Conditions: Healthy Obese
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 141
Healthy volunteers: yes

Brief summary:
Obesity and its adverse cardiometabolic consequences are major public health problems. Several features of obesity contribute to the associated cardiovascular risk and are potential targets for intervention. These include insulin resistance and beta cell dysfunction, reduced metabolic rate, and impaired aerobic capacity.The purpose of this study is to examine if the phosphodiesterase type 5A inhibitor tadalafil improves cardiometabolic health in individuals who are obese and insulin resistant.

Detailed description:
Obesity is a risk factor for nearly all cardiovascular (CV) disease including coronary artery disease, hypertension, and heart failure. Increased CV risk in obese individuals appears to depend largely on the degree of metabolic dysregulation and metabolic risk factors (glucose intolerance, dyslipidemia, etc.). Notably, interventions that improve insulin sensitivity and cardiorespiratory fitness can reduce CV risk in obese individuals, even in the absence of weight loss.

The cyclic guanylate monophosphate pathway (cGMP) is involved in energy homeostasis and systemic metabolism. Multiple lines of evidence suggest that increasing cGMP activity is beneficial from a metabolic standpoint. Tadalafil is a clinically-available drug that inhibits the enzyme that breaks down cGMP.

The study investigators hypothesize that chronic PDE5 inhibition in obese, insulin-resistant adults will improve cardiometabolic health.

Aim 1: To examine the effect of PDE5 inhibition on energy expenditure. Aim 2: To examine the effect of PDE5 inhibition on insulin sensitivity and secretion.

Aim 3: To examine the effect of PDE5 inhibition on cGMP tone and circulating mediators of cardiometabolic risk.

Arms:
arm 1: Subjects will be randomized to one of two arms. 100 obese adult subjects will be randomized to the Tadalafil arm following the screening visit. Beginning at their baseline visit, they will receive an oral daily dose of Tadalafil (20mg) that they will take for 12 weeks (through their completion of the study). After randomization has occurred, the active comparator subjects will undergo the following visit protocol: baseline visit (two half-days), an interim visit (6 weeks post-baseline), and a 12-week visit (two half-days). arm 2: Subjects will be randomized to one of two arms. 100 obese adult subjects will be randomized to the placebo arm following the screening visit. Beginning at their baseline visit, they will receive an oral daily dose of a placebo pill (20mg) that they will take for 12 weeks (through their completion of the study). After randomization has occurred, the placebo comparator subjects will undergo the following visit protocol: baseline visit (two half-days), an interim visit (6 weeks post-baseline), and a 12-week visit (two half-days).

Interventions: intervention 1: Tadalafil intervention 2: Placebo
Intervention descriptions: intervention 1: Tadalafil is an FDA approved, clinically-available drug that inhibits the enzyme phosphodiesterase type 5A (PDE5). Subjects who are randomized to this arm will be provided with 20mg of Tadalafil to take every day for 12 weeks, beginning at their baseline visit. intervention 2: 100 patients will be randomized to receive a placebo pill. Subjects in this arm will be provided with 20mg Placebo to take every day for 12 weeks, beginning at their baseline visit.

Protocol document:
Prot_SAP_000.pdf: 
Protocol Version #: 7 
1 
Protocol Date: 04/30/2019 
PDE5 Inhibition for Obesity-Related Cardiometabolic 
Dysfunction 
NCT02819440
Protocol Date: 4/30/2019
Principal Investigator: 
Evan Brittain, MD, MSCI 
Co-Investigators: 
Thomas Wang, MD 


Protocol Version #: 7 
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Protocol Date: 04/30/2019 
Table of Contents: 
1.0 
Background 
2.0 
Rationale and Specific Aims 
3.0 
Animal Studies and Previous Human Studies 
4.0 
Inclusion/Exclusion Criteria 
5.0 
Enrollment 
6.0 
Study Procedures 
7.0 
Optional Genetic and Future Research on Biological Samples 
8.0 
Reporting of Adverse Events or Unanticipated Problems involving Risk to 
Participants or Others 
9.0 
Study Withdrawal/Discontinuation 
10.0 Statistical Considerations 
11.0 Privacy/Confidentiality Issues 
12.0 Follow-up and Record Retention 
13.0 Literature cited 


 
 
Protocol Version #: 7 
 
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Protocol Date: 04/30/2019 
 
1.0 
Background 
 
Obesity is a risk factor for nearly all cardiovascular (CV) diseases including coronary artery 
disease, hypertension, and heart failure. Obesity and its adverse cardiometabolic sequelae 
represent major and worsening public health burdens. In 2000, the World Health Organization 
declared obesity as the greatest threat to the health of Westernized nations.1 In the U.S. Obesity 
accounts for over 400,000 deaths per year and affects 35% of the adult population. 
 
Increased cardiovascular risk in obese individuals appears to depend largely on the degree of 
metabolic dysregulation. Obese individuals without other metabolic risk factors (such as glucose 
intolerance, dyslipidemia, or central adiposity) have cardiovascular outcomes similar to those of 
lean individuals. In contrast, obese individuals with a high burden of metabolic risk factors are at 
very high risk of future events. Notably, interventions that improve insulin sensitivity2 and 
cardiorespiratory fitness3 can reduce cardiovascular risk in obese individuals, even in the 
absence of weight loss. 
 
The poor effectiveness of lifestyle-based interventions highlights the need for new strategies to 
improve cardiometabolic health in obese individuals. Estimates suggest that over half of all 
Americans are currently attempting weight loss, and another quarter are attempting to maintain 
achieved weight loss. Despite these efforts, many people never achieve significant weight 
reduction and weight loss recidivism at one year approaches 90%.4,5 Thus, lifestyle-based 
interventions alone are unlikely to achieve significant reductions in obesity-related 
cardiovascular risk at the population level. These data underscore the potential value of 
physiologic interventions directly targeting metabolic fitness in obesity. 
 
2.0 
Rationale and Specific Aims 
 
Obesity is frequently accompanied by abnormalities in energy homeostasis, cardiopulmonary 
function, and peripheral insulin resistance. A large body of experimental and clinical data 
suggests that these abnormalities contribute to the increased cardiovascular and metabolic risk 
in obese individuals, making them potentially attractive targets for intervention. 
 
Energy expenditure is a major determinant of weight gain and loss.6 The primary components of 
total energy expenditure are physical activity (30%), the thermic effect of food (10%), and 
resting energy expenditure (REE, 60%). Obese individuals have approximately 50% lower REE, 
indexed to body weight, compared with normal weight adults.1 Weight loss is associated with a 
further reduction in REE, an adaptive mechanism that resists maintenance of a reduced body 
weight and ultimately contributes to weight regain. Adjunctive strategies to complement diet and 
exercise by safely increasing metabolic rate during rest and physical activity may assist in 
efforts at weight loss and weight loss maintenance. 
 
Obesity is associated with reduced cardiopulmonary capacity. Maximal oxygen consumption 
during exercise is inversely related to body mass index and fasting insulin level.7,8 Furthermore, 
exercise capacity is strongly related to clinical outcomes in obese individuals, highlighting its 
value as an endpoint in this population.9,10 
 
Increasing cGMP activity can be achieved pharmacologically by inhibiting phosphodiesterase 
type 5A (PDE5), which catabolizes cGMP. The clinically-available drugs sildenafil and tadalfil 
are specific inhibitors of PDE5 and multiple studies suggest that they have beneficial 
cardiometabolic effects. 


 
 
Protocol Version #: 7 
 
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Strategies to increase energy expenditure may improve the success of lifestyle interventions in 
obese individuals. In an animal model of obesity, PDE5 inhibition increased energy expenditure 
for the same amount of physical activity. PDE5 inhibition improves insulin sensitivity and beta 
cell function in experimental models and small human studies. Prior studies have been limited 
by small sample size and less sensitive methods for assessing insulin sensitivity and secretion. 
PDE5 inhibition increases intracellular cGMP tone. Prior studies have used plasma or urine 
concentrations of cGMP as an index of “target-organ” responsiveness to NP, proposing the ratio 
of cGMP to natriuretic peptide (NP) concentrations as an index of NP sensitivity. 
 
We propose to evaluate energy expenditure in response to a metabolic intervention in which 
weight loss is not a direct target.  We hypothesize that PDE5 inhibition will increase resting 
energy expenditure, improve insulin sensitivity, and increase the cGMP/NP ratio in obese 
adults. 
 
Aim 1.  
To examine the effect of PDE5 inhibition on energy expenditure in obese  
 
 
adults. 
 
 
Subjects will undergo a 4-hour metabolic chamber protocol at baseline and after  
 
 
12 weeks of placebo or tadalafil therapy to measure resting and sub-maximal  
 
 
exercise energy expenditure. The primary endpoint is resting energy expenditure  
 
 
at 12 weeks, adjusted for baseline values. 
 
Aim 2.  
To examine the effect of PDE5 inhibition on insulin sensitivity. 
 
 
Subjects will undergo FS-IVGTTs which will provide a validated estimate of  
 
 
insulin sensitivity and the acute insulin response to glucose. The primary   
 
 
endpoint is insulin sensitivity at 12 weeks, adjusted for baseline values. 
 
Aim 3.  
To examine the effect of PDE5 inhibition on cGMP tone and circulating  
 
 
mediators of cardiometabolic risk. 
 
 
Subjects will undergo fasting blood draw at baseline and 12 weeks to  
 
 
 
measure cGMP and NPs. The primary endpoint is cGMP/NP ratio at 12 weeks,  
 
 
adjusted for baseline value 
 
 
We may perform interim unblinded analyses of select secondary endpoints (e.g. fat mass, blood 
pressure) after getting approval from the data safety monitor and the study biostatistician. We 
will not perform any unblinded analyses of primary endpoints until study procedures are 
complete. 
 
 
 
3.0 
Animal Studies and Previous Human Studies 
 
Prior experimental work 
 
In a high-fat diet mouse model of obesity and insulin resistance, Dr. Wasserman showed that 
sildenafil increases resting and ambulatory energy expenditure, independent of activity level. 
Sildenafil also conferred resistance to weight gain from high fat diet over 12 weeks. Finally, 
PDE5 inhibition improved insulin sensitivity and muscle glucose uptake assessed by 
hyperinsulinemic-euglycemic clamp. These observations support measuring energy expenditure 
and insulin sensitivity as primary endpoints in a human trial of PDE5 inhibition. 
 
Prior observational studies 


 
 
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We have performed metabolomic profiling in more than 3,000 individuals in the Framingham 
Heart Study and Malmo Diet and Cancer Study. In prior studies, we have shown associations of 
branched chain amino acids, aromatic amino acids, tryptophan metabolites, nucleotides, 
glutamine, and glutamate with obesity, insulin resistance, and other metabolic traits.28 In pre-
diabetic individuals, we have shown that branched chain and aromatic amino acids predict 
incident diabetes up to 12 years later.29 We have also demonstrated the association of a lysine 
metabolite, 2-aminoadipic acid, with future diabetes, and shown that 2-aminoadipic acid 
modulates glucose homeostasis in vivo.33 In the Malmo Diet and Cancer Study, we have 
demonstrated that many of these metabolites predict cardiovascular events and subclinical 
atherosclerosis.30 In analyses of >110 lipid species, we have demonstrated that lipids of lower 
carbon number and double-bond content are associated with a higher risk of diabetes.34 
 
We examined metabolite profiles before and after OGTT in 377 non-diabetic Framingham 
Offspring cohort participants.35 Dynamic changes were observed in multiple pathways 
demonstrating the feasibility of altering metabolomic profiles in response to a metabolic 
intervention (glucose administration). We identified metabolite responses that distinguished 
individuals with and without insulin resistance, including in pathways not associated with insulin 
or glucose action. Findings from these studies support the use of metabolomic profiling to 
assess the effect of interventions such as PDE5 inhibition on pathways associated with 
cardiometabolic risk. Exploratory analyses may also allow the identification of additional 
metabolites related to increased cGMP signaling. 
 
Pilot Studies of PDE5 inhibition 
 
We conducted a pilot study of tadalafil versus placebo in insulin-resistant individuals with BMI > 
27 kg/m2. In double-blind fashion, individuals were randomized to receive tadalafil (20mg/day) 
or placebo for 3 months (n=53). During follow up, 3 subjects from the tadalafil arm compared 
with 2 subjects from the placebo arm withdrew from the study. Reasons for withdrawal in the 
tadalafil arm included headache (n=1) and muscle aches (n=2). There were no instances of 
hypotension, visual disturbances, or serious adverse events among individuals randomized to 
tadalafil. 
 
In the pilot study, we found improvement in the oral disposition index, a composite measure of 
insulin release and resistance (p=0.009). We also observed a trend toward improvement in the 
Matsuda index (p = 0.05). HOMA-IR improved significantly in subjects with BMI above the 
median (36 kg/m2) assigned to tadalafil (p=0.02 for comparison with placebo), but not in the 
entire sample (which included overweight individuals). There was no significant change in 
weight in either group. In additional preliminary work, we have performed hyperglycemic clamps 
in subjects with impaired glucose tolerance randomized to sildenafil (n=21) 25 mg TID or 
placebo (n=21) for 3 months. PDE5 inhibition improved the insulin sensitivity index and 
disposition index, measured by clamp (both p<0.05). 
 
Experience with metabolic techniques 
 
This proposal brings together a multi-disciplinary and collaborative team to leverage some of 
Vanderbilt’s unique institutional and intellectual resources. 
 
Energy Balance Laboratory. 
, an NIH-funded expert in the quantification and 
interpretation of energy expenditure and physical activity data, directs the lab and Metabolic 


 
 
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Protocol Date: 04/30/2019 
 
Core 
. The group has extensive experience 
using whole room indirect calorimetry (metabolic chamber) to assess resting and physical 
activity related energy expenditure and Vanderbilt is one of only 13 institutions in the U.S. with 
this technology available. 
 will also consult on the use of accelerometry to 
quantify physical activity.39-41 
 
Assays of insulin and glucose homeostasis. 
, an NIH-funded expert in 
metabolic studies in humans and mice has extensive experience conducting IVGTTs, 
hyperinsulinemic-euglycemic clamps, and hyperglycemic clamps.42,43  He and his team have 
completed over 200 in the past 5 years and have expertise in the software and statistical 
analyses for FS-IVGTT studies.  
 
Cyclic GMP signaling and metabolomic profiling. Our group has experience in the quantification 
of metabolites involved in cGMP signaling and broad-based metabolomics platforms. Dr. Wang 
has performed several cohort based studies using a targeted mass spectrometry-based 
platform that quantifies approximately 300 metabolites.28-30,35 Dr. Brittain has also participated in 
metabolomics studies using plasma and tissue. 
 
4.0 
Inclusion/Exclusion Criteria 
 
Inclusion criteria: 
 
1) Adults (ages 21-50) 
 
2) Obesity (BMI ≥ 30 kg/m2) 
 
3) Evidence of abnormal glucose metabolism at screening visit, defined as any of the  
 
following: 
 
. 
-     Hemoglobin A1C between 5.7-6.4 
- 
Fasting glucose between 100-125  
- 
HOMA IR greater than 2.5 
 
Rationale for Inclusion Criteria: This study is designed to target individuals at the highest risk 
for the development of diabetes and subsequent adverse CV outcomes. The upper age cutoff is 
specified to reduce the likelihood of enrolling individuals with subclinical cardiac disease, which 
could influence the cardiopulmonary endpoints. 
 
Exclusion criteria: 
 
1. Age <21 or > 50 
 
2. BMI < 30 kg/m2 
 
3. Systolic blood pressure (SBP) < 100, > 150 mmHg 
 
4. Current anti-hypertensive medication use, including diuretics 
 
5. Current use of organic nitrates 
 
6. Current use of PDE-5 inhibitors (sildenafil, tadalafil, vardenafil) 
 
7. History of reaction to PDE-5 inhibitors 
 
8. Known HIV infection 
 
9. Use of medications that strongly alter CYP3A4 activity 
 
10. History of myocardial infarction, angina, uncontrolled cardiac arrhythmia, stroke, 
 
transient ischemic attack, or seizure 
 
11. Known non-arteritic ischemic optic retinopathy (NAIOR) 
 
12. History of hearing loss 
 
13. Estimated glomerular filtration rate (eGFR) < 60 ml/min/1.73 m2 by the modified diet 
 
in renal disease (MDRD) equation 


 
 
Protocol Version #: 7 
 
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Protocol Date: 04/30/2019 
 
 
14. Hepatic transaminase (AST and ALT) levels greater than three times the upper limit 
 
of normal 
 
15. Known pregnancy or breastfeeding or those unwilling to avoid pregnancy during the 
 
course of the study 
 
16. History of priapism 
 
17. Use in excess of four alcoholic drinks daily 
 
18. History of diabetes mellitus or use of anti-diabetic medications 
 
19. Known anemia (men, Hct < 38% and women, Hct <36%) 
 
20. Menopause 
 
21. Inability to exercise on a bicycle 
 
 
 
Rationale for Exclusion Criteria: 
1) Given the increasing prevalence of cardiovascular disease and hypertension in older obese 
subjects, we will exclude individuals over age 50 from the study. Our institutional IRB defines 
adults as 19 years and older. 
2) Given our intent to study the effects of this medication in clinically obese individuals, we will 
exclude individuals with BMI < 30 kg/m2. 
3) Given the potential for mild changes in blood pressure and the potential interaction of tadalafil 
with antihypertensive medications, we will exclude subjects with low-normal blood pressure and 
those requiring treatment with anti-hypertensive medication during the study. 
4) Given the possibility for hypotension when tadalafil and anti-hypertensive medications are 
combined, we will exclude subjects currently taking anti-hypertensive medications. 
5) Given the potential for severe hypotension when combining tadalafil and nitrates, we will 
exclude individuals taking nitrates. 
6) Given the possibility for interaction between two medications from the same class and the 
intent to study the native effects of the medication, we will exclude individuals already taking a 
PDE-5 inhibitor. 
7) Given the increased likelihood of a second reaction in those who have already experienced a 
prior side effect of a PDE-5 inhibitor, we will exclude those who have had a previous reaction to 
a PDE-5 inhibitor (sildenafil, tadalafil, vardenafil). 
8) Given the inhibition of CYP3A4 and subsequent elevation of tadalafil levels by protease 
inhibitors, which are used only to treat HIV infection, we will exclude individuals with known HIV 
infection. 
9) Given their potential effects on CYP3A4 activity and, as a result, tadalafil levels, we will 
exclude individuals taking oral ketoconazole, itraconazole, erythromycin, rifampin, phenytoin, or 
carbamazepine; topical preparations of antifungal or antibiotic medications will not be 
contraindicated as these do not appear to significantly alter systemic CYP3A4 activity. 
10) Given the temporal association of myocardial infarction, angina, uncontrolled cardiac 
arrhythmia, stroke, transient ischemic attack, or seizure with tadaladil in post-market 
surveillance (which has not been directly attributed to the drug itself), we will exclude those with 
a past history of these conditions. 
11) Given the temporal association of NAIOR with tadalafil in post-marketing surveillance, we 
will exclude those with this condition from the study. 
12) Given the temporal association of hearing loss with tadalafil in post-marketing surveillance, 
we will exclude those with this condition from the study. 
13) Given the altered metabolism of tadalafil in subjects with renal dysfunction, we will exclude 
those with abnormal renal function (by eGFR as measured by MDRD). 
14) Given the altered metabolism of tadalafil in subjects with hepatic dysfunction, we will 
exclude those with transaminase levels more than three times the upper limit of normal. 


 
 
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Protocol Date: 04/30/2019 
 
15) Given the untested effects of tadalafil on pregnant or breastfeeding women, we will exclude 
women who are pregnant or breastfeeding or who are able to become pregnant and not 
practicing an acceptable method of birth control during the study (including abstinence). 
16) Given the increased risk of priapism with use of tadalafil, individuals with a prior history of 
priapism will be excluded. 
17) Given that alcohol use in excess of four drinks at one time combined with tadalafil 20mg can 
precipitate hypotension, individuals who consume more than this amount daily will be excluded. 
18) Given that we are interested in determining the effect of tadalafil on insulin resistance and 
metabolism, we will exclude individuals who are taking or are at risk of starting medication for 
diabetes treatment, which would affect these measures. 
19) Given that blood samples will be taken at three time points over the 3 month study, we will 
exclude individuals with known anemia. 
20) Given the menopause affects energy balance in females, we will exclude menopausal 
women, determined via interview 
21) Given that one of the objectives of our study is to investigate the effect of tadalafil on 
exercise capacity, we will exclude individuals who are unable to exercise on a bicycle. 
 
5.0 
Enrollment 
 
Subjects will be recruited from Vanderbilt’s Research Notification Distribution List, Research 
Match, Subject Locator, and the Vanderbilt Nutrition and Diet Assessment Research Core. 
Within the distributions, a brief description of the study will be provided and a contact number for 
further information. Upon contacting the study staff, the potential participant will receive a 
complete explanation of the protocol procedures by telephone. 
 
6.0 
Study Procedures 
This study will consist of a screening visit and study testing at 3 time points over the course 
of 14 weeks. Patients will be consented over the phone prior to their screening visit and will 
sign the consent in-person during their screening visit at the Clinical Research Center. At 
screening, to determine if subjects have evidence of abnormal glucose metabolism, the 
following tests will be conducted: Hemoglobin A1C, fasting glucose, and fasting insulin. 
HOMA-IR will be calculated from fasting glucose and insulin. The patient will also be 
screened to determine if C-peptide, metabolic and blood counts are within normal limits.  
 
Subjects meeting exclusion criteria based on abnormal laboratory values due to chronic, 
stable conditions may be enrolled at the discretion of the PI in consultation with the subject’s 
treating physician. 
 
Urine testing: A urine sample will be collected at screening, and if patient qualifies, at baseline 
and at 12 weeks, and a urine microalbumin will be measured. Samples may be stored for 
additional analyses. 


 
 
Protocol Version #: 7 
 
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Baseline Testing: Baseline testing will take place over 2 half-days. Each half-day visit will 
last approximately 4 hours. Determined by the randomization outcome, subjects will also 
receive a 7 week supply of tadalafil or the placebo at the second half-day visit. The study 
staff will discuss how to take the study medication and answer any questions. The following 
protocols, tests, and surveys will be performed at baseline: 
 
Metabolic Chamber Protocol: The room calorimeter (2.6 m x 4 m; ~19 m3 volume) assures 
high-precision measurements of physiological variables (e.g. oxygen consumption, heart rate) in 
a controlled environment and semi-naturalistic conditions (i.e. not wearing a breathing mask or a 
mouthpiece). The room is equipped with a bed, a television, a toilet, a sink, and an exercise 
bicycle. Accuracy and precision of the system for measuring EE is tested monthly using an 
alcohol combustion test. Precision averages 99.2 ± 0.5% (mean ± SD) over 24 hours and 98.6 ± 
2.1% over 30 minutes. The system reliably detects short-term changes in metabolic rate to 2.7% 
over 30 min and 0.6% over a 2 h measurement period.50,55 In a previous study in adults 
conducted in our room calorimeter, mean short-term within-subject variability in resting energy 
expenditure was 3.5±2.2%. The operation of the room calorimeter is controlled and data are 
collected minute-by-minute using a customized computer program. Subjects will arrive to the 
CRC after an overnight (~12 hour) fast, have vital signs and a blood sample taken before 
entering the chamber. Participants will enter the chamber at 8:00 am and remain there until 
12:00 pm. After entering the room, subjects will lie on the bed for 30 minutes. Resting energy 
expenditure will then be averaged over the next 30 minutes while the patient remains on the 
bed. At approximately 9am, the patient will consume a meal containing approximately 25% of 
individual daily energy needs and containing 55% of energy from carbohydrates, 30% from fat, 
and 15% from protein. Diet composition will be determined using the Nutrition Data System for 
Research (NDSR) software. 
 
Cardiopulmonary Exercise Test Protocol: The test will be conducted inside the metabolic 
chamber at the CRC according to the American College of Sports Medicine guidelines using an 
electrically-braked cycle ergometer. We have chosen the ergometer test because it may be 
better tolerated than the exercise treadmill by obese adults, mechanical efficiency of individuals 
varies less, measurement of work performed is more accurate, and fatigue-induced change in 
work efficiency is feasible to measure. Heart rate and rhythm (12-lead electrocardiogram), blood 
pressure, breath-by-breath gas exchange, and power will be monitored or measured. Peak VO2 
uptake will be calculated (ml O2/kg/min) and the anaerobic threshold will be measured. The 
pedal cadence of ~50 revolutions per minute will be maintained via feedback from a display on 
the ergometer until voluntary termination of the task (i.e. reaching tolerable fatigue level). Based 
on our experience the test will last up to 45 minutes. The following measures will be calculated:  
• 
Energy expenditure (EE) rate (kcal/min): Oxygen consumption (VO2), carbon-dioxide 
production, air flow rate, temperature, barometric pressure, and air humidity will be 
measured minute-by-minute and integrated to calculate respiratory quotient (RQ = 
VCO2/VO2) and EE using the Weir equation.56,57  
• 
Resting EE rate (kcal/min; REE): Calculated as the average EE during a 30-min period 
while the subject is lying in bed having fasted for ~12 hours.  
• 
Physical activity-induced EE (kcal/day): EE related to physical activity will be calculated 
as total EE above the resting level while performing the ergometer test.  
 
 
Fasting intravenous glucose tolerance test (FS-IVGTT) Protocol: We will conduct insulin 
modified FS-IVGTTs, which provide a validated estimate of insulin sensitivity and the acute 
insulin response to glucose (AIRg).7   The AIRg is a measurement of insulin secretion (i.e. beta 


 
 
Protocol Version #: 7 
 
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Protocol Date: 04/30/2019 
 
cell function). The disposition index accounts for the hyperbolic relationship between AIRg and 
SI and is associated with risk of diabetes. After an overnight ~12-hour fast, subjects will assume 
a supine position and we will place an intravenous catheter in each arm, one for sampling and 
one for infusion, and allowed to rest for 15 minutes. 
 
We will collect baseline samples at t=-15 min and t=-5 min for measurement of glucose and 
insulin. At t=0, we will bolus 300 mg D-glucose/kg in a 25% glucose-saline solution over 1 
minute. At t=20 min, we will give a bolus of 0.02 units/kg of regular insulin followed by a 5 mL 
0.9% saline flush (Actrapid, Novo Nordisk, Princeton, NJ) intravenously, to improve the 
accuracy of the FS-IVGTT in patients in whom insulin responses are reduced or absent.8-13  Use 
of low dose insulin avoids saturation of insulin binding sites and maintains insulin concentrations 
in a range where insulin sensitivity is independent of insulin level.14 Blood will be collected for 
measurement of glucose and insulin at t=2, 3, 4, 5, 6, 8, 10, 14, 16, 19, 22, 25, 30, 40, 50, 60, 
70, 90, 110, 130, 150, 170, and 180 min. We will estimate insulin sensitivity (SI), glucose 
effectiveness at basal insulin (SG), glucose disappearance constant (Kg), AIRg, and disposition 
index using the MINMOD program (MINMOD Millennium v. 6.02).15-17 Plasma glucose will be 
determined immediately after bedside plasma centrifugation, using the glucose oxidase method 
(YSI 2300, Yellow Springs Instruments). Plasma insulin will be measured by RIA using samples 
stored at -80 °C until assay.  
 
Physical Activity (PA): Total PA in the free-living environment will be measured using tri-axial 
accelerometers ActiGraph-GT9X+ (Actigraph, Pensacola, FL) for 7 days. Participants will be 
asked to maintain their regular sleep-wake schedule and wear the monitor for consecutive 7 
days. If subject wears the monitor on their wrist, subject will be instructed to wear the monitor on 
their non-dominant wrist. The raw data will be collected at 30 Hz, preprocessed and converted 
to vector magnitude counts. After assessing monitor wear/no-wear time, the outcomes of 
interest will be total number of counts and time and spent in sedentary, light, moderate and 
vigorous activity intensities. Intra- and inter-individual differences in the amount and patterns of 
physical activity will be assessed using methods described in previous studies in adults. 
Subjects will be asked to wear the accelerometer for 7 days after their second baseline half-day, 
for 7 days before their interim visit, and for 7 days before their 12-week visit. 
 
Quality of Life: Quality of life will be assessed using the Medical Outcomes Study Short-Form 
Health Survey (SF-36). The SF-36 is a generic, multi-item scale with eight domains that include: 
physical functioning, bodily pain, mental health, social functioning, vitality, general health 
perceptions, role limitations-physical, and role limitations-emotional. Individual subscale scores, 
a mental component summary score, and a physical component score summary score are 
calculated.64 The SF-36 has been widely used to measure quality of life among patients with 
obesity and is sensitive to changes in weight and BMI.65,66 The International Index of Erectile 
Function (IIEF) will be used to measure sexual function in men. The IIEF addresses four 
domains of male sexual function (erectile function, orgasmic function, sexual desire, intercourse 
satisfaction, and overall satisfaction) and is the gold standard for measuring erectile 
dysfunction.67 The Female Sexual Function Index will be used to measure sexual function in 
women. The FSFI is comprised of five domains: desire and subjective arousal, lubrication, 
orgasm, satisfaction, and pain/discomfort and is the most widely used measurement of female 
sexual function.68  A nurse practitioner 
) with extensive experience 
administering quality of life surveys will perform these assessments. 
 


 
 
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Dual-Energy X-ray Absorptiometry (DEXA): Body composition will be assessed at baseline 
and at 12 weeks.  Participants will lie flat on a table while a machine takes pictures of different 
areas of their bodies. This test will last about 15 minutes. 
 
Interim Visit: This visit will take place approximately 6 weeks after the baseline visits. At this 
visit, we will discuss any noted side effects, conduct a pill count, perform a blood draw, and give 
subjects their next 6-week supply of the study medication. 
 
12 Week: The tests performed at baseline (described above) will be performed approximately 
12 weeks after beginning the study medication.  
 
 
7.0 
Optional Genetic and Future Research on Biological Samples 
 
As part of the informed consent, participants will be asked whether blood, not used in this 
analysis, may be stored for future analysis. If the participant does not consent to this, samples 
will be used only for the present study and excess samples discarded. 
 
8.0 
Reporting of Adverse Events or Unanticipated Problems involving Risk to 
Participants or Others 
Risks and Discomfort 
Tadalafil is a medication with proven safety. This drug was initially approved by the FDA in 2003 
for the treatment of erectile dysfunction (under the trade name CIALIS) at a starting dose of 
2.5mg and a maximum dose of 5mg for daily use and at a starting dose of 10mg and a 
maximum dose of 20 mg up to once daily for as needed use. Tadalafil 20mg daily has been 
used without serious adverse effects in a recent study of healthy men with mild erectile 
dysfunction. Safety data are provided in the package inserts and Physician Desk 
Reference (PDR through MICROMEDEX) entry and summarized here. In 2009, the FDA 
approved use of this medication at a dose of up to 40mg daily for management in pulmonary 
arterial hypertension (under the trade name ADCIRCA) based on Phase III clinical trial data. In 
pre-clinical safety data, tadalafil 20mg daily produced no significant decrease in supine or 
standing systolic/diastolic blood pressure when compared to placebo (supine decreased by 
1.6/0.8 mm Hg and standing decreased by 0.2/4.6 mm Hg).  
 
Contraindications for the use of tadalafil are concomitant use of organic nitrates (because of 
severe hypotension) and a history of hypersensitivity reaction to tadalafil; individuals with those 
characteristics are excluded from this study. Individuals using anti-hypertensive medications are 
at increased risk of having low blood pressure when using tadalafil, and these individuals are 
excluded from this study as well. Safety testing and post-marketing surveillance have identified 
serious potential side effects for which study subjects could be at risk. These include myocardial 
infarction, unstable angina, heart failure, uncontrolled arrhythmias, stroke, sudden visual loss, 
sudden hearing loss, and priapism. These conditions are all rare, and whether they are directly 
related to tadalafil use is unclear. Individuals with a history of these conditions are excluded 
from the protocol. Study subjects are advised to stop taking tadalafil and seek medical attention 
immediately should they experience any of the above or symptoms that may indicate any of the 
above. The most common adverse reactions experienced during pre-clinical safety trials and 
post-surveillance marketing for individuals taking tadalafil 20mg daily are: headache (15%), 
dyspepsia (10%), back pain (6%), myalgia (3%), nasal congestion (3%), flushing (3%), and pain 
in a limb (3%). In a study of otherwise healthy men with mild erectile dysfunction, tadalafil 20mg 
daily for 9 months was associated with the following adverse events: headache (16%), back 


 
 
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pain (14%), dyspepsia (8%), gastroesophageal reflux disease (8%), myalgia (8%), pain (4%), 
pain in an extremity (4%), dizziness (3%), and nasopharyngitis (3%) [23]. 
 
Because headache is a relatively common side effect of the medication, we will ask subjects to 
contact us if they experience a persistent headache. If it is determined by the study team that 
the duration and severity of the subject’s symptoms indicate a need to decrease the subject’s 
dosage, the dosage may be decreased to 10mg daily. After adjusting the dose of the 
medication, study personnel will communicate with subjects and the dose may be readjusted as 
symptoms decrease. Board certified Internist/Cardiologists (the MPIs) are available by pager 
and telephone at all times, if not physically present, during the study.  
 
Venipuncture  
The risks of drawing blood are uncommon and may include bleeding, minor infection and 
bruising. Commonly, having blood drawn is painful, and rarely can lead to infection at the site of 
the blood draw. The amount of blood drawn is small, and represents an exceedingly small 
percentage of the amount of the total blood volume and will not represent a significant risk to the 
subject. 
 
Subjects will be removed from the study if symptoms or medical issues arise that are deemed 
clinically significant by the PI or NP. A few examples are listed here:  
 
1) If the participant has or develops clinically significant contraindications to taking the 
study drug. 
 
2) If the participant has or develops clinically significant lightheadedness, particularly 
when standing at any blood pressure. 
 
3) If the participant develops systolic blood pressure less than 100 mm Hg when seated 
 
or standing. 
 
4) If clinically significant anemia is identified 
 
As stated in the consent form and reviewed at the time of enrollment, subjects may end 
participation at any time without prejudice. 
 
Adverse Events 
Investigators will report all serious, unexpected, and study-related adverse events to the Data 
Safety Monitor and the local Institutional Review Board in a timely manner according to IRB 
Policies and Procedures. 
 
Adverse event reporting: It is proposed that events possibly related to the study will be graded 
by the investigators in the following manner: 
 
Adverse Event (AE) Grading Scale: 
 
0= No Adverse Event or within normal limits 
 
1= Mild Severity: Transient laboratory test alterations; discomforts noted but no 
 
disruption of daily activities; no therapy, or only symptomatic therapy required 
 
2= Moderate Severity: Laboratory test alterations indicating injury without long-term risk; 
 
discomfort sufficient to modify normal daily activity; specific therapy required (i.e., more 
 
than symptomatic) 
 
3= Serious Severity: Laboratory test indicating a serious health threat or permanent 
 
injury; incapacity, inability to work, inability to perform normal daily activity; 
 
hospitalization required or prolonged; emergency treatment required; life-threatening 
 
events; death 
 


 
 
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f) Breech in confidentiality that may involve risk to that individual or others; 
g) Complaint of a participant that indicates an unanticipated risk or which cannot be resolved by 
the research staff; or 
h) Other event that is unanticipated, involved risk to participants or others and was possibly 
related to the research procedures. 
 
Adverse events of a serious severity thought to be related to the research protocol will be 
reported to the NIH, the Vanderbilt IRB, the DSM and Drs. Wang and Brittain 24 hours of 
occurrence and the specific study will cease to perform the suspect procedure until review. The 
IRB will have the responsibility to confirm the severity of the adverse event, determine if it is 
likely that the adverse event was related to the study, and make recommendations for 
continuation, modification or cessation of the study. Data monitoring will routinely be performed 
by Drs. Wang and Brittain and in response to the reports of serious adverse events. 
 
9.0 
Study Withdrawal/Discontinuation 
 
We plan to monitor closely for adverse effects and remove a subject for symptoms deemed 
related to study drug. The inability to tolerate tadalafil or hypersensitivity to drug will trigger 
withdrawal. Furthermore, the DSM has the ability to stop the study for safety concerns. 
If at any time during the study, participants decide they do want their blood samples or 
metabolic chamber data analyzed or included in the study results, they can contact the PI via 
phone or in writing (all contact information is provided in the consent form). At that point, the 
patient’s data would be removed from the electronic database. 
 
10.0 
Statistical Considerations 
Aim 1: 
Statistical analysis: We will test the effects of tadalafil versus placebo on the primary endpoint 
(resting energy expenditure) and the secondary endpoints, in similar fashion. We will use a 
generalization of the Wilcoxon test – the proportional odds ordinal logistic model – with the week 
12 value of resting energy expenditure as the dependent variable. Based on our pilot study, we 
will assume dropouts to be infrequent. Predictor variables will include the baseline resting 
energy expenditure, age, gender, BMI, race, and a treatment group indicator. The effects of the 
baseline value and age will be assumed to be smooth but not linear by using restricted cubic 
spline functions. A sensitivity analysis will assign Week 12 non-completers to the worst rank and 
then apply the proportional odds model. In exploratory analyses, we will also study whether 
treatment effects differ by patient subgroups. These pre-defined analyses will be performed for 
sex, race (white or non-white), and BMI. We will also perform sensitivity analyses excluding 
those non-compliant with study pills (missing ≥ 2/3 doses). 
 
Statistical power: Power analysis is based on an approximation to the proportional odds 
analysis controlling for baseline by subtracting (analysis of change) instead of regression. Data 
on the within-individual standard deviation (SD) of resting energy expenditure are limited, 
particularly from the metabolic chamber. Thus, as an approximation we used the between-
individual SD (364 kcal) from our prior studies of 108 obese (BMI 32 kg/m2, 56% female) 
individuals. We estimate the correlation between pre- and post-treatment measurements to be 
0.5 so that the SD of the within-subject change is also 364 kcal.  We model several scenarios, 
up to a dropout rate of 15%, a high estimate. Even with a dropout of 15%, we will have excellent 
power (84%) to detect a relatively modest 182 kcal difference between tadalafil and placebo 
groups, with a type I error of 0.025. We have set the p-value threshold at 0.05/2 = 0.025, to 
account for the 2 primary clinical endpoints in the proposal (corresponding to Specific Aims 1 


 
 
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and 2). 182 kcal represents ~10% of the total daily energy expenditure for an obese adult from 
our prior studies. 
 
Aim 2: 
Statistical analysis: The analytic approach will parallel that described for Aim 1 (substituting the 
endpoints from Aim 2). The primary analyses will focus on the effect of PDE5 inhibition on 
insulin sensitivity (SI). Secondary analyses include measures of insulin secretion (AIRg, and 
disposition index), body composition, and quality of life. 
 
Statistical Power: We selected the sample size based on the baseline insulin sensitivity (SI) 
measured by FS-IVGTT in a prior study of patients with insulin resistance and on the magnitude 
of effect of PDE5 inhibition on insulin sensitivity in our preliminary data. A higher SI indicates 
better insulin sensitivity. We plan to enroll 100 subjects per arm and allow up to a 15% dropout 
rate over 3 months. With 85 completed subjects per arm, the study will have 90% power with 
alpha 0.025 to detect a 23% increase in SI from a baseline of 5.89±2.75 x10-4•min-1 per mU/L 
(mean ± SD) versus no change in the placebo arm. We observed a 30% increase in Si in our 
pilot data using a lower equivalent dose of PDE5 inhibitor over a shorter study duration. 
Therefore, we will have excellent power to detect a modest effect of PDE5 inhibition. The 
baseline AIRg from our prior studies in a similar population was 577.3±259.8. With 85 
completed subjects per arm, we will have 90% power to detect a 25% increase in AIRg, the 
other component of disposition index. In prior studies we observed within-subject correlations of 
0.7-0.8 for repeated IVGTT measurements (SI, AIRg), but we based our power estimates on a 
more conservative within-subject correlation of 0.5 for both variables to ensure adequate 
statistical power. We and others have observed that the acute (AIRg)- and late-phase insulin 
secretion and disposition index are significantly increased in African American subjects 
independent of body mass index or insulin sensitivity.21,22 We will address this by stratifying for 
race (in addition to gender) at the time of randomization. 
 
Aim 3: 
Statistical analysis: The analytic approach will parallel that described for Aim 1 (substituting the 
endpoints from Aim 3). We will also assess the correlation of the change in cGMP/NP ratio with 
the change in the primary endpoints (energy expenditure, insulin sensitivity) in the active arm 
and in the overall sample. 
 
Although the metabolomics studies are exploratory, we will adjust the statistical significance 
threshold for metabolite comparisons using a conservative Bonferroni correction (p = 0.05/300). 
As a secondary analysis we will do variable clustering to empirically organize the metabolites 
into clusters such that within-cluster correlations are high and between-cluster correlations of 
individual metabolites are low. Each cluster will be summarized by its first principal component 
and these components will be tested for treatment effects using the Wilcoxon test. This reduces 
multiplicity problems and focuses power. 
 
Statistical power: Based on our pilot study, the within-individual SD of cGMP/NP over 12 weeks 
in the absence of intervention is 3.6 pmol/pg. With 85 completed subjects per arm (assuming 
15% dropout), we will have 90% power with alpha 0.05 to detect a 19% increase in cGMP/NP. 
We observed an increase of 85% in cGMP/NP in tadalafil-treated subjects in our smaller pilot 
study, with a smaller sample. Thus, we will have excellent power to detect modest effects. 
Analysis of metabolites and VEGF-A165b measurements are considered exploratory; therefore, 
power calculations were not specifically performed for those endpoints. 
 


 
 
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11.0 
Privacy/Confidentiality Issues 
 
Consent forms, medical history data, and study data are stored in secured files, either in locked 
file cabinets or in a locked room separate from medical records and coded such that all subject 
identifiers have been removed. As an additional precaution all HIPAA regulated information is 
stored in an electronic file separate from other study data. Only approved study staff 
(determined by Drs. Wang and Brittain) will be given authorization to access the database. Bio-
specimens are processed and labeled with barcode labels that include the subjects 
electronically generated study code and date of sample collection. The bio-specimens are 
stored in locked freezers in the study Laboratory; only approved study staff has access to the 
keys for each freezer. Access to the electronic freezer inventory of the specimens is kept on a 
secure password protected computer. 
 
 
12.0 
Follow-up and Record Retention 
 
The duration of this study is estimated to be 4 years.  The duration of record retention will be at 
least 6 years after study completion, but the possibility exists for indefinite archival of study 
information via the REDCap database.  Should the outcome of this study prompt future 
investigations, the participants may be contacted to obtain follow-up information and invited to 
participate in additional studies. 
 
13.0 
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