Clinical trial NCT03289481
Conditions: Heart Failure With Normal Ejection Fraction
Keywords: Heart failure HFpEF Nitrate supplement
Sponsor: MaineHealth
Start date: 2017-06-29; Completion date: 2018-08-22
Enrollment: 13 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
The objective of this project is to determine if Neo40, a nitric oxide generating lozenge, when consumed twice daily by subjects with HFpEF, will increase exercise tolerance, decrease symptoms and improve quality of life for patients.

Detailed description:
Heart failure (HF) is the most common principal diagnosis for hospital admission in patients over 65 years old. There are two types of HF, those with reduced ejection fraction (HFrEF) and those with preserved ejection fraction (HFpEF). Approximately half of patients with the clinical syndrome of HF have preserved systolic function. HEpEF is becoming more prevalent with aging of the population and obesity. There are only two class I recommendations for the treatment of HFpEF, which are controlling blood pressure and the use of diuretics to relieve symptoms. Exercise training is another approach to improving symptoms, however it may be poorly tolerated.

Nitrate supplement in the form of concentrated beetroot juice was recently shown to improve exercise tolerance in patients with HFpEF. (1) Beetroot juice contains high concentration of nitrate (NO3). This is metabolized to nitrite (NO2). It enters the blood stream, where it is further reduced to nitric oxide (NO) resulting in intense vasodilation.

Patients with diastolic dysfunction are often asymptomatic at rest but complain of dyspnea with exertion. Increase in heart rate with exercise causes reduced diastolic filling time and increases left sided filling pressure. Borloug, et al demonstrated this with right heart catheterization and supine exercise in patients with diastolic dysfunction. Infusion of NO2 resulted in decreased filling pressures and increased cardiac output. (2)

Neo40 is a new product made from concentrated beetroot juice in the form of a lozenge designed to dissolve on the tongue. NO3 supplement causes vasodilatation only in the setting of hypoxia and acidosis resulting in targeted vasodilatation.

Arms: arm 1: Subject will take active lozenge (Vitamin C, Vitamin B12, Nitric Oxide Blend, L-citrulline, Sodium Nitrite) for one week, perform cardiac testing then take placebo lozenge for one week and perform cardiac testing. arm 2: Subject will take placebo lozenge for one week, perform cardiac testing then take active lozenge (Vitamin C, Vitamin B12, Nitric Oxide Blend, L-citrulline, Sodium Nitrite) for one week and perform cardiac testing.
Interventions: intervention 1: Active lozenge intervention 2: Placebo
Intervention descriptions: intervention 1: nitric oxide generating lozenge intervention 2: placebo tablet

Protocol documents:
Prot_000.pdf: 
Protocol version date: March 20, 2017 
 
Page 1 of 6 
 
Treatment of HFpEF with nitrate supplement: A double-blind, placebo controlled 
trial including patients with atrial fibrillation 
Principal Investigator: Ralph Hamill, MD 
Sub Investigators: Rodney Mayhorn, MD 
INTRODUCTION 
Heart failure (HF) is the most common principal diagnosis for hospital admission in patients over 65 
years old. 10 According to the 2015 American Heart Association Statistical Update, one in nine deaths has 
HF mentioned on the death certificate.1 It is estimated that 6.5 million Americans >20 years of age have 
heart failure. Projections show the prevalence of HF will increase 46% from 2012 to 2030. 11 There are 
two types of HF, those with reduced ejection fraction (HFrEF) and those with preserved ejection fraction 
(HFpEF). Approximately half of patients with the clinical syndrome of HF have preserved systolic 
function. HEpEF is becoming more prevalent with aging of the population and obesity. 1 
While there are nine American Heart Association class I recommendations for the treatment of HFrEF, 
there are only two class I recommendations for the treatment of HFpEF, which are controlling blood 
pressure and the use of diuretics to relieve symptoms.3 Spironolactone may also be particularly 
effective. 4 
Exercise training is another approach to improving symptoms, however it may be poorly tolerated. 
Nitrate supplement in the form of concentrated beetroot juice was recently shown to improve exercise 
tolerance in patients with HFpEF.5  Beetroot juice contains high concentration of NO3 which is absorbed 
in the gut, concentrated in the salivary glands and secreted in saliva. This is metabolized to NO2 by 
symbiotic bacteria on the tongue. NO2 is swallowed, absorbed and enters the blood stream, where, in 
the presence of deoxyhemoglobin and acidosis, it is further reduced to NO resulting in intense 
vasodlitation and mitochondrial activation. 
Patients with diastolic dysfunction are often asymptomatic at rest but complain of dyspnea with 
exertion. Increase in heart rate with exercise causes reduced diastolic filling time and increases left sided 
filling pressure. Borloug, et al demonstrated this with right heart catheterization and supine exercise in 
patients with diastolic dysfunction. Infusion of NO2 resulted in decreased filling pressures and increased 
cardiac output. 9 
Neo40  is a new product made from concentrated beetroot juice in the form of a lozenge designed to 
dissolve on the tongue. This bypasses the enterosalivary circulation resulting in immediate production of 
NO instead of a delay of several hours seen when drinking beetroot juice.  It also caontains nitrite 
reductase which promotes the final step in NO production. 
Nitroglycerine in the form of isosorbide mononitrate is oxidized by endothelial nitric oxide synthetase 
causing widespread vasodilatation and hypotension. It was recently shown to be ineffective in treating 


Protocol version date: March 20, 2017 
 
Page 2 of 6 
 
HFpEF. 6 NO3 supplement causes vasodilatation only in the setting of hypoxia and acidocis resulting in 
targeted vasodilatation.  
NO may have direct effect on diastolic function via titin. Titin is a giant multi-functional sarcomeric 
filament that is important in maintaining the structural integrity of the contracting sarcomere7  and 
together with the extracellular matrix for defining diastolic stiffness.8  Titins elasticity is dependent on 
phosphorilation which in turn is dependent on NO.  
We propose to study the effect of Neo40 on patients with HFpEF. 
STUDY OBJECTIVES 
The objective of this project is to determine if NEO40, when consumed twice daily by subjects with 
HFpEF, will increase exercise tolerance, decrease symptoms and improve quality of life for patients.  
Primary endpoint: 
- 
Exercise Echocardiogram 
o Total time on the treadmill  
o Metabolic equivalents (METS)6  
o Average E/E’ 
o Estimation of right ventricular systolic pressure using standard echo measurements 
o Quality of life questionnaire changes  
Secondary endpoints: 
- 
Change in blood pressure measurements 
- 
Change in heart rate measurements 
- 
Change in 6 minute walk test results 
STUDY DESIGN 
This is a double blind cross over study. Each subject will consume the NEO40 for two weeks; one week 
with NEO40 and one week with nitrate free NEO40 (placebo). The project will randomly assign which the 
subject consumes first. 
 
Randomization 
visit
1 week of Neo40
Stress echo
1 week of nitrate 
free Neo40
Stress echo
1 week of nitrate 
free Neo40
Stress echo
1 week of Neo40
Stress echo


Protocol version date: March 20, 2017 
 
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Approximately 25 subjects will be enrolled at Pen Bay Medical. Interim analysis will be done when 12 
subjects have completed the study or at approximately six months, whichever comes first. The protocol 
will be reevaluated to determine if any amendment should be made as well as to assess safety and 
efficacy of the NEO40.  
Inclusion Criteria 
1. Males and females aged 18 years or greater 
2. Diagnosis of HFpEF, defined as: 
a. Symptomatic with one or more of the following: orthopnea, paroxysmal 
nocturnal dyspnea, lower-extremity edema, dyspnea on exertion; and 
b. Ejection fraction > 50%; and 
c. Ratio of early mitral inflow velocity to septal tissue Doppler velocity >8; and  
d. One or more of the following: 
i. Left atrium measurement > 34 mL/m2 
ii. Elevated N-terminal pro-brain natriuetic peptide level within the past 12 
months 
iii. Long-term loop diuretic use for control of symptoms 
iv. Elevated filling pressures on prior cardiac catheterization 
3. Stable medical therapy, defined as: no change in cardiac medications within 30 days 
4. Willing to comply with the protocol and provide written informed consent 
 
Exclusion Criteria: 
1. Non-cardiac condition causing limitation of exercise tolerance 
2. Acute coronary syndrome, myocardial infarction or cardiac revascularization within 60 
days 
3. Clinically significant valvular disease, defined as moderate-severe or severe stenosis or 
insufficiency 
4. Significant ischemia seen on stress testing within the past 12 months which was not 
revascularized 
5. Subjects who have taken an investigational medication within the past 30 days 
6. History of allergic reaction to beets  
7. Unwilling to comply with the protocol requirements 
8. Systolic blood pressure of <100 at screening  
9. The subject has a significant medical condition and/or conditions that would interfere 
with treatment, safety or compliance with the protocol 
 
 
 
 
 
 
STUDY PROCEDURES 
Screening Visit:  After the subject has provided informed consent, the Study Coordinator will collect 
basic demographic information; the patient’s cardiovascular history and measure subject’s heart rate 
and blood pressure. The subject will complete the World Health Organization Quality of Life (WHOQOL) - 


Protocol version date: March 20, 2017 
 
Page 4 of 6 
 
BREF questionnaire. The subject will then be randomized by an unblinded pharmacy staff member to 
either receive the NEO40 or the nitrate-free NEO40 for the first week and 16 doses will be dispensed to 
the subject, along with a dosing diary and written instructions regarding the dosing and diary.  
Visit 2: Seven days (+/- 2 days) later, the subject will return for the second study visit. Blood pressure 
and heart rate will be measured. The quality of life questionnaire will be re-administered and the diary 
will be collected at this visit. The exercise echocardiogram will be performed by one of the investigators. 
The exercise echocardiogram will be done 2-3 hours after the subject ingests their last dose of NEO40 
for this study period. Another 16 doses of either NEO40 or nitrate-free NEO40 will be dispensed to the 
subject along with another diary.  
During the exercise testing, the Borg scale of perceived exertion will be utilized and recorded with each 
subject approximately one minute prior to each incremental stage increase. The goal will be to have 
each subject exercise to a perceived “ten” on the one-to-ten scale.  
Visit 3: Seven days (+/- 2 days) later, the subject will return for the end-of-study visit. Blood pressure and 
heart rate will be measured. The quality of life questionnaire will be re-administered and the second 
diary will be collected. The exercise echocardiogram will be performed by one of the investigators. The 
exercise echocardiogram will be done within 30 minutes of the subject ingests their last dose of NEO40 
for this study period.  
RISKS 
Exercise Echocardiogram 
The risks of the exercise echocardiogram include fainting, disorders of heartbeats, abnormal blood 
pressure response, chest pain and, very rarely, a heart attack. A cardiologist will be in attendance during 
the exercise echocardiogram.  
Neo40 
A subject could be allergic to beets.  
CONFIDENTIALITY 
Subject identity will remain confidential. Records will be kept in the locked Clinical Research. No reports 
which come from this study will identify the subjects in any way. Only basic demographics, such as age 
and gender will be reported.  
STORAGE OF STUDY MATERIALS 
NEO40 will be stored at room temperature in the pharmacy of Penobscot Bay Medical Center.   
 
 


Protocol version date: March 20, 2017 
 
Page 5 of 6 
 
SUBJECT STIPENDS OR PAYMENTS AND COSTS 
Subjects will not be compensated for participation in this study. Neo40 will be provided free of charge to 
the subjects. There will be no charge to the subjects, nor their insurance plan, for any procedures 
related to this study.  
 


Protocol version date: March 20, 2017 
 
Page 6 of 6 
 
REFERENCES 
1. 
Mozaffarian, et al (2015). Heart Disease and Stroke Statistics – 2015 Update. 
Circulation. 2015;131:e29-e322. 
2. 
Borlaug, B.A. (2014). The pathophysiology of heart failure with preserved ejection 
fraction. Nat. Rev. Cardiol. 11, 507-515 
3. 
Yancy, C.W. (2013). 2013 ACCF/AHA Guideline for the Management of Heart Failure: 
Executive Summary. Cirulation. 128, 1810-1852. 
4. 
Lewis, E.F., et al (2016). Impact of Spironolactone on Longitudinal Chages in Health-
Related Quality of Life in the Treatment of Preserved Cardiac Function Heat Failure With an 
Aldosterone Antagonist Trial. Circ Heat Fail,9(3).  
5. 
Eggebeen, J., et al (2016). One Week of Daily Dosing With Beetroot Juice Improves 
Submaximal Edurance and Blood Pressure in Older Patients With Heart Failure and Preserved 
Ejection Fraction. JACC: Heart Failure, 4(6), 428-437. 
6. 
Redfield, M.M., et at (2015). Isosorbide Mononitrate in Heart Failure with Preserved 
Ejection Fraction. N Engl J Med, 373(24), 2314-2324. 
7. 
Horowits, R. and Rodolsky, R.J. (1987). The Positional Stability of Thick Filaments in 
Activated Skeletal Muscle Depends on Sarcomere Length: Evidence for the Role of Titin 
Filaments. Journal of Cell Biology, 105, 2217-2223. 
8. 
Granzier, H.L. and Irving, T.C. (1995). Passive Tension in Cardiac Muscle: Contribution of 
Collagen, Titin, Microtubules, and Intermediate Filaments. Biophysical Journal, 68, March, 1027-
1044. 
9. 
Borlaug, B.A., Koepp, K.E., and Melenovsky, V. (2015). Sodium Nitirite Improves Exercise 
Hemodynamics and Ventricular Performance in Heart Failure with Preserved Ejection Fraction. 
Journal of the American College of Cardiology 66/15, 1672-1682. 
10. 
Pfuntner, A., Wier, L.M. and Stocks, C. (2013). Most frequent conditions in U.S. hospital, 
2010. Healthcare Cost and Utilization Project Statistical Brief #148. Jan, 2013. 
11. 
Benjamin, E., et al (2017). Heart Disease and Stroke Statistics – 2017 Update, A Report 
From the American Heart Association. Circulation 135:00 1-459. 

SAP_001.pdf: 
Document date: March 20, 2019 
 
Treatment of HFpEF with nitrate supplement: A double-blind, placebo controlled 
trial including patients with atrial fibrillation 
Principal Investigator: Ralph Hamill, MD 
NCT #03289481 
 
 


Document date: March 20, 2019 
 
A t-test was utilized to examine the difference between the change in outcome measures for the 
exercise echocardiogram: diastolic dysfunction (E/E’), estimated right ventricular systolic pressure (TR), 
metabolic equivalents (METS), and time on the treadmill among the treatment and the placebo groups. 
The change in outcome measures were calculated by subtracting the pre-stress test measure from the 
post-stress test measure at the time of the patient encounter. T-values were considered significant at 
the .05 level. Analysis was done using SAS Enterprise Guide 7.1.