A Safety and Clinical Efficacy Study Measuring Echocardiographic Composite Comparing Ambrisentan (Letairis®) After a Switch From Bosentan (Tracleer®) or Macintentan (Opsumit®) in Treatment of Pulmonary Arterial Hypertension (PAH)
Phase: PHASE4
Conditions: Pulmonary Arterial Hypertension
Sponsor: Medical University of South Carolina

A 24-Week-Study to Evaluate the Safety and Clinical Efficacy As Measured By an 
Echocardiographic Composite Comparing Ambrisentan (Letairis®) After a Switch 
From Bosentan (Tracleer® ) or Macitentan (Opsumit®)In the Treatment of 
Connective Tissue Disease Associated Pulmonary Arterial Hypertension 
Investigators: 
 
 
Protocol Version 3: Date 08/12/2016 
 
IRB approved: 8/18/2016 
 
NCT02885012 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
J. Terrill Huggins, M.D. 
Associate Professor of Medicine 
Director, Interstitial Lung Disease Program 
Division of Pulmonary, Critical Care, Allergy and Sleep Medicine 
Medical University of South Carolina, Charleston, SC 
 
David Gregg, MD 
Assistant Professor of Medicine 
Division of Cardiology 
Medical University of South Carolina, Charleston, SC 
Stacy Mandras, MD 
 
Director, Pulmonary Hypertension Program 
Division of Cardiology 
Oschner Clinic Foundation 
New Orleans, LA 

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Introduction:
Pulmonary arterial hypertension (PAH) is a serious and progressive 
condition associated with significant morbidity and mortality.1,2 Although PAH complicating 
systemic sclerosis occurs at the highest frequency when compared to other rheumatic 
diseases, virtually all forms of connective tissue diseases (CTD) can be complicated with 
the development of PAH.3,4 Several pathophysiologic processes have been identified in 
CTD-PAH and include: 1) endothelial proliferation with plexiform lesions, 2) microthrombi
formation, 3) intimal and medial hyperplasia with luminal narrowing, 4) immune-mediated 
vasculopathy, 5) obliterative vasculopathy, and 6) veno-occlusive disease.  The net result 
is ongoing pulmonary vascular remodeling which leads to progressive obliteration of
pulmonary arteries. The result is progressive increases in pulmonary vascular resistance, 
which leads to right ventricular failure and death.5-7
Although histologically indistinguishable from idiopathic PAH, CTD-PAH is thought to have 
differences in pathogenesis with a more pronounced inflammatory component. Studies 
have described a mortality difference between IPAH and CTD-PAH, with CTD-PAH being 
associated with a poorer prognosis and having a more recalcitrant response to therapy 
when compared to IPAH. Mortality differences may be 4 fold higher in CTD-PAH, with a
median survival as low as 12 months in untreated CTD-PAH patients.
The therapeutic approach in CTD-PAH is similar to IPAH, with a caveat that theresponse 
rates and prognosis does differ. To date, several randomized control trials have lookedat 
PAH therapies as it relates to CTD-PAH. Most available data on efficacy of PAH
medications (prostacyclin analogues, endothelin receptor antagonists, and
phosphodiseterase inhibitors) are largely based on studies of IPAH or PAH irrespectiveof 
the underlying cause. Therefore, connective tissue disease is an ideal group whichshould 
be studied. There are sub-group analysis of CTD-PAH patients with bosentan, 
ambrisentan, epoprostenol, and sildenafil.8-18 In regards to endothelin receptor antagonists 
(ERA) therapy, 1 year survival with bonsentan was 85.9% compared to 81.2 % versus85.5 
for 3 mg versus 10 mg of macitentan respectively; while 1 year survival with ambrisentan 
was 90.3%.13,17 In terms of six minute walk response at 1 year, mean increase with 
bosentan was 14.7m versus baseline,compared to mean increase of 7.4m to 16.8m for 
3mg versus 10 mg of macitentan; while mean increase with ambrisentan (dose 10 mg 
daily) was 27.2 m versus baseline.13,17,28 These data would suggest a superioradvantage 
of ambrisentan compared to bosentan and macitentan in the treatment of CTD-PAH. 
Ambrisentan has been evaluated in two Phase 3 randomized, double-blind, placebo-
controlled, multicenter trials (ARIES-1 and ARIES-2). In ARIES-1, a total of 202 PAH
patients were randomized to three treatment groups for a 12-week studyduration.
Significant improvements in the 6MWD change from baseline were noted at 12 weeks. 
Improvements in WHO functional class and Borg dyspnea index was noted.17 In ARIES-2, 
192 patients with PAH were randomized to receive either ambrisentan 2.5mg, 5mg, or 
placebo for 12 weeks.17 Again, ambrisentan significantly improved 6MWD and delayed 
time to clinical worsening. A combined analysis of both trials confirmed adose-dependent
increase in 6MWD with ambrisentan treatment.17

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All pivotal trials in the treatment of PAH have relied on end points, such as 6MWD as their 
primary endpoint. Although, one may argue that a 6MWD could be considered a surrogate 
of cardiac output and mortality, it is not with recognized limitations of the six-minute walk 
(6MWT). In addition, the 6MWT assessing exercise capacity cannot account for patterns 
of sedentary time (time at <1.5 metabolic equivalents), which has been associated with 
adverse cardiovascular health.18-21 Accurately assessing physical activity and increased 
sedentary behavior may contribute to a reduced quality of life in patients with CTD-PAH, 
improving methods to access physical activity in these patients and sedentary behavior are 
needed for possible trial end points.
Hemodynamics measurements with right heart catheterization are considered to be gold 
standard for diagnosis and useful in following response to therapy. In terms of bosentan 
treatment as it relates to the hemodynamic response in WHO Group 1, bosentan (125mg 
BID for 12 weeks) has been shown to decrease mean pulmonary artery pressure (mPAP) -
1.6 mmHg, decrease pulmonary vascular resistance (PVR) -223 dynes and increase 
cardiac index (CI) 0.5L/min as compared to baseline.13 In terms of macitentan treatment 
as it relates to the hemodynamic response, macitentan (10mg daily for 6 months) was 
shown to decrease PVR by -227 dynes and increase CI 0.3L/min compared to baseline. 
Treatment with ambrisentan (10mg daily for 16 weeks), resulted in a decrease mPAP -13.3 
mmHg, decrease PVR -345 dynes, and increase CI of 0.37 L/min.17,28 ERAs clearly  have 
a favorable change in overall hemodynamics with time. Due to the fact that RHC are 
invasive procedures with associated morbidity, CTD-PAH patients may potential benefit 
form non-invasive hemodynamic measurements in response totherapy.
The advantages of ambrisentan when compared to bosentan and macitentan are related to 
daily dosing, less drug-drug interactions, reduced liver toxicity, and potential survival 
advantage, the current study will evaluate the safety and efficacy of ambrisentan in the 
treatment of CTD-PAH. Tolerance and safety with the use of ambrisentan will 
be 
correlated to the development of edema, nasal congestion, and use of diuretic therapy 
over 24 weeks. Similarly, efficacy will be correlated to worsening WHO functional class,
>20% decline in 6MWTD, need for chronic prostanoid therapy due to clinical worsening of 
PAH, and death.
Due to the potential advantages of ECHO, efficacy will be measured serially starting with 
stable bosentan and macitentan patients of at least 3 months duration and monitored at 4, 
12, 18, and 24 weeks after switch to ambrisentan. Spirometry,  diffusing  capacity for 
carbon monoxide, and NT-probrain naturetic peptide(NT-proBNP) will be collected at 
baseline, 12, and 24 weeks as secondary end-points. ECHO will be performed with 
worsening WHO functional class, >20% decline in 6MWTD, and the need for chronic 
prostanoid therapy due to clinical worsening of PAH

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Hypothesis: This study is designed to assess safety and efficacy of ambrisentan 
(Letairis®) in patients with CTD-PAH who were previously treated with bosentan or 
macitentan.
Clinical efficacy will be determined by  an 
echocardiographic 
composite, which includes Doppler assessment of stroke volume, pulmonary 
vascular resistance, and Speckle Doppler to assess RV strain and strainrates.
Primary Endpoint:  ECHO Composite
SVI/CI (measurements of both the RV and LV)
Estimation of PVR (Peak Tricuspid Valve regurgitant velocity/ velocity time integral (VTI) of 
the RVOT; VTI of LVOT may be used as a surrogate to RVOT if not able to beacquired)
Secondary Endpoints:
WHO functional class 
Dyspnea as measured by 
EmPHasis-10 Questionnaire 
6 minute walk test
FVC and DLCO
NT-proBNP
Time to clinical worsening (defined by the initiation of ambrisentan treatment to the first 
occurrence of death, lung transplantation, hospitalization for pulmonary
arterial 
hypertension, atrial septostomy, a change in chronic prostanoid or increasing sildenafil or 
tadalafil treatment due to protocol defined worsening criteria or study withdrawal due to 
additional of other clinically approved PAH therapeuticagents.
Tolerance of ambrisentan as it relates to edema, nasal congestion, and use of diuretic 
therapy.
ECHO data collected
TAPSE
PA Acceleration time 
Peak PRVSP
IVC size and variability index with estimation of right atrial pressure 
Myocardial performance index
Eccentricity index (measurement of the degree of septaldyskinesia)
Quantitative assessment of LV diastolic properties (PW Doppler assessment  of mitral 
inflow with tissue Doppler assessment of mitral annulus; LV strain and strainrates)
LV systolic function (EF, SVI/CI) 
Mean Pulmonary Artery Pressure
RV strain and strain rates measured by Speckle Doppler techniques

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Inclusion criteria:
Diagnosis of a CTD as defined by ACR proposed criteria 
Age 18-80
Previous RHC demonstrating PAH (mPAP > 25 mmHg and PAOP ≤15 mmHg) 
Forced vital capacity (FVC) > 50%
DLCO > 50%
WHO functional class II or III
Able to perform a 6 minute walk test
Stable dose of antihypertensive medications 
Non-pregnant females
Have to be currently on stable dose of bosentan or macitentan for at least 3 months 
Adequate acoustic images to allow for transthoracic echocardiography to be performed 
(PLEASE NOTE: FVC and DLCO will not exclusionary for patients with lower FVC and DLCO who
have been tolerating bosentan or macitentan for at least 3 months prior to switch)
Exclusion criteria:
Exercise limitation related to a non-cardiopulmonary reason (e.g.arthritis) 
Severe systemic hypertension > 170/95
Patients with a prior history of cardiovascular disease, including LV systolic dysfunction, 
CAD, CAGB, and aortic and mitral valvular disease
WHO functional class IV status
Patients with severe other organ disease felt by investigators to impact on survivalduring 
the course of the study.
FVC < 50% of predicted
DLCO < 50% of predicted
Study Design:
Patients will be recruited into an open label, switch trial comparing the clinical efficacy of 
bosentan or macitentan compared to ambrisentan. This study will be conducted over 24 
weeks. Patients meeting inclusion criteria will have a diagnosis of CTD-PAH. These 
patients maybe currently receiving treatment with bosentan or representative of 
a
treatment naïve patient cohort at which time ERA therapy is started. For the treatment 
naïve cohort, patients will have to receive at least 3 months of bosentan or macitentan 
prior to switch to ambrisentan. If diuretic therapy is currently not part of their medical 
regimen at screen visit, diuretic therapy will be permissible during the study period for 
edema control. At entry to the study following the screen visits, patients will be switched to 
ambrisentan to complete a 24 week trial. The table below summarizes the studydesign.
Patients will have previously undergone right heart catheterization (RHC) to confirm a
diagnosis of PAH. At the screening visit, patients will undergo informed consent followed 
by a complete history and physical examination, oxygen saturation, six minute walk 
(6MWT), pulmonary function tests (spirometry and diffusion), ECHO and phlebotomy. The
6 minute walk test will be performed on the patient’s dose of supplemental oxygen. 
Patients meeting the inclusion criteria will proceed to the baseline (time 0) visit. The 
baseline and screening visit must be within 30 days of each other, but can be conducted 
the same day as baseline assessment if baseline testing can be conducted with 24 hours. 
At the baseline visit, patients will undergo a complete history and physical examination, 
oxygen saturation, six minute walk, pulmonary function studies (spirometry and diffusion), 
ECHO, complete the Borg Dyspnea index and EmPHasis 10 questionnaire;.
Patients will be started on ambrisentan 5mg daily. At week 4, ambrisentan will be up-
titrated  to  10  mg  daily  if   the   subject  is  tolerating the medication.

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Week
S
0
4
12
18
24
Consent
X
Inclusion/Exclusion Criteria
X
History and Physical Exam
X
X
X
EmPHasis-10 Questionnaire
X
X
X
Six minute walk
X
X
X
Resting room air oxygen saturation
X
X
X
Oxygen saturation with exercise
X
X
X
Borg score (pre and post walk)
X
X
X
Echocardiography
X
X
WHO Functional Class
X
X
X
Spirometry
X
X
X
DLCO
X
X
X
Dispense drug
X
X
Drug Return & Accountability
X
X
Compliance with therapy evaluation
X
X
X
X
NT-proBNP
X
X
X
Urine Pregnancy test (childbearing 
monthly)
X
X
X
X
X
Telephone Call by study coordinator
X
X
Screen and baseline visit can be combined if all testing can be performed within 24 
hours.
Monthly pregnancy testing will be conducted over the 24 weeks for child bearing 
females who have not received tubal sterilization procedures or received approved 
intrauterine devices as required by the FDA.
Liver function testing will not be conducted for the ambrisentan treatmentperiod.
Patients will return at 12 and 24 weeks for history and physical, functional class 
assessment, compliance and drug dispensation (at week 12 only). ECHO will be 
performed at Baseline and Week 24 visits; NT-proBNP, six minute walk testing and PFT 
assessments will be conducted at Baseline, 12 and 24 weeks. ECHO will also be 
performed at any point of the study for subjects who have defined clinical worsening of 
PAH as previously described. At week 4 for patients receiving ambrisentan, patients will be 
increased to ambrisentan (Letairis®) 10mg if tolerating the 5 mg dose. All 6 minute walk
tests
will
be
performed
on same
oxygen
concentration
as
the
Baseline
walk.
Compliance will be determined by patient report and pill counts. Study coordinator will
conduct brief telephone contact to enrolled subjects in order to improve drug compliance 
and minimize potential toxicity related to ERA therapy at weeks 4 and 18.
During the course of the study, subjects will be continued on treatment for theirCTD-PAH. 
We will not change medication protocols as it relates to defined PAH therapy, exceptin 
cases where there is a decompensation of disease. We will define a clinical worseningof
PAH to include 2 of the 3 following criteria: 1) a decrease from baseline of at least 20%in
the 6MWD; 2) an increase of 1 or more in WHO functional class: and 3) worseningright 
ventricular failure (increased jugular venous pressure, and new/worseninghepatomegaly, 
ascites or peripheral edema).

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Rationale for Clinical Measurements:
All pivotal trials in the treatment of PAH have relied on end points, such as 6MWD as their 
primary endpoint. Although, one may argue that a 6MWD could be considered a surrogate 
of cardiac output and mortality, there are well described limitations of the six-minute walk 
(6MWT). In addition, the 6MWT assessing exercise capacity cannot account for patterns 
of sedentary time (time at <1.5 metabolic equivalents), which has been associated with 
adverse cardiovascular health.18-23
Although ECHO in the diagnosis and management of PAH is a heavily debated area, we 
should consider more meaningful ECHO-derived variables which do not need to take into 
account the complexity of the RV geometry. More importantly, it is clear PAH is a
pulmonary vascular resistance problem that leads to progressive RV failure, which is 
clinically manifested by an increasing right atrial pressure, worsening lower extremity 
edema, and ascites. Due to the progressive decline in forward flow (a falling SV), multi-
organ failure and progressive renal dysfunction ensues. Once overt right heart failure 
occurs, restoration of adequate cardiac output is dismal without the need of ionotropic 
support, which often fails to restore RV function.
Furthermore, one should also consider that an optimal cardiac performance is governed 
more so by stroke volume (SV) than CO. To understand this concept, one should consider
the determinates of CO, heart rate and SV. CO may be maintained at the expense of a
falling SV by compensatory increases in heart rate, which is not ideal. Similarly, a
decrease in CO may be seen despite an increase in SV due to a reduced heart rate. It is 
obvious that the later scenario places a patient in a better physiologically advantage and a
potential survival advantage, despite a reduction in CO. It has been previously been 
shown that cardiac output as measured by right heart catheterization has a strong 
correlation to ECHO-derived CO.
Tricuspid annular excursion (TAPSE) predicted 2-year survival in patients with PAH.24 In a
follow-up study looking at 50 consecutive patients with SSc-related PAH, it was shown that 
a TAPSE ≤1.7 cm conferred nearly a 4-fold increased risk of death.25.
Pulmonary artery acceleration time, tissue strain and strain rate of the RV measured by 
speckle doppler both have potential promise as meaningful ECHO-derived parameters in 
the assessment of overall RV function. Based on a prior study, systolic velocity and strain 
best correlated with invasively determined right ventricular stroke volume and tracked 
changes in right ventricular function during vasodilator infusion.26 As pulmonary vascular 
resistance increases, pulmonary acceleration times begin to decrease. Similarly, the RV
myocardial performance index is another potential marker for overall RV function and
circumvents the need to account for the complex, geometric shape of theRV.
Finally, it is well known that CTD-PAH is a disease of increased pulmonary vascular 
resistance (PVR) and changes of pulmonary artery pressure may not truly reflect changes 
in PVR. As PAH progresses, it is reflected in increases in PVR and declining SV. 
Noninvasive assessment of PVR by Doppler ECHO has been recently validated when 
compared to invasive PVR calculation.27 In 150 consecutive patients, the results of the 
study showed a good correlation (r=0.79) between PVR derived by RHC andDoppler

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ECHO. Furthermore, their findings establish Doppler Echo is a reliable method to identify 
patients even in the setting of significantly elevated PVR (PVR >6 Woods units). 27
The design of the current study will allow us assess the efficacy of ambrisentan in the 
treatment of CTD-PAH, who were previously treated with bosentan or macitentan. 
Ambrisentan efficacy in CTD-PAH treatment will be longitudinally assessed with pre-
determined ECHO defined end-points, QOL questionnaire and 6MWT change over 24
weeks.
Sample size and statistical analysis: The primary end point of the study will be to 
determine the efficacy, as measured by an ECHO composite score, for combination 
therapy with ambrisentan in the treatment of CTD-PAH after switch from bosentan or 
macitentan. Secondary endpoints include: 1) safety and tolerability of ambrisentan in the 
treatment of CTD-PAH; 2) 6MWD, 3) WHO Functional Class; 4) EmPHasis
10 
questionnaire; 5) pulmonary function as measured by FVC and DLCO; 6) NT-proBNP; 7) 
compliance with treatment; 8) time to clinical worsening; and 9) defined ECHO variables as 
previously described.
We plan to enroll 50 patients with CTD-PAH at the Medical University of South 
Carolina and the Oschner Clinic. A power analysis is attached in Appendix 1, which 
justifies this number of subjects (sample size needed to detect a 2 Wood units difference 
and a 20% improvement in cardiac index is 42 and 20 respectively). Primary comparisons 
will be pre/post treatment (24 weeks) comparisons of study outcomes (six minute walk
distance, time to clinical worsening, pulmonary function, ECHO-defined variables, FVC,
and DCLO) using a separate paired t-test for each outcome. The paired t-test accounts for
the dependent nature of the data collected on the same subjects at different points intime.
Patient recruitment: Patients will be recruited from the Pulmonary Hypertension and 
Interstitial Lung Disease Clinic of the Medical University of South Carolina and the Ochsner 
PAH clinic in New Orleans. At the Medical University of South Carolina (MUSC), we have 
approximately 30 patients with CTD-PAH who are currently on bosentan or macitentan and 
are potential candidates for the proposed switch study.
Estimated time to complete the study: We estimate that the study will require 12 months 
to recruit 50 patients, which with the expected drop out of zero, will yield 50 full evaluable 
patients and 24 weeks to complete after the last patient is enrolled. The study is designed 
to allow for inclusion of additional sites to decrease enrollment time and increase the 
number of recruited patients into the study. We anticipate the study will require 21 months 
to fully complete.
Mechanism for ensuring patient safety: Patients will be monitored closely by study 
physicians throughout the study. Data from individual tests will be monitored prospectively 
as completed and group data reviewed every 3 months. Pulmonary function testing, six 
minute walk and ECHO will be performed by professionals following standard protocols to 
minimize risks to the subjects. Any time a serious treatment-related adverse event occurs, 
the principal investigator will contact the Institutional Review Board and sponsor. Study 
data will be collected and managed using REDCap (Research Electronic Data Capture) 
electronic data capture tools hosted at MUSC. REDCap is a secure, web-basedapplication

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designed to support data capture for research studies, and our faculty and coordinatorsare 
familiar with its use.
The study coordinator will set up and manage the database, and quality assurance will be 
coordinated by the PI in conjunction with the biostatistician
Early termination: Subjects will be terminated prior to completion of the study for any of 
the following reasons:
Development of pregnancy.
20 % decline in 6-minute walk distance from baseline on two successivevisits. 
Severe lower extremity edema.
Evidence of significant patient noncompliance (ingestion of less than 80% of study 
medication)
Patient desire to terminate the study.
Investigator opinion that continuation in the study would pose undue risk to thesubject.

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Appendix 1
Six Minute Walk Test
The 6MWT will be performed at Screening, Baseline (Week 0), Week 12 and Week 24. It willbe
performed according to ATS Guidelines including the preparation (rest and vitals), use of thepre 
and post-walk Borg Scale for dyspnea and fatigue and heart rate and pulse oximetry recordings 
per lap. The 6MWD will also be recorded and assessed for protocol-defined decline. Each6MWT 
assessment should be administered using the same 02 concentration used at the Baseline visit. If 
the subject is unable to complete the walk, the distance walked will be recorded, as well as the
time of duration of the test.
Pre Borg Scale
0 Nothing at all
0.5 Very, very slight (just noticeable) 
1 Very slight
2 Slight (light)
3 Moderate
4 Somewhat severe
5 Severe (heavy)
6
7 Very severe
8
9
10 Very, very severe (maximal)
Post Borg Scale
0 Nothing at all
0.5 Very, very slight (just noticeable) 
1 Very slight
2 Slight (light)
3 Moderate
4 Somewhat severe
5 Severe (heavy)
6
7 Very severe
8
9
10 Very, very severe (maximal)

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Appendix 2
alpha = 0.50, 2-sided hypothesis testing
Sample SizeRequirements
Detectable change with 80%power
Detectable change
(dyn*sec/cm^5)
Estimated SD ofchange*
Sample size needed
1 Wood unit change inPVR
80
357.8
159
2 Wood units change inPVR
160
357.8
42
3 Wood units change inPVR
240
357.8
20
* based on pooling SD estimates from Klinger & Galie (10mg) 
This assumes no change during first 6 months, followed by the change listed above in the second 6 months
Detectable change with 80%power
Detectablechange(l/min/m^2)**
Estimated SD ofchange*
Sample size needed
10% improvement from baseline in cardiac index
0.25
0.75
73
20% improvement from baseline in cardiac index
0.50
0.75
20
30% improvement from baseline in cardiac index
0.75
0.75
10
** based on pooling baseline estimates from Klinger & Galie (10mg)

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Appendix 3

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