Conditions: Cystic Fibrosis
Keywords: arterial stiffness flow-mediated dilation endothelial function pulse wave velocity inflammation oxidative stress pulmonary function test Cystic Fibrosis Lung Disease Nitric Oxide Exercise Capacity CF Muscle Function Sildenafil Viagra Revatio
Allocation: RANDOMIZED | Intervention model: CROSSOVER
Enrollment: 19
Healthy volunteers: yes

Brief summary:
Cystic fibrosis (CF) has many health consequences. A reduction in the ability to perform exercise in patients with CF is related to greater death rates, steeper decline in lung function, and more frequent lung infections. However, the physiological mechanisms for this reduced exercise capacity are unknown. The investigators laboratory recently published the first evidence of systemic vascular dysfunction in patients with CF. Therefore, it is reasonable to suspect that the blood vessels are involved with exercise intolerance in CF. This study will look at how 1) blood flow and 2) artery function contribute to exercise capacity in CF.

Detailed description:
The most disturbing aspect of Cystic Fibrosis (CF) is the associated premature death. Low exercise capacity predicts death in patients with CF and is also associated with a steeper decline in lung function and more lung infections. A critical barrier to improving exercise tolerance in patients with CF is the investigators lack of knowledge regarding the different physiological mechanisms which contribute to their lower exercise capacity. We have compelling data to indicate that the blood vessels may contribute to the low exercise capacity in CF. The impact of this proof of concept investigation will test Phosphodiesterase Type 5 inhibitors (PDE5) inhibitors as a potential therapy in CF and will explore blood flow and endothelial function as potential mechanisms which contribute to exercise intolerance in CF. Improvements in exercise capacity will not only contribute to a better quality of live for patients with CF, it will also increase longevity in these patients.

Arms:
arm 1: In randomized order, on two separate days, endothelial function and exercise capacity will be determined 1 hour following a single dose of sildenafil (50 mg) or placebo. arm 2: In randomized order, on two separate days, endothelial function and exercise capacity will be determined 1 hour following a single dose of sildenafil (50 mg) or placebo. arm 3: Following the acute study, patients will be instructed to take 20 mg of sildenafil, three times a day, for 4 weeks. Endothelial function will be determined within 48 hours following the last dose.

Interventions: intervention 1: Sildenafil (Acute-1 hour) intervention 2: Sildenafil (Subchronic-4 weeks) intervention 3: Placebo
Intervention descriptions: intervention 1: Vascular function will be assessed 1 hour following oral ingestion of sildenafil (50 mg) intervention 2: Vascular function will be assessed 4 weeks following 20 mg three times per day (TID) of sildenafil for four weeks intervention 3: Sugar pill designed to mimic the sildenafil treatment

Protocol document:
Prot_SAP_000.pdf: 
Role of blood flow on artery function and exercise capacity in cystic fibrosis 
 (CF-FLOW) 
 
NCT02057458 
 
January 27, 2014 


SPECIFIC AIMS 
     Cystic Fibrosis (CF) is the most common fatal genetic disease in North America.  The most disturbing 
aspect of CF is the associated premature death. Clinical manifestations of CF include not only lung 
dysfunction, but many systemic consequences as well.   
     
     Among the systemic consequences, exercise intolerance has been shown to predict mortality in 
patients with CF independent of lung function.  Exercise capacity (VO2 peak), an objective measurement of 
exercise tolerance, drops approximately 5-8% per year in patients with CF.  This excessive decay in exercise 
capacity not only represents a 5-8 fold decline compared to healthy sedentary adults, it also leads to 
deterioration of lung function and more pulmonary infections. It is important to recognize that decreases in 
lung function (FEV1) do not always contribute to reductions in VO2 peak and that less than 2% of patients 
who have an FEV1 greater than 50% predicted will have a significant drop in 
hemoglobin oxygen saturation (SpO2) during maximal exercise.  These data 
rule out the role of lung function induced hypoxemia to exercise intolerance 
in CF. There have been major advances in research and medical therapies 
to target improvements in lung function; however, non-pulmonary factors 
have been neglected. Based on our recent discovery that patients with CF 
have systemic endothelial dysfunction, it is reasonable to suspect the 
involvement of the blood vessels in the pathophysiology of exercise 
intolerance in CF - a concept that has yet to be examined. Preventing the 
excessive annual reductions in exercise capacity is essential to 
increasing the quality of life and longevity of patients with CF; however, 
a critical barrier to improving exercise capacity in CF is our lack of 
knowledge regarding the different physiological mechanisms that contribute to exercise intolerance. 
 
     In general, Phosphodiesterase type 5 (PDE5) inhibitors increase microvascular O2 delivery and improve 
endothelial function. We have recently published the first evidence of systemic endothelial dysfunction in a 
cohort of young, healthy patients with CF. Vascular function is in part responsible for blood flow regulation, and 
preliminary data from our lab suggests that patients with CF also have an impairment in blood flow regulation 
during exercise when compared to healthy controls. Thus, the overall goals of this proposal are to 1) provide 
proof of concept for using PDE5 inhibition to improve exercise capacity in patients with CF and 2) investigate 
blood flow per se and endothelial dysfunction as potential mechanisms that contribute to exercise intolerance 
in CF. Our central hypothesis is that PDE5 inhibition will improve blood flow (acute treatment) and endothelial 
function (chronic treatment) leading to an increase in exercise capacity (Figure 1).  We will test our central 
hypothesis with the following specific aims: 
 
Aim 1.  Test the hypothesis that acute PDE5 inhibition will increase systemic blood flow and contribute 
to an improvement in exercise capacity in patients with CF.   
     Exercise capacity (VO2 peak) and indices of gas exchange (O2 uptake kinetics, expired CO2, VE/VO2, 
VE/VCO2), will be determined using a graded maximal exercise test on a cycle ergometer at baseline and 1 
hour following the ingestion of a single dose of sildenafil (PDE5 inhibitor, 50 mg) or placebo (randomized order) 
in patients with CF.  Secondary outcomes will include the non-invasive patented technology of the Physioflow 
Enduro, which will be utilized to examine differences in cardiovascular hemodynamics (heart rate, stroke 
volume, cardiac output, systemic vascular resistance, and ejection fraction) between patients and healthy 
controls during maximal cycling exercise. This acute experiment transiently increase blood flow and test 
blood flow per se as a mechanism that will improve exercise capacity in CF.    
 
Aim 2.  Test the hypothesis that chronic PDE5 inhibition will improve endothelial function and 
contribute to an improvement in exercise capacity in patients with CF.   
    Following completion of Aim 1, endothelial function, exercise capacity (VO2 peak), and the indices of gas 
exchange (indicated above), will be performed, only in patients with CF, at baseline and following 4 weeks of 
sildenafil, 20 mg thrice daily.  Secondary outcomes will include measurements of arterial stiffness (pulse wave 
velocity), the Physioflow Enduro, and biomarkers of nitric oxide bioavailability.  This chronic experiment will 
test the mechanism that an increase in endothelial function will improve exercise capacity in CF. 
This proof of concept trial is the first to explore the therapeutic benefit of PDE5 inhibitors on exercise 
capacity in CF. This high impact proposal will also target blood flow and endothelial function as potential 
mechanisms that contribute to lower exercise capacity in CF.  Findings from this proposal will provide the 
foundation to stimulate additional strategies for the treatment and prevention of exercise intolerance in CF. 
Figure 1.  Working hypothesis. 


Figure 2.  Factors that 
may contribute to 
exercise intolerance. 
A.  SIGNIFICANCE 
     Over 20 years ago, exercise intolerance was shown to predict mortality in patients with CF 
independent of lung function1.  Exercise intolerance is a common complaint associated with CF, and the 
preservation of exercise capacity is essential for survival in this population.  A VO2 peak threshold of < 32 
ml/kg/min in patients with CF is predictive of a 60% chance of mortality in the subsequent 8 years following 
testing2. Exercise capacity falls approximately 5-8% per year (~2.1 ml/kg/min)2 in CF patients, which 
represents a 5-8 fold decrease compared to healthy sedentary adults.  Even in the presence of fairly healthy 
spirometric function, data from our lab demonstrates that young patients with CF have a lower exercise 
capacity compared to apparently healthy controls3.  Additionally, it is important to emphasize that decreases in 
FEV1 do not always contribute to reductions in VO2 peak4 and very few patients with CF (only ~1.6%) who 
have an FEV1 > 50% predicted will have a significant drop in hemoglobin oxygen saturation (SpO2) during 
maximal exercise.  Collectively, these data suggest that it is possible to observe exercise intolerance in 
patients with CF even when lung function and gas exchange are preserved.   
     Lower exercise capacity is associated with greater mortality, steeper decline in pulmonary function, 
and more pulmonary infections5.  There are many identified barriers associated with exercise intolerance in 
CF6, 7 and the reasons for “why exercise is medicine in CF” has recently been published8. However, a critical 
barrier to improving exercise capacity in patients with CF is our lack of knowledge regarding the different 
physiological mechanisms that contribute to exercise intolerance. There have been major advances in 
research and medical therapies to target improvements in lung function; however, non-pulmonary factors have 
been neglected. Based on our recent discovery of vascular endothelial dysfunction in CF3, it is reasonable to 
suspect the involvement of the blood vessels in the pathophysiology of exercise intolerance in CF - a concept 
that has yet to be explored.  Very little work using PDE5 inhibitors in human patients with CF has been 
conducted and a very recent review on the topic highlights the potential use for PDE5 inhibitors in CF 
treatment9.   
     There is a desperate need to understand the mechanisms which contribute to exercise intolerance in CF, 
and our recent findings3 support further investigations into blood vessel dysfunction in the context of CF-
mediated exercise intolerance. The impact of this proof of concept investigation will test PDE5 inhibitors as a 
potential therapy in CF and will explore blood flow and endothelial function as potential mechanisms which 
contribute to exercise intolerance in CF.   
      
B.  INNOVATION 
     It is well established that patients with CF exhibit exercise intolerance, however, the physiological 
mechanisms for the reduced exercise capacity in CF is unknown.  Innovation of this proposal is evident by: 
 
 
The novel concept that impaired blood flow and vascular dysfunction 
contributes to exercise intolerance in patients with CF.  Figure 2 represents a 
simplistic schematic of the potential systems that could, in general, contribute to 
exercise intolerance. By maintaining a constant partial pressure of oxygen and 
knowing that lung function-mediated hypoxemia during exercise is unlikely in the 
proposed CF cohort4. Blood vessels are downstream from air and lungs and 
upstream from skeletal muscle (Figure 2); therefore, we can begin our reductionist 
approach with investigation into the role of the blood vessels on exercise capacity 
in CF.  We have recently identified endothelial dysfunction at rest3 and now have 
compelling evidence that patients with CF exhibit impaired blood flow regulation 
during exercise (preliminary data Figure 4).  
 
This is the first study that uses PDE5 inhibitors to target improvements in exercise capacity in 
cystic fibrosis. The proposed investigation not only represents an innovative proof of concept trial for the 
role of PDE5 inhibitors in CF, we are using PDE5 inhibition as a novel tool to test blood flow and 
endothelial dysfunction as two potential mechanism that contribute to exercise intolerance in CF. 
 
We have employed state-of-the-art methodology.  Using the FMD test, we were the first to identify 
systemic endothelial dysfunction in patients with CF.  We will use FMD to assess endothelial function 
following PDE5 inhibition.  In addition, we will use the innovative assessment of monitoring cardiovascular 
hemodynamics during maximal exercise using the Physioflow Enduro. Physioflow is the first and only 
system that is fully validated for use during exercise and has never been used in patients with CF.    
 
This proposal, through the integration of exercise physiology, vascular biology, and pharmacology, 
represents a major breakthrough in the approach to begin understanding the mechanisms which contribute 
to exercise intolerance in this deadly disease. Accordingly, this project has high clinical significance that will 


help stimulate strategies for the treatment and prevention of exercise intolerance in CF that will impact clinical 
practice and patient care.  Regardless of the outcomes, findings from this study will provide the foundation to 
conduct full-scale, multicenter clinical trials that will target chronic improvements in 1) exercising blood flow 
regulation, 2) endothelial function, and 3) exercise capacity in CF. 
 
C.  APPROACH 
C1. Approach Overview.  We are proposing a randomized, placebo controlled, acute experiment (aim 1) 
followed by an open label 4 week treatment trial (aim 2).  Findings from aim 1 will isolate the transient increase 
in blood flow delivery as a mechanism to improve exercise capacity in CF, whereas findings from aim 2 will 
target an improvement in endothelial function and therefore, the improvement in exercise capacity will likely be 
greater in magnitude and sustainable.  Nonetheless, the current proposal will shift current research and 
bring new knowledge about the mechanisms associated with exercise intolerance in patients with CF. 
 
C2. Research Design and Methods.  Figure 3 
illustrates our experimental design. On Visit 1 
(preliminary day), all eligible participants (and 
parents of minors) will come to the Laboratory of 
Integrative 
Vascular 
and 
Exercise 
Physiology 
(LIVEP) to provide written consent/assent.  Visit 1 
will also be utilized to assess 1) resting oxygen 
saturation, 2) body composition using DXA, 3) 
pulmonary function test (PFT), 4) diffusion capacity 
of the lung for carbon monoxide (DLCO), and 
maximal exercise capacity on a cycle ergometer.  
Aim 1 will be comprised of experimental days 1 and 
2.  A fasting blood sample will be collected during 
Day 1, pre-test only.  The Pre-test will consist of an 
FMD 
test 
and 
pulse 
wave 
velocity 
(PWV) 
assessment. One hour following sildenafil or placebo 
(randomized order), Post-test assessments of FMD, 
PWV and maximal exercise capacity will be 
determined.  Aim 2 will be comprised of experimental day 3, in which a fasting blood sample, FMD, PWV, and 
exercise capacity will be determined 24-48 hours following the completion of 4 weeks of sildenafil treatment 
(20 mg thrice daily). 
 
C3. Study Participants.  A total of 40 patients with CF (20 children and 20 adults) and 40 demographically- 
matched healthy controls will be recruited for this investigation.  Age in patients with CF is most often related to 
disease severity. Accordingly, we plan to recruit both children (9-17 years old who likely have less 
complications) and adults (>18 years old who likely have more complications) with CF. Both children and 
adults accompanied with or without multi-organ pathophysiology (i.e. CFRD, pancreatic insufficiency, etc.) will 
be included.  All patients will be instructed to maintain their daily routine for airway clearance and inhaled 
therapies.  Healthy control subjects will be recruited and demographically-matched for age, sex, and BMI of the 
patients with CF.  Healthy controls will not undergo any treatment. The treatment response to sildenafil 
observed in the patients will be compared to the healthy controls baseline data.    Inclusion and Exclusion 
criteria for all subjects are presented below: 
 
Inclusion Criteria: 
 
Diagnosis of CF and healthy controls   
 
Men and women (> 18 yrs. old) 
 
Boys and girls (9-17 yrs. old) 
 
FEV1 percent predicted > 50% 
 
Resting oxygen saturation (room air) >90% 
 
Patients with or without CF related diabetes 
 
Traditional CF-treatment medications 
 
Ability to perform reliable/reproducible PFTs 
 
Clinically stable for 2 weeks (no exacerbations 
or need for antibiotic treatment within 2 weeks 
of testing or major change in medical status) 
 
Exclusion Criteria: 
 
Children 8 yrs. old and younger 
 
FEV1 percent predicted < 50% 
 
Resting oxygen saturation (room air) < 90% 
 
Clinical diagnosis of heart disease, PAH 
 
Febrile illness within two weeks of visit 
 
Currently smoking, pregnant, or nursing  
 
Individuals on vaso-active medications (i.e. 
nitrates, beta blockers, ACE inhibitors, etc.) 
 
Patients with B. cepacia (only ~3% of our CF 
center patient population) 
 
Medications that interfere with Sildenafil
Figure 3.  Proposed experimental approach 


Figure 4. Brachial artery retrograde velocity 
at baseline and during sub-max workloads 
in patients with CF (n=14) and controls 
(n=14). Data are mean ± SEM.  *Significant 
from control. 
 
C4.1. Rationale for Aim 1.  PDE5 inhibitors prolong NO-mediated vasodilation and increase microvascular O2 
delivery10.  A single dose of Sildenafil, the most popular PDE5 inhibitor, has been shown to increase exercise 
capacity in Fontan patients11, systolic heart failure12 and congestive heart failure13.  Our study design proposes 
to compare exercise capacity obtained on different days.  In support, the reproducibility of repeated maximal 
exercise testing on a cycle ergometer in patients with CF over a 28-day period has been documented14. 
 
C4.2. Pilot Data for Aim 1.  Data from our lab not only confirms that 
young patients with CF exhibit exercise intolerance3, patients also 
exhibit a greater ventilatory equivalent for CO2 compared to 
demographically matched healthy controls (VE/VCO2; 33.2±0.8 vs. 
30.8±0.98, respectively; p=0.022).  In addition, we have generated 
preliminary data that suggest that patients with CF exhibit an 
impairment in blood flow regulation during sub-maximal exercise 
when compared to healthy controls (Figure 4).  Blood flow regulation 
was determined by assessing brachial artery retrograde velocity at 
baseline and during progressive, sub-maximal cycling (lower leg) 
exercise at 20%, 40%, and 60% of maximal workload in patients and 
healthy controls. No differences in age (p=0.201) BMI (p=0.502), or 
FEV1 (p=0.508) were observed between patients and healthy 
controls. Impaired blood flow regulation may represent a 
potential mechanism for exercise intolerance in patients with CF and acute PDE5 inhibition represents a 
novel method to increase blood flow and muscle oxygenation to test our stated hypothesis. 
 
C4.3. Aim 1 Experimental Design.  Exercise capacity (VO2 peak) and indices of gas exchange (O2 uptake 
kinetics, expired CO2, VE/VO2, VE/VCO2), will be determined using a graded maximal exercise test on a cycle 
ergometer (Godfrey Protocol) 1 hour following the ingestion of either  a single dose of sildenafil (50 mg) or 
placebo (in randomized order) in patients with CF.  We will allow for a washout period of at least 1 week (but 
no more than 2 weeks) in between treatments. Post-test values will be compared to pre-test values; however, 
exercise capacity will be compared to values obtained during visit 1. Secondary outcomes will include the 
patented technology of the Physioflow Enduro15, 16, will be utilized to examine differences in cardiovascular 
hemodynamics (heart rate, stroke volume, cardiac output, systemic vascular resistance, and ejection fraction) 
non-invasively between case and healthy controls during maximal cycling exercise.  We will also perform pre 
and post assessments of FMD and pulse wave velocity (PWV), non-invasive assessments of endothelial 
function and arterial stiffness, respectively.  In addition, a fasting blood sample will be taken during pre-test of 
experimental day 1 to assess baseline clinical labs (lipid panel, high sensitivity C-reactive protein (hsCRP), 
Polymorphonuclear neutrophil (PMN), glucose, insulin, HbA1c, estradiol, progesterone, testosterone, 
hematocrit, and hemoglobin) and biochemical research assays (endothelial nitric oxide synthase (NOS3) 
expression and levels of cyclic guanosine monophosphate (cGMP) in platelets, and 8-isoprostane, 
nitrate/nitrite in plasma). 
 
C4.4. Aim 1 Expected Results. Based on our published3 and preliminary data (Figure 4), we predict that 
patients with CF will exhibit exercise intolerance compared to healthy controls.  In addition, we predict that 
acute treatment with Sildenafil will increase systemic blood flow such that we expect that baseline brachial 
artery blood flow and cardiac output will be increased. Accordingly, we predict that a significant transient 
improvement in exercise capacity in following sildenafil will be observed, whereas placebo will elicit no change.  
We also predict that acute sildenafil treatment will result in more efficient ventilatory equivalents17 and 
improvements in VO2 at the anaerobic threshold (AT)18.  Findings from this acute experiment will test the 
mechanism that an increase in blood flow per contributes to an improvement in exercise capacity in CF.    
 
C4.5. Aim 1 Statistical Analyses Plan.  Mixed-effects regression models (MRMs)19 will be used to perform the 
standard analyses required for a two-period, two-treatment (AB/BA) cross-over design. In this analysis, MRM's 
will be used to compare Sildenafil vs. placebo in terms of the primary outcomes of exercise capacity and 
indices of gas exchange, all of which will be measured at baseline and 1 hour following ingestion of either a 
single dose of sildenafil or placebo. The dependent variable of interest will be the change in each outcome 
between baseline and post-ingestion for each treatment. The secondary outcomes (cardiac output, FMD, 
C4.  Specific Aim 1.  Test the hypothesis that acute PDE5 inhibition will increase systemic blood 
flow and contribute to an improvement in exercise capacity in patients with CF.   


PWV, etc.) will be analyzed in the same way. If necessary, adjustment will be made for the potential 
confounders for this aim, including age group (child vs. adult), spirometric function, diffusion limitation, 
presence of CFRD or pancreatic insufficiency, etc. Independent group’s t-tests will be used to compare the CF 
patients in the sildenafil treatment phase with the healthy control participants for all outcomes measured.  All 
analyses for Aims 1 and 2 involving MRMs will be performed using the MIXED procedure in SAS 9.3 (SAS 
Institute, Cary, NC, 2010) and a significance level of 0.05 will be used for all statistical tests. 
 
C4.6. Aim 1 Power Analysis.  The power calculation for Aim 1 is based on the anticipated effect size for 
exercise capacity, the primary outcome for this Aim.  A sample size of n=20 in each sequence (AB and BA) of 
our cross-over design will yield 87% power to detect a difference in the mean change in VO2 peak between 
sildenafil and placebo as small as that found in the cross-over study by Bocchi et al.13 . It will also yield over 
99% power to detect a similar effect size for VE/VCO2
13, another key outcome for Aim 1. 
 
C4.7. Aim 1 Potential Pitfalls and Alternatives.  It is possible that the state-of-the-art methodology using the 
Physioflow Enduro will not be sensitive to determine changes in cardiac output following acute sildenafil 
treatment.  Accordingly, we plan to use ultrasound to confirm increases in basal brachial artery blood flow 
before and after treatment.  In addition, we can measure femoral artery blood flow as an alternative.  Patients 
with CF may have slight day to day variations in lung function and resultant spirometry.  On each testing day 
we will measure spirometry and diffusion capacity and if significant changes between testing days are 
observed, we will statistically control for those differences where appropriate.  Because we anticipate a 
heterogeneous sample with respect to disease severity and concomitant systemic consequences, we will 
document all patient characteristics and therapies, and will statistically control for potential confounders. 
Further, there are equivocal data that support both an improvement in FMD20, 21 as well as no change in FMD20, 
21 following acute sildenafil treatment.  Therefore, it is plausible that a single dose of sildenafil will improve 
systemic blood flow and transiently improve endothelial function making interpretation somewhat difficult.  We 
plan on assessing FMD pre- and post- acute treatment; however, the acute improvement in FMD (if any) is 
likely 1) transient and not representative of a true improvement in vascular function, and 2) confounded by 
drug-mediated changes in baseline diameter and shear stress, both of which discount the “improvement” in 
FMD. Taken together, we are confident that changes in exercise capacity following acute sildenafil will be 
mediated by an increase in blood flow and microvascular O2 delivery and not improvements FMD.   
 
 
C5.1. Rationale for Aim 2. Through production of NO, endothelial dependent vasodilation is an important 
function of the endothelium. The flow mediated dilation (FMD) test represents a non-invasive assessment of 
NO bioavailability and endothelial function22.  Chronic PDE5 inhibition is routinely shown to improve endothelial 
function (in as little as 4 weeks) in patients with vascular dysfunction23-25.  
  
C5.2. Pilot Data for Aim 2.  We have recently published in Chest that 
young, fairly healthy patients with CF exhibit endothelial dysfunction 
compared to healthy, demographically-matched controls3 (Figure 1).  
Additionally, data supports improvements in endothelial function in 
populations who exhibit vascular dysfunction, in as little as 4 weeks 
following treatment with PDE5 inhibitors23-25.  Further, we and others 
have reported the reproducibility of the FMD test26-28; however, there are 
no FMD reproducibility data in patients with CF. To address this 
knowledge gap, FMD was performed in 7 patients with CF (20±9 years, 
BMI = 20.9±2.9 kg/m2, FEV1 93±17 % predicted) during 2 sequential 
visits, separated by at least 14 days (mean 29±7 days).  The intra-class 
correlation coefficients and p values for baseline diameter, FMD, and 
FMD/shear were 0.984; p<0.001, 0.949; p=0.001, and 0.885; p=0.009, respectively.  These data indicate that 
patients with CF have endothelial dysfunction, the FMD test is reproducible in CF, and PDE5 inhibitors 
are likely to cause an increase in FMD in patients with CF.  
 
C5.3. Aim 2 Experimental Design. Brachial artery FMD, exercise capacity (VO2 peak) and indices of gas 
exchange (O2 uptake kinetics, expired CO2, VE/VO2, VE/VCO2) will be determined at baseline and following 4 
weeks of oral sildenafil treatment (20 mg thrice daily).  Secondary outcomes will include assessment of PWV 
Figure X. Endothelial dysfunction in CF3.  
*Significant from controls. 
C5.  Specific Aim 2.  Test the hypothesis that chronic PDE5 inhibition will improve endothelial 
function and contribute to an improvement in exercise capacity in patients with CF.   


and the patented technology of the Physioflow Enduro. In addition, a fasting blood sample will be taken to 
determine post-treatment clinical labs and biochemical research assays to compare to baseline values. 
 
C5.4. Aim 2 Expected Results. At baseline, we predict patients with CF to exhibit endothelial dysfunction and 
exercise intolerance compared to their control counterparts.  In addition, we predict that FMD and exercise 
capacity in patients with CF will be restored to healthy control values following 4 weeks of thrice daily sildenafil 
treatment. Moreover, we expect to see concomitant improvements in oxygen uptake kinetics, VO2 at the AT, 
and more efficient ventilatory equivalents. Findings from this aim will not only provide proof of concept for the 
use of PDE5 inhibitors as therapy in patients with CF, they will also elucidate endothelial dysfunction as a 
potential mechanism that contributes to exercise intolerance in CF.   
 
C5.5. Aim 2 Statistical Analyses Plan.  Mixed-effects regression models19 will be used to perform a within-
subject repeated measures analysis of variance (ANOVA) in terms of mean change from baseline to 4 weeks 
for each of the primary outcomes associated with this aim (FMD, exercise capacity, and indices of gas 
exchange). One of the advantages of the MRM methodology is that one is able to incorporate subject-specific 
parameters into the within-subject analyses. In all MRM analyses, adjustment will be made for any significant 
covariates (age group, changes in spirometry or diffusion capacity, presence of CFRD or pancreatic 
insufficiency, etc.). The same analysis will be used for each of the secondary outcomes (PWV, Physioflow 
Enduro measurements, NOS3, cGMP, 8-isoprostane, nitrate/nitrite, etc.). 
 
C5.6. Aim 2 Power Analysis.  The power calculation for Aim 2 is based on the anticipated effect size for 1) 
FMD23 and 2) exercise capacity12 following 4 weeks of PDE5 inhibition. A sample size of n=40 will yield 97% 
power to detect a difference in the mean change in VO2 peak between baseline and 4 weeks as small as that 
found by Lewis et al.12.  A sample size of n=40 will also yield >99% power for detecting a mean change in FMD 
between baseline and 4 weeks as small as that found by Rosano et al.23. 
 
C5.7. Aim 2 Potential Pitfalls and Alternatives.  PDE5 inhibitors are generally less effective in the presence of 
down regulated NOS3 and very little is known about NOS3 expression in patients with CF.  Therefore the 
efficacy of sildenafil in CF may be patient/genotype specific depending on NO bioavailability. Accordingly, we 
plan on assess NOS3 expression and cGMP levels in circulating platelets at baseline and following treatment 
which will complement the proposed use of FMD and provide further insight in the unlikely event that sildenafil 
does not improve either FMD or exercise capacity.  It is important to note that insignificant findings for FMD or 
exercise capacity are just as important and it is plausible that a change in exercise capacity will be observed 
with no change in endothelial function (and vice versa).  These findings will lend support for future 
investigations into the skeletal muscle as a barrier to improve exercise capacity in CF.  It is important to 
mention that the FDA has recently issued a statement warning against the use of sildenafil to treat pulmonary 
arterial hypertension in children due to the increased risk of death29.  However, this elevated risk was only seen 
in the high dose treatment group (up to 80 mg TID), and only following >2 years of treatment.  No increased 
risk of death was observed using the dose we are proposing.  Further, we are proposing the use of 
sildenafil to look at a mechanism, and our trial will 1) use a smaller dose, 2) only last 4 weeks, and 3) will 
exclude patients with PAH.  Accordingly, we fell that our proposed use of sildenafil is safe. 
 
C6. Subject Recruitment.  The PI has developed a strong collaboration with Dr. McKie, the Georgia Regents 
University (GRU) CF center director and both Dr. McKie and Dr. Forseen (adult CF center director) are very 
enthusiastic about the current proposal (See letters from Drs. McKie and Foreseen). The GRU CF center 
currently cares for approximately 95 pediatric and 85 adult patients with CF, the majority of whom would qualify 
for the study.  In fact, over the past 2 summers (5 months total) we have successfully recruited/ tested 42 
patients with CF.   Thus, we are confident we can recruit enough patients and achieve our targeted goals.   
     The main source of control subject recruitment will be the subjects that are either enrolled or previously 
enrolled in other studies at the Georgia Prevention Center (GPC). Last year, the GPC saw approximately 
12,000 subject visits, which included approximately 50% children and 50% adults.  In addition, we will list the 
study on the GRU website under “Studies in Progress,” place classified ads in local newspapers, and utilize 
free online sources which attracts participants from the GRU community and Augusta as a whole. We 
anticipate through all the aforementioned recruitment strategies we will be able to meet our recruitment goals.   
 
C7. Subject Safety.  The overall proposal is associated with minimal risk and great benefit. A complete 
description of our Data Safety Monitoring Plan (DSMP) is located in the Protection of Human Subjects section. 


C8. Role of the Study Physician.  Dr. McKie will play an integral role in the successful completion of this 
project.  Not only has she been instrumental in the collection and interpretation of our preliminary data, she 
brings clinical expertise and experience in CF patient care and research.  Dr. McKie will help screen patients 
with CF prior to enrollment and verify they meet the inclusion/exclusion criteria.  In addition, Dr. McKie will 
continue to assist in subject recruitment, data analysis and interpretation, data dissemination, and provide 
medical oversight for the entire proposed investigation. 
 
C9. Study Timetable. Table 1 illustrates a 
conservative number of subject visits per month 
throughout the project period. One preliminary 
day is required for all subjects.  For patients, the 
completion of both aims will require a total of 4 
visits (1 preliminary day and 3 experimental 
days; 40 patients x 4 visits = 160 visits), 
whereas healthy controls will only require 2 subject visits (1 preliminary day and 1 experimental day; 40 healthy 
controls x 2 visits = 80 visits). Completion of both aims would require a total of 240 subject visits.  
     The proposed timeline is based on the 1) number of patients/healthy controls who can be recruited and 
studied each month, and 2) the number of visits needed for study completion.  In our experience, recruitment 
of children is more effective when school is out; therefore, we will plan to test more subjects during the summer 
months (June, July, and August). Research assays will be batched and likely performed during the second 
year.  Abstracts will be presented and manuscripts will be prepared when applicable.  We have allowed for a 
buffer period during the last 3 months allowing for subject visit overflow and finalization of the study.      
 
C10. Collaboration among the Investigative Team.  Our group has been working diligently over the past 2 ½ 
years to uncover the recently discovered vascular phenotype at rest3 and during exercise (Figure 4). Together, 
we have published our FMD findings in Chest3 and presented our research at the 201130 and 201231 NACFC 
conference, and the 2012 American College of Sports Medicine32 and 2012 Experimental Biology33 national 
meetings.  We are committed to research aimed to improve the quality of life and longevity in patients with CF. 
 
C11. Consultant Arrangements.  For the current proposal, we have arranged to include Dr. Clinton Webb, PhD 
as a consultant. Dr. Webb holds the Kupperman Chair in Cardiovascular disease and is the Department chair 
of Physiology at GRU.  Dr. Webb is internationally recognized as an expert on PDE5 inhibition and vascular 
function34-36 and has provided invaluable suggestions to strengthen our experimental approach.  In addition, Dr 
Webb has agreed to assist interpretation throughout our study (See letter of support from Dr. Webb).  
 
C12. Specific Techniques  
     Maximal Exercise Capacity Test. Subjects will perform the maximal exercise tests on an electronically 
braked cycle ergometer using the Godfrey protocol.  Expired gasses will be collected using a Parvo Medics 
True One metabolic cart for determination of exercise capacity (VO2 peak), CO2 retention, O2 uptake kinetics, 
ventilatory equivalents, and AT. In addition, blood pressures (Suntech Tango) will be assessed during each 
stage of exercise, SpO2 (Nonin) will also be monitored throughout the exercise tests, and we will use the 
Physioflow Enduro  to monitor cardiovascular hemodynamics during the maximal exercise tests in all subjects. 
 
     Pulmonary Function Testing.  Pulmonary function testing will be repeated several times (according to ATS 
standards37) to ensure that lung function is not changing over time. The National Health and Nutrition 
Examination Survey (NHANES) III spirometric reference standards will be used to determine the % predicted 
data set.  The diffusing capacity of carbon monoxide (DLCO) test will be used to determine diffusion limitations.  
Briefly, subjects will exhale to RV and then take a deep breath which will include a mixture of gas (10% He, 
21% O2, 0.3% CO, and balance N2) and will be held for 10 seconds.   Concentrations of CO will be analyzed.  
All values will be presented as % predicted and normalized for TLC and hemoglobin. 
 
     Brachial Artery FMD.  The brachial artery FMD test will be performed according to the recent tutorial on the 
ultrasonic assessment of FMD22 and shear rate will be calculated as the stimulus of the vasodilatory response. 
 
     Arterial Stiffness.  SphygmoCor Pulse Wave Analysis System (SphygmoCor, PWV Medical, Sydney, 
Australia), which uses applanation tonometry, will be used for accurate, non-invasive assessment of arterial 
stiffness.  Aortic augmentation index will also be determined and is an index of arterial stiffness. 
 
     Physioflow Enduro.  The Physioflow Enduro device uses the patented technology of signal Morphology-
based impedance cardiography. This non-invasive, wireless, device produces continuous, sensitive, and 
reproducible assessments of cardiovascular hemodynamics (i.e. HR, SV, CO, SVR, and EF) that has been 
validated against the invasive thermodilution Swan-Ganz catheter technique.  


LITERATURE CITED 
1. 
Nixon PA, Orenstein DM, Kelsey SF, Doershuk CF. The prognostic value of exercise testing in patients 
with cystic fibrosis. The New England journal of medicine. 1992;327:1785-1788 
2. 
Pianosi P, Leblanc J, Almudevar A. Peak oxygen uptake and mortality in children with cystic fibrosis. 
Thorax. 2005;60:50-54 
3. 
Poore S BB, Eidson D, McKie KT, Harris RA. Evidence of vascular endothelial dysfunction in young 
patients with cystic fibrosis. Chest. In Press 
4. 
Moorcroft AJ, Dodd ME, Webb AK. Long-term change in exercise capacity, body mass, and pulmonary 
function in adults with cystic fibrosis. Chest. 1997;111:338-343 
5. 
van de Weert-van Leeuwen PB, Slieker MG, Hulzebos HJ, Kruitwagen CL, van der Ent CK, Arets HG. 
Chronic infection and inflammation affect exercise capacity in cystic fibrosis. Eur Respir J. 2012;39:893-
898 
6. 
Wilkes DL, Schneiderman JE, Nguyen T, Heale L, Moola F, Ratjen F, Coates AL, Wells GD. Exercise 
and physical activity in children with cystic fibrosis. Paediatric respiratory reviews. 2009;10:105-109 
7. 
Swisher AK, Erickson M. Perceptions of physical activity in a group of adolescents with cystic fibrosis. 
Cardiopulm Phys Ther J. 2008;19:107-113 
8. 
Wheatley CM, Wilkins BW, Snyder EM. Exercise is medicine in cystic fibrosis. Exercise and sport 
sciences reviews. 2011;39:155-160 
9. 
Noel S, Dhooghe B, Leal T. Pde5 inhibitors as potential tools in the treatment of cystic fibrosis. Front 
Pharmacol. 2012;3:167 
10. 
Sperandio PA, Oliveira MF, Rodrigues MK, Berton DC, Treptow E, Nery LE, Almeida DR, Neder JA. 
Sildenafil improves microvascular o2 delivery-to-utilization matching and accelerates exercise o2 
uptake kinetics in chronic heart failure. American Journal of Physiology - Heart and Circulatory 
Physiology. 2012;303:H1474-H1480 
11. 
Giardini A, Balducci A, Specchia S, Gargiulo G, Bonvicini M, Picchio FM. Effect of sildenafil on 
haemodynamic response to exercise and exercise capacity in fontan patients. Eur Heart J. 
2008;29:1681-1687 
12. 
Lewis GD, Shah R, Shahzad K, Camuso JM, Pappagianopoulos PP, Hung J, Tawakol A, Gerszten RE, 
Systrom DM, Bloch KD, Semigran MJ. Sildenafil improves exercise capacity and quality of life in 
patients with systolic heart failure and secondary pulmonary hypertension. Circulation. 2007;116:1555-
1562 
13. 
Bocchi EA, Guimaraes G, Mocelin A, Bacal F, Bellotti G, Ramires JF. Sildenafil effects on exercise, 
neurohormonal activation, and erectile dysfunction in congestive heart failure: A double-blind, placebo-
controlled, randomized study followed by a prospective treatment for erectile dysfunction. Circulation. 
2002;106:1097-1103 
14. 
McKone EF, Barry SC, FitzGerald MX, Gallagher CG. Reproducibility of maximal exercise ergometer 
testing in patients with cystic fibrosis. Chest. 1999;116:363-368 
15. 
Ferreira EM, Ota-Arakaki JS, Barbosa PB, Siqueira AC, Bravo DM, Kapins CE, Silva CM, Nery LE, 
Neder JA. Signal-morphology impedance cardiography during incremental cardiopulmonary exercise 
testing in pulmonary arterial hypertension. Clin Physiol Funct Imaging. 2012;32:343-352 
16. 
Welsman J, Bywater K, Farr C, Welford D, Armstrong N. Reliability of peak vo(2) and maximal cardiac 
output assessed using thoracic bioimpedance in children. Eur J Appl Physiol. 2005;94:228-234 
17. 
Oudiz RJ, Roveran G, Hansen JE, Sun XG, Wasserman K. Effect of sildenafil on ventilatory efficiency 
and exercise tolerance in pulmonary hypertension. Eur J Heart Fail. 2007;9:917-921 
18. 
Goldberg DJ, French B, McBride MG, Marino BS, Mirarchi N, Hanna BD, Wernovsky G, Paridon SM, 
Rychik J. Impact of oral sildenafil on exercise performance in children and young adults after the fontan 
operation: A randomized, double-blind, placebo-controlled, crossover trial. Circulation. 2011;123:1185-
1193 
19. 
Jones B, Kenward M. Design and analysis of cross-over trials. Boca Raton, FL: Chapman & Hill/CRC; 
2003. 
20. 
Hryniewicz K, Dimayuga C, Hudaihed A, Androne AS, Zheng H, Jankowski K, Katz SD. Inhibition of 
angiotensin-converting enzyme and phosphodiesterase type 5 improves endothelial function in heart 
failure. Clin Sci (Lond). 2005;108:331-338 
21. 
Katz SD, Balidemaj K, Homma S, Wu H, Wang J, Maybaum S. Acute type 5 phosphodiesterase 
inhibition with sildenafil enhances flow-mediated vasodilation in patients with chronic heart failure. J Am 
Coll Cardiol. 2000;36:845-851 


22. 
Harris RA, Nishiyama SK, Wray DW, Richardson RS. Ultrasound assessment of flow-mediated dilation. 
Hypertension. 2010;55:1075-1085 
23. 
Rosano GM, Aversa A, Vitale C, Fabbri A, Fini M, Spera G. Chronic treatment with tadalafil improves 
endothelial function in men with increased cardiovascular risk. Eur Urol. 2005;47:214-220; discussion 
220-212 
24. 
Deyoung L, Chung E, Kovac JR, Romano W, Brock GB. Daily use of sildenafil improves endothelial 
function in men with type 2 diabetes. J Androl. 2012;33:176-180 
25. 
Burnett AL, Strong TD, Trock BJ, Jin L, Bivalacqua TJ, Musicki B. Serum biomarker measurements of 
endothelial function and oxidative stress after daily dosing of sildenafil in type 2 diabetic men with 
erectile dysfunction. J Urol. 2009;181:245-251 
26. 
Harris RA, Padilla J, Hanlon KP, Rink LD, Wallace JP. Reproducibility of the flow-mediated dilation 
response to acute exercise in overweight men. Ultrasound Med Biol. 2007;33:1579-1585 
27. 
Harris RA, Padilla J, Rink LD, Wallace JP. Variability of flow-mediated dilation measurements with 
repetitive reactive hyperemia. Vasc Med. 2006;11:1-6 
28. 
Welsch MA, Allen JD, Geaghan JP. Stability and reproducibility of brachial artery flow-mediated 
dilation. Med Sci Sports Exerc. 2002;34:960-965 
29. 
Barst RJ, Ivy DD, Gaitan G, Szatmari A, Rudzinski A, Garcia AE, Sastry BK, Pulido T, Layton GR, 
Serdarevic-Pehar M, Wessel DL. A randomized, double-blind, placebo-controlled, dose-ranging study 
of oral sildenafil citrate in treatment-naive children with pulmonary arterial hypertension. Circulation. 
2012;125:324-334 
30. 
Harris RA, K.T. McKie, D. Eidson, K. Lange, T.S. Poore, V. Hudson. Interaction among lung function, 
vascular function, and exercise capacity in patients with cystic fibrosis. Pediatric pulmonology. 
2011;46:335 
31. 
Harris RA, D. Eidson, J.S. Pollock, G.W. Davison, J. Quindry, V. Hudson, K.T. McKie. Inflammation and 
oxidative stress during exercise in patients with cystic fibrosis. Pediatric pulmonology. 2012;47:369 
32. 
Harris RA, B. Berry, S. Poore, D. Eidson, G.W. Davison, K.T. McKie. Blood flow regulation is blunted 
during exercise in patients with cystic fibrosis. Medicine and science in sports and exercise. 
2012;44:S413 
33. 
Harris RA, S Poore, B. Berry, D. Eidson, J.S. Pollock, K.T. McKie. Flow-mediated dilation is attenuated 
in young patients with cystic fibrosis. Journal of the Federation of American Societies for Experimental 
Biology. 2012;26:1130 
34. 
Teixeira CE, Priviero FB, Webb RC. Differential effects of the phosphodiesterase type 5 inhibitors 
sildenafil, vardenafil, and tadalafil in rat aorta. J Pharmacol Exp Ther. 2006;316:654-661 
35. 
Giachini FR, Lima VV, Carneiro FS, Tostes RC, Webb RC. Decreased cgmp level contributes to 
increased contraction in arteries from hypertensive rats: Role of phosphodiesterase 1. Hypertension. 
2011;57:655-663 
36. 
Toque HA, Priviero FB, Zemse SM, Antunes E, Teixeira CE, Webb RC. Effect of the 
phosphodiesterase 5 inhibitors sildenafil, tadalafil and vardenafil on rat anococcygeus muscle: 
Functional and biochemical aspects. Clin Exp Pharmacol Physiol. 2009;36:358-366 
37. 
American Thoracic Society. Standardization of spirometry, 1994 update. American thoracic society. 
American journal of respiratory and critical care medicine. 1995;152:1107-1136