
Ana Mora, MD 
Marc Simon, MD 
Nicole Helbling, MS, RN 
Manisha Jhamb, MD, MPH 
 
 
Study Monitor: 
Education and Compliance Office for Human Subject Research 
Research Conduct and Compliance Office 
University of Pittsburgh 
3500 Fifth Avenue, Suite 303 
Pittsburgh, PA 15213 
 
Research Facilities: 
UPMC Montefiore Hospital CTRC 
3459 Fifth Ave.  
Pittsburgh, PA 15213 
 
UPMC Montefiore Hospital Endocrinology and Metabolism 
Research Center (EMRC) 
3459 Fifth Ave.  
Pittsburgh, PA 15213 
 


Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
4 
 
Pepper Center –SMART Center 
Kaufmann Medical Building, Suite 1200 3471 Fifth Ave. 
Pittsburgh, PA 15213 
 
UPMC Presbyterian/Montefiore  Echocardiography lab 
200 Lothrop St 
Pittsburgh PA  15213 
 
Clinical Laboratories: 
UPMC Presbyterian University Hospital 
3477 Euler Way 
Pittsburgh, PA 15213 
 
Study Rationale: 
Heart failure (HF) is epidemic with aging and prevalence of HF 
is steadily increasing (1) as the population of older adults 
expands. While age stands out as a leading risk factor for HF 
incidence as well as for poor HF prognosis (2), few HF trials 
focus specifically on aging physiology as a key determinant of 
the disease and as a pertinent therapeutic target. Consistently, 
HF trials have tended to focus primarily on central mechanisms 
of cardiac pumping dysfunction (3) despite the fact that HF-
outcomes are strongly related to functional decrements that are 
largely mediated by age- (4) and disease-related (5) peripheral 
manifestations. HF-related skeletal muscle myopathy is a 
component of HF that diminishes physical function, and which 
is likely exacerbated by sarcopenia, vascular stiffening, and 
other aspects of aging such that exercise intolerance is 
disproportionate among older HF populations as well as its 
insidious clinical implications (6). In a pilot investigation, we will 
study older (age ≥70 years) adults, including patients with HF 
with reduced ejection fraction (HFrEF), patients with HF with 
preserved ejection fraction (HFpEF), and age-matched healthy 
controls, to study benefits of enteral nitrite therapy (in addition 
to established standards of HF care) to improve physical 
function. In this pilot analysis we will focus on the utility of daily 
nitrite supplements to moderate aerobic (maximal and 
submaximal) and strength (maximal, endurance, and power) 
indices as well as underlying skeletal muscle mechanisms 
(skeletal muscle mitochondrial performance, gene expression, 
and capillarity). Studies have demonstrated nitrite therapy 
increases ATP synthesis in skeletal muscle mitochondria 
concomitant with reduced whole-body oxygen cost during 
steady state exercise (15). Our own work has demonstrated 
safety and efficacy of a FDA-IND approved sodium nitrite (10 
mg) tablet, and its utility to upregulate the SIRT3-AMP pathway 
of skeletal muscle of younger HF patients (16).  
Study Objectives: 
Studies have demonstrated nitrite therapy increases ATP 
synthesis in skeletal muscle mitochondria concomitant with 
reduced whole-body oxygen cost during steady state exercise 
(15). Our own work has demonstrated safety and efficacy of a 
FDA-IND approved sodium nitrite (10 mg) capsule, and its utility 
to upregulate the SIRT3-AMPpathway of skeletal muscle of 
younger HF patients (16). It now seems exceptionally logical 


Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
5 
 
and opportune to apply these insights to older HF patients and 
to delineate mechanisms of disease and aging that respond to 
nitrite therapy.     
Study Hypothesis: 
Oral supplementation with nitrite will intrinsically improve 
skeletal muscle performance in HF as a vital means to improve 
physical function and moderate effects of disease itself as well 
as well as to frailties and enfeeblements associated with the 
disease. This will potentially improve efficacy and quality of care, 
and also potentially mitigate the skyrocketing costs associated 
with aggregate HF management.  
Study Aims: 
 
1.  
To demonstrate that oral nitrite pills provide skeletal 
muscle physiological benefit in old HFrEF and HFpEF 
patients.   
2.  
To show that oral supplements are manifest as 
increased plasma nitrite. 
3.  
To show that increased plasma nitrite is associated with 
improved skeletal muscle (mitochondrial respirometry) and 
platelet (Seahorse XF) metabolism. 
4.  
To demonstrate that improved metabolism is associated 
with shifts in skeletal muscle anabolic gene expression 
(FNDC5, PGC1α, Sirtuin 3) as well as reduced catabolic gene 
expression (ubiquitin [MuRF, Atrogin1]) and inflammation 
(TNFα, IL-1β, IL-6). 
5.  
To demonstrate that improved skeletal physiology 
achieved using oral nitrate pills is associated with improved 
clinical indices in old HFrEF and HFpEF patients.  
6. 
To show that oral nitrite supplements increase efficiency 
of work, i.e., reduced oxygen uptake (VO2) required for the 
same work intensity. 
Study Design: 
The study aims to assess the safety and feasibility of oral nitrite 
in adults with HFrEF, HFpEF or healthy control. 
 
Subjects who meet the Inclusion Criteria and none of the 
Exclusion Criteria will receive oral sodium nitrite for 4 weeks. We 
will stratify dosing as below: 
Study drug to be initiated and prescribed as below:   
a. 20 mg tid to subjects who 
i. 
are ≥80 years of age or, 
ii. 
are age 70 to <80 years with recent history of 
low blood pressure or other factors that PI 
judges to be at higher risk for hypotensive 
response  
b. 40 mg tid to subjects who 
i. 
are age 70 to <80 with no recent history of low 
blood pressures or PI judges to be at lower risk 
for hypotensive response  
 
c. Subjects to ingest drug once in the morning and again 
in the early afternoon and again in the evening, 
approximately 6 hours apart. 


Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
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Subjects will be evaluated as outpatients with the first dose of 
study drug. Blood pressure, heart rate, methemoglobin levels 
and plasma nitrite will be measured in the first two hours after 
dosing. Every week safety visits will occur by phone to assess 
symptoms, adverse events (AE), interval histories and 
medication compliance review. 
 
Subjects will also be contacted by the study investigator and/or 
the study coordinator by phone for a follow-up assessment one 
week following completion of Visit 5. 
 
Planned Sample Size:  
30 subjects 
Duration of Treatment: 
4 weeks 
 
Major Inclusion Criteria:  Inclusion Criteria HF Subjects 
• 
NYHA class II or III for the previous three months despite a 
minimum of 6 weeks of treatment. Echo criteria will be 
confirmed as part of the initial study assessment. 
• 
Age ≥70 years 
• 
HFrEF patients left ventricular ejection fraction [LVEF] 
<40%  
• 
HFpEF patients LVEF ≥40%, may also include E/E′ >8, left 
atrial size>40 mL/m2 
• 
Optimal therapy according to AHA/ACC and HFSA HFrEF 
guidelines, including treatment with ACEI and beta-blocker 
therapy (for at least 6 weeks), or have documented reason 
for variation, including medication intolerance, 
contraindication, patient preference, or personal physician's 
judgment. 
• 
Patients using aspirin (ASA) will be eligible, but asked to 
hold the medication for 48 hours prior to biopsy. This 
technique has previously been used with consistent safety. 
Patients will also be asked to avoid non-steroidal anti-
inflammatory medications (NSAIDs) for 48 hours prior to 
the biopsy. 
• 
Patients using anti-thrombin and anti-platelet therapy will 
plan to modify prior to muscle biopsies individually in 
coordination with the participant's primary provider or 
cardiologist. 
• 
Baseline BP ≤170/95:  subjects with either systolic or 
diastolic over stated level are ineligible. 
• 
Subjects who report using PDE-5s will be asked to hold the 
medication 24 hours before initial nitrite dose and for the 
entire period on study drug. 
  
Inclusion Criteria Age-Matched Control Population 
• 
Age ≥70 years 
• 
Absence of clinically significant chronic disease processes 
(including cardiovascular disease, diabetes, cancer, CKD, 
liver disease, COPD, and/or osteoarthritis). Non-significant 


Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
7 
 
cardiac rhythm abnormalities and stable hypertension are 
acceptable for inclusion 
• 
Baseline BP ≤170/95:  subjects with either systolic or 
diastolic over stated level are ineligible. 
• 
Subjects who report using PDE-5s will be asked to hold the 
medication 24 hours before initial nitrite dose and for the 
entire period on study drug. 
• 
Patients using aspirin (ASA) will be eligible, but asked to 
hold the medication for 48 hours prior to biopsy. This 
technique has previously been used with consistent safety. 
Patients will also be asked to avoid non-steroidal anti-
inflammatory medications (NSAIDs) for 48 hours prior to 
the biopsy. 
• 
Patients using anti-thrombin and anti-platelet therapy will 
plan to modify prior to muscle biopsies individually in 
coordination with the participant's primary provider or 
cardiologist. 
 
 
Exclusion Criteria All Participants 
Major Exclusion 
Criteria: 
• 
Allergy to lidocaine 
• 
Anemia:  Hgb <11.0 in men and <10.0 in women 
• 
Dementia  
• 
End-stage malignancy 
• 
Orthopedic exercise limitation 
• 
Chronic use of oral corticosteroids or other medications 
that affect muscle function. 
• 
Chronic ETOH or drug dependency.   
• 
Any bleeding disorder that would contraindicate biopsy 
such as history of clinically significant bleeding diathesis 
(e.g., Hemophilia A or B, Von Willebrand's Disease or 
congenital Factor VII deficiency). 
• 
Psychiatric hospitalization within the last 3 months 
• 
Chronic nitrate use  
• 
Baseline BP <100/60 (either value) 
• 
Refusal to participate in muscle biopsy 
 
• 
Exclusion Criteria HF Population  
• 
Major cardiovascular event or procedure within the prior 6 
weeks. 
• 
HF secondary to significant uncorrected primary valvular 
disease (except mitral regurgitation secondary to left 
ventricular dysfunction). If valve replacement has been 
performed, patient may not be enrolled for 12 months after 
this procedure. 
• 
Severe valvular heart disease 
• 
Mechanical valve replacement requiring warfarin 
• 
Currently taking clopidogrel or other anti-platelet 
medication for a recent stent placement and/or a complex 
atherosclerotic lesion such that holding clopidogrel (or 
similar medications) creates disproportionate risk. This 


Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
8 
 
decision will always be made by the patient’s own primary 
provider or cardiologist.  
• 
ICD device with heart rate limits that prohibit exercise 
assessments. Referring physicians will be provided with an 
opportunity to reprogram devices so that patients can 
participate.  
 
Study Endpoints: 
Overall, study endpoints pre- and post- nitrites will include:  
1. Skeletal muscle bioenergetics: Mitochondrial function 
will be assessed using respirometry. Polymerase chain 
reaction (PCR) to assess pertinent gene expression 
(ubiquitin [MuRF, Atrogin1, FoxO], FNDC5, PGC1α, 
Sirtuin 3).   
2. Serology: plasma BNP, inflammatory markers, plasma 
nitrite and platelet bioenergetics (using Seahorse XF 
analysis) (40), i.e., including glycolytic as well as basal 
and maximal respiratory rates.   
3. Measures of physical function will include CPX indices, 
gait speed, handgrip strength, and balance.   
4. Quality of life: Kansas City Cardiomyopathy 
Questionnaire or SF-12. 
  
 
1. 
OBJECTIVE, SPECIFIC AIMS, BACKGROUND, AND SIGNIFICANCE 
 
 
1.1 OBJECTIVE 
 
Studies have demonstrated nitrite therapy increases ATP synthesis in skeletal muscle 
mitochondria concomitant with reduced whole-body oxygen cost during steady state exercise 
(15). Our own work has demonstrated safety and efficacy of a FDA-IND approved sodium nitrite 
(10 mg) capsule, and its utility to upregulate the SIRT3-AMP pathway of skeletal muscle of 
younger HF patients (16). It now seems exceptionally logical and opportune to apply these 
insights to older HF patients and to delineate mechanisms of disease and aging that respond to 
nitrite therapy. 
 
1.2 SPECIFIC AIMS 
 
Hypothesis:  
Oral supplementation with nitrite will intrinsically improve skeletal muscle performance in HF as a 
vital means to improve physical function and moderate effects of disease itself as well as well as 
to frailties and enfeeblements associated with the disease. This will potentially improve efficacy 
and quality of care, and also potentially mitigate the skyrocketing costs associated with aggregate 
HF management. 
Specific Aims: 
 
Aim 1:  To demonstrate that oral nitrite pills provide skeletal muscle physiological benefit in old 
HFrEF and HFpEF patients 
 
Aim 2:  To show that oral supplements are manifest as increased plasma nitrite. 


Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
9 
 
 
Aim 3:  To show that increased plasma nitrite is associated with improved skeletal muscle 
(mitochondrial respirometry) and platelet (Seahorse XF) metabolism. 
 
Aim 4:  To demonstrate that improved metabolism is associated with shifts in skeletal muscle 
anabolic gene expression (FNDC5, PGC1α, Sirtuin 3) as well as reduced catabolic gene 
expression (ubiquitin [MuRF, Atrogin1]) and inflammation (TNFα, IL-1β, IL-6). 
 
Aim 5: To demonstrate that improved skeletal physiology achieved using oral nitrite pills is 
associated with improved clinical indices in old HFrEF and HFpEF patients:  
 
Aim 6: To show that oral nitrite supplements increase efficiency of work, i.e., reduced oxygen 
uptake (VO2) required for the same work intensity. 
 
BACKGROUND 
 
1.3. Longevity is increasing in the United States and much of the world, and the older adult 
population is growing as more people are surviving into old age (13). Since aging is also 
associated with predictable morphological and physiological changes that are conducive to 
cardiovascular disease (CVD), prevalence of CVD is increasing as the older adult population 
expands (6,17). Consistently, prevalence of heart failure (HF) with reduced ejection fraction 
(HFrEF) and HF with preserved ejection fraction (HFpEF) are both growing (18). HFrEF and 
HFpEF are associated with high morbidity and mortality among older adults, as well as with 
decreased quality of life and increased dependency. Vulnerability to and consequences from 
exercise intolerance are especially significant in older HF patients. Both HFrEF and HFpEF are 
associated with reduced type 1 skeletal muscle fibers (7,8,9), and clinical manifestations are 
compounded by age-related reductions of type 2 muscle fibers (19) as well as associated 
predispositions to increased interstitial fat and inflammation in skeletal muscle (20,21). Exercise 
intolerance in HFrEF and HFpEF is common and insidious, and predisposes to HF’s notoriously 
poor outcomes and associated (high) costs (stemming from increased hospitalizations, increased 
dependency, and increased frailty). Interventions to intrinsically improve skeletal muscle health 
may prove vital therapeutic benefit and also help moderate today’s mounting healthcare 
expenditures.  
 
Peripheral mechanisms of exercise intolerance in HFrEF have been recognized for many years. 
Seminal work by Sullivan, Massie, and Hambrecht (6) and other preeminent investigators have 
demonstrated diminished type I skeletal muscle fibers, mitochondria (size and density), and 
capillarity in HF patients. Related work has demonstrated increased inflammation (particularly 
TNFα) (22) and increased in ubiquitin proteolysis, myostatin, and other catabolic factors in HFrEF 
(23). While skeletal muscle changes associated with HFpEF have often seemed more ambiguous 
than in HFrEF, recent work by Kitzman et al. demonstrates decrements in Type I fibers in HFpEF 
similar to those seen in HFrEF, and correlation to similar reductions in peak oxygen uptake (VO2) 
(9). 
 
There has been much enthusiasm about the utility of exercise training to modify HF skeletal 
muscle changes (24, 25). Benefits have been established in HFrEF and HFpEF, but training for 
either is also similarly confounded by the high prevalence of comorbidities, especially those who 
are older and who are also inherently prone age-related pulmonary disease (COPD, pulmonary 
hypertension, sleep apnea), metabolic disease (diabetes), osteoarthritis depression, and other 
morbidities, as well as to geriatric syndromes (frailty, falls, incontinence, sensory impairments, 
poor sleep) and logistic impediments (transportation, financial) (13). Even HF-ACTION, a well-


Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
10 
 
funded trial that included significant behavioral components to reinforce exercise compliance in a 
large HFrEF study population, achieved notably suboptimal exercise adherence (14).  
 
Nitrite therapy is a relatively novel concept to potentially modify skeletal muscle bioenergetics and 
which may thereby improve physical function. Shiva et al demonstrated the capacity of nitrite to 
be converted nitric oxide (NO) in response to ischemic stress. NO then can bind to cytochrome c 
oxidase of the mitochondrial electron transport chain, reducing electron transport chain and 
utilization (26, 27). Key clinical correlates in multiple animal and human trials include increased 
exercise time and decrease oxygen uptake (VO2) efficiency (i.e., reduced VO2 for an equivalent 
workload). In one such trial Larsen et al. compared healthy men before and after dietary nitrate 
supplements. Gross efficiency increased from 19.7 ±1.6 to 21.1±1.3%, with no differences in heart 
rate, lactate, ventilation (VE), VE/VO2, or the respiratory exchange ratio (RER) indicating this was 
mediated purely as an intrinsic skeletal muscle change (28). Nitrite rich beetroot juice 
supplementation has been demonstrated to improve time required to achieve a pre-specified work 
level for several sports and to also improve performance during intense intermittent exercise (29). 
 
1.4 SIGNIFICANCE 
 
The proposed research responds to a critical need to elucidate peripheral mechanisms that 
contribute to functional limitations of older HF in older adults (i.e., abnormal skeletal muscle 
bioenergetics) and to assess the related benefits of a novel therapy that can potentially mitigate 
these abnormalities, and thereby enhance vital functional attributes of HF patients.  
Theoretically, both HFrEF and HFpEF patients may benefit.  Potential benefits pertaining to 
HFpEF patients are particularly exciting to consider, as currently there is no established therapy 
for this common disease. 
 
2.   RESEARCH DESIGN AND METHODS 
 
2.1 CLASSIFICATION AND METHODOLOGICAL DESIGNS 
 
The study is a 4-week open-label, single-center, non-randomized drug treatment study of 3 
groups: HFpEF, HFrEF, healthy age-matched controls.  
 
2.2 DETAILED DESCRIPTION OF STUDY DESIGN 
 
Subjects will undergo a screening visit to determine that eligibility requirements are met. Prior to 
receiving the study drug, subjects will be scheduled within 4 weeks of screening for a baseline 
assessment. 
 
Subjects who meet the Inclusion Criteria and none of the Exclusion Criteria will be provided study 
drug. The study population (n=30) will receive oral sodium nitrite combination for 4 weeks: 
 
Drug: oral formulation of sodium nitrite 20 or 40 mg oral three times daily or TID (once in 
the morning and again in the early afternoon and again in the evening) 
 
Subjects will complete a 4-week outpatient treatment period. During the treatment period, subjects 
will be evaluated at weekly intervals. Throughout the study period, subjects will be asked to remain 
diligent in recording the time of study drug dosing and any associated symptoms (diary cards will 
be pre-dated). 
 
2.2.1. Study Drug Preparation and Distribution 


Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
11 
 
The nitrite formulations will be prepared at and obtained from the Investigational Drug Pharmacy 
Service at the National Institutes of Health (NIH-IDS). The University of Pittsburgh Medical Center 
Investigational Drug Pharmacy Service (UPMC-IDS) will be utilized for dispensing of the study 
drug. 
 
The daily study drug is: 
 
14N Sodium nitrite  
Standard sodium nitrite will be supplied as one or two 20 mg capsules, three times daily oral 
administration at the dose strength of 60 or 120 mg qd. 
 
2.2.2. Dose Selection 
The Joint Food and Agriculture Organization (FAO)/World Health Organization (WHO) Expert 
Committee on Food Additives (JECFA) proposed an Acceptable Daily Intake (ADI) for nitrate in 
1990 based on harmless daily intakes of ≤500 mg of sodium nitrate/per kg of body weight in rats 
and dogs. They divided this dose by an uncertainty factor of 100 to yield an ADI of 5 mg of sodium 
nitrate or 3.7 mg nitrate ion per kg of body weight. An ADI for nitrite ion was derived and revised 
in 2003 based on intakes of 10 mg of sodium nitrite or 6.7 mg nitrite ion per kg of body weight in 
rats. They divided this dose by an uncertainty factor of 100 and rounded to yield an ADI for sodium 
nitrite of 0.1 mg/kg of body weight.  
 
A recent modeling of the fruit and vegetable consumption patterns of The Dietary Approaches to 
Stop Hypertension (DASH) diet was done choosing foods that were low or high in nitrate. The 
“high nitrate” DASH diet pattern would result in consumption of 1,222 mg nitrate ion and 0.351 
mg nitrite ion daily. The “low nitrate” DASH diet would result in consumption of 174 mg nitrate ion 
and 0.41 mg nitrite ion daily. The hypothetical high-nitrate DASH diet pattern exceeds the WHO’s 
ADI for nitrate ion by 550% for a 60-kg adult. Subjects on this diet exhibited lower blood pressure 
with no adverse effects.  
 
In a recent study, single dose potassium nitrate (KNO3) capsule ingestion in healthy adult 
volunteers in doses of 2,424 mg KNO3 (or 1,488 mg nitrate) and 1,212 mg KNO3 (or 744 mg 
nitrate) resulted in dose dependent reductions in BP over a 24 hour period without adverse effects.  
 
Hunault et al. reported an open-label pharmacokinetic crossover study in 9 healthy adult subjects 
who received two single high dose oral sodium nitrite solutions equivalent to 290-380 mg and 
140-190 mg sodium nitrite, respectively, and one intravenous sodium nitrite dose of 290-380 mg. 
Mild headache occurred in 44-55% and up to 22% experienced nausea, which subsided within 
half an hour during each treatment session. Lowered blood pressure accompanied by increased 
heart rate were observed after each higher dose treatment, whereas the lower dose did not induce 
notable changes. 
 
Chronic dietary supplementation of inorganic sodium nitrate in obese, hypertensive, insulin 
resistant mice lacking the gene for endothelial NO synthase (eNOS) (20) was studied. With a 10 
week treatment of a modest dose of sodium nitrate of 0.1 mmol/kg/day (8.5 mg/kg/day) in the 
drinking water, significant reductions in blood pressure, fasting blood glucose, HbA1C, and 
proinsulin to insulin ratio were demonstrated, indicating reduced demands on the β-cell for insulin 
secretion. 
 
Sodium nitrate drug is considered a source of nitrite through salivary nitrate reduction. Despite 
the plasma nitrite rise following a single oral sodium nitrate dose, Drs. Gladwin, Hughan and Shiva 
recently demonstrated in a phase I PK/PD study in 10 normal volunteers that no persistent blood 
pressure reduction with sodium nitrate 1,000 mg. In contrast, in the same phase I PK/PD study in 


Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
12 
 
10 normal volunteers, they showed blood pressure decline in systolic, diastolic and mean arterial 
pressures over 3 hours with sodium nitrite 20 mg. The oral sodium nitrite half-life was about 40-
minutes and the effects of nitrite only lasted ≤6 hours. This was demonstrated by the plasma 
nitrite levels returning to baseline levels between 3 and 6 hours after nitrite dosing (i.e. the drug 
was given and gone out of the circulation within 6 hours). Specifically, mean baseline (pre-nitrite 
dosing) blood nitrite concentrations were 0.129 micromolar with a peak at 30 minutes of 5.5 
micromolar, and return to baseline levels between the 3- (0.176 micromolar) and 6-hour (0.103 
micromolar) monitoring time points. In addition, there were no serious adverse events when these 
normotensive individuals received sodium nitrite. 
 
Next, a recently published study (Mohler III ER, Hiatt WR, Gornik HL, Kevil CG, Quyyumi A, 
Haynes WG, Annex BH. Sodium nitrite in patients with peripheral artery disease and diabetes 
mellitus: safety, walking distance and endothelial function. Vascular Medicine 2014; 19(1):10-17) 
randomized adults 1:1:1 to oral sodium nitrite 40 mg BID vs. 80 mg BID vs. placebo for 10 weeks 
in >50 adults (age 35-85 years) with peripheral arterial disease +/- diabetes. The dose was 
escalated after the 10 weeks for 1 additional week in all 3 groups (dose-doubling) to 80 mg BID 
vs. 160 mg BID vs. doubling of placebo, respectively. The dosing was well tolerated, especially in 
the 40 mg BID and 80 mg BID randomized dosings. (Our proposed study dosing of 20-40 mg TID 
(60-120 mg total daily) falls just below and in the middle of the above two total daily doses of 80 
mg and 160 mg daily.) Safety was monitored throughout all aspects of the study. No serious 
adverse events occurred in the two sodium nitrite groups. There were no imbalances in GI, 
musculoskeletal, respiratory, vascular or skin disorders nor shifts in liver function or creatinine 
levels. Pertinent safety outcomes revealed that methemoglobin changes never exceeded 3% in 
any nitrite dose regimen and no clinically significant change in resting blood pressure.  
 
A current phase II study of inhaled nitrite in patients with pulmonary arterial hypertension (PAH) 
at the University of Pittsburgh uses a dose of 80 mg, and is approved by the University of 
Pittsburgh IRB and FDA. Also, phase II studies went as high as 90 mg three times a day, approved 
by the FDA. So, our dose of 20-40 mg TID will not be out of line of approved dosing.  
 
Nine of 10 control subject enrollees into this pilot study have tolerated 40 mg po tid without 
incident; the one exception was a subject who had a decrease in systolic BP at pK but was 
asymptomatic. In the three heart failure subjects enrolled to date we have observed a vasoactive 
response at the pK with the 40 mg dose, such that low blood pressures or symptoms have been 
observed in the 2 hour monitoring period. This includes subjects with preserved and reduced 
ejection fraction. Therefore, we postulate the heart failure subjects appear to have a greater 
vasodilatory response to nitrite and could require a stratified dose based on age and baseline risk 
for hypotension. To further ameliorate risk, we will serve a standardized meal prior to drug dosing 
at Visit 3 so that lack of po intake is not a causative factor of hypotension. 
 
In summary, based on the above findings, inclusive of: 
• 
The Joint FAO/WHO ADI for sodium nitrate and nitrite, 
• 
the above DASH diet patterns, 
• 
the recently observed human and rodent effects of nitrate and nitrite on blood pressure 
and glucose homeostasis, including our PK/PD trial of single doses of oral nitrate and 
nitrite, 
• 
the open label trial safety and BP findings following a total of 10 subjects' completion, 
• 
the existing inhaled nitrite trial, 
• 
the preliminary findings of the current nitrite protocol with regard to blood pressure in heart 
failure subjects,  


Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
13 
 
the study investigators believe that a safe and likely therapeutically effective dose of sodium nitrite 
will be 20-40 mg three times daily (~0.21-0.42 mg/kg body weight per day for a 95 kg subject). 
The 40 mg sodium nitrite dose selection is equivalent to 12.6 times the ADI.  
 
2.2.3. Treatment Period 
Subjects will undergo study treatment duration of 4 weeks of 14N sodium nitrite 20-40 mg three 
times daily. This is considered appropriate to study benefits of oral nitrite therapy to improve 
physical function in patients with HFrEF and HFpEF and age-matched controls. 
 
2.2.4. Safety Monitoring: 
Subjects will be contacted by phone weekly and this will include symptom review, assessing 
adverse events, interval histories and medication compliance. 
 
In addition to these phone 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 the subject will be asked to return to the EMRC or SMART Center as soon as 
possible for evaluation. Evaluations will be planned on an individual basis (depending on each 
individual’s circumstances) and may include weight, vital signs, methemoglobin %, medication 
compliance review, and assessment by the study physician. If appropriate, based upon the 
evaluation, medical treatments will be provided to subjects, including appropriate referral to 
physicians or other services at the UPMC.  
 
2.2.5. Medication Compliance 
Subjects will self-administer their first dose of study medication under the supervision of the CTRC 
nurse (physician investigator will be immediately available) in the outpatient CTRC during Visit 3, 
following completion of all baseline clinic assessments. Subjects will also be dispensed their study 
medication (42-day supply) for home use, including four 7-day prefilled and labelled pill boxes 
and drug/symptom diary cards to help with compliance and to help reduce burden or confusion. 
(Visit 4 may be scheduled up to and on the 42nd day from visit 3 (25-35±5 days); additional drug 
will be distributed with the initial dispensation if needed to meet the need.) Subjects will return the 
previously dispensed study medication when they return for Visit 4 approximately 4 weeks later 
(+1 week if need be for flexibility of scheduling) and medication compliance will be assessed. The 
study coordinator will review the daily diary card with each phone call evaluation and also assess 
medication compliance with queries regarding empty pill compartments in the current 7-day box 
and remaining boxes. In the event that the compliance rate is <80% (subjects’ report of doses 
taken/total doses prescribed at that date & time *100), subjects will be re-educated on medication 
compliance. If medication compliance repeatedly falls outside of the acceptable range, the study 
investigators will discuss subject eligibility for continued participation in the study.  
 
2.2.6. Medication Accountability 
The study investigators or the study coordinator will document the amount of study drug 
dispensed and/or administered to subjects, the amount returned by subjects, and the amount 
received from and returned to the UPMC-IDS. The study drug accountability records will be 
maintained throughout the course of the clinical trial. 
 
Dose reduction 
There is a rare possibility that subjects will have a high methemoglobin level or low blood pressure 
from the study drug dose during visit 3 or while they are on the drug. If this happens, they may 
experience lightheadedness or dizziness.   
 
In CTRC: 


Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
14 
 
If this occurs in the CTRC, care will be administered, including fluids and monitoring, to ensure 
that the problem resolves and patient is stable. The physician investigator will factor relevant 
subject symptoms and hemodynamics and then decide whether it is appropriate for the subject 
to proceed with study participation prior to discharge from CTRC. This will include what dose is 
appropriate and drug dispensing. The same process will be applied to subjects who are initially 
initiated on 20 mg; however, in these subjects there is relatively greater consideration of 
withdrawal as no lower dosing options are available. 
 
As outpatient: 
Blood pressure and related symptoms. If subjects experience lightheadedness or dizziness after 
visit 3 while they are on the drug, we request that they call the study coordinator who will inventory 
their symptoms, compare to baseline levels (measured at pK), and discuss need for interim visit 
with study physician. If a visit is indicated, we will measure blood pressure, RR, heart rate, and 
check methemoglobin level by a finger probe. A study physician will evaluate each subject, 
including: 
1. review of all clinical measures,  
2. subject history of symptoms, 
3. blood pressures pre- and while on drug.  
The physician will confirm a management plan including the possibility of lowering dose to 20 mg 
tid or need to discontinue and drop from the trial. Subjects will be reassessed via phone each day 
for the next 2 days thereafter to ensure that the patient is stable on the new dose.  If stable, 
subjects will continue with the half dose.  However if there are continued symptoms on lower 
doses, the physician investigator will reevaluate the patient’s suitability for continued participation 
based on symptoms and/or hemodynamics.  
If subjects initiated at Visit 3 on the 20 mg dose report symptoms as described above, the same 
process will be followed; however, the physician may have to withdraw the subject since a dose 
lower than 20 mg tid is not an option. 
 
 
Methemoglobin. If subjects report change in skin tone and dizziness or other signs of 
methemoglobinemia for >24 hours, an interim visit will be requested. Methemoglobin will be 
assessed. If greater than 5%, a nitrite dose reduction is proposed from 40 mg three times daily to 
20 mg three times daily.  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 methemoglobin testing after 3 days.  If 
level persists above 5%, participation in the study for the subject will be discontinued.  
 
Safety of holding anti-platelet or antithrombotic medications 
The research physician and the staff under his auspice will guide holding of the antiplatelet and 
anti-thrombotic medications. In each case, this will be discussed doctor to doctor by the research 
doctor and the patient’s PCP or cardiologist.  
 
 
2.3 STUDY ASSESSMENT 
 
The complete study assessment and procedures are outlined in Table 1.  
 
2.3.1. Study Visits and Procedures 
 
Visit 1 (Screening) 
 


Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
15 
 
The outpatient screening will take place in either the Montefiore Clinical and Translational 
Research Center (CTRC) or the Endocrinology Metabolism Research Center (EMRC) at the 
UPMC Montefiore Hospital and will last approximately 2-3 hours. 
 
• Obtain written informed consent 
• Complete history and physical examination to include body weight and height 
• Medical history obtained prior to visit will be reviewed with subject. Subjects will be asked 
questions about any heart disease and related diseases in first degree relatives; no identifying 
information will be obtained.Dr. Forman will review risk factors for hypotension in subjects who 
are 70 to less than 80 years of age at the physical exam, in preparation for dose selection. 
Subjects will be asked while they are in the study to report promptly any changes in their health 
or medications so that the PI can evaluate any implications with study drug or participation. 
• Hemoglobin and hematocrit to assess for anemia  
• Blood pressure and other vital signs (temperature, pulse oximetry, breathing rate, heart rate) 
Blood pressure may be re-measured multiple times if out of inclusion range. This is to allow for 
influences of medication activation, subject nervousness, lack of fluid intake, and known factors 
which contribute to clinical variation.  
• Transthoracic Echocardiogram (performed by trained study staff) - results of echo will confirm 
group placement. If subjects have had an echocardiogram within 4 weeks of the consent date, 
the records will be obtained prior to visit 1 and the test will be reviewed and interpreted by a study 
physician. Trained study staff doing echos will consist of cardiologists doing advanced echo 
training at UPMC under the supervision of Dr. Gorcsan. Drs. Gorcsan, Simon and Forman are 
responsible for all echocardiography procedures and interpretations. The subject may be 
transported to a lab in the UPMC Presbyterian/Montefiore system for the echocardiogram. 
 
Screening evaluations will include a hemoglobin and hematocrit by the Presbyterian Hospital 
clinical automated testing lab and are to be covered by research funds. The total volume of blood 
drawn is approximately 1 teaspoon (4 mL). If laboratory results exclude low hemoglobin (<11.0 
g/dL males, <10.0 females) the subject will be called to arrange the baseline assessments. 
 
Subjects who fail to meet applicable inclusion/exclusion criteria based upon the results of the 
screening assessment will be excluded from further study participation.  
 
An important component of the protocol is the muscle biopsy. Subjects who refuse the first biopsy 
will be excluded at screening. Participants who undergo the first biopsy at Visit 3 may refuse a 
second pass. (Participants may also refuse a second biopsy pass at Visit 5). However, 
participants who refuse to complete a second biopsy at Visit 5 (prior to starting sodium nitrite) will 
be withdrawn from the study. Alternatively, patients who refuse the 2nd biopsy after they have 
completed the 4 weeks of nitrite therapy will still be included. 
 
Visit 2 (First Baseline Assessment Visit)  
 
Subjects will be scheduled for an outpatient EMRC and Pepper SMART Center visit within 4 
weeks of screening. The subject will be asked to refrain from caffeine the morning of this visit and 
to limit breakfast to a lighter meal prior to coming in. The visit will last approximately 3 hours. 
 
Interval history and brief physical exam: temperature, blood pressure, pulse oximetry, breathing 
rate, heart rate and body weight 
 
Physical Function Battery: 
● Cardiopulmonary Stress Exercise Testing: Exercise testing in association with air-gas-
exchange, an optimal gauge of aerobic capacity will be conducted by trained research exercise 


Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
16 
 
physiologists. Dr. Forman or another physician will be immediately available if needed. A 
computerized cycle ergometer using a ramp protocol will be used to generate a symptom limited 
exercise stimulus. A lightweight disposable pneumotach device face mask will be positioned over 
the subject’s mouth and nose during the exercise for gas exchange assessments of VO2, 
VE/VCO2 slope, and respiratory exchange ratio (RER) will be measured as well as 
hemodynamics (max heart rate [HR] and blood pressure [BP]), time, and ECG waveforms). Any 
unexpected abnormalities will be reported to a patient’s cardiologist or primary care physician; 
continued participation in the study will require physician clearance. Both the Borg Rate of 
Perceived Exertion (RPE) and the Modified Borg Scale for Perceived Dyspnea (Shortness of 
Breath) will be completed during the CPX. The EMRC has performed similar tests in more than 
1,000 volunteers ranging from elderly women in their 80’s to highly trained athletes. 
 
 
● Short Physical Performance Battery (SPPB): a brief performance battery is used to assess how 
well older persons perform simple movements that represent the building blocks of daily activities 
that require good lower extremity function. It is based on timed 4 meter walk, repeated chair 
stands, and a set of balance tests. 
 
● Fatigability will be assessed based on rate of perceived exertion (RPE 6-20 scale) reported 
after a steady state walking on a treadmill. Patents will walk 1.5 miles/hour (0.67 m/sec) for 5 
minutes on a treadmill as per techniques validated by Simonsick, et al. (J Am Geriatr Soc. 
2014;62:347-51). Those with RPE ≥10 will be categorized as highly fatigued. Our prior work in 
HF patients indicates this steady state walking provocation is feasible for the patients we 
anticipate we will enroll, and that it will (appropriately) generate a wide range of perceived 
fatigability responses for meaningful analyses of underlying mechanisms. Aerobic capacity will be 
assessed with gas-exchange (as noted above) during the steady state walk for VO2, VE/VCO2 
slope, and respiratory exchange ratio (RER).  
 
● Hand Grip Strength Assessment will be performed to measure muscle strength. Participants 
will be asked to sit in a chair without arms and squeeze a device as hard as they can with one 
hand at a time, rest and switch to the other hand. 
 
● The Kansas City Cardiomyopathy Questionnaire will be used to measure disease-specific 
health-related quality of life (QOL). This 23-item instrument produces the following scores: 
Physical Limitation, Symptoms, Symptom Stability, Social Limitation, Self-efficacy, Quality of Life, 
and two summary measures, and is widely used as a standard by which quality of life, self-
efficacy, and other personal perspectives related to functional capacity at baseline and over time. 
Subjects in the healthy elderly control group without HF will be asked to complete the SF-12 which 
is a practical, reliable and valid measure of physical health. 
 
● 24-hour dietary recall. The study coordinator will collect a 24-recall of all foods eaten the day 
before from subjects for analysis for calories, macronutrients, sodium, nitrates and nitrites. 
 
Visit 3 (Second Baseline Assessment Visit) 
 
This visit involves a percutaneous muscle biopsy, blood draw, first dose of study medication and 
dispensing of study medication. To prevent acute effects of exercise on muscle mitochondria 
function, this fasting visit will occur 2-7 days after Visit 2. This visit will take place at the MUH-
CTRC and will last 4-5 hours. Subjects will be asked to observe 8 hours fasting prior to arrival. 
Upon arrival subjects will be weighed and then served a standardized breakfast [15% of 30 
calories/kg current body weight (CBW) and 15% of 1.0 gm protein/kg CBW, low nitrite/nitrate] in 


Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
17 
 
the CTRC. A dietitian will review and adjust subjects’ food choices as needed to meet the 
standardized diet prescription.  
Interval history and brief physical exam: temperature, blood pressure, pulse oximetry, breathing 
rate, heart rate and body weight. Blood pressures may be re-measured to allow for clinical 
variation. Prior to the biopsy, study staff will call indicated subjects to hold their anti-platelet and 
anti-coagulation medications for 1-5 days (depending on the different medications and relative 
half-lives). In each instance a subject is on an anti-platelet or anti-coagulation medication, the 
plan to hold the medication prior to the biopsy will first be cleared by the study physician with the 
subject’s own primary care provider or cardiologist. Physician orders for CTRC will include 
confirmation with subject upon arrival. 
 
• Blood sampling via IV line to include:  research blood for inflammatory peptides (IL-1, IL-6, IL-
15, TNFα), platelet sample and nitrite levels. Blood samples will be drawn into a 1) heparinized 
syringe (baseline and approximately 30 mins. and 2 hours post study drug administration) and 2) 
a cell preparation tube (CPT) (baseline and approximately 30 mins and 2 hours post study drug 
administration). Heparinized blood will be immediately centrifuged at bedside to separate plasma 
and the plasma and red cells separately flash frozen for later nitrite measurements (via reductive 
chemiluminescence to determine that nitrite levels were increased in the muscle as described. 
The second tube of blood (CPT tube) will be immediately subjected to differential centrifugation 
in order to isolate platelets. Platelets will then be subjected to Seahorse extracellular flux analysis 
in order to measure bioenergetic profile including basal, non-ATP producing, maximal and non-
mitochondrial respiratory rates along with glycolytic rate. Extra aliquots will be stored for use in 
future studies. Two 10 ml purple top and 2 10 ml red top tubes will be used to collect blood for 
inflammatory peptides. Total volume of blood drawn will be 88 ml (6 tablespoons). 
 
• Skeletal Muscle Biopsies:  Patients will lie comfortably in the supine position for biopsy of the 
vastus lateralis muscle of the non-dominant leg. Biopsy sites will be prepped with betadine. 2% 
xylocaine (without epinephrine) will then be administered, superficially initially and then more 
deeply into the muscle, using a small gauge needle. Subjects who have report a history of poor 
pain control or low threshold will be offered the use of a topical anesthetic cream prior to the 
xylocaine. Ultrasound may be used to enhance visualization of the site for the needle insertion. A 
small superficial incision will next be completed using a #11 blade scalpel. A 5 mm Bergstrom 
muscle biopsy needle will be inserted through the skin incision and advanced into the muscle. 
Suction will be generated using a syringe attached to the outside portion of the needle, to thereby 
suck skeletal muscle into a hole on the side of the needle positioned in the muscle tissue; this 
draws a small piece of muscle tissue (about 150 mg of muscle tissue) into the hollow shaft of the 
needle, which is then cut with a cutting trochar that slides through the shaft to cut the tissue drawn 
within its core. After harvesting the first sample, the needle will be rotated 90 degrees, and a 
second sample extracted to maximize yield for analysis. Additional passes of the needle may be 
necessary for an adequate muscle sample. The wound site will be closed with steri-strips and a 
sterile pressure bandage. Muscle specimens will be immediately processed, and stored. One 
fragment will be placed in sterile cryotubes containing RNA preservation solution (RNAlater) and 
immediately frozen in liquid nitrogen for measurement of mRNA expression. A second fragment 
will have the muscle fibers aligned for microscopy, placed in OCT, then cooled in a thawing 
isopentane slurry and placed in a cryotube and frozen in liquid nitrogen for morphological analysis. 
One specimen will immediately be used for high resolution respirometry. Briefly, muscle fibers will 
be dissected and immediately placed in an Oroboros respirometry. Basal respiratory rate will be 
measured followed by respiration after the administration of oligomycin A to assess non-
productive respiration and FCCP to measure maximal respiratory capacity. A separate muscle 
specimen will be saved for Western blots and to measure nitrite concentrations by reductive 
chemiluminescence. 
 


Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
18 
 
Subjects will rest for approximately 30 minutes post biopsy while wound is dressed and prior to 
baseline vitals prior to dosing. Study staff will record a baseline level of symptoms, including 
shortness of breath, dizziness, lightheadedness for use during weekly phone calls, prior to dosing. 
 
Following biopsy, subjects will receive their first dose of study drug as follows: 
• 14N sodium nitrite, 20 or 40 mg per physician order 
• Blood pressure (sitting and then standing after 3 minutes), HR, RR, pulse oximeter and 
methemoglobin will be measured to ensure they are at a steady state before taking the study drug 
(baseline) and then approximately every 15 minutes for the 2 hours post-drug administration. Any 
perturbations of concern will be addressed per the Dose Reduction protocol described in Section 
2.2.6. 
• AE assessment 
• Provide a 42-day supply of study drug, pre-filled in 4 weekly pill boxes and an extra bottle and 
drug diary cards to assist with compliance and reduce burden or confusion. Study staff will review 
medication administration and use of the drug diary card with all subjects. Study staff or CTRC 
RN will review biopsy site care with subjects and if indicated, remind subjects to resume their 
normal anti-coagulation and anti-platelet medications the following day (as indicated). 
 
● 24-hour dietary recall. The study coordinator will collect a second 24-recall of all foods eaten 
from subjects for analysis for calories, macronutrients, sodium, nitrates and nitrites. 
 
Subjects will be reminded before departure to report promptly any changes in their medication, 
health or symptoms of concern, not to wait for the weekly call. 
 
Visit 4 (First Completion Assessment visit to be scheduled 25-35±7 days following first dosing of 
20-40 mg oral nitrite) Subjects who need more than 42 days’ supply of drug due to scheduling 
later in window will be issued additional at visit 3. 
 
This visit will take place in the EMRC and/or Pepper SMART Center and is identical to visit 2 
(approximately 3 hours): 
 
Interval history and brief physical exam: temperature, blood pressure, pulse oximetry for 
methemoglobin %, breathing rate, heart rate and body weight; additionally, AE and symptom 
assessment will be collected and medication compliance via drug diary cards and pill boxes will 
be assessed.  
 
Physical Function Battery: 
● Cardiopulmonary Stress Exercise Testing: Exercise testing in association with air-gas-
exchange, an optimal gauge of aerobic capacity will be conducted by trained research exercise 
physiologists. Dr. Forman or another physician will be immediately available if needed. A 
computerized cycle ergometer using a ramp protocol will be used to generate a symptom limited 
exercise stimulus. A lightweight disposable pneumotach device face mask will be positioned over 
a subject’s mouth and nose during the exercise for gas exchange assessments  VO2, VE/VCO2 
slope, and respiratory exchange ratio (RER) will be measured as well as hemodynamics (max 
heart rate [HR] and blood pressure [BP]), time, and ECG waveforms). Any unexpected 
abnormalities will be reported to a patient’s cardiologist or primary care physician; continued 
participation in the study will require physician clearance. Both the Borg Rate of Perceived 
Exertion (RPE) and the Modified Borg Scale for Perceived Dyspnea (Shortness of Breath) will be 
completed during the CPX. The EMRC has performed similar tests in more than 1,000 volunteers 
ranging from elderly women in their 80’s to highly trained athletes. 
 


Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
19 
 
● Short Physical Performance Battery (SPPB): a brief performance battery is used assess how 
well older persons perform simple movements that represent the building blocks of daily activities 
that require good lower extremity function. It is based on timed 4 meter walk, repeated chair 
stands, and a set of balance tests. 
 
● Fatigability will be assessed based on rate of perceived exertion (RPE 6-20 scale) reported 
after a steady state walking on a treadmill. Patents will walk 1.5 miles/hour (0.67 m/sec) for 5 
minutes on a treadmill as per techniques validated by Simonsick, et al. (J Am Geriatr Soc. 
2014;62:347-51). Those with RPE ≥10 will be categorized as highly fatigued. Our prior work in 
HF patients indicates this steady state walking provocation is feasible for the patients we 
anticipate we will enroll, and that it will (appropriately) generate a wide range of perceived 
fatigability responses for meaningful analyses of underlying mechanisms.  
 
● Hand Grip Strength Assessment will be performed to measure muscle strength. Participants 
will be asked to sit in a chair without arms and squeeze a device as hard as they can with one 
hand at a time, rest and switch to the other hand. 
 
● The Kansas City Cardiomyopathy Questionnaire will be used to measure disease-specific 
health-related quality of life (QOL). This 24-item instrument produces the following scores: 
Physical Limitation, Symptoms, Symptom Stability, Social Limitation, Self-efficacy, Quality of Life, 
and two summary measures, and is widely used as a standard by which quality of life, self-
efficacy, and other personal perspectives related to functional capacity at baseline and over time. 
Subjects in the healthy elderly control group will be asked to complete the SF-12. 
 
● 24-hour Dietary recall. The study coordinator will collect a post 24-recall of all foods eaten from 
subjects for analysis for calories, macronutrients, sodium, nitrates and nitrites. 
 
Visit 5 (Final Completion Visit - 2-7±7 days following Visit 4)  
 
Subjects will be fasting and take their last dose of study drug at home on their usual schedule 
prior to scheduled testing time. Upon arrival to the CTRC, subjects will be served a standardized 
breakfast as described in visit 3, then interval history and brief physical exam; temperature, blood 
pressure, pulse oximetry for methemoglobin %, breathing rate, heart rate and body weight will be 
documented; additionally, AE and symptom assessment, medication compliance via drug diary 
cards and pill boxes, and confirmation of last dose will be performed prior to assessment 
procedures. Prior to the biopsy, study staff will call indicated subjects to hold their anti-platelet 
and anti-coagulation medications for 1-5 days (depending on the different medications and 
relative half-lives). This visit will last approximately 2 hours. 
 
• Blood draw to include: research blood for inflammatory peptides (IL-1, IL-6, IL-15, TNFα), platelet 
sample and nitrite levels: Two blood samples will be drawn into a 1) heparinized syringe and 2) a 
cell preparation tube (CPT). Heparinized blood will be immediately centrifuged at bedside to 
separate plasma and the plasma and red cells separately flash frozen for later nitrite 
measurements (via reductive chemiluminescence to determine that nitrite levels were increased 
in the muscle as described. The second tube of blood (CPT tube) will be immediately subjected 
to differential centrifugation in order to isolate platelets. Platelets will then be subjected to 
Seahorse extracellular flux analysis in order to measure bioenergetic profile including basal, non-
ATP producing, maximal and non-mitochondrial respiratory rates along with glycolytic rate. Extra 
aliquots will be stored for use in future studies. Total volume of blood drawn will be 56 ml (4 
tablespoons) 
 


Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
20 
 
• Skeletal Muscle Biopsies:  Patients will lie comfortably in the supine position for biopsy of the 
vastus lateralis muscle of the non-dominant leg. Biopsy sites will be prepped with betadine. 2% 
xylocaine (without epinephrine) will then be administered, superficially initially and then more 
deeply into the muscle, using a small gauge needle. Subjects who have report a history of poor 
pain control or low threshold will be offered the use of a topical anesthetic cream prior to the 
xylocaine. Ultrasound may be used to enhance visualization of the site for the needle insertion.A 
small superficial incision will next be completed using a #11 blade scalpel. A 5 mm Bergstrom 
muscle biopsy needle will be inserted through the skin incision and advanced into the muscle. 
Suction will be generated using a syringe attached to the outside portion of the needle, to 
thereby suck skeletal muscle into a hole on the side of the needle positioned in the muscle 
tissue; this draws a small piece of muscle tissue (about 150 mg of muscle tissue) into the hollow 
shaft of the needle, which is then cut with a cutting trochar that slides through the shaft to cut 
the tissue drawn within its core. After harvesting the first sample, the needle will be rotated 90 
degrees, and a second sample extracted to maximize yield for analysis. Additional passes of the 
needle may be necessary for an adequate muscle sample. The wound site will be closed with 
steri-strips and a sterile pressure bandage. Muscle specimens will be immediately processed, 
and stored. One fragment will be placed in sterile cryotubes containing RNA preservation 
solution (RNAlater) and immediately frozen in liquid nitrogen for measurement of mRNA 
expression. A second fragment will have the muscle fibers aligned for microscopy, placed in 
OCT, then cooled in a thawing isopentane slurry and placed in a cryotube and frozen in liquid 
nitrogen for morphological analysis. One specimen will immediately be used for high resolution 
respirometry. Briefly, muscle fibers will be dissected and immediately placed in an Oroboros 
respirometry. Basal respiratory rate will be measured followed by respiration after the 
administration of oligomycin A to assess non-productive respiration and FCCP to measure 
maximal respiratory capacity. A separate muscle specimen will be saved for Western blots and 
to measure nitrite concentrations by reductive chemiluminescence. 
 
  
• Transthoracic Echocardiogram. The subject may be transported to UPMC Presbyterian for the 
echocardiogram. 
 
● 24-hour Dietary recall. The study coordinator will collect a second post 24-recall of all foods 
eaten the day before from subjects for analysis for calories, macronutrients, sodium, nitrates and 
nitrites. 
 
End of Trial and Follow-Up Period  
 
Post Study Drug Telephone Assessment (about 1 week following Visit 5) 
• Telephone assessment for interval histories, muscle biopsy follow up and AEs.


Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
21 
 
Table 1. Study Assessment and Procedures 
 
 
Screening 
Baseline 
 
Final 
FU 
 Visit 
1 
2 
3 
Phone call follow-ups during 
intervention time 
4 
5 
Phone call 
 
 
 
 
1 
2 
3 
4 
 
 
1 
Week of Treatment 
- 
-4~-1 
-4~-1 
1 
2 
3 
4 
5 
5 
6 
Day of Treatment 
- 
 
 
7 
~14 
~21 
~28 
~25-
35 
V4+2-
7d 
 
Time Window 
 
V1+not
>4 wks. 
V2+ 
2-7d 
±2d 
±7d 
±7d 
±7 
±7d 
 
About 1 week 
following V5 
Visit Type 
OP 
OP 
OP 
Phone 
Phone 
Phone 
Phone 
OP 
OP 
Phone 
Informed Consent / Eligibility Criteria  
x 
 
 
 
 
 
 
 
 
 
History & Physical Exam 
x 
 
 
 
 
 
 
 
 
 
Body Weight, Height (V1 only) 
x 
x 
x 
 
 
 
 
x 
x 
 
Vital signs 
x 
x 
x 
 
 
 
 
x 
x 
 
Methemoglobin % 
 
 
x 
 
 
 
 
x 
x 
 
Research Labs 
 
 
x 
 
 
 
 
 
x 
 
Two Hour Safety Blood Sampling 
 
 
x 
 
 
 
 
 
 
 
Two Hour HR, BP (sitting and after  
standing 3 mins.), Methemoglobin 
Monitoring:  baseline & q15 min. 
 
 
x 
 
 
 
 
 
 
 
Hemoglobin & hematocrit 
x 
 
 
 
 
 
 
 
 
 
Echocardiogram 
x 
 
 
 
 
 
 
 
x 
 
Muscle Biopsy 
 
 
x 
 
 
 
 
 
x 
 
Physical Function Testing* 
 
x 
 
 
 
 
 
x 
 
 
Questionnaire-KCCQ or SF-12 
 
x 
 
 
 
 
 
x 
 
 
Meal/snack 
 
 
xs 
 
 
 
 
 
xs 
 
AE Assessment 
 
 
x 
x 
x 
x 
x 
x 
x 
x 
Medication Compliance Review 
 
 
 
x 
x 
x 
x 
x 
x 
 
Interval History Assessment  
 
x 
x 
x 
x 
x 
x 
x 
x 
X 
Brief Physical Exam 
 
x 
x 
 
 
 
 
x 
x 
 
Symptom review 
 
 
x 
x 
x 
x 
x 
x 
x 
x 
24 hour dietary recall 
 
x 
x 
 
 
 
 
x 
x 
 
sStandard kitchen meal/snack 
 
*includes nCPET, Steady State Walking, SPPB and Hand-grip strength


Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
22 
 
2.4 STUDY ENDPOINTS 
 
Overall, study endpoints pre- and post- nitrites will include:  
1. Skeletal muscle bioenergetics: Mitochondrial function will be assessed using 
respirometry. Polymerase chain reaction (PCR) to assess pertinent gene expression 
(ubiquitin [MuRF, Atrogin1, FoxO], FNDC5, PGC1α, Sirtuin 3).   
2. Serology: plasma BNP, inflammatory markers, plasma nitrite and platelet bioenergetics 
(using Seahorse XF analysis) (40), i.e., including glycolytic as well as basal and maximal 
respiratory rates.   
3. Measures of physical function will include CPX indices, gait speed, strength (endurance, 
power), and balance.   
4. Quality of life: Kansas City Cardiomyopathy Questionnaire. 
 
 
2.5 STATISTICAL ANALYSIS 
 
Sample Size Justification: We believe our proposed sample size is adequate for successfully 
addressing the aims of this initial investigation. Numerous published pilot studies leading to 
subsequent successful larger trials have used similar sample sizes (47, 48). Our prior data 
indicate between-subject standard deviation of approximately 32 pmol O2/min/106 cells in platelet 
mitochondrial respiration rate change subsequent to administration of nitrite (49).  Based on this 
estimate, with the proposed sample sizes, we will be able to detect statistical significance of a 
within-group change as small as 32 pmol O2/min/106 cells, and a between-group difference as 
small as 42 pmol O2/min/106 cells with 80% statistical power in two-tailed tests conducted at 
α=0.05.  These correspond to large effect sizes by Cohen’s d criteria (50), and thus afford an 
adequate level of statistical sensitivity in our pilot investigation. 
Data Analysis: Our analytic strategy will involve formal statistical analyses, but in our 
interpretation, we will also pay attention to descriptive statistics, graphical summarizations of 
individual participant data such as needle plots (51,52), and magnitudes of parameter estimates 
from modeling in addition to their statistical significance based on p-values and a strict criterion 
based on α=0.05.  Such an interpretive strategy is appropriate in an initial study such as ours, 
where the cost of a missed finding is far greater than a false finding.  We will fit a series of linear 
mixed models using the SAS® MIXED procedure (SAS Institute, Inc., Cary, North Carolina) with 
each of the skeletal muscle, platelet, gene expression, inflammatory, oxygen uptake and physical 
function related measures as the dependent variable; participant group (HFrEF/HFpEF/control), 
time point (baseline/4-week) and their interaction as fixed effects of interest; and a participant 
random effect to account for multiple measurements from the same participants.  We will 
appropriately create means contrasts to estimate (a) between time changes in each of the groups 
corresponding to periods of usual care and nitrite usage, and (b) compare the said changes 
across the three groups.  We will use Pearson/Spearman correlations to quantify strength of 
associations between changes in measures of skeletal physiology and those of strength, gait 
speed and balance.  Finally, in addition to drawing preliminary conclusions, we will use our 
descriptive statistics for formal sample size computations in planning a larger subsequent trial to 
more definitively examine the impact of nitrites. 
 
 
 
 
3.  HUMAN SUBJECTS 


Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
23 
 
 
 
3.1 SUBJECT POPULATION 
 
The anticipated age range for the study population will be >70 years of age based upon the target 
disease population. Thirty subjects with any ethnic background who have a confirmed diagnosis 
of HFrEF/HFpEF/healthy control will be eligible for enrollment. All subjects must provide written 
informed consent prior to participation.    
 
3.1.1 Inclusion of Women and Minority 
Women who meet the inclusion criteria, and have none of the exclusion criteria, will be enrolled 
without restriction as dictated by the study protocols. We will make efforts to enroll participants in 
this research in a distribution that mirrors the study population of the Pittsburgh area.   
 
3.1.2 Inclusion of Children 
This investigation will not enroll children based upon the target disease population and lack of safety 
data in adults. 
 
3.2 INCLUSION CRITERIA 
 
Inclusion Criteria HF Population 
- NYHA class II or III for the previous three months despite a minimum of 6 weeks of treatment. 
Echo criteria will be confirmed as part of the initial study assessment. 
- Age ≥70 years 
- HFrEF patients left ventricular ejection fraction [LVEF] <40% 
- HFpEF patients LVEF ≥40%, may also include E/E′ >8, left atrial size>40 mL/m2 
- Optimal therapy according to AHA/ACC and HFSA HFrEF guidelines, including treatment with 
ACEI and beta-blocker therapy (for at least 6 weeks), or have documented reason for variation, 
including medication intolerance, contraindication, patient preference, or personal physician's 
judgment. 
- Patients using aspirin (ASA) will be eligible, but asked to hold the medication for 48 hours prior 
to biopsy. This technique has previously been used with consistent safety. Patients will also be 
asked to avoid non-steroidal anti-inflammatory medications (NSAIDs) for 48 hours prior to the 
biopsy. 
- Patients using anti-thrombin and anti-platelet therapy will plan to modify prior to muscle 
biopsies individually in coordination with the participant's primary care provider or cardiologist. 
- Baseline BP ≤170/95: subjects with either systolic or diastolic over stated level are ineligible. 
- Subjects who report using PDE-5s will be asked to hold the medication 24 hours before initial 
nitrite dose and for the entire period on study drug. 
 
 
Inclusion Criteria Age-Matched Control Population 
- Age ≥70 years 
- Absence of clinically significant chronic disease processes (including cardiovascular disease, 
diabetes, cancer, CKD, liver disease, COPD, and/or osteoarthritis). Non-significant cardiac 
rhythm abnormalities and stable hypertension are acceptable for inclusion. 
- baseline BP ≤170/95: subjects with either systolic or diastolic over stated level are ineligible. 
- Patients using aspirin (ASA) or NSAIDs will be eligible, but asked to hold the medication for 48 
hours prior to biopsy. This technique has previously been used with consistent safety. Patients 


Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
24 
 
will also be asked to avoid non-steroidal anti-inflammatory medications (NSAIDs) for 48 hours 
prior to the biopsy. 
- Patients using anti-thrombin and anti-platelet therapy will hold these medications prior to 
muscle biopsies in coordination with each subject’s primary care provider or cardiologist. 
- Subjects who report using PDE-5s will be asked to hold the medication 24 hours before initial 
nitrite dose and for the entire period on study drug. 
 
 
3.3 EXCLUSION CRITERIA 
 
Exclusion Criteria for All participants   
- Allergy to lidocaine 
- Anemia (Hgb <11.0 males, <10.0 females) 
- Dementia  
- End-stage malignancy 
- Orthopedic exercise limitation 
- Chronic use of oral corticosteroids or other medications that affect muscle function. 
- Chronic ETOH or drug dependency.   
- Any bleeding disorder that would contraindicate biopsy such as history of clinically significant 
bleeding diathesis (e.g., Hemophilia A or B, Von Willebrand's Disease or congenital Factor VII 
deficiency). 
- Psychiatric hospitalization within the last 3 months 
- Chronic nitrate use  
- Baseline BP <100/60, either value 
- Refusal to participate in muscle biopsy 
 
Exclusion Criteria HF Population  
- Major cardiovascular event or procedure within the prior 6 weeks. 
- HF secondary to significant uncorrected primary valvular disease (except mitral regurgitation 
secondary to left ventricular dysfunction). If valve replacement has been performed, patient may 
not be enrolled for 12 months after this procedure. 
- Severe valvular heart disease 
- Mechanical valve replacement requiring warfarin 
- Currently taking clopidogrel or other anti-platelet medication for a recent stent placement 
and/or a complex atherosclerotic lesion such that holding clopidogrel (or an equivalent 
medication) creates disproportionate risk. This decision will always be determined by the 
patient’s own primary care provider or cardiologist. 
- ICD device with heart rate limits that prohibit exercise assessments. Referring physicians will 
be provided with an opportunity to reprogram devices so that patients can participate. 
 
 
4.  RECRUITMENT AND INFORMED CONSENT PROCEDURES 
 
 
4.1 RECRUITMENT METHODS 
 
Heart failure patients can be identified by screening of the clinics of the Heart and Vascular 
Institute, Cardiology, and Geriatrics in which the PI provides clinical services, by their referral from 
UPMC cardiologists or by flyers placed in the clinic areas.  


Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
25 
 
Dr. Forman will seek approval from physicians in those clinics to have the study coordinator pre-
screen the clinic schedules for eligible candidates on an ongoing basis. On the day of the clinic 
visit of identified candidates, the study coordinator will reaffirm with the subject's physician that 
recruitment of the subject is medically appropriate and that she has permission to speak with the 
candidate. After speaking with the subject, if the potential participant is interested in the study, 
he/she will be asked if it is acceptable to review the medical records further and finalize eligibility. 
Recruitment and enrollment procedures will then follow, including apprising PCP or cardiologist 
of their patient's involvement, (and managing hold of anti-coagulants per protocol, if needed). If 
due to unforeseen circumstances, the coordinator is unable to meet with the subject on the day 
of clinic visit, he/she will contact the subject (phone, email, postal mail). This will only be done if 
the attending physician has approved contact; the contact will always be initiated by first informing 
the subject that their physician has approved the contact.  
Subjects can also be recruited using flyers and advertisements including the use of the CTSI 
research registry and OCR study listing website. Potential subjects will be instructed to contact 
the investigators via a telephone number/email address that is provided in these advertisements. 
They will then be explained the study (with approved script) and with their permission, screened 
on the phone to make sure they comply with the eligibility criteria. Subjects’ medical records will 
be accessed prior to visit 1 to confirm that they meet eligibility criteria (request for access for 
screening purposes only, without consent to be obtainedIf the subjects are deemed eligible and 
are interested in the study, an outpatient screening visit (Visit 1) will be scheduled where the 
study, risks and potential benefits, and rights as a research subject will be described in detail and 
where eligibility will be confirmed. Screening data will be de-identified and stored in an on line 
document that is password protected and on a secure drive with access only by study PI and 
coordinator. Codes linking names and IDs will be on a separate document, also password 
protected on secure drive. 
 
4.2 INFORMED CONSENT PROCEDURES 
 
Subjects must provide informed consent. The information about this study will be given to the 
subject in language understandable to them. Either the physician investigator or a non-physician 
member of the research team will present the study. They will verbally present a general outline 
of the research plan, including inclusion and exclusion criteria, to the prospective participant. The 
consent form, outlining the design of the study, will include the risks and benefits of participating, 
and will be reviewed and the physician investigator and/or non-physician member of the research 
team will answer any questions. Prospective participants may take as much time as required to 
make an informed decision. Written informed consent will be obtained from each participant and 
the investigator prior to performing any research study procedures. 
 
 
5.  POTENTIAL RISKS AND BENEFITS 
 
 
5.1 POTENTIAL RISKS 
5.1.1 Risk of Experimental Drug Intervention 
Numerous studies have evaluated acute, subacute and chronic drinking water exposures of 
nitrate and nitrite in laboratory animals and drinking water and dietary exposures in humans. 
Recent studies are available using high doses of nitrite by oral route in the form of beet root juice. 
Recent studies have evaluated acute exposures of oral preparations of nitrite and nitrate 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 


Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
26 
 
emergency treatment of cyanide poisoning31,32. It is also notable that neutrapharmaceutical 
preparations are currently being sold with levels of nitrite (12.7 mg per tablet) and nitrate (3.9 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 (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 (300 mg) used in the emergency 
treatment of cyanide poisoning – do not produce methemoglobin levels greater than 3% and have 
not been associated with clinically significant hypotension. There have been no adverse events 
noted in the 80 treated normal human volunteers and patients with sickle cell disease 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. 
 


Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
27 
 
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 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 much lower doses of nitrite than used safely in any of these 
studies. Our nitrite dose of 20-40 mg three times daily is lower than the dose used on the 
cardiovascular IND and <40% of the dose for cyanide poisoning.  
 
Nine of 10 control subject enrollees into this pilot study have tolerated 40 mg po tid without 
incident; the one exception was a subject who had a decrease in systolic BP at pK but was 
asymptomatic. In the three heart failure subjects enrolled to date we have observed a vasoactive 
response at the pK with the 40 mg dose, such that low blood pressures or symptoms have been 
observed in the 2 hour monitoring period. This includes subjects with preserved and reduced 
ejection fraction. Therefore, we postulate the heart failure subjects appear to have a greater 
vasodilatory response to nitrite and could require a stratified dose based on age and baseline risk 
for hypotension. To further ameliorate risk, we will serve a standardized meal prior to drug dosing 
at Visit 3 so that lack of po intake is not a causative factor of hypotension. 
 
 
 
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 


Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
28 
 
• 
Rare:  methemoglobinemia 
Cardiovascular 
• 
Common: none 
• 
Frequent: hypotension in subjects with heart failure 
• 
Rare:  flushing, tachycardia, hypotension in healthy control subjects 
Neurologic 
• 
Common: none 
• 
Frequent: none 
• 
Rare:  headache, dizziness, seizure, coma 
Respiratory 
• 
Common: none 
• 
Frequent:  none 
• 
Rare:  tachypnea, dyspnea, cyanosis 
 
 
5.1.2 Risk of Study Procedures 
 
The collection of the research data from history and physical examination such as body weight, 
height and vital signs carries minimal risk. Phlebotomy or intravenous blood sampling risks of 
bruising, infection, anemia and fainting are common.  
 
The risks of percutaneous muscle biopsy are bleeding and infection (both rare), bruising 
(infrequent) and discomfort (likely). With the use of a local anesthetic, our subjects describe the 
discomfort of muscle biopsy as about twice that of intravenous cannulation. Some residual 
stiffness in the vastus lateralis can persist for 1-2 days (common). An elastic wrap and ice bag 
are applied post-biopsy to decrease the risk of bruising and for an anesthetic effect. In our 
experience using similar protocols, subjects have not experienced any adverse effects from these 
procedures other than a small amount of residual localized soreness at the biopsy site. Additional 
risk includes any unusual reaction to the elastic bandage wrap and ice, i.e. leg numbness which 
would indicate the elastic bandage had been applied too tightly or the ice left on too long; or any 
skin redness, irritation, and chafing from the applied steri-strips.  
 
Use of anesthetic cream 
There is a risk of reaction to the anesthetic cream (lidocaine and prilocaine). The use of anesthetic 
cream has a risk of methemoglobinemia when used with larger areas or times, or on certain meds 
including subjects taking nitrites. To prevent adverse reactions, the cream will only be used in 
subjects with a history of poor pain control. The PI will carefully screen subjects including 
medications with potential contraindications and evaluate the risk/benefit of use. In subjects who 
require it, use will be limited to no more than 2 applications of 2.5 grams as per package 
instructions, approximately 5-6 weeks apart to an area 2-4 cm on the thigh. The cream will be 
applied, and then biopsy will not proceed for an hour. Initial nitrite drug dosing occurs a minimum 
of 30 minutes after the biopsy. The subjects are then monitored for % methemoglobinemia for 2 
hours. Screening of subject medication profiles, using the minimal required amount of the cream, 
spacing with drug dosing, and monitoring of methem% of subjects allows for safe use in those 
subjects for which it is indicated. 
 
 
Risk of holding blood thinners, anti-clotting or anti-inflammatory medications 
There is a risk of increased pain if the anti-inflammatory medication is taken for arthritis or other 
pain related diagnosis. There is a risk of cardiac event if the medication is taken for anti-clotting 
prophylaxis related to cardiovascular disease. The risk is managed by individually evaluating the 


Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
29 
 
risk for subjects and working with their PCP or cardiologist, implementing bridging therapy if 
needed, and keeping the number of days the medication is held to the minimum necessary to 
prevent bleeding during the biopsy. 
 
The exercise test (VO2max) and subsequent exercise training sessions may cause muscle 
soreness or fatigue, but in adults without a known history of heart disease, the risk of heart attack 
or death from maximal or sub-maximal exercise bouts is rare. The relative risk of exercise testing 
for obese adults has not been clearly defined. However, a survey of more than 2,000 clinical 
exercise testing laboratories, in which more than 600,000 tests were performed, showed a death 
rate of approximately 0.5 per 10,000.  
American College of Sports Medicine (ACSM) criteria will be used to halt exercise testing. In 
addition to ACSM criteria, the exercise test will be stopped if the subject has either signs or 
symptoms of cardiovascular compensation, e.g. hypotensive response to exercise. These 
exercise tests will be interpreted by a physician at the University of Pittsburgh Medical Center. 
The staff conducting maximal exercise tests are ACLS certified. The Exercise Physiology 
Laboratory has a crash cart with emergency equipment (including a defibrillator-monitor, airway 
equipment, IV sets and fluids, syringes, needles, Lidocaine, Epinephrine, Nitroglycerine, etc.) and 
will be present during maximal exercise testing.  
 
Heart rate monitoring may result in skin chafing or irritation from the EKG electrodes used 
during exercise testing. 
 
The local anesthesia (buffered lidocaine) used for muscle biopsy may cause an anaphylactic 
reaction to 2% which could result in a severe allergic reaction that may be simple or severe 
enough to cause shortness of breath, swelling of the throat, inflammation of the skin, hypotension 
and death.  
 
 
Echocardiograms present no direct risks, but subjects will need to remain quietly at supine rest 
as a small plastic probe is placed against their chest. A gel is also applied to increase transmission 
of sound waves for optimal imaging quality. In rare instances, some people may find this to be 
anxiety producing. Dr. Gorcsan, who has advanced training in echocardiography, will oversee 
trained study staff performing echocardiograms to assure these are reliably completed with a 
minimum of patient distress. Likewise the ultrasound which may be used to guide the muscle 
biopsy carries no direct risk and may improve experience for subject. 
 
Patients will answer questionnaires as part of the study assessments, i.e., requiring responses 
about daily activities and quality of life which in some cases may be a source of emotional distress. 
Efforts will be made to keep the environment and support by staff to be reassuring and pleasant. 
 
There are no health risks of fasting for 8 hours but subjects may feel hungry, tired, or irritable until 
the standardized meal is provided in the CTRC upon arrival.  
 
There is a potential risk of stress and anxiety while participating in this study. It is likely that at 
least mild anxiety will occur while the subject is hospitalized to participate in this study.  There is 
a potential of developing an infection (viral or bacterial) while in the hospital because of exposure 
to sick patients. We will try to not pair participants to share a hospital room with a sick patient. 
Participants will not have tests that have more than a minimal risk of causing an infection. In our 
experience, the risk of developing an infection while at the CTRC is minimal and should be even 


Oral Nitrite for Older Heart Failure Patients 
Sponsor: Mark T. Gladwin, MD 
______________________________________________________________________________ 
 
30 
 
lower in the EMRC or Pepper Center where there are lower numbers of subjects seen and where 
only non-invasive procedures are performed.  
 
5.2 ALTERNATIVE TREATMENTS 
 
The alternative treatments for the subjects participating in this investigation are to continue their 
medical care under the direction of their primary physicians. 
 
 
5.3 POTENTIAL BENEFITS 
 
There may be no direct medical benefit to subjects in the study. It is hoped that researchers will 
learn more about the effectiveness of using oral nitrite to improve strength, gait speed and 
balance. Oral nitrite may also increase efficiency of work by reducing their oxygen uptake 
required for the same work intensity. We hope the information learned from this study will 
benefit other heart failure patients in the future. 
 
5.4 DATA SAFETY MONITORING PLAN   
 
5.4.1 Data Safety Monitoring Plan 
 
Monitoring of safety and data quality in the proposed study will be the responsibility of all 
personnel on the project, with primary responsibility and supervision by the PI. Both the 
Institutional Review Board will approve the Statement of Informed Consent for the study and 
provide institutional oversight of data and safety issues. The study protocol will be approved prior 
to recruiting or consenting any participants. Moreover, the study will be reviewed on an annual 
basis by the IRB committee. Each participant will sign the Informed Consent Form described 
above prior to participating in the study. To ensure participant safety, once participants are 
enrolled in the study, study staff will immediately report all adverse and serious adverse events 
to one of the PIs. The PI will, per standardized procedures, report them to the IRB for their review. 
With regard to monitoring of data quality and protected health information, all required personnel 
proposed for this project will have the required human subjects and confidentiality training, which 
includes information about maintaining data integrity and security. Confidentiality will be guarded 
using established procedures such as storing data in locked cabinets within locked offices or 
locked data rooms, coding by study identification numbers rather than any personally identifying 
information to avoid revealing the identity of subjects, and aggregating data across participants. 
The key linking names and study identification numbers will be kept separately from the data sets 
with limited access by study personnel. Only study personnel will have access to the data sets on 
protected servers. In order to maintain the highest standard of data entry quality, all data will be 
double-entered, with discrepancies highlighted so that they can be reviewed by the project 
coordinator. Oversight of all aspects of data management will occur with the PIs. 
 
Data Monitoring Plan. Data will be collected using standardized forms and will only be identified 
using the participant’s ID number (no names or identifying information will be on the forms). The 
codes that link the names of participants and their ID numbers will be kept confidential by the PI 
located on a password protected document on a secured drive. These data will only be accessible 
to the PI and staff working directly with data with the study. All data will be entered on-line, by the 
project coordinator. Data will be double entered in the computer independently by trained study 
staff, and data entry discrepancies will be monitored and corrected by the project coordinator, 
based on source documents. The quality of the data will be monitored on an ongoing basis. Data 


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quality will be monitored by inspection of the completed forms by the study coordinator and any 
problems detected will be discussed with the PIs. 
 
In the proposed study we will use the following University of Pittsburgh IRB definition of reportable 
events for the reporting of adverse events.  
1. Internal Adverse Events that are (i) Unexpected, (ii) Related or Possibly Related to the 
Research Intervention, and (iii) serious or otherwise suggests that the research places 
the subject or others at a greater risk of harm (including physical, psychological, 
economic or social harm) than was previously known or recognized. 
2. External Adverse Events that are (i) Unexpected; (ii) Related to the Research Intervention 
and (iii) Serious or otherwise suggests that the research places subjects or others at 
greater risk than was previously recognized 
 
Any event meeting #1 or #2 above will be reported immediately to the IRB in their required format. 
In the event that a participant either withdraws from the study or the PIs decide to discontinue a 
participant due to a SAE, the participant will be monitored by the co-PIs until (a) a resolution is 
reached (e.g., the problem has resolved or stabilized with no further change expected), (b) the 
SAE is determined to be clearly unrelated to the study intervention, or (c) the SAE results in death. 
Outcomes of SAEs will be regularly reported to the IRB. A summary of the SAEs that occurred 
during the previous year will be included in the annual progress report as well as in the annual 
IRB renewal. Suicidal ideation and AEs will be formally assessed immediately after treatment and 
referrals for further care will be made as needed. In the Informed Consent Form, we will provide 
specific information about emergency contacts. Participants are instructed to contact Dr. Forman 
during working hours. After hours participants will be provided the information on how to contact 
the cardiology fellow on call. 
 
Stopping Rule: 
For safety reasons, we propose to discontinue participation for any subject who experiences any 
of the following: 
 
1. Methemoglobin greater than 5% after dose lowering  
A nitrite dose reduction is proposed from 40 mg three times daily to 20 mg three times 
daily if this methemoglobin % is reached at any study interim visit, as described in 
section 2.2.6 (phone report of change in skin tone and dizziness or other symptom 
suggestive of methemoglobinemia). Follow up symptom assessment for 48 hours and 
repeat methemoglobin in 3 days will be implemented. If methemoglobin is greater than 
5% on the lower nitrite dose, participation in the study for the subject will be discontinued. 
 
2. Blood pressure drop after dose lowering 
Blood pressure and related symptoms.  
 
If subjects experience lightheadedness or dizziness after visit 3 while they are on the 
drug, we request that they call the study coordinator who will inventory their symptoms, 
compare to baseline (measured at pK) levels, and discuss need for interim visit with 
study physician. If a visit is indicated, we will measure blood pressure, RR, heart 
rate, and check methemoglobin level by a finger probe. A study physician will evaluate 
each subject, including: 
a. review of all clinical measures,  
b. subject history of symptoms, 


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c. blood pressures pre- and while on drug.  
The physician will confirm a management plan including the possibility of lowering dose 
to 20 mg tid or need to discontinue and drop from the trial. Subjects will be reassessed 
via phone each day for the next 2 days thereafter to ensure that the patient is stable on 
the new dose.  If stable, subjects will continue with the half dose.  However if there are 
continued symptoms on lower doses, the physician investigator will reevaluate the 
patient’s suitability for continued participation based on symptoms and/or 
hemodynamics.   
If subjects initiated at Visit 3 on the 20 mg dose report symptoms as described above, 
the same process will be followed; however, the physician may have to withdraw the 
subject since a dose lower than 20 mg tid is not an option. 
 
 
3. Biopsy refusal 
Subjects who refuse the first biopsy will be excluded at screening. Participants who 
underwent the first biopsy at Visit 3 may refuse a second pass. (Participants may also 
refuse a second biopsy pass at Visit 5). However, participants who refuse to complete a 
second biopsy at Visit 5 (prior to starting sodium nitrite) will be withdrawn from the study. 
Alternatively, patients who refuse the 2nd biopsy after they have completed the 4 weeks 
of nitrite therapy will still be included.  
 
4. Medication compliance 
In the event that the medication compliance rate is <80%, subjects will be re-educated 
on medication compliance. If medication compliance repeatedly falls outside of the 
acceptable range, the study investigators will discuss subject eligibility for continued 
participation in the study. 
 
 
5.4.3 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 (e.g., 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. 
 
• 
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: 
 


Oral Nitrite for Older Heart Failure Patients 
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• 
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. 
 
5.4.4 Frequency of Monitoring 
The Investigator will review subject safety data as it is generated. The Investigator, sub-
investigators, and the research staff will meet on a two week interval 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 it be re-evaluated and 
changed. 
  
5.4.5 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.    
 
5.4.6 Adverse Events Reporting Timeline 
 
Life-threatening or fatal unexpected adverse events associated with the use of the study drug or 
procedures must be reported to 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. 


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Serious and unexpected adverse events associated with the use of the study drug or procedures 
must be reported to 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 findings will be prepared and submitted to the regulatory agencies. 
 
5.5 RISKS MANAGEMENT PROCEDURES 
 
5.5.1 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 and/or legal 
representative decide to withdraw or be withdrawn from study participation, they may request that 
the study data and samples be destroyed. 
 
5.5.2 Protection Against Potential Risks of Experimental Intervention 
 
• 
Involvement by trained staff / investigators with experience in the administration of the 
study drug 
 
• 
Continuous monitoring by the Data and Safety Monitoring Board 
 
• 
Required Education in the Protection of Human Research Participants 


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The Investigator and all sub-investigators listed on University of Pittsburgh Institutional Review 
Board approved protocol are required to participate in a course entitled The Education and 
Certification Program in Research & Practice Fundamentals (RPF).  This web based tutorial is a 
requirement of the IRB for protocol submission.  
 
6.  COSTS AND PAYMENTS 
 
6.1 COSTS 
 
Study drug and all research testing will be supported by ongoing research grants. All medications, 
lab tests, and any procedures described will not be billed to the subjects and/or their health 
insurance company.  
 
6.2 PAYMENTS 
 
Subjects will be reimbursed $200 for the baseline study visits after completion of Visit 3 [Visits 1 
($50), Visit 2 ($75) and Visit 3 ($75)]. Subjects will be reimbursed an additional $370 following 
the completion of Visit 5 [Visit 4 ($125), Visit 5 ($145) and Study drug compensation ($100)]. 
The total compensation will be $570.00 and will be paid via the University’s WePay debit card 
system. Subjects will also be provided a voucher for outpatient UPMC Montefiore parking at all 
visits. In the event that a subject drops out due to an AE, the subject will be reimbursed for the 
individual study visits that they completed to date per the above reimbursement schedule. 
 
7.  QUALIFICATIONS AND SOURCE OF SUPPORT 
 
 
7.1 QUALIFICATIONS OF THE INVESTIGATORS 
Sponsor: 
 
Mark Gladwin, MD, is Professor of Medicine, University of Pittsburgh. Dr. Gladwin is the 
Chairman of the Department of Medicine and Director of the 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: 
 
Daniel Forman, MD, is a Professor of Medicine at UPMC and Chair, Geriatric Cardiology Section, 
Divisions of Geriatrics and Cardiology. Dr. Forman has a well-established track record of 
translational work focused on the interplay between skeletal muscle and physical function in older 
heart failure (HF) patients. Moreover, he is an expert in functional assessments in older HF 
patients. He is responsible for all aspects of this investigation, and will work directly with research 
staff to screen patients, coordinate data collection and quality, and he will personally complete all 
muscle biopsies and supervise all functional evaluations. He will play a primary role in data 
analysis and publications. Dr. Forman’s effort will be supported by the Department of Medicine. 
Sub-Investigators: 


Oral Nitrite for Older Heart Failure Patients 
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Mark Gladwin, M.D., is Professor and Chairman of Medicine, University of Pittsburgh School of 
Medicine and Director of the 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. 
 
Sruti Shiva, Ph.D., is an Associate Professor in the Department of Pharmacology and Chemical 
Biology and Vascular Medicine Institute (VMI) at the University of Pittsburgh. Her research lab 
focuses on the mechanisms by which reactive nitrogen species (particularly nitrite and nitric oxide) 
regulate mitochondrial function, the factors that influence this regulation and the implications of 
this regulation on pathology. 
 
John Gorcsan, III, MD is a Professor of Medicine and Director of Echocardiography Laboratory. 
He will provide his expertise and two-decade experience in quantification of left ventricular 
function by echocardiographic methods. He has extensive experience in whole organ ventricular 
physiology and quantitative echocardiography in patients with both depressed and preserved 
ejection fraction, having directed echocardiography core labs for multi-center studies in the past.  
 
Marc Simon, MD is an Assistant Professor of Medicine at the University of Pittsburgh Vascular 
Medicine Institute-Medicine, Bioengineering, and Clinical Translational Science including 
Director, Heart Failure Research / Clinical Hemodynamics Core Facility and Director, Vascular 
Clinical and Translational Research Center. He brings expertise in elucidating the mechanisms 
by which mitochondrial function is regulated in cardiovascular physiology and pathology, and 
thus evaluation of the data from this study concerning the mitochondrial analysis. As a 
cardiologist who specializes in heart failure, he brings added expertise to the protocol and can 
serve to assist Dr. Forman in evaluating, consenting and monitoring subjects. 
 
Ana Mora, MD, is an Assistant Professor of Medicine, Division of Pulmonary, Allergy and Critical 
Care Medicine, UPMC. Dr. Mora will participate in data analysis and publications. Her effort will 
be supported by the UPMC Heart and Vascular Institute. 
 
 
Nicole Helbling, MS RN, is a registered nurse with extensive clinical and translational research 
experience in the EMRC. She has been a research coordinator within the EMRC and is well 
trained to review study consent, obtain medical histories, assist with muscle biopsy procedures 
and exercise testing and day to day clinical trial execution. 
 
Manisha Jhamb, MD is an Assistant Professor at the University Of Pittsburgh School Of 
Medicine, Department of Medicine, Renal-Electrolyte Division and a clinician at UPMC 
Presbyterian and Magee Women’s Hospital.  She has clinical trial experience in the renal 
population including in the areas of quality of life and fatigue, outcome parameters in this pilot. 
She is being added to the protocol to support Dr. Forman in evaluating subjects for enrollment, 
consenting, performing physicals, evaluating adverse events and monitoring subjects. 
 
 
7.2 SOURCES OF SUPPORT 
 
2014 Mitochondria, Aging and Metabolism/Basic Biology Aging Pilot Project Program 
 
 


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Oral Nitrite for Older Heart Failure Patients 
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Oral Nitrite for Older Heart Failure Patients 
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______________________________________________________________________________ 
 
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51. VanSwearingen JM, Perera S, Brach JS, Cham R, Rosano C, Studenski SA.  A 
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