Effects of Liothyronine on Energy Expenditure and Cardiovascular Function
Phase: PHASE1
Conditions: Healthy
Sponsor: Virginia Commonwealth University

 
 
Title: In vivo characterization of the non-genomic effects of T3 on endothelial and cardiovascular function 
 
NCT #: NCT03098433 
 
Document date: 01/19/2017 
 
Document Type: Protocol and Statistical Analysis Plan 
 
 
 

In vivo characterization of the non-genomic effects of T3 on endothelial and cardiovascular function 
 
Study protocol 
Research Description 
Hypothyroidism is a common condition caused by failure of the thyroid gland or as a result of thyroid 
surgery for benign or malignant lesions. Hypothyroidism is treated by administering levothyroxine (LT4) 
a synthetic inactive form of the thyroid hormone. This drug is then metabolized by the organism into its 
active form, T3.  
Several lines of evidence indicate that LT4 alone may not be sufficient in assuring adequate tissue levels 
of T3 and some studies suggest that the addition of liothyronine (LT3) the synthetic form of T3 to LT4 
can improve the symptoms and signs associated with hypothyroidism. LT3 has a short half life and it is 
usually administered in two-three doses daily. There is also evidence supporting the hypothesis that T3 
exerts rapid, “non genomic” effects, particularly on the vessels. If this is the case a tight regulation of the 
LT3 dose to maintain the serum concentration of T3 becomes of primary importance. 
To assess the non genomic effect of LT3 we will perform a proof-of-concept, double blind randomized 
crossover study in twenty healthy volunteers aimed to measure the acute cardiovascular and energy 
expenditure changes following a single equimolar dose of LT3 or LT4.  
Specific aims or goals. 
Aim 1: To compare the effects of a single dose of 0.7mcg/kg in liquid formulation (equivalent to a dose 
of 50 mcg in a 70 Kg individual) of LT3 to an equimolar 0.86 mcg/kg single dose of LT4 in liquid 
formulation, and to placebo on cardiovascular system. We will record the following parameters: heart 
rate, blood pressure, cardiac output, flow-mediated dilation. 
Aim 2: To compare the effects of a single dose of 0.7mcg/kg of LT3 in liquid formulation to an 
equimolar single dose of LT4 in liquid formulation, and to placebo on the energy metabolism and 
substrate utilization. We will record the following parameters: energy expenditure by whole room 
indirect calorimeter, respiratory quotient, skin and core temperature. 
Aim 3: To measure the changes in circulating thyroid hormone following a single dose of 0.7mcg/kg of 
LT3 in liquid formulation to an equimolar single dose of LT4 in liquid formulation, and to placebo. We 
will record at multiple time points the following parameters: free T3, free T4, TSH. 
Background and significance. 
Thyroid hormone (TH) affects development, growth and function on the majority of tissues of the 
organism and the specificity of the signaling is assured by a complex, multilevel system that assures 
time- and tissue-specificity of the hormonal signal relatively independent from the circulating levels(1). 
Among the various systems, the peripheral metabolism of pro-hormone T4, the main product of the 
thyroid, into the hormonally active T3 plays a crucial role in assuring the tissue-specific delivery of the 
hormonal signal(2). Two enzymes, type-1 (D1) and type-2 (D2) deiodinase, convert T4 into T3.  Of note, 
D1 is thought to be responsible for a substantial fraction of circulating T3(3). Hypothyroidism is an 
extremely prevalent condition affecting approximately up to 10% of the adult population and is 

associated with dyslipidemia, cognitive impairment, hypertension, and increased cardio-vascular 
morbidity and mortality(4, 5). Autoimmune thyroid disease (AITD) is the most common etiology of 
hypothyroidism in the US and it is characterized over a period of years by progressive loss of functional 
parenchyma. Since the thyrotroph is exquisitely sensitive to minimal changes in circulating TH, a 
compensatory increase in Thyroid Stimulating Hormone (TSH) secretion occurs while levels of TH are still 
well within the range of normality. This is illustrated by the classical linear-logarithmic relationship 
between free T4 and TSH(6). As the disease progresses, while the circulating T4 decrease to frankly 
pathologic levels, T3 tends to remain within normal range until extreme hypothyroidism ensues(7, 8). 
Similar conditions are observed in iodine insufficiency, and in experimental models of perturbation of 
the thyroid homeostasis(9). In aggregate, these observations suggest that multiple homeostatic 
mechanisms have developed to protect the circulating, and by extension the intracellular levels, of T3. 
Of interest, some of the hormonal actions of T3 are exerted via a rapid non-genomic pathway, which 
appears to play an important role in some of the cardiovascular effects of TH(10, 11). The therapy of 
hypothyroidism consists in administration of LT4, which in turn is converted into the active metabolite 
T3 in the peripheral tissues by D1 and D2. The therapeutic goal is a TSH within normal range, indicating 
the achievement of euthyroidism at the level of the thyrotroph on the assumption that a state of 
pituitary euthyroidism equates to adequate T3 concentration in all the tissue targets of the hormonal 
action(12). This assumption has been challenged by clinical observations(13), clinical trials(14), and 
mechanistic studies(15). The results of clinical studies aimed to measure the effects of TH replacement 
on “hard” endpoints (i.e. mortality and cardiovascular events) have been marginal or disappointing, 
particularly in subclinical hypothyroidism and in the elderly(16). This is surprising since the cross-
sectional data provide compelling evidence of the association between hypothyroidism and indexes of 
cardiovascular risk. Collectively these data question the utility of TH replacement, its therapeutic goals, 
the target population who could benefit from the intervention, and the modality of the treatment, and 
indeed there is active discussion on this very topic within the thyroidologist community(17). The 
attempt to provide a more “physiologic” form of therapy has led to the experimentation of 
combinationLT3 /LT4 regimens(13, 18-29), or to the use of thyroid extracts(30). Furthermore, some 
observations suggest that carriers of a common polymorphism of the D2 gene (Thr92Ala) (31)may 
benefit from combination therapy, suggesting a pharmacogenomics component in the therapeutic 
response(14, 32). This information indicate that the current therapy for hypothyroidism is costly and 
suboptimal, and there is need for a formal characterization of the pathophysiologic state of 
hypothyroidism during replacement treatment. 
This project challenges the established notion that LT4 generates identical biological effects on the 
pituitary and on the end-organ tissues, and pituitary euthyroidism (i.e. a normal TSH) is 
pharmacodynamically equivalent to production of TH from the normal thyroid gland. We will interrogate 
the clinical relevance of non-genomic effects of TH by assessing the short-term effects of a single-dose 
of LT3 on endothelial and cardiovascular function. This translational project will provide important 
information on the need (or lack thereof) of maintaining stable circulating levels of T3. In this clinical 
protocol, we chose as primary endpoints measurable and clinically relevant indices of TH action, which 
in turn could be used in the future as outcomes for large intervention studies. Specifically, beside 
measurements of circulating TH, we will assess energy expenditure(33, 34) and endothelial (35, 36)and 
cardiovascular function(37). The results of the proposed experiments will shed light on the interaction 
between the pathophysiologic state of hypothyroidism and administration of therapy, providing new 

insights on the clinical rationale and utility of targeting the circulating levels of T3. Collectively, these 
studies will expand the knowledge base necessary to design a rationale treatment of hypothyroidism. 
Study design. 
Proof-of-concept, randomized double blind cross-over study in healthy volunteers. Twenty adult healthy 
volunteers will be invited to participate in the study.  
A) Screening visit. Age >18<45 year, BMI >20<30 kg/m2 TSH >0.5<5.0 mcIU/mL and no evidence of 
thyroid autoimmunity by history or anti-thyroid peroxidase (TPO) antibodies measurement. Exclusion 
criteria will be: pregnancy; hypothyroidism or autoimmune thyroid disease by history or presence of anti 
TPO antibodies; use of prescription drugs; diabetes mellitus; dyslipidemia; coronary artery disease; 
hypertension (systolic blood pressure > 140 mm/Hg, and/or diastolic blood pressure > 90 mm/Hg); 
anemia; renal insufficiency; liver disease or ALT >2.5 x the upper laboratory reference limit; psychiatric 
conditions; and current tobacco use. During the screening visit, the following procedures will be 
performed: informed consent, history and physical examination, EKG, CBC, glucose, creatinine, ALT, hCG 
(in females), TSH, freeT4, TPO, and genomic DNA.  
B) Study visit #1-3. Study participants will be evaluated in the morning after an overnight fast during 
three consecutive outpatient visits, with at least a 24-hour interval between. Upon arrival to the Clinical 
Research Service Unit (CRSU) and abbreviated history and physical examination with recording of 
anthropometrics will be obtained; an i.v. cannula will be inserted in the left arm. Study volunteers will 
be fitted with electrodes for EKG Holter recording, and a cuff on the third finger of the right hand to 
record blood pressure and heart rate, cardiac output, systemic vascular resistance and endothelial 
function.  An echocardiogram will be recorded at the beginning and at the end of each study session. 
Study volunteers will then enter in the whole room (“Flex room”) a 4’ x 8’ x 7’ indirect calorimeter with a 
temperature set at 24°C and rest on a bed. The Flex room has a window and a TV monitor, and is 
connected to the nursing station via an interphone system. An i.v. port is located on the door of the Flex 
room allowing drawing of samples without compromising the recording of energy expenditure. After 40 
minutes (time 0’), study volunteers will receive either 0.7mcg/kg (equivalent to a dose of 50 mcg in a 70 
Kg individual) of LT3 to an equimolar 0.86 mcg/kg single dose of LT4, and to placebo in liquid form. The 
doses were chosen to be within the usual therapeutic range prescribed in preparation for radioiodine 
ablation. Blood samples will be obtained at the following time points: 0’, 60’ 120’ 180’ 240’ for total and 
free T3, total and free T4, TSH and 500 l aliquots of serum TSH will be recorded at times 0’ and 240’ of 
each Study visit. Energy expenditure and respiratory quotient will be recorded throughout the duration 
of the stay in the Flex room. The first 20 minute of the recording will be discarded to allow for 
equilibration. At the end of the session the i.v. cannula will be removed, vital signs recorded and the 
volunteer will be discharged provided with a coupon for a meal in the hospital cafeteria. The same 
procedures will be repeated in the identical order and timing for the other formulations. 
Justification for the sample size: 
The primary analysis will be performed by two-tailed paired t-test with an 0.05  error as a threshold for 
statistical significance on the assumption that LT3 administration, compared to LT4 will result in a 30% 
increase in FMD, corresponding to 0.75 SD. Using this experimental conditions and assumptions, a 20 
volunteers will provide 88% power to detect a significant difference, while assuming a 20% drop-out 
rate the power will be 80%. 

List the study inclusion criteria: 
Healthy  volunteers, both sexes 
Age range 18-45 year 
Body Mass Index range 20- 30 kg/m2  
Ability to provide informed consent to the participation in the study 
List the study inclusion criteria: 
Abnormal TSH (>0.5<5.0 mcIU/mL) 
Thyroid disease or autoimmunity by history or anti-thyroid peroxidase (TPO) antibodies.  
Pregnancy or lactation 
Chronic use of prescription drugs 
Diabetes mellitus (by history or fasting glucose >126 mg/dl) 
Dyslipidemia (total cholesterol >240 mg/dl) 
Hypertension ( by history or screening systolic blood pressure > 140 mm/Hg, and/or diastolic blood 
pressure > 90 mm/Hg) 
Cardiac arrhythmia (by history or screening EKG) 
Anemia (Hb<13.3 g/dl M, 12.0 g/dl F)  
Renal insufficiency by history or estimated creatinine clearance ≤ 60 mL/min/1.73m2 BSA. 
Liver disease or transaminases >2.5 x the upper laboratory reference limit.  
Current tobacco use. 
Criteria for withdrawing an individual participant from the study: 
Volunteers may withdraw from the study at any time. The investigators can withdraw any volunteer at 
any time from the study if medically necessary.  
Medical reasons for withdraw from the study: 
1) Tachycardia (heart rate greater than 100/min) 
2) Anxiety 
3) Development of hypertension (systolic >160 mmHg, diastolic > 90 mmHg) following 
administration of the study drug 
4) Development of hypotension (systolic <90 mmHg, diastolic < 60 mmHg) following administration 
of the study drug 
Adverse events mitigation: 
All AEs and SAEs will be followed until resolution, until the condition stabilizes, until the event is 
otherwise explained, whichever occurs first. All AEs and SAEs documented at a previous visit/contact 

and designated as ongoing, will be reviewed at subsequent visits/contacts, where the designation may 
remain ongoing. The investigator will ensure that the follow-up includes any supplemental investigations 
as may be indicated to elucidate the nature and/or causality of the SAE. This may include additional 
laboratory tests or investigations, histopathological examinations, or consultation with other health care 
professionals. SAEs that are ongoing at the time of the subjects final study visit/contact will be 
documented as ongoing. 
DSMP. 
The PI will be responsible for data and safety monitoring during the study. A physician not affiliated with 
the research proposal will serve as independent compliance officer and will review quarterly compliance 
with inclusion/exclusion criteria, gender and minority, adverse events. The results of the review will be 
reported to the PI and the IRB. 
 
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