TRPV2 Agonists in Fontan Circulation Patients
Phase: PHASE4
Conditions: Fontan
Sponsor: Children's Hospital Medical Center, Cincinnati

 
 
 
PROBENECID IMPROVES CARDIAC FUNCTION IN SUBJECTS WITH A FONTAN 
CIRCULATION AND AUGMENTS CARDIOMYOCYTE CALCIUM HOMEOSTASIS 
 
 
 
NCT#: 03965351 
 
 
 
DATE: 1/25/21 

 
 
Protocol v8 15Nov2018 
 
 
 
 
 
 
 
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TRPV2 AGONISTS IN FONTAN CIRCULATION PATIENTS 
Study Site:  
Cincinnati Children’s Hospital Medical Center 
 
Principal Investigator:  
Ryan Moore, MD  
Sub-Investigators:  
Jack Rubinstein, MD 
 
 
Jessica Woo, PhD 
 
 Gruschen Veldtman, FRCP, MBChB 
Collaborators:  
Harry Rossiter, PhD 
 
Wayne Mays, R-CPT 
 
Ashley Nebbia, PharmD, BCPPS 
1. 
ABSTRACT 
Patients with single ventricle physiology (either a single morphologic left or right ventricle) have 
a form of very complex congenital heart disease that is fatal without intervention.  The 
development of the Fontan procedure to separate the systemic from the pulmonary circulation has 
resulted in improved survival well into adulthood; however, this procedure is palliative and results 
in a circulation that is ultimately fraught with multisystem complications related to high central 
venous pressure, low cardiac output, valvular and ventricular dysfunction (both systolic and 
diastolic). At least 30% have occult diastolic disease. 
 
There are currently very few proven pharmacologic options available for these patients. The recent 
discovery of transient receptor potential vanilloid (TRPV) channels, particularly TRPV2 channels, 
in the cardiovascular system is promising as a potential pathway for pharmacologic intervention 
for Fontan patients.  Probenecid, a drug best known as a treatment for gout or as a penicillin 
adjunct, acts as a TRPV2 agonist and has recently become the subject of study as a model therapy 
for the treatment of cardiomyopathy due to its positive inotropic and lusitropic effects.  The 
purpose of this pilot study is to determine if probenecid will improve magnetic resonance (MRI) 
parameters of systolic and/or diastolic dysfunction as well as associated symptoms in patients with 
a Fontan circulation.  We will quantitatively assess functional improvement with pre- and post-
treatment cardiopulmonary exercise testing. 
2. 
INTRODUCTION 
2.1. 
Background 
Thirty-five of every 100 000 live births are complicated by the presence of single ventricle 
physiology.  Some malformations consist of a single left ventricle (i.e. tricuspid atresia) while 
others result in a single right ventricle (i.e. hypoplastic left heart syndrome).  Although there are 
inherent differences in lifelong functionality based on the morphology of a single right versus a 
single left ventricle, the prognosis of single ventricle patients as a whole was transformed by the 
introduction of the Fontan operation.  This palliative procedure has certainly improved survival, 
but patients with a Fontan circulation experience a vast array of multisystem physiologic 
consequences related to high central venous pressure and valvular and ventricular dysfunction 

 
 
Protocol v8 15Nov2018 
 
 
 
 
 
 
 
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(both systolic and diastolic). Despite early palliation, the ventricle in Fontan circulations remains 
particularly vulnerable to failure.  Up to 40% of adult Fontan patients exhibit heart failure during 
late follow-up.  
 
Although management of heart failure in adults with structurally normal hearts has been well-
studied, the same does not apply to heart failure in congenital heart disease.  Many of the same 
drugs are used by congenital cardiologists, although somewhat empirically, since the body of 
evidence to support the long-term efficacy of such drugs is lacking.  Transient receptor potential 
vanilloid (TRPV) channels are Ca2+ channels that have been fairly recently discovered in the 
cardiovascular system, including in cardiac myocytes, where TRPV2 channels are more abundant 
than TRPV1 channels.  Probenecid, an FDA-approved drug that has been in use for other purposes 
(i.e. gout) for more than 40 years, activates TRPV2 channels in cardiomyocytes and increases 
intracellular Ca2+.  This results in improved myocardial contractility and relaxation.  Importantly, 
it does not induce apoptosis, malignant arrhythmias, or adverse changes in peripheral vascular 
resistance. These findings suggest that TRPV2 activation, via probenecid, could represent a novel 
and clinically relevant approach for treating cardiac dysfunction in adolescents and adults with 
Fontan palliation for single ventricle congenital heart defects. 
2.2. 
Preliminary Studies 
Probenecid has been shown to induce a TRPV2 dependent cytosolic influx of calcium resulting in 
increased cell contractility in isolated cardiomyocytes.  This effect has also been shown ex vivo in 
hearts and in vivo in mice.  There are ongoing studies in vivo in mice and pigs, and investigators 
have proposed trials in humans; however, to date, we are not aware of any published data using 
Probenecid for the treatment of heart failure in humans.  There are also no studies looking at the 
application of Probenecid as a heart failure management drug in patients with a Fontan.  These 
endeavors are a practical next step given that Probenecid is already an FDA-approved drug and 
has a long history of use for other medical purposes with a low side-effect profile.  Probenecid has 
also been safely used in children as an adjunct to treatment with penicillin and other antibiotics.  It 
has also been used as an adjunct in the treatment of malaria in children, with a defined dosing 
regimen, and no evidence of drug intolerance. 
3. 
PURPOSE OF THE STUDY 
The primary objective of this study is to determine if Fontan patients treated with probenecid for 
four weeks will experience increased systolic and diastolic function (as measured via standard and 
advanced MRI parameters) compared with four weeks of placebo.   
Secondary objectives of this study include the following:  
• Fontan patients treated with probenecid for four weeks will experience improved exercise 
performance compared with four weeks of placebo.  
• Determine if there is a difference in the effect probenecid has on patients with a single right 
versus a single left ventricle after a Fontan procedure. 

 
 
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4. 
STUDY DESIGN 
4.1. 
Study Description 
This will be a prospective randomized, placebo-controlled cross-over pilot study of up to 22 
patients who have had a prior Fontan operation for single ventricle physiology who are ≥ 10 years 
old.  Including teenagers younger than 18 years old is important for the following reasons: 1) The 
adult Fontan patient population is small, 2) Most adult Fontan patients > 18 years old have single 
left ventricle physiology; therefore, single right ventricle patients (i.e. with Hypoplastic Left Heart 
Syndrome (HLHS)) would be under-represented, as surgical palliation allowing survival beyond 
infancy was reported to be successful in 1981, with many initial problems with early connections 
that were created; and 3) Patients in the 10-18 year old age group could potentially also benefit 
from probenecid with little risk associated risk.  
4.2. 
Study Duration 
The entire study duration is estimated to be 3 years total to allow sufficient time to recruit patients 
and conduct the study (estimated 2 years), and analyze the data (estimated 1 year). From the time 
of study consent to the time of study completion, we expect study participation to be 12 weeks for 
each participant. 
5. 
SELECTION AND RECRUITMENT OF PARTICIPANTS 
We will select up to 22 patients meeting the criteria below.  Potentially eligible patients will be 
identified from existing patients of the Fontan Management Clinic or the Adult Congenital Heart 
Disease clinic, and will be evaluated for eligibility criteria prior to enrollment; eligibility screen 
failures will be noted in the enrollment log. Patients will be recruited at the time of a clinic visit, 
hospital stay or over the phone following a recruitment letter.  Letters will be sent 2 weeks prior 
to calling potential participants so they are informed about the study and can opt-out if desired. 
 
Investigators may reach out to colleagues at nearby institutions to present an overview of the study. 
IRB approved promotional material will be provided to these cardiologists to disperse to their 
potentially eligible/interested Fontan patients. The cardiologists from outside our institution will 
refer these potential subjects to our study team. Study staff will engage the potential subjects and 
begin the screening/recruitment process. 
5.1. 
Inclusion Criteria 
Subjects who meet all of the following criteria will be eligible for the study: 
1. ≥ 10 years old  
2. Single ventricle congenital heart disease status post Fontan procedure. 
3. Impaired ventricular function as assessed by preexisting echocardiographic studies and any 
available MRI studies. 
a. LV inclusion criteria: Ejection fraction by cMRI or echo assessment of <50% or 
moderate to severe dysfunction. 

 
 
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b.   RV inclusion criteria: Ejection fraction by cMRI of <45% or moderate to severe 
dysfunction. Or, given the ASE recommendation to avoid use of 2-D imaging 
quantification for assessment of right ventricular systolic function, a peak global 
longitudinal strain value as assessed by a single reviewer with a value greater than -
17% will also be included. Peak global longitudinal strain analysis will be performed 
for all eligible single right ventricles noted by subjective echo reports to have 
abnormal systolic function if no qualifying cardiac MRI assessment of ejection 
fraction is available. 
5.2. 
Exclusion Criteria 
Subjects who meet any of the following criteria will be excluded from the study: 
1. Clinically unstable or ongoing illness. 
2. Evidence of untreated Fontan pathway obstruction. 
3. Presence of uncontrolled arrhythmias. 
4. Evidence of moderate or greater atrioventricular valve regurgitation. 
5. Pregnancy. 
6. History of sulfonamide allergy 
7. Known G6PD deficiency 
8. Patients on certain drugs that have potentially dangerous interactions with probenecid other 
than for prophylactic reasons: doripenem, zalcitabine, deferiperone, citalopram, 
methotrexate, ciprofloxacin, amoxicillin, cefprozil, cefpodoxime, cefotaxime, meropenem, 
ertapenem, valganciclovir, ganciclovir, ziovudine ketorolac, cefdinir, cephalexin, dapsone, 
indomethacin, and piperacillin.Each subject’s medication list will be reviewed prior to 
study participation.  
9. Impaired renal function as defined by a GFR < 60mL/min/1.73 m2 within the last year.  
10. Patients at a higher risk for arrhythmia including those with a prior history of arrhythmia 
including atrial and ventricular dysrhythmia or those on established anti-arrhythmic 
therapy. 
11. Admission to the hospital due to a clinically significant arrhythmia within the previous 
month.  
12. Greater than moderate atrioventricular regurgitation as denoted on most recent echo report.  
13. Patients with atrio-pulmonary Fontan 
14. Currently enrolled in an interventional drug trial or completed an interventional drug trial 
within the past 30 days.  
15. Not appropriate for MRI screening due to having an implanted device.  

 
 
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6. 
STUDY PROCEDURES 
Once patients are enrolled, they will be randomized to receive either probenecid or placebo as the 
first treatment.. Randomization procedures are described below under “Statistical Analysis”. 
 
All patients will have a baseline assessment immediately preceding initiation of probenecid or 
placebo. This will include a physical exam to assess any clinical symptoms, review of cardiac 
symptoms, blood work, pregnancy test for those girls/women of child bearing potential, PEDS QL 
questionnaire completion, exercise testing and MRI, as described in detail below.  Patients will 
then receive either probenecid or placebo for 28(±7 days) of treatment according to their 
randomization group, after which the same testing will be repeated. Following 28(±7 days) of 
washout, patients will undergo the same testing procedures as during the first period of study but 
with the alternate treatment. To assist in achieving congruence between clinical testing performed 
and desired pre-intervention testing, we will coordinate with each patient and his/her cardiologist 
prior to the clinical visit.  
 
Patients will receive a one month supply of oral probenecid therapy with the following dosing 
regimen: 
• For patients (adults and pediatric) ≥ 40 kg: 500 mg by mouth twice daily. 
• For children 10-17 years old and < 40 kg: 250 mg by mouth twice daily 
 
Note: This dosing regimen is based upon available literature of those used in both adults and 
children and with the guidance of a clinical pharmacist.  A dose of 20-25 mg/kg/day divided in 2 
daily doses has been well-tolerated in children for 3 days in 1 study (Sowunmi, A. et al. May 2004. 
Tropical Medicine and International Health; Craft JC, et al. Antimicrobial Agents and 
Chemotherapy. 1979). The standard maximum adult dose is 2000 mg/day. Probenecid is available 
as a 500 mg tablet, and there is no commercially available compound. 
 
During the placebo treatment arm, patients will receive 28(±7 days) of placebo therapy. Oral 
placebo therapy will be twice daily, with an identical dosing regimen as the placebo. 
 
The investigators will review all clinical data to assess for adverse experiences during the period 
from enrollment through the end of study. The investigators will evaluate any changes in clinical 
symptomatology, and determine if the change is clinically important and different from expected 
in the course of treatment of Fontan patients. This will be assessed by a clinical research 
coordinator with a direct telephone call to the study participant at 4 time points during the 28(±7 
days) of therapy with the first call taking place within 2 days of commencement of each treatment 
(probenecid or placebo) and weekly thereafter. Patients who manifest new symptoms will be asked 
to return to the CCHMC Heart Institute for further assessment. All subjects will also be clinically 
evaluated before and after each arm of study by a clinician. 
 
 
6.1 Quality of Life Assessment 
All subjects will complete the Pediatric Quality of Life Assessment 4.0 Generic Core Module 
(PedsQLCore) at each visit. The PedsQL Core questionnaire is a modular instrument for 

 
 
Protocol v8 15Nov2018 
 
 
 
 
 
 
 
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measuring health-related quality of life in adolescents through adults ages 10 and up.  
Participants will complete the designated Peds QL Core for their respective age group. 
 
 
 
 
 
 
 
 
Schedule of Procedures 
 
Treatment Period A 
Wash 
Out 
Treatment Period B 
Procedures 
Before 
treatment 
28 
(±7days) 
Immediately 
After 
Treatment 
28 
(±7days) 
Before 
Treatment 
28 
(±7days) 
Immediately 
After 
treatment 
Informed consent 
X 
 
 
 
 
 
 
Demographics, medical 
history, concomitant 
medication 
X 
 
 
 
X 
 
 
Physical Exam 
X 
 
X 
 
X 
 
X 
Review of symptoms 
X 
 
X 
 
X 
 
X 
Blood sample - NT-
proBNP, CBC, CMP, 
LDH and, GGT 
X 
 
X 
 
X 
 
X 
Pregnancy Test (Urine) 
X 
 
X 
 
X 
 
X 
Cardiopulmonary 
Exercise Tests (CPET) 
X 
 
X 
 
X 
 
X 
MRI 
X 
 
X 
 
X 
 
X 
Drug 
dispensing/administration 
X 
 
 
 
X 
 
 
Adverse events 
X 
X 
X 
X 
X 
X 
X 
Drug adherence check 
 
 
X 
 
 
 
X 
PEDS QL 
X 
 
X 
 
X 
 
X 
 
 
6.2 Process of Obtaining Informed Consent 
The Fontan database at CCHMC (IRB 2016-2425) will be used to screen for this study. Patients 
meeting eligibility criteria will be approached for participation during a regularly scheduled clinic 
visit at CCHMC. Alternatively, a letter of introduction approved by the IRB will be mailed to 
potential participants along with a consent document. In the letter, patients will be given the 
opportunity to opt-out of the study by calling the study line. After ten days, study personnel will 
call patients to further explain the study. If there is interest in the study, study personnel will 
schedule a research visit for the patient.  

 
 
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When the subject arrives to the visit, study personnel (research coordinator or MD) will again 
provide a verbal explanation about the study and discuss the risks and benefits that may come with 
participation.  Adequate time will be given for the participant or legal guardian to read the informed 
consent and all questions will be answered. If the participant and, if applicable, a legally authorized 
representative, agree to participate in the study, informed consent will be obtained.  Assent will be 
obtained from all participants between the ages of 10-17, inclusive. 
 
We will stress that non-participation in the study will not affect the subject’s ability to obtain care 
at Cincinnati Children’s Hospital.  
 
No research procedures will be done prior to obtaining assent/consent. 
6.3 Laboratory Procedures 
Each participant will have blood drawn for assessing NT-proBNP, CBC, CMP, GGT, and LDH 
both before and immediately after each 4 week drug-treatment period. The blood will be 
processed through the CCHMC clinical laboratory. 
     6.4 Imaging Procedures: MRI 
Image acquisition: Cardiac magnetic resonance imaging will be performed on all eligible study 
patients at baseline and repeated at the intervals specified above. Studies will be performed on 
non-sedated patients utilizing a Philips Ingenial 1.5T clinical magnet. Patients will have weight 
and height measured to calculate body surface area. Studies will be terminated in case of patient 
instability or development of claustrophobia. A resuscitation cart is available. Patients are 
monitored by ECG and respiratory gating within the presence of a physician and an MRI 
technician.  
 
CMR Scanning: Studies will be ECG gated with leads placed prior to scanning. The multichannel 
body array and spine coils will be used. The patient will be placed head first into the MRI scanner. 
Parallel imaging will be used on all scans. The breath holding approach will be used for sections 
3 and greater; if the patient is unable to cooperate, multiple signal averages will be used instead.  
A standard protocol for image acquisition will include localizer images for planning purposes 
followed by single sequence free-precession (SSFP) cine imaging performed in the vertical long 
axis as well as horizontal long axis and short axis stacks at a slice thickness of 6-8 mm with 
sufficient coverage to quantify atrial and ventricular volumes in both systole and diastole. Phase-
encoded velocity mapping will be performed at the level of the AV valve leaflet tips at end-diastole 
as well as at the ventricular outlet for verification of volumetric analysis. Velocity encoding will 
be optimized to avoid flow aliasing while maximizing flow velocity fidelity. Additional phase 
contrast imaging through the branch pulmonary arteries and/or pulmonary veins will be performed 
to quantify relative pulmonary and systemic blood flow.  During the initial scan, a SSFP 
respiratory-navigated 3D whole-heart sequence will also be performed during late diastole to 
characterize any additional extracardiac abnormalities which might influence image interpretation.  
 
The CMR techniques and scans to be used in this study are as follows: 

 
 
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1. Static steady state free precession (SSFP) imaging: Contiguous axial images forming a full 
volume data set of the thorax will be obtained. These images will be used to assess general 
cardiovascular anatomy to ensure that no unexpected lesions are present. 
2. Multi-planar reformatting: The axial images in #1 will be reformatted to calculate the exact 
slice position and orientation to obtain the 4-chamber and short axis ventricular views for 
cine images and perpendicular to flow in the main (MPA) and branch pulmonary arteries 
(BPA) and across the aortic valve for PC-MRI. 
3. Cine imaging: Off-axis, retrospectively gated cine images are obtained in the 4-chamber 
view and as a stack of ventricular short axis images from base to apex for ventricular 
performance parameters (20-30 phases, depending upon the heart rate). 
4. Phase-encoded velocity mapping (VMAP): “Through-plane” retrospective, phase-encoded 
velocity maps will be obtained at the aortic annulus and in the MPA and branch pulmonary 
arteries. Initially, a velocity encoding (VENC) of 150 cm/s will be utilized. After each 
acquisition, images will be checked and if velocities exceeded the VENC, the images will 
be repeated with an increased VENC. 
5. Native T1: The modified Look-Locker inversion recovery (MOLLI) sequence will be used 
to obtain a non-contrast native T1 value at 1 short axis level (mid ventricle).  
Imaging Data Analysis:   All study images will be reviewed and interpreted by a qualified 
investigator with expertise in congenital cardiac MRI.   Institutional post-processing software 
(CMR42, Circle Cardiovascular Imaging, Calgary, Canada) will be utilized for off-line image 
analysis.  Contouring of the single ventricle at end-systole and end-diastole will be performed to 
quantify ventricular volumes and calculation of ejection fraction as a measure of global systolic 
function.   Phase contrast quantification of flow including blood volume and diastolic parameters 
such as deceleration time, E and A wave velocities, and pulmonary vein flow patterns will be 
assessed. Flow volumes will be utilized to verify accuracy of volumetric analysis and to generate 
flow patterns for temporal analysis to quantify diastolic function as previously described33,34. 
Tissue velocities obtained by phase contrast imaging will also be analyzed at the level of the AV 
valve annulus for measurement of E’ velocity and additional diastolic parameters.   Additional 
off-line processing of horizontal long-axis tissue strain and strain rate will be performed via 
feature-tracking software (CMR42, Circle Cardiovascular Imaging, Calgary, Canada)) for further 
characterization of myocardial mechanics. 
Images will be analyzed for global ventricular function, blood flow, ventricular strain and diffuse 
fibrosis (native T1 value).  
 
Global ventricular function:  From the cine images of the ventricular short axis, the endocardial 
and epicardial borders will be traced (semi-automatically) and ventricular performance parameters 
computed using standard analysis packages (e.g. CMR42, Circle Cardiovascular Imaging, 
Calgary, Canada). Ventricular volumes will be determined by multiplying the slice thickness with 
the segmented area. The volumes of each slice will be integrated along the ventricular longitudinal 
axis using Simpson’s rule to extract the total volume of the ventricle in a cardiac phase. This will 
be applied at end diastole (for end-diastolic volume - EDV) and at end-systole (for end systolic 
volume - ESV). Stroke Volume=EDV–ESV. Ejection Fraction=Stroke Volume/EDV. Cardiac 
output=stroke volume multiplied by heart rate. Ventricular mass=1.05 * (epicardial volume minus 
the endocardial volume). 

 
 
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PC-MRI:  Integration of the velocities of each voxel of the vessel cross-section represents the 
flow at a given phase of the cardiac cycle. Further integration of all PC-MRI data over the cardiac 
cycle represents flow in one heartbeat and multiplying by heart rate yields cardiac output. 
Regurgitant fraction (%) = ([reverse flow]/[forward flow])X100. 
 
Ventricular Strain:  From the cine images of the ventricular short axis and long axis, 
endocardial and epicardial borders will be traced (semi-automatically)_ and circumferential, 
radial, and longitudinal strain measures will be obtained. In addition, strain rate, rotational 
dispersion, time to maximum strain rate will also be recorded. 
 
Native T1:  Standard analysis packages (e.g. CMR42, Circle Cardiovascular Imaging, Calgary, 
Canada) will be utilized to analyze both regional and global diffuse fibrosis as a signal intensity 
map at the mid-ventricular level from the native MOLLI sequence. The raw signal intensity on 
these specialized sequences are the T1 relaxation times of the myocardium. 
 
6.5. Other Non-invasive procedures 
6.5.1       Cardiopulmonary Exercise Tests (CPET) 
Each participant will undergo both a pre- and post-intervention cardiopulmonary exercise test 
at CCHMC.    Participants will perform the clinical maximal ramp-incremental exercise test.  
The CCHMC Exercise cart will be used for cardiopulmonary exercise testing in all cases.   
 
 
6.5.2 Pregnancy Testing: 
Females of childbearing potential include any female who has experienced menarche.  Urine 
pregnancy test (for all females of child-bearing potential) will be conducted at all visits at 
CCHMC.  A positive pregnancy test will exclude the subject from further participation in the 
study.  Pregnant subjects who are discontinued from the study will be transitioned into an 
alternate therapy at the discretion of the Investigator. 
 
6.5.3. Clinical Data 
The following data will be collected via either chart review with participant verification, or direct 
patient questioning. 
• Age 
• Gender 
• Anthropometric data 
• Cardiac diagnoses; type of congenital heart disease 
• Previous cardiac surgeries; date and type 
• Previous cardiac catheterizations; date and type 
• Other major medical problems 
• Current medications 
• Clinical status (New York Heart Association (NYHA) 

 
 
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7. 
  
INVESTIGATIONAL AGENT 
7.1 Description 
Probenecid is a uricosuric and renal tubular transport blocking agent that inhibits the tubular 
reabsorption of urate and increasing the urinary excretion of uric acid.  It also inhibits the tubular 
secretion of penicillin and increases plasma penicillin levels.  Probenecid is FDA approved in 
adults and children >2 years of age as adjuvant to therapy with penicillin, ampicillin, methicillin, 
oxacillin, cloxacillin, or nafcillin.  Additionally, it is FDA approved for the treatment of 
hyperuricemia associated with gout and gouty arthritis in adults.  Studies have shown that 
probenecid also activates the TRPV2 channel.  Activation of the TRPV2 channel results in 
increased intracellular calcium levels and increased cell contractility in isolated cardiomyocytes. 
Probenecid reaches peak serum concentrations in 1-5 hours after oral administration in adults and 
in 3-9 hours in children. Probenecid is highly protein bound and is extensively metabolized in the 
liver (oxidation and glucuronide conjunction) to active and inactive metabolites. Approximately 
75-88% is excreted in the kidney and the remainder is excreted unchanged in the urine.  The half-
life of probenecid is 3 to 17 hours. 
Probenecid is contraindicated in patients with a history of allergic reaction to probenecid, children 
<2 years of age, it is not recommended in patients with known blood dyscrasias or uric acid kidney 
stone, and in patients with an acute gout attack. 
There are two main types of drug interactions with probenecid.  The first type of interaction 
involves probenecid and decreased clearance of acidic medications and resulting plasma 
concentrations of the following medications: doripenem, zalcitabine, deferiperone, citalopram, 
methotrexate, ciprofloxacin, amoxicillin, cefprozil, cefpodoxime, cefotaxime, meropenem, 
ertapenem, valganciclovir, ganciclovir, ziovudine and ketorolac.  We will exclude patients on these 
drugs from the study unless they are on these drugs short-term for prophylaxis . 
The second type of interaction involves the use of probenecid and the reversal of the uricosuic 
effects of the following medications: magnesium salicylate, sodium thiosulfate, aspirin, choline 
magnesium trisalicylate and bismuth subsalicylate.  As probenecid is not being utilized for its 
properties of urinary excretion of uric acid, it is unlikely that these drug interactions of this type 
will be of clinical significance in the patient population being studied.  Probenecid’s inhibition of 
secretion of acidic medications and clinical impact on its potential use as an inotropic agent is 
unknown.   
Adverse effects reported with probenecid include alopecia, puritis, urticaria, and Stevens Johnson-
syndrome.   Gastrointestinal effects including nausea, vomiting, and anorexia have been reported. 
Isolated cases of aplastic anemia, leukopenia, neutropenia, thrombocytopenia and hepatic necrosis 
have been reported.  Probenecid is categorized as a sulfonamide without an aromatic amine and 
therefore the incidence of cross-allergenicity between classes of sulfonamides is low; however, 
anaphylaxis and hypersensitivity reaction have been reported.  Precipitation of gout attacks and 
uric acid stones with or without hematuria have been reported. Nephrotic syndrome is rare. 
Headache, dizziness and fever have been reported.   
 
7.1.1 How Supplied 

 
 
Protocol v8 15Nov2018 
 
 
 
 
 
 
 
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Probenecid is available as a 500 mg tablet.  It will be supplied by Cincinnati Children’s Hospital 
Medical Investigational Pharmacy. Each participant will be given a one month supply in a bottle 
with an attached MEMS® cap. The MEMS® cap is an adherence monitoring device that will be 
used to determine participant compliance with the medication. Each time the cap is removed 
from the bottle, it will record the date and time. If the cap is removed from the bottle more than 
once within fifteen minutes, the cap will only record the first dispensing action. Along with the 
utilization of the MEMS® caps, a tablet count will be completed by a designated study staff 
member prior to dispensing the one month supply as well as after the 28 (±7days) period to 
ensure consistency in the data obtained. Participants will also be provided with a pocket diary, in 
the event that they go on vacation and are unable to take their bottle with them. In this case, they 
will be instructed to record how many tablets they removed from the bottle at once so as to have 
a record of why the cap data is discrepant.  
 
7.1.2 Storage 
Probenecid is stored at room temperature, 20° to 25°C (68° to 77°F) and should be protected 
from light. 
7.1.3 Proposed Dose 
• For patients (adults and pediatric)≥ 40 kg: 500 mg by mouth twice daily 
• For children 10-17 years old and < 40 kg: 250 mg by mouth twice daily 
 
8. DATA COLLECTION AND MANAGEMENT 
All data will be recorded on study-specific case report forms (CRFs). Participants will be assigned 
a study identification number that will be reported on all CRFs and source documents to protect 
patients’ confidentiality.  
Data will be collected by designated research coordinator(s) and the physicians participating in the 
study, with paper forms stored securely in locked file cabinets. A study database will be designed 
in the Research Electronic Data Capture (REDCap) system, a secure, HIPAA-compatible online 
platform housed on secure research servers behind the CCHMC institutional firewall, and available 
free to all UC and CCHMC investigators. REDCap includes user-specific access rights to prevent 
unauthorized access beyond study personnel. 
 
All data will be recorded on study specific case report forms (CRFs).  Participants will be given a 
study identification number that should be reported on all CRFs and source documents.  The key 
which links this study number to participant’s direct identifying information will be stored in a 
separate, secure location with access to only authorized individuals. 
The following information will be collected for this study:  
Demographics: 
a. Date of birth 

 
 
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b. Gender 
c. Race / Ethnicity 
 
Clinical Data/Assessments: 
a. Height / Weight, Heart rate, Blood pressure, O2 Saturation 
b. Cardiac diagnosis; type of congenital heart disease 
c. Major medical history 
d. Previous surgeries 
e. Concomitant Medications 
f. NYHA classification 
 
a. Physical Exam/Review of Systems Pertinent to Congenital Heart Disease and 
Fontan PopulationPresence of cough 
b. Presence of wheeze 
c. Chest pain presence 
d. Shortness of breath presence 
e. Heart palpitations, heart murmur, S3/S4 gallop presence 
f. Swelling presence in legs 
g. Varicose vein presence 
h. Current illnesses 
i. Dizziness or syncope presence 
j. Ascites presence 
k. Presence of jugular vein distention 
l. Palpability liver and spleen 
m. Presence of weight loss, change in appetite, loose stools, blood in stool 
n. Recent stroke symptoms 
 
Laboratory Assessment: 
a. NT-proBNP 
b. CBC  
c. CMP 

 
 
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d. LDH 
e. GGT  
Cardiopulmonary Exercise Tests (CPET): 
a. Spirometry 
b. Blood pressure 
c. Resting EKG  
d. Power output 
e. Gas exchange 
f. Ventilation 
g. Pulse oximetry 
 
MRI: 
a. Ventricular volumes 
b. Ejection fraction 
c. Ventricular mass 
a. Cardiac output 
b. Cardiac index 
c. SVC flow 
d. IVC flow 
e. Aortic flow 
f. Mitral inflow 
d. Strain 
 
a. Global circumferential  
 
b. Global longitudinal 
e. T1 Mapping 
 
Patient Disposition 
 
Data will be collected by designated research coordinator(s) and the physicians participating in the 
study, with paper forms stored securely in locked file cabinets. Data will be managed by an 
experienced team of clinical data specialists and data coordinators using Data Management Center 
(DMC) standard operating procedures and guidelines.    Appropriate data cleaning methods will 

 
 
Protocol v8 15Nov2018 
 
 
 
 
 
 
 
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be applied and final analysis dataset(s) with any necessary calculations/derivations will be 
prepared by a SAS programmer before sending the data to the project statistician.   
A study database will be designed in the Research Electronic Data Capture (REDCap) system, a 
secure, HIPAA-compatible online platform housed on secure research servers behind the CCHMC 
institutional firewall, and available free to all UC and CCHMC investigators. REDCap includes 
user-specific access rights to prevent unauthorized access beyond study personnel. 
We will take the following precautionary measures to protect the privacy and confidentiality of 
the participants’ research and/or medical record: 
1. Computerized study data will be stored on a secure network that prohibits access by 
unauthorized users. 
2. A password will be required to access the computerized study data. 
3. Only approved individuals associated with the research project will have access to the study 
records. 
 
9. STATISTICAL ANALYSIS 
9.1 Study Endpoints 
The primary endpoints are change in systolic and diastolic function on probenecid vs. placebo 
(measured via standard and advanced MRI parameters).  
The secondary endpoint will be exercise performance on probenecid vs. placebo.  
9.2  Statistical Analyses 
Randomization and blinding: The randomization plan will be conducted according to Standard 
Operating Procedure (SOP) ST-43.01.01. Briefly, the study statistician will prepare a 
randomization plan to stratify randomization and sequence of treatments, based on the study 
protocol, and will test the randomization protocol to ensure study parameters are met. An 
independent (non-study) statistician will produce the final randomization list and will provide it to 
the investigational pharmacy who will be responsible for implementing the randomization and 
maintaining this randomization list until the study is unblinded. 
All primary analyses will be conducted as intention to treat (ITT), with all outcomes tested between 
probenecid and placebo for all randomized patients. Safety analyses will, however, include a 
modified ITT, including any participant taking at least one study dose. The main outcomes will be 
pre- to post-treatment changes in EF and E/E’ ratio; pre- to post-treatment changes in exercise 
performance between probenecid and placebo groups will also be evaluated. Transformations of 
measurements (e.g., log transformation or nonparametric rank transformation) will be considered 
as necessary to meet the assumptions of the models. The analysis will consider the sequence of 
treatments, the patient effect (within sequence), the period of study (first or second arm), and the 
treatment effect (probenecid or placebo) as potential factors impacting the pre-to-post change in 
the outcomes of interest. Mixed modeling will be used to test these effects, with consideration of 
repeated measures within patient represented by the cross-over design. 

 
 
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An exploratory analysis will test for differences in drug response between left and right single 
ventricle patients, recognizing this analysis will not be well-powered. Because the two physiologic 
groups will not be randomized relative to one another, demographic and clinical covariates that 
may differ between these strata will also be considered in mixed modeling. An interaction term 
will be introduced into the model and tested to evaluate whether treatment effects statistically 
differ by physiologic group (e.g., treatment*group). For all analyses, two-sided p<0.05 will be 
considered significant.  
9.3  Sample Size Calculation 
Power estimates for this study were based on preliminary data for EF, from the randomized cross-
over designed study of adult patients with HFrEF. In the full analysis sample of 15 patients, the 
difference in the EF response between probenecid and placebo treatment in the 1-week cross-over 
design was normally distributed with mean difference of 7.7% and standard deviation (SD) of 7.6. 
Assuming power of 80%, α=0.05, and a 2-sided test, a sample size of 17 patients would be required 
to complete both arms of the study to detect this magnitude of effect. This sample size would also 
provide 73% power to detect the observed differences in E/E’ ratio noted in the pilot study after 1 
week of treatment. 
To conservatively account for potential differences in drug effect between adults and children and 
other stochastic variability, as well as the potential for participant dropout during the crossover 
design, this study will enroll up to 22 patients, which would allow for up to 30% dropout. 
10. 
RISKS AND BENEFITS 
10.1 Potential Benefits 
Patients who participate in this study may experience improvement in their ventricular function, 
exercise performance and tolerance, and symptoms of heart failure (if present) as a result of 
treatment with probenecid.  If this occurs, they will be considered for possible continuation of 
therapy.   
10.2  Potential Risks 
There is minimal risk associated with this study.  Risks are only related to cardiopulmonary 
exercise testing and to the potential side effects of probenecid therapy.   
Probenecid has a long and reassuring safety record; however the following are potential adverse 
reactions: 
Serious reactions: Hemolytic anemia, aplastic anemia, leukopenia, thrombocytopenia, 
hepatic necrosis, anaphylaxis, nephrotic syndrome 
Common but mild/reversible reactions: Headache, dizziness, anorexia, nausea, vomiting, 
gingival soreness, urinary frequency, renal colic, anemia, dermatitis, pruritus, flushing, 
fever, gout exacerbation. 
Care will be taken to avoid potential drug interactions by reviewing each subject’s existing 
medication list prior to enrolling in the study.  Probenecid has numerous known and well 
documented drug interactions, most of which will not be relevant to our study population.  Patients 

 
 
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receiving any of the following medications will be excluded from the study: doripenem, 
zalcitabine, deferiperone, citalopram, methotrexate, ciprofloxacin, amoxicillin, cefprozil, 
cefpodoxime, cefotaxime, meropenem, ertapenem, valganciclovir, ganciclovir, ziovudine 
ketorolac, cefdinir, cephalexin, dapsone, indomethacin, and piperacillin.  
Exercise testing also carries some risks, including mechanical trauma (i.e. from a fall), 
arrhythmias, syncope, and cardiac arrest. To minimize these risks, a trained exercise physiologist 
will perform this procedure.  If the patient is considered moderate to high risk, a physician will 
also be present  In addition, heart rhythm, vital signs, and perceived exertion will be monitored 
continuously throughout the test.  Commonly reported symptoms include fatigue, shortness of 
breath, and muscle soreness; however, these symptoms do not typically result in any real risk to 
the patient.  With the cardiopulmonary testing, patients frequently complain of discomfort or 
claustrophobia wearing the face mask, but again, this does not carry a true clinical risk. Chest pain 
is occasionally a complaint, but the risk of myocardial infarction is extremely rare in our patient 
population.  The test will be discontinued if there is any concern for a pathologic arrhythmia, 
coronary ischemia, other evidence that the patient cannot safely proceed with the test, or if the 
patient expresses discomfort with proceeding due to symptoms or overwhelming anxiety. 
Pregnancy: 
Female participants 
Pregnancy is an exclusion criteria for the study.  Participants will have pregnancy testing at all 
study visits to CCHMC prior to any study procedures.  Participants will also be instructed to use 
birth control for the duration of the study.  Acceptable methods of birth control will be discussed 
with the patient by the PI or designee. 
 
Loss of Confidentiality: There is a minimal risk for loss of confidentiality associated with this 
study. Precautions will be taken to minimize the possibility of disclosure of personal 
information. This risk will be minimized by utilizing traditional precautions for the storage of 
paper records (lock and key) and electronic records (password protection and use of secure 
servers).  These precautions are outlined in the Privacy and Confidentiality section of this 
protocol. Patient identifiers will not be used in reports or publications from this study. 
10.3  Risk/Benefit Analysis   
The proposed study has minimal risks associated with it, which are reasonable in relation to the 
knowledge that will be gained and used to create interventional programs in this high-risk 
population. This study is being listed as “more than minimal risk” due to the very rare possibility 
of serious adverse reactions to probenecid. 
 
Minimal risk but with direct benefit to participants 
 
Minimal risk but without direct benefit to participants 
 
More than minimal risk but with direct benefit to participants 
 
More than minimal risk but without direct benefit to participant 

 
 
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11 
ASSESSMENT OF SAFETY 
11.1 Adverse Events 
An adverse effect (AE) is any untoward medical occurrence associated with the use of a drug in 
humans, whether or not considered drug related. An AE can be any unfavorable and unintended 
sign (e.g., an abnormal laboratory finding), symptom, or disease temporally associated with the 
use of a drug, without any judgment about causality. An AE can arise from any use of the drug 
(e.g., off-label use, use in combination with another drug) and from any route of administration, 
formulation, or dose, including an overdose. Adverse events will be recorded from the time of the 
first dose of study drug until resolution (or study completion).  
Events that are unexpected and considered to be related or possibly related to the study procedures 
as well as breaches of confidentiality, and protocol violations must be reported to the IRB as soon 
as possible after discovery of the event.  All adverse events will be classified by the PI for 
relationship (Related, Possibly Related, Unlikely Related, Not Related); severity (Mild, Moderate, 
or Severe); and expectedness (Expected or Unexpected).  Events not meeting the criteria for 
prompt reporting will be reported to the IRB at the time of continuing review. 
 
11.2 Serious Adverse Events 
A Serious Adverse Event (SAE) is any undesirable occurrence associated with the use of a drug 
in a patient occurring at any dose, whether or not considered drug related, when the outcome is:  
• Death,  
• Life-threatening (i.e., participant was at substantial risk of dying at the time of the AE), 
• Inpatient hospitalization or prolongation of existing hospitalization,  
• A persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions or resulted in a significant, persistent or permanent change, 
impairment, damage or disruption in the patient’s body function/structure, physical 
activities and/or quality of life,  
• Congenital anomaly/birth defect in the offspring of a subject who received study 
medication, or 
• Other serious (important) medical events that may jeopardize the participant and may 
require medical or surgical intervention to prevent one of the other outcomes are also 
considered to be SAEs. 
11.2.1 Relationship 
All adverse events will be classified by the PI for relationship to the study drug as: Related, 
Possibly Related, Unlikely Related, Not Related. 
11.2.2 Severity 
All adverse events will be classified for severity by the PI as: Mild, Moderate, or Severe. 
11.2.3 Expectedness 
All adverse events will be classified for expectedness by the PI as: Expected or Unexpected.  
Very rare but potentially expected events associated with the use of probenecid include: 

 
 
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• Nausea 
• Vomiting 
• Dizziness 
• Headache 
• Anorexia 
• Gingival soreness 
• Urinary frequency 
• Renal colic 
• Anemia 
• Dermatitis 
• Pruritus 
• Flushing 
• Fever  
11.3 Adverse Event Reporting 
Expedited Reporting 
SAEs require expedited reporting when meeting the following criteria: 
• Serious 
• Unexpected 
• At least possibly related to the study agent or other protocol specific activity 
SAEs meeting the above criteria are required to be reported to the FDA as follows: 
• If characterized as fatal or life-threatening, within 7 calendar days of the sponsor’s initial 
receipt of the information  
• If non-fatal or non-life threatening, within 15 calendar days.  
 
Events that are considered unexpected and possibly related to the conduct of the study will be 
reported to the IRB within seven days of discovery.  
The DSMB will review SAEs within 48 hours after initial receipt of the information by the 
investigator(s) to review the PIs assignment of SAE as related or unrelated to treatment; to confirm 
the grading of toxicity, and assure that the study may continue. 
Significant unplanned deviations from the protocol will also be reported as stipulated above.  All 
other serious adverse events and non-serious adverse events will be reported at the time of 
submission of annual reports.  Likewise, minor deviations will be reported in annual reporting. 
12 DATA SAFETY AND MONITORING PLAN  
Data will be collected electronically or on data collection forms that will not be labeled with the 
patient’s name, only the study number. The list of matching codes to the patient’s name will be 
handled and retained by the study coordinator and investigator. To ensure proper use and continued 
protection of these data, the data collection sheets will not be given to any individuals except those 
co-investigators and study coordinators performing data-entry.  

 
 
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Electronic participant binders will be maintained with names of the subject, pertinent clinical data, 
and files with signed, scanned and certified consent forms and copies of pertinent clinical and 
research related forms. All participant binders and study records will be stored on a password 
protected network accessible to only research personnel. A separate enrollment database linking 
subject identification number to identifying information (first name, last name, medical record 
number, and date of birth) will also be maintained on a password-protected computer network. 
Access to this information will be restricted to the PI, research staff and the IRB.  
13 DATA SAFETY AND MONITORING BOARD (DSMB)  
A Data Safety and Monitoring Board (DSMB) will be assembled to monitor for adverse outcomes.   
It will be comprised of 2 certified cardiologists with expertise in clinical trials and one 
biostatistician and will periodically review and evaluate the accumulated data for enrollment, 
eligibility criteria, and safety data, and will make recommendations for any alterations to the 
protocol or any safety concerns.  The DSMB will meet after 2, 12, and 22 patients have completed 
the study protocol, or at least annually, and will also meet if a serious adverse event occurs.  
 
Recruitment into the study will be temporarily halted under the following conditions: 
1) If two subjects have the same Grade 3 or above AE that is thought to be related to study 
participation 
2) Three or more subjects have a Grade 3 or 4 Adverse Event  
3) One SAE occurs that is thought to be related to study participation 
 
The DSMB charter provides additional details regarding the DSMB. 
14 PRIVACY & CONFIDENTIALITY 
The privacy and confidentiality of patient information will be maintained in accordance with 
Health Insurance Portability and Accountability Act (HIPAA) regulations. All research personnel 
who work on this study must complete HIPPA and the Collaborative Institutional Training 
Initiative module on human research with direct subject interaction. No identifying data will be 
used in any publications that were a result from this work.  
15 PARTICIPATION COST AND PAYMENTS 
There is no cost to participate in this study. Participants will not be charged for the tests that are 
done for research purposes however, participants will still be responsible for the usual costs of 
medical care.  To compensate for time and travel, each participant will receive up to $240 total for 
full study participation. For attending each study visit, the participant will receive $50. However, 
if the participant also returns their MEMS bottle and cap with any remaining medication at visit 2 
and visit 4, they will receive an additional $20 at each of those visits. Participants will also be 
reimbursed for travel at the federal mileage rate when traveling ≥ 50 miles one way and lodging 
at the discretion of the study team. 

 
 
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