clinical track pharmacokinetics and pharmacodynamics of

DOI: 10.1161/CIRCRESAHA.116.309832 1

CLINICAL TRACK

Pharmacokinetics and Pharmacodynamics of Inorganic Nitrate in Heart Failure With Preserved Ejection Fraction

Payman Zamani1, Victor Tan1, Haideliza Soto-Calderon1, Melissa Beraun1, Jeffrey A. Brandimarto1, Lien Trieu2, Swapna Varakantam1, Paschalis-Thomas Doulias3, Raymond R. Townsend4, Jesse Chittams5, Kenneth B. Margulies1, Thomas P. Cappola1 David C. Poole, Harry Ischiropoulos3, Julio A. Chirinos1

1Division of Cardiovascular Medicine; Hospital of the University of Pennsylvania; Philadelphia, PA;

2Rowan University School of Osteopathic Medicine, Stratford, NJ; 3Children's Hospital of Philadelphia Research Institute, Philadelphia, PA; 4Division of Nephrology/Hypertension, Perelman School of

Medicine, University of Pennsylvania, Philadelphia, PA; 5Office of Nursing Research, School of Nursing, University of Pennsylvania, Philadelphia, PA; 6Departments of Kinesiology, Anatomy, and Physiology,

Kansas State University, Manhattan, KS.

Running title: Inorganic Nitrate in HFpEF

Subject Terms: Endothelium/Vascular Type/Nitric Oxide Heart Failure Exercise Address correspondence to: DR. Payman Zamani Hospital of the University of Pennsylvania 3400 Spruce Street, 9026 Gates Philadelphia, PA 19104 pH: (215) 662-6192 fax: (215) 349-8017 [email protected] In November 2016, the average time from submission to first decision for all original research papers submitted to Circulation Research was 15.7 days.

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DOI: 10.1161/CIRCRESAHA.116.309832 2

ABSTRACT Rationale: Nitrate-rich beetroot juice has been shown to improve exercise capacity in Heart Failure with Preserved Ejection Fraction (HFpEF), but studies using pharmacologic preparations of inorganic nitrate are lacking. Objectives: To determine: (1) the dose-response effect of potassium nitrate (KNO3) on exercise capacity; (2) the population-specific pharmacokinetic and safety profile of KNO3 in HFpEF. Methods and Results: We randomized 12 subjects with HFpEF to oral KNO3 (n=9) or potassium chloride (KCl, n=3). Subjects received 6mmol twice-daily during Week-1, followed by 6mmol thrice-daily during Week-2. Supine cycle ergometry was performed at baseline (Visit 1) and after each week (Visits 2&3). Quality of life (QOL) was assessed with the Kansas City Cardiomyopathy Questionnaire (KCCQ). The primary efficacy outcome, peak O2-uptake, did not significantly improve (P=0.13). Exploratory outcomes included exercise duration and quality of life. Exercise duration increased significantly with KNO3 (Visit 1: 9.87 [95%CI=9.31-10.43]; Visit 2: 10.73 [95%CI=10.13-11.33]; Visit 3: 11.61 [95%CI=11.05-12.17] minutes, P=0.002). Improvements in the KCCQ total symptom (Visit 1: 58.0 [95%CI=52.5-63.5], Visit 2: 66.8 [95%CI=61.3-72.3]; Visit 3: 70.8 [95%CI=65.3-76.3], P=0.016) and functional status scores (Visit 1: 62.2 [95%CI=58.5-66.0], Visit 2: 68.6 [95%CI=64.9-72.3], Visit 3: 71.1 [95%CI=67.3-74.8]; P=0.01) were seen after KNO3. Pronounced elevations in trough levels of nitric oxide metabolites (NOm) occurred with KNO3 (Visit 2: 199.5 [95%CI=98.7-300.2]; Visit 3: 471.8 [95%CI=377.8-565.8]) versus baseline (Visit 1: 38.0 [95%CI=0.00-132.0] μM; P<0.001). KNO3 did not lead to clinically-significant hypotension or methemoglobinemia. Following 6 mmol of KNO3, systolic blood pressure was reduced by a maximum of 17.9 (95%CI -28.3-[-7.6]) mmHg 3.75 hours later. Peak NOm concentrations were 259.3 (95%CI 176.2-342.4) μM 3.5 hours after ingestion, and the median half-life was 73.0 (IQR 33.4-232.0) minutes. Conclusions: KNO3 is potentially well-tolerated and improves exercise duration and QOL in HFpEF. This study reinforces the efficacy of KNO3 and suggests that larger randomized trials are warranted. ClinicalTrials.gov: NCT02256345; https://www.clinicaltrials.gov/ct2/show/NCT02256345 Keywords: Heart failure, exercise capacity, nitric oxide. Nonstandard Abbreviations and Acronyms: KNO3 potassium nitrate KCl potassium chloride VTI velocity time integral LVOT left ventricular outflow tract AIx augmentation index VO2,peak peak oxygen uptake NIRS near infrared spectroscopy SS steady state KCCQ Kansas City Cardiomyopathy Questionnaire SBP systolic blood pressure EDV end-diastolic volume



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INTRODUCTION The prevalence of heart failure with preserved ejection fraction (HFpEF) is rising.1, 2 Recent tissue data demonstrate decreased cyclic GMP (cGMP) concentration, and increased oxidative stress in subjects with HFpEF,3, 4 suggesting impaired nitric oxide (NO) signaling. However, efforts at increasing NO bioavailability, either through phosphodiesterase-5 inhibition or the administration of organic nitrate, have not led to improvements in exercise capacity in HFpEF.5, 6 Traditionally, NO synthesis has been known to occur via the enzymatic oxidation of L-arginine by nitric oxide synthases (NOS), in the presence of molecular oxygen, to yield citrulline and NO.7 Given its dependence on oxygen, NO synthesis from NOS may be inefficient in the setting of hypoxia. Inorganic nitrate, ingested in the diet or via supplementation, is rapidly absorbed in the gastrointestinal tract, concentrated in the salivary glands, and then released into the oral cavity where bacteria facilitate its reduction to nitrite. Nitrite is then reabsorbed into the circulation and can be reduced to NO by deoxyhemoglobin.8 Conversion of nitrite to NO is potentiated by hypoxia and acidosis,9, 10 as would be seen in exercising muscle,11 leading to vasodilation and increased perfusion at sites of greater metabolic need.12 Thus, circulating nitrate/nitrite may serve as an alternative pool for bioactive NO, which can sustain/enhance NO signaling when the traditional pathway for NO generation is less functional.9, 13 Indeed, nitrate/nitrite supplementation has been shown to increase blood flow to exercising muscle in rats with14, 15 and without16, 17 heart failure. NO-induced vasodilation during exercise may facilitate the matching of perfusion to metabolic activity.18 Recently, our group demonstrated that acute supplementation with beetroot juice, which is rich in inorganic nitrate, improves vasodilatory reserve and peak oxygen uptake in HFpEF subjects.19 A more recent study demonstrated improvements in endurance during submaximal exercise following one-week of supplementation with beetroot juice.20 However, studies using pharmacologic preparations of inorganic nitrate are lacking. The current study was an in-depth pharmacodynamic and pharmacokinetic study of oral KNO3 in HFpEF with the specific goals of: (1) Determining the safety profile of short-term (2-week) inorganic nitrate supplementation, administered as potassium nitrate (KNO3) capsules; and (2) Determine the physiologic effects and the dose-response associated with KNO3 administration over 2-weeks on exercise capacity and key physiologic adaptations to exercise; and (3) Assess the pharmacokinetic profile of KNO3 in this population. METHODS Participants. Inclusion criteria included symptomatic heart failure in the context of a preserved (≥50%) ejection fraction. Subjects were required to have evidence of elevated filling pressures, demonstrated by at least one of the following: (1) Elevated invasively-measured filling pressures; (2) Septal E/e’>15; (3) Septal E/e’>8 with either elevated natriuretic peptide levels within the past year, left atrial enlargement, or chronic loop diuretic use for control of symptoms. Exclusion criteria included any rhythm other than sinus with native conduction, inability to exercise, ≥ moderate valvular disease, known hypertrophic/infiltrative/inflammatory cardiomyopathy, pericardial disease, current angina, acute coronary syndrome or coronary intervention within the past 2 months, primary pulmonary arteriopathy, clinically-significant lung disease, current ischemia, chronic treatment with organic nitrates or allopurinol, significant liver disease, eGFR<30 mL/min/1.73 m2 or creatinine > 2.5 mg/dL, current smoking, alcohol dependency, Barrett’s esophagus, G6PD deficiency, and a baseline methemoglobin >3%. Background medications were not altered. The study was approved by the Institutional Review Board of the University of Pennsylvania



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and conducted under an FDA IND (#123,785). Written informed consent was obtained from all subjects. This study was registered on ClinicalTrials.gov (NCT02256345). Study medication and dosing regimen. This was a randomized trial of KNO3 given in two dosing regimens: 6 mmol twice daily for one week followed by dose-escalation to 6 mmol thrice daily for one week. While a primary goal of the study was to assess the safety of KNO3 and within-group changes in various endpoints in KNO3-treated subjects, a small number of placebo-treated (PB, n=3) subjects were included, only to assess for any potential training-effect on repeated exercise and KCCQ measurements.21 Potassium chloride (KCl), given in equivalent doses, was used as the PB to account for differences in blood pressure or flow that could be attributed to potassium.22 Design. The study was initially designed to be single-blinded, to allow the Principal Investigator to be aware of arm allocation due to potential concerns for methemoglobinemia with drug administration. As described below; however, no clinically-significant elevations in methemoglobin occurred. One investigator (PZ), who was the Primary Investigator responsible for supervising all visits and measurements during the study, remained blinded to treatment allocation throughout the entirety of the study. All physiologic and imaging data were analyzed in a double-blind manner. Study protocol. Subjects presented to the Clinical and Translational Research Center at the University of Pennsylvania in the fasted state. Resting blood pressure was obtained using an automated oscillometric device (Accutorr Plus, Datascope Corp., Paramus, NJ). Blood was obtained for serum nitric oxide metabolites (NOm) and nitrite determination, basic chemistries, NT-pro-BNP determination (Cobas e411 Analyzer, Roche Diagnostics, Indianapolis, IN), and measurement of hemoglobin and methemoglobin (ABL800 Flex Analyzer, Radiometer America, Brea, CA). Subjects refrained from using mouthwash and were provided with a list of nitrate-rich foods to avoid during the study. A registered dietician met with subjects at each study visit to reinforce dietary restrictions. During the baseline visit (Visit 1), subjects proceeded directly to the exercise testing protocol after verification of inclusion/exclusion criteria, a physical examination, echocardiography with arterial tonometry, and laboratory testing. During Visit 2 and Visit 3, study drug was administered with a standardized low-nitrate meal. Two hours later, orthostatic blood pressure measurements were made, followed by echocardiography, arterial tonometry, and the exercise protocol, as described below. The Kansas City Cardiomyopathy Questionnaire was administered at each study visit.23 Echocardiography and arterial tonometry. Subjects underwent resting echocardiography using a GE Vivid I machine (GE Healthcare, Fairfield, CT). Metrics were quantified in triplicate, according to the American Society of Echocardiography guidelines.24 Left atrial volumes were quantified using the Area-Length method and indexed to body-surface area (BSA). Left ventricular outflow tract (LVOT) Doppler velocity-time integrals (VTI) were obtained from the 5-chamber view to obtain stroke volume (LVOT cross-sectional area * VTI) at rest and at peak exercise. Resting arterial tonometry (Millar Instruments, Houston, TX) was performed at the left radial artery and calibrated using brachial oscillometric blood pressures (Scholar III507EL, Criticare Systems, Inc., Waukesha, WI). Tonometric signals were processed using Sphygmocor software (AtCor Medical, Australia) to derive central pressures. Augmentation index (AIx) was calculated as the ratio of the amplitude of the second peak to the first peak (P2/P1 x 100%). Exercise studies. As described previously,19 subjects underwent peak effort exercise testing using supine cycle ergometry with gas exchange measurements (Parvo Medics, Sandy, UT). Peak oxygen uptake (VO2,peak) was defined as the average value obtained during the last 30 seconds of exercise. Exercise economy was estimated as



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the ratio of total work performed (kilojoules) to total oxygen consumed (liters) above baseline during the entirety of the exercise study. Prior work demonstrates an increased benefit for inorganic nitrate supplementation at higher exercise intensities, when Type II muscle fibers are utilized to a greater degree.16,

25-28 In post hoc analysis, we quantified total oxygen uptake and work performed during the last 3- and 6-minutes of exercise, when subjects were exercising at higher intensities, and obtained estimates of exercise economy over those time periods. After at least 15 minutes of rest, subjects exercised at 25W for 6 minutes with steady-state VO2 measured during the final minute of exercise. Radial tonometry and LVOT VTIs were obtained at rest and during steady-state to determine input impedance, as described previously.29 VO2 recovery kinetics (τ) were modeled according to a mono-exponential decay. Skeletal muscle mitochondrial oxidative function. We used near infrared spectroscopy (NIRS) to determine skeletal muscle mitochondrial oxidative function after a standardized exercise protocol, as described by Ryan et al.,30 and reported previously by our group.19 Safety and pharmacokinetic determination. On Visit 1 and Visit 2, subjects were given study medications (either 6 mmol KNO3 or 6 mmol KCl) with a standardized nitrate-free meal following exercise testing. For the next 5 hours, blood pressure was measured every 15 minutes, and serum was drawn every 30 minutes for determination of NOm and methemoglobin. The elimination rate (Ke) was the negative of the slope from the linear regression of log concentration versus time over the last 3 time points and used to calculate the half-life (t1/2 = ln 2/Ke). Serum NOm quantification. Serum levels of NOm were measured as reported previously using vanadium (III)/ hydrochloric acid reduction, heated at 95°C, and coupled with chemiluminescence detection using the Sievers-Nitric Oxide Analyzer (Sievers-NOA 280, Nitric Oxide Analyzer, Sievers Instruments, Boulder, CO). This method quantifies nitrate, as well as nitrite, NO-metal and heme complexes, low molecular weight thiol-NO adducts and protein cysteine-NO adducts.19 NOm samples for 3 individuals were repeated in each of the 9 batches performed. The intra-class correlation coefficient for NOm was 0.98 (95%CI 0.93-1.00; P<0.001), demonstrating excellent reproducibility. Serum nitrite levels were quantified using reduction with potassium iodide/crystalline iodide and the same Sievers instrument. Nitrite samples were measured in each of the 6 batches performed. The intra-class correlation coefficient for nitrite was 0.94 (95%CI 0.77-1.00; P<0.001). Activity monitoring and 24-hour blood pressure measurements. At the conclusion of Visit 1 and Visit 2, subjects were given an ambulatory blood pressure monitor (Mobil-O-Graph PWA, I.E.M. GmbH, Stolberg, Germany) and an activity monitor (wActiSleep-BT Monitor, Actigraph, LLC., Pensacola, FL). Blood pressure measurements were obtained every 30 minutes over a 24-hour period. Actigraphy was used to obtain daily steps taken and estimated kilocalorie expenditure. Because follow-up visits were scheduled during the last 1-2 days of each week, data was abstracted for days 1-5 in all subjects. Values for days 3-5 were averaged to obtain estimates corresponding to steady-state drug effects (SS). Study outcomes. The primary efficacy endpoint was the change in VO2,peak. Secondary endpoints included changes in vasodilatory reserve (percent reduction in systemic vascular resistance during exercise, compared to the resting measurement), changes in mitochondrial oxidative function using NIRS, and changes in AIx. Exploratory endpoints included other metrics of exercise capacity, and changes in the KCCQ. Pharmacokinetics were assessed via measurement of NOm after single-dose administration of 6 mmol and with trough levels after repeated twice- and thrice-daily administration.



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Statistical methods. Demographic data are presented as n(%) or means(SD). Mixed-effects models were generated in STATA (Stata/SE 13.1, StataCorp, College Station, TX), incorporating the correlation between repeated measurements in the same individual. No assumption of linearity was made. As pre-specified in the protocol, only within group comparisons were performed. Marginal means with 95% confidence intervals (CI) were determined from the mixed models and presented in the tables and figures. An overall P-value<0.05 was taken to be significant for each model, with post-hoc comparisons between visits performed subsequently. No adjustment for multiple comparisons was made. The elimination half-life was determined using the PK package in STATA. Linear regression was performed to assess the correlation between changes in serum NOm and nitrite levels and the change in exercise metrics. Regression coefficients are presented as standardized units describing the standard deviation change in the endpoint for each standard deviation change in the predictor variable. In a previous trial, we demonstrated a 1.0±1.2 mL/min/kg improvement in peak VO2 following a single-dose (12.9 mmol) of inorganic nitrate, given in the form of beetroot juice.19 Using a correlation between studies of 0.80, beta=0.80, and alpha=0.05, we estimated that 7 subjects would be needed to demonstrate a similar change. We thus planned for 9 subjects in the active arm to allow for drop-out. Randomization was performed by the University of Pennsylvania Investigational Drug Pharmacy. Randomization tables were constructed using a 3:1 KNO3:PB ratio in blocks of 4 (www.randomization.com). RESULTS

From 703 subjects screened, a total of 15 subjects were enrolled (Figure 1). An alternative explanation for dyspnea was identified during baseline testing in 3 individuals, who were subsequently excluded. Twelve individuals met all inclusion/exclusion criteria and completed the study (Table 1). Of these 12, 7 (58%) previously had elevated invasively-determined intra-cardiac pressures. The remaining individuals had an elevated E/e’ ratio>8 along with at least one of the following: (a) an elevated NT-pro-BNP previously (n=1); (b) left atrial enlargement (n=1); (c) or the need for chronic loop diuretics for control of symptoms (n=3). Mean (SD) age of participants was 62.5 (5.8) years, 4 (33%) were males, and 3 (25%) were African-American. Most participants had comorbidities typical for HFpEF: hypertension (100%), obesity (67%), hyperlipidemia (83%), diabetes (58%), obstructive sleep apnea (50%), coronary disease (33%), and chronic renal insufficiency (33%). Maximal effort exercise test.

VO2,peak, the primary efficacy endpoint, tended to increase, without reaching statistical significance (P=0.13). Vasodilatory reserve did not change with supplementation (P=0.72). The exploratory endpoint of exercise duration was significantly increased in the KNO3 group (P=0.002; Table 2, Figure 2). Both total work performed (P=0.02) and total oxygen uptake (P=0.019) increased. Additional hemodynamic data from the maximal effort study are presented in Supplemental Table I. Interestingly, the change in VO2,peak from the baseline to final visit correlated with the change in serum NOm levels: Standardized β=0.74; P=0.006. The change in nitrite levels did not correlate with any metrics of exercise capacity.

In post hoc analysis, we evaluated oxygen uptake and work performed during the final 3 and final 6 minutes of exercise (i.e. heavy and severe intensity). Work performed in the final 3 minutes increased significantly (P=0.002), as did total O2 uptake in the final 6 minutes (P=0.041) and total work performed in the final 6 minutes (P=0.002; Figure 2). Overall exercise economy in the final 3-minute (P=0.007) and 6-minute (P=0.004) periods increased (Supplemental Table II). The increase in oxygen uptake during the



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final 3 minutes of exercise correlated with the increase in serum NOm levels (Standardized β=0.64; P=0.024). Pharmacodynamics following administration of 6 mmol of KNO3.

Blood pressure was measured every 15 minutes, and serum collected every 30 minutes, following the administration of 6 mmol of KNO3. The maximal reduction in SBP occurred at 3 hours and 45 minutes (-17.9 [95%CI -28.3-[-7.6]] mmHg; Figure 3). We observed a statistically-significant, though clinically-irrelevant, increase in methemoglobin concurrent with the reduction in SBP (Figure 3). Peak NOm concentrations were 259.3 (95%CI 176.2-342.4) μM occurring 3.5 hours after ingestion. The median half-life was 73.0 (IQR 33.4-232.0) minutes. Tolerability of KNO3.

KNO3 was well-tolerated, with all subjects completing the dosing and study protocols. Five individuals experienced side-effects during the study (Supplemental Table III): 4 (44%) in the KNO3 group and 1 (33%) in PB. Gastrointestinal symptoms and headache were the most common side-effect. There were numerically fewer side-effects in the KNO3 group during Week 2 (18 mmol/d of KNO3). Serum NOm, blood pressure and laboratory testing at each visit.

KNO3 significantly increased fasting serum NOm concentration in a dose-dependent manner (Table 3). This primarily reflects an increase in nitrate levels, as nitrite levels did not change (data not shown). We observed a reduction in systolic blood pressure (SBP) with KNO3 (Table 3). Importantly, percent methemoglobin was no different between visits (Table 3). Likely due to the serial blood sampling during the safety/pharmacokinetics portions of Visit 1 and Visit 2, hemoglobin was significantly reduced in the KNO3 group (Table 3). Orthostatic blood pressure measurements.

Orthostatic measurements were performed 2 hours following dose administration. Inorganic nitrate caused an asymptomatic reduction in SBP following 2 minutes of standing from a supine position, particularly at the highest doses (Visit 1: +1.2 [95%CI -6.1-8.4]; Visit 2: -1.1 [95%CI -7.8-5.6]; Visit 3: -11.6 [95%CI -18.3-[-4.9]] mmHg, P=0.051). Kansas City cardiomyopathy questionnaire.

The KNO3 group experienced an improvement in HF symptoms over the 2-week study, as evidenced by higher scores on the KCCQ in several domains: symptom stability (P=0.03), symptom burden (P=0.049), total symptom score (P=0.016), and the functional status score (P=0.01; Figure 4). The overall summary score tended to improve with KNO3 (P=0.067). Resting echocardiography.

Diastolic function and left-ventricular end-diastolic volumes were quantified at rest. Diastolic function was not impacted by KNO3. Additionally, EDV did not change (P=0.99, Supplemental Table IV). Oxygen uptake and input impedance during submaximal exercise.

Using radial tonometry and LVOT flow, input impedance was determined at rest and while at steady-state during submaximal (25W) exercise. No changes in input impedance were observed



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(Supplemental Table V). Neither steady-state VO2 (P=0.85) nor economy (P=0.98) were impacted by KNO3 (Supplemental Table VI). Ambulatory BP.

During the 24-hours following Visit 1 (first day of 6 mmol twice daily) and Visit 2 (first day of increase to 6 mmol thrice daily), no differences in 24-hour ambulatory blood pressure were seen (data not shown). Actigraphy.

KNO3 did not alter activity. Specifically, neither average daily steps taken during each week (Week 1: 9244.1 [95%CI 8435.8-10052.4]; Week-2: 9469.8 [95%CI 8661.5-10278.1] steps; P=0.71) nor estimated daily kilocalories expended (Week-1: 1683.3 [95%CI 1555.5-1811.1]; Week-2: 1678.7 [95%CI 1550.9-1806.4]; P=0.96) were altered with KNO3. Augmentation index and mitochondrial oxidative function.

There was no difference in AIx or mitochondrial oxidative function between visits in either the KNO3 or the PB group (P=NS for all). Lack of training effect in PB-Treated subjects.

There were no significant changes in any metric of exercise with PB. Furthermore, KCCQ scores did not change in these subjects during the course of the study, suggesting lack of a training effect on repeated testing. Data on study-endpoints for the PB-treated subjects can be found in the online supplement (Supplemental Table VII). DISCUSSION

In subjects with HFpEF, the principal findings of this investigation are that KNO3 (1) appears to be potentially safe at a dose of up to 18 mmol/day; (2) did not significantly improve the primary efficacy endpoint of peak VO2, although a non-significant trend was observed; (3) significantly increased the exploratory endpoint of exercise duration; (3) significantly improved several domains of the Kansas City Cardiomyopathy Questionnaire; (4) did not worsen outpatient activity; and (5) this preparation demonstrates favorable pharmacokinetics, with high trough levels of NO metabolites achieved with doses of 6 mmol twice daily and substantially higher levels with thrice daily dosing, but without significant methemoglobinemia for either dosing interval.

KNO3 appeared to be potentially safe up to a dose of 18 mmol/day, and no subject prematurely discontinued study medications for any reasons. The side-effects experienced were mild and were not more frequent at the higher dose. Similar to other studies, we demonstrate that KNO3 mildly reduced resting blood pressure.20, 31 We did, however, note a greater decrease in orthostatic SBP at the higher dose of 18 mmol/day, suggesting that this approaches the maximum tolerated dose. The reduction of nitrite to NO leads to the generation of methemoglobin, raising the theoretical concern for methemoglobinemia with KNO3 administration. Consistent with this biology, we demonstrate a clinically-insignificant rise in methemoglobin immediately after dose administration. However, there was no elevation in steady-state methemoglobin levels at any study visit, suggesting that the endogenous mechanisms of hemoglobin reduction operate fast enough to prevent any sustained rise in methemoglobin.



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In contrast to NO production via NOS, which requires molecular oxygen, KNO3 may provide an

alternative source of NO that causes vasodilation and increased perfusion within hypoxic exercising muscle, leading to improved exercise duration. During our progressive ramp protocol, exercise duration increased via improved tolerance to heavy/severe exercise. As the intensity of exercise increases, a greater proportion of Type II fibers is activated.25 Type II fibers, with their lower capillary volume density and increased dependence on glycolytic pathways for ATP production, reside in a relatively more hypoxic and acidotic milieu, particularly during exercise thus providing an ideal environment for the reduction of nitrite to NO.25 Our findings are in line with recent evidence for the preferential activation of nitrite by Type II fibers16 and at higher exercise intensities.26-28

Participants randomized to KNO3 experienced an improvement in HF-related symptoms, as

evidenced by higher total symptom, symptom burden, and functional status scores. The total symptom score has previously been demonstrated to be the most responsive metric for the evaluation of HF symptoms, and lower functional status scores have been associated with an increased incidence of death or HF hospitalization.23 Importantly, the magnitude of the changes seen in our study were generally >5 points, suggesting the potential for clinical relevance.32 Given the substantial morbidity associated with HFpEF,33 strategies that improve quality of life and exercise represent important progress for this patient population that has few therapeutic options. Beyond the short-term effects demonstrated in this trial, inorganic nitrate might enable HFpEF patients to better tolerate the initiation/maintenance of an exercise prescription, improving the adherence to, and efficacy of, an exercise regimen.

Recently, a randomized placebo controlled crossover study of an organic nitrate, isosorbide

mononitrate (ISMN), was performed in HFpEF patients.5 This study demonstrated a reduction in physical activity with ISMN. In our study, no decrease in activity was identified, suggesting that KNO3 may not have the same harmful impact. Limitations.

There are several limitations to our study. First, the sample size was small because a larger population could not be justified prior to assessing safety. Given the small number of patients exposed to active agent and the very small number of placebo patients, the study had low power to detect safety signals. Gathering more robust safety data is a goal of the ongoing registered larger clinical trial that was supported by our present findings (KNO3CK OUT HFpEF Trial; ClinicalTrials.gov: NCT02840799). Second, the pharmacokinetics portions of our study required serial blood draws, which caused a significant decrease in hemoglobin concentration from 13.6 g/dL to 12.8 g/dL (-5.9%). Because VO2,peak is limited by oxygen delivery, particularly in heart failure subjects,18 the decrease in hemoglobin may have constrained the increase in VO2,peak and reduced the magnitude of the improvement in exercise capacity and quality of life. Indeed, in our current study, we found a 3.4% improvement in VO2,peak. Had the hemoglobin remained constant, mathematically VO2,peak would be expected to improve by ~6%, leading to a similar magnitude of change as demonstrated in our prior study where a single acute dose of 12.9 mmol of inorganic nitrate improved VO2,peak by approximately 8%.19 Differences in patient characteristics may also have contributed to the non-significant increase in VO2,peak. Our prior study included a greater number of African-American males. It is conceivable that hypertensive African-Americans represent a unique population with particularly deranged NOS function,34 and stand to benefit more from interventions that increase NO bioavailability, as demonstrated in HFrEF.35 Furthermore, there may be differences in KNO3 responsiveness between genders, as females demonstrate less reduction in blood pressure following inorganic nitrate supplementation despite greater increases in serum nitrate concentration.36 Given the forced up-titration of study medications, we cannot exclude the possibility that the improvement in exercise duration was due to longer exposure to study medications versus the higher dose administered. That the change in VO2,peak and oxygen uptake in the last 3 minutes of exercise correlated with the change in serum



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NOm levels suggests that the supplementation dose is also important. Additionally, we did not obtain serial urine and saliva samples during the pharmacokinetics segment of the study, precluding more detailed assessments. Finally, it is possible that more PB-treated subjects were needed to demonstrate a training effect; however, we were limited in the number of subjects to be performed in this initial pilot study. Our group is currently conducting a follow-up cross-over study of KNO3 versus PB in HFpEF subjects, which will be adequately powered to detect most relevant treatment-related differences and will also specifically address possible gender and race differences in the response to KNO3 (KNO3CK OUT HFpEF Trial; ClinicalTrials.gov: NCT02840799). Conclusions.

KNO3 improves exercise duration and QOL in subjects with HFpEF. KNO3, at a dose of 18 mmol/day, appears to be potentially safe with only mild reduction in blood pressure, and no elevation in methemoglobin.

ACKNOWLEDGEMENTS The authors thank the Clinical and Translational Research Center at the University of Pennsylvania for the financial, equipment, and personnel support that made this study possible. SOURCES OF FUNDING PZ: Institute for Translational Medicine and Therapeutics of the University of Pennsylvania (5UL1TR000003-09 from the National Center for Research Resources), 5-T32-HL007843-17, and 1-K23-HL-130551-01. The project described was supported by Grant Number UL1RR024134 from the National Center for Research Resources and by Grant Number UL1TR000003 from the National Center for Advancing Translational Sciences, National Institutes of Health; the content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. JAC: 5R21AG043802-02, 1R56HL124073-01A1, and 1 R01 HL121510-01A1. HI: NIH HL054926 and the Gisela and Dennis Alter Research Professor of Pediatrics. KBM: Research Grant; Modest; Juventis Therapeutics, Celladon Corporation, Thoratec Corporation, and Innolign Biomedical, LLC. Research Grant; Significant; Merck. DISCLOSURES JAC: Consulting fees from Bristol-Myers Squibb, OPKO Healthcare, Fukuda Denshi, Microsoft Research and Merck. JAC received research grants from National Institutes of Health, American College of Radiology Network, Fukuda Denshi, Bristol-Myers Squibb, Microsoft Research and CVRx Inc, and device loans from Atcor Medical. He is named as inventor in a University of Pennsylvania patent application for the use of inorganic nitrates/nitrites for the treatment of Heart Failure and Preserved Ejection Fraction. KBM: Consultant/Advisory Board; Modest; Janssen, Merck, Glaxo-Smith-Kline, Pfizer, Ridgetop Research, AstraZeneca, NovoNordisk (unpaid). TPC: Consultant/Advisory Board; Modest; Novartis. RRT: Consultant for Medtronic, Fukuda Denshi, Relypsa.



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REFERENCES 1. Steinberg BA, Zhao X, Heidenreich PA, Peterson ED, Bhatt DL, Cannon CP, Hernandez AF,

Fonarow GC, Get With the Guidelines Scientific Advisory C, Investigators. Trends in patients hospitalized with heart failure and preserved left ventricular ejection fraction: Prevalence, therapies, and outcomes. Circulation. 2012;126:65-75

2. Tsao CW, Lyass A, Larson MG, Vasan RS. Abstract 23: Divergent temporal trends in the incidence of heart failure with preserved and reduced ejection fraction. Circulation. 2015;131:A23

3. van Heerebeek L, Hamdani N, Falcao-Pires I, Leite-Moreira AF, Begieneman MP, Bronzwaer JG, van der Velden J, Stienen GJ, Laarman GJ, Somsen A, Verheugt FW, Niessen HW, Paulus WJ. Low myocardial protein kinase g activity in heart failure with preserved ejection fraction. Circulation. 2012;126:830-839

4. Franssen C, Chen S, Unger A, Korkmaz HI, De Keulenaer GW, Tschope C, Leite-Moreira AF, Musters R, Niessen HW, Linke WA, Paulus WJ, Hamdani N. Myocardial microvascular inflammatory endothelial activation in heart failure with preserved ejection fraction. JACC Heart Fail. 2016;4:312-324

5. Redfield MM, Anstrom KJ, Levine JA, Koepp GA, Borlaug BA, Chen HH, LeWinter MM, Joseph SM, Shah SJ, Semigran MJ, Felker GM, Cole RT, Reeves GR, Tedford RJ, Tang WH, McNulty SE, Velazquez EJ, Shah MR, Braunwald E, Network NHFCR. Isosorbide mononitrate in heart failure with preserved ejection fraction. The New England journal of medicine. 2015;373:2314-2324

6. Redfield MM, Chen HH, Borlaug BA, Semigran MJ, Lee KL, Lewis G, LeWinter MM, Rouleau JL, Bull DA, Mann DL, Deswal A, Stevenson LW, Givertz MM, Ofili EO, O'Connor CM, Felker GM, Goldsmith SR, Bart BA, McNulty SE, Ibarra JC, Lin G, Oh JK, Patel MR, Kim RJ, Tracy RP, Velazquez EJ, Anstrom KJ, Hernandez AF, Mascette AM, Braunwald E, Trial R. Effect of phosphodiesterase-5 inhibition on exercise capacity and clinical status in heart failure with preserved ejection fraction: A randomized clinical trial. Jama. 2013;309:1268-1277

7. Lundberg JO, Weitzberg E, Gladwin MT. The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics. Nature reviews. Drug discovery. 2008;7:156-167

8. Liu C, Wajih N, Liu X, Basu S, Janes J, Marvel M, Keggi C, Helms CC, Lee AN, Belanger AM, Diz DI, Laurienti PJ, Caudell DL, Wang J, Gladwin MT, Kim-Shapiro DB. Mechanisms of human erythrocytic bioactivation of nitrite. The Journal of biological chemistry. 2015;290:1281-1294

9. Maher AR, Milsom AB, Gunaruwan P, Abozguia K, Ahmed I, Weaver RA, Thomas P, Ashrafian H, Born GV, James PE, Frenneaux MP. Hypoxic modulation of exogenous nitrite-induced vasodilation in humans. Circulation. 2008;117:670-677

10. Modin A, Bjorne H, Herulf M, Alving K, Weitzberg E, Lundberg JO. Nitrite-derived nitric oxide: A possible mediator of 'acidic-metabolic' vasodilation. Acta physiologica Scandinavica. 2001;171:9-16

11. Cosby K, Partovi KS, Crawford JH, Patel RP, Reiter CD, Martyr S, Yang BK, Waclawiw MA, Zalos G, Xu X, Huang KT, Shields H, Kim-Shapiro DB, Schechter AN, Cannon RO, 3rd, Gladwin MT. Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation. Nature medicine. 2003;9:1498-1505

12. Jones AM. Dietary nitrate supplementation and exercise performance. Sports medicine. 2014;44 Suppl 1:S35-45

13. Ferguson SK, Glean AA, Holdsworth CT, Wright JL, Fees AJ, Colburn TD, Stabler T, Allen JD, Jones AM, Musch TI, Poole DC. Skeletal muscle vascular control during exercise: Impact of nitrite infusion during nitric oxide synthase inhibition in healthy rats. J Cardiovasc Pharmacol Ther. 2016;21:201-208

14. Glean AA, Ferguson SK, Holdsworth CT, Colburn TD, Wright JL, Fees AJ, Hageman KS, Poole DC, Musch TI. Effects of nitrite infusion on skeletal muscle vascular control during exercise in rats



ownloaded from


DOI: 10.1161/CIRCRESAHA.116.309832 12

with chronic heart failure. American journal of physiology. Heart and circulatory physiology. 2015;309:H1354-1360

15. Ferguson SK, Holdsworth CT, Colburn TD, Wright JL, Craig JC, Fees A, Jones AM, Allen JD, Musch TI, Poole DC. Dietary nitrate supplementation: Impact on skeletal muscle vascular control in exercising rats with chronic heart failure. Journal of applied physiology. 2016;121:661-669

16. Ferguson SK, Hirai DM, Copp SW, Holdsworth CT, Allen JD, Jones AM, Musch TI, Poole DC. Impact of dietary nitrate supplementation via beetroot juice on exercising muscle vascular control in rats. The Journal of physiology. 2013;591:547-557

17. Colburn TD, Ferguson SK, Holdsworth CT, Craig JC, Musch TI, Poole DC. Effect of sodium nitrite on local control of contracting skeletal muscle microvascular oxygen pressure in healthy rats. Journal of applied physiology. 2016:jap 00367 02016

18. Poole DC, Hirai DM, Copp SW, Musch TI. Muscle oxygen transport and utilization in heart failure: Implications for exercise (in)tolerance. American journal of physiology. Heart and circulatory physiology. 2012;302:H1050-1063

19. Zamani P, Rawat D, Shiva-Kumar P, Geraci S, Bhuva R, Konda P, Doulias PT, Ischiropoulos H, Townsend RR, Margulies KB, Cappola TP, Poole DC, Chirinos JA. Effect of inorganic nitrate on exercise capacity in heart failure with preserved ejection fraction. Circulation. 2015;131:371-380; discussion 380

20. Eggebeen J, Kim-Shapiro DB, Haykowsky M, Morgan TM, Basu S, Brubaker P, Rejeski J, Kitzman DW. One week of daily dosing with beetroot juice improves submaximal endurance and blood pressure in older patients with heart failure and preserved ejection fraction. JACC Heart Fail. 2016;4:428-437

21. Russell SD, McNeer FR, Beere PA, Logan LJ, Higginbotham MB. Improvement in the mechanical efficiency of walking: An explanation for the "placebo effect" seen during repeated exercise testing of patients with heart failure. Duke university clinical cardiology studies (duccs) exercise group. American heart journal. 1998;135:107-114

22. Haddy FJ, Vanhoutte PM, Feletou M. Role of potassium in regulating blood flow and blood pressure. American journal of physiology. Regulatory, integrative and comparative physiology. 2006;290:R546-552

23. Green CP, Porter CB, Bresnahan DR, Spertus JA. Development and evaluation of the kansas city cardiomyopathy questionnaire: A new health status measure for heart failure. Journal of the American College of Cardiology. 2000;35:1245-1255

24. Nagueh SF, Appleton CP, Gillebert TC, Marino PN, Oh JK, Smiseth OA, Waggoner AD, Flachskampf FA, Pellikka PA, Evangelista A. Recommendations for the evaluation of left ventricular diastolic function by echocardiography. J Am Soc Echocardiogr. 2009;22:107-133

25. Jones AM, Ferguson SK, Bailey SJ, Vanhatalo A, Poole DC. Fiber type-specific effects of dietary nitrate. Exerc Sport Sci Rev. 2016;44:53-60

26. Bailey SJ, Varnham RL, DiMenna FJ, Breese BC, Wylie LJ, Jones AM. Inorganic nitrate supplementation improves muscle oxygenation, o(2) uptake kinetics, and exercise tolerance at high but not low pedal rates. Journal of applied physiology. 2015;118:1396-1405

27. Coggan AR, Leibowitz JL, Kadkhodayan A, Thomas DP, Ramamurthy S, Spearie CA, Waller S, Farmer M, Peterson LR. Effect of acute dietary nitrate intake on maximal knee extensor speed and power in healthy men and women. Nitric oxide : biology and chemistry / official journal of the Nitric Oxide Society. 2015;48:16-21

28. Coggan AR, Leibowitz JL, Spearie CA, Kadkhodayan A, Thomas DP, Ramamurthy S, Mahmood K, Park S, Waller S, Farmer M, Peterson LR. Acute dietary nitrate intake improves muscle contractile function in patients with heart failure: A double-blind, placebo-controlled, randomized trial. Circulation. Heart failure. 2015;8:914-920

29. Chirinos JA, Segers P. Noninvasive evaluation of left ventricular afterload: Part 2: Arterial pressure-flow and pressure-volume relations in humans. Hypertension. 2010;56:563-570



ownloaded from


DOI: 10.1161/CIRCRESAHA.116.309832 13

30. Ryan TE, Southern WM, Reynolds MA, McCully KK. A cross-validation of near-infrared spectroscopy measurements of skeletal muscle oxidative capacity with phosphorus magnetic resonance spectroscopy. Journal of applied physiology. 2013;115:1757-1766

31. Kapil V, Khambata RS, Robertson A, Caulfield MJ, Ahluwalia A. Dietary nitrate provides sustained blood pressure lowering in hypertensive patients: A randomized, phase 2, double-blind, placebo-controlled study. Hypertension. 2015;65:320-327

32. Flynn KE, Pina IL, Whellan DJ, Lin L, Blumenthal JA, Ellis SJ, Fine LJ, Howlett JG, Keteyian SJ, Kitzman DW, Kraus WE, Miller NH, Schulman KA, Spertus JA, O'Connor CM, Weinfurt KP, Investigators H-A. Effects of exercise training on health status in patients with chronic heart failure: Hf-action randomized controlled trial. Jama. 2009;301:1451-1459

33. Nolte K, Herrmann-Lingen C, Wachter R, Gelbrich G, Dungen HD, Duvinage A, Hoischen N, von Oehsen K, Schwarz S, Hasenfuss G, Halle M, Pieske B, Edelmann F. Effects of exercise training on different quality of life dimensions in heart failure with preserved ejection fraction: The ex-dhf-p trial. European journal of preventive cardiology. 2015;22:582-593

34. Zamani P, French B, Brandimarto JA, Doulias PT, Javaheri A, Chirinos JA, Margulies KB, Townsend RR, Sweitzer NK, Fang JC, Ischiropoulos H, Cappola TP. Effect of heart failure with preserved ejection fraction on nitric oxide metabolites. The American journal of cardiology. 2016

35. Gupta D, Georgiopoulou VV, Kalogeropoulos AP, Marti CN, Yancy CW, Gheorghiade M, Fonarow GC, Konstam MA, Butler J. Nitrate therapy for heart failure: Benefits and strategies to overcome tolerance. JACC Heart Fail. 2013;1:183-191

36. Kapil V, Milsom AB, Okorie M, Maleki-Toyserkani S, Akram F, Rehman F, Arghandawi S, Pearl V, Benjamin N, Loukogeorgakis S, Macallister R, Hobbs AJ, Webb AJ, Ahluwalia A. Inorganic nitrate supplementation lowers blood pressure in humans: Role for nitrite-derived no. Hypertension. 2010;56:274-281



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FIGURE LEGENDS Figure 1: CONSORT diagram. Summary of the screened subjects, including reasons for exclusions. Figure 2: Maximal Effort Exercise Study Results . Figure 3: Pharmacokinetics following initial administration of 6 mmol of KNO3. Figure 4: Kansas City Cardiomyopathy Questionnaire Results.



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DOI: 10.1161/CIRCRESAHA.116.309832 15

NOVELTY AND SIGNIFICANCE What Is Known?

Heart Failure with Preserved Ejection Fraction (HFpEF) is associated with marked reductions in quality of life and exercise tolerance.

Although nitric oxide (NO) bioavailability is reduced in HFpEF, prior efforts to increase NO using organic nitrate or phosphodiesterase-5 inhibitors have not been beneficial.

Inorganic nitrate, which is reduced to nitrite by oral anaerobic bacteria, can increase NO signaling

via selective reduction of nitrite to NO at the site of exercising muscle, leading to increased muscle blood flow.

What New Information Does This Article Contribute?

Inorganic nitrate supplementation appears to be potentially safe and well-tolerated in HFpEF subjects.

Inorganic nitrate supplementation increases exercise duration and quality of life in HFpEF subjects in a dose-dependent fashion.

The benefits of inorganic nitrate in HFpEF subjects are most manifest at high exercise intensities.

We studied the population-specific safety and efficacy data for inorganic nitrate in HFpEF. We performed detailed phenotypic and pharmacokinetic assessments of inorganic nitrate in HFpEF. We found that a dose of up to 18 mmol/day of inorganic nitrate potentially appears to be safe, with dose-dependent improvements in exercise duration and quality of life. In contrast to organic nitrate therapy, inorganic nitrate did not reduce physical activity over the course of our 2-week study. These findings strengthen the rationale for performing a larger trial of inorganic nitrate in HFpEF patients.



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Table 1 – Demographic, Laboratory, and Echocardiographic Characteristics of the Study Population at Baseline

Variable Overall KNO3 Placebo n=12 n=9 n=3

Age (years), mean (SD) 62.5 (5.8) 62.4 (5.5) 62.7 (8.0) Male, n (%) 4 (33.3) 3 (33.3) 1 (33.3) Race, n (%) White 8 (66.7) 5 (55.6) 3 (100) African-American 3 (25.0) 3 (33.3) 0 (0) Pacific Islander 1 (8.3) 1 (11.1) 0 (0) NYHA Class, n (%) Class II 10 (83.3) 8 (88.9) 2 (66.7) Class III 2 (16.7) 1 (11.1) 1 (33.3) Body Mass Index (kg/m2), mean (SD) 34.4 (7.0) 33.4 (6.3) 37.4 (9.9) Obese, n (%) 8 (66.7) 5 (55.6) 3 (100) Hypertension, n (%) 12 (100) 9 (100) 3 (100) Hyperlipidemia, n (%) 10 (83.3) 7 (77.8) 3 (100) Coronary Artery Disease, n (%) 4 (33.3) 3 (33.3) 1 (33.3) History of Atrial Fibrillation, n (%) 1 (8.3) 0 (0) 1 (33.3) Diabetes, n (%) 7 (58.3) 5 (55.6) 2 (66.7) Obstructive Sleep Apnea, n (%) 6 (50.0) 3 (33.3) 3 (100) Current CPAP use, n (%) 5 (41.7) 2 (22.2) 3 (100) Obstructive Lung Disease, n (%) 3 (25.0) 2 (22.2) 1 (33.3) Medical Therapy Beta-Blockers, n (%) 9 (75.0) 7 (77.8) 2 (66.7) Ace-I/ARB, n (%) 9 (75.0) 7 (77.8) 2 (66.7) Mineralocorticoid Receptor Antagonists, n (%)

3 (25.0) 2 (22.2) 1 (33.3)

Calcium-Channel Blockers, n (%) 4 (33.3) 3 (33.3) 1 (33.3) Loop Diuretics, n (%) 9 (75.0) 7 (77.8) 2 (66.7) Thiazide Diuretics, n (%) 4 (33.3) 3 (33.3) 1 (33.3) Statin, n (%) 7 (58.3) 5 (55.6) 2 (66.7) Baseline Laboratories eGFR (mL/min/1.73m2), mean (SD) 69.4 (12.6) 71.0 (14.1) 64.7 (5.9) eGFR<60 mL/min/1.73m2, n(%) 4 (33.3) 3 (33.3) 1 (33.3) NT-pro-BNP (pg/mL), mean (SD) 111.0 (89.0) 108.3 (94.7) 119 (87.0) Elevated NT-pro-BNP, n (%) 5 (41.7) 3 (33.3) 2 (66.7) Hemoglobin (g/dL), mean (SD) 13.6 (0.8) 13.6 (0.8) 13.5 (1.0) Methemoglobin (%), mean (SD) 1.0 (0.3) 1.0 (0.2) 1.0 (0.5) Baseline Echocardiographic Data (mean, SD)

LV Mass (g) 172.32 (66.36) 174.43 (77.09) 165.98 (19.27) LV Mass Index (g/m2), mean (SD) 78.88 (22.46) 79.78 (26.01) 76.18 (7.33) Relative Wall Thickness 0.47 (0.09) 0.47 (0.09) 0.46 (0.09) Mitral Early Inflow Velocity (E; cm/s) 77.33 (21.91) 74.53 (24.75) 85.73 (7.04) Mitral Atrial Inflow Velocity (cm/s) 75.21 (20.99) 70.74 (20.06) 88.61 (21.28) Septal Tissue Doppler Early Velocity (e’; mm/s)

76.41 (19.26) 70.64 (12.45) 93.71 (28.65)

Septal E/e’ Ratio 10.42 (3.40) 10.70 (3.76) 9.60 (2.39) Left Atrial Volume Index (mL/m2) 27.74 (7.15) 27.20 (7.87) 29.38 (5.33) Left Ventricular Ejection Fraction (%) 64.23 (9.27) 65.84 (7.74) 59.41 (13.66)

NYHA = New York Heart Association; CPAP = continuous positive airway pressure; ACE-I = angiotensin converting enzyme inhibitor; ARB = angiotensin receptor blocker; elevated NT-pro-BNP defined as >125 pg/mL; eGFR calculated using the MDRD formula



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Table 2 – Peak Effort Gas Exchange Data Marginal Mean (95% CI)

KNO3 (n=9)

Visit 1 Visit 2 Visit 3 P-value

Ventilatory Threshold Ventilatory Threshold (L/min)

0.75 (0.69-0.81)

0.68 (0.61-0.74)

0.74 (0.68-0.80)

0.23

iVentilatory Threshold (mL/kg/min)

8.18 (7.65-8.72)

7.51 (6.94-8.08)

8.07 (7.53-8.60)

0.24

Peak Exercise Peak VO2 (L/min) 1.16

(1.13-1.19) 1.20

(1.17-1.24) 1.20

(1.17-1.23) 0.13

Peak iVO2 (mL/kg/min)

12.50 (12.18-12.83)

12.83 (12.49-13.18)

12.87 (12.55-13.20)

0.26

Exercise Duration (min)

9.87 (9.31-10.43)

10.73 (10.13-11.33)

11.61†

(11.05-12.17) 0.002

Total Work Performed (kJ)

23.22 (18.82-27.61)

26.92 (22.22-31.63)

33.02†

(28.63-37.41) 0.02

Total Oxygen Consumed (L)

8.58 (7.72-9.44)

10.00*

(9.08-10.92) 10.54†

(9.67-11.40) 0.019

Total Exercise Economy (kJ/L O2 Consumed)

2.52 (2.31-2.72)

2.61 (2.39-2.83)

2.85 (2.65-3.06)

0.10

RER 1.08 (1.06-1.10)

1.09 (1.06-1.11)

1.09 (1.07-1.11)

0.83

VE/VCO2 Slope 34.37 (32.75-35.98)

36.10 (34.38-37.83)

33.81 (32.20-35.42)

0.18

RER = respiratory exchange ratio; kJ = kilojoules * p<0.05 V1 vs. V2; † p<0.05 V1 vs. V3; ‡p<0.05 V2 vs. V3



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Table 3 – Vital Signs and Laboratory Data Marginal Means (95% CI)

KNO3 (n=9)

Vital Signs Visit 1 Visit 2 Visit 3 P Weight (kg) 95.0

(94.6-95.4) 95.1

(94.7-95.5) 95.0

(94.6-95.4) 0.94

SBP (mmHg) 127.2 (121.1-133.3)

118.6 (112.5-124.6)

115.0†

(108.9-121.1) 0.037

DBP (mmHg) 72.6 (69.5-75.6)

68.3 (65.2-71.4)

69.9 (66.8-73.0)

0.19

Heart Rate (bpm) 65.1 (59.9-70.3)

65.4 (60.2-70.7)

67.3 (62.1-72.5)

0.82

Laboratory Data Serum Sodium (mEq/dL)

137.8 (137.1-138.5)

137.0 (136.3-137.7)

136.4 (135.7-137.2)

0.06

Serum Potassium (mEq/dL)

3.8 (3.6-4.0)

4.0 (3.8-4.2)

4.1 (3.9-4.3)

0.28

eGFR (mL/min/1.73m2)

71.0 (66.9-75.1)

70.5 (66.4-74.6)

69.6 (65.5-73.7)

0.89

NT-pro-BNP (pg/mL)

108.3 (84.0-132.6)

117.8 (93.5-142.1)

93.2 (68.9-117.5)

0.39

Hemoglobin (g/dL) 13.6 (13.4-13.8)

12.9*

(12.7-13.1) 12.8†

(12.6-13.0) <0.001

Methemoglobin (%) 1.04 (0.92-1.17)

1.19 (1.06-1.32)

1.18 (1.05-1.31)

0.25

Serum NOm (μM) 38.0 (0.00-132.0)

199.5*

(98.7-300.2) 471.8†,‡

(377.8-565.8) <0.001

* p<0.05 V1 vs. V2; † p<0.05 V1 vs. V3; ‡p<0.05 V2 vs. by guest on April 1, 2018


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703 Subjects Screened

15 SubjectsConsented

12 Subjects CompletedStudy

Reason Exclusions

Any rhythm other than sinus 75Inability to Exercise 31

Significant Valvular Disease 54Hypertrophic or Infiltrative Cardiomyopathy

22

Pericardial Disease 3Pulmonary Arterial Hypertension

19

Recent Ischemia 1Non‐Cardiac Dyspnea 64

Uncontrolled HTN 1Hemoglobin < 10 g/dL 20Cirrhosis 6Current Ischemia or angina 19Therapy with PDE5‐I 2Use of Nitrates 23eGFR<30/Hemodialysis 42Reduced LVEF 52Prior reduced LVEF, now recovered

29

No Symptoms of HF 67Deceased 54No recent records available to review

32

Eligible but Not Interested 72Total 688

3: Alternative causeof dyspnea identified during baseline visit

FIGURE 1

by guest on April 1, 2018 http://circres.ahajournals.org/ Downloaded from


1.1

1.1

51

.21

.25

O2

Up

take

, L

/min

(9

5% C

I)

1 2 3Visit

Peak Oxygen Uptake

910

11

12

Min

ute

s (9

5%

CI)

1 2 3Visit

Exercise Duration6

.26

.46

.66

.87

7.2

O2

Up

take

, L

/min

(9

5%

CI)

1 2 3Visit

Final 6 Min: Oxygen Uptake

16

18

202

22

4kJ

(9

5%

CI)

1 2 3Visit

Final 6 Min: Work Performed

Overall P=0.13 Overall P=0.002

P=0.058P=0.001

P=0.055

Overall P=0.041

P=0.049P=0.019

P=0.72

Overall P=0.002

P=0.071

P=0.001

P=0.043

FIGURE 2



*P<0.05 compared to baseline

Overall P=0.007

* * ***

** *

* * **

Overall P=0.054

Overall P=0.014

* * * ** *

Overall P=0.029

* * * * * **

*

FIGURE 3



40

5060

70

80

Me

an

Sco

re (

95%

CI)

1 2 3Visit

Symptom Burden Score

505

560

6570

75

Me

an S

core

, (9

5%

CI)

1 2 3Visit

Total Symptom Score55

60

65

707

5M

ea

n S

core

(9

5%

CI)

1 2 3Visit

Functional Status Score

55

60

6570

75

Mea

n S

core

(9

5% C

I)

1 2 3Visit

Overall Summary Score

Overall P=0.049

P=0.066P=0.02

P=0.55

Overall P=0.016

P=0.042P=0.32

P=0.005

Overall P=0.013

P=0.031

P=0.005

P=0.37

Overall P=0.067

FIGURE 4



Kenneth B Margulies, Thomas P Cappola, David C Poole, Harry Ischiropoulos and Julio A ChirinosTrieu, Swapna Varakantam, Paschalis-Thomas Doulias, Raymond R Townsend, Jesse Chittams,

Payman Zamani, Victor X Tan, Haideliza Soto-Calderon, Melissa Beraun, Jeffrey Brandimarto, LienPreserved Ejection Fraction

Pharmacokinetics and Pharmacodynamics of Inorganic Nitrate in Heart Failure With

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 2016 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

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1

Expanded Methods and Supplementary Materials

Title: Pharmacokinetics and Pharmacodynamics of Inorganic Nitrate in Heart Failure with Preserved

Ejection Fraction

Short Title: Zamani, Inorganic nitrate in HFpEF

Authors: Payman Zamani, MD, MTR*; Victor Tan, BA*; Haideliza Soto-Calderon, BA*; Melissa Beraun,

MD*; Jeffrey A. Brandimarto, MS*; Lien Trieu, BS†; Swapna Varakantam, MD*; Paschalis-Thomas

Doulias‡, PhD; Raymond R. Townsend, MD§; Jesse Chittams, MS||; Kenneth B. Margulies, MD*; Thomas

P. Cappola, MD, ScM*; David C. Poole, PhD, DSc**; Harry Ischiropoulos, PhD‡; Julio A. Chirinos, MD,

PhD*

Affiliations:

* Division of Cardiovascular Medicine; Hospital of the University of Pennsylvania; Philadelphia,

PA

† Rowan University School of Osteopathic Medicine, Stratford, NJ

‡ Children's Hospital of Philadelphia Research Institute, Philadelphia, PA

§ Division of Nephrology/Hypertension, Perelman School of Medicine, University of

Pennsylvania, Philadelphia, PA

|| Office of Nursing Research, School of Nursing, University of Pennsylvania, Philadelphia, PA

** Departments of Kinesiology, Anatomy, and Physiology, Kansas State University, Manhattan,

KS

Corresponding Author:

Payman Zamani, MD, MTR

Hospital of the University of Pennsylvania

3400 Spruce Street, 9026 Gates

Philadelphia, PA 19104

pH: (215) 662-6192

[email protected]

2

Study Protocol

Subjects presented to the Clinical and Translational Research Center at the University of Pennsylvania in

the fasted state. Resting blood pressure was obtained using an automated oscillometric device (Accutorr

Plus, Datascope Corp., Paramus, NJ). Blood was obtained for serum nitric oxide metabolites (NOm) and

nitrite determination, basic chemistries, NT-pro-BNP determination (Cobas e411 Analyzer, Roche

Diagnostics, Indianapolis, IN), and measurement of hemoglobin and methemoglobin (ABL800 Flex

Analyzer, Radiometer America, Brea, CA). Subjects refrained from using mouthwash and were provided

with a list of nitrate-rich foods to avoid during the study. A registered dietician met with subjects at each

study visit to perform a 24-hour dietary recall and to reinforce dietary restrictions. During the baseline

visit (Visit 1), subjects proceeded directly to the exercise testing protocol after verification of

inclusion/exclusion criteria, a physical examination, and laboratory testing. During Visit 2 and Visit 3,

study drug was administered with a standardized low-nitrate meal. Two hours later, orthostatic blood

pressure measurements were made, followed by echocardiography, arterial tonometry, and the exercise

protocol, as described below. The Kansas City Cardiomyopathy Questionnaire was administered at each

study visit.1

Echocardiography and Arterial Tonometry

Subjects underwent resting echocardiography using a GE Vivid I machine (GE Heathcare, Fairfield, CT).

Metrics were quantified in triplicate, according to the American Society of Echocardiography guidelines.2

Left atrial volumes were quantified using the Area-Length method and indexed to body-surface area

(BSA). Left ventricular outflow tract (LVOT) Doppler velocity-time integrals (VTI) were obtained from

the 5-chamber view to obtain stroke volume (LVOT cross-sectional area * VTI) at rest and at peak

exercise.

Resting arterial tonometry (Millar Instruments, Houston, TX) was performed at the left radial

artery and calibrated using brachial oscillometric blood pressures (Scholar III507EL, Criticare Systems,

3

Inc., Waukesha, WI). Tonometric signals were processed using Sphygmocor software (AtCor Medical,

Australia) to derive central pressures. Augmentation index (AIx) was calculated as the ratio of the

amplitude of the second peak to the first peak (P2/P1 x 100%).

Exercise Studies

We used a supine cycle ergometer designed for stress echocardiography (Stress Echo Ergometer

1505, Medical Positioning, Inc., Kansas City, MO), as described previously.3 Subjects underwent expired

gas analysis using a ParvoMedics True One 2400 device (Parvomedics, Utah, USA). Subjects performed

a maximal exertion-limited exercise test using a graded-exercise protocol. Resistance began at 15 Watts

(W) for 3 minutes, increasing to 25W for 3 minutes, then increasing by 25W every 3 minutes thereafter.4

Breath-by-breath information was recorded. Auscultatory blood pressure, 12-lead EKG (XScribe 5,

Mortara Instruments, Milwaukee, Wisconsin), and oxygen saturation were monitored during the test.

Limited echocardiography was performed during each stage of exercise. Verbal encouragement was given

to all subjects. Doppler LVOT VTI was acquired at peak exertion immediately at the cessation of

exercise.

Custom-designed software was programmed in MATLAB (Version R2011b, MathWorks, Natick,

MA) for processing and quantification of CPET data, as described previously.3 All data quantification

was blinded to treatment. Breath-by breath CPET data was visually assessed, and aberrant breaths were

excluded. A Savitzky–Golay filter was used to remove high-frequency breath-by-breath noise in an

operator-independent manner. Peak oxygen uptake (VO2) was determined as the average value during the

final 30 seconds of exercise. The gas exchange/ventilatory threshold (VT) was determined using both the

V-slope and ventilatory equivalent methods, with the results of the two measurements averaged.5

VE/VCO2 slope was calculated from the beginning of exercise to peak effort.6 Respiratory exchange ratio

(RER) was calculated as the ratio of VCO2 to VO2 at end-exercise.

Exercise economy was estimated as the ratio of total work performed (kilojoules) to total oxygen

consumed (liters) above baseline during the entirety of the exercise study. Prior work demonstrates an

4

increased benefit for inorganic nitrate supplementation at higher exercise intensities, when Type II muscle

fibers are utilized to a greater degree.7-11 In post hoc analysis, we quantified total oxygen uptake and work

performed during the last 3- and 6-minutes of exercise, when subjects were exercising at higher

intensities, and obtained estimates of exercise economy over those time periods.

After at least 15 minutes of rest, subjects exercised at 25W for 6 minutes with steady-state VO2

measured during the final minute of exercise. Radial tonometry and LVOT VTIs were obtained at rest and

during steady-state to determine input impedance (described in detail below).12 VO2 recovery kinetics (τ)

were modeled according to a mono-exponential decay.

Central arterial hemodynamics during the submaximal (25W) exercise study

Radial tonometry and cardiac flow, obtained from the 5-chamber LVOT Doppler profile, were obtained at

rest and during the last minute of constant-intensity (25W) exercise. Custom-designed software was

programmed using MATLAB (R2014b, MathWorks, Natick, MA) to derive input impedance. Central

pressure measurements, derived from radial tonometry, were ensemble-averaged and time-aligned with

LVOT flow such that the upstroke of pressure and flow occurred simultaneously, peak flow was

coincident with the first systolic peak or inflection point in the pressure waveform, and flow ceased at the

dicrotic notch. Characteristic impedance (Zc) was quantified in the frequency domain as the average

modulus at higher frequencies. Total vascular resistance (TVR) was quantified as the ratio of mean

pressure to mean flow. Total arterial compliance (TAC) was determined using the pulse-pressure

method.12 Linear wave separation was performed to obtain the amplitude of the forward (Pf) and

backward (Pb) pressure waves. Reflection magnitude was defined as the ratio of Pb to Pf (Pb/Pf).

Skeletal muscle mitochontrial oxidative capacity

We performed skeletal muscle mitochondrial function testing using the technique developed by Ryan et

al. which has been validated against 31P-MRI and in situ measurements of mitochondrial respiration.13-15

Fifteen minutes following the end of the constant intensity exercise protocol, subjects were asked to sit

5

comfortably with their arms raised on a table to the level of the heart. The elbows were placed in mild

flexion at approximately 30 degrees to avoid venous pooling. NIRS devices were then placed over the

flexor digitorum superficialis bilaterally. A blood pressure cuff was placed on the dominant arm and

connected to a rapid inflator (E20 Rapid Cuff Inflator, D.E. Hokanson, Inc., Bellevue, WA), which was

connected to a large-volume compressor (Hokanson AG101 Cuff Inflator Air Source, D.E. Hokanson,

Inc., Bellevue, WA). Three initial high-pressure inflations (200 mm Hg) for 10s were performed to

measure the rate of local O2 muscle oxygen consumption (MVO2) at rest. During arterial occlusions, the

inflow of oxygenated blood into muscle tissue is interrupted; thus, the rate of decrease in oxyhemoglobin

concentration is solely proportional to mVO2.14 Subjects were then instructed to perform 8 maximal

contractions at a rate of 0.5 Hz using a dynamometer in the dominant hand. Following the last

contraction, a series of rapid cuff inflations (200 mm Hg) was performed in the following sequence: Cuff

occlusions 1-5: 5 second on/5 seconds off, Cuff occlusions 6-10: 5 seconds on/10 seconds off; Cuff

occlusions 11-15: 10 seconds on/20 seconds off.16 A two-minute rest period was then given before the

entire protocol was repeated. A custom-made video was used to standardize the cadence and timing of

contractions and occlusions between experiments.

The recovery of mVO2 after brief exercise was assessed, as previously described in detail.3, 13, 17

Oxy- and deoxyhemoglobin signals were corrected based on the principle that total blood-volume remains

constant during arterial occlusions. This correction is performed under the assumption that decreases in

oxyhemoglobin occur in a 1:1 ratio with increases in deoxyhemoglobin and that the rate of this change is

proportional to local muscle oxygen consumption (mVO2).17 Second, to account for differences in the

adipose layer overlying the muscle bed of interest, signals were normalized to the minimum values

obtained during 5-minutes of brachial artery occlusion. These methods have been shown to remove the

variability caused by differing degrees of adipose thickness, as well as differences due to the depth of

interrogation.17 Finally, the linear slope of the decrease in oxyhemoglobin over time was calculated for

each arterial occlusion with linear regression, using a custom-made software interface programmed in

Matlab. Such slopes plotted over time have been shown to follow a mono-exponential recovery described

6

by a time constant (τ, tau). This recovery corresponds with PCr recovery kinetics measured with MRI

spectroscopy and quantifies mitochondrial oxidative capacity.13, 15, 18 In some cases, slopes taken early

after the cessation of exercise showed a rapid increase in mVO2, followed by a monoexponential

recovery. In these cases, only the monoexponential recovery portion was fitted to assess τ.

Safety and Pharmacokinetic Determination

On Visit 1 and Visit 2, subjects were given study medications (either 6 mmol KNO3 or 6 mmol KCl) with

a standardized nitrate-free meal following exercise testing. For the next 5 hours, blood pressure was

measured every 15 minutes using an automated oscillometric device (Accutorr Plus, Datascope Corp.,

Paramus, NJ), and serum was drawn every 30 minutes for determination of NOm (described in detail

below) and methemoglobin. The elimination rate (Ke) was the negative of the slope from the linear

regression of log concentration versus time over the last 3 time points and used to calculate the half-life

(t1/2 = ln 2/Ke).

Serum NOm Quantification

Samples were passed through a filter (AmiconUltra-0.5 Centrifugal Filter Unit, EMD Millipore) to

remove proteins with molecular weight >30 kilodalton. Samples were then injected into a custom-made

ice-water cooled reaction chamber containing vanadium(III)/hydrochloric acid solution heated to 95°C.

The NO generated from the reduction of NOm was quantified using chemiluminescence (Nitric Oxide

Analyzer 280, Sievers Instruments, Boulder, CO). Signal peaks (mV) were manually integrated, and the

corresponding areas were used for the quantification of NOm concentration. A 10-point standard curve

was generated by the injection of nitrate in the range of 0 to 50 μmol/L. The detection limit of this assay

was 1.6 μmol/L. NOm samples for 3 individuals were repeated in each of the 9 batches performed. The

intra-class correlation coefficient for NOm was 0.98 (95%CI 0.93-1.00; P<0.001), demonstrating excellent

reproducibility. Serum nitrite levels were quantified using reduction with potassium iodide/crystalline

iodide and the same Sievers instrument. Authentic nitrite in the range of 0.16-10 μmol/L was injected into

7

the system, and a 7-point standard curve was generated by plotting area against concentration. Nitrite

samples were measured in each of the 6 batches performed. The intra-class correlation coefficient for

nitrite was 0.94 (95%CI 0.77-1.00; P<0.001).

Activity Monitoring and 24-hour Blood Pressure Measurements

At the conclusion of Visit 1 and Visit 2, subjects were given an ambulatory blood pressure monitor

(Mobil-O-Graph PWA, I.E.M. GmbH, Stolberg, Germany) and an activity monitor (wActiSleep-BT

Monitor, Actigraph, LLC., Pensacola, FL). Blood pressure measurements were obtained every 30 minutes

over a 24-hour period. Actigraphy was used to obtain daily steps taken and estimated kilocalorie

expenditure. Because follow-up visits were scheduled during the last 1-2 days of each week, data was

abstracted for days 1-5 in all subjects. Values for days 3-5 were averaged to obtain estimates

corresponding to steady-state drug effects (SS).

Study Outcomes

The primary efficacy endpoint was the change in VO2,peak. Secondary endpoints included changes in

vasodilatory reserve (percent reduction in systemic vascular resistance during exercise, compared to the

resting measurement), changes in mitochondrial oxidative function using NIRS, and changes in AIx.

Exploratory endpoints included other metrics of exercise capacity, and changes in the KCCQ.

Pharmacokinetics were assessed via measurement of NOm after single-dose administration of 6 mmol and

with trough levels after repeated twice- and thrice-daily administration.

Statistical Methods

Demographic data are presented as n(%) or means(SD). Mixed-effects models were generated in STATA

(Stata/SE 13.1, StataCorp, College Station, TX), incorporating the correlation between repeated

measurements in the same individual. No assumption of linearity was made. As pre-specified in the

protocol, only within group comparisons were performed. Marginal means with 95% confidence intervals

8

(CI) were determined from the mixed models and presented in the tables and figures. An overall P-

value<0.05 was taken to be significant for each model, with post-hoc comparisons between visits

performed subsequently. No adjustment for multiple comparisons was made. Pharmacokinetic data was

analyzed using the PK package in STATA. Linear regression was performed to assess the correlation

between changes in serum NOm and nitrite levels and the change in exercise metrics. Regression

coefficients are presented as standardized units describing the standard deviation change in the endpoint

for each standard deviation change in the predictor variable. In a previous trial, we demonstrated a

1.0±1.2 mL/min/kg improvement in peak VO2 following a single-dose (12.9 mmol) of inorganic nitrate,

given in the form of beetroot juice.16 Using a correlation between studies of 0.80, beta=0.80, and

alpha=0.05, we estimated that 7 subjects would be needed to demonstrate a similar change. We thus

planned for 9 subjects in the active arm to allow for drop-out. Randomization was performed by the

University of Pennsylvania Investigational Drug Pharmacy. Randomization tables were constructed using

a 3:1 KNO3:PB ratio in blocks of 4 (www.randomization.com).

9

References

1. Green CP, Porter CB, Bresnahan DR, Spertus JA. Development and evaluation of the kansas city

cardiomyopathy questionnaire: A new health status measure for heart failure. Journal of the

American College of Cardiology. 2000;35:1245-1255

2. Nagueh SF, Appleton CP, Gillebert TC, Marino PN, Oh JK, Smiseth OA, Waggoner AD,

Flachskampf FA, Pellikka PA, Evangelista A. Recommendations for the evaluation of left

ventricular diastolic function by echocardiography. J Am Soc Echocardiogr. 2009;22:107-133

3. Zamani P, Rawat D, Shiva-Kumar P, Geraci S, Bhuva R, Konda P, Doulias PT, Ischiropoulos H,

Townsend RR, Margulies KB, Cappola TP, Poole DC, Chirinos JA. Effect of inorganic nitrate on

exercise capacity in heart failure with preserved ejection fraction. Circulation. 2015;131:371-380;

discussion 380

4. Haykowsky MJ, Brubaker PH, Stewart KP, Morgan TM, Eggebeen J, Kitzman DW. Effect of

endurance training on the determinants of peak exercise oxygen consumption in elderly patients

with stable compensated heart failure and preserved ejection fraction. J Am Coll Cardiol.

2012;60:120-128

5. American Thoracic S, American College of Chest P. Ats/accp statement on cardiopulmonary

exercise testing. Am J Respir Crit Care Med. 2003;167:211-277

6. Balady GJ, Arena R, Sietsema K, Myers J, Coke L, Fletcher GF, Forman D, Franklin B, Guazzi

M, Gulati M, Keteyian SJ, Lavie CJ, Macko R, Mancini D, Milani RV, American Heart

Association Exercise CR, Prevention Committee of the Council on Clinical C, Council on E,

Prevention, Council on Peripheral Vascular D, Interdisciplinary Council on Quality of C,

Outcomes R. Clinician's guide to cardiopulmonary exercise testing in adults: A scientific

statement from the american heart association. Circulation. 2010;122:191-225

10

7. Ferguson SK, Hirai DM, Copp SW, Holdsworth CT, Allen JD, Jones AM, Musch TI, Poole DC.

Impact of dietary nitrate supplementation via beetroot juice on exercising muscle vascular control

in rats. The Journal of physiology. 2013;591:547-557

8. Jones AM, Ferguson SK, Bailey SJ, Vanhatalo A, Poole DC. Fiber type-specific effects of dietary

nitrate. Exerc Sport Sci Rev. 2016;44:53-60

9. Bailey SJ, Varnham RL, DiMenna FJ, Breese BC, Wylie LJ, Jones AM. Inorganic nitrate

supplementation improves muscle oxygenation, o(2) uptake kinetics, and exercise tolerance at

high but not low pedal rates. Journal of applied physiology. 2015;118:1396-1405

10. Coggan AR, Leibowitz JL, Kadkhodayan A, Thomas DP, Ramamurthy S, Spearie CA, Waller S,

Farmer M, Peterson LR. Effect of acute dietary nitrate intake on maximal knee extensor speed

and power in healthy men and women. Nitric oxide : biology and chemistry / official journal of

the Nitric Oxide Society. 2015;48:16-21

11. Coggan AR, Leibowitz JL, Spearie CA, Kadkhodayan A, Thomas DP, Ramamurthy S, Mahmood

K, Park S, Waller S, Farmer M, Peterson LR. Acute dietary nitrate intake improves muscle

contractile function in patients with heart failure: A double-blind, placebo-controlled, randomized

trial. Circulation. Heart failure. 2015;8:914-920

12. Chirinos JA, Segers P. Noninvasive evaluation of left ventricular afterload: Part 2: Arterial

pressure-flow and pressure-volume relations in humans. Hypertension. 2010;56:563-570

13. Ryan TE, Southern WM, Reynolds MA, McCully KK. A cross-validation of near-infrared

spectroscopy measurements of skeletal muscle oxidative capacity with phosphorus magnetic

resonance spectroscopy. Journal of applied physiology. 2013;115:1757-1766

14. Van Beekvelt MC, Colier WN, Wevers RA, Van Engelen BG. Performance of near-infrared

spectroscopy in measuring local o(2) consumption and blood flow in skeletal muscle. Journal of

applied physiology. 2001;90:511-519

11

15. Ryan TE, Brophy P, Lin CT, Hickner RC, Neufer PD. Assessment of in vivo skeletal muscle

mitochondrial respiratory capacity in humans by near-infrared spectroscopy: A comparison with

in situ measurements. The Journal of physiology. 2014;592:3231-3241

16. Ryan TE, Brizendine JT, McCully KK. A comparison of exercise type and intensity on the

noninvasive assessment of skeletal muscle mitochondrial function using near-infrared

spectroscopy. J Appl Physiol (1985). 2013;114:230-237

17. Ryan TE, Erickson ML, Brizendine JT, Young HJ, McCully KK. Noninvasive evaluation of

skeletal muscle mitochondrial capacity with near-infrared spectroscopy: Correcting for blood

volume changes. J Appl Physiol (1985). 2012;113:175-183

18. Hamaoka T, McCully KK, Quaresima V, Yamamoto K, Chance B. Near-infrared

spectroscopy/imaging for monitoring muscle oxygenation and oxidative metabolism in healthy

and diseased humans. Journal of biomedical optics. 2007;12:062105

12

Supplemental Table I - Maximal Effort Exercise Study Hemodynamic Data in KNO3-Treated Subjects Marginal Means (95% CI)

KNO3 (n=9)

Visit 1 Visit 2 Visit 3 P Baseline Heart Rate (beats/min)

60.2 (56.0-64.4)

59.9 (55.4-64.4)

59.8 (55.6-64.0)

0.99

Brachial SBP (mmHg)

128.3 (121.3-135.4)

127.2 (120.2-134.3)

125.6 (118.5-132.6)

0.86

Central SBP (mmHg)

119.4 (112.8-126.1)

119.1 (112.5-125.7)

116.4 (109.8-123.1)

0.79

Central MAP (mmHg)

92.8 (89.0-96.5)

92.1 (88.4-95.9)

90.3 (86.6-94.1)

0.65

Stroke Volume (mL)

72.7 (66.4-78.9)

71.5 (65.3-77.7)

67.1 (60.9-73.3)

0.45

Cardiac Output (L/min)

4.3 (3.9-4.8)

4.3 (3.9-4.8)

3.9 (3.5-4.4)

0.37

SVR (Wood Units)

22.3 (19.9-24.6)

23.5 (21.2-25.8)

24.0 (21.7-26.3)

0.58

Peak Exercise Heart Rate (beats/min)

131.3 (126.5-136.0)

127.9 (122.8-133.0)

131.2 (126.4-135.9)

0.58

Brachial SBP (mmHg)

206.1 (195.2-216.9)

200.3 (189.5-211.2)

194.5 (183.6-205.3)

0.37

Central SBP (mmHg)

169.2 (161.5-176.9)

168.5 (160.8-176.2)

170.0 (162.3-177.7)

0.97

Central MAP (mmHg)

129.4 (123.8-134.9)

133.4 (127.9-139.0)

138.6 (133.0-144.1)

0.11

Stroke Volume (mL)

67.0 (61.8-72.3)

67.7 (62.4-72.9)

71.3 (66.4-76.2)

0.46


8.9 (8.1-9.7)

8.8 (7.9-9.7)

9.3 (8.6-10.1)

0.62

SVR (Wood Units)

16.1 (14.9-17.3)

16.5 (15.2-17.8)

16.2 (15.0-17.4)

0.91

Reserves Heart Rate Reserve (%)

120.1 (103.6-136.5)

119.7 (100.6-138.8)

128.1 (111.7-144.6)

0.74

Stroke Volume Reserve (%)

-6.2 (-18.5-6.0)

-2.1 (-14.3-10.2)

5.9 (-5.6-17.3)

0.38

Cardiac Output Reserve (%)

112.3 (84.4-140.2)

133.0 (102.8-163.2)

145.0 (119.1-170.9)

0.27

Vasodilatory Reserve (%)

-25.6 (-40.9-[-10.4])

-27.1 (-43.6-[-10.5])

-34.2 (-49.4-[-18.9])

0.72

HR = heart rate; SBP = systolic blood pressure; DBP = diastolic blood pressure; MAP = mean arterial pressure; SVR = systemic vascular resistance * p<0.05 V1 vs. V2; † p<0.05 V1 vs. V3; ‡p<0.05 V2 vs. V3

13

Supplemental Table II – Oxygen Uptake/Work Performed in the Last 3 and 6 Minutes of Exercise Marginal Means (95% CI)

KNO3 (n=9)

Visit 1 Visit 2 Visit 3 P-value Oxygen Uptake in Last 3 minutes (L of O2)

3.46 (3.37-3.56)

3.61 (3.50-3.71)

3.61 (3.51-3.71)

0.11

Work Performed in Last 3 minutes (kJ)

10.29 (9.45-11.13)

11.59 (10.69-12.49)

12.90†

(12.06-13.74) 0.002

Economy in Last 3 minutes (kJ/L O2)

2.81 (2.59-3.04)

3.08 (2.84-3.32)

3.42†

(3.19-3.64) 0.007

Oxygen Uptake in Last 6 minutes (L of O2)

6.48 (6.24-6.71)

6.86*

(6.60-7.11) 6.92†

(6.69-7.16) 0.041


16.88 (15.31-18.46)

19.18 (17.50-20.87)

21.79†,‡

(20.22-23.37) 0.002

Economy in Last 6 minutes (kJ/L O2)

2.44 (2.27-2.62)

2.67 (2.48-2.85)

2.95†,‡ (2.78-3.13)

0.004

* p<0.05 V1 vs. V2; † p<0.05 V1 vs. V3; ‡p<0.05 V2 vs. V3 kJ=kilojoules

14

Supplemental Table IIIa – Any Side Effect During Study N (%) Total

(n=12) KNO3 (n=9)

Placebo (n=3)

Any Side Effect 5 4 (44) 1 (33) GI Symptoms* 4 3 (33) 1 (33) Headache 2 2 (22) 0 (0) Palpitations 1 1 (11) 0 (0) Fatigue 1 1 (11) 0 (0) Mild Lower Extremity Edema

1 0 (0) 1 (33)

Tooth Infection

1 1 (11) 0 (0)

* Gastrointestinal (GI) symptoms include: GI upset, nausea, vomiting, flatulence Supplemental Table IIIb – Any Side Effect During Week 1 N (%) Total

(n=12) KNO3 (n=9)

Placebo (n=3)


0 0 (0) 0 (0)

Tooth Infection

0 0 (0) 0 (0)

Supplemental Table IIIc – Any Side Effect During Week 2 N (%) Total

(n=12) KNO3 (n=9)

Placebo (n=3)


1 0 (0) 1 (33)

Tooth Infection

1 1 (11) 0 (0)

15

Supplemental Table IV – Resting Echocardiography in KNO3-Treated Subjects Marginal Means (95% CI)

KNO3 (n=9)

Visit 1 Visit 2 Visit 3 P Mitral E (cm/s) 74.5

(68.9-80.2) 81.2

(75.5-86.9) 79.1

(73.4-84.7) 0.28

Mitral A (cm/s) 70.7 (66.6-74.9)

68.2 (64.0-72.3)

66.2 (62.0-70.3)

0.33

E/A Ratio 1.17 (0.94-1.40)

1.48 (1.25-1.71)

1.45 (1.22-1.68)

0.15

Septal e’ (cm/s) 7.06 (6.32-7.81)

7.80 (7.05-8.55)

7.69 (6.94-8.44)

0.36

E/e’ Ratio 10.70 (9.50-11.90)

10.80 (9.60-12.00)

10.59 (9.39-11.79)

0.97

LAVI (mL/m2) 30.34 (27.36-33.32)

33.55 (30.35-36.74)

32.18 (29.19-35.16)

0.38

LV EDV (mL) 79.39 (73.73-85.05)

78.81 (73.15-84.46)

78.78 (73.12-84.43)

0.99

RV FAC (cm2) 51.95 (47.05-56.84)

51.55 (46.41-56.69)

54.47 (48.85-60.10)

0.73

TAPSE (cm) 2.19 (1.99-2.40)

2.29 (2.10-2.49)

2.38 (2.19-2.57)

0.43

LAVI = left atrial volume index; LV EDV = Left ventricular end diastolic volume; RV FAC = right ventricular fractional area change; TAPSE = tricuspid annular plane systolic excursion * p<0.05 V1 vs. V2; † p<0.05 V1 vs. V3; ‡p<0.05 V2 vs. V3

16

Supplemental Table V – Input Impedance during SubMaximal (25W) Exercise in KNO3-Treated Subjects Marginal Means (95% CI)

KNO3 (n=9)

Baseline Visit 1 Visit 2 Visit 3 P Zc, mmHg/ mL/s

0.07 (0.06-0.08)

0.09* (0.08-0.10)

0.07‡ (0.05-0.08)

0.029

TAC, mL/mmHg 1.02 (0.82-1.21)

0.89 (0.70-1.09)

1.30‡ (1.11-1.50)

0.032

RM 0.44 (0.39-0.49)

0.48 (0.43-0.53)

0.47 (0.42-0.52)

0.62

Pf, mmHg 27.4 (22.6-32.2)

33.6 (28.8-38.4)

25.4 (20.6-30.2)

0.077

Pb, mmHg 11.8 (9.5-14.0)

15.9*

(13.6-18.1) 11.8‡

(9.6-14.0) 0.036

Augmentation Index (%)

121.3 (113.8-128.7)

129.5 (122.0-136.9)

130.1 (122.6-137.5)

0.22

Steady State Zc, mmHg/ mL/s

0.11 (0.09-0.13)

0.11 (0.09-0.13)

0.10 (0.08-0.12)

0.88

TAC, mL/mmHg 0.54 (0.43-0.66)

0.62 (0.52-0.73)

0.63 (0.53-0.74)

0.53

RM 0.46 (0.40-0.52)

0.45 (0.39-0.50)

0.51 (0.46-0.57)

0.20

Pf, mmHg 47.8 (41.2-54.5)

47.6 (41.4-53.7)

45.3 (39.2-51.5)

0.83

Pb, mmHg 21.4 (18.0-24.8)

20.5 (17.3-23.6)

22.1 (19.0-25.3)

0.77


123.2 (115.5-130.8)

123.1 (116.0-130.2)

121.3 (114.2-128.4)

0.92

Percent Change Zc 4.65

(-18.5-27.8) -14.9

(-36.3-6.5) -1.8

(-23.2-19.6) 0.48

TAC -49.4 (-65.6-[-33.2])

-29.7 (-44.7-[-14.8])

-46.7 (-61.6-[-31.7])

0.19

RM 12.7 (-7.1-32.5)

-4.6 (-23.0-13.7)

11.2 (-7.1-29.6)

0.39

Pf 84.7 (42.3-127.0)

43.8 (4.7-83.0)

80.6 (41.5-119.8)

0.33

Pb 100.4 (63.0-137.8)

37.8 (3.2-72.4)

90.8 (56.2-125.4)

0.06

Augmentation Index 3.0 (-5.4-11.4)

-5.4 (-13.1-2.4)

-7.1 (-14.9-0.6)

0.23

Zc = Characteristic Impedance of the Proximal Aorta; TAC = total arterial compliance; RM = reflection magnitude * p<0.05 V1 vs. V2; † p<0.05 V1 vs. V3; ‡p<0.05 V2 vs. V3

17

Supplemental Table VI – SubMaximal (25W) Exercise Data in KNO3-Treated Subjects Marginal Mean (95% CI)

KNO3 (n=8)

Visit 1 Visit 2 Visit 3 P-value

Baseline Cardiac Output (L/min)

5.0 (4.2-5.9)

5.0 (4.2-5.8)

4.9 (4.1-5.7)

0.97

VO2 (L/min) 0.29 (0.26-0.32)

0.28 (0.25-0.31)

0.27 (0.24-0.30)

0.50

Steady State Cardiac Output (L/min)

8.0 (7.1-8.9)

8.3 (7.5-9.2)

7.7 (6.9-8.6)

0.61

VO2 (L/min) 1.04 (1.00-1.08)

1.06 (1.02-1.09)

1.05 (1.02-1.09)

0.85

Economy (kJ/L O2 Consumed)

2.18 (1.78-2.57)

2.17 (1.78-2.56)

2.23 (1.83-2.62)

0.98

τ, off kinetics (s) 15.6 (14.0-17.2)

15.6 (14.0-17.2)

13.5 (11.9-15.1)

0.15

kJ = kilojoules; s = seconds * p<0.05 V1 vs. V2; † p<0.05 V1 vs. V3; ‡p<0.05 V2 vs. V3

18

Supplemental Table VII – Study Data for PB-Treated Subjects Marginal Means (95% CI)

Placebo (n=3)

Vital Signs and Laboratory Data Vital Signs Visit 1 Visit 2 Visit 3 P Weight (kg) 105.2

(104.6-105.7) 104.7

(104.0-105.4) 105.2

(104.7-105.8) 0.52

SBP (mmHg) 121.3 (108.6-134.1)

127.0 (114.3-139.7)

113.3 (100.6-126.1)

0.41

DBP (mmHg) 63.3 (60.5-66.2)

68.3 (65.5-71.2)

64.3 (61.5-67.2)

0.14

Heart Rate (bpm) 59.7 (54.3-65.1)

63.3 (57.9-68.7)

65.3 (59.9-70.7)

0.42

Serum Sodium (mEq/dL)

137.7 (136.6-138.8)

135.3 (134.2-136.4)

135.7 (134.6-136.8)

0.08

Serum Potassium (mEq/dL)

4.0 (3.8-4.2)

3.8 (3.6-4.0)

4.1 (3.9-4.3)

0.23

eGFR (mL/min/1.73m2)

64.7 (59.4-70.0)

66.1 (60.7-71.4)

70.4 (65.1-75.8)

0.39

NT-pro-BNP (pg/mL)

119.0 (103.3-134.7)

125.0 (109.3-140.7)

123.0 (107.3-138.7)

0.87

Hemoglobin (g/dL) 13.5 (13.0-13.9)

13.0 (12.5-13.4)

12.5 (12.0-12.9)

0.10

Methemoglobin (%) 0.97 (0.63-1.30)

1.13 (0.80-1.47)

0.97 (0.63-1.30)

0.74

Serum NOm (μM) 11.9 (3.5-20.4)

30.5 (22.0-39.0)

18.1 (9.6-26.6)

0.09

Maximal Effort Exercise Test Visit 1 Visit 2 Visit 3 P Baseline Heart Rate (beats/min)

66.2 (60.6-71.7)

63.5 (57.9-69.0)

60.3 (54.8-65.9)

0.43

Brachial SBP (mmHg)

133.7 (123.0-144.3)

118.0 (107.4-128.6)

117.7 (107.0-128.3)

0.17

Central SBP (mmHg)

119.7 (105.4-133.9)

107.7 (93.4-121.9)

103.3 (89.1-117.6)

0.35

Central MAP (mmHg)

93.7 (81.1-106.2)

82.7 (70.1-95.2)

79.0 (66.4-91.6)

0.34

Stroke Volume (mL)

99.7 (88.9-110.6)

92.7 (81.8-103.6)

93.4 (82.6-104.3)

0.65


6.7 (5.4-7.9)

6.0 (4.7-7.2)

5.7 (4.5-7.0)

0.60

SVR (Wood Units)

14.9 (13.7-16.1)

14.8 (13.7-16.0)

14.7 (13.5-15.9)

0.97

Peak Exercise Heart Rate (beats/min)

136.0 (130.2-141.8)

134.3 (128.6-140.1)

133.7 (127.9-139.4)

0.85

Brachial SBP (mmHg)

201.1 (190.5-211.7)

199.6 (191.3-208.0)

192.6 (184.3-201.0)

0.46

Central SBP 157.7 167.4 161.4 0.46

19

(mmHg) (146.8-168.6) (158.9-176.0) (152.9-170.0) Central MAP (mmHg)

122.6 (113.0-132.3)

131.6 (124.0-139.2)

127.6 (120.0-135.2)

0.46

Stroke Volume (mL)

90.8 (81.7-99.9)

99.5 (87.9-111.1)

91.9 (82.8-101.0)

0.56


12.2 (11.4-13.0)

13.4 (12.4-14.4)

12.3 (11.5-13.1)

0.29

SVR (Wood Units)

9.6 (9.0-10.1)

9.5 (9.0-10.1)

10.3 (9.9-10.7)

0.21

Reserves Heart Rate Reserve (%)

112.5 (96.8-128.2)

116.7 (101.0-132.4)

128.5 (112.8-144.2)

0.42

Stroke Volume Reserve (%)

-8.1 (-26.5-10.3)

17.4 (-6.0-40.8)

0.1 (-18.3-18.5)

0.38

Cardiac Output Reserve (%)

96.0 (46.8-145.2)

171.7 (109.2-234.2)

137.4 (88.2-186.6)

0.31

Vasodilatory Reserve (%)

-25.8 (-31.4-[-20.3])

-30.6 (-36.2-[-25.1])

-20.0 (-24.2-[-15.8])

0.17

Peak Effort Gas Exchange Data Visit 1 Visit 2 Visit 3 P Ventilatory Threshold (L/min)

0.80 (0.69-0.91)

0.73 (0.62-0.85)

0.76 (0.64-0.87)

0.71

iVentilatory Threshold (mL/kg/min)

7.88 (6.58-9.18)

7.30 (5.65-8.95)

7.44 (6.14-8.74)

0.85

Exercise Duration (min)

16.07 (12.94-19.21)

15.00 (11.87-18.13)

17.72 (14.58-20.85)

0.54

Total Work Performed (kJ)

65.72 (34.70-96.75)

60.95 (29.92-91.97)

87.64 (56.61-118.66)

0.51

Peak VO2 (L/min)

1.49 (1.43-1.54)

1.44 (1.39-1.49)

1.44 (1.39-1.50)

0.47

Peak iVO2 (mL/kg/min)

14.92 (14.37-15.47)

14.67 (13.98-15.37)

14.46 (13.92-15.01)

0.57

Total Oxygen Consumed (L)

15.51 (11.15-19.87)

18.41 (14.04-22.77)

19.95 (15.58-24.31)

0.44

Total Exercise Economy (kJ/L O2 Consumed)

3.67 (3.08-4.27)

2.69 (2.10-3.29)

3.50 (2.91-4.10)

0.16

RER 1.07 (1.02-1.12)

1.16 (1.11-1.21)

1.11 (1.06-1.16)

0.16

VE/VCO2 Slope 35.61 (33.30-37.92)

32.87 (30.55-35.18)

32.89 (30.58-35.20)

0.28

Oxygen Uptake and Work Performed in the last 3 and 6 minutes of Exercise Visit 1 Visit 2 Visit 3 P Oxygen Uptake in Last 3 minutes (L of O2)

4.52 (4.35-4.69)

4.39 (4.22-4.56)

4.38 (4.21-4.55)

0.50


19.60 (14.90-24.30)

17.99 (13.29-22.69)

22.06 (17.36-26.76)

0.54

20

Gross Economy in Last 3 minutes (kJ/L O2)

4.06 (2.99-5.13)

3.77 (2.69-4.84)

4.71 (3.64-5.78)

0.52

Oxygen Uptake in Last 6 minutes (L of O2)

8.78 (8.31-9.25)

8.59 (8.13-9.06)

8.55 (8.08-9.02)

0.78


34.84 (25.52-44.17)

31.89 (22.57-41.22)

39.63 (30.30-48.95)

0.56

Gross Economy in Last 6 minutes (kJ/L O2)

3.65 (2.56-4.75)

3.36 (2.26-4.45)

4.28 (3.19-5.37)

0.54

SubMaximal (25W) Exercise Data Visit 1 Visit 2 Visit 3 P Baseline Cardiac Output (L/min)

5.8 (5.3-6.3)

6.4 (5.7-7.1)

6.8 (6.3-7.3)

0.17

VO2 (L/min) 0.26 (0.22-0.29)

0.32 (0.28-0.35)

0.25 (0.21-0.28)

0.09

Steady-State Cardiac Output (L/min)

10.6 (9.0-12.2)

8.6 (6.5-10.6)

10.1 (8.5-11.7)

0.42

VO2 (L/min) 1.19 (1.09-1.29)

1.21 (1.10-1.31)

1.16 (1.06-1.27)

0.84

Gross Economy (kJ/L O2 Consumed)

1.66 (1.55-1.78)

1.82 (1.70-1.93)

1.66 (1.55-1.78)

0.21

τ, off kinetics (s) 13.9 (12.8-15.0)

14.8 (13.7-15.9)

14.7 (13.7-15.8)

0.49

Input Impedance During SubMaximal Exercise Baseline Visit 1 Visit 2 Visit 3 P Zc, mmHg/ mL/s

0.04 (0.03-0.06)

0.05 (0.03-0.07)

0.06 (0.04-0.07)

0.46

TAC, mL/mmHg 1.52 (1.29-1.76)

1.43 (1.13-1.73)

1.49 (1.25-1.72)

0.90

RM 0.50 (0.37-0.63)

0.37 (0.21-0.54)

0.39 (0.26-0.52)

0.50

Pf, mmHg 23.0 (19.4-26.6)

25.2 (20.6-29.8)

30.2 (26.6-33.8)

0.14

Pb, mmHg 10.4 (8.3-12.5)

9.7 (6.9-12.4)

11.0 (8.8-13.1)

0.79


113.7 (105.0-122.3)

123.9 (112.9-134.8)

121.3 (112.7-129.9)

0.41

Steady State Zc, mmHg/ mL/s

0.06 (0.03-0.08)

0.07 (0.03-0.10)

0.05 (0.02-0.07)

0.67

TAC, mL/mmHg 1.03 (0.42-1.64)

0.70 (-0.07-1.47)

1.34 (0.73-1.94)

0.52

RM 0.42 0.46 0.48 0.84

21

(0.29-0.55) (0.29-0.63) (0.34-0.61) Pf, mmHg 37.6

(25.0-50.1) 41.5

(25.6-57.5) 31.5

(19.0-44.0) 0.65

Pb, mmHg 16.2 (10.1-22.3)

17.6 (9.8-25.3)

15.5 (9.4-21.6)

0.93


110.6 (102.9-118.2)

109.8 (100.1-119.5)

121.6 (113.9-129.2)

0.22

Percent Change Zc -4.1

(-66.3-58.1) -1.8

(-80.9-77.3) 18.1

(-44.1-80.3) 0.87

TAC -33.9 (-59.7-[-8.0])

-49.6 (-82.4-[-16.7])

-16.1 (-41.9-9.8)

0.40

RM -3.6 (-55.2-48.0)

13.8 (-51.8-79.4)

25.9 (-25.7-77.5)

0.75

Pf 73.3 (6.7-140.0)

64.0 (-20.7-148.7)

0.65 (-66.0-67.3)

0.39

Pb 52.4 (0.13-104.6)

77.0 (10.6-143.4)

29.6 (-22.7-81.8)

0.60

Augmentation Index

-2.3 (-11.5-6.8)

-11.9 (-23.5-[-0.2])

-0.2 (-9.4-9.0)

0.41

Resting Echocardiography Visit 1 Visit 2 Visit 3 P Mitral E (cm/s) 85.7

(75.5-96.0) 90.8

(80.6-101.1) 96.5

(86.2-106.7) 0.43

Mitral A (cm/s) 88.6 (76.4-100.8)

75.7 (63.5-87.9)

86.7 (74.5-98.9)

0.38

E/A Ratio 1.01 (0.85-1.18)

1.26 (1.09-1.42)

1.13 (0.97-1.29)

0.24

Septal e’ (cm/s) 9.37 (7.90-10.85)

9.27 (7.79-10.75)

10.52 (9.05-12.00)

0.49

E/e’ Ratio 9.60 (7.70-11.49)

10.74 (8.84-12.63)

9.23 (7.33-11.12)

0.56

LAVI (mL/m2) 32.32 (27.12-37.53)

31.12 (24.51-37.74)

29.94 (24.73-35.14)

0.83

LV EDV (mL) 99.00 (80.98-117.02)

98.67 (80.65-116.68)

98.17 (80.15-116.18)

1.00

RV FAC (cm2) 43.64 (34.43-52.86)

49.45 (40.24-58.67)

61.24 (49.53-72.95)

0.22

TAPSE (cm) 2.35 (1.93-2.77)

2.18 (1.77-2.60)

2.53 (2.12-2.95)

0.56

Kansas City Cardiomyopathy Questionnaire Visit 1 Visit 2 Visit 3 P Symptom Stability

50.0 (33.7-66.3)

58.3 (42.0-74.7)

41.7 (25.3-58.0)

0.44

Symptom Burden 33.3 (14.6-52.1)

58.3 (39.6-77.1)

61.1 (42.4-79.8)

0.19

Total Symptom Score

37.5 (26.7-48.3)

50.7 (39.9-61.5)

46.2 (35.4-56.9)

0.33

Overall Summary Score

40.8 (27.8-53.8)

56.1 (43.1-69.1)

50.1 (37.1-63.1)

0.36

Functional Status 45.8 57.3 52.3 0.50

22

Score (33.5-58.2) (44.9-69.7) (39.9-64.6) HR = heart rate; SBP = systolic blood pressure; DBP = diastolic blood pressure; MAP = mean arterial pressure; SVR = systemic vascular resistance; kJ = kilojoules; Zc = Characteristic Impedance of the Proximal Aorta; TAC = total arterial compliance; RM = reflection magnitude; LAVI = left atrial volume index; LV EDV = Left ventricular end diastolic volume; RV FAC = right ventricular fractional area change; TAPSE = tricuspid annular plane systolic excursion * p<0.05 V1 vs. V2; † p<0.05 V1 vs. V3; ‡p<0.05 V2 vs. V3

clinical track pharmacokinetics and pharmacodynamics of

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