A Phase 2, Open-label, Clinical Trial of Fluoxetine, a Selective Serotonin Reuptake Inhibitor, in the Treatment of Pulmonary Arterial Hypertension
Phase: PHASE2
Conditions: Pulmonary Arterial Hypertension
Sponsor: University of Texas Southwestern Medical Center

1 
 
Title: A phase 2, open-label, clinical trial of fluoxetine, a selective serotonin 
reuptake inhibitor, in the treatment of pulmonary arterial hypertension  
 
Short title: Fluoxetine in PAH Trial 
 
Location: UT Southwestern Medical Center,  
 
Protocol author and supervising physician: Kelly Chin, MD 
 
Sponsor: Investigator initiated, with partial funding from NIH K23 
 
Protocol version 4.1 04/01/2019 
 
Data monitoring physician: Vaidehi Kaza, MD 
 
 
 

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Table of contents 
 
1. Title page 
2. Table of contents 
3. Aims, Introduction and Purpose 
4. Background 
 
Introduction 
 
Preliminary data 
 
Fluoxetine background 
 
Sub studies  
5. Concise summary 
6. Study measures (primary / secondary) 
7. Study procedures 
8. Study Subjects – PAH and controls 
9. Recruitment 
10. Risks 
11. Data and safety monitoring 
12. Statistical analysis 
13. Rationale for IND exemption 
14. Appendix - 5HIAA testing – food interactions 
15. References 
1 
2 
3 
4-8 
 
4 
 
5-6 
 
7 
 
7-8 
8 
8-10 
11 
12-13 
14 
14-16 
17 
17 
18 
19 
20 
 
 
 
 

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Title: A phase 2, open-label, clinical trial of fluoxetine, a selective serotonin reuptake inhibitor, in 
the treatment of pulmonary arterial hypertension (PAH) 
 
Short trial name: Fluoxetine in PAH Trial 
 
Introduction and Purpose 
PAH is a devastating, progressive condition.  A better understanding of the role for serotonin may 
lead to improved treatments and potentially to improved survival beyond the current average of 
five years.  This protocol describes a phase 2 clinical trial of fluoxetine in PAH looking at change 
in pulmonary vascular resistance (PVR) as the primary endpoint; if the trial were positive this 
could lead to larger more definitive studies and novel therapies.   
 
Primary aim / Aim 1: To determine whether fluoxetine lowers pulmonary vascular 
resistance in patients with pulmonary arterial hypertension by conducting a phase 2, open-
label, clinical trial   
 
Primary Hypothesis: Fluoxetine treatment for 24 weeks will lead to significantly lower 
pulmonary vascular resistance in 18 patients with PAH  treated in an open-label clinical trial. 
 
Study: In this clinical trial, 18 patients with PAH will be on fluoxetine for 24 weeks.  A Right 
Heart Catheterization will be performed at baseline and 24 weeks.  Change in PVR will be the 
primary endpoint; other hemodynamic endpoints, quality of life, QIDS-SR depression scale, 
functional class and six-minute walk distance will also be evaluated.   
 
Aim 2: To determine whether baseline 5HIAA / creatinine levels predict response to 
fluoxetine.   
We hypothesize that 5HIAA will be elevated in PAH, will correlate with baseline PVR and will 
correlate inversely with change in PVR after 24 weeks of fluoxetine.  
 
Aim 3: To determine whether fluoxetine leads to a reduction in 5HIAA levels.   We 
hypothesize that fluoxetine lowers 5HT production and turnover systemically, as measured by 
5HIAA levels.   
 
Aim 4: To determine whether fluoxetine reduces markers of platelets and endothelial 
activation in PAH.   
 
Safety endpoints: Safety and tolerability will be assessed by tabulating adverse events and by 
determining the number of patients tolerating each dose of fluoxetine.   
 
 
Background 
Pulmonary arterial hypertension (PAH) is a life threatening pulmonary vascular condition.  
Average survival is five to six years, even with current therapies.  For patients with persistent 
functional class III symptoms after monotherapy, average survival is 2-3 years [1,2].  Novel 
therapies involving alternative pathways are needed.  This study seeks to determine whether 
fluoxetine, a selective serotonin reuptake inhibitor, will improve hemodynamics in PAH. 
 
Serotonin (5HT) has long been considered a potential contributor to PAH, based on its ability to 
promote pulmonary vasoconstriction and vascular cell growth.  Enthusiasm for clinical studies 
waned after antagonists to the serotonin 2A receptor failed to significantly improve pulmonary 

4 
 
hemodynamics.  However, recent progress in the molecular pharmacology of 5HT signaling 
suggests that other 5HT signaling pathways including both the serotonin transporter and other 
serotonin receptors may be relatively more important (table 1). 
 
Specifically, the selective serotonin reuptake inhibitors have been studied in animal models of 
pulmonary hypertension and were found to be effective, and were studied in observational studies 
in human subjects, as below. 
 
 
Table 1: Serotonin Receptor and Transporter Antagonists 
 
Hypoxic PH 
Monocrotaline PH 
Human data:  
1B receptor  
antagonist 
Mixed results – at 
best 
moderately 
effective[3-5] 
No studies 
Triptans (agonists) increase PAP 
25% in normals[6]; no studies in 
PAH  
2A receptor 
antagonist 
Failed 
to 
prevent 
hypoxic PH[3] 
Prevented but did not 
reverse 
monocrotaline PH[7] 
Ketanserin lowered PVR moderately 
but caused hypotension, and long-
term studies were not done[8,9]  
2B receptor 
antagonist 
Prevented 
hypoxic 
PH[10]  
No studies of isolated 
2B antagonism  
ATS Abstract 2008 – antagonist 
improved exertional PVR in lung 
disease PH 
Transporter  
antagonist 
(SSRI) 
Prevented 
hypoxic 
PH[3] 
Prevented 
and 
reversed 
monocrotaline 
PH[11] 
SSRIs 
were 
associated 
with 
improved survival in observational 
studies 
in 
PAH 
(p<0.05 
in 
one)[12,13] 
Table: SSRIs are effective in monocrotaline and hypoxic PH, are able to both prevent and reverse 
monocrotaline PH, and are more effective than 5HT 2A and 1B receptor blockers in head to head 
studies[3]. 
 
The serotonin transporter is also over-expressed in PAH, and over-expression may be harmful 
[14] 
 
Over-expression causes PH spontaneously in animals [15,16] 
 
Over-expression worsens fenfluramine (diet-pill) associated PH in animals [17].   
 
Standard of care: Standard of care in PAH currently involves the use of one or more approved 
PAH therapies from three classes.  These medications improve exercise capacity and symptoms 
at 12-16 weeks vs placebo, and improve long-term survival vs. historical survival.  A longer-term 
placebo-controlled study was also recently completed, and it suggests macitentan, an endothelin-
1 antagonist, reduces longer-term clinical worsening and reduces the combined endpoint of death 
or hospitalization for worsening disease.  Combination therapy studies have also been completed, 
with some positive results.  The most positive study compared oral sildenafil + intravenous 
epoprostenol vs. epoprostenol alone for 12-weeks; combination oral therapy studies have been 
less convincing.  As such, combination therapy is recommended for moderately ill patients 
symptomatic on monotherapy, but participation in a clinical trial is also felt to be appropriate.  
Combination therapy is more strongly recommended for patients with advanced disease who are 
willing to receive continuous intravenous therapy delivered via an external pump.  Triple 
combination therapy (oral ERA, PDE5 and iv, sc or inhaled PGI2) has not been studied in 
randomized trials, but case reports / small series suggest this is tolerable and could be beneficial. 
 
Alternatives to this study: All enrolled patients will be on  one, two or three PAH therapies, 
which is considered standard of care. Alternatives to enrollment include adding additional 
approved therapies, if not already on maximal therapy. Patients on one, two or three approved 
PAH therapies will be allowed as long as the drug regimen (type of meds) is unchanged for 12 

5 
 
weeks and the dose stable for 4 weeks. Lung transplantation is another alternative; it is not 
recommended unless all medical alternatives have been exhausted and survival is not anticipated 
to exceed 1-2 years. Transplant should be considered when right ventricular failure is seen 
clinically with right heart catheterization findings of RA pressure above 15 mmHg and / or cardiac 
index below 2.4 L/min/m2, though not all patients with these findings will be appropriate for 
transplant.  All patients will be informed of these treatment options during the informed consent 
process. 
 
Preliminary Data:  
Pilot Study – reserpine and reserpine + fluoxetine:  
In an earlier study at UT Southwestern, reserpine and fluoxetine were evaluated in 5 patients with 
pulmonary arterial hypertension. Reserpine is a 5HT depleting agent, and the idea was that by 
giving reserpine combined with fluoxetine we could more globally reduce serotonin signaling.  
Specifically, our hypothesis was that the use of fluoxetine to block the 5HT transporter combined 
with reserpine to deplete stored pulmonary and platelet 5HT (and reduce 5HT receptor signaling) 
would have a highly favorable effect on PVR. Funding was obtained through the North and Central 
Texas Clinical and Translational Science Initiative (NCTCTSI). The original protocol called for 
reserpine first, and three patients received open label reserpine for up to 12 weeks.  All three 
patients reported increased dyspnea, lower extremity edema and / or had elevated JVD during 
weeks 2-8.  One patient withdrew from the study, while symptoms gradually improved in the other 
two patients.  Hemodynamic evaluation at 12 weeks in those two patients showed a decline in 
PVR - marked (40%) in in one patient, and small (3%) in the other.  The confusing symptoms and 
clinical course (early worsening, but with possibly late stability or improvement) was reviewed 
carefully with an independent study safety physician and the UTSW IRB and two modifications 
were made: fluoxetine would be given first and the dose of reserpine would be decreased 50% in 
the hopes that this would improve tolerability. The next two patients tolerated fluoxetine for 12 
weeks without difficulty, but both developed dyspnea within four weeks of starting reserpine, and 
one patient had exertional syncope. At this point the reserpine arm of the study was halted.   
 
The cause for this early worsening remains unclear; however, patients with left-sided heart failure 
have been reported to experience worsening heart failure following initiation of reserpine, 
attributed to catecholamine depletion (reserpine depletes both serotonin and catecholamine).  
The overall risk-benefit profile appeared to be likely unfavorable, and the reserpine arm of the 
study was then permanently discontinued.   
 
Pilot – fluoxetine only: 
A modified fluoxetine-only protocol was then continued, and an additional four patients received 
fluoxetine for 12 weeks. This made a total of six patients who had received fluoxetine:  4 fluoxetine 
alone, and the 2 above who had a fluoxetine only period for the first 12 weeks, all on top of 
background PAH therapy. Fluoxetine was begun at 20 mg and increased to 40 mg at two weeks 
and 80 mg at four weeks. No serious adverse events were seen. Five patients completed the 
study, with two patients on the maximum dose and three on lower doses (40 mg in two, 20 mg in 
one) related to mild but sustained symptoms such as anxiety, poor sleep, or general unease. The 
one patient withdrawing reported anxiety and trouble sleeping even on the lowest dose, and also 
was worried about taking a “psychiatric drug”; he chose to withdraw rather than try lower doses 
and his symptoms immediately resolved.    
 
Hemodynamic results for 5 patients are shown in the figures; PVR declined 16% (p=NS) and CI 
improved 19% (p<0.05) with no significant change in PA mean; the sixth patient who had 
withdrawn left during week 2 and did not want to undergo a repeat right heart catheterization so 
soon.   

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Three of five patients completing the study reported improved symptoms in a general sense, but 
a formal PH symptom scale was not utilized. The QIDS-SR depression scale also improved by 
>50% in two patients (50% is considered significant) despite the absence of reported depression 
at study entry.  However, although the scale is depression focused, some depressive symptoms 
(fatigue, low energy, even poor mood) are sometimes difficult to distinguish from the physical 
problems in PH. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Interpretation of preliminary results / modifications to current protocol based on finding: 
This open label pilot study suggests a possible beneficial effect based on the change in PVR 
(16%) and CI (19%) which, if seen in a larger study, would be close to other approved PAH 
therapies (PVR improvement range for effective therapies 12%-39%) 
 
There were no serious adverse events during fluoxetine administration, but tolerability was a 
concern as most patients did not tolerate the full dose. As such, a result, several changes are 
made: 
 
The dose titration will be slowed such that patients will not be given the full dose until week 
12 in the hope that this may improve tolerability of the higher doses.   
 
The study duration will be increased to 6 months based on the slower up-titration, so as 
to allow 3 months at the maximal tolerated dose.   
 
A more careful dose response analysis will be complete, so that we will better understand 
whether pushing the higher doses is required.  This will include a comparison of dose (mg 
/ day) vs. change in PVR as well as assessment of fluoxetine blood levels vs. change in 
PVR.   
 
Separately, we will add quality of life measures to better detect overall change in clinical status, 
in addition to continued use of a depression scale. The maximum dose of fluoxetine is still targeted 
because higher doses are more effective in animals. 
 
Fluoxetine: Fluoxetine is FDA-approved for depression and is widely used today in the United 
States.  It functions as a selective inhibitor of serotonin uptake, acting on the serotonin transporter.  
Its safety in PAH is unknown, but SSRI use for depression is felt to be acceptable in PAH, and 
two epidemiological studies have shown use rates in the 13-15% range. A theoretical concern 
with fluoxetine is that it could acutely increase circulating serotonin levels and cause pulmonary 
vasoconstriction via signaling at the serotonin receptor. However, sustained elevations in 
serotonin levels (circulating) are not seen with SSRI use [18] (CNS serotonin levels after SSRI 

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are more variable and depend on the study and region of the brain). Additionally, serotonin release 
from storage in the lungs will gradually decrease, due to reduced uptake into both pulmonary 
neuroendocrine cells and platelets.  Finally, the two retrospective studies in PH found a hazard 
ratio for mortality with SSRI use that was less than one (HR 0.53, p=NS[12] and HR 0.56, 
p=0.003[13]) – though of uncertain significance, there was certainly no evidence of harm with 
SSRI use noted in these studies. Side effects may include: Common (>10%): nausea, diarrhea, 
dry mouth, anxiety, and somnolence.  Less common but serious: chest pain, suicidal ideation and 
suicide attempts. Full prescribing information is available at 
http://www.accessdata.fda.gov/drugsatfda_docs/label/2011/018936s091lbl.pdf  
 
Rationale: we propose that the 5HT transporter plays an important role in the pathophysiology of 
PAH, related to the growth promoting effects of serotonin when it is internalized by the transporter.  
This hypothesis is supported by the findings that 5HT promotes growth and remodeling via the 
5HT transporter through uptake and internalization of serotonin. After internalization, growth is 
subsequently promoted via serotonylation of intracellular proteins as well as the generation of 
reactive oxygen species [19,20]. Selective serotonin reuptake inhibitors block serotonin uptake 
and reduce pulmonary artery smooth muscle cell growth in vitro and lower pulmonary arterial (PA) 
pressure and reduce vascular remodeling in animals[14,21]. Of note, while human studies in PAH 
are lacking, observational data suggests that SSRI use may be associated with longer survival in 
PAH, though data is limited [12,13]  
 
SUBSTUDIES: 
Are 5HIAA levels (1) Increased in PAH and (2) Predictive of response to fluoxetine?  
Serotonin is metabolized to 5HIAA. Individuals with higher 5HT production have higher 5HIAA 
levels.  Prior small studies suggest 5HIAA is higher in PAH vs. normal controls and in congenital 
heart disease with PAH vs. congenital heart disease without PH. We plan to further evaluate 
5HIAA levels before and after SSRI use.  We propose that patients with PAH will have  
1. Elevated 5HIAA levels 
2. 5HIAA levels that correlate with disease severity by right heart catheterization 
3. Those with higher levels will also have a better response to fluoxetine.   
 
This is based on the finding that 5HT production and turnover appear to be increased in PAH, 
and that 5HT promotes vascular cell growth. The cause for increased turnover is unclear, but 
studies have shown that PAH endothelial cells make more 5HT, PAH neuroendocrine cells and 
platelets take up and store more 5HT (and platelets release it more easily), and 5HIAA, the 
primary 5HT metabolite, is increased levels in small PAH case series[22-29].   
 
Separately, we propose that fluoxetine will lower 5HIAA levels – though we are not particularly 
confident of this. Circulating 5HT levels increase acutely with SSRI therapy, and then normalize 
(based on animal studies) [18]. How this occurs is not clear – either upregulated alternative 
clearance, or reduced production. Since almost all 5HT is metabolized to 5-HIAA, measurement 
of 5HIAA should reflect total body production / metabolism (at steady state).   
 
Study specifics: 5HIAA can be measured as either a 24 hour urine test, a spot urine 5HIAA level 
/ creatinine or as a serum test.  The spot urine / creatinine test was chosen for simplicity and cost 
reasons.  1st am urine for 5HIAA and creatinine will be obtained at study entry and exit.  
Specimens will be frozen; testing is valid as long as performed within 365 days.   
 
A group of 16 healthy volunteers will be recruited as well to serve as normal controls.  All normal 
controls will complete the following:  
1. A questionnaire about their health and their current medications, 

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2. Blood pressure, pulse, height and weight will be checked, 
3. Completion of the QIDS-SR and the SF-36 questionnaires,  
4. A blood sample (4 teaspoons) and one urine sample will be collected.  
 
Platelet and Inflammation: 
Are markers of platelet and endothelial activation increased in PAH, and are they altered 
by therapy with fluoxetine?   
Platelet and endothelial cell activation is seen in PAH, may correlate with PAH disease severity, 
and can fall with treatment of PAH [30-34].  We hypothesize that platelet activation will fall with 
SSRI therapy, because SSRIs reduce platelet activation in other settings and because SSRIs 
deplete platelet 5HT [35].  However, platelet aggregation tests have been inconsistent in PAH[32].  
We plan to bank plasma for future measurement of indirect markers of platelet and 
endothelial cell activation such as soluble p-selectin, soluble CD40 ligand and beta-
thromboglobulin.  Prior studies suggest these markers are elevated in PAH, and they are readily 
measured [30,36]. Evaluations will be performed at baseline and week 24 in all patients in the 
clinical trial and at baseline in the 16 controls.   
 
CONCISE SUMMARY OF PROJECT 
This is a phase II single open-label study enrolling 18 patients with PAH. Patients will be on 
fluoxetine for 6 months, undergoing testing right heart catheterization at baseline or just prior to 
baseline and at 6 months. An estimated 3 years will be required to complete enrollment.  
Fluoxetine dose will be initiated at 20 mg, with dose titration every 4 weeks to a maximum of 80 
mg daily, as tolerated. The primary objective will be to determine whether fluoxetine leads to 
improvement in PVR; secondary endpoints include 6MWD, quality of life, functional class and 
safety.   
 
Study Methods:  
Medication: 18 patients will receive fluoxetine.  Dosing will be as descripted below: 
 
Week 1-4: 20 mg daily  
 
Week 5-8: 40 mg daily  
 
Week 9-12: 60 mg daily  
 
Week 13-24: 80 mg daily  
 
Fluoxetine details:  
Compounding: Prescription for fluoxetine will be sent to Walgreens pharmacy here at UTSW at 
the time of patient enrollment. 20 mg pills will be procured initially, and dosing will therefore be 1, 
2, 3 or 4 pills daily. Fluoxetine is also available in a 40 mg pill, so that once a stable dose is 
reached patients will be transitioned to 20 mg daily (one 20 mg pill), 40 mg daily (one 40 mg pill), 
60 mg daily (one 20 mg and one 40 mg pill) or 80 mg daily (two 40 mg pills). The maximum 
(fastest) rate of titration is the schedule above. Patients will follow the schedule as long as having 
no more than mild side effects. Slower up-titration will be allowed, and down-titration will also be 
allowed.  However, by 16 weeks, a stable and final treatment dose must be reached.  At this point 
no additional upward titrations will be allowed; down- titration for new onset side effects will still 
be permissible but this is anticipated to be uncommon. 
 
Dose selection: Eighty mg is the maximum approved dose of fluoxetine, and this dose was chosen 
based on animal studies showing better responses at higher doses [11]. We anticipate that this 
will be tolerated in approximately 50% of fluoxetine patients, and that most of the remaining 
patients will tolerate one of the lower doses with ~10-20% drop out rate.  These estimates are 
based on the tolerability of these medications in general, drop-out rates in PAH studies, and our 

9 
 
findings in the pilot study.  Maximum tolerated dose was 20 mg in one patient, 40 mg in two and 
80 mg in two.  We are hoping to increase tolerability of the higher doses by slowing the up-titration 
rate.  Even higher doses were also considered, but based on our pilot study tolerability of even 
the currently planned dose may be limited.   
 
Early withdrawal: Patients may exit the study for medication side effects, withdrawal of consent, 
worsening symptoms or other reasons.  Patients exiting the study early will be asked to complete 
all or some of the week 24 procedures (either all procedures or if unwilling, all procedures except 
right heart catheterization). This includes those who request study discontinuation (for side effects 
or other reasons) and those who worsen during the study and require additional medications. 
Discontinuation will ideally be gradual, to be accomplished over up to 4 weeks if possible, as 
recommended for SSRIs in general. However, the long half-life of fluoxetine significantly lessens 
the chance of withdrawal symptoms and in fact few withdrawal symptoms were seen in a sudden 
withdrawal study with fluoxetine (unlike sertraline and paroxetine) in patients undergoing 
treatment for depression [37]. 
 
Study Outcome Measures 
Primary endpoint: Change in PVR between baseline and follow-up will be utilized as the primary 
endpoint. PVR will be calculated as [(PA mean – wedge) / Fick CO] PVR was chosen because it 
is a sensitive measure of improved hemodynamics in PAH, and improvement in PVR has also 
been strongly associated with improved exercise capacity and survival.  PVR has also improved 
with all of the current FDA-approved PAH therapies.  Fick CO was chosen over thermodilution 
because Fick appears to have greater precision (but not accuracy).  Since we are most interested 
in a signal suggesting that there is a change in PVR with this therapy, then use of the Fick CO 
will improve power by minimizing random variability. Clinical studies have suggested that 
outpatient resting oxygen consumption tends to be stable over modest (6-12 months) periods of 
time in most individuals.   
 
Secondary endpoints include change in: RA, PA and wedge pressure, CO, SvO2, PVR 
(thermodilution), QIDS-SR, SF-36, 3 other symptoms scales (single item scales), systemic BP 
and HR, functional class and change in 6MWD.   
Safety endpoints: will be assessed by tabulating adverse events. Following the study, a 30-day 
safety window will be observed.  We will also assess longer-term outcomes by chart-review after 
1 and 2 years. 
 
Measurements: 
Right Heart Catheterization: Right Heart Catheterizations will be performed in the CUH 
catheterization laboratory using the internal jugular or femoral veins for venous access.  
Procedures will be conducted per catheterization-lab usual routine, including standard safety 
monitoring (EKG, oximetry, blood pressure).  Ultrasound and fluoroscopy will be used in catheter 
placement.  Measurements will include end-expiratory right atrial (RA), PA and wedge, systemic 
noninvasive blood pressure, heart rate, cardiac output (CO, by thermodilution, performed in 
triplicate and averaged), pulmonary (SvO2) oxygen saturation, systemic pulse-ox oxygen 
saturation, hemoglobin.  Calculations will include PVR and Fick CO (using estimated VO2)[38].    
 
Six minute walk distance: Six minute walk distance (6MWD) is the distance a patient can walk 
in 6 minutes using a long (50 meters) hallway.  6MWD correlates with VO2 max, predicts survival 
in PAH, and is an important efficacy endpoint in pivotal PAH clinical trials.   
 
Quality of Life, Depression and WHO functional class  
1. Single item symptom severity scales 

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a. PGIS: Patient global impression of severity – symptoms 
i. 
None 
ii. Very mild 
iii. Mild 
iv. Moderate 
v. Severe 
b. CGIS: Clinician global impression of severity – symptoms 
i. 
None 
ii. Very mild 
iii. Mild 
iv. Moderate 
v. Severe 
c. CGI change: Clinical global impression of change 
i. 
Very much better 
ii. Moderately better 
iii. A little better 
iv. No change 
v. A little worse 
vi. Moderately worse 
vii. Very much worse 
2. WHO functional class is a symptom scale with 4 classes: no symptoms (class I) or symptoms 
with ordinary activity (II), less than ordinary activity (III) or at rest (IV).   
3. The SF-36 is a widely utilized quality of life scale 
4. The QIDS-SR depression scale is a 16-item depression scale that is shorter than and 
correlates well with the HAM-D[39] 
 
Labs:  
1. Fluoxetine level at week 24 will be performed. This will require at least 1 ml of blood  
2. Urine pregnancy will be performed in female patients of child bearing potential 
3. Urine for spot urine 5-HIAA and creatinine will be collected (see substudy below) 
4. Plasma and serum will be banked for future non-genetic studies including p-selectin, soluble 
CD 40L, beta-thromboglobulin.  A total of 20 ml will be drawn for these studies at visit 24 or 
end of study 
 
Study procedures – timeline 
During screening, inclusion/exclusion criteria will be reviewed and if met, informed consent will be 
obtained.  Patients will be seen by the MD or nurse practitioner and coordinator at each visit, with 
tests as indicated in the chart.  All visits and study procedures should be within +/- 7 days.   
 
 
 
 
Table: Study Visits for Clinical Trial of Fluoxetine 
 
 
Base
-line 
Week 1 
Week 
4 
Week 
8 
Week 12  Week 18 Week 24 
Phone call 
 
X 
 
 
 
 
 
Clinic visit / vitals / 
exam 
X 
 
X 
X 
X 
X 
X 
6MWD, WHO Class 
X 
 
X 
X 
X 
X 
X 
QIDS-SR 
X 
 
 
 
X 
 
X 

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SF-36 
X 
 
 
 
X 
 
X 
Other QOL 
X 
 
 
 
X 
 
X 
24-hr Urine / blood-
work  
X  
 
 
 
  
 
X 
Right 
Heart 
Catheterization 
X 
 
 
 
 
 
X 
Fluoxetine (mg) 
Start 
20 
mg 
20 mg 
40 mg 
60 mg 
80 mg 
80 mg 
80 mg 
Table: Medication doses increase every 4 weeks, as tolerated.  Pregnancy testing (when 
appropriate) will also be done at entry and prior to caths.  6MWD: six minute walk distance.  WHO: 
World Health Organization.  QIDS-SR: Depression Scale.   
 
 
Controls: will undergo a spot urine 5-HIAA – creatinine level and blood banking 
 
Early withdrawal: Patients exiting the study early for any reason will complete the week 24 
procedures, whenever possible. This includes those who request study discontinuation (for side 
effects or other reasons) and those who worsen during the study and require additional 
medications. Discontinuation will ideally be gradual, to be accomplished when possible over up 
to 4 weeks.   
 
Routine end of study: patients will be offered treatment with open-label fluoxetine.   
 
Sub-Study Procedures:  
Urine 5HIAA and creatinine - 18 clinical trial patients and 16 controls (unrelated family members) 
will undergo spot urine 5HIAA, tested in the clinical laboratory. Trial participants will undergo a 
repeat test at week 24. 5HIAA and creatinine are clinical tests that can be run at Mayo. Total 
volume will be 1 urine sample (at least 10 ml) and 1 blood sample for all (20 ml); for clinical trial 
patients this will be repeated at end of study. 
 
Comparisons will be: 
1. 5HIAA level: IPAH vs. control.   
2. Correlations: baseline 5HIAA vs. baseline PVR and vs. change in PVR at 24 weeks, IPAH 
patients.   
3. Change in 5HIAA level: baseline vs. week 24, IPAH patients.   
 
Blood – platelet and endothelial activation and fluoxetine 
18 clinic patients and 16 controls.   
1. 20 ml blood will be obtained for plasma and serum. This will be placed in a red-top tube 
(serum, at least 1 ml) and blue-top tube.   
2. For the plasma tests, a plasma volume of 750 L is required. Samples will be sent together, 
as a batch of 50 is required, on dry ice via overnight courier. Plasma will be obtained by 
drawing blood into a blue-top citrated tube, inverting the tube 6 times, and then centrifuging 
at 2000g for 10 minutes. The platelet poor plasma will be drawn off, and then re-centrifuged 
for 10 minutes.  Then freeze. 
Kalyn Sowell 
Myriad RBM, Inc 
3300 Duval Road 
Austin, TX 78759 
512-835-8026  

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3. Serum levels are required for fluoxetine.  This requires a red top with at least 1 ml (resulting 
in at least 0.5 ml serum). Should be separated to serum within 2 hours of blood draw.  
Fluoxetine levels are a clinical test and can be run at Mayo. 
 
 
Study Subjects:  
Number of patients to be consented is planned at 18 in the clinical trial, and 16 controls for the 
blood draw study. Potential subjects will be patients of the investigators. Recruitment is 
anticipated to require 3-4 years 
 
1. PAH patients: patients with PAH and persistently elevated PVR after treatment with one or 
more approved therapies will be eligible, including endothelin-1 receptor antagonists 
(ambrisentan or bosentan), phosphodiesterase-5 inhibitors (sildenafil or tadalafil) and 
prostacyclins (epoprostenol, treprostinil, iloprost).  See I/E criteria below 
 
2. Controls for 5HIAA substudy: additionally, 16 unrelated controls will be recruited for the 24 
hour 5HIAA tests and platelet tests: these will be age-matched and will be recruited through 
PH clinic.  These subjects will complete a brief questionnaire and a 24-hour urine collection 
and a blood draw.  Subjects must be willing to comply with urine collection and dietary 
restriction prior to testing.  Subjects will be excluded for known heart disease (unless 
asymptomatic and with echo within 1 year showing no pulmonary hypertension), known 
pulmonary hypertension 
 
Control inclusion / exclusion criteria 
1. Not genetically related to a PH patient 
2. No significant heart or lung disease 
3. No history of carcinoid 
4. Not on an SSRI or TCAWilling to avoid the following medications  72 hours prior samples 
collection: Aspirin, Corticotropin, Ethanol, Isoniazid, Homogentisic acid, Imipramine, 
Monoamine oxidase inhibitors (MAOIs), Methyldopa, Promethazine, Phenothiazines, 
Perchlorperazine, Octreotide, Acetaminophen, Diazepam, Ephedrine, 5-Fluorouracil, 
Guaifenesin, Melphalan, Naproxen, Nicotine, Phenobarbitone, Phentolamine, Reserpine 
5. Age 16-80 
6. Willing to follow diet recommendation  72 hours prior to samples collection: No avocados, 
bananas, caffeine, butternuts, cantaloupe, dates, eggplant, grapefruit, hickory nuts, honeydew 
melon, kiwifruit, melons, nuts, pecans, pineapple, plantains, plums, tomatoes and tomato 
products, or walnuts for 72 hours before collection. 
 
 
Table 2: Inclusion / Exclusion Criteria 
Inclusion 
1. WHO Group I PAH subtypes of idiopathic PAH and PAH associated with drugs / toxins, 
connective tissue disease, repaired congenital heart disease and unrepaired atrial 
septal defect, diagnosed by complete work-up (VQ or CTA, echo, labs, PFTs, right 
heart catheterization)* 
2. Age 16-80 
3. WHO Functional Class II or III (definition in “procedures” below) 
4. Right Heart Catheterization within 3 weeks of study entry with mPAP  25 mmHg, 
wedge ≤ 15 mmHg, and PVR ≥ 3 Wood units.   

13 
 
5. Contraception use, (-) urine pregnancy test, not breast feeding (women of childbearing 
potential)† 
6. One, two or three approved PAH therapies for 3 months, no change in dose for 1 
month (endothelin-1 antagonist, phosphodiesterase-5 inhibitor, prostacyclin / 
prostacyclin analog).  Novel approved therapies in one of the three existing classes will 
also be acceptable as background therapy if they become available during the course 
of the study; other medication classes are excluded  
 
Exclusion 
 
 
7. WHO Functional Class IV or listed for lung transplant (Reason: may be too ill / 
unstable) 
8. Moderate or greater obstructive lung disease: FEV1/FVC <70% and FEV1 <60% 
9. Moderate or greater restrictive lung disease: TLC or FVC <60% (if 50-60%: OK if TLC 
or FVC ≥50% + PFT stable x1 year + CT with no more than mild lung disease) 
10. Other cause for pulmonary hypertension: all other WHO group I diseases (including but 
not limited to liver disease, HIV), and WHO Groups II-V (i.e. left heart disease, lung 
disease, chronic PE and miscellaneous causes)24.   
a. High probability VQ or positive CTA  
b. Left ventricular ejection fraction <40% 
11. Depression  
12. Severe liver, renal or other medical or physical disease preventing completion of the 
study procedures  
13. Use of antidepressants within 3 months  
*ACCP consensus conference definitions; these sub-types in general respond similarly to 
PAH therapies25. 
 †Contraceptive pill, implant, vaginal ring, IUD, abstinent, vasectomised partner or combination 
barrier method.   
 
The entry criteria above were chosen because:  
1. Patients with PAH should be on one or more approved PAH background therapies, because 
monotherapy has been shown to be beneficial. Combination therapy is also permitted.  
Epoprostenol (an intravenous therapy) has shown a survival benefit in a randomized clinical 
trial; the other approved medications have led to improved exercise capacity and symptoms 
and in aggregate, a survival benefit has been suggested by meta-analysis.   
2. Class II – III patients are included because these patients may benefit from additional therapy 
/ Class IV patients are excluded due to high mortality rates and frequent need for transplant; 
they also make up <5% of our patient population. 
3. Pregnant women are excluded because patients with PAH should not become pregnant, and 
mortality is 30-40% during the pregnancy.   
4. Children under 16 are excluded because of possibly greater risks with SSRIs and definitely 
greater risk with the study right heart catheterizations, due to the need for sedation.  Children 
16 years and older are included because the severity of their condition warrants evaluation 
with novel medical therapies when possible. 
5. Patients over 80 are excluded due to high rates of comorbidities and because of higher rates 
of misdiagnosis, even in the rare patient who is initially thought to have idiopathic PAH.   
 
Sources of research material 
Existing electronic medical records (Epic) will be utilized to obtain medical history, diagnosis, prior 
right heart catheterization and other records required for inclusion / exclusion criteria. Data 
generated during the study will include results from the right heart catheterization, walk distances, 
survey results and AE and SAE reports.   

14 
 
 
Recruitment:  
Patients will be identified in one of two ways:  
1) Identification during routine clinical visits that do not involve specific chart review outside 
of routine clinical practice.  This occurs as the MDs and NPs in clinic are sub-investigators 
on this study and most research patients in PAH are identified in this manner.   
2) Screening of charts of patients with pulmonary arterial hypertension by research 
coordinators: an estimated 200 charts will be reviewed; patients will not be approached 
unless treating MD agrees.  
 
Patients will be informed that this is an early phase clinical trial, that participation is voluntary and 
not required as a part of their care, and that there are generally other options to consider such as 
combinations of approved PAH therapies (if not already on triple therapy). Informed consent will 
be obtained by the enrolling physician or nurse practitioner. Patients are provided with a written 
copy of the informed consent, generally at a screening rather than enrollment visit. This allows 
them time to review the consent and ask any questions.  After questions are answered, the 
consent and HIPAA documents will be signed and the patient will be given a copy of the signed 
consent to keep.  The consent form includes detailed information about the nature of the research, 
the study procedures, the risks involved, and alternatives to participation.  A study visit schedule 
is also included.  For minors 16-17, patient assent plus parental consent (at least one parent) will 
be obtained.  Because the children included are near the age of consent, these patients will be 
fully included in the discussion of risk vs. benefit, including review and an “assent” signature on 
the consent form and any answering any questions they may have. Minors will not be reconsented 
when they turn 18 as they will be old enough to understand that their samples will be banked. 
However, it will be the responsibility of the parent/Legally Authorized Representative to inform 
them of this fact. Patients  18 years old and over are legally able to consent for themselves in our 
location.   
 
Controls will be identified through clinic – genetically unrelated family members (like spouses and 
step siblings) of patients with PAH will be eligible for the substudy (urine and blood sample). 
These controls will be recruited when they come in with the PAH patients for clinic visit. 
 
 
 
Potential risks 
Risks in the study involve the performance of one or two extra right heart catheterizations 
(depending on whether the baseline right heart catheterization is routine care), the use of 
fluoxetine, an FDA approved antidepressant, and the performance of other study procedures 
including walk distances, blood draws and the administration of surveys.   
 
Safety concerns: fluoxetine 
Although RCT in PAH are lacking, in other cardiac conditions (coronary artery disease or heart 
failure), no concerning safety signals for SSRIs have been identified.  Cohort studies in PAH also 
suggest that users of SSRI have a survival as good or better than the survival of nonusers of 
SSRI therapy.  We will monitor patients closely with visits every 4-6 weeks, as above.  Patients 
will be observed closely for expected SSRI side effects (gastrointestinal symptoms, anxiety and 
other psychiatric symptoms) and for worsening PH.  No patients with active depression will be 
enrolled, and patients who develop signs of depression will be withdrawn and undergo psychiatric 
evaluation.  A local independent data-safety monitoring physician will review all SAEs at 
the time of the event, and will review the overall study (AEs, SAEs, other data) every 6 
months.   

15 
 
 
Medication: 
1. Anxiety, insomnia, gastrointestinal upset, and sexual dysfunction have been reported with 
fluoxetine and other SSRIs: In order to minimize this, patients experiencing bothersome side 
effects who are willing to continue in the study may have their dose reduced to the prior 
tolerated dose including use of a 10 mg dose if required. 
2. Suicidal ideation has been reported with fluoxetine when used for depression.  As such, all 
patients will be warned of the signs and symptoms of depression and suicidal ideation, and 
we will monitor for symptoms of depression during monthly visits.  Patients will also be called 
during their first week of therapy and a review of any treatment emergent side effects will be 
completed.  A baseline and follow-up QIDS-SR depression scale will be performed.  Patients 
who develop symptoms of depression during the study will be referred for psychiatric 
evaluation.  Note that this risk is based on depression studies; in particular, the suicidal 
ideation risk in a non-depression study is unknown.  The drug-placebo risk within depression 
studies varied by patient age: increased risk if under 25 (14 and 5 additional cases per 1000 
exposed for <18 and 18-24 years old, respectively) and decreased risk if 25 and over (1 and 
6 fewer cases per 1000 for those 25-64 and >64 years, respectively).  Since each individual 
patient may react positively or negatively, close follow-up will be made 
3. Pregnancy and lactation: fluoxetine is pregnancy category C and possibly unsafe with 
lactation.  A negative pregnancy test and effective contraception will be required.   
 
Risks based on depression studies: 
Risk 
Typical Severity  
Frequency 
Fluoxetine risks 
 
 
Anxiety 
Mild-moderate 
Common 
Trouble sleeping 
Mild-moderate 
Common 
GI upset or poor appetite 
Mild-moderate 
Common 
Sexual dysfunction 
Mild-moderate 
Common 
Dry mouth 
Mild-moderate 
Common 
Tremor 
Mild-moderate 
Common 
Rash 
Variable 
Common 
Increased or new suicidal 
ideation 
Severe 
Infrequent 
Serotonin syndrome, 
particularly if taken with 
medications (see below) known 
to increase risk 
Severe 
Rare 
   
 
 
 
 
 
 
 
 
 
 
 
Common: >1% (common mild side effects only reported if >5%), Infrequent: 0.1-1%, Rare <0.1% 
 
The risk of serotonin syndrome, an increased levels of serotonin and toxicity related to drug 
interactions, is increased with use of triptans, TCAs, fentanyl, lithium, tramadol, trypotphan, 
buspirone, St. Johns wart, MAOIs, linezolid and methylene blue.   
Linezolid, methylene blue and MAOIs are contraindicated; the other medications on this list  
should only be used if required and with extra caution. 
 
Right Heart Catheterization risks:  

16 
 
Right Heart Catheterizations are performed routinely in pulmonary hypertension for diagnosis and 
monitoring for disease progression, typically at least yearly for several years.  Right Heart 
Catheterization is also often repeated after ~3 months of major change in therapy.  Participating 
in this will result in a right heart catheterization at baseline (generally a routine care right heart 
catheterization) plus another right heart catheterization at six months, meaning one or two extra 
right heart catheterizations may be performed beyond what would normally have taken place for 
clinical reasons.  Risk of right heart catheterization includes pneumothorax, injury to vascular 
structures and death.  Risk will be minimized by routine use of ultrasound during internal jugular 
access, and use of fluoroscopy and EKG monitoring during catheter placement.  In experienced 
centers, the complication rate is low (<1%), and the serious complication rate is very low 
(<0.1%)[40].  Serious adverse event rates are likely lower in the current era due to the universal 
use of ultrasound, avoidance of systemic vasodilators for vasodilator testing, and avoidance of 
systemic sedation when possible.   
 
Other risks 
Risks related to the other study procedures (blood drawing, six minute walk testing, quality of life 
surveys) are generally low and will be minimized by following routine precautions for blood 
drawing, by monitoring oxygen saturation, heart rate and patient wellbeing during walk testing, 
and by reminding patients that continued participating in the study is voluntary.  Patients are asked 
to stop walking if they feel dizzy, develop severe dyspnea, or develop chest pain, or if they appear 
unsteady.  They are monitored closely by a trained operator during the test.  Patients will also be 
told they can refuse to answer any questions that make them uncomfortable. 
 
Confidentiality:  
Protected health information will be collected during the study. This will be protected in locked 
cabinets within locked offices, and by use of password protected computers in locked offices.  
Study samples will be stored within research freezers and identified with subject study numbers 
only. 
 
Decompensation related or unrelated to study medication: worsening pulmonary hypertension 
is always a possibility and a concern.  Patients will be receiving closer than usual monitoring 
through more frequent visits and right heart catheterizations.  However, decompensation may 
occur.  Any patient with clinical worsening who requires additional therapies will be withdrawn 
from the study, after undergoing an early “end of study” visit, when it is felt to be safe to do so and 
when they are willing.  Additional therapy, as appropriate, will then be considered. 
 
Other Potential Problems: Completion: we need 16 patients to undergo a post-treatment right 
heart catheterization and other week 24 assessments out of a planned enrollment of 18 patients.  
This allows for 2 (12.5%) patients to drop out without a post-Rx catheterization and still have 
sufficient power. Based on past experience, this should be achievable: even patients leaving early 
typically allow end of study assessments. However, if a higher dropout rate is seen then 
enrollment will be increased so as to allow 16 patients to complete. Safety: because these patients 
are sick at baseline, adverse events from the disease are not unexpected. We will regularly review 
adverse events (AE) in order to identify any concerns, and a data safety monitory committee will 
review the AE and SAE data prior to each IRB continuing review..   
 
 
Data and safety monitoring plans:  
Monitoring will include: 

17 
 
1. Principal investigator: As the principal investigator, I will have the primary responsibility for the 
safe conduct of the study. This will include careful conduct of the study, close monitoring for 
any adverse effects, and frequent follow-up.   
2. Institutional review board (IRB): the UTSW IRB will monitor the studies through approval of 
the protocol prior to any patient enrollment, through routine review of any serious adverse 
events, and through general oversight of all research conducted at UT Southwestern. 
3. Internal safety physician: A local safety monitoring physician is planned. This physician will 
be independent from the conduct of the study. A charter outlining responsibilities will be 
created.  All SAEs will be reviewed.  Every 6 months, all SAEs along with a tabulated chart of 
all AEs every 6 months (tabulated and stratified by group assignment).  Adverse events will 
also be reported to the NIH and IRB, as required. 
 
Statistical Analysis  
Aim 1: To determine whether fluoxetine lowers pulmonary vascular resistance in patients 
with pulmonary arterial hypertension by conducting an open-label clinical trial     
1. Study design and measurements: 18 patients will receive on fluoxetine for \24 weeks.  
Patients will undergo a right heart catheterization, 6MWD testing, QIDS-SR depression scale 
testing, SF-36, other quality of life (as above) and functional class assessment pre-treatment 
and after 24 weeks.   
2. Efficacy endpoints: Primary endpoint: Change in PVR between baseline and end of study.  
Secondary endpoints include change in: RA, PA and wedge pressure, CO, SvO2, PVR (Fick), 
QIDS-SR, SF-36, systemic BP and HR, functional class and change in 6MWD.   
3. Safety and tolerability endpoints: will be assessed by tabulating adverse events and by 
determining the number of patients tolerating each dose of fluoxetine.   
4. Open label hemodynamics after one year will also be presented descriptively and vs. baseline 
among patients who undergo a one-year right heart catheterization. 
5. Comparisons and evaluation of the data: All statistical testing will be baseline vs. follow-up 
at 24 weeks. Student’s t-tests will be used for significance testing if the data are normally 
distributed with no missing data. If data are missing due to dropouts or lack of study 
completion, patients with worsening symptoms will be assigned worst rank, and a Wilcoxon 
rank sum test will be used. The Wilcoxon rank sum test will also be used to evaluate the 
ordinal results from the QIDS-SR, the SF-36, and changes in WHO functional class.   
6. Assumptions and anticipated magnitude of treatment effect and power:  Baseline PVR 
is estimated to be 9 wood units, based on preliminary data PVR of 8.4 wood units as well as 
other similar trials. Effect size: this study will be powered to detect a 20% improvement in 
fluoxetine patients with 16 of 18 patients undergoing an end of study right heart 
catheterization.  In earlier studies, approved PAH therapies (endothelin-1 antagonists, PDE-
5 inhibitors and prostacyclins) have lowered PVR 12-39% (all except one: 20-39%).  We also 
anticipate a standard deviation of 20% of baseline PVR. 
  Summary: With 16 patients completing, this study will have 80% power (=0.05) to detect a 
between-group difference assuming 20% improvement with fluoxetine (References for SD 
and other assumptions: [41-47]) 
 
Safety and tolerability endpoint: An important secondary endpoint will be safety and dosing 
tolerability.  This will be evaluated both qualitatively and quantitatively. We anticipate that ≥80% 
of patients will be able to complete the study (completing either 24 weeks or undergoing early end 
of study) and ≥70% of enrolled patients will complete it without suffering a serious adverse event 
judged as possibly or likely related to medication (SAE; death, hospitalization, risk to life or 
permanent disability). The determination of “likely” related is to be made by an independent 

18 
 
physician who will review the data for all SAEs. These modest goals are chosen because all 
patients will be seriously ill.     
 
Investigational New Drug (IND) Exemption:  
The fluoxetine clinical trial should be IND exempt based on our discussions with the FDA.  For 
the pilot study (completed) we proposed exemption, but the UTSW IRB requested IND submission 
to be certain criteria 3 was met; this was done and the FDA classified it as IND exempt.  We 
therefore anticipate similar classification with the slightly larger study as it will still be small and 
not seeking a new indication.  Specific criteria we meet include: 
 
IND exemption criteria: 
(1) it is not intended to be reported to FDA in 
support of a new indication for use or to 
support any other significant change in the 
labeling for the drug; 
As a small study looking at a surrogate 
endpoint (change in PVR), this will not 
support a label change or new indication. 
(2) it is not intended to support a significant 
change in the advertising for the product;  
Same as response to (1)  
(3) it does not involve a route of 
administration or dosage level, use in a 
subject population, or other factor that 
significantly increases the risks (or decreases 
the acceptability of the risks) associated with 
the use of the drug product; 
The pilot study of fluoxetine and reserpine 
(see preliminary data section) met this 
criteria, per FDA response to our IND 
application.  The fluoxetine study alone has a 
similar or lower risk (because only fluoxetine 
is used) and should therefore be similarly  
exempt.   
(4) it is conducted in compliance with the 
requirements for IRB review and informed 
consent [21 CFR parts 56 and 50, 
respectively];  
IRB review is being obtained for the clinical 
trial. 
(5) it is conducted in compliance with the 
requirements concerning the promotion and 
sale of drugs [21 CFR 312.7];  
Use of fluoxetine will be investigational; 
promotional claims of safety and 
effectiveness for PAH will not be made 
(6) it does not intend to invoke 21 CFR 50.24.  We will obtain informed consent  
 
 
 
Appendix: 5HIAA testing interactions.   
Subjects will be instructed to avoid the following for 72 hours prior to the test: 
Medications/factors that decrease 5-HIAA levels are as follows: 
 
Aspirin 
 
Corticotropin 
 
Ethanol 
 
Isoniazid 
 
Homogentisic acid 
 
Imipramine 
 
Monoamine oxidase inhibitors (MAOIs) 
 
Methyldopa 
 
Promethazine 
 
Phenothiazines 
 
Perchlorperazine 
 
Octreotide 

19 
 
Medications/factors that increase 5-HIAA levels are as follows: 
 
Recent surgery 
 
Stress 
 
Acetaminophen 
 
Caffeine 
 
Diazepam 
 
Ephedrine 
 
5-Fluorouracil 
 
Guaifenesin 
 
Melphalan 
 
Naproxen 
 
Nicotine 
 
Phenobarbitone 
 
Phentolamine 
 
Reserpine 
Foods that interfere with test results are as follows: 
 
Avocados 
 
Various herbal remedies 
 
Bananas  
 
Butternuts  
 
Cantaloupe  
 
Dates  
 
Eggplant  
 
Grapefruit  
 
Hickory nuts  
 
Honeydew melon  
 
Kiwifruit  
 
Melons  
 
Pecans 
 
Pineapple  
 
Plantains  
 
Plums  
 
Tomatoes and tomato products  
 
Walnuts 
 
 
 
 
 
 
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