Carnitine Consumption and Augmentation in Pulmonary Arterial Hypertension
Phase: PHASE1
Conditions: Pulmonary Arterial Hypertension, Familial Primary Pulmonary Hypertension, Primary Pulmonary Hypertension, Lung Diseases, Carnitine Nutritional Deficiency
Sponsor: Vanderbilt University Medical Center

Carnitine 
 
 
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Protocol Version #: 2.1 
 
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Carnitine Consumption and Augmentation in  
Pulmonary Arterial Hypertension 
 
Protocol 
September 7, 2021 
Version: 2.1 
 
 
Anna R. Hemnes, MD 
Vanderbilt University Medical Center 
 
Co-Investigators 
Evan L. Brittain, MD, MSCI 
Ivan Robbins, MD 
 
 
 
 
 
 
 
 
 
 
Revision History: 
Version 1.0 
 
 
4/26/2021 
Version 2.0 
 
 
5/27/2021 
Version 2.1 
 
 
9/7/2021 
 
 
 
 
 
 
 

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Brief Table of Contents 
Contents .......................................................................................................................................................... 3 
Abstract ........................................................................................................................................................... 5 
Protocol Summary ........................................................................................................................................... 6 
1 
Background & Significance ....................................................................................................................... 9 
2 
Objectives and Specific Aims .................................................................................................................. 11 
3 
Screening and Subject Selection ............................................................................................................. 11 
4 
Data Collection ...................................................................................................................................... 12 
5 
Assessments of Efficacy and Outcome Measures .................................................................................... 16 
6 
Statistical Considerations....................................................................................................................... 19 
7 
Study Risks and Discomforts .................................................................................................................. 20 
8 
Quality Control...................................................................................................................................... 21 
9 
Participant Safety and Confidentiality .................................................................................................... 22 
References ..................................................................................................................................................... 28 
 
 
 
 

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Contents 
Abstract ........................................................................................................................................................... 5 
Protocol Summary ........................................................................................................................................... 6 
1 
Background & Significance ....................................................................................................................... 9 
1.1 
RV Failure in Pulmonary Arterial Hypertension .......................................................................................... 9 
1.2 
PAH As A Systemic Metabolic Disease With RV Manifestation .................................................................. 9 
1.3 
Carnitine Metabolism in Health and Disease: Focus on the heart .............................................................. 9 
1.4 
Background Ingestion of Carnitine Varies by Diet. ................................................................................... 10 
1.5 
Carnitine Supplementation in Humans ..................................................................................................... 10 
2 
Objectives and Specific Aims .................................................................................................................. 11 
2.1 
Objectives .................................................................................................................................................. 11 
2.2 
Specific Aims.............................................................................................................................................. 11 
3 
Screening and Subject Selection ............................................................................................................. 11 
3.1 
Recruitment of Study Sample .................................................................................................................... 11 
3.2 
Inclusion/Exclusion Criteria ....................................................................................................................... 11 
3.3 
Supplement Dosing ................................................................................................................................... 12 
4 
Data Collection ...................................................................................................................................... 12 
4.1 
Consent ..................................................................................................................................................... 12 
4.2 
Study Visits and Contacts .......................................................................................................................... 13 
4.2.1 
Pre-visit Consent Telephone Contact (minimum -4 days) ................................................................... 13 
4.2.2 
Visit 1 Visit (0 Weeks) ........................................................................................................................... 13 
4.2.4 
Study Day – Visit 2 (Week 12) .............................................................................................................. 14 
4.2.5 
Study Day- Visit 3 (Week 12+1 day) ..................................................................................................... 14 
4.2.6 
Telephone Contact (Week 13) ............................................................................................................. 14 
4.2.7 
Study Day- Visit 4 (Week 14) ................................................................................................................ 14 
4.3 
Study Schedule of Endpoints and Procedures ........................................................................................... 15 
5 
Assessments of Efficacy and Outcome Measures .................................................................................... 16 
5.1 
Assessments of Efficacy............................................................................................................................. 16 
5.2 
Secondary Outcome Measures ................................................................................................................. 17 
6 
Statistical Considerations....................................................................................................................... 19 
6.1 
Study Design .............................................................................................................................................. 19 
6.2 
Statistical Analysis..................................................................................................................................... 19 
6.3 
Subjects’ retention and supplement compliance ...................................................................................... 19 
6.4 
Interim Monitoring Guidelines .................................................................................................................. 20 
6.5 
Protocol Violations .................................................................................................................................... 20 

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6.6 
Safety and masking analysis ..................................................................................................................... 20 
7 
Study Risks and Discomforts .................................................................................................................. 20 
7.1 
Echocardiography ..................................................................................................................................... 20 
7.2 
Venipuncture ............................................................................................................................................. 20 
7.3 
Carnitine .................................................................................................................................................... 21 
8 
Quality Control...................................................................................................................................... 21 
8.1 
Personnel training ..................................................................................................................................... 21 
8.2 
Data Quality .............................................................................................................................................. 21 
8.3 
Independent Safety Officer ....................................................................................................................... 21 
9 
Participant Safety and Confidentiality .................................................................................................... 22 
9.1 
Consent ..................................................................................................................................................... 22 
9.2 
Safety and Adverse Event.......................................................................................................................... 22 
9.2.1 
Definitions of Adverse Events .............................................................................................................. 22 
9.2.2 
Classifying AEs ...................................................................................................................................... 23 
9.2.3 
Interpretation of Definitions ................................................................................................................ 25 
9.2.4 
Subject withdrawal............................................................................................................................... 25 
9.3 
Confidentiality ........................................................................................................................................... 25 
9.4 
Privacy ....................................................................................................................................................... 26 
9.5 
Follow-up and Record Retention ............................................................................................................... 26 
9.6 
Ethical Considerations ............................................................................................................................... 26 
References ..................................................................................................................................................... 28 
 
 
 

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Abstract 
 
 
Right ventricular (RV) failure is the most common cause of death in pulmonary arterial 
hypertension (PAH). No RV-specific therapies are available, in part because the underlying 
mechanisms of RV failure are poorly understood. A growing body of evidence suggests that 
metabolic abnormalities may underlie RV dysfunction in PAH. Interventions against metabolic 
dysfunction in PAH may protect against RV failure. We have identified abnormalities in fatty acid 
(FA) metabolism in PAH that overlap considerably with disorders of carnitine deficiency. 
Carnitine links to an acyl group, which is required to transport FAs across the mitochondrial 
membrane to undergo beta-oxidation, the predominate source of ATP production in the human 
heart. Inborn errors of carnitine metabolism and acquired carnitine deficiency are associated 
with cardiomyopathy. Acquired deficiency primarily occurs via binding of carnitine to excess 
circulating fatty acids or renal wasting. Carnitine deficiency and PAH are both associated with 
insulin resistance, myocardial lipotoxicity, and mitochondrial oxidative stress. Carnitine 
supplementation in humans and animal models of cardiometabolic dysfunction reverses these 
abnormalities but has not been studied in PAH. In published work, we found that in RV samples 
from humans with PAH, there is a marked (up to 300-fold) reduction in acylcarnitines along with 
increased long-chain fatty acids. We also a found a two-fold increase in circulating fatty acids 
FAs in humans with PAH, indicating increased delivery to the myocardium. As a consequence 
of unchecked fatty acid accumulation, we observed 7-fold higher RV lipid content and markers 
of lipotoxicity. These observations suggest there is inadequate carnitine substrate to bind fatty 
acids and facilitate their transport across the mitochondrial membrane in the human PAH RV.  
 
Our overarching hypothesis is that in human PAH, RV function can be improved by 
augmenting carnitine substrate availability to improve outcomes. In preparation for a future 
mechanistic study, we now propose to test the specific hypothesis that carnitine consumption is 
not reduced in PAH, that plasma carnitine levels are stable over time in PAH and that carnitine 
supplementation in PAH can increase plasma carnitine and thereby delivery of carnitine to the 
RV and possibly improve RV function. We propose three aims in humans to test this 
mechanistic hypothesis, 1) Measure the oral consumption of carnitine in human PAH. This aim 
will use food diaries and carnitine supplement use questionnaires in PAH patients to test the 
hypothesis that carnitine supplementation is uncommon in PAH and food consumption is 
adequate. Aim 2) Measure the stability over time in plasma carnitine levels in PAH patients. This 
aim will test the hypothesis that plasma carnitine is not affected by disease severity and is 
stable over time in PAH patients. We will measure plasma carnitine concentration and markers 
of fatty acid oxidation at Visit 1 and Visit 2. 3) Perform a mechanistic pilot study using carnitine 
supplementation to enhance circulating carnitine in PAH. This small pilot study will test the 
hypothesis that carnitine supplementation increases plasma carnitine (primary endpoint) and will 
test for physiologic effects using six minute walk testing, echocardiography and plasma markers 
of lipid metabolism.    
 
 
 
 
 
 

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Protocol Summary 
OBJECTIVES: 
The primary objective of this study is to evaluate the effect 
of standard L-carnitine supplementation in PAH patients on 
plasma carnitine levels.  
Secondary objectives include: 
• 
to measure the oral consumption of carnitine in human 
PAH 
• 
to measure the stability over time in plasma carnitine 
levels in PAH patients 
STUDY DESIGN: 
 
This is a single-center, prospective study enrolling 10 PAH 
patients. All eligible participants will be given carnitine 
supplements for 2 weeks. 
STUDY POPULATION:      
 
 
 
Inclusion criteria: 
• 
Adults aged 18 or older. 
• 
Diagnosed with idiopathic, heritable, simple congenital 
heart disease, or drug- or toxin-associated pulmonary 
arterial hypertension (PAH) according to World Health 
Organization consensus recommendations.  
• 
Stable PAH-specific medication regimen for three 
months prior to enrollment. Subjects with only a single 
diuretic adjustment in the prior three months will be 
included. Adjustments in IV prostacyclin for side effect 
management are allowed. 
• 
FEV1> or = 60% predicted and no more than mild 
abnormalities on lung imaging 
• 
WHO Functional Class I-III 
• 
Ambulatory 
Exclusion criteria: 
• 
Prohibited from normal activity due to wheelchair 
bound status, bed bound status, reliance on a 
cane/walker, activity-limiting angina, activity-limiting 
osteoarthritis, or other condition that limits activity 
• 
Pregnancy 
• 
Diagnosis of PAH etiology other than idiopathic, 
heritable, simple congenital heart disease, or 
associated with drugs or toxins 
• 
Drug and toxin associated PAH patients with active 
drug use 

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• 
Prior diagnosis of cirrhosis 
• 
Malignancy 
• 
eGFR by MDRD <60mL/min 
• 
Known allergy to carnitine                                                  
PRIMARY ENDPOINT: 
Difference in plasma carnitine concentration from Visit 1 
to Visit 2 and Visit 3 to Visit 4. 
SECONDARY ENDPOINTS: 
 
• 
Quantify the prevalence of carnitine supplement use 
and measure oral ingestion of carnitine in PAH 
patients. 
• 
Correlation of carnitine ingestion with six-minute walk 
test. 
• 
Correlation of carnitine ingestion with functional class. 
• 
Correlation of carnitine ingestion with plasma BNP. 
• 
Difference in plasma carnitine concentration from Visit 
1 to Visit 2. 
• 
Correlation of plasma carnitine concentration with 
TMAO concentration. 
• 
Difference in plasma acylcarnitines from Visit 1 to Visit 
2. 
• 
Difference in lipid metabolites from Visit 1 to Visit 2. 
• 
Difference in RV function with L-carnitine 
supplementation. 
• 
Difference in plasma carnitine concentration from Visit 
1 to Visit 2. 
• 
Difference in plasma acylcarnitine profile from Visit 3 
to Visit 4. 
• 
Difference in lipid metabolites from Visit 3 to Visit 4. 
• 
Correlation of change in plasma carnitine with change 
in Functional class. 
• 
Correlation of change in plasma carnitine with change 
in six-minute walk distance. 
• 
Correlation of change in plasma carnitine with change 
in BNP. 
• 
Correlation of change in plasma carnitine 
concentration with TMAO concentration 
• 
Correlation of change in plasma carnitine with change 
in markers of RV function including TAPSE and RV 
fractional area change. 
• 
Markers of tolerability including presence of side 
effects, adverse events and serious adverse events. 
• 
Pill counts at Visit 4 for adherence. 
 
STUDY OBSERVATIONS: 
 
 

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• 
Subjects will be evaluated in person at Visit 1, Visit 2, 
Visit 3, and Visit 4.  
• 
BNP, carnitine assay, lipid metabolites, carnitine, 
acylcarnitine, and other biomarkers will be assessed 
at Visit 1, Visit 2, Visit 3, and Visit 4.  
• 
Subjects will have six-minute walk testing at Visit 1, 
Visit 2, and Visit 4. 
• 
Subjects will have a transthoracic echocardiogram at 
Visit 2 and Visit 4. 
• 
Subjects will submit food and supplement diary at Visit 
1, Visit 2, and Visit 4. 
SAMPLE SIZE AND POWER: 
 
 
In a study of 28 PAH patients6 the mean SD of plasma 
carnitine data was 38.611.1 uM. The data appeared to be 
normally distributed. In a pilot study21, L-carnitine 
supplementation increased free plasma carnitine 
concentration by 22% (Table 3 in21). A sample size of 10 
will have 80% power to detect a within subject change in 
means of 8.5 (increase from Visit 1 mean of 38.6 to 47.1 
after 2 weeks of treatment, representing a 22% increase) 
using a paired t-test with a 5% two-sided significance level. 
The standard deviation of within-subject differences is 
assumed to be 8.6 and it is calculated using SD of 11.1 
assuming a moderate within-subject correlation 0.7. We 
plan to study 10 PAH patients in this proof-of-concept 
study. The study has 80% power to detect a within-subject 
change of effect size 1, i.e. change measured as 1 times of 
the SD.  
DATA ANALYSIS: 
The primary endpoint and other continuous endpoints will 
be summarized using the mean, standard deviation, the 
median, and interquartile range. We will use either the 
paired t-test or the Wilcoxon-signed rank test to assess the 
change from Visit 1 on these endpoints. Estimate of mean 
change along with its 95% confidence interval will be 
reported to quantify the treatment effect. To assess 
tolerability of carnitine, we will closely monitor any adverse 
events. For these binary events, proportion of subjects who 
experience the event will be calculated along with its 95% 
confidence interval. We will use the open-source statistical 
package R48 for analyses. 
 
 

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1 Background & Significance  
1.1 
RV Failure in Pulmonary Arterial Hypertension  
PAH is a progressive and incurable disease characterized by obliteration of the 
pulmonary vasculature, elevated pulmonary vascular resistance (PVR), and eventual 
right ventricular (RV) failure often within 7-10 years of diagnosis1. Mortality in patients 
with PAH predominantly results from RV failure and its complications2. However, no RV-
specific therapy exists because the underlying determinants of RV failure are poorly 
understood. The importance of targeting RV function directly is highlighted by the 
observation that outcomes in PAH more closely mirror changes in RV function than 
pulmonary hemodynamics3. Identification of additional molecular contributions to RV 
failure in PAH would advance the pursuit of RV-specific therapies and carnitine 
supplementation holds promise to improve RV function in this deadly disease.  
1.2 
PAH As A Systemic Metabolic Disease with RV Manifestation  
PAH is associated with systemic abnormalities in glucose and lipid metabolism. The first 
clue in humans was a study showing that patients with PAH have an elevated 
triglyceride to high density lipoprotein ratio, a marker of insulin resistance. Subsequently, 
we found a prevalence of almost 60% of unrecognized glucose intolerance in our PAH 
patients, independent of body mass index (BMI)4. The lack of an association with BMI 
suggests that the mechanism of insulin resistance in PAH may be unrelated to increased 
adiposity, per se. In addition, we found elevated plasma free fatty acids and long-chain 
acylcarnitines in humans with PAH compared with matched controls (see Preliminary 
Data)5. More recently, we showed that insulin resistance in PAH specifically involves the 
lipid axis and is detected through peripheral blood abnormalities in triglycerides and lipid 
metabolism6. While many organs may be affected by insulin resistance, the RV is highly 
reliant on fat metabolism and thus susceptible to metabolic disorders. We have 
previously published that the RV in humans with PAH, both alive and at autopsy, has 
enhanced lipid content5,7. We have also demonstrated that this lipotoxicity is due to 
increased lipid import via CD36 and reduced fatty acid oxidation in cardiomyocytes8. We 
also demonstrated in human samples an increase in plasma free fatty acid delivery to 
the PAH RV and a marked (100-300 fold) reduction in myocardial long-chain 
acylcarnitine concentration, suggesting reduced fatty acid oxidation5. These findings 
provide strong evidence that metabolic dysregulation, and specifically lipid metabolism, 
is an important feature of PAH and that the RV appears impacted by this altered 
metabolism.  
1.3 
Carnitine Metabolism in Health and Disease: Focus on the heart  
Carnitine is a nutrient required for the transport of long-chain fatty acids as acylcarnitines 
across the mitochondrial membrane to undergo beta-oxidation and subsequent ATP 
production9. Carnitine is particularly abundant in skeletal muscle and the myocardium, 
where fatty acids are the predominant substrate for energy production10. The presence 
of adequate carnitine in myocardial cells is essential for normal fatty acid metabolism. 
Reduced availability of carnitine from any cause leads to impaired fatty acid oxidation 
and muscle dysfunction. When carnitine binds fatty acids, acylcarnitines are formed and 
then normally undergo beta-oxidation. In conditions of excess circulating free fatty acids 
(e.g. diabetes and heart failure), acylcarnitines are formed in excess of mitochondrial 
oxidative capacity. This results in a depletion of carnitine, reduced fatty acid oxidation, 
oxidant stress, and accumulation of toxic lipids. Excess acylcarnitines are excreted from 
cells and elevated levels can be detected in the plasma in conditions of abnormal 

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carnitine metabolism9. Oleoylcarnitine and three other long-chain acylcarnitines are 
strongly associated cardiovascular mortality in a dialysis population11; the same four 
acylcarnitines were most elevated in our PAH population compared to healthy subjects5.  
Cardiomyocytes cannot synthesize carnitine, which must be acquired exogenously. 
Thus, deficiencies, even relative, in carnitine have particularly adverse effects in 
cardiomyocytes. Inborn errors of carnitine metabolism involve abnormal transporter 
function and present with severe, often fatal, cardiomyopathy12. Secondary carnitine 
deficiency occurs because the available carnitine is bound to excess fatty acids or due to 
renal wasting13. Acquired carnitine deficiency has been described in dialysis therapy, 
diabetes, and heart failure, among other conditions14-16. Chronic carnitine deficiency is 
associated with insulin resistance, lipid-induced cardiomyopathy, and increased 
mitochondrial oxidative stress16. Carnitine supplementation in humans and experimental 
models appears to mitigate these defects. In humans with heart failure, carnitine 
improves diastolic and systolic dysfunction and may reduce cardiovascular events15,17. In 
diabetics, carnitine reduces myocardial free FA uptake and circulating long-chain 
acylcarnitines. In mice exposed to doxorubicin, carnitine reduces ceramide production 
and apoptosis. Thus, multiple lines of evidence suggest that maintenance of intracellular 
carnitine may be beneficial for cardiometabolic function.  
1.4 
Background Ingestion of Carnitine Varies by Diet.  
A typical Western diet is high in meat and dairy intake and thus provides adequate 
carnitine ingestion to prevent depletion18. The Food and Nutrition Board has not 
established a recommended dietary allowance for carnitine and it is not considered an 
essential nutrient19. Nonetheless, relative carnitine deficiency develops in numerous 
inborn and chronic diseases, indicating that background ingestion is not sufficient to 
prevent depletion in some states13,18,20 and that normal plasma carnitine 
concentration may not be adequate for tissue needs depending on the demand21. 
This observation suggests that supplementation may be required to prevent or reverse 
depletion in at risk individuals despite normal dietary consumption. There are no 
published data on frequency of vegetarian or vegan diet in PAH that may predispose to 
deficiency or use of carnitine supplementation in this disease state.  
1.5 
Carnitine Supplementation in Humans 
Given its safety and role in energy production, carnitine has been studied extensively 
and even touted as a performance enhancer for professional athletes. Although the liver 
and kidneys in a healthy human can metabolize the amino acids lysine and methionine 
to meet daily carnitine needs19, carnitine supplements are widely available. Carnitine 
supplementation has primarily been in the form of L-carnitine, or levocarnitine, which is 
the active form in the body22, that has a bioavailability of approximately 25%22,23. After an 
oral dose of 30-100mg/kg body weight, peak plasma carnitine concentration occurs at 3-
6 hours24. In a study of the effect of long-term supplementation with L-carnitine, healthy 
adult men were fed similar doses (2g/day) of L-carnitine per day or placebo for 15 days. 
Subjects in the supplementation group maintained total plasma concentrations that were 
about 25 umol/L higher than in the control group, thus demonstrating the ability of 
carnitine supplementation to increase plasma carnitine over two weeks22. While there 
are no data on the effect of oral carnitine supplementation on cardiac carnitine content, a 
2g/day oral L-carnitine supplement can modesty increase the skeletal muscle carnitine 
concentration by about 13% over four weeks25. Carnitine supplementation has been tried 
in ageing, where it has had no effect on muscle strength21, however after acute 
myocardial infarction, a meta-analysis demonstrated that L-carnitine supplementation 

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reduced mortality, ventricular arrhythmias and angina over two months26. L-carnitine can 
be metabolized by gut microbiota to proatherogenic trimethylamine-N-oxide (TMAO) 
which is associated with increased cardiovascular risk27. However, limited prospective 
data exist to support this notion and none related to PAH or RV failure, which is not an 
atherosclerotic disease. Moreover, survival in PAH is generally fewer than 10 years28, 
thus the relevance of TMAO to PAH is unclear.  
2 Objectives and Specific Aims 
2.1 
Objectives 
This is a single-center, prospective study enrolling 10 PAH patients. All eligible 
participants will be given carnitine supplements for 2 weeks. 
2.2 
Specific Aims 
Primary Aim:  
1. The primary objective of this study is to evaluate the effect of standard L-
carnitine supplementation in PAH patients on plasma carnitine levels.  
Secondary Aims:  
1. to measure the oral consumption of carnitine in human PAH 
2. to measure the stability over time in plasma carnitine levels in PAH 
patients 
3 Screening and Subject Selection 
3.1 
Recruitment of Study Sample 
Patients will be recruited from the Center for Pulmonary Vascular Disease (CPVD) at 
Vanderbilt University Medical Center (Nashville, TN). Potentially eligible subjects will be 
pre-screened by study personnel through medical record review and informed about that 
study to determine if they have an interest in enrolling. After the initial conversation by 
telephone, the subject will be provided informed consent and a link to the e-consent will 
be emailed directly to them. Informed consent will occur before any study procedures 
are performed. 
3.2 
Inclusion/Exclusion Criteria 
Inclusion criteria: 
• Adults aged 18 or older. 
• Diagnosed with idiopathic, heritable, simple congenital heart defect, 
or drug- or toxin-associated pulmonary arterial hypertension (PAH) 
according to World Health Organization consensus 
recommendations.  
• Stable PAH-specific medication regimen for three months prior to 
enrollment. Subjects with only a single diuretic adjustment in the 
prior three months will be included. Adjustments in IV prostacyclin 
for side effect management are allowed. 
• FEV1> or = 60% predicted and no more than mild abnormalities on 
lung imaging 

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• WHO Functional Class II-IV 
• Ambulatory 
Exclusion criteria: 
• Prohibited from normal activity due to wheelchair bound status, bed 
bound status, reliance on a cane/walker, activity-limiting angina, 
activity-limiting osteoarthritis, or other condition that limits activity 
• Pregnancy 
• Diagnosis of PAH etiology other than idiopathic, heritable, simple 
congenital heart defect, or associated with drugs or toxins 
• Drug and toxin associated PAH patients with active drug use 
• Prior diagnosis of cirrhosis 
• Malignancy 
• eGFR by MDRD <60mL/min 
• Known allergy to l-carnitine supplements 
3.3 
Supplement Dosing 
 A 14-19-day supply of Carnitine supplements will be provided to the subject at Visit 2. 
The VUMC Pharmacy for Research will supply Nature’s Bounty L-Carnitine 500mg 
caplets to be taken by mouth. For subjects weighing <90kg, l- carnitine will be taken at 
1.5g twice a day. For subjects weighing >90kg, l-carnitine will be taken at 50 mg/kg/day 
split between 2 doses. Dosing for these participants will be rounded to the next whole 
pill to achieve approximately 50 mg/kg per day. Dosing will be incrementally titrated by 
25- 50% in the event of intolerance and/or side effects limiting subject’s compliance to 
prescribed regimen. 
4 Data Collection 
4.1 
Consent 
Written consent or e-consent will be obtained for enrollment from participants. For each 
consent process, study personnel will discuss the details of the study, the risks and 
benefits, and the subject’s rights and responsibilities if they choose to participate in the 
trial and their right to refuse to participate. It will be made clear that their clinical care 
will not be affected by their decision. Written consent or e-consent will be obtained 
before Visit 1.  
For e-consent, the patient consent process will be conducted using a REDCap-based 
electronic consent form.  The consent form has been developed in REDCap, a secure, 
web-based, HIPAA-compliant, data collection platform with a user management system 
allowing project owners to grant and control varying levels of access to data collection 
instruments and data (e.g. read only, de-identified-only data views) for other users.  
Potential participants will participate in the consent process by either: 
1.)  Being approached in-person at a Vanderbilt Clinic and accessing the REDCap 
survey via iPad or other portable electronic device and/or 
2.) Self-initiated access of consent forms on personal portable electronic devices 
using posted QR codes or web-links on study posters, brochures, or websites.  
Self-initiated accessing of consent forms may occur in clinic or at home. 

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During the in-person consent process, patients will be consented by a member of the 
key study personnel.  
For self-initiated consent, contact information will be provided (email and phone) for 
prospective patients to contact a member of the key study personnel with questions, 
prior to consent. 
Patient signatures will be obtained using a typed signature.  Upon completion of the 
consent, patients will be provided with a copy of their version of the consent document 
by printing a pdf copy of the consent form in clinic or at their Visit 1 visit.  
4.2 
Study Visits and Contacts 
This study will consist of four visits: Visit 1 (0 week), Visit 2 (Week 12), Visit 3 (Week 
12+1 day) and Visit 4 (Week 14). The Visit 1 and Visit 3 are both estimated to take 
approximately 2 hours each. Visit 2 and Visit 4 are both estimated to take approximately 
4 hours each. Patients will be consented by the PI, a clinical research coordinator, or a 
research nurse. Patient’s written consent and study procedures will take place at the 
Pulmonary Vascular Disease Clinic (CPVD), the Clinical Research Center (CRC), there 
will be no changes to a patient’s medication regimen. 
4.2.1 
Pre-visit Consent Telephone Contact (minimum -4 days) 
Prior to subject’s first CRC visit, the subject will consent by REDCap e-consent 
process and complete a 3-day ASA24 food and supplement diary. 
4.2.2 
Visit 1 Visit (0 Weeks) 
This visit will last approximately 2 hours and occur at the Clinical Research Center 
at VUMC. At Visit 1, written consent will be obtained from subjects after all 
questions are adequately answered. The following procedures will be performed: 
• 
written informed consent  
• 
review inclusion/exclusion criteria  
• 
review of medical history 
• 
demographic data 
• 
review of current medications 
• 
orientation to food and supplement diary (ASA24) 
• 
WHO functional class 
• 
blood collection 
• 
symptom assessment 
• 
vital signs 
• 
physical exam 
• 
six-minute walk test 
After fasting research labs have been drawn, the subject will have the opportunity 
to eat a snack. The investigator or research nurse will take a medical history, 
perform a physical examination including checking vital signs, and review current 
medications. The subject will perform the six-minute walk test. 
4.2.3 
Telephone Contact (Week 11) 
The research coordinator will call the subject 1 week prior to Visit 2 to remind them 
to complete the 3-day ASA24 food and supplement diary. Study compliance will be 
assessed and reinforced. 

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4.2.4 
Study Day – Visit 2 (Week 12) 
All tests done at the Visit 1 visit will be repeated at the Visit 2 Visit. The following 
procedures will be performed:  
• 
review of interim medical history 
• 
review of current medications 
• 
WHO functional class 
• 
blood collection 
• 
urine pregnancy test (as necessary) 
• 
symptom assessment 
• 
vital signs 
• 
physical exam 
• 
review of food and supplement diary (ASA24) 
• 
six-minute walk test 
• 
transthoracic echocardiography 
• 
Dosing of Carnitine supplement 
After fasting research labs have been drawn, the subject will have the opportunity 
to eat a snack. The investigator or research nurse will take an interim medical 
history, perform a physical examination including checking vital signs, and review 
current medications. The subject will perform the six-minute walk test. An 
experienced research sonographer will perform the transthoracic 
echocardiography.  
4.2.5 
Study Day- Visit 3 (Week 12+1 day) 
The day following Visit 2, a brief visit will be performed with the following 
procedures: 
• 
blood collection 
• 
symptom assessment 
• 
vital signs 
• 
review of food and supplement diary (ASA24) 
• 
2 weeks supply of carnitine supplement provided 
4.2.6 
Telephone Contact (Week 13) 
The research coordinator will call the subject a week after beginning the carnitine 
supplement to review and record symptoms, potential side effects, and changes in 
medications. Study compliance will also be assessed and reinforced. 
4.2.7 
Study Day- Visit 4 (Week 14) 
All tests done at the Visit 2 visit will be repeated at the Visit 4 Visit. The following 
procedures will be performed: 
• 
review of interim medical history 
• 
review of current medications 
• 
WHO functional class 
• 
blood collection 

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• 
symptom assessment 
• 
vital signs 
• 
physical exam 
• 
review of food and supplement diary (ASA24) 
• 
six-minute walk test 
• 
transthoracic echocardiography 
• 
pill count of carnitine supplement 
After fasting research labs have been drawn, the subject will have the opportunity 
to eat a snack. The investigator or research nurse will take an interim medical 
history, perform a physical examination including checking vital signs, and review 
current medications. The subject will perform the six-minute walk test. An 
experienced research sonographer will perform the transthoracic 
echocardiography. 
4.3 
Study Schedule of Endpoints and Procedures 
  
The table below summarizes the study endpoint assessments and procedures. 
 
Phone 
Call 
Visit 1 
Phone 
Call 
Visit 2 
Visit 3 
Phone 
Call 
Visit 4 
 
 
Observational 
Period 
Supplement Period 
Day 
-3 
0 
80 
84 (+/- 
5 days) 
85 
92 (+/- 5 
days) 
99 (+/- 5 
days) 
Week 
-1 
0 
11 
12 
12 
13 
14 
Informed Consent 
x 
 
 
 
 
 
 
History and Physical  
 
 
 
 
 
 
 
Vital Signs 
 
x 
 
x 
x 
 
x 
Medical History 
 
x 
 
x 
 
 
x 
Medication review 
 
x 
 
x 
 
x 
x 
Physical Exam 
 
x 
 
x 
 
 
x 
Functional Class 
 
x 
 
x 
 
 
x 
Symptom Assessment 
 
x 
 
x 
x 
x 
x 
Testing 
 
 
 
 
 
 
 
Phlebotomy 
 
 
 
 
 
 
 
      CBC 
 
x 
 
x 
x 
 
x 
      CMP 
 
x 
 
x 
x 
 
x 
      BNP 
 
x 
 
x 
x 
 
x 
      Carnitine assay 
 
x 
 
x 
x 
 
x 
      Lipid metabolites 
 
x 
 
x 
x 
 
x 
      Carnitine 
 
x 
 
x 
x 
 
x 
      Acylcarnitine 
 
x 
 
x 
x 
 
x 
6MWTD 
 
x 
 
x 
 
 
x 
Echo 
 
 
 
x 
 
 
x 

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Table 1. Study Procedures 
5 Assessments of Efficacy and Outcome Measures 
5.1 
Assessments of Efficacy  
Primary Endpoint: Difference in plasma carnitine concentration from Visit 1 to 
Visit 4. 
Secondary Endpoints: 
• 
Quantify the prevalence of carnitine supplement use 
• 
Measure oral ingestion of carnitine in PAH patients. 
• 
Difference in six-minute walk test from Visit 1 to Visit 2. 
• 
Difference in functional class from Visit 1 to Visit 2. 
• 
Difference in plasma BNP from Visit 1 to Visit 2 
• 
Difference in plasma carnitine concentration from Visit 1 to 
Visit 2. 
• 
Correlation of plasma carnitine concentration with TMAO 
concentration. 
• 
Difference in plasma acylcarnitines from Visit 1 to Visit 2. 
• 
Difference in lipid metabolites from Visit 1 to Visit 2. 
• 
Change in plasma carnitine from Visit 2 to Visit 3. 
• 
Difference in RV function from Visit 2 to Visit 4. 
• 
Difference in plasma acylcarnitine profile from Visit 2 to Visit 4. 
• 
Difference in lipid metabolites from Visit 2 to Visit 4. 
• 
Correlation of change in plasma carnitine with change in 
Functional class. 
• 
Correlation of change in plasma carnitine with change in six 
minute walk distance. 
• 
Correlation of change in plasma carnitine with change in BNP. 
• 
Correlation of change in plasma carnitine concentration with 
TMAO concentration 
• 
Correlation of change in plasma carnitine with change in 
markers of RV function including TAPSE and RV fractional 
area change. 
• 
Markers of tolerability including presence of side effects, 
adverse events and serious adverse events. 
Urine Pregnancy Test 
 
 
 
x 
 
 
 
Study Procedures 
 
 
 
 
 
 
 
ASA-24 Dietary 
Assessment 
x 
x 
x 
x 
 
 
 
Carnitine supplement 
(3g/day x 2 weeks) 
 
 
 
x 
x 
x 
 
Adverse events 
 
 
 
x 
x 
x 
x 
Supplement 
compliance 
 
 
 
 
x 
x 
x 

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• 
Pill counts at Visit 4 for adherence
 
5.2 
Secondary Outcome Measures 
5.2.1 
Six Minute Walk Distance 
Walking is the most basic form of exercise and is integral to daily activities. The 
6MWT is a standardized, times submaximal test of unencouraged, self-
determined distance walked which is reliable and valid54. Standardized test 
methods and scripted and timed statements have been established in prior 
studies of PAH28,29-35. The 6MWT will be administered according to the 
Thoracic Society guidelines36,54. The 6MWT will be performed at Visit 1, Visit 2 
Visit 3 and Visit 4. The subject will be instructed to wear comfortable clothing 
and shoes. The test will be performed at approximately the same time of day at 
each visit. In addition, Borg dyspnea Score, oxygen saturation, and heart rate 
will be recorded at the beginning and conclusion of each test. 
5.2.2 
The Borg Dypsnea Score 
In order to assess exercise capacity, the Borg score for dyspnea and overall 
fatigue will be recorded before and after subjects complete the six-minute walk, 
performed at Visit 1, Visit 2 visits and Visit 4 visits. This score is a measure the 
physical activity intensity level based on the subject's perceived exertion. 
Subjects will rate at resting and peak exercise. 
5.2.3 
WHO Functional Class 
The WHO functional classification for PAH has been modified from the 
well-known New York Heart Association functional classification. This 
functional classification is based on symptoms, with Class I being defined 
by no symptoms, Class II as having mild limitation in physical activity, 
Class III as having markedly limited physical activity and Class IV as being 
unable to perform any physical activity. The WHO functional class will be 
assessed at Visit 1, Visit 2 visits and Visit 4 visits. 
5.2.4 
Blood Biomarkers 
5.2.4.1 
Lipid Metabolism 
Fasting plasma lipid metabolites will be evaluated via non-targeted 
metabolomics screen. Mass spectroscopy metabolites such as 
LDL, HDL, and triglycerides will be performed at Visit 1, Visit 2, 
Visit 3 and Visit 4. 
5.2.4.2 
Fatty Acid Metabolism 
Elevated circulating fatty acids and acylcarnitines contribute to RV 
lipotoxicity. To evaluate the effect of this intervention on fatty acid 
metabolism, we will measure fasting long-chain fatty acid and 
acylcarnitine profiles using mass spectrometry at Visit 1 and Visit 
2.  
5.2.4.3 
Plasma NT-proBNP 

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Plasma NT-proBNP reflects right ventricular function in PAH and is 
a strong predictor of outcome in PAH. BNP will be assessed at 
Visit 1, Visit 2, Visit 3, and Visit 4. 
5.2.4.4 
TMAO 
Trimethylamine-N-oxide (TMAO) is associated with increased 
cardiovascular risk27 and presents as a detrimental carnitine effect. 
As a safety endpoint, TMAO will be measured via mass 
spectroscopy. 
5.2.5 
Transthoracic Echocardiographic Measures 
We will assess RV free wall longitudinal strain and other parameters as 
secondary endpoints via transthoracic echocardiography. The ultimate 
determinant of outcome in subjects with PAH is right ventricular function, 
so we have focused on metrics, which are predictors of survival in PAH, 
including RV strain and qualitative RV function. The following metrics will 
be obtained: 1) tricuspid annular plane systolic excursion (TAPSE); 2) RV 
& LV ejection fraction51; 3) RV fractional area change; 4) tricuspid annular 
velocity (S’); 5) tricuspid regurgitant (TR) velocity; 6) RV systolic pressure 
(estimated from the equation: RV systolic pressure = 4(maximum TR 
velocity)61 + right atrial pressure); 7) right atrial pressure59; 8) RV & LV 
diastolic function53; 9) RV free wall longitudinal strain54,60. All 
measurements will be performed according to the American Society of 
Echocardiography guidelines51,53,59. 
5.2.6 
Other Feasibility Endpoints 
a) Drop Out Rates 
b) Side effects and tolerability of supplementation including carnitine 
5.2.7 
Hospitalization 
A hospital admission due to PAH progression and/or right-sided heart failure 
will be defined as a hospitalization because of lower extremity edema or 
dyspnea and/or PAH symptoms (e.g., syncope) refractory to outpatient 
increases in dose or frequency of diuretics or specific PAH medications. 
We will record all hospitalizations during the time of the study. Records from 
each hospitalization will be obtained by the local study coordinator. These 
records will be reviewed by the Data and Safety Monitoring Board (DSMB), 
which is unrelated to the study. 
5.2.8 
Death 
 
Cardiovascular Death: We define as cardiovascular death:  
1) 
sudden death 
2) 
death preceded by either one of the following: 
a) cardiogenic shock defined by either: 
i. hypotension resulting in a failure to maintain normal renal 
ii. cerebral function for >15 minutes prior to death) 
b) heart failure symptoms or signs requiring one of the following: 
i. intravenous therapy or oxygen in the hospital  

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ii. confinement to bed in the absence of secondary causes (such as 
systemic infection or dysfunction of intravenous or subcutaneous 
medication delivery devices) or alternative causes of death.  
Non-Cardiovascular Death: A death which does not meet the criteria above will 
be considered a non-cardiovascular death. 
6 Statistical Considerations 
6.1 
Study Design 
In a study for 28 PAH patients6 the mean  SD of plasma carnitine data was 
38.611.1 uM. The data appeared to be normally distributed. In a pilot study21, 
L-carnitine supplementation increased free plasma carnitine concentration by 
22% (Table 3 in21). A sample size of 10 will have 80% power to detect a within 
subject change in means of 8.5 (increase from Visit 1 mean of 38.6 to 47.1 
after 2 weeks of treatment, representing a 22% increase) using a paired t-test 
with a 5% two-sided significance level. The standard deviation of within-subject 
differences is assumed to be 8.6 and it is calculated using SD of 11.1 
assuming a moderate within-subject correlation 0.7. We plan to study 10 PAH 
patients in this proof-of-concept study. The study has 80% power to detect a 
within-subject change of effect size 1, i.e. change measured as 1 times of the 
SD. 
6.2 
Statistical Analysis 
The primary endpoint and other continuous endpoints will be summarized 
using the mean, standard deviation, the median, and interquartile range. We 
will use either the paired t-test or the Wilcoxon-signed rank test to assess the 
change from Visit 1 on these endpoints. Estimate of mean change along with 
its 95% confidence interval will be reported to quantify the treatment effect. To 
assess tolerability of carnitine, we will closely monitor any adverse events. For 
these binary events, proportion of subjects who experience the event will be 
calculated along with its 95% confidence interval. We will use the open-source 
statistical package R57 for analyses. 
6.3 
Subjects’ retention and supplement compliance 
We will enforce subject retention in several ways. We will record extensive 
contact information for each subject at their enrollment in the trial. This will 
include home, work, and cellular telephone numbers. The research 
coordinator will call before each study visit to remind the subject to attend. 
Subjects will be reimbursed for time at the clinic for research procedures 
and reasonable travel expenses necessary for their participation in the 
study. Subjects will receive $75 each for Visit 1 and Visit 2, $100 for Visit 3, 
and $150 for Visit 4. The total reimbursement for completing all study 
procedures will be $400. 
The research coordinator and physician will explain the importance of 
compliance with the study protocol at each subject contact. If a subject fails 
to comply with a study visit, the coordinator will contact him or her by 
telephone. If this fails, the coordinator will send two letters, one week apart, 
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We have considered how to minimize difficulties with adherence to the 
supplement. We will strongly emphasize the importance of complying with 
the supplement regimen. Nonetheless, we will perform pill counts at visits 
and record episodes when the supplement is withheld for any reason. If a 
subject wishes to drop-out from the supplement phase of the study or has a 
serious adverse event (SAE) (whether related to study drugs or not), we 
will continue to follow-up with the subject for study assessments to assist 
with safety monitoring and to avoid the problems introduced by missing 
data. The inclusion of such follow-up data will allow for analysis by 
intention-to-treat. 
If a subject is withdrawn from the supplement portion of the study for any 
reason, the subject will be strongly encouraged to continue with the 
remainder of the study assessments, as scheduled. 
6.4 
Interim Monitoring Guidelines 
We have not planned formal interim analyses for efficacy and therefore there 
are no stopping rules for efficacy for this trial. This project will have an 
independent safety officer who may consider whether to stop the trial or not if 
there is an increased risk of adverse events or toxicity. 
6.5 
Protocol Violations 
Serious protocol violations such as discontinuation of the intervention 
unrelated to AEs will be carefully recorded and regularly reviewed by the 
Principal Investigator. Remedial changes in procedure will be recommended 
where feasible to reduce the incidence of such violations. When known, the 
causes and circumstances of all violations will be documented for future 
secondary analyses and interpretation. Because all primary analyses will be 
intent-to-treat, it is essential that violations be kept to a minimum especially 
where it is possible to influence their rate of occurrence.  
6.6 
Safety and masking analysis 
All subjects will be assessed for toxicity and included in the safety analysis. 
This analysis will include summaries of the incidence and grade of toxicities. 
Safety interim analyses will be performed and reported to the DSMB. Subjects 
will be evaluated for SAEs. 
7 Study Risks and Discomforts 
7.1 
Echocardiography 
There are no risks to this ultrasound procedure aside from minor discomfort 
related to placement of the ultrasound probe on the chest. 
7.2 
Venipuncture 
The protocol requires patients to have blood drawn for research purposes. 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 
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7.3 
Carnitine 
Given its safety and role in energy production, carnitine has been studied 
extensively and is widely available. In long-term administration of oral L-
carnitine, mild gastrointestinal side effects have been reported; these include 
transient nausea and vomiting, abdominal cramps, and diarrhea. Notable, are 
some reported cases of a “fishy” body odor while taking carnitine 
supplements. Mild myasthenia is reported in uremic patients; however, this 
patient population has been excluded from enrolling in our study. In rare 
cases, patients with or without pre-existing seizure activity have reported 
seizures to occur, in addition to increase in frequency and/or severity of 
seizures. 
8 Quality Control 
Design strategies and monitoring activities throughout the study will ensure the integrity 
and high quality of the data. Design strategies include training and certification of 
personnel. The rigorous monitoring program includes data queries and performance 
monitoring over the time of the trial. 
8.1 
Personnel training 
Prior to enrolling the first subject in the study protocol, study coordinators and 
research assistance who will perform the outcome assessments will have 
completed appropriate training. The purpose of training is to ensure that study 
personnel are carrying out the protocol in a consistent way and are adhering to 
a good clinical practice guidelines. Staff will have current Human Subjects 
Training Certification on file.  
8.2 
Data Quality 
The PI and study coordinator will constitute the first line of monitoring of the 
safety of the human participants. They will perform continuous monitoring of 
data quality and completion of CRFs. Surveillance for AEs will consist of 
questioning subjects about potential AEs at every study contact, having 
subjects report any adverse event to the study team, and having subjects 
undergo vital sign checks and physical exams during each study visit.  
Project team members at Vanderbilt listed as Key Study Personnel with 
existing electronic health record (HER) system access rights will make use of 
REDCap Clinical Data Pull (CDP) tools. These tools are designed to enable 
transfer of relevant study-related data from the Vanderbilt Research Derivative 
and/or directly from the HER into REDCap. 
8.3 
Independent Safety Officer 
Dr. Meredith Pugh will serve as an independent safety officer and will monitor 
the trial. The aims of the officer are to safeguard the interests of the trial’s 
participants, potential participants, and investigators, to ensure the safety of 
the trial’s interventions, to monitor the trial’s overall conduct, and protect the 
trial’s validity and credibility. Dr. Pugh serves as an attending physician in the 
Vanderbilt Medical Intensive Care Unit, the inpatient consult service, and the 
pulmonary hypertension service. Dr. Pugh has outpatient clinics where she 
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She will be invited to attend weekly meetings with the PI and study personnel. 
She will be available to discuss adverse events with the PI and respond to 
safety concerns from subjects or study personnel.  
9 Participant Safety and Confidentiality 
9.1 
Consent 
Written consent will be obtained for enrollment for participants. For each 
consent process, study personnel will discuss the details of the study, the risks 
and benefits, and the subject’s rights and responsibilities if they choose to 
participate in the trial and their right to refuse to participate. It will be made 
clear that their clinical care will not be affected by their decision. Subjects will 
be permitted to provide verbal consent over the phone prior to being scheduled 
for a screening visit. A consent script will be provided, and documentation of 
verbal consent will be noted. Written or e-consent will be obtained at the Visit 1 
visit or before.  
9.2 
Safety and Adverse Event 
9.2.1 
Definitions of Adverse Events 
9.2.1.1 
Unanticipated Problem (UP): any incident, experience, or 
outcome that meets all of the following criteria: 
1) unexpected (in terms of nature, severity, or frequency) given a) the 
research procedures that are described in the protocol-related 
documents, such as the IRB-approved research protocol and informed 
consent document; and b) the characteristics the subject population 
being studied; 
2) related or possibly related to participation in the research (Possibly 
related to participation in the research means there is a reasonable 
possibility that the AE, experience, or outcome may have been 
caused by the procedures involved in the research); and 
3) suggests that the research places subjects or others at a greater 
risk of harms (including physical, psychological, economic, or social 
harm) that was previously known or recognized. 
9.2.1.2 
Adverse Event (AE): Any symptom, sign, illness, or experience 
that develops or worsens in severity during the course of the 
study. Intercurrent illnesses or injuries should be regarded as 
AEs. Abnormal results of diagnostic procedures are considered 
to be AEs if the abnormality: 
• 
Results in study withdrawal 
• 
Is associated with a serious AE 
• 
Is associated with clinical signs or symptoms  
• 
Leads to additional treatment or to further diagnostic tests 
• 
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9.2.1.3 
Serious Adverse Event (SAE): Adverse reactions are classified 
as serious or non-serious. A serious adverse event is any AE 
that is: 
• 
Fatal 
• 
Life-threatening 
• 
Requires or prolongs hospital stay 
• 
Results in persistent or significant disability or incapacity 
• 
A congenital anomaly or birth defect 
• 
An important medical event 
Important medical events are those that may not be immediately life 
threatening but are clearly of major clinical significance. They may 
jeopardize the subject and may require intervention to prevent one of the 
other serious outcomes noted above. For example, drug overdose or 
abuse, a seizure that did not result in in-subject hospitalization or intensive 
treatment of bronchospasm in an emergency department would typically 
be considered serious. All AEs that do not meet any of the criteria for 
serious should be regarded as non-serious AEs. 
9.2.1.4 
Suspected Adverse Reaction: any adverse event for which 
there is a reasonable possibility that the drug caused the 
adverse event. For reporting purposes, “reasonable possibility” 
means there is evidence to suggest a causal relationship 
between the drug/investigational product and the adverse event. 
9.2.2 
Classifying AEs 
9.2.2.1 
Severity: the intensity of the AE is classified according to the 
CTCAEv4.0. Grade refers to the severity (intensity) of the AE: If 
the intensity of an AE worsens during study drug administration, 
only the worst intensity should be reported on the AE page. If 
the AE lessens in intensity, no change in the severity is required. 
CTCAEv4 Grade 1: mild; asymptomatic or mild symptoms; clinical or 
diagnostic observations only; intervention is not indicated. 
CTCAEv4 Grade 2: moderate; minimal, local, or noninvasive intervention 
is indicated; limiting to age-appropriate instrumental activities or daily living 
(ADL; instrumental ADL refers to preparing meals, shopping for groceries 
or clothes, using the telephone, managing money, etc). 
CTCAEv4 Grade 3: severe or medically significant but not immediately 
life threatening; hospitalization or prolongation of hospitalization is 
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bathing, dressing and undressing, feeding self, using the toilet, taking 
medications, and not bedridden) 
CTCAEv4 Grade 4: life-threatening consequences; urgent intervention is 
indicated. 
CTCAEv4 Grade 5: death due to an AE.  
In this grading system, severity is not equivalent to seriousness. For 
example, a SAE would be any events which is life-threatening or disabling 
(Grade 4) or fatal (Grade 5) or was moderate-severe (Grade 2-3) and 
required or prolonged hospitalization. 
9.2.2.2 
Expectedness: AEs must be assessed as to whether they were 
expected to occur or were unexpected, meaning not anticipated 
based on current knowledge found in the protocol, investigator 
brochure, product insert, or label. 
Expected: an AE known to be associated with the intervention or 
condition under study. OHRP defines an unexpected AE as any AE 
occurring in one or more subjects participating in a research protocol, 
the nature, severity, or frequency of which is not consistent with either: 
1) the known or foreseeable risk of AEs associated with the 
procedures involved in the research that are described in a) the 
protocol-related documents, such as the IRB-approved research 
protocol, any applicable investigator brochure, and the current 
IRB-approved informed consent document, and b) other relevant 
sources of information, such as product labeling and package 
inserts; or  
2) the expected natural progression of any underlying disease, 
disorder, or condition of the subject(s) experiencing the AE and 
the subject’s predisposing risk factor profile for the AE. 
9.2.2.3 
Relatedness 
1) Definite: the AE is clearly related to the research procedures 
2) Probably: the AE is likely related to the research procedures 
3) Possible: the AE may be related to the research procedures 
4) Unlikely: the AE is doubtfully related to the research procedures 
5) Unrelated: the AE is clearly not related to the research procedures 
Possibly related to participation in the research: There is a reasonable 
possibility that the adverse event experience, or outcome may have been 
caused by the procedures involved in the research. 
For each identified AE, an AE entry on the appropriate form will be 
completed using the above classifications as soon as possible, updating 
as necessary. Reporting procedures should be started immediately (within 
24 hours) upon learning of a SAE or UP. 

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9.2.3 
Interpretation of Definitions 
9.2.3.1 
AE and UP Reporting Period 
The study period during which AEs must be reported is normally defined 
as the period from the initiation of any study procedures to the end of the 
study treatment follow-up. For this study, the study treatment follow-up is 
defined as 14 days following the last administration of study treatment.  
9.2.3.2 
Expedited Reporting Procedures  
Any adverse event that occurs will be reported to Dr. Anna R. Hemnes, 
MD, or Dr. Evan Brittain, MD, within 24 hours of the event using a required 
form or as a written report of the event.  
9.2.4 
Subject withdrawal 
A subject has the right to withdraw from the study entirely at any time for 
any reason without prejudice to future medical care by the investigator or 
other physician. The investigator also has the right to withdraw subjects 
from the study in the event of concurrent illness, AEs, or other reasons 
deemed to be in the subject’s best interest. A subject should be withdrawn 
from the study if there is: 
• 
Withdrawal of consent 
• 
Termination of study 
• 
PI determination that the subject should be withdrawn for safety. 
In order to preserve the integrity of the intention-to-treat analysis, even if 
the subject is withdrawn from the treatment portion of the protocol (either 
due to subject, physician, or investigator decision), it is imperative to 
continue with the scheduled follow-up assessments both for the safety of 
the subject and for completeness of data collection. This will be explained 
to potential subjects at the time of informed consent. The importance of 
compliance with study visits will be reinforced throughout the trial.  
9.3 
Confidentiality 
In this study, each patient will be assigned a unique Participant ID number 
(PID) when his/her demographic and race/ethnicity information is entered for 
the first time. Follow-up data are subsequently entered as needed when a 
patient has a clinic visit. The unique PID number remains with each patient 
permanently and is matched with all new data entered. The PID number and 
patient identifiers are directly linked in the study database.  
 
Study personnel will also generate a Global Unique Identifier (GUID) for each 
subject using a NIH tool client. This is an identifying code assigned to a single 
research participant so that data can be compiled between research studies 
without using personally identifiable information (PII), even if the data are 
collected at different locations or by different studies. The GUID is created 
using PII (including, current name, legal given name given at birth (first, middle, 
and last), date of birth, city of birth, state of birth, country of birth, and physical 
sex at birth). Data including the GUID (without other identifiers) is considered 

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de-identified by the NIH and OHRP. Personal identifying information used to 
generate the GUID will be erased by the study staff after the GUID is created at 
the end of the study. 
 
The potential for data sharing has been included in the informed consent. Data 
releases to investigators for approved research purposes and analyses (after 
review and approval by the Publications and Presentations Committee and 
approval by IRB and execution of a Data Use Agreement) will be stripped of 
identifiers using a “Safe Harbor” approach. If an approved investigator has 
conducted a separate study in which a shared participant has also consented 
to use of GUID then this will be retained in the data release; however, for all 
other data releases the GUID will be removed.  
 
Information about study subjects will be kept confidential and managed 
according to the requirements of the Health Insurance Portability and 
Accountability Act of 1996 (HIPAA). Those regulations require a signed subject 
authorization informing the subject of the following: 
• 
What protected health information (PHI) will be collected from subjects 
in this study 
• 
Who will have access to that information and why 
• 
Who will use or disclose that information 
• 
The rights of a research subject to revoke their authorization for use of 
their PHI 
9.4 
Privacy 
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 the 
PI) 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.  
9.5 
Follow-up and Record Retention 
The duration of this study is estimated to be 1 year. 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.  
9.6 
Ethical Considerations 
This study is to be conducted in accordance with applicable US government 
regulations and international standards of Good Clinical Practice, and 
applicable institutional research policies and procedures.  

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This protocol and any amendments will be submitted to a properly constituted 
IRB in agreement with local legal prescriptions for formal approval of the study 
conduct. The decision of the IRB concerning the conduct of the study will be 
made in writing to the investigator and a copy of this decision will be provided 
to the NIH before commencement of this study. 
 
All subjects for this study will be provided a consent form describing this study 
and providing sufficient information for subject to make an informed decision 
about their participation in this study. This consent form will be submitted with 
the protocol for review and approval by the IRB for the study. The formal 
consent of a subject, using the IRB-approved consent form, must be obtained 
before that subject undergoes any study procedure. The consent form must be 
signed by the subject or legally acceptable surrogate, and the personnel 
obtaining the consent.  
 
 

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