Conditions: Subclinical Hypothyroidism
Keywords: Levothyroxine Liothyronine Thyrotropin Releasing Hormone Aging Hypothyroidism Thyroid diseases
Allocation: RANDOMIZED | Intervention model: CROSSOVER
Enrollment: 14
Healthy volunteers: no

Brief summary:
Subclinical hypothyroidism, defined as an elevated TSH in the setting of normal thyroid hormone levels, is a common diagnosis in the elderly. The purpose of this study is to examine the hypothalamic-pituitary-thyroid axis in men and women aged 70 years and older with persistent subclinical hypothyroidism. To evaluate the mechanism behind this condition, participants will undergo thyrotropin releasing hormone stimulation testing at 3 visits: baseline and while taking two different thyroid hormone preparations, levothyroxine and liothyronine. The investigators will also assess physiologic responses to these two different thyroid hormone medications to help us understand how the thyroid works in advanced age.

Detailed description:

Arms:
arm 1: Participants will start on the thyroid hormone Levothyroxine prior to crossing over to Liothyronine arm 2: Participants will start on the thyroid hormone Liothyronine prior to crossing over to Levothyroxine

Interventions: intervention 1: Levothyroxine intervention 2: Liothyronine intervention 3: Thyrotropin-Releasing Hormone
Intervention descriptions: intervention 1: Oral levothyroxine with a total starting dose of 0.7mcg/kg/day split into three daily doses will be titrated to a target TSH level of 0.5 -1.5mU/L intervention 2: Oral liothyronine with a starting dose of 1/3 of the weight-based LT4 dose divided into three daily doses titrated to a target TSH level of 0.5 -1.5mU/L intervention 3: 200 µg intravenous TRH will be given at study visit 1 (baseline), study visit 2 (on first thyroid treatment), and study visit 3 (on second thyroid treatment).

Protocol document:
Prot_SAP_000.pdf: 
 
CONFIDENTIAL 
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sponsor 
CLINICAL RESEARCH 
 
 
 
NCT02399475 
 
 
 
The Thyroid Axis in Older Individuals with Persistent Subclinical 
Hypothyroidism: a Mechanistic, Randomized, Double-Blind, 
Cross-Over Study of Levothyroxine and Liothyronine 
Administration 
 
 
Regulatory Sponsor: 
Anne R. Cappola, MD, ScM 
Division of Endocrinology, Diabetes, and Metabolism 
12-136 Translational Research Center 
3400 Civic Center Blvd, Bldg 421 
Philadelphia, PA 19104-5160 
(215) 573 5359 
 
 
Funding Sponsor: 
National Institute on Aging, NIH 
 
Medications Used: 
Thyrotropin Releasing Hormone (TRH) 
Levothyroxine (LT4) 
Liothyronine (LT3) 
Protocol Number: 
821564 
IND Number: 
125167 
Initial version:[11/26/2014]               Amended [7/22/2015]  
Amended: 
 [12/18/2014]               Amended [9/9/2015] 
Amended: 
 [1/2/2015]  
    Amended [8/25/2016] 
Amended: 
 [3/26/2015]                Amended [12/23/2016] 
Amended: 
 [5/13/2015]  
    Amended [3/24/2017] 
Amended:       [06/04/2015]               Amended [9/26/2017] 
Amended:  
 [6/10/2015]                 Amended [12/18/2017] (additional next page) 
Amended [12/18/18]     
 
 
 
 
 
 


LT4 and LT3 in Subclinical Hypothyroidism  
 
 
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Table of Contents 
 
STUDY SUMMARY ......................................................................................................... 1 
1 
INTRODUCTION ...................................................................................................... 3 
1.1 
BACKGROUND ..................................................................................................... 3 
1.2 
INTERVENTIONAL AGENTS .................................................................................... 4 
1.3 
PRECLINICAL DATA .............................................................................................. 4 
1.4 
CLINICAL DATA TO DATE ...................................................................................... 5 
1.5 
DOSE RATIONALE AND RISK/BENEFITS .................................................................. 6 
2 
STUDY OBJECTIVES ............................................................................................. 8 
3 
STUDY DESIGN ...................................................................................................... 9 
3.1 
GENERAL DESIGN ................................................................................................ 9 
3.2 
PRIMARY STUDY ENDPOINTS .............................................................................. 10 
3.3 
SECONDARY STUDY ENDPOINTS ......................................................................... 10 
3.4 
PRIMARY SAFETY ENDPOINTS ............................................................................ 10 
4 
SUBJECT SELECTION AND WITHDRAWAL ...................................................... 11 
4.1 
INCLUSION CRITERIA .......................................................................................... 11 
4.2 
EXCLUSION CRITERIA ........................................................................................ 11 
4.3 
SUBJECT RECRUITMENT AND SCREENING ............................................................ 12 
4.4 
EARLY WITHDRAWAL OF SUBJECTS .................................................................... 12 
4.4.1 
When and How to Withdraw Subjects ...................................................... 12 
4.4.2 
Data Collection and Follow-up for Withdrawn Subjects ........................... 12 
5 
STUDY DRUGS ..................................................................................................... 13 
5.1 
DESCRIPTION .................................................................................................... 13 
5.2 
TREATMENT REGIMEN ........................................................................................ 13 
5.3 
METHOD FOR ASSIGNING SUBJECTS TO TREATMENT GROUPS .............................. 15 
5.4 
PREPARATION AND ADMINISTRATION OF STUDY DRUG .......................................... 15 
5.5 
SUBJECT COMPLIANCE MONITORING ................................................................... 16 
5.6 
PRIOR AND CONCOMITANT THERAPY ................................................................... 16 
5.7 
PACKAGING ...................................................................................................... 16 
5.8 
BLINDING OF STUDY DRUG ................................................................................. 16 
5.9 
RECEIVING, STORAGE, DISPENSING AND RETURN ................................................ 17 
5.9.1 
Receipt of Drug Supplies ......................................................................... 17 
5.9.2 
Storage .................................................................................................... 17 
5.9.3 
Dispensing of Study Drug ........................................................................ 17 
5.9.4 
Return or Destruction of Study Drug ........................................................ 17 
6 
STUDY PROCEDURES ......................................................................................... 17 
6.1 
SCREENING VISIT (DAY -60 TO DAY -1) ............................................................... 17 
6.2 
VISIT 1 (DAY 1) ................................................................................................. 18 
6.3 
DAY 2-WEEK 4 .................................................................................................. 19 
6.4 
MEDICATION TITRATION (Q4WEEKS) .................................................................. 19 
6.5 
VISIT 2 ............................................................................................................. 19 


LT4 and LT3 in Subclinical Hypothyroidism  
 
 
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6.6 
VISIT 3 ............................................................................................................. 20 
6.7 
PHONE CONTACTS ............................................................................................ 20 
6.8 
ASSAYS ............................................................................................................ 20 
7 
STATISTICAL PLAN ............................................................................................. 21 
7.1 
SAMPLE SIZE DETERMINATION ............................................................................ 21 
7.2 
STATISTICAL METHODS ...................................................................................... 21 
7.3 
SUBJECT POPULATION(S) FOR ANALYSIS ............................................................. 21 
8 
SAFETY AND ADVERSE EVENTS ....................................................................... 21 
8.1 
DEFINITIONS ..................................................................................................... 21 
8.2 
RECORDING OF ADVERSE EVENTS ...................................................................... 24 
8.3 
REPORTING OF SERIOUS ADVERSE EVENTS AND UNANTICIPATED PROBLEMS ........ 24 
8.3.1 
Investigator reporting: notifying the study sponsor ................................... 24 
8.3.2 
Investigator reporting: notifying the Penn IRB .......................................... 25 
8.3.3 
Sponsor reporting: Notifying the FDA ...................................................... 26 
8.4 
UNBLINDING PROCEDURES ................................................................................. 27 
8.5 
STOPPING RULES .............................................................................................. 27 
8.6 
MEDICAL MONITORING ....................................................................................... 28 
9 
DATA HANDLING AND RECORD KEEPING ....................................................... 28 
9.1 
CONFIDENTIALITY .............................................................................................. 28 
9.2 
SOURCE DOCUMENTS ........................................................................................ 28 
9.3 
CASE REPORT FORMS ....................................................................................... 28 
10 
STUDY MONITORING, AUDITING, AND INSPECTING ................................... 29 
10.1 
STUDY MONITORING PLAN ................................................................................. 29 
10.2 
AUDITING AND INSPECTING ................................................................................. 30 
11 
ETHICAL CONSIDERATIONS ........................................................................... 30 
12 
STUDY FINANCES ............................................................................................ 31 
12.1 
FUNDING SOURCE ............................................................................................. 31 
12.2 
CONFLICT OF INTEREST ..................................................................................... 31 
12.3 
SUBJECT STIPENDS OR PAYMENTS ..................................................................... 31 
13 
PUBLICATION PLAN ........................................................................................ 31 
14 
REFERENCES ................................................................................................... 31 
15 
ATTACHMENTS ................................................................................................ 33 
16 
APPENDIX ......................................................................................................... 33 
16.1 
APPENDIX 1 ...................................................................................................... 33 
16.2 
APPENDIX 2 ...................................................................................................... 34 
16.3 
APPENDIX 3 ...................................................................................................... 34 
 
 
 
 
 


LT4 and LT3 in Subclinical Hypothyroidism  
 
 
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List of Abbreviations 
 
 
AE 
Adverse event 
AUC 
Area under the curve 
BMI 
Body mass index 
CHS 
Cardiovascular Health Study 
CTRC 
Clinical and Translational Research Center 
CVD 
Cardiovascular disease 
ECG 
Electrocardiogram 
FDA 
FT4 
Food and Drug Administration 
Free T4 
HUP 
Hospital of the University of Pennsylvania 
IDS 
Investigational Drug Service 
IRB     
Institutional review board 
IV              Intravenous 
LFTs         Liver function tests 
LT3 
Liothyronine  
LT4 
Levothyroxine 
mRNA 
Messenger ribonucleic acid 
NHANES National Health and Nutrition Examination Survey 
NIA 
National Institute on Aging 
NIDDK 
National Institute of Diabetes and Digestive and Kidney Diseases 
NIH 
National Institutes of Health 
PHI            Protected health information 
REE 
Resting energy expenditure 
SAE 
SD            
Severe adverse event 
Standard deviation 
SEM 
SF 
Standard error of the mean 
Short form 
T3 
Triiodothyronine 
T4 
Thyroxine 
TPO 
Thyroid peroxidase  
TRH 
Thyrotropin releasing hormone  
TSH          Thyroid stimulating hormone 
TSHmax 
Maximum serum TSH concentration  


LT4 and LT3 in subclinical hypothyroidism  
 
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Study Summary 
Title 
The Thyroid Axis in Older Individuals with Persistent Subclinical 
Hypothyroidism: a Mechanistic, Randomized, Double-Blind, 
Cross-Over Study of Levothyroxine (LT4) and Liothyronine (LT3) 
Administration 
Short Title 
 LT4 and LT3 in Subclinical Hypothyroidism 
Protocol Number 
821564   
Phase 
 
Methodology 
Randomized, Double-Blind, Cross-Over Study 
Study Duration 
Three years 
Study Center(s) 
Single-center 
Objectives 
To examine the pituitary-thyroid axis in older individuals with 
persistent subclinical hypothyroidism utilizing dynamic testing 
with thyrotropin releasing hormone (TRH) stimulation under 
different therapeutic conditions of either LT4 or LT3. We will 
measure thyroid stimulating hormone (TSH) area under the curve 
(AUC), TSHmax, change in TSH bioactivity, and change in free T4 
and total T3 levels. All outcomes will be assessed at baseline and 
after achieving target TSH levels with each thyroid preparation.  
Number 
of 
Subjects 
Thirty 
Diagnosis 
and 
Main 
Inclusion 
Criteria 
Men and women aged 70 and older with persistent subclinical 
hypothyroidism 
Study 
Product, 
Dose, 
Route, 
Regimen 
1. Intravenous TRH 200 µg at study visit 1 (baseline), study visit 
2 (on first thyroid treatment), and study visit 3 (on second thyroid 
treatment) 
2. Oral LT4 with a total starting dose of 0.7mcg/kg/day split into 
three daily doses and titrated to a target TSH level of 0.5 -1.5mU/L 
3. Oral LT3 with a starting dose of 1/3 of the weight based LT4 
dose divided into three daily doses titrated to a target TSH level 
of 0.5 -1.5mU/L  
Duration 
of 
administration 
1. Three single administrations of TRH (at baseline study visit 1, 
study visit 2 on the first thyroid treatment, and study visit 3 on the 
second thyroid treatment) 
 
2. LT4 taken as divided doses three times daily  
  
3. LT3 taken as divided doses three times daily  
4. For LT4 and LT3, there will be medication adjustments every 
four weeks as needed until the TSH level is at goal.  


LT4 and LT3 in subclinical hypothyroidism  
 
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Reference therapy 
None 
Statistical 
Methodology 
Changes in TRH stimulation test parameters and other outcomes 
will be compared from baseline for each thyroid hormone 
preparation (LT4 or LT3) via paired t-tests and between treatment 
arms via two-tailed tests. AUC for TRH stimulated TSH response 
will be calculated. Nonparametric tests will be employed as 
needed. 
 


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1 Introduction 
This document is a protocol for a human research study. This study is to be conducted 
according to US and international standards of Good Clinical Practice (FDA Title 21 part 
312 and International Conference on Harmonization guidelines), applicable government 
regulations, and Institutional research policies and procedures.  
1.1 Background 
Thyroid hormone is important for multiple physiologic 
functions, and insufficiency and excess of thyroid hormone 
are each pathologic conditions. Thyroid hormone is tightly 
regulated through a feedback loop between the hypothalamic 
hormone thyrotropin releasing hormone (TRH), the pituitary 
hormone thyroid stimulating hormone (TSH), and the thyroid 
hormones thyroxine (T4) and triiodothyronine (T3) (Fig. 1, 
from [1]). Individuals with normal thyroid function maintain 
TSH levels in a narrow range, though the setpoint for thyroid 
hormone levels varies between individuals.  
 
With increasing age, there is a progressive shift 
toward higher TSH levels in people who have no 
detectable underlying thyroid disease  
(Fig. 2, from [2]). This leads to a higher prevalence of 
subclinical 
hypothyroidism 
in 
older 
people. 
Subclinical hypothyroidism is defined as elevated 
TSH levels with normal levels of thyroid hormones. It 
is a common clinical diagnosis affecting 10-15% of 
individuals aged 65 years and older and up to 20% of 
those 85 years and older.[1-5] The purpose of this 
study is to examine mechanisms to explain the age-
related increase in TSH levels and resulting 
subclinical hypothyroidism.  
 
In addition, it is not clear if individuals with subclinical hypothyroidism benefit from 
replacement of thyroid hormone. Some argue that subclinical hypothyroidism should be 
treated as a form of mild hypothyroidism, whereas others argue that it may be adaptive. 
The question is whether, in older people, a TSH level just outside the reference range 
should be considered a pathologic finding. We hypothesize that slightly elevated TSH 
results from an age-related decrease in the function of TSH that leads to an adaptive shift 
in the pituitary-thyroid setpoint, rather than an early sign of thyroid failure. That is, the 
TSH that is produced by the pituitary gland may become less biologically active with older 
age. More TSH would then be required to keep thyroid hormone level production 
constant. 
 
There is evidence to support the biologic plausibility of a decline in TSH bioactivity with 
age. TSH with decreased bioactivity has been detected using cell-based methodologies, 
and has been reported in conditions of thyroid axis dysfunction, ranging from primary and 
Figure 2 
Figure 1 


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secondary hypothyroidism to TSH-producing pituitary adenomas.[3-6] An inverse 
correlation between TSH bioactivity and age was also found in 11 healthy men and 
women aged 28-52 years.[4]  
 
Changes in TSH structure including glycosylation patterns affect its biologic activity.[7] 
These changes in glycosylation could be due to alterations in TRH output from the 
hypothalamus, as is found with age for other hypothalamic hormones such as GHRH and 
GnRH. [8, 9] Unfortunately, TRH cannot be measured directly; however, TSH bioactivity 
can be measured before and after TRH stimulation testing to provide indirect evidence of 
this mechanism. We can then create perturbations to the system such as adding back 
thyroid hormones to understand these hormonal feedback loops. Together the TRH 
stimulation testing and the crossover design with T4 and T3 add back will provide insight 
into the dynamics of the hypothalamic-pituitary-thyroid axis in older people with subclinical 
hypothyroidism. 
1.2 Interventional Agents 
TRH, a naturally occurring hypothalamic neuropeptide in humans, is an endogenous 
ligand for the TRH receptor. It is composed of three amino acids and stimulates the 
release of TSH and prolactin from the anterior pituitary. The release of TSH then 
stimulates the release of thyroid hormones, T4 and T3 (Fig. 1).  Synthetic TRH has been 
used for over 40 years in the diagnostic assessment of pituitary or hypothalamic 
dysfunction.  
 
Intravenous TRH has a short half-life with a mean of approximately 5 minutes in normal 
subjects. Bassiri and Utiger administered 400 µg TRH to 8 healthy men and noted that 
plasma TRH levels rose from <0.4 ng/mL to 33 ± 7 ng/mL (mean ± SEM) 2 minutes after 
injection and linearly fell to 2.9 ng/mL 20 min after injection. The mean half-life of TRH 
was found to be 5.3 ± 0.5 min.[10]. TSH levels rise rapidly; reach a peak, and later decline 
after TRH administration. Anderson et al administered 100-800 µg TRH to 79 healthy men 
and found that TRH-stimulated TSH levels peaked after 30 min and fell to baseline levels 
in approximately 3 hours.[11] 
 
Levothyroxine (LT4) and liothyronine (LT3) are synthetic forms of T4 and T3, which are 
endogenous hormones produced by follicular cells of the thyroid gland in response to 
stimulation by TSH from the anterior pituitary. The majority of circulating thyroid hormone 
in the blood is in the form of T4. T4 is then converted to T3 in target tissues, with T3 being 
the more physiologically active hormone. Both T4 and T3 bind to the thyroid receptor in 
cell nuclei and regulate DNA transcription and protein synthesis. Human studies show 
that thyroid hormone levels can also alter TSH bioactivity. [4, 6, 12] T4 is metabolized 
slowly with eighty percent of circulating T3 derived from T4 through deiodination. The 
half-life of LT4 is six to eight days. The half-life of LT3 is one to two days.[13-15]  
 
1.3 Preclinical Data 
Slightly elevated TSH may result from an age-related alteration in the function of TRH, 
analogous to the age-related alterations found in other hypothalamic hormones such as 


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GHRH and GnRH.[8, 9] Preclinical data show that alterations to the structure and 
packaging of the TSH molecule may affect its function. TSH is a glycoprotein composed 
of an alpha and a beta subunit that requires glycosylation of three asparagine residues in 
order to have maximal function at the TSH receptor.[16] When the glycosylation pattern 
is abnormal, TSH binding to its receptor is less efficient, resulting in decreased bioactivity. 
These changes in glycosylation could be due to alterations in TRH output from the 
hypothalamus, as suggested by animal models.[7, 17-19] Taylor et al. showed that 
hypothalamic as compared to primary hypothyroidism caused by lesions in the rat 
paraventricular nuclei alters carbohydrate structure of secreted thyrotropin. Taylor and 
Weintraub later showed that this alteration in TSH carbohydrate structure could be 
corrected by administering TRH in vivo.[19] Hypothalamic TRH output cannot be 
measured directly. However, TSH bioactivity can be measured before and after TRH 
stimulation testing to provide indirect evidence of this mechanism.  
 
In addition to TRH, there is evidence that thyroid hormones themselves can alter the 
bioactivity of thyroid stimulating hormone. Thyroid hormone status has been shown to 
modulate glycosyltransferase mRNA levels in mouse thyrotrophs.[20] This has been 
postulated to result from a direct action of T3 on thyroid hormone responsive elements in 
the upstream regulatory region of the sialyltransferase gene. If so, TSH glycosylation 
could be affected by the observed age-associated decline in T3 levels.  
 
Rodent data indicate that replacement therapy with LT4 alone does not achieve adequate 
levels of T3 in all tissues.[21] However, combined replacement therapy of LT3 to LT4 
completely restored euthyroidism in thyroidectomized rats at much lower doses of LT4 
than those needed to normalize LT3 in most tissues when L-T4 alone was used. The LT4-
LT3 cross-over design will give us insight into this mechanism.  
 
1.4 Clinical Data to Date 
In men and women from the Cardiovascular Health Study with a mean age of 74 years at 
the time of the initial TSH, we found a gradual increase in TSH levels over a 13-year 
period, with an average increase of 0.28 mU/L (p<0.01) and no change in free T4 levels, 
suggesting a shift in the pituitary-thyroid setpoint. These data parallel the 0.32 mU/L TSH 
increase over a 13-year period published in a younger population (mean age 58 years at 
the second TSH), also without change in free T4 levels.[22] NHANES data also illustrate 
a progressive shift toward higher TSH levels with increasing age [2] and another study 
has shown higher TSH in people with exceptional longevity.[23] 
 
The hypotheses that there is a decline in TSH bioactivity with age and that thyroid 
hormone supplementation can alter bioactivity are supported by data in humans [4, 6, 
12]. Oliveira et al. evaluated in vitro serum TSH bioactivity and found a strong inverse 
correlation between serum TSH bioactivity and age in 11 normal controls. This study also 
showed a decrease in TSH bioactivity in patients with primary hypothyroidism. The 
bioactivity improved with levothyroxine supplementation and was positively correlated 
with change in T3 levels but not free T4 levels. Evaluation of TSH glycosylation patterns 
showed a higher proportion of sialylated TSH molecules in patients with decreased 


LT4 and LT3 in subclinical hypothyroidism  
 
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bioactivity, which decreased after LT4 replacement.[4] Increase in sialylation of the TSH 
molecule was also shown in patients with central hypothyroidism due to Sheehan’s 
syndrome [3] and chronic TRH administration in patients with central hypothyroidism has 
been shown to improve TSH bioactivity.[24] These data suggest that hypothalamus-
pituitary-thyroid feedback loops are regulated by TSH bioactivity and thyroid hormone 
levels. 
 
1.5 Dose Rationale and Risk/Benefits 
TRH stimulation testing:  
TRH has a short half-life and does not exhibit a sustained biological effect after a single 
intravenous bolus. For decades, TRH has been intravenously administered to a variety 
of study populations, including patients with thyroid, pituitary, and hypothalamic 
disorders.[10, 11, 25] In this study we will be using the standard clinical dose of 200 µg 
at three time points. This dose has been used in studies of dynamic pituitary testing 
utilizing TRH stimulation testing.[25, 26] Side effects have been reported in about 50% of 
patients, but side effects are generally minor and persist for only a few minutes following 
injection.  
 
These side effects include[27]: 
 
Most frequent: transient nausea, flushing, palpitations (without change in heart rate), 
lightheadedness, bad taste in mouth, abdominal discomfort, dry mouth, and an urge to 
urinate 
 
Less frequent: anxiety, sweating, tightness in throat (without change in oxygenation or 
respiration), pressure in chest, tingling sensation, and drowsiness 
 
Uncommon: change in blood pressure, seizure  
 
The mean elevation of blood pressure reported was 16 mm of mercury. The peak blood 
pressure elevation occurred between one and five minutes, with a return to baseline blood 
pressure levels within ten minutes.[11] Participants with uncontrolled blood pressure or 
history of ischemic cardiac disease or stroke will be excluded. 
 
Seizures have only been reported in those with a history of seizure disorder. Individuals 
with a history of seizure will be excluded. 
 
No direct benefits are expected to result from TRH administration. 
 
 
LT4 and LT3:  
 
LT4 and LT3 have each been in therapeutic use for more than 50 years. Adverse 
reactions to either drug are exclusively related to excessive thyroid hormone, which can 
produce iatrogenic thyrotoxicosis. No serious adverse events were reported in a 


LT4 and LT3 in subclinical hypothyroidism  
 
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published LT4/LT3 crossover study, and no symptoms of hyperthyroidism or 
hypothyroidism were reported.[25, 28] Participants will be asked about hyperthyroid 
symptoms, and a TSH level will be checked prior to the planned date if a patient reports 
any hyperthyroid symptoms.  
 
Administration and monitoring of LT4 and LT3 will be modeled off of a published 
protocol.[28] The dosing of thyroid hormone will be based on published studies of 
thyroid hormone replacement in subclinical hypothyroidism. The literature supports a 1 
mcg/kg/day dose for initiation of therapy.[29-34]  We will tailor the thyroid hormone dose 
to each subject using a weight-based dosing scheme that is also based on how far the 
subject’s TSH level is from goal. The dosing scheme is conservative. 1mcg/kg is the 
dose reported in the literature. The initial dose in our study will be lower than that - 
0.7mcg/kg/day given that we are studying subjects 70 years and older. We will use 
thrice daily dosing due to the short half-life of LT3.  
 
LT4 
Each participant will start on 0.7mcg/kg/day divided into three daily doses. 
The dose will be adjusted according to subject weight and how far the TSH level is from 
goal.  There will be a medication cap such that no dose adjustment will exceed 25 mcg 
per day. 
 
LT3 
The starting dose of liothyronine will be 1/3 of that of levothyroxine divided into three 
daily doses. We will titrate the dose according to subject weight and how far the TSH 
level is from goal.  No dose adjustment will be greater than 8.3 mcg per day.  
 
The total daily dose of study medications will be adjusted to a TSH of 0.5-1.5 mU/L 
using the adjustment scheme to avoid excess thyroid hormone replacement.  
 
At the time of crossover of study drug, participants will undergo a 4 week wash-out period 
from the study drug. This is to avoid over-replacement when switching from LT4 to LT3, 
given the longer half-life of LT4. Following Visit 2, participants will stop all study drug for 
4 weeks. At the end of 4 weeks they will begin the second study drug at the equivalent 
dose as they were taking at Visit 2, divided into 3 equal doses. TSH level will not be 
checked at the end of the wash-out period.  A TSH level will be checked 4 weeks after 
crossing over to the new study drug. All further TSH level evaluations and medication 
adjustments will follow the titration protocol. 
 
No direct benefits are expected to result from LT4 and LT3 administration. 
 
The weight based dosing scheme and examples are displayed below: 
 
LT4 Titration Scheme:  
 


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TSH (mU/L) 
>4.50 
3.51-4.50 
2.51-3.50 
1.51-2.50 
0.50-1.50 
0.20-0.49 
<0.20 
Dose change 
(mcg/kg/day) 
0.25 
0.21 
0.17 
0.1 
GOAL: 
No 
change 
-0.08 
-0.09 
 
Example of LT4 titration in a 50, 70, & 100 kg individual 
 
 
TSH 
(mU/L) 
>4.50 
3.51-
4.50 
2.51-
3.50 
1.51-2.50 
0.50-1.50 
0.20-
0.49 
<0.20 
Weight 
(kg) 
Start dose 
(0.7mcg/k
g/day) 
Dose Change: Total mcg change per day 
50 
35 
12.5 
10.5 
8.5 
5 
GOAL: No 
change 
-4 
-4.5 
70 
49 
17.5 
14.7
11.9 
7 
-5.6 
-6.3 
100 
70 
25 
21 
17 
10 
-8 
-9 
 
 
LT3 Titration Scheme: LT4 Dose scheme divided by 3  
 
TSH (mU/L) 
>4.50 
3.51-4.50 
2.51-3.50 
1.51-
2.50 
0.50-1.50 
0.20-0.49 
<0.20 
Dose change 
(mcg/kg/day) 
0.08 
0.07 
0.06 
0.03 
GOAL: 
No 
change 
-0.027 
-0.03 
 
 
Example of LT3 titration in a 50, 70, & 100 kg individual 
 
 
TSH 
(mU/L) 
>4.50 
3.51-
4.50 
2.51-
3.50 
1.51-
2.50 
0.50-
1.50 
0.20-0.49 
<0.20 
Weight (kg) 
Start dose  
1/3 LT4 Dose 
Dose Change: Total mcg change per day 
50 
11.7 
4.17 
3.5 
2.83 
1.67 
GOAL: 
No 
change 
-1.33 
-1.50 
70 
16.3 
5.83 
4.9
3.97 
2.33 
-1.87 
-2.10 
100 
23.3 
8.3 
7 
5.67 
3.33 
-2.67 
-3.00 
 
2 Study Objectives 
To understand the function of the thyroid axis in subclinical hypothyroidism, by examining 
the thyroid axis in older individuals with persistent subclinical hypothyroidism under 
dynamic conditions.  
 
Primary Objective: 


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To assess the adaptability of the thyroid axis to perturbations from TRH stimulation or 
thyroid hormone supplementation via assessment of TRH stimulation test parameters 
(TSH AUC, TSHmax, change in TSH bioactivity, change in free T4, change in total T3) at 
baseline and after achieving target TSH levels with each thyroid preparation. 
 
Secondary Objectives: 
To assess changes in between baseline and each thyroid preparation in: 
1) unstimulated TSH bioactivity 
2) resting energy expenditure (REE) 
3) body temperature 
4) weight 
5) body composition 
6) lipids  
7) bone density 
8) heart rate 
9) physical activity  
10) hypothyroid and hyperthyroid symptoms  
11) health status and well-being assessment with SF-36 
12) cognitive testing with digit symbol substitution test 
13) arterial tonometry, doppler echocardiography, and arterial load assessment 
3 Study Design 
3.1 General Design 
The study design is a randomized, double-blind, cross-over study in which we will 
examine the pituitary-thyroid axis in older subclinical hypothyroid individuals in response 
to TRH stimulation and to daily LT4 or LT3 supplementation over a minimum period of 14 
consecutive weeks. Physiologic responses to LT4 and LT3 supplementation will also be 
examined. Thirty participants will receive a total of three TRH stimulation tests and one 
course each of LT4 and LT3 in randomized order.  
 
 
All study visits will be at the University of Pennsylvania’s Clinical and Translational 
Research Center (CTRC). There will be a screening visit (Day -60 to Day -1), a baseline 
Figure 4 


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study visit (Visit 1; Day 1), and a final study visit while taking their first thyroid preparation 
(Visit 2). Following the completion of Visit 2, participants will cross-over to the second 
thyroid preparation and complete Visit 3, as above.  
 
On Day 1 (baseline study visit, Visit 1) the participants will undergo their first TRH 
stimulation test and receive written instruction on thyroid hormone dosing by a study team 
member. The participant will begin oral thyroid supplementation as divided doses three 
times per day for four weeks starting the morning after the baseline study visit. After four 
weeks, the participants will have their TSH level checked at a Penn outpatient laboratory, 
after which LT4 or LT3 will be titrated to a target TSH level of 0.5-1.5 mU/L by a blinded 
study investigator. TSH levels will continue to be checked at four-week intervals until the 
TSH level is at goal, after which, Visit 2 will be scheduled. At Visit 2, a TRH stimulation 
test will be performed and participants will cross-over to the other thyroid preparation. The 
sequence of TSH assessment and thyroid preparation titration will occur with this thyroid 
preparation. At Visit 3, a TRH stimulation test will be performed and the participant will 
complete the study. Physiologic endpoints will be measured at each study visit.  
3.2 Primary Study Endpoints 
Changes in TRH stimulation test parameters (listed below) will be measured at each study 
visit: 
1. TSH area under the curve (AUC)  
2. TSHmax 
3. Free T4 
4. Total T3 
5. TSH bioactivity measured by an in-vitro cell-based bioactivity assay [35]  
3.3 Secondary Study Endpoints 
The following endpoints will be measured at each study visit: 
1. Unstimulated TSH bioactivity 
2. Resting energy expenditure (REE) evaluation by indirect calorimetry  
3. Weight  
4. Body composition including body fat (percent and total), free fat mass, and bone 
density measured by dual energy x-ray absorptiometry (DXA) 
5. Lipid profile via laboratory measurements 
6. SF-36 for health status, hypothyroid symptoms (Appendix 2) 
7. Heart rate and physical activity via actigraphy 
8. Digit symbol substitution test 
9. Body temperature using rectal temperature measurement 
10. Optional exploratory genetic testing (one study visit only) 
11. arterial tonometry, Doppler echocardiography, and arterial load assessment 
3.4 Primary Safety Endpoints 
Participants will undergo ECG testing at the screening visit so that a baseline EKG is on 
file. Hyperthyroid symptoms will be assessed to monitor participant safety. Symptoms of 
palpitations and shortness of breath will be assessed every two weeks by telephone while 


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the participant is on study drug. See Appendix 1 for the hyperthyroid symptom lists for 
phone contacts and study visits.  
4 Subject Selection and Withdrawal 
4.1 Inclusion Criteria 
We will include men and women aged 70 and older who are able to provide informed 
consent and have a TSH between 4.5 and 19.9 mU/L in conjunction with a normal free 
thyroxine (FT4) level.  
4.2 Exclusion Criteria 
Laboratory Tests: 
1. thyroid peroxidase (TPO) antibody positive 
3. abnormal liver function tests (LFTs >3 x upper limit of normal) 
4. hemoglobin <11 g/dL 
 
Surgeries or Procedures: 
 
1. thyroid surgery 
2. pituitary surgery 
3. bariatric surgery 
4. Bowel resection involving the jejunum and upper Ileum  
5. radioactive iodine therapy 
6. radiation treatments to head or neck 
 
Medical Conditions: 
1. diagnosis of pituitary disease 
2. diagnosis of amyloidosis, sarcoidosis, hemochromatosis 
3. diagnosis of adrenal insufficiency 
4. obesity with BMI > 35 mg/kg2 
5. history of stroke 
6. chronic or ongoing angina, Class II or higher congestive heart failure, or uncontrolled 
hypertension with current blood pressure greater than 160/100 
7. diabetes mellitus with hemoglobin A1C level greater than 8.0% in the past six months 
8. celiac sprue, Crohn’s disease, ulcerative colitis, Zollinger-Ellison syndrome 
9. renal insufficiency with calculated glomerular filtration rate <45 cc/min 
10. cognitive impairment with Mini Mental State Exam[36] <24/30 
11. history of any seizures 
12. unstable medical or psychological condition in the judgment of the principal 
investigator 
 
Medications:  
 
1. thyroid hormone preparations 
2. antithyroid drugs  
3. medications that interfere with the absorption or metabolism of thyroid hormone 


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4. medications that interfere with the TRH stimulation test 
5. proton pump inhibitors 
 
Please see Appendix 3 for full list of medications 
 
Exogenous estrogen and testosterone is not an exclusion, but all participants will be 
required to remain on the same dose throughout the study.  
4.3  Subject Recruitment and Screening 
Study participants will be recruited from the general medicine, geriatrics, and endocrine 
clinics of the University of Pennsylvania Health System. We will use the Penn Data Store, 
a data warehousing and reporting entity containing patient related information extracted 
from five major clinical electronic systems. The Data Store can be queried by tailored 
request using multiple different parameters, including age, diagnosis, medication, 
laboratory result, and clinic over a specified time frame. Selected providers of individuals 
who meet our inclusion criteria and are not taking thyroid medication will be contacted by 
the Investigator about the study protocol and recruitment efforts in order to obtain 
potential participants.  Penn Research Trial Advisory (electronic alerting in EPIC) will also 
be used for contacting potential participants. The pop-up would appear in EPIC screens 
of participating practice providers during patient visits. If the potential subject agrees, their 
information would be relayed to the study coordinator via the EPIC system.  I-connect, a 
novel Clinical Trial search and match technology will also be utilized.  A study recruitment 
poster will be displayed on University bulletin boards and at University physician 
practices. The Penn Medicine biobank will be utilized as a source of subject identification. 
Once identified by way of these recruitment methods, verbal contact with potential 
participants, utilizing incoming and outgoing telephone recruitment scripts will be 
followed.  
4.4 Early Withdrawal of Subjects 
4.4.1 When and How to Withdraw Subjects 
The protocol defined follow-up period per subject will range from 14-78 weeks. A subject 
may be withdrawn from the study prior to his or her expected completion date for the 
following reasons: 
 
1. If the investigator discovers a condition(s) which indicates unacceptable risk to the 
subject 
2. If the subject fails to adhere to the protocol requirements 
3. If the subject withdraws consent from participation in the study 
4.4.2 Data Collection and Follow-up for Withdrawn Subjects 
Attempts will be made to obtain at least survival data on these subjects up to the end of 
the subject follow-up period. In the case where the subject withdraws consent from 
participation in the study, permission to collect survival data will be obtained first. A 
subject will be labeled as “Lost to Follow-Up” only after the following has been 
documented: 


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1. Failure to reach the subject by telephone on 3 separate days 
2. Failure to reach the subject’s next of kin on 3 separate days 
 
5 Study Drugs  
5.1 Description 
TRH is an endogenous hormone that is a ligand for the TRH receptor and is involved in 
regulating TSH release from the pituitary. TSH stimulates release of T4, which can be 
converted to T3, a more active form. Both T4 and T3 regulate metabolism in humans. 
 
TRH will be purchased from American Peptide, which will produce a pharmaceutical 
grade (cGMP) formulation of TRH appropriate for human use. The TRH we will use is a 
tri-peptide and has a molecular weight of 362.3. TRH is supplied as a white to clear 
crystalline and powder. Syringes containing 200 mcg of TRH will be made by the 
University of Pennsylvania Investigational Drug Service. TRH is intended for intravenous 
administration. 
 
Synthetic forms of T4 (levothyroxine, or LT4) and T3 (liothyronine, or LT3) will be 
purchased and shipped to the IDS pharmacy at Penn. Each of these medications is FDA 
approved for treatment of conditions of hypothyroidism. LT4 has a molecular weight of 
776.87 and LT3 has a molecular weight of 650.98. LT4 and LT3 will be supplied as dose-
tailored capsules filled with pharmaceutical grade powder. The capsules will be provided 
by the IDS.  
5.2 Treatment Regimen 
On Day 1 (Visit 1) participants will receive an IV injection of 200 µg TRH by a CTRC nurse 
plus verbal and written instructions on thyroid hormone dosing and sufficient study 
medication for 6 weeks. On the same day, participants will receive LT4 or LT3 in 
randomized order. Each will be titrated to a target TSH level of 0.5-1.5mU/L. 
Administration of LT4 and LT3 and monitoring in this study will be performed in a similar 
fashion to a published protocol and based on the literature of thyroid hormone 
replacement in subclinical hypothyroidism.[28-34] 
 
LT4 
Each participant will start on 0.7mcg/kg/day divided into three daily doses. 
The dose will be adjusted according to subject weight and how far the TSH level is from 
goal.  There will be a medication cap such that no dose adjustment will exceed 25 mcg 
per day. 
 
LT3 
The starting dose of liothyronine will be 1/3 of that of levothyroxine ( 0.23 mcg/kg/day of 
LT3) divided into three daily doses. We will titrate the dose according to subject weight 
and how far the TSH level is from goal.  No dose adjustment will be greater than 8.3 
mcg per day.  


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The total daily dose of study medications will be adjusted to a TSH of 0.5-1.5 mU/L 
using the adjustment scheme to avoid excess thyroid hormone replacement.  
 
 
At the time of crossover of study drug, participants will undergo a 4 week wash-out period 
from the study drug. This is to avoid over-replacement when switching from LT4 to LT3, 
given the longer half-life of LT4. Following Visit 2, participants will stop all study drug for 
4 weeks. At the end of 4 weeks they will begin the second study drug at the equivalent 
dose as they were taking at Visit 2, divided into 3 equal doses. TSH level will not be 
checked at the end of the wash-out period.  A TSH level will be checked 4 weeks after 
crossing over to the new study drug. All further TSH level evaluations and medication 
adjustments will follow the titration protocol. 
 
 
No direct benefits are expected to result from LT4 and LT3 administration. 
 
The weight based dosing scheme and examples are displayed below: 
 
LT4 Titration Scheme:  
 
TSH (mU/L) 
>4.50 
3.51-4.50 
2.51-3.50 
1.51-2.50 
0.50-1.50 
0.20-0.49 
<0.20 
Dose change 
(mcg/kg/day) 
0.25 
0.21 
0.17 
0.1 
GOAL: 
No 
change 
-0.08 
-0.09 
 
Example of LT4 titration in a 50, 70, & 100 kg individual 
 
 
TSH 
(mU/L) 
>4.50 
3.51-
4.50 
2.51-
3.50 
1.51-2.50 
0.50-1.50 
0.20-
0.49 
<0.20 
Weight 
(kg) 
Start dose 
(0.7mcg/k
g/day) 
Dose Change: Total mcg change per day 
50 
35 
12.5 
10.5 
8.5 
5 
GOAL: No 
change 
-4 
-4.5 
70 
49 
17.5 
14.7
11.9 
7 
-5.6 
-6.3 
100 
70 
25 
21 
17 
10 
-8 
-9 
 
 
LT3 Titration Scheme: LT4 Dose scheme divided by 3  
 
TSH (mU/L) 
>4.50 
3.51-4.50 
2.51-3.50 
1.51-
2.50 
0.50-1.50 
0.20-0.49 
<0.20 
Dose change 
(mcg/kg/day) 
0.08 
0.07 
0.06 
0.03 
GOAL: 
-0.027 
-0.03 


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No 
change 
 
 
Example of LT3 titration in a 50, 70, & 100 kg individual 
 
 
TSH 
(mU/L) 
>4.50 
3.51-
4.50 
2.51-
3.50 
1.51-
2.50 
0.50-
1.50 
0.20-0.49 
<0.20 
Weight (kg) 
Start dose  
1/3 LT4 Dose 
Dose Change: Total mcg change per day 
50 
11.7 
4.17 
3.5 
2.83 
1.67 
GOAL: 
No 
change 
-1.33 
-1.50 
70 
16.3 
5.83 
4.9
3.97 
2.33 
-1.87 
-2.10 
100 
23.3 
8.3 
7 
5.67 
3.33 
-2.67 
-3.00 
 
Identical coded dose-tailored capsules containing LT4 or LT3, will be produced at Penn’s 
Investigational Drug Service (IDS). All therapeutic adjustments will be performed every 
four weeks by a blinded study physician and an IDS pharmacist. Identical amounts of 
study medication will be provided in each of the three daily doses. Subjects will receive 1 
capsule per dose, and the dose of each capsule will depend on the total daily dose 
requirement. New study medication will be mailed overnight to participants within 5 days 
of the receipt of interim lab results. Interim TSH level checks will continue every four 
weeks until the target TSH is achieved.  
After the targetTSH level is achieved, participants will attend Visit 2. They will receive a 
second IV injection of 200 µh TRH. Subjects will stop taking study drug for 4 weeks after 
the study visit 2. During this wash-out period once every two weeks a phone contact will 
continue to be made by one of the study team members to inquire if the participant has 
experienced any adverse events. Specifically, the participant will be asked if they have 
any symptoms of palpitations or shortness of breath. A six week supply of crossover study 
drug will either be sent home with the participant from study visit 2 or it will be mailed to 
them. Subjects will be instructed to be beginning this study drug 4 weeks after study visit 
2. TSH level will be checked 4 weeks after starting the new study drug.  All study 
measures for the other thyroid preparation will be repeated as above and Visit 3 will be 
identical to Visit 2, without administration of additional thyroid medication. 
5.3 Method for Assigning Subjects to Treatment Groups 
The order in which LT4 and LT3 are received will be randomized. A randomization list will 
be created by the IDS at Penn using a web-based program. The IDS will subsequently 
perform randomization assignment, and all study investigators, staff, and participants with 
direct contact with participants will be masked to treatment assignment.  
5.4 Preparation and Administration of Study Drug 
TRH in powder form must be stored at -20 degrees Celsius or cooler. LT4 and LT3 must 
be stored at room temperature. Storage, preparation, labeling and dispensing of all study 
drugs (TRH, LT4, and LT3) will be done by the IDS (available at 215-349-8817 or, for 
after-hours emergencies, 800-670-3151) in accordance with HUP’s Pharmacy Policy and 


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Procedures. Each drug label will include the subject’s unique identification number, date 
dispensed, and directions for use. 
 
One prefilled syringe containing 200 µg TRH will be administered to participants by a 
CTRC nurse in the CTRC at each study visit. 
 
TRH should be administered with the patient in the supine position. It will be administered 
as an intravenous bolus over 1 minute. The patient should remain supine until all of the 
post-injection blood draws have been completed. The patient should urinate prior to the 
procedure as they may feel an urge to urinate after the TRH is administered.  
 
The LT4 and LT3 will be packaged in identical coded dose-tailored capsules containing 
either LT4 or LT3. The participants will be given sufficient study medication for 6 weeks 
at the start of each treatment regimen (LT4 or LT3). Participants will obtain TSH level 
checks at 4-week intervals, after which they will be provided with additional study 
medication as needed. If a dose change is required, new study medication will be mailed 
overnight to participants within 5 days of review of the lab results. Unused capsules will 
be returned to IDS at Visits 2 and 3. 
5.5 Subject Compliance Monitoring 
Adherence will be assessed by direct questioning of study participants. Any evidence of 
noncompliance will be recorded and subjects who are significantly noncompliant with the 
study treatment regimen may be withdrawn from the study. Determination of 
noncompliance will be based on investigator judgment. To avoid unnecessary 
adjustments, blinded study physicians involved in any medication adjustments will be 
made aware by study team members of the participants’ self-reported adherence to the 
regimen.  
5.6 Prior and Concomitant Therapy 
Prior and concomitant medication history will be collected for each subject. Medications 
that are used on a daily basis, and are used to treat medical conditions that do not meet 
exclusion criteria will be permitted. Excluded prescription medications include thyroid 
hormone preparations, antithyroid drugs, corticosteroids, medication that interferes with 
the TRH stimulation test, and medication that interferes with the absorption or metabolism 
of LT4 or LT3 (Appendix 3). 
5.7 Packaging 
TRH will be shipped in bulk. IDS will prepare syringes for intravenous injection. 
 
LT4 and LT3 will be shipped in bulk and packaged by IDS in identical coded capsules 
containing LT4 or LT3. 
5.8 Blinding of Study Drug 
LT4 and LT3 will be packaged by IDS in identical coded capsules. 


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5.9 Receiving, Storage, Dispensing and Return 
5.9.1 Receipt of Drug Supplies 
Upon receipt of the drug supplies from the manufacturers, an inventory will be performed 
by the IDS and a drug receipt log filled out and signed by the IDS staff person accepting 
the shipment. The designated study staff will count and verify that the shipment contains 
all the items noted in the shipment inventory. Any damaged or unusable study drug in a 
given shipment will be documented in the study files. 
5.9.2 Storage 
TRH will be stored in IDS at -20 degrees Celsius or cooler. LT4 and LT3 will be stored in 
a dry place and protected from light at room temperature. 
5.9.3 Dispensing of Study Drug 
TRH obtained from the IDS pharmacy will be given to patients as an injection in the CTRC. 
LT4 and LT3 obtained from the IDS pharmacy will be given to patients as oral capsules 
to take home. Regular study drug reconciliation will be performed to document drug 
assigned, drug consumed, and drug remaining. This reconciliation will be logged on the 
drug reconciliation form, and signed and dated by the study team. 
5.9.4 Return or Destruction of Study Drug 
All study participants will return unused capsules of study drug to study staff at Visits 2 
and 3, which will be given to IDS. At the completion of the study, there will be a final 
reconciliation of drug shipped, drug consumed, and drug remaining. This reconciliation 
will be logged on the drug reconciliation form, signed and dated.  Any discrepancies noted 
will be investigated, resolved, and documented prior to return or destruction of unused 
study drug.  Drug destroyed on site will be documented in the study files. 
6 Study Procedures 
6.1 Screening Visit (Day -60 to Day -1) 
Upon arrival at the CTRC, the participant will review and sign the IRB-approved consent 
form. The participant will also review an optional exploratory genetics research study IRB-
approved consent form and sign only if they agree to participate in the optional study. 
They will then be assigned a unique screening visit identification number. Participants will 
be asked for a thorough history detailing their past medical history, current medications, 
and social habits. They will perform the MMSE. The research nurse will measure weight, 
height, blood pressure, heart rate, and perform a 12 lead ECG. A study investigator will 
perform a physical examination. Subjects will have blood drawn to evaluate lab 
parameters for eligibility criteria. A free thyroxine (FT4) level in conjunction with a TSH 
level will be sent to confirm subclinical hypothyroidism. The thyroid stimulating hormone 
(TSH) test obtained during the screening visit is measured at least 4 weeks after the last 
elevated TSH. Instructions for use of the activity and heart rate monitors will be given to 
each participant. Participants will then be discharged to home. If participants are eligible 
for the study, an activity and heart rate monitor will be mailed to their home. Participants 
will be advised to wear the activity and heart rate monitor for 4 days) prior to the first study 


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visit. The heart rate band will be moistened with either water or gel per the activity monitor 
instructions. Participants will also be given a log to record when they place their heart rate 
and activity monitors on and off.  
6.2 Visit 1 (Day 1) 
Participants will arrive at 7:30 am after a 12-hour fast. Weight and vital signs including 
heart rate, rectal temperature, and blood pressure will be measured. The activity and 
heart rate monitors will be collected. An REE evaluation by indirect calorimetry will be 
completed.  
 
An IV will be inserted into the forearm and blood will be collected at times -5 and 0, 
followed by injection of 200 µg TRH.[28] Additional blood will be collected at 5, 10, 15, 
20, 30, 60, 120, and 180 minutes, immediately processed, and stored at -80 degrees for 
measures of TSH, free T4, and total T3 (all time points), prolactin, alpha subunit, reverse 
T3 (0, 60, and 180 minutes), and a lipid profile (time 0). Additional samples at the 0, 120, 
and 180 time points will be processed and saved for TSH bioactivity measurements and 
TSH structure evaluation. If the participant consents to the optional exploratory genetic 
research study, then a sample will be collected, processed and saved for genetic testing 
at one study visit only (time -5). 
 
Blood pressure will be monitored before administration of TRH, and at 5, 15, 30, 60, 120, 
180 minutes.  
 
Questions about health status, quality of life, and hypothyroid symptoms will be 
completed. Digit symbol substitution test will be completed.  
 
Participants will be provided with lunch and undergo a DXA scan.  
Participants will then undergo arterial tonometry and Doppler echocardiography testing. 
Assessment of arterial load will be made from this data.  
Arterial tonometry: We will use a commercially available system (SphygmoCor, AtCor 
Medical) that uses a high-fidelity applanation tonometer (Millar Instruments; Houston, 
TX). The tonometer will be used to record carotid, radial and femoral arterial pressure 
waveforms. Mean brachial pressure will be obtained from a radial waveform calibrated 
with brachial systolic and diastolic pressures, measured with a validated oscillometric 
device. Diastolic and mean pressures will then be used to calibrate the carotid pressure 
waveform. Carotid-femoral and carotid-radial pulse wave velocity (PWV) will be computed 
from the time delay between the waveform foot on these arterial sites and the distance 
between the sites (measured on the body surface), where PWV = distance/time.  
Doppler echocardiography: will be performed using a standard protocol, which will include 
parasternal views and apical views of the left ventricle. It will also include pulsed-wave 
Doppler interrogation of flow velocities in the LV outflow tract, mitral inflow and mitral 
annular velocities. Flow volume will be computed by multiplying LV outflow tract flow 
velocity by the LV outflow tract cross-sectional area measured with 3D echocardiography. 


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Diastolic function will be assessed using standard parameters from mitral inflow and mitral 
annular tissue velocity profiles.  
 Assessment of arterial load will be performed via pressure-flow analyses and will include 
assessments of proximal aortic characteristic impedance, total arterial compliance, and 
reflection magnitude computed using linear wave separation analysis, using central 
pressure and flow waveforms measured via arterial tonometry and Doppler 
echocardiography, respectively.  
 
Arterial tonometry is non-invasive and does not have any known risks.  
During various procedures (arterial tonometry, echocardiography), we will use 
 adhesive electrodes attached to the participant’s skin to record the electrical  
signal from the heart. These may occasionally cause skin itching and irritation. 
 
Arterial tonometry and Doppler echocardiographic examinations will only be performed 
by appropriately trained personnel as per our institutional standards. 
 
Prior to the end of the visit the participant will receive verbal and written instructions on 
thyroid hormone dosing and provided with sufficient study medication for 6 weeks. Then 
they will be discharged home. 
6.3 Day 2-Week 4 
Participants will consume coded capsules of either LT4 or LT3 supplements as divided 
doses three times per day in the morning, afternoon, and evening.  
6.4 Medication Titration (Q4Weeks) 
Participants will have TSH testing every 4 weeks (acceptable range 4-8 weeks) at a Penn 
outpatient laboratory facility until TSH is at goal, and then the participant will be scheduled 
for Visit 2. This visit can be scheduled up to four weeks from the date of the last TSH 
level.  
 
Symptoms of palpitations and shortness of breath will be ascertained at the reminder calls 
prior to TSH level checks. The total daily dose of study medications will be adjusted by 
blinded study investigators to a TSH of 0.5-1.5 mU/L. New study medication for 
adjustment dosing will be mailed overnight to participants within 5 days of receipt of the 
interim lab results. Once the TSH is at goal, an activity and heart rate monitor will be 
mailed to the participant to wear one week prior to the next study visit. 
6.5 Visit 2  
Participants will repeat all measures from Visit 1 at this study visit on their first thyroid 
preparation (LT4 or LT3). Subjects will stop taking study drug for 4 weeks after the study 
visit 2. During this wash-out period once every two weeks a phone contact will continue 
to be made by one of the study team members to inquire if the participant has experienced 
any adverse events. Specifically, the participant will be asked if they have any symptoms 


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of palpitations or shortness of breath. A six week supply of crossover study drug will either 
be sent home with the participant from study visit 2 or it will be mailed to them. Subjects 
will be instructed to be begin this study drug 4 weeks after study visit 2. TSH level will be 
checked 4 weeks after starting the new study drug, and all study procedures for 
medication titration and TSH checks will be repeated as above.  
 
6.6 Visit 3 
Participants will repeat all measures from Visit 2 at this final study visit on their second 
thyroid preparation (LT4 or LT3). The study will be complete.  
6.7 Phone Contacts 
Once during the first week, a phone contact will be made by one of the study team 
members to inquire if the participant has experienced any adverse events following LT4 
or LT3 supplementation. Thereafter, once every two weeks a phone contact will be made 
by one of the study team members to inquire if the participant has experienced any 
adverse events following drug supplementation. Specifically, the participant will be asked 
if they have any symptoms of palpitations or shortness of breath. Additionally, one day 
following each TRH administration (study Visits 1, 2, and 3) a phone contact will be made 
by one of the study team members to inquire if the participant has experienced any 
adverse events following study drug injection. If the participant experiences any adverse 
events following the final phone contact, they will have an emergency phone number to 
contact, which is 954-464-8588 as noted on the front of the informed consent document 
and utilized throughout the entire study.  
6.8 Assays 
All screening blood assays will be performed at William Pepper Laboratory, and will 
include a CBC, TSH, FT4, TPO antibodies, and a complete metabolic panel including liver 
function tests to exclude hyperglycemia, renal insufficiency, or hepatic insufficiency. Total 
blood volume drawn at the screening visit is approximately 12 ml. This will be drawn as 
two gold-serum separator tubes and one lavender-top tube each containing 4 ml of blood. 
If a hemoglobin A1C level has not been checked on diabetic particpants in the last 6 
months, a hemoglobin A1C level will be added on to the screening labs. 
 
During the baseline study visit and study visit on each treatment arm, blood will be 
collected and batched for biochemical assays for TSH, free T4, total T3, prolactin, and a 
lipid profile, to be performed by the Pepper laboratory. Reverse T3 and alpha-subunit will 
be measured using available kits. TSH bioactivity will be measured under the guidance 
of Dr. Gershengorn’s laboratory at NIDDK. A sample for genetic testing will be collected 
at one study visit if the participant consents to the optional genetic research study. At the 
completion of the subject participation portion of the study, the optional genetic 
specimens, de-identified, will be sent to Dr. Francesco Celi at Virginia Commonwealth 
University for analysis.  When complete, the samples and analysis will be sent back to 
Penn for incorporation into the study database.   
Total blood volume drawn at each study visit is approximately <90 mL. Total blood drawn 
at these three visits combined is approximately <270 mL. 


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For interim thyroid hormone titration, a TSH level will be performed every 4 weeks until 
TSH is at goal. These TSH levels have a turnaround time of <24 hours. Total blood drawn 
at each titration draw is approximately 4 mL and will not cumulatively exceed 52 mL.  
 
7 Statistical Plan 
7.1 Sample Size Determination 
We plan to enroll 30 subjects with an anticipated 15% dropout rate. With an alpha of 0.05 
and 80% power and 15% dropout, we will be able to detect a 0.58 standard deviation 
(SD) change in outcome from baseline. A 3 SD improvement in unstimulated TSH 
bioactivity has been reported after treatment of overt primary hypothyroidism.[4] No 
previous studies have measured TSH bioactivity in subclinical hypothyroidism. 
7.2 Statistical Methods 
All statistical analyses will be performed under consultation with Dr. Cucchiara, Ph.D., a 
CTRC biostatistician. Our primary goal will be to assess changes in TRH stimulation test 
parameters at baseline compared to each thyroid hormone supplementation (LT4 or LT3) 
and our secondary goal will be to compare physiologic responses to LT4 and LT3 therapy 
from baseline. The area under the curve (AUC) for TRH-stimulated TSH response will be 
calculated using the trapezoidal rule. The maximum serum TSH concentration following 
TRH administration (TSHmax) will be obtained by direct inspection of the serum 
concentration-time profiles. Changes in TSH bioactivity, free T4, and total T3 will be 
determined using the higher of the 120 or 180 minute post-TRH values. Paired t-tests will 
be used to examine change from baseline for each preparation, and two-tailed tests will 
be used to compare data between treatment arms. Nonparametric tests will be employed 
as needed. The primary outcomes will be the changes in TRH stimulation test parameters 
(AUC, TSHmax, ∆ TSH bioactivity, ∆ free T4, ∆ total T3). Additional outcomes will include 
changes in unstimulated TSH bioactivity, REE, body temperature, weight, body 
composition, lipids, physical actigraphy and heart rate, cognitive testing, symptoms, 
arterial tonometry, and Doppler echocardiography. 
7.3 Subject Population(s) for Analysis 
Any subject with study data at Baseline visit or later will be subjected to study analysis. 
8 Safety and Adverse Events 
8.1 Definitions 
Unanticipated Problems Involving Risk to Subjects or Others 
Any incident, experience, or outcome that meets all of the following criteria:  
 Unexpected in nature, severity, or frequency  (i.e. not described in study-related 
documents such as the IRB-approved protocol or consent form, the investigators 
brochure, etc.) 


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 Related or possibly related to participation in the research (i.e. possibly related 
means there is a reasonable possibility that the incident experience, or outcome 
may have been caused by the procedures involved in the research) 
 Suggests that the research places subjects or others at greater risk of harm 
(including physical, psychological, economic, or social harm). 
 
Adverse Event 
An adverse event (AE) is 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 adverse events.  Abnormal results of diagnostic procedures are 
considered to be adverse events if the abnormality: 
 results in study withdrawal 
 is associated with a serious adverse event 
 is associated with clinical signs or symptoms 
 leads to additional treatment or to further diagnostic tests 
 is considered by the investigator to be of clinical significance 
 
Serious Adverse Event 
Adverse events 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-patient hospitalization or 
intensive treatment of bronchospasm in an emergency department would typically be 
considered serious.  
 
All adverse events that do not meet any of the criteria for serious should be regarded as 
non-serious adverse events.  
 
Adverse Event Reporting Period 
The study period during which adverse events 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 30 days following 
the last administration of study treatment.  
 
Preexisting Condition 


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A preexisting condition is one that is present at the start of the study.  A preexisting 
condition should be recorded as an adverse event if the frequency, intensity, or the 
character of the condition worsens during the study period. 
 
General Physical Examination Findings 
At screening, any clinically significant abnormality should be recorded as a preexisting 
condition.  At the end of the study, any new clinically significant findings/abnormalities 
that meet the definition of an adverse event must also be recorded and documented as 
an adverse event.  
 
Post-study Adverse Event 
All unresolved adverse events should be followed by the investigator until the events are 
resolved, the subject is lost to follow-up, or the adverse event is otherwise explained.  At 
the last scheduled visit, the investigator should instruct each subject to report any 
subsequent event(s) that the subject, or the subject’s personal physician, believes might 
reasonably be related to participation in this study.  The investigator should notify the 
study sponsor of any death or adverse event occurring at any time after a subject has 
discontinued or terminated study participation that may reasonably be related to this 
study.  The sponsor should also be notified if the investigator should become aware of 
the development of cancer or of a congenital anomaly in a subsequently conceived 
offspring of a subject that has participated in this study.  
 
Abnormal Laboratory Values 
A clinical laboratory abnormality should be documented as an adverse event if any one 
of the following conditions is met:  
 The laboratory abnormality is not otherwise refuted by a repeat test to confirm the 
abnormality 
 The abnormality suggests a disease and/or organ toxicity 
 The abnormality is of a degree that requires active management; e.g. change of 
dose, discontinuation of the drug, more frequent follow-up assessments, further 
diagnostic investigation, etc. 
 
Hospitalization, Prolonged Hospitalization or Surgery 
Any adverse event that results in hospitalization or prolonged hospitalization should be 
documented and reported as a serious adverse event unless specifically instructed 
otherwise in this protocol.  Any condition responsible for surgery should be documented 
as an adverse event if the condition meets the criteria for and adverse event.  
 
Neither the condition, hospitalization, prolonged hospitalization, nor surgery are reported 
as an adverse event in the following circumstances: 
 Hospitalization or prolonged hospitalization for diagnostic or elective surgical 
procedures for a preexisting condition.  Surgery should not be reported as an 
outcome of an adverse event if the purpose of the surgery was elective or 
diagnostic and the outcome was uneventful. 
 Hospitalization or prolonged hospitalization required to allow efficacy measurement 
for the study. 


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 Hospitalization or prolonged hospitalization for therapy of the target disease of the 
study, unless it is a worsening or increase in frequency of hospital admissions as 
judged by the clinical investigator. 
8.2 Recording of Adverse Events 
At each contact with the subject, the investigator must seek information on adverse 
events by specific questioning and, as appropriate, by examination.  Information on all 
adverse events should be recorded immediately in the source document, and also in the 
appropriate adverse event module of the case report form (CRF).  All clearly related signs, 
symptoms, and abnormal diagnostic procedures results should recorded in the source 
document, though should be grouped under one diagnosis. 
 
All adverse events occurring during the study period must be recorded.  The clinical 
course of each event should be followed until resolution, stabilization, or until it has been 
determined that the study treatment or participation is not the cause.  Serious adverse 
events that are still ongoing at the end of the study period must be followed up to 
determine the final outcome.  Any serious adverse event that occurs after the study period 
and is considered to be possibly related to the study treatment or study participation 
should be recorded and reported immediately. 
8.3 Reporting of Serious Adverse Events and Unanticipated Problems 
Investigators and the protocol sponsor must conform to the adverse event reporting 
timelines, formats and requirements of the various entities to which they are responsible, 
but at a minimum those events that must be reported are those that are: 
 related to study participation, 
 unexpected, and  
 serious or involve risks to subjects or others  
(see definitions, section 8.1).   
If the report is supplied as a narrative, the minimum necessary information to be provided 
at the time of the initial report includes: 
 Study identifier 
 Study Center 
 Subject number 
 A description of the event 
 Date of onset 
 Current status 
 Whether 
study 
treatment 
was 
discontinued 
 The reason why the event is classified as 
serious 
 Investigator 
assessment 
of 
the 
association between the event and study 
treatment 
 
8.3.1 Investigator reporting: notifying the study sponsor 
Any study-related unanticipated problem posing risk of harm to subjects or others, and 
any type of serious adverse event, must be reported to the study sponsor by telephone 
within seven days of becoming aware of the event.  To report such events, a Serious 
Adverse Event (SAE) form must be completed by the investigator and emailed or faxed 


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to the study sponsor within 10 days of becoming aware of the event  The investigator will 
keep a copy of this SAE form on file at the study site.  Report serious adverse events by 
phone and email or facsimile to: 
 
Susan Zieman, MD, PhD,  Phone: 301-496-6949, email:  
ziemans@nia.nih.gov 
 
Within the following 48 hours, the investigator must provide further information on the 
serious adverse event or the unanticipated problem in the form of a written narrative.  This 
should include a copy of the completed Serious Adverse Event form, and any other 
diagnostic information that will assist the understanding of the event.  Significant new 
information on ongoing serious adverse events should be provided promptly to the study 
sponsor. 
8.3.2 Investigator reporting: notifying the Penn IRB 
This section describes the requirements for safety reporting by investigators who are 
Penn faculty, affiliated with a Penn research site, or otherwise responsible for safety 
reporting to the Penn IRB. The University of Pennsylvania IRB (Penn IRB) requires 
expedited reporting of those events related to study participation that are unforeseen and 
indicate that participants or others are at increased risk of harm.  The Penn IRB will not 
acknowledge safety reports or bulk adverse event submissions that do not meet the 
criteria outlined below.  The Penn IRB requires researchers to submit reports of the 
following problems within 10 working days from the time the investigator becomes aware 
of the event: 
 Any adverse event (regardless of whether the event is serious or non-serious, on-
site or off-site) that occurs any time during or after the research study, which in the 
opinion of the principal investigator is: 
Unexpected (An event is “unexpected” when its specificity and severity are not 
accurately reflected in the protocol-related documents, such as the IRB-approved 
research protocol, any applicable investigator brochure, and the current IRB-
approved informed consent document and other relevant sources of information, 
such as product labeling and package inserts.) 
AND 
Related to the research procedures (An event is “related to the research 
procedures” if in the opinion of the principal investigator or sponsor, the event was 
more likely than not to be caused by the research procedures.) 
 
Reporting Process 
Unanticipated problems posing risks to subjects or others as noted above will be reported 
to the Penn IRB using the form: “Unanticipated Problems Posing Risks to Subjects or 
Others Including Reportable Adverse Events” or as a written report of the event (including 
a description of the event with information regarding its fulfillment of the above criteria, 
follow-up/resolution and need for revision to consent form and/or other study 
documentation). 
 
Copies of each report and documentation of IRB notification and receipt will be kept in 
the Clinical Investigator’s study file. 


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Reporting Deaths:  more rapid reporting requirements 
Deaths that occur during the course of a research study and that are:  
 
Unexpected; AND  
 
Related to the research study; AND  
 
When other participants are believed to be at an increased risk of harm 
 
Must be reported to the IRB within 3 days from the time the investigator becomes aware 
of the death.  
 
Other Reportable events: 
For clinical drug trials, the following events are also reportable to the Penn IRB: 
 Any adverse experience that, even without detailed analysis, represents a serious 
unexpected adverse event that is rare in the absence of drug exposure (such as 
agranulocytosis, hepatic necrosis, Stevens-Johnson syndrome). 
 Any adverse event that would cause the sponsor to modify protocol or informed 
consent form, or would prompt other action by the IRB to assure protection of 
human subjects. 
 Information that indicates a change to the risks or potential benefits of the research, 
in terms of severity or frequency. For example:  
– An interim analysis indicates that participants have a lower rate of response to 
treatment than initially expected. 
– Safety monitoring indicates that a particular side effect is more severe, or more 
frequent than initially expected. 
– A paper is published from another study that shows that an arm of your research 
study is of no therapeutic value. 
 Change in FDA safety labeling or withdrawal from marketing of a drug, device, or 
biologic used in a research protocol. 
 Breach of confidentiality 
 Change to the protocol taken without prior IRB review to eliminate apparent 
immediate hazard to a research participant. 
 Incarceration of a participant when the research was not previously approved under 
Subpart C and the investigator believes it is in the best interest of the subject to 
remain on the study. 
 Complaint of a participant when the complaint indicates unexpected risks or the 
complaint cannot be resolved by the research team. 
 Protocol violation (meaning an accidental or unintentional deviation from the IRB 
approved protocol) that in the opinion of the investigator placed one or more 
participants at increased risk, or affects the rights or welfare of subjects. 
8.3.3 Sponsor reporting: Notifying the FDA 
The study sponsor is required to report certain study events in an expedited fashion to 
the FDA.  These written notifications of adverse events are referred to as IND safety 


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reports. The following describes the safety reporting requirements by timeline for 
reporting and associated type of event: 
 
 Within 7 calendar days 
Any study event that is: 
– associated with the use of the study drug 
– unexpected,  
– fatal or life-threatening, and  
 
 Within 15 calendar days 
Any study event that is: 
– associated with the use of the study drug, 
– unexpected, and 
– serious, but not fatal or life-threatening 
-or- 
– a previous adverse event that was not initially deemed reportable but is later 
found to fit the criteria for reporting (reporting within 15 calendar days from when 
event was deemed reportable). 
Any finding from tests in laboratory animals that:  
– suggest a significant risk for human subjects including reports of mutagenicity, 
teratogenicity, or carcinogenicity. 
 
Additional reporting requirements 
Sponsors are also required to identify in IND safety reports all previous reports concerning 
similar adverse events and to analyze the significance of the current event in light of the 
previous reports. 
 
Reporting Process 
Adverse events may be submitted on FDA Form 3500A or in a narrative format. If supplied 
as in a narrative format, the minimum information to be supplied is noted above at the 
beginning of section 8.3. The contact information for submitting IND safety reports is 
noted below: 
 
Contact information pending IND submission 
 
8.4 Unblinding Procedures 
Any intentional breaking of the blind prior to study completion, which may be necessary, 
for example, in the case of an SAE, will be done in conjunction with the IDS, whose 
responsibility it will be to notify a designated member of the research team of the individual 
subject’s treatment assignment. Any intentional or unintentional breaking of the blind prior 
to study completion will be documented and reported to the PI, CTRC, and the IRB within 
24 hours and the FDA within seven calendar days. 
8.5 Stopping Rules  
The study will be stopped if the following criteria occur: 


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1. The PI discovers a condition or conditions that indicate unacceptable risk to the 
subjects. 
2. There is evidence of harm of study-related treatments or procedures to the subjects. 
8.6 Medical Monitoring 
It is the responsibility of the Principal Investigator to oversee the safety of the study at 
his/her site.  This safety monitoring will include careful assessment and appropriate 
reporting of adverse events as noted above, as well as the construction and 
implementation of a site data and safety-monitoring plan (see section 9 Auditing, 
Monitoring and Inspecting).  Medical monitoring will include a regular assessment of the 
number and type of serious adverse events. Additional medical monitoring will be 
performed by an external study monitor who is not an investigator in this study. 
9 Data Handling and Record Keeping 
9.1 Confidentiality 
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.  
 
In the event that a subject revokes authorization to collect or use PHI, the investigator, by 
regulation, retains the ability to use all information collected prior to the revocation of 
subject authorization.  For subjects that have revoked authorization to collect or use PHI, 
attempts should be made to obtain permission to collect at least vital status (i.e. that the 
subject is alive) at the end of their scheduled study period. 
9.2 Source Documents 
Source data is all information, original records of clinical findings, observations, or other 
activities in a clinical trial necessary for the reconstruction and evaluation of the trial.  
Source data are contained in source documents.  Examples of these original documents, 
and data records include: hospital records, clinical and office charts, laboratory notes, 
memoranda, subjects’ diaries or evaluation checklists, pharmacy dispensing records, 
recorded data from automated instruments, copies or transcriptions certified after 
verification as being accurate and complete, microfiches, photographic negatives, 
microfilm or magnetic media, x-rays, subject files, and records kept at the pharmacy, at 
the laboratories, and at medico-technical departments involved in the clinical trial. 
9.3 Case Report Forms 
The study case report form (CRF) is the primary data collection instrument for the study.  
All data requested on the CRF must be recorded.  All missing data must be explained.  If 
a space on the CRF is left blank because the procedure was not done or the question 


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was not asked, write “N/D”.  If the item is not applicable to the individual case, write “N/A”.  
All entries should be printed legibly in black ink.  If any entry error has been made, to 
correct such an error, draw a single straight line through the incorrect entry and enter the 
correct data above it.  All such changes must be initialed and dated.  DO NOT ERASE 
OR WHITE OUT ERRORS.  For clarification of illegible or uncertain entries, print the 
clarification above the item, then initial and date it. 
 
10 Study Monitoring, Auditing, and Inspecting 
10.1 Study Monitoring Plan 
Internal medical monitoring will be performed by Dr. Cappola, who will oversee the safety 
and validity of the study, according to the University of Pennsylvania Sponsor-Investigator 
Standard Operating Procedures. In addition, an external medical monitor who is not a 
study investigator will perform safety monitoring. 
 
Safety monitoring will include careful real-time assessment and appropriate reporting of 
AEs and SAEs. Safety assessments will be performed by the internal medical monitor 
after each subject has completed the study (baseline study visit through final visit on 
treatment 2), and by the external medical monitor after the first, fifteenth, and thirtieth 
participants have completed the study. All subjects who experiences and/or discontinued 
due to AEs will be reviewed for key safety data. A final safety assessment will be 
performed within 3 months of when the last subject has completed the study. 
 
As the Study Sponsor, the PI assumes responsibility to assure that study activities are 
being carried out as planned so that deficiencies can be identified and corrected. This 
study will in addition be monitored by a clinical research professional from the staff of 
Clinical Research Coordinators working for other investigators at the University of 
Pennsylvania who will be responsible to complete a monitoring process for this study.  
The designated monitor is not an active member of this study team.  A CV for the monitor 
will be obtained and updated annually.  The CV will be kept on file in the Regulatory 
Binder.  The monitor will receive a current protocol, most recently approved Informed 
Consent Form, and Case Report Forms prior to each monitoring visit.     
 
All CRFs for the first two subjects completed will be 100% source data verified.  In 
addition, CRF’s for the 10th, 20th, and 30th subjects will be 100% source data verified 
upon study completion.  If a greater than 10% error rate is noted during the data review, 
the monitor will source data verify 100% of the data on a larger sample of active subjects 
following the monitoring visit.  The following variables will be 100% source data verified 
during the monitoring process:  
 Informed Consent 
 Inclusion/Exclusion Criteria 
 Serious and Non-Serious Adverse Events 
 Prohibited concomitant medications.    
 Drug accountability (IDS will be responsible for drug accountability. Research 
staff will return all unused study drugs to IDS. 


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In addition, all subjects who discontinued for “lost to follow-up” or other reasons will be  
100 % source data verified.  All monitoring activities completed by the internal study 
monitor will be recorded on the OHR approved subject review checklists. Monitoring 
activities cited above will be documented by the monitor in a written report to the PI so 
that appropriate corrective actions may be implemented as indicated and the report 
submitted will be filed in the Regulatory Binder. 
 
 
10.2 Auditing and Inspecting 
The investigator will permit study-related monitoring, audits, and inspections by the 
EC/IRB, the sponsor, government regulatory bodies, and University compliance and 
quality assurance groups of all study related documents (e.g. source documents, 
regulatory documents, data collection instruments, study data etc.).  The investigator will 
ensure the capability for inspections of applicable study-related facilities (e.g. pharmacy, 
diagnostic laboratory, etc.). 
 
Participation as an investigator in this study implies acceptance of potential inspection by 
government regulatory authorities and applicable University compliance and quality 
assurance offices. 
11 Ethical Considerations 
This study is to be conducted according to US and international standards of Good 
Clinical Practice (FDA Title 21 part 312 and International Conference on Harmonization 
guidelines), applicable government regulations and Institutional research policies and 
procedures. 
 
This protocol and any amendments will be submitted to a properly constituted 
independent Ethics Committee (EC) or Institutional Review Board (IRB), in agreement 
with local legal prescriptions, for formal approval of the study conduct.  The decision of 
the EC/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 sponsor before commencement of this 
study.  The investigator should provide a list of EC/IRB members and their affiliate to the 
sponsor. 
 
All subjects for this study will be provided a consent form describing this study and 
providing sufficient information for subjects to make an informed decision about their 
participation in this study.  See Attachment 1 for a copy of the Subject Informed Consent 
Form.  This consent form will be submitted with the protocol for review and approval by 
the EC/IRB for the study.  The formal consent of a subject, using the EC/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 
investigator-designated research professional obtaining the consent.  


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12 Study Finances 
12.1 Funding Source 
This study is financed through a grant from the US National Institutes of Health. 
12.2 Conflict of Interest 
Any investigator who has a conflict of interest with this study (patent ownership, royalties, 
or financial gain greater than the minimum allowable by their institution, etc.) must have 
the conflict reviewed by a properly constituted Conflict of Interest Committee with a 
Committee-sanctioned conflict management plan that has been reviewed and approved 
by the study sponsor prior to participation in this study.  All University of Pennsylvania 
investigators will follow the University conflict of interest policy. 
12.3 Subject Stipends or Payments 
Participants will not be paid for completing the initial screening visit, but will receive a 
validated parking stamp or transportation costs for this visit. Eligible participants who 
continue to participate in the study will receive validated parking stamps or transportation 
costs at each study visit plus $300 following completion of all study visits. 
13 Publication Plan 
Neither the complete nor any part of the results of the study carried out under this protocol, 
nor any of the information provided by the sponsor for the purposes of performing the 
study, will be published or passed on to any third party without the consent of the study 
sponsor.  Any investigator involved with this study is obligated to provide the sponsor with 
complete test results and all data derived from the study. Results will be available in Pub 
Med Central, as per NIH guidelines. 
14 References 
1. 
Faix, 
J.D. 
and 
L.M. 
Thienpont. 
2013; 
Available 
from: 
https://www.aacc.org/publications/cln/articles/2013/may/tsh-harmonization. 
2. 
Surks, M.I. and J.G. Hollowell, Age-specific distribution of serum thyrotropin and 
antithyroid antibodies in the US population: implications for the prevalence of 
subclinical hypothyroidism. J Clin Endocrinol Metab, 2007. 92(12): p. 4575-82. 
3. 
Oliveira, J.H., et al., Investigating the paradox of hypothyroidism and increased 
serum thyrotropin (TSH) levels in Sheehan's syndrome: characterization of TSH 
carbohydrate content and bioactivity. J Clin Endocrinol Metab, 2001. 86(4): p. 
1694-9. 
4. 
Oliveira, J.H., et al., Evidence for thyroid hormone as a positive regulator of serum 
thyrotropin bioactivity. J Clin Endocrinol Metab, 2007. 92(8): p. 3108-13. 
5. 
Persani, L., et al., Circulating thyrotropin bioactivity in sporadic central 
hypothyroidism. J Clin Endocrinol Metab, 2000. 85(10): p. 3631-5. 
6. 
Persani, L., et al., Circadian variations of thyrotropin bioactivity in normal subjects 
and patients with primary hypothyroidism. J Clin Endocrinol Metab, 1995. 80(9): p. 
2722-8. 
7. 
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15 Attachments 
16 APPENDIX 
16.1 Appendix 1 
 
Hyperthyroid Symptoms assessed with each telephone encounter while on study 
drug: 
 
1. palpitations 


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2. shortness of breath 
 
Hyperthyroid Symptoms assessed at each study visit:  
 
1. anxiety 
2. irritability 
3. muscle discomfort or weakness 
4. tremor 
5. heat intolerance 
6. palpitations 
7. fatigue 
8. weight loss  
9. frequent bowel movements 
10.  shortness of breath 
 
16.2 Appendix 2 
 
Hypothyroid symptoms assessed at each study visit: 
1. slower thinking 
2. depression 
3. muscle cramps 
4. dry skin 
5. cold intolerance 
6. fatigue 
7. weight gain 
8. constipation 
16.3 Appendix 3 
 
Thyroid hormone preparations: 
1. levothyroxine 
2. liothyronine 
3. desiccated thyroid extract 
 
Antithyroid medication: 
1. methimazole 
2. propylthiouracil 
 
Medication that interferes with thyroid hormone or TRH stimulation test: 
1. lithium 
2. amiodarone 
3. Kelp tablets 


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4. radiographic contrast agents within 16 days of thyroid testing (if participant has had 
radiographic contrast agent in this time frame, we will retest at least 16 days from the 
day the contrast was administered 
5. cholestyramine 
6. colestipol 
7. colesevelam 
8. interferon 
9. ipilimumab 
10.  alemtuzumab 
11.  pembrolizumab 
12.  sunitinib 
13.  sorafenib 
14.  bexarotene 
15.  phenytoin 
16.  carbamazepine 
17.  rifampin 
18.  phenobarbital 
19.  octreotide, somatostatin 
20.  dobutamine 
21. dopamine antagonists such as risperidone, olanzapine, quetiapine, ziprasidone, 
aripiprazole, 
asenapine, 
clozapine, 
Iloperidone, 
Lurasidone, 
Paliperidone, 
chlorpromazine, fluphenazine, haloperidol, loxapine, perphenazine, pimozide, 
Thioridazine, 
thiothixexe, 
trifluoperazine, 
droperidol, 
prochlorperzaine, 
metaclopramide 
 
Proton Pump inhibitors: 
1. dexlansoprazole 
2. esomeprazole 
3. lansoprazole 
4. omeprazole 
5. pantoprazole 
6. rabeprazole