Eltrombopag Added to Standard Immunosuppression in Treatment-Naive Severe Aplastic Anemia
Phase: PHASE1
Conditions: Severe Aplastic Anemia
Sponsor: National Heart, Lung, and Blood Institute (NHLBI)

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Neal S. Young, M.D.  3/18/2020 (Amendment UU/47)
CLINICAL RESEARCH PROJECT
Protocol # 12-H-0150
Drug Name:  eltrombopag (Promacta®)
IND: 104877
IND holder: NHLBI OCD
Date:
March 18, 2020
Title:
Eltrombopag added to standard immunosuppression in treatment-naïve severe aplastic anemia
Other Identifying Words: Immunosuppression, T-cells, hematopoiesis, autoimmunity, 
thrombocytopenia, neutropenia, pancytopenia.
Protocol Principal Investigator:
Neal S. Young, M.D., Chief, HB, NHLBI (E)
496-5093
Bldg. 10, CRC 3-5142
Subjects of Study:
Number 
Sex
Age-range 
Cohort 1
31
either
> 2 years and weight>12 kg
Cohort 2
33 (enrolled only 31)
either
> 2 years and weight >12 kg
Cohort 3
31
either
> 2 years and weight >12 kg
Extension Cohort
87
either
> 2 years and weight >12 kg
Laboratory Exploratory Cohort 25
either
> 2 years and weight >12 kg
Total
207
Project Involves Ionizing Radiation? 
Yes (medically indicated only)
Off-Site Project? 
No
Multi-center trial?
No
DSMB Involvement?
Yes
Tech Transfer: CRADA, MTA
Yes

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PRECIS
Severe aplastic anemia (SAA) is a life-threatening bone marrow failure disorder characterized by 
pancytopenia and a hypocellular bone marrow.  Allogeneic bone marrow transplantation offers the 
opportunity for cure in younger patients, but most are not suitable candidates for transplantation due to 
advanced age or lack of a histocompatible donor.  Comparable long-term survival in SAA is attainable with 
immunosuppressive treatment with horse anti-thymocyte globulin (h-ATG) and cyclosporine (CsA).  
However, of those patients treated with h-ATG/CsA, one quarter to one third will not respond, and 30-40% 
of responders relapse.  The majority of the hematologic responses observed following initial h-ATG/CsA 
are partial, with only a few patients achieving normal blood counts.  Furthermore, analysis of our own 
extensive clinical data suggests that poor blood count responses to a single course of ATG (non-robust 
responders), even when transfusion-independence is achieved, predicts a worse prognosis than when robust 
hematologic improvement is achieved (protocol 90-H-0146).  The explanation for partial recovery and 
relapse are not fully understood, but incomplete elimination of auto-reactive T cells and insufficient stem 
cell reserve are both possible.  Furthermore, 10-15% of SAA patients treated with standard 
immunosuppression will develop an abnormal karyotype in follow-up, with monosomy 7 being most 
common, which portends progression to myelodysplasia and leukemia. In contrast, malignant clonal 
evolution is rare in complete responders to immunosuppression. Although horse ATG/CsA represented a 
major advance in the treatment of SAA, refractoriness, incomplete responses, relapse, and clonal evolution 
limit the success of this modality. Thus, newer regimens are needed to address these limitations, and provide 
a better alternative to stem cell transplantation.
One approach to augment the quality of hematologic responses is to improve underlying stem cell function. 
Previous attempts to improve responses in SAA with hematopoietic cytokines including erythropoietin, G-
CSF, and stem cell factor, have failed. Thrombopoietin (TPO) is the principal endogenous regulator of 
platelet production. In addition, TPO also has stimulatory effects on more primitive multilineage 
progenitors and stem cells in vitro and in animal models. Eltrombopag (Promacta®), an oral 2nd generation 
small molecule TPO-agonist, is currently approved for treatment of chronic immune thrombocytopenic 
purpura (ITP), chronic hepatitis C-associated thrombocytopenia, and severe aplastic anemia who have had 
an insufficient response to immunosuppressive therapy. Eltrombopag increases platelets in healthy subjects 
and in thrombocytopenic patients with chronic ITP and hepatitis C virus (HCV) infection. Our Branch 
recently completed a pilot study of eltrombopag in refractory SAA. We saw encouraging clinical results in 
a cohort of patients who have failed on average two prior immunosuppressive regimens (Olnes et al. ASH 
Annual Meeting Abstracts, San Diego, CA, 2011, oral presentation and N Engl J Med 2012;367:11-9.1). Of 
the twenty-five SAA patients treated with eltrombopag by mouth for three months, eleven (44%) patients 
met protocol criteria of clinically meaningful hematologic responses, without significant toxicity.  Nine 
patients demonstrated an improvement in thrombocytopenia (>20k/L increase or transfusion 
independence), hemoglobin improved in two patients (>1.5g/dL or achieved transfusion independence, and 
four patients had a significant response in their neutrophil count. When responders continued the drug 
beyond three months, the hematologic response to eltrombopag increased; a trilineage response was 
observed in four patients, and a bilineage response occurred in another four, with median follow-up of 13 
months. These results suggest that stem cell depletion, a major component of the pathophysiology of SAA, 
might be directly addressed by eltrombopag administration. The aim of the current study would be to 
improve the hematologic response rate and its quality, as well as prevent late complications such as relapse 
and clonal progression, by addition of eltrombopag to standard immunosuppressive therapy.
This trial will evaluate the safety and efficacy of combining eltrombopag with standard hATG/CSA as first 
line therapy in patients with SAA. The primary endpoint will be the rate of complete hematologic response 
at six months. Secondary endpoints are relapse, robust hematologic blood count recovery at 3, 6, and 12 
1Olnes MJ et al. Eltrombopag and Improved Hematopoiesis in Refractory Aplastic Anemia. N Engl J Med 2012;367:11-9.

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Neal S. Young, M.D.  3/18/2020 (Amendment UU/47)
months, survival, clonal evolution to myelodysplasia and leukemia, marrow stem cell content, and 
hematological response of relapse patients that re-start treatment.

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TABLE OF CONTENTS
1.
OBJECTIVES.....................................................................................................................................................5
2.
BACKGROUND AND SCIENTIFIC JUSTIFICATION..................................................................................5
3.
STUDY DESIGN..............................................................................................................................................17
4.
ELIGIBILITY ASSESSMENT ........................................................................................................................19
4.1
Inclusion Criteria ......................................................................................................................................19
4.2
Exclusion Criteria .....................................................................................................................................19
5.
TREATMENT PLAN.......................................................................................................................................20
6.
CLINICAL MONITORING .............................................................................................................................27
7.
CRITERIA FOR RESPONSE ..........................................................................................................................30
8.
EXPLORATORY LABORATORY RESEARCH STUDIES..........................................................................31
9.
BIOSTATISTICAL CONSIDERATIONS.......................................................................................................32
9.1
Sample sizes..............................................................................................................................................32
9.2
Statistical Methods....................................................................................................................................34
9.3
Primary Endpoints ....................................................................................................................................34
9.4
Secondary Endpoints ................................................................................................................................35
9.5
Stopping Rules..........................................................................................................................................35
9.6
Off Study/Off Treatment Criteria .............................................................................................................38
9.7
Data Management .....................................................................................................................................39
10.
DATA AND SAFETY MONITORING.......................................................................................................39
10.1
Safety Monitoring .....................................................................................................................................39
10.2
Event Characterization and Reporting......................................................................................................40
10.3
Reporting of Pregnancy ............................................................................................................................44
10.4
Protocol Monitoring..................................................................................................................................45
11.
HUMAN SUBJECT PROTECTION............................................................................................................45
11.1
Rationale for Subject Selection.................................................................................................................45
11.2
Participation of Children...........................................................................................................................46
11.3
Exclusion of Pregnant Women and Nursing Mothers ..............................................................................46
11.4
Risks and Discomforts ..............................................................................................................................47
11.5
Risks in Relation to Benefit ......................................................................................................................56
11.6
Informed Consent Processes and Procedures ...........................................................................................57
11.7
Conflict of interest ....................................................................................................................................58
11.8
FWA Coverage Agreement.......................................................................................................................59
11.9
Collaboration(s) ........................................................................................................................................59
12.
PHARMACEUTICALS ...............................................................................................................................59
13.
REFERENCES .............................................................................................................................................63
APPENDIX A:  MEDWATCH FORM....................................................................................................................67
APPENDIX B:  NHLBI HEMATOLOGY BRANCH LABORATORY RESEARCH STUDIES v. 2.5.2013 ......68
APPENDIX C:  SUPPLEMENTAL FIGURES .......................................................................................................72
APPENDIX D:  PHARMACOKINETIC STUDIES................................................................................................75
APPENDIX E:  PROMIS QUESTIONNAIRE ........................................................................................................76
APPENDIX F:  SOP 14E, APPENDIX B, TABLE 1 ..............................................................................................87

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1.
OBJECTIVES
To determine the safety and efficacy of h-ATG/CsA + eltrombopag in untreated subjects with severe 
aplastic anemia (SAA).
2.
BACKGROUND AND SCIENTIFIC JUSTIFICATION
2.1
Pathophysiology of Aplastic Anemia
Aplastic anemia is a serious hematologic disease characterized by pancytopenia and a hypocellular bone 
marrow.  Although the exact etiology of aplastic anemia is not known, clinical experiences and laboratory 
data suggest that the primary mechanism leading to development of bone marrow failure is immune-
mediated destruction of hematopoietic stem and progenitor cells.1  Specific populations of effector T-cells 
are elevated and localized to the bone marrow in aplastic anemia, including activated cytotoxic T-cells 
expressing HLA-DR, the IL-2 receptor, and IFN-γ.2-4  The effects exerted by cytotoxic T-lymphocytes are 
mediated in part due to Fas ligand-induced apoptosis of hematopoietic progenitor cells; IFN-γ, in addition 
to its intrinsic inhibitory activity on hematopoietic progenitor and stem cells, can induce over-expression 
of Fas on target cells.5  High resolution VB CDR3 analysis in patients with aplastic anemia shows 
significantly increased nonrandom skewing of the VB-chain families of the T cell receptor, suggestive of 
disease specific clonal expansion.6 Immune-mediated marrow destruction with many similarities to the 
pathophysiology of human aplastic anemia can be modeled in the mouse.7
Despite its often acute presentation, aplastic anemia is now recognized as a chronic disease with frequent 
flares of the immune process and the need for long-term immunosuppression.  There is evidence that 
depletion of primitive hematopoietic stem and progenitor cells is profound, demonstrating that immune 
attack against the most primitive stem cells is paramount.8  Even with recovery of blood counts following 
successful immunosuppressive therapy, a significant quantitative stem cell defect persists, suggesting either 
ongoing immune destruction or persistent depletion of stem cells even in the absence of an active immune 
process.9
2.2
Clinical Consequences of Aplastic Anemia
Symptoms derive from low blood counts. Anemia leads to fatigue, weakness, lassitude, headaches, and in 
older patients dyspnea and chest pain, and these manifestations are most commonly responsible for the 
clinical presentation.  Thrombocytopenia produces mucosal bleeding: petechiae of the skin and mucous 
membranes, epistaxis, and gum bleeding are frequent and early complaints.  Bleeding can be brisk in the 
presence of accompanying physical lesions, as in gastritis and fungal infection of the lungs.  The most 
feared complication of thrombocytopenia is intracranial hemorrhage.  Bacterial and fungal infections in the 
setting of neutropenia are a major cause of morbidity and mortality, and most often the cause of death in 
refractory or untreated aplastic anemia.
2.3
Treatment of Aplastic Anemia
2.3.1
Allogeneic Hematopoietic Stem Cell Transplantation
Allogeneic bone marrow transplantation from a histocompatible matched sibling is curative therapy in the 
majority of aplastic anemia patients who undergo the procedure.10  Survival rates with allogeneic 
hematopoietic stem cell transplantation from a histocompatible sibling have been reported to be as high as 
90% from single institution studies, and approximately 70% in composite registry data, which more likely 
reflects the general experience.11,12  The frequency and severity of graft-versus-host disease correlates with 
patient age and continues to be the major limiting factor in terms of both morbidity and mortality as well 

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Neal S. Young, M.D.  3/18/2020 (Amendment UU/47)
as long-term quality of life.13  In general, adults have a lower survival rate compared to children, and most 
experts do not suggest allogeneic stem cell transplantation as first-line therapy, even from a fully matched 
sibling donor, for older SAA patients.  Allogeneic bone marrow transplantation is available to only a 
minority of patients, since 70% will lack a suitable matched sibling donor.  Alternative donor 
transplantation using a matched unrelated source is almost never the initial treatment, given the extended 
time period required to identify and recruit an appropriate matched unrelated donor, and the reported lower 
survival and higher rates of graft-versus-host disease compared to sibling donor transplants.  Cord blood 
transplants are even less frequently utilized in SAA, due to higher rate of delayed hematopoietic 
reconstitution and persistent immune dysfunction in SAA patients and resulting poor survival in published 
trials.
2.3.2.
Immunosuppressive Regimens
Horse Anti-thymocyte Globulin (ATGAM®; h-ATG) is currently approved for the treatment of aplastic 
anemia by the United States Food and Drug Administration.  h-ATG as a single agent resulted in response 
rates of 30-50% in SAA in several large studies carried out in the 1980s.14-16  The mechanism by which h-
ATG improves bone marrow failure in aplastic anemia is not fully understood.  h-ATG preparations contain 
a variety of antibodies recognizing human T-cell epitopes, many directed against activated T-cells or 
activation antigens.17,18  After treatment with h-ATG, circulating levels of lymphocytes drop to 10% of 
pretreatment level, through a variety of mechanisms; Fc receptor complement-dependent lysis, 
opsonization and phagocytosis by macrophages, and immunomodulation leading to long-term depletion via 
antibody dependent cell-mediated cytotoxicity and activation-induced apoptosis.  Although the decline in 
circulating levels of lymphocytes is transient, the number of activated T -cells is decreased for more 
prolonged periods of time; this effect is also reflected in decreased lFN-γ and possibly TNF production after 
h-ATG.19,20  The response to h-ATG may be mediated by circulating factors produced in this 
immunologically activated state, although response to ATG has not correlated with severity or presence of 
clinical serum sickness.
Tissue culture preparations of peripheral blood lymphocytes treated with ATG produce hematopoietic 
colony stimulating factors, suggesting a possible additional activity of ATG in stimulating hematopoiesis 
in vivo.  h-ATG binds to numerous other cell types in addition to lymphocytes, including cells of the bone 
marrow.21-23
Cyclosporine (CsA) is a major immunosuppressive drug and probably secondary only to corticosteroids 
in worldwide utilization in this role. In addition to its longstanding use in bone marrow and solid organ 
transplant recipients; CsA has been widely employed as an immunosuppressive drug in many autoimmune 
diseases.  In contrast to ATG, CsA has a selective inhibitory effect on T lymphocytes, suppressing early 
cellular response to antigenic and regulatory stimuli. By blocking expression of nuclear regulatory proteins, 
it leads to reduced T cell proliferation and activation with diminished release of cytokines such as 
interleukin-2 and interferon-γ. CsA binds to intracellular receptors termed immunophilins, inhibiting in turn 
the activity of calcineurin, which results in the blocking of interleukin-2 production and T cell activation 
and proliferation.  In vivo, CsA inhibits the release of IL-2 from activated T-cells and consequently 
decreases T cell proliferation.24,25
h-ATG/CsA is the current standard immunosuppressive regimen in SAA.1  The addition of CsA to ATG 
improved response rates to 60-70% and the 5-year survival in responding patients to 80-90%.26-28  With this 
regimen, relapses occur in 1/3 of responders and clonal evolution to myelodysplasia and leukemia in 10-
15% of cases overall.1  Although this non-transplant therapy represents a therapeutic success, the significant 
minority of unresponsive patients, frequent relapse, and progression to late clonal disease remain 
problematic and result in significant morbidity and mortality. Robust and rapid hematologic recovery has 

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been associated with better survival outcomes,27 and in our experience, complete hematologic responders 
are unlikely to evolve to myelodysplasia or leukemia (unpublished data).
Alternative immunosuppressive agents have been used with limited success.  At Johns Hopkins, high dose 
cyclophosphamide without stem cell rescue produced hematologic response rates similar to those seen with 
ATG combined with CsA in an initial small study, with no relapse or evolution to paroxysmal nocturnal 
hemoglobinuria (PNH) or myelodysplasia observed.29  However, a prospective randomized trial conducted 
at the NHLBI, which compared ATG and cyclosporine to cyclophosphamide and cyclosporine, was 
terminated prematurely due to excessive toxicity, severe fungal infections and deaths in the group that 
received cyclophosphamide.30  In contrast to the Hopkins’ experience, some of our patients relapsed or 
developed cytogenetic abnormalities.31  The explanation for the increased toxicity seen in the 
cyclophosphamide-treated patients is the serious immunosuppression and resulting neutropenia.  With the 
cyclophosphamide regimen (at 200 mg/kg) the rate of hematologic complete response (40-50%) appears 
higher than that of h-ATG regimen (10-15%).  In an attempt to circumvent the immediate toxicity with 
cyclophosphamide, we have investigated a lower dose (120 mg/kg) in treatment-naïve SAA patients in the 
past 15 months.  Thus far complete responses have been observed in accordance to our and the Hopkins 
experience, but there is continued concern with toxicities despite lower dose cyclophosphamide.
As the addition of CsA clearly improved outcomes compared to the use of ATG alone, other 
immunosuppressive drugs were predicted, based on their mode of action, animal studies, and experience in 
other human diseases and with organ transplant, to increase response rates or decrease relapse.  The addition 
of mycophenolate mofetil (MMF) to ATG and CsA in an NIH trial of 104 patients (protocol 00-H-0032) 
did not change hematologic response (about 62%), relapse (37%), or evolution rates.32  In a follow up study 
sirolimus was added as a third agent to standard h-ATG/CsA.  Again, when tested in a randomized protocol, 
results were not superior to standard h-ATG/CsA (protocol 03-H-0193).  More lymphocytotoxic agents that 
are active in the relapsed and refractory settings were studies as first line therapy in the context of a large 
randomized study (protocol 06-H-0034).  Results were disappointing. Both rabbit ATG and alemtuzumab 
yielded inferior response rates when compared to horse ATG.33  Thus, h-ATG plus CsA remains the 
standard immunosuppressive regimen in SAA.
2.3.3
Hematopoietic Cytokines
The isolation and cloning of hematopoietic growth factors was rapidly followed by testing of their clinical 
activity in aplastic anemia.  The rationale for use of cytokines that act on committed hematopoietic 
progenitors in specific differentiated lineages, such as erythropoietin and G-CSF, has been suspect.  First, 
their receptors are not present on primitive stem cells, at best might be expected to shorten time to recovery 
of blood counts following therapy, once immunosuppression or transplantation allows some recovery of 
stem cell numbers.  Second, endogenous erythropoietin levels are extremely elevated in SAA, calling into 
question the rationale for exogenous administration.  Clinical trials have examined the addition of GM-
CSF, G-CSF, and IL-3, all considered myeloid cell cytokines, alone or added to standard 
immunosuppressive therapy.  In this setting, G-CSF has been widely studied in combination with horse 
ATG plus CsA.  This regimen has not been shown to improve outcomes in several randomized trials.34-36  
The lack of benefit may be secondary to the activity of G-CSF on more committed myeloid progenitors, 
suggesting that a growth factor that acts on less differentiated progenitor cells might have activity.  A recent 
meta-analysis of 19 individual trials concluded that there was no impact on survival or response rate with 
the addition of these cytokines to standard immunosuppressive regimens.37
The relationship between extended treatment with G-CSF and clonal evolution of SAA to myelodysplasia 
and leukemia is unclear.  A Japanese study in pediatric patients with SAA found a relationship between the 
cumulative number of days of G-CSF and eventual progression to MDS/AML, however, the study was not 
randomized or controlled regarding whether G-CSF was given or not.38  A smaller case series in adults also 

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suggested a relationship.39  However, patients with SAA clearly have an increased risk of late clonal disease, 
and those who are non-responders or less robust responders are more likely to progress38 (our unpublished 
data).  Non-responding patients are more likely to receive G-CSF for prolonged periods of time, despite 
lack of efficacy, as some patients may show an increase in neutrophil counts with G-CSF, despite lack of 
overall survival or response benefit.  At present, there is no clear role for G-CSF in the treatment of patients 
with SAA, and the relationship of G-CSF to late clonal disease is remains uncertain.
2.4
Thrombopoietin and Hematopoiesis
Thrombopoietin (TPO) was purified, identified and cloned by independent research groups in academia 
and industry in the mid1990s, based on its activity as the primary factor stimulating maturation of 
megakaryocytes and platelet release, and its binding to the receptor c-mpl. TPO is a glycoprotein class 1 
hematopoietic cytokine, produced primarily in the liver.
A number of lines of evidence support a pleiotropic role for TPO in hematopoiesis, beyond function as the 
primary endogenous factor controlling platelet production.  The c-mpl receptor is expressed and functional 
on primitive hematopoietic stem and progenitor cells.40  Animals and patients with genetic defects in either 
TPO or c-mpl have significant reduction in HSC numbers and activity, along with profound defects in 
platelet production.41,42  In vitro expansion of functional and phenotypic HSCs can be stimulated by TPO, 
either alone or in combination with other cytokines.43
The control of TPO levels and TPO production in complex, and involves sensing of c-mpl receptor 
occupancy, with levels generally inversely proportional to megakaryocyte mass.  In early studies performed 
in our Branch, we demonstrated that TPO levels were extremely high in SAA and surprisingly low to normal 
in chronic ITP, comparing patients with these two conditions with equivalent platelet counts.44  More recent 
studies also confirm TPO levels to be high in SAA and moderately elevated in myelodysplastic syndromes 
compared to normal controls.45
A slightly modified form of recombinant TPO, termed megakaryocyte growth and development factor 
(MGDF), was in clinical development by Amgen in the late 1990s.  It clearly stimulated platelet production 
in vivo in healthy control individuals and in chemotherapy patients, but its development came to a halt 
when several normal volunteers receiving MDGF prior to donating platelets developed neutralizing 
antibodies, which reacted not only to MDGF but also to endogenous TPO, causing profound persistent 
thrombocytopenia.
2.5
Eltrombopag
Eltrombopag (SB-497115-GR,Promacta®),the bis-monoethanolamine salt form, is an orally bioavailable, 
small molecule 2nd generation thrombopoietin receptor (TPO-R) agonist, developed for the treatment of 
thrombocytopenia by scientists at GlaxoSmithKline.46  Studies conducted in vitro have shown that 
eltrombopag is an effective agonist binding to mpl, the thrombopoietin receptor (TPO-R), to stimulate 
thrombopoiesis.  It binds mpl at a position distinct from the ligand binding site, within the juxtamembrane 
domain of the receptor, and thus does not compete with TPO for binding to its receptor.47  The differences 
in binding to the receptor may theoretically result in activation of different signaling pathways from native 
thrombopoietin, however, to date, data indicates similar impact on megakaryocytes and HSCs to 
thrombopoietin.48
In vivo, eltrombopag increased platelet number in the chimpanzee (the only nonclinical species which is 
pharmacologically responsive to eltrombopag).48  These findings, coupled with supporting clinical efficacy 
data in humans, suggested that eltrombopag is an orally active TPO-R agonist that functions in a similar 
manner to endogenous thrombopoietin (TPO).  Initial clinical trials were carried out in normal volunteers, 

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and then in patients with chronic ITP, based on their inappropriately low or low-normal levels of 
endogenous thrombopoietin.44  The initial phase 1/ 2 and randomized, controlled phase 3 registration trials 
in chronic ITP were very encouraging, with little toxicity and much higher responses by comparison with 
placebo49-51, which led to its approval by the Food and Drug Administration (FDA) on November 20, 2008 
in patients with chronic ITP who have had an insufficient response to corticosteroids, immunoglobulins, or 
splenectomy.  Eltrombopag is the first oral thrombopoietin (TPO) receptor agonist approved for adult 
patients with chronic ITP.  A second TPO-agonist, romiplostim (Nplate) was developed by Amgen, is also 
available for the treatment of chronic ITP and is given subcutaneously weekly.
Longer follow-up in EXTEND suggests that eltrombopag remains well tolerated (EXTEND Trial, 
NCT00351468).  On December 6, 2011 the FDA agreed to modify eltrombopag’s Risk Evaluation and 
Mitigation Strategies to assure safety use and removed the requirement for healthcare professionals and 
institutions to enroll in the Promacta Care Program (Promacta Package Insert, 2011).  Continued monitoring 
of adverse events for eltrombopag will be monitored by post-marketing surveillance programs and ongoing 
clinical trials.
2.6
Eltrombopag for Refractory Severe Aplastic Anemia
Reasoning that eltrombopag stimulated primitive HSCs and progenitors and as there was a clear deficit in 
HSC numbers and function in SAA, in 2009 we initiated a single-arm dose escalation phase 1/2 trial for 
patients with refractory SAA.  For protocol entry, all patients had to have severe thrombocytopenia in 
addition to fulfilling criteria for SAA, and they must have failed at least one prior regimen of standard 
ATG-containing immunosuppressive therapy.  The primary endpoints were safety and clinically significant 
hematologic response.  The study design and response criteria are shown in Supplemental Figure 1.
At termination, 26 patients were enrolled in the protocol and 25 had received study drug.  As shown in 
Table 1, this patient population had very prolonged and serious cytopenias.  All were platelet transfusion-
dependent, and most also required frequent red blood cell transfusions, and were severely neutropenic and 
thus susceptible to life-threatening infections.  All had failed at least one prior cycle of high dose 
immunosuppression more than six months prior to study entry, with the majority failing two and some as 
many as four prior cycles of immunosuppression.  The median time since last immunosuppression was 14 
months, with a range of up to 117 months, excluding any chance that responses could be attributed to prior 
immunosuppressive therapy.
Table 1. Baseline Characteristics of Study Patients.
Number of patients
26
Age (median)
44
        Range
18-77
Race 
N (%)
        White
12 (46)
        African American
7 (27)
        Asian
1 (4)
        Hispanic
6 (23)
Male sex
14 (54)
Time from last IST (Mo.)
Median
14
        Range
6-117
Transfusion dependent
         PRBCs
23 (88)

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Number of patients
26
         Platelets
25 (100)
Baseline parameters
Median (range)
Platelets (K/μL)
9 (5-15)
Neutrophils (K/μL)
0.8 (0.07-2.8)
Hemoglobin (g/dL)
8.0 (6.0-13.8)
In all but one patient (in whom drug was discontinued at 125 mg/day due to possible cataracts as described 
below), drug was escalated to the maximum dose of 150 mg per day, and this maximum dosage was very 
well tolerated.  All severe adverse events (SAE), and all grade 2 and higher adverse events (AE) that were 
possibly, probably, or definitely attributed to eltrombopag treatment are listed in Table 2.  There was one 
SAE that was possibly related to eltrombopag treatment: a patient with a history of diabetic gastroparesis 
was hospitalized for recurrent abdominal pain while taking eltrombopag.  There were no grade 4 or 5 AEs.  
One patient developed acute hepatitis B infection with a grade 3 elevation of his hepatic transaminases to 
greater than 6X the upper limit of normal while on study, so the drug was discontinued; however, the 
transaminase elevation was almost certainly related to acute hepatitis B.  He was taken off study, and with 
recovery from hepatitis B infection, serum transaminase values returned to baseline.
In March 2014 data from this clinical trial was also published (Desmond, Townsley, et al. Blood 2014) 
reporting safety and efficacy data on a further 18 patients and long-term follow-up on the entire cohort of 
43 patients.  The overall response rate was 17 of 43 patients (40%) at 3 to 4 months, including trilineage 
and bilineage responses.  Most patients (14/17) continued to show trilineage improvement and 5 patients 
had drug discontinued for near normalization of blood counts without relapse.  Eight patients developed 
new cytogenetic abnormalities on eltrombopag, including 5 with chromosome 7 loss or partial deletion, but 
none evolved to leukemia.  Eltrombopag was immediately discontinued when cytogenetic abnormalities 
were observed.
In January 2014 eltrombopag (Promacta) gained Breakthrough Therapy designation status from the FDA 
and Priority Review in April 2014.  On August 26, 2014, the FDA approved the additional use of 
eltrombopag in patients with severe aplastic anemia who have had an insufficient response to 
immunosuppressive therapy.  The approval was based on results from the clinical trial done at the NIH 
described above, 09-H-0154 (NCT00922883).
Table 2. Adverse Events, and Grade 2 or Higher Non-Hematologic Adverse Events
Category
Event
N
Dose (mg)
Related to 
eltrombopag
Allergic
Cephalosporin 
reaction
1
100
Unlikely
Cardiovascular
Orthostasis
1
Off
Unlikely
Gastrointestinal
Abdominal pain
1
125
Possibly
Hematologic
Gingival bleeding
1
100
Unlikely

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Category
Event
N
Dose (mg)
Related to 
eltrombopag
Infection
C. difficile colitis
Neutropenic 
fever
Gastroenteritis
1
6
1
1
150
100, 150 x3, off x 2Off x 6
150
Unlikely
Unlikely
Unlikely
Unlikely
Constitutional
muscle weakness
-
1
-
Possibly
Dermatology/skin
Rash
-
1
-
Possibly
Metabolic
ALT, AST increased
-
1
-
Possibly
Psychiatric
Depression
-
1
-
Possibly
AE, adverse event; ALT, alanine aminotransferase; AST, aspartate aminotransferase; SAE, severe adverse 
event.
Prior studies in patients with chronic ITP have raised the concern that TPO mimetics, including 
eltrombopag, might increase bone marrow reticulin deposition.52  We performed bone marrow biopsies 
with reticulin staining at baseline and after three months of eltrombopag treatment to assess for fibrosis.  
Patients on the extended access protocol underwent bone marrow biopsies with reticulin staining every six 
months.  Reticulin deposition was graded by a single hematopathologist in a blinded manner on a scale of 
0-4 according to standard guidelines.  Two patients refused to have the 12-week response assessment bone 
marrow performed.  Among 23 patients assessed, there was no significant increase in reticulin staining 
either at three months, or on serial biopsies in patients on prolonged eltrombopag treatment (Supplemental 
Fig. 4).
Eltrombopag has an FDA warning for cataract formation based on a prior study in ITP patients.  We 
performed ophthalmologic examinations to assess for cataracts at baseline, after three months on 
eltrombopag, and every six months in patients on the extended access protocol.  One patient was found to 
have a new lens opacification prompting discontinuation of eltrombopag, but this finding was not confirmed 
on a second examination performed 2 months later, or on subsequent serial eye examinations.  No other 
patients had new cataract formation or worsening of existing cataracts after treatment with eltrombopag.
Among 25 evaluable patients 11 (44%) achieved protocol-defined hematologic response after 12 weeks of 
eltrombopag treatment (Supplemental Fig. 2).  Patients reaching a response at 12 weeks and maintaining it 
to 16 weeks were continued on an extension phase, and continued to receive eltrombopag 150 mg per day.  
All patients were platelet transfusion dependent at the time of enrollment, and nine patients achieved 
platelet transfusion independence after eltrombopag treatment, with patients on the extension phase 
continuing to show a gradual increase in platelet counts over time (Supplemental Fig. 3A).  Two patients 
achieved a hemoglobin response by 12 weeks, and 4 additional patients had improved hemoglobin levels 
on the extension phase (Supplemental Fig. 3B), with a median hemoglobin increase of 3.6 g/dL (range 1.5-
8.2 g/dL).  Four patients who were previously dependent on packed red blood cell transfusions achieved 
transfusion independence and two were able to be phlebotomized to treat transfusional iron overload.  Seven 
neutropenic patients had increased neutrophil counts after eltrombopag treatment (median increase 590K 

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cells/μL), including 4 patients who were severely neutropenic at baseline (Supplemental Fig. 3C).  It is 
notable that patients who reached response criteria and thus were continued on the drug continued to 
improve over time, suggest that some non-responders potentially could have achieved clinical improvement 
had the drug been given for more than 12 weeks.
The patient with an unconfirmed finding of cataracts at 12 weeks achieved a platelet response, and he has 
maintained platelet transfusion independence for 19 months since discontinuing eltrombopag.  This patient 
also improved his hemoglobin by 3g/dL, which enabled him to undergo therapeutic phlebotomy to treat his 
transfusional iron overload.  The events in this patient suggest that continued eltrombopag might not be 
required to maintain HSC recovery.  The remaining responders on the extension study will have their dose 
of eltrombopag tapered in order to determine whether responses more generally can be maintained off of 
drug. There have been no other suggestions of new cataracts in this study.
Marrow biopsies were performed at study entry, at the 12-week response assessment, and for responders at 
one year after study entry.  Supplemental Figure 4 shows pre-treatment and one-year biopsies.  There is 
striking normalization of cellularity in three of four responders.  There was no increase in reticulin on 12-
week biopsies in any patient, and no increase in fibrosis in follow-up marrows at 6 or 12 months in 
responders remaining on the extension phase.
Of the 14 non-responders, two patients died of disease progression, from complications of severe 
cytopenias.  Two patients showed morphologic changes and cytogenetic abnormalities (monosomy 7) 
consistent with progression to myelodysplasia; one patient ultimately died, and the other underwent 
allogeneic stem cell transplantation.  Progression to monosomy 7/MDS is concerning, given the controversy 
regarding whether chronic G-CSF increases progression to MDS in SAA as summarized above, and the 
warning regarding progression of MDS to AML for patients being treated with an alternative TPO-mimetic, 
romiplostim.  Of note, romiplostim directly binds to the thrombopoietin receptor (c-mpl) and competes with 
endogenous thrombopoietin; while eltrombopag binds to a transmembrane domain of c-mpl leading to 
signal transduction.  The distinct mechanism of action between the 2 thrombopoietin mimetics may account 
for differences in efficacy and safety profiles between them.  In our cohort of over 400 patients with SAA 
followed long-term, approximately 15% progress to clonal disease including myelodysplasia and leukemia, 
and therefore two patients out of 25 enrolled is not unexpected.  Our experience also suggests that patients 
with long-standing severe refractory disease and who lack of a robust response to initial 
immunosuppression, as were enrolled on our protocol, are the most likely to progress.
2.7
Rationale for Dose Selection
Eltrombopag 150 mg once daily has been selected as the starting dose for this study because this regimen 
has been safe and effective in increasing platelet counts in our recently-completed non-randomized, off 
label, pilot phase II study (NCT00922883) of eltrombopag as a single agent in patients with refractory SAA.  
25 patients (age range 18-77 years) received 50mg daily of eltrombopag with dose escalation every two 
weeks to a maximum dose of 150mg daily.  Patients were successfully escalated to the 150mg daily dose 
without observing any dose-limiting toxicities.  Hematologic responses were only observed while receiving 
the 150mg daily dosing; it is possible that patients would have responded to lower doses of drug had the 
dose not been escalated every two weeks.  Therefore, the duration of therapy in this setting has not been 
well defined.
There is preliminary safety data with doses up to 300 mg per day in a number of different patient 
populations.  In healthy subjects, a clear dose and exposure response was seen for eltrombopag doses of 
10 mg to 200 mg once daily for 5 days, with geometric mean AUC (0-τ) values of 302 μg/mL for the 
200 mg once daily regimen.53  Eltrombopag was well tolerated in healthy subjects at all dose levels. In a 
recently completed open label study for patients with soft tissue sarcomas (NCT00358540), eltrombopag 

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doses of up to 150 mg have been given in conjunction with chemotherapy, without significant side effects. 
The most extensive data on dosing and long-term side effects has been obtained in patients with chronic 
ITP.  An initial randomized phase 2 trial, followed by two randomized phase 3 trials all showed efficacy 
for eltrombopag compared to placebo for increasing the platelet count utilizing doses of up to 75 mg per 
day.49-51  In ITP subjects, there was a dose response for eltrombopag 30 mg to 75 mg once daily, with 
geometric mean AUC(0-τ) values of 169 μg/mL for the 75 mg once daily regimen.  There was no significant 
difference between the safety profile of ITP subjects receiving 30, 50 or 75 mg of eltrombopag.
A starting dose of 75 mg once daily in East Asian and South East Asian patients will be used.  Modified 
dosing for subjects of East Asian and South East Asian heritage (self-reported) has been implemented for 
the following reasons.  In healthy Japanese subjects, plasma eltrombopag AUC(0-τ) was approximately 80% 
higher when compared to non-Japanese healthy subjects who were predominantly Caucasian.  Similarly, in 
patients with ITP, plasma eltrombopag exposure was approximately 70% higher in East Asian and South 
East Asian subjects as compared to non–East Asian subjects who were predominantly Caucasian as higher 
drug exposure in East Asian and South East Asian subjects has been observed.
For pediatric subjects, there is a predicted higher weight-adjusted drug clearance than older children and 
adults based upon studies of several drugs approved for use in children, such as anticonvulsants, proton 
pump inhibitors, theophylline, and HIV protease inhibitors, have routinely demonstrated that young 
children have higher weight-adjusted drug clearance than older children and adults [Lamictal Package 
Insert, 2007;Trileptal Package Insert, 2007; Keppra Package Insert, 2008; Prilosec Package Insert, 2008; 
Kaletra Package Insert, 2007; Viracept Package Insert, 2007; Grygiel, 1983]. In the ongoing open-label 
phase of PETIT, a phase II pediatric chronic ITP study, subjects between 1 and 5 years received 1.2 – 2.5 
mg/kg eltrombopag once daily, while subjects between 6 and 17 years of age received and average daily 
dose of 58.5 mg daily (NCT00908037).  The maximum dose used in the PETIT trial among all age groups 
is 75 mg daily dose. Cohort 3 (ages 1 to 5 years) was opened for patient recruitment on 01 June 2011 and 
the initial group of 5 subjects has been enrolled.  Preliminary data have been evaluated for an initial group 
of 5 subjects aged 1 to <6 years enrolled in Cohort 3 of PETIT.  These subjects initiated dosing with 0.7 
mg/kg once daily and increased to at least 1.4 mg/kg once daily by the Week 12 visit.  Preliminary PK data 
collected for 3 subjects (ages ranging from 2 to 5 years) receiving eltrombopag 1.1 to 1.2 mg/kg once daily 
at Week 6 suggest that this regimen delivers plasma eltrombopag exposure similar to a 37.5 to 50 mg once 
daily regimen in adults.  No new pediatric specific safety signal has been identified thus far.  The available 
platelet count, safety, and PK data available for subjects enrolled in the PETIT trial support a starting dose 
of 2.5 mg/kg once daily for non-Asian subjects aged 2-5 years.
Thrombocytosis is a theoretical risk of eltrombopag treatment when high dosages are administered.  
However, thrombocytosis has not been observed in the 25 patients with refractory SAA who were treated 
with 150 mg per day.  It is possible although unlikely that patients with previously untreated SAA, who 
may have better residual hematopoietic stem cell function, could develop very high platelet counts on 
eltrombopag.  Thrombocytosis has been observed in healthy volunteers as well as in subjects with ITP, and 
there was a suggestion of a higher risk of thrombosis in patients on eltrombopag compared to placebo in 
the phase 3 randomized trials for chronic ITP.51  However, patients with ITP, in contrast to patients with 
SAA, have hyper-reactive platelets and an increased endogenous risk of thrombosis.  In an extensive 
analysis of ITP patients treated long-term with romiplostim, an alternative thrombopoietin mimetic, there 
was no evidence for increased thrombotic events in the romiplostim-treated patients compared to controls.54  
In a meta-analysis of randomized trials using either eltrombopag or romiplostim, there was a numerically 
but non-statistically significant trend to increase the occurrence of thromboembolisms compared to 
controls.55  In the current trial, based on concern regarding thrombosis, dose reductions of eltrombopag will 
be made if the platelet count reaches 200,000 per μL or greater.

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2.8
Rationale for Permitting Dose Interruption
The effect of dose interruption is unknown in the aplastic anemia population.  31% (34 ITP subjects) on the 
long-term extension study (EXTEND Trial, NCT00351468) had an interruption to eltrombopag dosing at 
some point in the study.  Of the subjects requiring a dose interruption, 7 had a dose interruption lasting 1 to 
7 days and 27 had a dose interruption lasting greater than 7 days.  Platelet counts decreased back to baseline 
within 1-2 weeks, although not associated with any bleeding complications.  However, the underlying 
pathophysiology of thrombocytopenia in ITP is very different from SAA, and eltrombopag is being utilized 
in that disorder to overproduce platelets in the bone marrow to compensate for increased antibody-mediated 
platelet destruction.  In SAA we postulate an effect on HSCs in the bone marrow, and a much more 
prolonged effect from eltrombopag, therefore our prediction would be that short or even longer term dose 
interruptions will not result in any sudden changes in blood counts.  We anticipate some patients on the 
current trial will be hospitalized for other disease-related issues such as fever and neutropenia and may 
require suspension of the study drug temporarily.
One patient in our recent trial of eltrombopag for refractory SAA had drug discontinued before the three 
month time point, despite meeting criteria for response, because of possible cataracts noted on eye 
examination (later deemed to have been a normal lens examination on repeat testing).  His response 
continued, now for over 18 months, with improvements in all three hematopoietic lineages despite no 
further eltrombopag treatment, suggesting that the effect of drug on HSC number or function is long-lasting 
and prolonged therapy may not be required in SAA.
2.9
Scientific and Clinical Justification of the Protocol
The combination of ATG, which lyses lymphocytes, and CsA, which blocks T-cell function, is responsible 
for survival rates comparable to those observed with transplant recipients.27,56  Hematology Branch efforts 
for the past 10-15 years have been focused on developing immunosuppressive regimens in SAA that would 
circumvent the limitations presently observed with h-ATG/CsA.  The failure of more intensive 
immunosuppressive regimens to improve outcomes suggests that even if immune attack on the bone 
marrow can be interrupted, deficits in the number or expansion ability of HSCs may limit hematopoiesis in 
these patients.  Clinical and laboratory data suggest that greatly diminished stem cell numbers limit the 
effectiveness of immunosuppression and contribute to relapse and evolution.57  It is reasonable therefore to 
conclude that the addition of a hematopoietic growth factor capable of expanding primitive HSCs and 
progenitors would be useful.
One such hematopoietic growth factor, TPO, is a potent endogenous cytokine and the principal regulator 
of platelet production, as summarized above.  A 2nd generation TPO-agonist, the nonpeptide mimetic 
eltrombopag, is administered orally, well tolerated and does not induce auto-antibodies, in contrast to first-
generation TPO-R agonists such as megakaryocyte growth and development factor (MDGF).  Eltrombopag 
has been shown to increase platelets in healthy subjects, patients with chronic immune thrombocytopenic 
purpura (ITP), and more recently shown to increase blood counts in patients with hepatitis C associated 
thrombocytopenia58,59 and now in SAA as single agent, in our recently completed trial which has been 
presented in abstract form and now published (N Engl. J. Med. 2012;367:11-9).60  These surprising results 
suggested that eltrombopag, like thrombopoietin itself, might act on the hematopoietic stem cell and offer 
real clinical benefit in SAA by increasing stem cell number.
Since outcome is strongly related to the presence of early recovery and to the quality of the blood cell count 
response at 3 months after receiving antithymocyte globulin, an agent like eltrombopag in enhancing the 
quality and speed of hematologic recovery could both shorten time at risk for infections, bleeding and 
transfusions as well as potentially reduce the rates of late events linked to a deficient stem cell compartment, 
and thus improve survival.  In a cohort of 122 patients with SAA at our institution treated with H-ATG and 

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CsA, survival was associated with early attainment of hematologic recovery (86% vs. 40% at 5 years, 
P<.001), and degree of blood count recovery at 3 months (90% vs. 42% for patients with less robust 
recovery).27  Therefore, the addition of eltrombopag to standard immunosuppressive therapy is likely to 
have an impact on survival if the quality of hematologic recovery can be improved during the first 3 - 6 
months of treatment.
The objectives of this trial are therefore to assess the effectiveness of eltrombopag alongside standard 
immunosuppressive therapy with h-ATG/CsA for treatment naïve severe aplastic anemia and to define the 
additive toxicity of eltrombopag with h-ATG/CsA.  A secondary objective for this study will be to describe 
the common aspects of health-related quality of life (HRQL) in the adult subjects pre- and post-study 
treatment which is recommended by the FDA61 and the IOM62 as indicators of treatment effects.
2.9.1 
Justification for adding Cohort 2 (August 2013)
In this study, we are observing more rapid hematologic improvement than observed with 
immunosuppression alone.  The overall response rate of 84% (11/13) at 6 months, with 100% of these 
responses observed by 3 months, is also higher than in historical data.  As subjects currently receive 
eltrombopag for 6 months but blood counts have improved by 3 months, it is possible that administration 
of eltrombopag may only be necessary for the shorter duration, lowering risk, cost, and inconvenience.  In 
particular, limiting exposure to eltrombopag may reduce the risk of cytogenetic evolution.  Further, in the 
event of relapse, expected with discontinuation of treatment, we can now not distinguish whether 
cyclosporine or eltrombopag is responsible, as both are administered concurrently. Therefore, we propose 
to treat another cohort (cohort 2) of 31 subjects, with no change to this protocol other than a reduction in 
eltrombopag administration from 6 to 3 months.  The study objectives, primary and secondary endpoints, 
statistics – sample size, stopping rules, eligibility, monitoring and ancillary studies will remain unchanged.  
Responses at 3 months can be assessed for the two cohorts, providing a confirmation cohort for our early 
results, whereas new information will be provided by comparison of long-term outcomes between the two 
cohorts.  These data can be used to inform design of a future randomized, multicenter phase III trial.  Note 
amendment 17 was submitted on October 23, 2014 to increase Cohort 2 sample size to 33 subjects in order 
to allow subjects to be enrolled and treated while amendment 16 to add Cohort 3 was being reviewed.  Note, 
only 31 subjects were enrolled into Cohort 2 because the Cohort 3 amendment was approved prior to 
needing to enroll the 2 additional subjects into Cohort 2.
2.9.2
Justification for adding Cohort 3 (September 2014)
As of September 15, 2014, 57 subjects have been enrolled on the protocol (31/31 in cohort 1, and 26/31 in 
cohort 2) and the combination of cyclosporine and eltrombopag has been safe and well tolerated.  Higher 
overall response rates with eltrombopag compared to immunosuppression alone are a consistent observation 
from both the first and second cohort.  Most subjects respond within 2-3 weeks of the first dose of 
eltrombopag alleviating the transfusion burden sooner than our historical cohort.  In cohort 2, where 
eltrombopag is discontinued at 3 months in all subjects, all subjects have either maintained or normalized 
their blood counts between 3 and 6 months.  In cohort 1 and cohort 2 eltrombopag was implemented to 
begin on Day 14 to avoid any potential hepatotoxicity from the combination of cyclosporine and h-ATG.  
However, now that we have ample data indicating the combination of cyclosporine and eltrombopag is safe, 
the earlier initiation of eltrombopag on Day 1 in combination with hATG should be tested in a third cohort.  
Therefore, we propose to treat another cohort (cohort 3) of 31 subjects, where eltrombopag will be 
administered without delay, starting Day 1, with the intention of accelerating hematologic recovery. 
Eltrombopag will be discontinued at the 6-month landmark visit.

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2.9.3
Justification for adding Extension Cohort
An Extension Cohort of 87 subjects was added to the protocol once enrollment was completed in cohort 3 
in order to allow additional subjects to be enrolled and treated while a new SAA treatment naïve protocol 
is developed and approved, and to obtain more precise data on secondary endpoints, and more 
pharmacokinetic data with the treatment regimen.  We anticipate enrollment will be completed before we 
have a new SAA treatment naïve protocol developed and approved.
2.9.4
Justification for adding Laboratory Exploratory Cohort
We currently have accrued 178/182 patients to our protocol, 12-H-0150. Among these patients, 86 compose 
an extension cohort; the primary aim of extension of the study was to obtain more precise data regarding 
important secondary endpoints and to collect more pharmacokinetic samples.
We now propose an extension to add 25 subjects to perform novel laboratory experiments using fresh bone 
marrow samples obtained at the time of diagnosis and prior to institution of definitive therapy. These 
experiments will not be feasible in our upcoming treatment naïve SAA protocol because we plan to 
initiate treatment remotely, and thus will not be collecting baseline marrow/blood samples for 
research.
T cell mediated immune destruction of hematopoietic stem and progenitor cells (HSPCs) is the pathophysiology 
of SAA, but precise mechanisms have not been characterized, nor have biomarkers been identified. We intend to 
elucidate the pathophysiology of SAA using advanced single-cell methodologies (scRNA-seq), a technique that 
allows high resolution whole transcriptome profiling of the individual cells, and time of flight cytometry 
(CyTOF), a novel deep-phenotyping technique using multiparameter mass cytometry. ScRNA-seq will be used to 
study the transcriptome of HSPCs and enriched T cells, and T cell clonality in patients with newly diagnosed 
severe aplastic anemia (SAA). CyTOF will enable high resolution characterization of HSPCs, immune cells 
especially regulatory T cell subsets, and other marrow populations simultaneously based on a range of cell surface 
and intracellular markers, to explore the relationship of bone marrow microenvironment and hematopoiesis in 
patients with SAA. If a biomarker or genetic signature is identified from these experiments, further experiments 
could be performed on larger cohort of frozen samples using simpler methods, such as standard flow cytometry or 
multiplex polymerase chain reaction (PCR).
Our aims are to:
(1) Identify characteristic cell population in treatment naïve SAA, provide a comprehensive view of active 
marrow process in SAA patients at both cellular and molecular levels
(2) Infer potential autoantigens from the expanded clonal T cells, perform integrated analysis of receptors and 
ligands of T cells and HSPCs
(3) Expand our understanding of the interaction between HSPCs and the microenvironment of the marrow of 
SAA patients by interrogating immune mechanisms in detail (cytotoxic T cells, T regs, MDSC, 
macrophages) 
(4) Characterize transcriptomes programs in HSPCs in treatment naïve SAA
(5) Identify biomarkers for clinical response and long term outcomes, particularly clonal evolution 
(6) Explore mechanisms by which eltrombopag (EPAG) stimulates hematopoiesis and modulates the immune 
system
We plan to perform this experiment with fresh bone marrow samples from 25 subjects at baseline and at 6 
months’ time point. These patients will not be included in the analysis on secondary endpoints of the current 
extension cohort. Fresh samples will be used for flow cytometry, particularly mass spectrometry, due to the 
impact of cryopreservation on fragile cell types and certain proteins. A study with human tumor specimen found 

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significant reduction of expression levels of multiple markers upon cryopreservation [70]. Fresh samples are 
desirable in single cell experiments using CD34 cells from SAA patients due to low cell numbers, which are 
inevitably decreased with cryopreservation. Additionally, there are data suggesting that cryopreservation results in 
molecular alterations in some cells including DNA damage, gene expression, and epigenetic changes[72, 73].
3.
STUDY DESIGN
This study is designed as a non-randomized, pilot phase I/II study in which 182 subjects with severe aplastic 
anemia who have not received prior definitive immunosuppressive therapy will be treated with a standard 
regimen of h-ATG/CsA, combined with eltrombopag as experimental therapy.  The first 31 subjects will 
be enrolled to cohort 1.  Then up to 33 subjects will be enrolled to cohort 2 while up to 31 subjects will be 
enrolled in cohort 3. Subjects enrolled to cohort 1 will receive eltrombopag for 6 months whereas subjects 
enrolled to cohort 2 will receive eltrombopag for 3 months.  Subjects enrolled in cohort 3 will initiate h-
ATG, CsA and eltrombopag on Day 1.  The primary clinical endpoint of this study is the quality of 
hematologic response at 6 months.  In cohorts 1 and 2 eltrombopag will be initiated on day 14 to avoid 
overlap with the known transient hepatotoxicities associated with ATG and cyclosporine.  Cohort 3 will 
initiate h-ATG, CsA, and eltrombopag on Day 1 if there are no significant hepatotoxicities seen in cohorts 
1 and 2. Patient-reported outcome questionnaires (Appendix E) will be collected initially when the subject 
enrolls on-study (pre-ATG/CSA), pre-eltrombopag, at 3 and 6 months, and annually for 5 years.
Up to an additional 87 subjects may be enrolled into the Extension Cohort in order to allow subjects to be 
enrolled and treated while a new SAA treatment naïve protocol is developed and approved.  In addition, 25 
subjects will be enrolled for Laboratory Exploratory Cohort. The treatment schedule for both the Extension 
& Laboratory cohorts will be the same as Cohort 3.
Protocol Design for Cohort 1
31 SAA
tx naïve
horse-ATG  x 4 days
CsA starting day 1 for 6 mo
Off study
Eltrombopag 
daily
starting day 14 
for 6 mo
Follow up 3 
mo, 6 mo, 
annually for 
up to 5 yrs

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Protocol Design for Cohort 2 added August 2013
33 SAA
tx naïve
subjects pts
horse-ATG x 4 days
CsA starting day 1 for 6 mo*
 
Off study
Eltrombopag
daily
starting day 14
for 3mo
Follow up 3
mo, 6 mo,
annually for
up to 5 yrs
*Cohort 2 will continue CsA administration month 6 to month 24 at 2.0mg/kg per amendment number 15
Protocol Design for Cohort 3 added September 2014
 31 SAA
tx naïve
Off 
study
• 
Horse-ATG x 4 days
• 
CsA Day 1 X 6 mo 
• 
Eltrombopag Day 1 X 
6mo
Day 1
3-month 
landmark
6-month 
landmark
Responder
Non-responder
CsA 2.0mg/kg/Day 
to month 24
Off tx
Follow-up 
annually for up 
to 5 yrs
Protocol Design for Extension Cohort and Laboratory Exploratory Cohort
112 SAA 
tx naive

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4.
ELIGIBILITY ASSESSMENT
All subjects age 2 and older with SAA who have not received prior ATG-based immunosuppressive therapy 
and lack a suitable matched sibling marrow donor, or are not allogeneic transplantation candidates due to 
patient choice, advanced age, or infeasibility of transplantation will be considered for enrollment.
Eligibility will be determined on another National Heart Lung and Blood Institute (NHLBI) screening 
protocol (97-H-0041) or any other active Hematology branch protocol.  The time between determination 
of eligibility and signing consent to participate on this protocol and initiate treatment on this protocol will 
not exceed 90 days.  Due to the nature of SAA, counts may fluctuate depending on transfusions.  Because 
of this, the lowest clinical laboratory result (ANC, platelet, and/or absolute reticulocyte count) obtained 
within 60-days prior to treatment initiation will be used for eligibility determination.
4.1
Inclusion Criteria
4.1.1
Severe aplastic anemia characterized by 
Bone marrow cellularity <30% (excluding lymphocytes) *
AND
At least two of the following:
• Absolute neutrophil count< 500/ μL
• Platelet count< 20,000/ μL
• Absolute reticulocyte count<60,000/ μL
4.1.2
Age > 2 years old
4.1.3
Weight > 12 kg
*If a range is provided instead of the overall bone marrow cellularity the median value of that range will be used for 
this inclusion criterion.
4.2
Exclusion Criteria
4.2.1
Known diagnosis of Fanconi anemia
4.2.2
Evidence of a clonal disorder on cytogenetics performed within 12 weeks of study entry. 
Patients with super severe neutropenia (ANC < 200 /μL) will not be excluded initially if 
cytogenetics are not available or pending.  If evidence of a clonal disorder consistent with 
myelodysplasia is later identified, the patient will go off study – see section 9.6
4.2.3
Prior immunosuppressive therapy with any ATG, alemtuzumab, or high dose 
cyclophosphamide
4.2.4
SGOT or SGPT >5 times the upper limit of normal
4.2.5
Subjects with known liver cirrhosis in severity that would preclude tolerability of 
cyclosporine and eltrombopag as evidenced by albumin < 35g/L
4.2.6
Hypersensitivity to eltrombopag or its components
4.2.7
Infection not adequately responding to appropriate therapy

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4.2.8
Moribund status or concurrent hepatic, renal, cardiac, neurologic, pulmonary, infectious, 
or metabolic disease of such severity that it would preclude the patient’s ability to tolerate 
protocol therapy, or that death within 7-10 days is likely
4.2.9
Potential subjects with cancer who are on active chemotherapeutic treatment or who take 
drugs with hematological effects will not be eligible
4.2.10 Current pregnancy, or unwillingness to take oral contraceptives or use a barrier method of 
birth control or practice abstinence to refrain from pregnancy if of childbearing potential 
during the course of this study
4.2.11 Inability to understand the investigational nature of the study or to give informed consent 
or does not have a legally authorized representative or surrogate that can provide 
informed consent per section 11.6.
5.
TREATMENT PLAN
5.1
Horse Anti-thymocyte Globulin (h-ATG) Administration
A single treatment course of h-ATG will be administered at a dose of 40 mg/kg/day for 4 consecutive days. 
Dose will be calculated based on actual body weight.  H-ATG will be infused intravenously for 
approximately 4 hours.  The infusion time may vary based on the patient and circumstances.   Infusion 
times may be extended up to 24 hours to improve tolerance of infusional side effects such as fever, chills 
and hypotension if necessary.  Serum sickness prophylaxis with oral prednisone (or intravenous 
methylprednisolone as clinically indicated) at 1 mg/kg/d will begin prior to the first dose of h-ATG, and 
will be continued for 10 days total and then tapered over the subsequent 7-14 days.  Those subjects who 
develop serum sickness may require a longer tapering schedule and will be dosed individually as clinically 
indicated.
5.2
Cyclosporine (CsA)
Day 1 to Month 6 dosing:
For subjects ≥ 12 years of age, cyclosporine will be started on day 1 at 3 mg/kg/dose by mouth administered every 
12 hours (total daily dose of 6 mg/kg/day).  Dosing will be based on actual body weight except in obese subjects. 
For obese subjects (defined as a body mass index > 35 in adult subjects [> 20 years of age] and > 95th percentile in 
subjects 12 to 20 years of age), cyclosporine dosage will be based on an adjusted body weight that is calculated as 
the midpoint between the ideal body weight and actual body weight (see below for definition of ideal body weight).
For subjects < 12 years of age, cyclosporine will be started on day 1 at 6 mg/kg/dose by mouth administered every 
12 hours (total daily dose of 12 mg/kg/day).  Dosing will be based on actual body weight except in obese subjects.  
For obese subjects (defined as a body mass index > 95th percentile in subjects 12 to 20 years of age), cyclosporine 
dosage will be based on an adjusted body weight that is calculated as the midpoint between the ideal body weight 
and actual body weight (see below for definition of ideal body weight).
Cyclosporine dosing will be adjusted, at the investigator's discretion, to aim for a therapeutic trough level 
between 200 and 400 mcg/L.  In subjects who had a therapeutic CsA level established prior to protocol 
enrollment, the same CsA dose will be initiated with the h-ATG and adjusted accordingly.  Cyclosporine 
dose may be interrupted or adjusted as clinically indicated at the discretion of the investigator for side 
effects.  Interruptions will only be recorded in the medical record.
In between NIH landmark visits, only CsA levels will be tracked and recorded into the clinical database.
Ideal Body Weight Definitions:

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Ideal body weight (adult male, age > 20): 50 kg + 2.3 kg per inch over 5 feet
Ideal body weight (adult female, age > 20): 45.5 kg + 2.3 kg per inch over 5 feet
Ideal body weight (pediatrics, ages 2–20): 50th percentile weight based on CDC growth curves.
Month 6 to Month 24 cyclosporine dosing:
On review of the data as of September 17, 2014 where a trend towards a higher than expected number of 
relapses after the 6 month landmark visit were observed and salvaged by resuming cyclosporine, the 
protocol was amended to prolong administration of cyclosporine beyond 6 months per the following:
At the 6-month landmark visit, responders will have the CsA dose reduced to 2.0mg/kg/day administered 
orally at a fixed dose through the 24 month timepoint.  Previous anecdotal experience suggested that a 
gradual taper of CsA may avoid relapse, but when we adopted this strategy prospectively in our clinical 
trials (03-H-0193 and 06-H-0034) the rate of relapse was unchanged (Scheinberg, Rios, et al. AJH 201467).  
However, time to relapse was delayed by 1 year with the longer course of CsA and may be dose-dependent.  
The CsA dose at the time of relapse during the taper phase was at a median of 1.32 (range: 0.52-4.18) 
mg/kg/day and a mean of 1.8 mg/kg/day.  This suggests that a CsA dose level above these levels might 
preclude relapse and improve tolerability of chronic maintenance.  For remaining subjects due for the 6 
month timepoint on cohort 2 we will prolong administration of cyclosporine beyond 6 months.  CsA will 
continue to be administered from Day 1 to 6 months, but from 6 months to 2 years CsA will be administered 
at a lower fixed daily dose (2 mg/kg/day) in an attempt to improve relapse, a secondary endpoint, among 
responders in the study.
Medication dosing errors, dose delays or dosing interruptions:
Interruptions such as delays in request for medication refills or medication errors by subjects will be 
recorded in the medical record.
5.3
Eltrombopag
Subjects will initiate eltrombopag at a starting daily dose as detailed in Table 3, according to age and 
ethnicity. Subjects between 12 and 17 years of age will receive the adult dose of 150 mg.  Those between 
6 and 11 will start at 75 mg, and children between 2 and 5 years of age will be started at 2.5 mg/kg (Table 
3).  To adjust for the higher expected exposure in children of East Asian and South East Asian ancestry, the 
starting dose for East Asian and South East Asian subjects between 12 and 17 years of age will be 75 mg 
once daily.  For East Asian and South East Asian subjects between 6 and 11 years of age, the starting dose 
will be 37.5 mg once daily, and for children between 2 and 5, the starting dose will be 1.25 mg/kg (Table 
3).  Eltrombopag will be administered orally as tablets or an oral suspension from sachet of eltrombopag 
powder.  Each 25 mg Pfos sachet contains eltrombopag olamine equivalent to 25 mg of eltrombopag free 
acid.  If a child’s dose is based on body weight and needs a dose of 28 mg, then dose only single sachet that 
provides 25 mg dose.  However, if the child needs a dose of 29 mg or greater, then the suggestion is to start 
using the second sachet.  This is mainly suggested to prevent the wastage of medicine by opening a second 
sachet to meet the additional 1-3 mg dose.  Dosing 25 mg where a patient needs 28 mg should not have a 
significant impact on PD response.  If an adult receives the oral suspension administration of eltrombopag, 
the required number of sachets will be used to administer the below adult doses.
Table 3. Dosing according to age and ethnicity
Age groups
Daily dose
Non-Asian
12-85
150 mg
6-11
75 mg

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Age groups
Daily dose
2-5
2.5 mg/kg
East Asian, South East Asian
12-85
75 mg
6-11
37.5 mg
2-5
1.25 mg/kg
Eltrombopag may be taken on an empty stomach (1 hour before or 2 hours after a meal) or with food 
containing little (<50 mg) or preferably no calcium or dairy products.  Allow at least a 4-hour interval 
between eltrombopag and other medications or products containing polyvalent cations (e.g. calcium, 
magnesium, aluminum, zinc, selenium or iron) such as antacids, dairy products, and mineral supplements 
to avoid significant (70-75%) reduction in eltrombopag absorption due to chelation.
Dose delay or dosing interruptions:
Cohort 1:
Eltrombopag dose may be interrupted when clinically indicated at the discretion of the investigator. 
Interruptions will be recorded in the medical record.
Cohort 2, 3, Extension Cohort and Exploratory Laboratory Cohort:
The first dose of eltrombopag may be delayed if it is clinically indicated or other unforeseen events affect 
the start of eltrombopag (for example weather related) at the discretion of the investigator.
Eltrombopag dose may be interrupted when clinically indicated at the discretion of the investigator (for 
example if the patient is in the intensive care unit and unable to take PO medications).  For subjects on 
cohort 2, if eltrombopag dosing is delayed or interrupted for more than 1 week (7 days) and the interruption 
is not the result of a severe adverse event related to eltrombopag, then the subject will receive additional 
eltrombopag in order to receive a total of 10 weeks as originally planned for cohort 2.
Medication dosing errors, dose delays or dosing interruptions:
Interruptions such as delays in request for medication refills or medication errors by subjects will be 
recorded in the medical record.
5.4
Dose Adjustments of Eltrombopag
The daily dose of eltrombopag will be decreased according to the following rules:
Platelet Count 
Dose Adjustment or Response 
>200,000/ μL (untransfused) at any time on study
Decrease dosage by 25mg every 2 weeks to 
lowest dosage that maintains platelet count 
≥ 50,000/ μL. In children under 12, the dose 
will be decreased by 12.5 mg.
>400,000/ μL (untransfused) at any time on study
Discontinue eltrombopag for one week, if 
platelets fall to <200,000/ μL; restart at 
dosage decreased by 25 mg/day (or 12.5 mg 
in children under 12).

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5.5
Dose Delays, Modifications or Discontinuation of Eltrombopag for Non-Hematologic Side 
Effects
5.5.1
Infection:  Subjects who experience an infection requiring intravenous antibiotics will not 
have eltrombopag discontinued.  If the subject experiences infection severe enough to require 
vasopressors or intubation, the drug will be withheld until the subject is stable.
5.5.2
Liver function abnormalities:
Cohorts 1 and 2:
In the event of an increase in the ALT level to > 6 times the ULN, subjects will return to clinic or 
have blood tests drawn by their home physician every 3-4 days. If the ALT remains > 6 times the 
ULN on a second blood test, eltrombopag will be discontinued until ALT is < 5 times the ULN.  
Eltrombopag will be restarted at a dose level 25 mg/day lower than the prior dose. If liver test 
abnormalities return to an ALT of > 6 times ULN on this reduced dose, the eltrombopag dose with 
be reduced by 25 mg/day until there is reduction of ULN to < 5 ULN.
Cohort 3 Extension Cohort and Exploratory Laboratory Cohort:
Transient hepatotoxicity is an expected and common side effect of h-ATG that occurs during the 
first 14 days following the start of h-ATG administration.  In the event of an increase in the ALT 
level to > 6 times the ULN during Days 1 – Days 14, eltrombopag will be held until ALT is < 5 
times the ULN and then resumed at the same dose.  If the ALT rises to > 6 times ULN after 
resuming eltrombopag (and is not attributable to other inciting factors such as serum sickness, 
sepsis, or azole antifungal agents) then the ALT will be monitored at least every 3-4 days.  If the 
ALT remains > 6 times the ULN on repeat blood tests, eltrombopag will be stopped until the ALT 
is < 5 times the ULN.  Then eltrombopag will be restarted at a dose level that is 25 mg/day lower 
than the prior dose.  If liver test abnormalities return to an ALT of > 6 times ULN on this reduced 
dose, the eltrombopag dose with be reduced by 25 mg/day until there is reduction of ULN to < 5 
ULN.
5.6
Dose Delays, Modifications or Discontinuation of Eltrombopag for Hematologic Side Effects 
5.6.1
Thrombosis/Embolism:  Subjects who experience a deep venous thrombosis (other than a 
line-related upper extremity thrombosis) or a pulmonary embolus, a TIA or stroke, or a 
myocardial infarction at any time while on eltrombopag will discontinue eltrombopag but 
remain on CsA and hATG. Subjects with platelet counts of > 50,000/ μL at the time of 
thrombosis will be treated with enoxaparin or another appropriate anticoagulant as clinically 
indicated until the platelet count drops below 20,000/ μL or they complete a standard 3-6 
month course of anticoagulation.
5.7
Extended Access to Study Drug
Responders:
Cohorts 1 and 2:  Study drug (eltrombopag) administration will be discontinued at 6 months for subjects in 
cohort 1 and at 3 months* for subjects in cohort 2; cyclosporine will be discontinued at 6 months for both 
cohorts.
Cohorts 1 and 2 Subjects that Relapse:
In cohort 2, if a subject who received eltrombopag for 3 months* has evidence of relapse as defined in 
Section 7, eltrombopag and/or cyclosporine can be restarted at the clinical investigator’s discretion.  Such 
cohort 2 subjects, who require treatment prior to month 6, will be denoted as non-responders at 6 months 

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for statistical purposes.
Subjects in cohort 1 or 2 that relapse (defined in section 7) after the end of 6-month treatment period while 
in follow-up may have eltrombopag and/or cyclosporine restarted at the clinical investigator’s discretion.  
Subjects in cohort 2 that received the reduced CsA dose (2mg/kg/day) starting after the 6-month landmark 
visit who relapse despite the prolonged reduced dose of CsA may have the CsA dose increased to achieve 
therapeutic levels (trough levels 200-400) at the clinical investigator’s discretion.  Subjects can remain on 
the drugs under this protocol until the end of the 5 year follow-up period.
*Study drug (eltrombopag) administration will be discontinued at 3 months for subjects in cohort 2, except 
for subjects experiencing dose delays/interruptions as outlined in Section 5.3.
Cohort 3 and Extension Cohort:
Subjects that relapse (defined in section 7) after the end of 6-month treatment period while in follow-up 
may have eltrombopag restarted at the clinical investigator’s discretion.  In addition, the cyclosporine dose 
may be increased to achieve a therapeutic level.  Subjects can remain on the drugs under this protocol until 
the end of the 5-year follow-up period.
Laboratory Exploratory Cohort: 
Follow up for subjects in this cohort will be the same as Cohort 3 and Extension Cohort (above).
5.8
Pre-medications and Management of Infusion Reactions
Subjects will receive pre-medication approximately 30 minutes prior to infusion of ATG as follows:
•
oral diphenhydramine 1 - 1.5 mg/kg/dose (NTE 50 mg) administered orally or intravenously, 
and;
•
oral acetaminophen 10-15 mg/kg/dose (NTE 650 mg)
Oral prednisone at 1 mg/kg/d will begin prior to the first dose of h-ATG for serum sickness prophylaxis, 
and will be continued for 10 days total and then tapered over the subsequent 7-14 days.  Infusion reactions 
will be treated symptomatically (e.g., antiemetics, IV fluid hydration, acetaminophen, antihistamines, 
inhaled bronchodilators, meperidine).  Prednisone dose will be calculated based on actual body weight.
In case of moderate or severe reactions hydrocortisone will be given and the infusion will be discontinued 
and restarted at a slower rate once the symptoms have subsided.  If a subject has a persistent severe infusion 
reaction that does not respond to measures to ameliorate the signs/symptoms associated with the infusion, 
the h-ATG infusion will be discontinued (see section off study criteria) and subjects will go off study.
5.9
Permitted Supportive Care
•
Transfusion support (blood and platelets) as clinically indicated.
•
Hematopoietic growth factors (e.g., G-CSF, GM-CSF, or erythropoietin) if deemed necessary 
by the investigator or treating physician. Romiplostim (N-Plate) or IL-11 (Neumega) should 
not be administered.
•
Estrogens or combination OCP’s as indicated for uterine bleeding
5.10 Concurrent Medications:
Cyclosporine/magnesium:  Subjects will be on chronic CsA therapy targeting a stable drug level as long 

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as eltrombopag is administered 4 hours after oral magnesium given to counteract magnesium-wasting on 
CsA.  Magnesium supplementation will not be given concurrently with eltrombopag as it may interfere 
with eltrombopag’s absorption.  The drug-drug interaction potential between eltrombopag and CsA is 
unknown.  Both CsA and eltrombopag are inhibitors of OATP and BCRP drug transporters, and 
eltrombopag is a substrate of BCRP in vitro.  It is not known if the combination will result in any PK 
changes to either drug.  Subjects will be monitored for signs of CsA toxicity during the study, and 
therapeutic drug monitoring can be instituted as required.  In the event of liver function abnormalities as a 
consequence of a drug-drug interaction between eltrombopag and CsA, eltrombopag will be dose-reduced 
according to Section 5.5.2.
Rosuvastatin:  In vitro studies demonstrated that eltrombopag is not a substrate for the organic anion 
transporter polypeptide, OATP1B1, but is an inhibitor of this transporter in vitro and as evidenced by 
increased plasma rosuvastatin levels when eltrombopag and rosuvastatin were co-administered in a clinical 
drug interaction study.  When co-administered with eltrombopag, a reduced dose of rosuvastatin should be 
considered and careful monitoring should be undertaken.  In clinical trials with eltrombopag, a dose 
reduction of rosuvastatin by 50% was recommended for co-administration of rosuvastatin and eltrombopag.  
Concomitant administration of eltrombopag and other OATP1B1 substrates should be undertaken with 
caution.
Inhibitors of cytochrome p450:  In vitro studies demonstrate that CYP1A2 and CYP2C8 are involved in 
the oxidative metabolism of eltrombopag.  Trimethoprim, gemfibrozil, ciprofloxacin, fluvoxamine and 
other moderate or strong inhibitors of CYPs may therefore theoretically result enhanced activity of 
eltrombopag, however these interactions have not yet been established in clinical studies.  All subjects on 
cyclosporine require prophylaxis against PCP and will be given inhaled pentamidine instead of TMP/SULF.  
NIH SAA patients are routinely placed on pentamidine instead of TMP/SULF for PCP prophylaxis to avoid 
potential marrow-suppressive effects of TMP/SULF anyway.  Subjects aged 5 years and over will receive 
pentamidine for PCP prophylaxis but children under 5 years of age (approximate) are often not able to 
complete the inhalation treatment with pentamidine and will receive dapsone or another prophylactic 
regimen.  Other CYP inhibitors can be used concomitantly but with careful attention to possible increased 
eltrombopag activity and toxicity.
Other medications:  Subjects may continue on any of the medications that they were prescribed prior to 
study enrollment for co-morbid conditions, with the exception of N-Plate and Neumega (see 5.9).
5.11 Infection Prophylaxis and Monitoring
Pneumocystis jiroveci prophylaxis:  Aerosolized pentamidine will be used as prophylaxis against 
Pneumocystis jiroveci, 300 mg approximately every 4 weeks by inhalation beginning the first month of 
therapy and to continue for 6 months total for subjects 5 years of age and older for all cohorts.  Dapsone or 
another prophylactic regimen against Pneumocystis jiroveci may be substituted at the discretion of the PI.  
Bactrim (TMP/SULF) will be avoided because trimethoprim is a moderate to strong inhibitor of CYPs that 
may theoretically result in enhanced activity of eltrombopag.  Children under 5 years of age (approximate) 
are often not able to complete the inhalation treatment with pentamidine and will receive dapsone or another 
prophylactic regimen at the discretion of the PI.
Antiviral prophylaxis:  Valacyclovir, 500 mg once daily, will be administered for at least 1 month in all 
subjects regardless of HSV serology status.  Pediatric subjects less than 40 kg, will receive acyclovir (or 
equivalent) at 20mg/kg PO q12h to a maximum dose of 800mg q12h.  Prophylaxis may be extended at the 
discretion of the PI.
Antibacterial and antifungal prophylaxis will not be included systematically with the 

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immunosuppressive regimen, but may be administered at the discretion of the PI or treating physician on a 
case-by-case basis.
5.12
Management of Fever in Neutropenic Subjects (All subjects)
Subjects with a single temperature of 38.5 °C or two readings of 38.0 °C or greater will be evaluated for 
infection including cultures of blood and urine and any other suspicious sites prior to beginning empiric 
therapy.  Antibiotics will be initiated following current infectious disease guidelines.
5.13
Instructions to Subjects
Special Instructions regarding CsA:
Regarding concomitant medications:  Certain other medications can change the level of cyclosporine in the blood.  
Some of these medications are erythromycin, ketoconazole, diltiazem, rifampin, phenytoin and phenobarbital.  
We will ask subjects to inform us of any medication taking concomitantly while on the study.
Regarding prohibited foods:  Grapefruit and grapefruit juice may increase the effects of CsA by increasing 
the amount of this medicine in the body.  Subjects will be advised not to eat grapefruit or drink grapefruit 
juice while taking this medicine.
Regarding Immunizations:  While taking CsA and for at least three months following discontinuation, 
immunizations should be avoided, and any planned immunization should be discussed with study 
investigators.  There is almost no possibility that a vaccination given during this time period will be effective 
in stimulating immunity.  Any live or attenuated vaccine may result in an infection, due to compromised 
immunity on CsA and h-ATG.  Subjects should also avoid close household contact with individuals 
receiving the live oral polio vaccine for at least 72 hours following administration.
Special Instructions regarding eltrombopag:
Timing in relation to food:  Subjects will be advised to take eltrombopag on an empty stomach (1 hour 
before or 2 hours after a meal), or adhere to a restricted diet of low calcium (dairy products) and polyvalent 
cations at least 4 hours apart from co-administration of eltrombopag.
Timing in relation to antacids and polyvalent cations:  Because co-administration of eltrombopag with 
antacids decreased plasma AUC of eltrombopag by 70%, subjects will be advised to take the eltrombopag 
at least 4 hours apart from antacids and other products containing polyvalent cations (i.e. aluminum, 
calcium, magnesium, iron, selenium and zinc) such as mineral supplements and dairy products.
5.14
HRQL Questionnaires
The relevant dimensions of HRQL being assessed in this study include (1) PROMIS Global Health, Sleep 
Disturbance, Applied Cognition-Abilities, Anxiety and Depression and (2) FACT- Anemia, 
Thrombocytopenia and Neutropenia.
Patient-Reported Outcomes Measurement Information System (PROMIS®), is an initiative based on an 
NIH grant to establish and provide the public a free, reliable and validated commonly used measures of 
patient-reported outcomes.63
The FACT instruments64 are a health assessment instrument designed to measure multi-dimensional quality 
of life in chronic illness and its associated therapy.  The different subscales selected for this study are 

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specific for patients with diseases or treatments with hematological effects.  All measures will be offered 
to adult subjects who read English or Spanish.  Any survey time-point that is missed due to the subject’s 
clinical status (e.g. critically ill) will be reported as a protocol deviation at time of continuing review.
6.
CLINICAL MONITORING
Samples will be ordered and tracked through the CRIS Screens.  Should a CRIS screen not be available, 
the NIH form 2803-1 will be completed and will accompany the specimen and be filed in the medical 
record. HRQL surveys will be mailed to the subject if they are not required to return to the NIH CC for 
study evaluations.
6.1
Pre-study Evaluation - PI may accept results from studies done outside of NIH.
Baseline studies will be conducted as follows:
 Medical history and physical examination
 Concurrent medication review
 HRQL survey administration (Surveys will not be required for the Extension and Exploratory 
Laboratory Cohort)

Baseline clinical studies
•
Complete blood count with differential
•
Reticulocyte count
•
DAT (direct antiglobulin test)
•
Type and screen
•
Acute Care (Na, K, Cl, CO2, Creatinine, Glucose, and Urea Nitrogen), Mineral 
(Phosphorus, Magnesium, Albumin, and Calcium), Hepatic (Alk Phosphatase, ALT, AST, 
Total Bilirubin, and Direct Bilirubin), and Other (Total Protein, CK, Uric Acid, and LD 
panel)
•
Coagulation and thrombosis screens (PT, PTT, D-dimer)
•
Thyroid function tests
•
Viral serologies for hepatitis A, B, C, HIV, HSV, EBV and CMV
•
EBV and CMV PCR
•
PPD (at risk subjects only, based on history or geography)
•
Folate level
•
B12 level
•
Iron panel (ferritin, transferrin, % saturation)
•
HLA typing (if not already available)
•
Pregnancy test (urine or blood HCG in women of child bearing potential)
•
Bone marrow aspiration and core biopsy, to be stained for standard morphologic analysis 
and quantitation of cellularity with hematoxylin and eosin, and special stains to assess 
reticulin 
and 
collagen, 
primitive 
stem 
and 
progenitor 
cells 
via 
CD34 
immunohistochemistry, and other lineage-specific or special stains as indicated to classify 
any abnormalities.
•
Bone marrow chromosomal analysis via standard cytogenetic techniques
•
Telomere length of leukocytes
•
Flow cytometry of the peripheral blood to quantitate GPI-negative cells
•
Lymphocyte peripheral blood phenotyping (analysis of T, B, and NK subsets via flow 
cytometry)
•
Chest X-ray- this does not need to be performed if a clinically indicated chest CT is 
performed to reduce the radiation burden

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Neal S. Young, M.D.  3/18/2020 (Amendment UU/47)
•
EKG
•
Placement of a central line if subject does not have a pre-existing indwelling central venous 
catheter
6.2
On Study Monitoring, Day 1 of h-ATG Through Hospital Discharge
On treatment monitoring will consist of the following, unless the hospitalization is interrupted due to the 
participant leaving the hospital “on pass” but not to exceed more than 7 days:

Pregnancy test (urine or blood HCG in women of child bearing potential) does not need to be 
repeated if done within two weeks (Day 0 to Day -14)

CBC with differential (daily)

Acute care, Mineral, Hepatic and Other panel (every other day)

Reticulocyte count (weekly +/- 3 days)

Vital signs (daily)

CsA blood level will be monitored every week (+/- 3 days) while inpatient in the hospital and 
continued per section 6.3.  The blood level will be monitored to ensure therapeutic range of 
200 – 400 ng/ml is achieved. CsA dosage will be adjusted to target this range.  More frequent 
drug serum levels may be obtained as needed to achieve target therapeutic levels and avoid 
toxicity.
6.3
On Study Monitoring (hospital discharge through 6 months)
After completing h-ATG administration, subjects will remain hospitalized until clinically stable.  Post-
discharge, subjects may be followed by their home physician or at the Clinical Center.  Progress notes and 
laboratory results from home physicians will be faxed to the research nurse.  Standard of care tests will be 
done as needed and may include the tests listed below.  Changes in frequency of the tests below will be 
performed as clinically indicated at the investigators discretion.

Complete blood counts with differential every 1-2 weeks.  Only the following parameters will be 
recorded in the clinical database because of their relevance to the underlying disease and study 
treatment: absolute neutrophil count (ANC), or neutrophil percentage and white blood cell count if an 
absolute neutrophil count is not provided, hemoglobin, platelets, and absolute reticulocyte count (or 
reticulocyte percentage and red blood cells [RBC] if an absolute reticulocyte count is not provided).

Chemistry panel (NIH Acute care, Mineral, Hepatic and Other panel, home laboratory chemistry panel 
must include electrolytes, hepatic transaminases, urea nitrogen (BUN), serum creatinine, and total 
bilirubin).  Only the following parameters will be recorded in the clinical database because of their 
relevance to the underlying disease and study treatment: aspartate aminotransferase (AST), alanine 
aminotransferase (ALT), alkaline phosphatase (ALP), total and direct bilirubin, creatinine, blood urea 
nitrogen (BUN), glucose, sodium, potassium, CO2 and chloride.

CsA blood level will be monitored every week for the first month and then every other week for the 
remainder of the treatment period once levels are stabilized in the therapeutic range of 200 – 400 ng/ml.  
CsA dosage will be adjusted to target this range unless participants cannot tolerate therapeutic dosing 
of CsA.  More frequent drug serum levels may be obtained as needed to achieve target therapeutic 
levels and avoid toxicity, and conversely less frequent levels may be obtained as needed in individuals 
unable to tolerate therapeutic dosing of CsA.  The dose may not necessarily be adjusted following every 
out of range CsA level, rather the decision to adjust the dose will be determined by the clinician 
judgement based on multiple data sources, that could include but not limited to time of measurement, 
prior trend of CsA levels, estimation of steady state, concomitant medications that may affect CsA 
metabolism, and renal function.  CsA monitoring will be discontinued at 6 months for all cohorts

HRQL survey administration (pre-eltrombopag; within 2 days of day 14 post ATG on cohorts 1 and 2) 

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Neal S. Young, M.D.  3/18/2020 (Amendment UU/47)
(Surveys will not be required for the Extension and Exploratory Laboratory Cohort.)
Landmark 3-month and 6-month monitoring
Subjects must be evaluated at the NIH Clinical Center at the 3- and 6-month (+/-10 days) time points

History and physical examination

Complete blood counts with differential

Acute care, Mineral, Hepatic and Other-panel

Reticulocyte count

Urine pregnancy test (woman of childbearing age only)

Bone marrow aspiration and core biopsy, to be stained for standard morphologic analysis and 
quantitation of cellularity with hematoxylin and eosin, and special stains to assess reticulin and 
collagen, primitive stem and progenitor cells via CD34 immunohistochemistry, and other lineage-
specific or special stains as indicated to classify any abnormalities

Bone marrow chromosomal analysis via standard cytogenetic techniques

Flow cytometry of the peripheral blood to quantitate GPI-negative cells

Lymphocyte peripheral blood phenotyping (analysis of T, B, and NK subsets via flow cytometry)

Peripheral blood for pharmacokinetic sampling (3-month, cohort 1; also, samples may be drawn any 
day between day 8 through day 30 on the extension cohort starting with subject #120). Pharmacokinetic 
sampling will not be collected for Exploratory Laboratory Cohort.

HRQL survey administration (Surveys will not be required for the Extension and Exploratory 
Laboratory Cohort.)

EKG (required at month 3 visit, may be performed at other time points)
6.4
Long Term Follow Up (12 months to 5 years)
After the 6 month visit, subjects must be evaluated at the Clinical Center at 12 months (+/- 30 days) and 
then yearly thereafter to 5 years (+/- 90 days for Years 1 & 2; +/- 1 Year for Years 3 & 4).  Subjects will be 
seen by their home physician as clinically indicated and the Hematology Branch investigators and home 
physicians will remain in communication. Home physicians will monitor blood counts and other clinical 
parameters as clinically indicated.  The following tests will be performed at each Clinical Center visit.  At 
the clinical investigator’s discretion, participants may be evaluated more frequently if medically indicated 
based on disease status. If a subject is unable to present to CC for landmark Year 3 or 4 follow up due to 
circumstances beyond their control and their peripheral blood counts are available and stable compared to 
last visit at CC, the subject may remain on the study and follow up as soon as able but no longer than 1 year 
from the missed visit.

Complete blood counts with differential

Acute care, Mineral, Hepatic and Other panel

Reticulocyte count

Bone marrow aspiration and core biopsy, to be stained for standard morphologic analysis and 
quantitation of cellularity with hematoxylin and eosin, and special stains to assess reticulin and 
collagen, primitive stem and progenitor cells via CD34 immunohistochemistry, and other 
lineage-specific or special stains as indicated to classify any abnormalities.

Bone marrow chromosomal analysis via standard cytogenetic techniques

Flow cytometry of the peripheral blood to quantitate GPI-negative cells

Lymphocyte peripheral blood phenotyping

HRQL survey administration (Surveys will not be required for the Extension and Exploratory 
Laboratory Cohorts.)

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6.5
Extended Access for Relapse
Indicated below are the procedures that can be performed on subjects that re-start drug due to relapse. At 
the clinical investigator’s discretion, participants may be evaluated as medically indicated based on disease 
status.  The procedures may be performed by the subjects’ home physician or at the Clinical Center. If 
testing is done by home physician, progress notes and laboratory results from home physician will be sent 
to the research team.  Subjects will be seen at the Clinical Center as clinically indicated while on extended 
access for relapse, but no less than annually until 5 years.  Below is the list of procedures that will be 
performed as medically indicated.
Procedures that may be performed when drugs are re-started

History and physical examination

Pregnancy test (urine or blood HCG in women of child bearing potential)

Complete blood counts with differential

Chemistry panel (NIH Acute care, Mineral, Hepatic and Other panel, home laboratory chemistry panel 
must include electrolytes, hepatic transaminases, urea nitrogen (BUN), serum creatinine, total bilirubin, 
and reticulocyte count)

CsA blood level will be monitored until levels are stabilized in the therapeutic range of 200 – 400 
ng/ml. CsA dosage will be adjusted to target this range.  More frequent drug serum levels may be 
obtained as needed to achieve target therapeutic levels and avoid toxicity.

Bone marrow aspiration and core biopsy, to be stained for standard morphologic analysis and 
quantitation of cellularity with hematoxylin and eosin, and special stains to assess reticulin and 
collagen, primitive stem and progenitor cells via CD34 immunohistochemistry, and other 
lineage-specific or special stains as indicated to classify any abnormalities.

Bone marrow chromosomal analysis via standard cytogenetic techniques Flow cytometry of 
the peripheral blood to quantitate GPI-negative cells

Lymphocyte peripheral blood phenotyping
7.
CRITERIA FOR RESPONSE
Response is defined as blood counts no longer meeting the standard ("Camitta") criteria for severe 
pancytopenia in SAA (see section 4.1), equivalent to 2 of the following values obtained on 2 serial blood 
count measurements at least one week apart at landmark time points (3, and 6 months)
•
Absolute neutrophil count ≥500/ μL
•
Platelet count ≥ 20,000/ μL
•
Reticulocyte count ≥ 60,000/ μL
A complete response (CR) will be defined as (all 3 must be met):
•
Absolute neutrophil count ≥1,000/ μL
•
Platelet count ≥100,000/ μL
•
Hgb≥10 g/dL
A partial response will be defined as blood counts that do not meet criteria for severe pancytopenia but are 
not sufficient for a CR.
Improvement in counts that are dependent upon exogenously administered growth factors or transfusion 
will not be considered as fulfilling response criteria.
The presence of evolution to PNH will be defined by flow cytometric detection of > 1% GPI-deficient 

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neutrophils at baseline and landmark time points through 5 years.  Evolution to myelodysplasia and/or acute 
leukemia will be assessed at landmark time points, or as clinically indicated between landmarks by 
examination of peripheral blood and bone marrow and diagnosis and classification according to the WHO 
criteria.  Evolution to clonal hematopoiesis will be defined by detection of new bone marrow cytogenetic 
abnormalities at landmark time points.
Relapse:  Clinical definition determined by observation of a decline in blood counts not explained by 
another clinical process (e.g. acute infection) that is either (a) a substantial decline in one or more blood 
counts, or (b) a progressive decline in one or more blood counts on at least two consecutive blood draws.  
The date of relapse will be recorded into the clinical database as the date the participant resumed 
immunosuppression with CsA or required higher doses of CsA if on the low dose prior to the 2-year 
landmark visit.  A description of how the clinical assessment was made by the PI will be recorded in the 
clinical database.  To corroborate the relapse, the most recent blood counts collected prior to the date of 
relapse will be recorded into the clinical database with the following parameters: absolute neutrophil count, 
or neutrophil percentage and white blood cell count if ANC is not available, hemoglobin, absolute 
reticulocyte count (or reticulocyte percentage and RBC if absolute reticulocyte count is not available) and 
platelets.
8.
EXPLORATORY LABORATORY RESEARCH STUDIES
8.1
Collecting, tracking and disposition of samples
Intended use:  During the course of participating on this study, 60 cc of blood (3 ml/kg not to exceed 60 
ml of blood for pediatric subjects) at baseline and at landmark visits at 3, 6, 12 months and annually 
thereafter, and 5 cc of bone marrow aspirate (baseline, 3 months, 6 months, 12 months, and annually 
thereafter) will be obtained for the following correlative laboratory research studies.  Up to an additional 
10 cc of blood (3 ml/kg not to exceed 10 ml of blood for pediatric subjects) may be collected during follow 
up every 1-6 months (according to how often they get their counts checked) for correlative laboratory 
studies.  The protocol will cover shipment materials/costs for these additional samples to be returned to the 
NIH since they are for research use (no results given).  Baseline samples may be obtained on another 
protocol, such as 04-H-0012.  These studies are not used in assessing the primary endpoint but are 
undertaken as descriptive or exploratory ancillary studies.  Some or all may be performed on each subject, 
and they may be correlated with response.

Thrombopoietin level

CD34 cell number in whole blood and bone marrow aspirate by flow cytometry

T cell receptor V-beta profile in the marrow and peripheral blood

Extended peripheral blood flow cytometric phenotyping for cell surface or intracellular proteins

Evaluation for the presence of abnormalities of the telomere repair complex including telomere 
length and genetic testing of genes associated with the telomere repair complex

Evaluation for the presence of abnormalities of genes associated with hematopoiesis, via genetic 
testing or gene expression analysis

Evaluation for the presence of abnormalities of genes associated with inherited bone marrow 
failure

Serum cytokine, chemokines and soluble receptor levels

Serum (or plasma) and cells for viral analyses

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
Hematopoietic progenitor colony, long term-culture-initiating cell, and immunodeficient mouse 
engraftment assays for primitive cell content and function

Pharmacokinetic studies of eltrombopag kinetics in Cohort 1 and extension cohort starting with 
subject # 120 (Appendix D). Pharmacokinetic sampling will not be collected for Exploratory 
Laboratory Cohort.

Single cell RNAseq on bone marrow specimens to detect chromosomal aneuploidy before and 
after treatment

Telomere length on peripheral blood leukocytes by flow-FISH before and after treatment

In the event there is any extra sample, these will be stored with the subject’s permission for other 
exploratory laboratory research studies reviewed and approved by the IRB and listed in Appendix 
B.
Storage:  Research samples will be stored coded in the secure laboratory of Dr. Young.
Tracking:  Samples will be ordered and tracked through the CRIS Research Screens.  Should a CRIS screen 
not be available, the NIH form 2803-1 will be completed and will accompany the specimen and be filed in 
the medical record.  Specimens will be entered in the NHLBI Biospecimen Inventory System (BSI).  Bone 
marrow samples obtained for cytogenetic studies will be submitted to Quest Laboratories under a fee-for-
service contract.  Coded/linked biospecimens to be shared outside of NIH for future research use requires 
an executed Material Transfer Agreement (MTA) and may require IRB approval if results will be returned 
and re-identified. Identifiable samples will not be sent outside of NIH without IRB approval and an MTA.
End of study procedures:  Samples from consenting subjects will be stored until they are no longer of 
scientific value or if a subject withdraws consent for their continued use, at which time they will be 
destroyed.
Loss or destruction of samples:  Should we become aware that a major breech in our plan for tracking and 
storage of samples has occurred, the IRB will be notified.
9.
BIOSTATISTICAL CONSIDERATIONS
9.1
Sample sizes
The statistical section of this protocol has been amended to first (amendment B) increase the accrual ceiling 
to 31 subjects from 25 subjects to increase the likelihood to have at least 25 evaluable subjects at the 6-
month time point for analysis.  Next, with amendment (8), submitted on August 22, 2013, to add a second 
cohort with a reduced eltrombopag schedule while keeping the primary endpoints unchanged.  Next, with 
amendment (16), submitted on October 2, 2014, to add a third cohort revising the administration schedule 
to allow for h-ATG, CsA, and eltrombopag to all be administered starting Day 1 while keeping the primary 
endpoints unchanged.  Eltrombopag will be administered for 6 months and the primary endpoint will remain 
the same, response at month 6.
Our past experience with h-ATG/CsA suggests that the CR probability at 6 months for previously untreated 
patients given this regimen is approximately 10%~12%.  Our data supports that among those in which a 
CR is observed, evolution to monosomy 7 and leukemia did not occur.  Therefore, CR will be used as a 
surrogate for late events for the purpose of study design.  We hypothesize that the actual CR probability 
using this treatment would reach 30% or more and a CR probability of 10% or less would warrant 
terminating the treatment on this patient population.  Let p be the CR probability at 6 months for this patient 
population.

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In the original protocol, approved on June 13, 2012, our sample size was determined by testing the null 
hypothesis, H0: p≤10%, versus the alternative, H1: p ≥30%, at 0.05 significance level and 0.80 of the power.  
We intended to test this treatment using a small number of patients, and terminate the study if early evidence 
suggests that the CR probability fell below the benchmark rate of 10%.  We determined the sample size 
using the Two-Stage Minimax Design of Simon (1989), since it required a smaller total number of subjects 
(n=25) compared to the Two-Stage Optimal Design (n=29).  At the first stage, 15 subjects would be accrued 
and the null hypothesis would be accepted (i.e., the treatment would be terminated) if no more than 1 subject 
had a CR to the treatment within 6 months.  If 2 or more subjects had a CR to the treatment within 6 months 
at the first stage, 10 additional subjects would be accrued.  The null hypothesis of p≤10% would be accepted 
if the total number of patients having CR within 6 months was 5 or less.
With amendment B, approved on December 3, 2012, we increased the accrual ceiling to 31 subjects from 
25 subjects to increase the likelihood to have at least 25 evaluable subjects at the 6-month time point for 
analysis of secondary endpoints while maintaining the statistical power for the primary endpoint of the 
study.  This modified the statistical design as follows: Let p be the CR probability at 6 months for this 
patient population.  Our sample size is determined by testing the null hypothesis, H0: p≤10%, versus the 
alternative, H1: p ≥30%, at 0.05 significance level and 0.865 for power.  We would like to test this treatment 
using a small number of patients, and terminate the study if early evidence suggests that the CR probability 
falls below the benchmark rate of 10%.  We determine the sample size using the Two-Stage Minimax 
Design of Simon (1989), since it requires a smaller total number of subjects (n=31) compared to the Two-
Stage Optimal Design (n=34).  At the first stage, 24 subjects will be accrued and the null hypothesis will 
be accepted (i.e., the treatment will be terminated) if no more than 2 subjects demonstrates a CR to the 
treatment within 6 months.  If 3 or more subjects have a CR to the treatment within 6 months at the first 
stage, an additional 7 subjects will be accrued.  The null hypothesis of p≤10% will be accepted if the total 
number of subjects having a CR within 6 months is 6 or less.  The “ph2simon” function in the “Clinfun” 
package “Clinical Trial Design and Data Analysis Function, Version 1.0.5” of the statistical software R was 
used to compute the numerical results of the Two-Stage Design.
The current pilot study design has a cohort of 31 subjects receiving eltrombopag starting day 14 and ending 
at the 6 month time point (cohort 1).  With amendment (8), submitted on August 22, 2013, once accrual 
to cohort 1 has been completed without reaching any study stopping rules we propose to treat another cohort 
(cohort 2) of 33 subjects whereby they would be treated with the same regimen except that they would 
receive a reduction in exposure to eltrombopag from 6 to 3 months.  The primary and secondary endpoints, 
objectives, eligibility, statistics, including sample size and stopping rules, monitoring and ancillary studies 
apply the same way for cohort 2 as for cohort 1.  With amendment 16, submitted on October 2, 2014, once 
accrual to cohort 2 has been completed without reaching any stopping rules we propose to treat another 
cohort (cohort 3) of 31 subjects whereby they would be treated with the same regimen except that they 
would start h-ATG, CsA, and eltrombopag on Day 1.  Eltrombopag will be administered up to the 6-month 
landmark visit.  The primary and secondary endpoints, objectives, eligibility, statistics, including sample 
size and stopping rules, monitoring and ancillary studies apply the same way for cohort 3 as for cohorts 1 
and 2 with the exception of one additional secondary objective (relapse rate between month 6 to month 24 
when receiving low CsA during this time period).
Sample size (n)
Eltrombopag
Primary Efficacy Endpoint
Statistical Design
Cohort 1
31
D 14 – 6 months
CR rate at 6 months
Simon Two-Stage
Cohort 2
33
D14 – 3 months
CR rate at 6 months
Simon Two-Stage
Cohort 3
31
D1 – 6 months
CR rate at 6 months
Simon Two-Stage
Starting with Amendment V, an extension cohort was added of 55 subjects in order to have data to improve 
precision of exploratory analysis of secondary endpoints, and to obtain more pharmacokinetic data on the 

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combined regimen.  Then with Amendment FF, the extension cohort ceiling is raised to 87 subjects with 
the aim of having an additional 32 subjects to provide fresh research samples for 1) detection of cytogenetic 
abnormalities by single cell RNAseq methods in our laboratory, 2) measurement of telomere length before 
and after treatment by flow-FISH, and 3) additional pharmacokinetic data.  The entire extension cohort 
(n=87) will be included in the analysis of secondary endpoints, but not in the primary endpoint analysis of 
cohort 3.  We plan to use descriptive statistics, not hypothesis testing, in the analysis of the extension cohort.
In this revision, we have estimated our sample size (n=32) on the need for fresh samples for ancillary 
studies.  Specifically, using our newly developed method to detect clonal evolution using RNAseq (will 
provide manuscript), we predict that we will require at least 3 patient samples with clonal evolution.  We 
estimate that between 10% to 40% of patients will clonally evolve (Townsley et al. NEJM 2017).  Assume 
that the expected proportion of patients with clonal evolution is 21%.  Based on the exact calculation using 
Binomial distributions, the requested sample size of (n=32) will lead to a probability of 97.6% to obtain 3 
or more samples.  The true proportions of clonal evolution and their precision will be estimated using 
sample proportions, standard errors and confidence intervals.
9.2
Statistical Methods
For each cohort, the planned analyses will include descriptive statistics on the proportions of responses (% 
subjects with partial or complete response), cumulative incidence of response with death considered to be 
a competing risk and the time to complete response.  The response probabilities, including complete 
response probability and partial response probability, will be estimated using the sample proportions, and 
their inferences, including confidence intervals and hypotheses testing, will be evaluated using Binomial 
distributions.  Survival analysis will include methods based on Kaplan-Meier estimates and Cox regression.  
Although we have a small sample size, if it is appropriate, we will consider additional analyses for the 
primary and secondary endpoints using the analysis of variance, multiple regression and logistic regression 
models.  Graphical tools will be used to display the appropriate estimates (i.e. estimated proportions and 
Kaplan-Meier curves) and their corresponding 95% confidence intervals.
Results for each cohort will be compared separately to the historical data from the large numbers of patients 
treated at the NIH previously with either a single or repeat course of immunosuppression from prior NHLBI 
studies:  the h-ATG/CsA protocol (90-H-0146); the h-ATG/CsA vs. Cytoxan/CsA protocol (97-H-0117); 
the h-ATG/CsA/MMF protocol (00-H-0032); the h-ATG/CsA/Rapamune protocol (03-H-0193); the r-ATG 
vs. alemtuzumab protocol (03-H-0249); and h-ATG/CsA vs. r-ATG/CsA protocol (06-H-0034). More 
specifically, results from the current trial will be compared to our large historical experience with a horse 
ATG based regimen in nearly 400 patients (from the aforementioned studies) of a hematologic response 
rate of 60-70%, a complete response rate of 10-15%, a relapse rate of 30-40%, a clonal evolution rate (any 
clonal abnormality) of 15-20%, and a high risk evolution rate (to monosomy 7, high risk MDS, leukemia, 
or complex cytogenetics) of 10-15%.
9.3
Primary Endpoints
The primary objective of this phase I/II study is to evaluate the safety and activity profile of h-
ATG/CsA/eltrombopag in treatment naïve SAA.

The primary safety endpoint will be toxicity profile in the 6 months following h-
ATG/CsA/eltrombopag.

The primary efficacy endpoint is CR rate at 6 months (see section 7 for CR definition).
Subjects who drop out, who have failed to respond and opt for alternative therapy (e.g. bone marrow 

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Neal S. Young, M.D.  3/18/2020 (Amendment UU/47)
transplant), or who require eltrombopag to be restarted (cohort 2 only) before the 6-month evaluation will 
be counted as non-responders.
9.4
Secondary Endpoints
Secondary endpoints will also be evaluated for the study to include: (a) hematological response at 3 and 12 
months and yearly thereafter; (b) relapse (c) clonal evolution to PNH, clonal chromosomal population in 
bone marrow, myelodysplasia by morphology, or acute leukemia; (d) survival; (e) health-related quality of 
life; (f) hematological response of relapse subjects that re-start treatment; and (g) affects of a 2.0mg/kg/day 
CsA dose starting month 6 for 18 months until month 24 on the rate of relapse of subjects deemed 
responders at month 6.
Subjects will be followed up to 60 months so that long-term disease-free and overall survival can be 
estimated.  Response and toxicity comparisons will also be made with the results obtained from prior 
NHLBI studies:  the h-ATG/CsA protocol (90-H-0146); the h-ATG/CsA vs. Cytoxan/CsA protocol (97-H-
0117); the h-ATG/CsA/MMF protocol (00-H-0032); the h-ATG/CsA/Rapamune protocol (03-H-0193); the 
3-arm randomized study (06-H-0034) and the r-ATG vs. alemtuzumab (03-H-0249) trials.
9.5
Stopping Rules
Evolution to clonal hematopoiesis will be monitored and documented in this study.  Currently, there are no 
standards to determine if evolution to clonal hematopoiesis with respect to this study is a serious adverse 
event or treatment related serious adverse event.  Therefore, evolution to clonal hematopoiesis will not be 
included in stopping rules.  Currently, none of the active SAA NHLBI Hematology Branch treatment 
protocols includes evolution to clonal hematopoiesis as a stopping rule, including all protocols 
administering eltrombopag.
Cohorts 1, 2 & 3
The study will be monitored to ensure that the occurrence of a specified set of treatment related serious 
adverse events (TRSAEs) within 6 months of treatment in the trial does not substantially exceed an 
anticipated rate.  The following TRSAEs will be monitored for early stopping of the study:

Death considered to be probably or definitely related to eltrombopag.

Any grade IV toxicity considered to be probably or definitely related to eltrombopag.

Grade IV thrombosis/embolism
The study will be monitored using the stopping rules as outlined below for early stopping if the number of 
subjects in the study who have developed one or more of the above specified TRSAEs is over a pre-specified 
threshold value.  TRSAEs are those attributed as definitely or probably related to eltrombopag.  Dr. John 
Tisdale will serve as the independent monitor who reviews the attribution of TRSAEs.
From our experience using this agent in other clinical settings, we anticipate the rate of developing at least 
one of the above specified TRSAEs for this patient population to be 20% or less.  Following Geller et al. 
2004, (Nancy L. Geller, Dean Follmann, Eric S. Leifer, and Shelly L. Carter “Design of Early Trials in 
Stem Cell Transplantation:  A Hybrid Frequentist-Bayesian Approach” Chapter 2, pp 41-52, Advances in 
Clinical Trial Biostatistics, 2004, Marcel Dekker: New York), our stopping rule is determined by a Bayesian 
approach.  The stopping boundary for the study is reached if the Bayesian posterior probability that the true 
probability of developing one or more of the above specified TRSAEs exceeds this benchmark rate of 20% 
is at least 90%.  We take our prior distribution to be a beta distribution with the sum of the two beta 

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Neal S. Young, M.D.  3/18/2020 (Amendment UU/47)
parameters to be 3, i.e. the parameters of the beta prior distribution are 0.60 and 2.40.  Since we have seen 
in the past that the first few subjects to be accrued are possibly sicker than the rest of the subjects in the 
sample, we will start safety monitoring the study when 3 or more subjects have developed specified 
TRSAEs.  The following tables summarize the threshold numbers for stopping the study:
Number of Tx naïve SAA 
subjects enrolled
Stop if the number of Tx naïve SAA subjects who 
develop any of the specified TRSAEs reaches:
≤ 6
3
≤ 9
4
≤ 13
5
≤ 17
6
≤ 21
7
≤ 25
8
≤ 29
9
≤ 31
10
≤ 37
11
≤ 41
12
≤ 45
13
≤ 49
14
≤ 54
15
For the stopping rule we generated a study with 25 independent Bernoulli trials, each had a probability p 
for having the above TRSAE and q=1-p for not having such TRSAE and compared the TRSAE outcomes 
with the above stopping boundary to determine whether the study was stopped.  We repeated the simulation 
100,000 times and computed the proportion of stopped studies (i.e. “number of stopped studies”/100,000), 
which were stopped using the above stopping rule.  The following table summarizes the proportions of 
stopped studies under a number of scenarios for p:
Probability of 
“Monitored” TRSAE= p
5%
10%
15%
20%
25%
40%
Proportion of stopped 
studies
0.27%
2.45%
9.19%
22.57%
41.44%
89.81%
Average number of Tx 
naive SAA subjects
24.95
24.58
23.59
21.78
19.30
11.02
Average number of Tx 
naive SAA with a 
specified TRSAE
1.25
2.47
3.53
4.35
4.83
4.41
These results suggest that the above stopping rules have a low probability of stopping a study when the 
proportion of the corresponding specified TRSAE is below the benchmark value of 20%, and the probability 
of stopping a study is high when the true proportion of the above specified TRSAE exceeds this benchmark 
value.  Based on these results, we believe that our Bayesian stopping rules have satisfactory statistical 
properties.
Extension Cohort
Following Amendment FF, we updated the TRSAE stopping rule for this expanded cohort of N=87 patients 
(55 patients in the original cohort, 32 additional patients) the TRSAE stopping rule is updated using the 

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same Bayesian approach (Geller et al., 2004) with the same assumptions.  Specifically, the assumptions 
are:
a)
The rate of developing one or more of the specified TRSAEs is 20% or less.
b)
The stopping boundary is reached if the Bayesian posterior probability that the true probability of 
developing one or more of the specified TRSAEs exceeds this benchmark rate of 20% is at least 
90%.
c)
The prior distribution is a beta distribution with the sum of the two beta parameters to be 3, i.e. the 
parameters of the beta prior distribution are 0.60 and 2.40.
d)
The safety monitoring starts when 3 or more subjects have developed the specified TRSAEs.
The following tables summarize the threshold numbers for stopping the study:
Number of Tx naïve SAA 
subjects enrolled
Stop if the number of Tx naïve SAA subjects who 
develop any of the specified TRSAEs reaches:
≤ 6
3
≤ 9
4
≤ 13
5
≤ 17
6
≤ 20
7
≤ 24
8
≤ 29
9
≤ 33
10
≤ 37
11
≤ 41
12
≤ 45
13
≤ 49
14
≤ 54
15
≤ 58
16
≤ 62
17
≤ 66
18
≤ 71
19
≤ 75
20
≤ 80
21
≤ 84
22
≤ 87
23
We performed a simulation study to verify the appropriateness of this stopping rule. In each simulation run, 
we generated a study with 87 independent Bernoulli trials, each had a probability p for having the above 
TRSAE and q=1-p for not having such TRSAE and compared the TRSAE outcomes with the above 
stopping boundary to determine whether the study was stopped.  We repeated the simulation 100,000 times 
and computed the proportion of stopped studies (i.e. “number of stopped studies”/100,000), which were 
stopped using the above stopping rule.  The following table summarizes the proportions of stopped studies 
under several different scenarios for the true value of p:
Probability of 
“Monitored” TRSAE= p
5%
10%
15%
20%
25%
40%

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Neal S. Young, M.D.  3/18/2020 (Amendment UU/47)
Proportion of stopped 
studies
0.27%
2.38%
10.35%
32.83%
66.73%
99.90%
Average number of Tx 
naive SAA subjects
86.78
86.13
79.57
66.31
47.15
12.58
Average number of Tx 
naive SAA with a 
specified TRSAE
4.34
8.50
11.93
13.27
11.79
5.04
These results suggest that the above stopping rule has a low probability of stopping a study when the 
proportion of the corresponding specified TRSAE is below the benchmark value of 20%, and the probability 
of stopping a study is high when the true proportion of the above specified TRSAE exceeds this benchmark 
value.  Based on these results, we believe that our Bayesian stopping rule has satisfactory statistical 
properties.
9.6
Off Study/Off Treatment Criteria
Subject choice:  Subjects may withdraw from the study at any time.  The risks of withdrawing will be 
discussed, as will alternative treatment options.  The subject will be informed that his/her condition poses 
a high mortality rate.  The risks of not receiving further therapy include a high mortality rate with supportive 
care only.  Those subjects who choose to withdraw will be strongly encouraged to continue to have blood 
counts monitored until initiation of alternative SAA therapy or through the 6 month off study medication 
to assess for late occurring adverse events.
Principal investigator decision:  Should any of the following events occur, study drug administration will 
be discontinued.  The subject will be followed until resolution of the event and laboratory values monitored 
through 6 months or until initiation of alternative SAA therapy:
•
Intolerance of eltrombopag not resolved by dose reduction
•
Thrombosis/embolism (DVT, PE, stroke or TIA, myocardial infarction) other than central line 
thrombosis
•
Persistent hepatotoxicity as defined in section 5.3
•
Infusion-related h-ATG reactions refractory to all appropriate supportive measures
•
Life threatening acute hypersensitivity reactions
•
Pregnancy or unwillingness to refrain from pregnancy
•
Initiation of additional immunosuppressive therapy other than steroids or cyclosporine (if beyond 
6 months then mycophenolate mofetil can also be administered per PI discretion in patients who 
cannot tolerate cyclosporine and who require immunosuppression) or if beyond 2 years, sirolimus 
per protocol 17-H-0019
•
Evidence of a clonal disorder identified in subjects with super severe baseline neutropenia (ANC < 
200/μL) who were not initially excluded because cytogenetics was not available or pending.
There are risks to pregnancy for patients with aplastic anemia or a history of aplastic anemia, and it
is generally advised against, regardless of participation in a research trial.  For this protocol, if
pregnancy occurs when a patient is not actively receiving medical drugs, it will not be cause for
withdrawal from study.
Completion of protocol participation:  After 5 years, protocol participation will be deemed complete. Once 
off study, subjects will be referred back to the referring physician or consented to the Hematology Branch 
evaluation and treatment protocol (94-H-0010) or evaluated for eligibility for another Branch protocol, 
depending on the wishes of the subject and availability of appropriate additional clinical trials at the NIH.

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Death:  Death of Subject
9.7
Data Management
The Principal Investigator will be responsible for overseeing entry of data into an in-house password 
protected electronic system and ensuring data accuracy, consistency and timeliness.  The Principal 
Investigator, associate investigators, Hematology Branch fellows, research nurses and/or a contracted data 
manager will assist with the data management efforts.  Data will be abstracted from Clinical Center progress 
notes as well as from progress notes forwarded from home physician. Laboratory data from NIH will be 
imported electronically from CRIS into an in-house clinical trial database.  Laboratory values from referring 
home physicians will be entered into the system.  Identifiable data will not be sent outside of the NIH 
without IRB approval and an executed agreement.
All human subjects personally identifiable information (PII) will be recorded according to the NIH policy.  
Primary data obtained during the conduct of the protocol will be kept in secure network drives or in 
approved alternative sites that comply with NIH security standards.  Primary and final analyzed data will 
have identifiers so that research data can be attributed to an individual human subject participant, e.g., 
unique code, or minimum PII required for subject identification.  Study data will be housed in the 
Hematology Branch P Drive, a secure limited access drive.
Novartis will receive quarterly accrual and toxicity information as detailed in the CRADA.  In order to 
maintain subject confidentiality, all communications relating to the study will identify participants by 
assigned subject study numbers.  No personally identifiable information will be sent to Novartis.  In 
accordance with local and federal regulations, the Investigator will allow Novartis personnel or their 
designee, access to all pertinent medical records in order to verify the data gathered and to audit the data 
collection process.
The US Food and Drug Administration (FDA) may also request access to all study records, including 
source documentation for inspection.
End of study procedures:  Data will be stored in locked cabinets and in a password protected database 
until it is no longer of scientific value.
Loss or destruction of data:  Should we become aware that a major breech in our plan to protect subject 
confidentiality and trial data has occurred, the IRB will be notified.
10. DATA AND SAFETY MONITORING
10.1
Safety Monitoring
Principal Investigator:  Accrual, efficacy and safety data will be monitored by the Principal Investigator, 
Neal S. Young, M.D.
NIH Intramural IRB: Prior to implementation of this study, the protocol and the proposed patient consent 
and assent forms will be reviewed and approved by the properly constituted Institutional Review Board 
(IRB) operating according to Title 45 CFR 46.  This committee will also approve all amendments to the 
protocol or informed consent, and conduct continuing annual review so long as the protocol is open to 
accrual, in follow up of subjects or in data analysis.

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NHLBI DSMB:  The NHLBI Data Safety and Monitoring Board will review the protocol at 6 to 12 month 
intervals.  A progress report will be forwarded to the DSMB at these times and their recommendations will 
be expeditiously implemented.  The DSMB may recommend early termination of the study for 
considerations of safety and efficacy.
FDA: An annual progress report, any amendments to the protocol, and any change in the status of the 
protocol will be forwarded to the FDA:
CDER Therapeutic Biological Products Document Room
Center for Drug Evaluation and Research, Food and Drug Administration
5901 B Ammendale Road, Beltsville, MD 20705-1266
(301) 796-0683
Novartis:  An annual progress report, any amendments to the protocol, and any change in the status of the 
protocol will be forwarded to:
Kelly Haines
Clinical Research Manager
Novartis Pharmaceuticals Corporation
One Health Plaza
East Hanover, NJ 07936-1080
USA
Phone    +1 862 778 3640
Mobile    +1 201 452 8479
Fax         +1 973 781 2116
kelly.haines@novartis.com
10.2
Event Characterization and Reporting
Events include Adverse Events (AE), Serious Adverse Events (SAE), Protocol Deviations (PD), 
Unanticipated Problems (UP), and non-compliance.  The characterization (e.g., seriousness, expectedness, 
etc.) of the event will determine the reporting requirements.
The principal investigator will review all events (AEs, PDs, UPs, SAEs) to determine the seriousness, 
expectedness, and reportability of the event.  As required and/or needed, the principal investigator will 
review the events with the Sponsor to make the final determination of seriousness and reportability.
10.2.1 Assessment of Safety
Definitions
Please refer to Policy 801 “Reporting Research Events” for current definitions.
10.2.2 Protocol Deviation (PD) Characterization
It is anticipated that approximately 50% of the clinical monitoring laboratory testing performed during the 
first 6 months will not be performed within +/- 3 days of the required time points, with the exception of the 
month 3 and 6 landmark visits.  If the clinical laboratory monitoring occurs outside the +/- 3 day window, 
these events will be captured in the database.  If the number of events per subject or per cumulative enrolled 
subjects exceeds 50%, which is a frequency greater than what is expected, this will be reported at time of 
continuing review.  Please note, subjects and providers are reminded of the importance of the timely 

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completion of the clinical laboratory testing.
Questionnaires that are not completed at the required time point due to clinical status (e.g. critically ill), 
will be recorded in the database.
Interruptions in eltrombopag dosing that are clinically indicated per section 5.3, will be recorded in the 
medical record.   
In addition, CsA interruptions, such as delay in request for medication refills or medication errors by 
subjects, will be recorded in the medical record.
10.2.3 Adverse Events (AEs) Characterization
Non-hematologic abnormal laboratory findings used to evaluate the safety of this protocol regimen will 
include any change from laboratory assessments done prior to first dose of study medication that result in 
a progression to a grade 3 or 4 laboratory toxicity or are characterized by any of the following:
•
Results in discontinuation from the study
•
Is associated with clinical signs or symptoms
•
Requires treatment or any other therapeutic intervention
•
Is associated with death or another serious adverse event, including hospitalization
•
Is judged by the Investigator to be of significant clinical impact
•
If any abnormal laboratory result is considered clinically significant, the investigator will provide 
details about the action taken with respect to the test drug and about the patient’s outcome.
The AEs will be attributed (unrelated, unlikely, possibly, probably or definitely related) to study medication 
and/or disease.  The AEs will be graded by severity utilizing CTCAE version 4.0.
Duration of adverse event collection:  The collection and recording  of events in the database will begin 
on the first day of initiation of the study drug and will continue for 30 days after the 6 month landmark visit 
or for 30 days after treatment is discontinued if before the 6 month landmark visit, after which the events 
will be captured in the medical record but not abstracted or recorded in the database or toxicity tables.
AEs that are captured in the database will be followed until satisfactory resolution as long as the subject 
remains on study.
Exclusions to AE data capturing:
•
In view of the underlying illness, severe aplastic anemia all patients will enter the study with 
abnormally low blood counts that would meet criteria as grade 3 or more commonly grade 4 
toxicity, and requiring frequent platelet and/or red cell transfusions, and thus AEs regarding 
hematologic lab values including thrombocytopenia or platelet-transfusion dependence, anemia or 
red cell transfusion dependence, neutropenia, lymphopenia, or leukopenia will not be captured in 
the research database.  Thus, we will collect hematologic laboratory values in the subject’s source 
documents, but will not record or report these abnormalities as adverse events in the research 
database.
•
In addition, the following non-hematologic AEs will be captured only in the source documents and 
will not be recorded in the database:
o
Because CsA, h-ATG (ATGAM®) and eltrombopag (Promacta®) are FDA approved drugs 
with known toxicity profiles, any observed or volunteered adverse events that are listed on 
the package insert will not be captured in the research database unless (1) the adverse event 
is more severe or occurs at a higher frequency than on the package insert; or (2) meets the 

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criteria for a serious adverse event.  The collection of AEs information will begin on the 
first day of initiation of therapy.
•
Cohorts 1 and 2:  Because grade 3 and 4 laboratory abnormalities which do not result in any clinical 
action frequently occur during h-ATG administration, only those events that result in a clinical 
action (i.e., dose reduction/discontinuation, prolongation of hospitalization, etc.) will be recorded 
in the database during the first week of h-ATG treatment.
•
Cohort 3 the Extension Cohort, and Laboratory Exploratory Cohort:  Because grade 3 and 4 adverse 
events which do not result in any clinical action frequently occur during h-ATG administration, 
only those events that result in a clinical action (i.e., dose reduction/discontinuation, prolongation 
of hospitalization, etc.) will only be recorded in the database from the start of h-ATG to 14 days 
post-treatment.  The exception to this will be if the event, which did not result in a clinical action, 
can be associated with both eltrombopag and h-ATG, and the event attribution cannot be assigned 
with a high level of certainty to only h-ATG, then these events will be recorded in the database and 
reported to the IRB per Policy 801.
•
Cohort 3, the Extension Cohort, and Laboratory Exploratory Cohort:  Grade 3 and 4 LFTs will be 
recorded in the medical record, and only recorded in the database as adverse events from the start 
of h-ATG to 14 days post treatment.
•
All grade 1 events listed as expected in the protocol, consent forms, or other applicable protocol 
documentation.
10.2.4 Serious Adverse Events Characterization
Serious adverse events will be attributed as definitely (clearly related to the research), probably (likely 
related to the research), possibly (may be related to the research), unlikely (doubtfully related to the 
research) and unrelated (clear not related to the research).
TRSAEs are those attributed as definitely or probably.  As detailed in section 9.5 stopping rules, TRSAE 
that will be monitored and considered for early stopping the study according to statistically determined 
criteria include Death and any grade IV toxicity considered to be probably or definitely related to study 
medication.
Hospitalizations for administrative issues (e.g., to receive a transfusion after hours) or movement to the 
ICU for routine monitoring per administrative requirements or nosocomial isolation will be captured in the 
database.
Duration of Serious Adverse Event collecting and reporting:  The collection or recording of SAEs in 
the database will begin on the first day of initiation of the study drug and will continue as long as the subject 
is on study.
Serious adverse event recording for events that are unexpected and/or definitely, probably or possibly 
related to the study drug will continue as long as the subject remains on study.
10.2.5 Event Reporting
All events will be reported to Neal S. Young, M.D., Principal Investigator of this study:
Neal S. Young, M.D.
Bldg. 10, Room CRC 3E-5142
Phone:  (301) 496-5093
E-mail:  youngns@nhlbi.nih.gov

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10.2.5.1
NIH Intramural IRB and NHLBI Clinical Director (CD) Reporting
Expedited Reporting
Events requiring expedited reporting will be submitted to the IRB per HRPP Policy 801 “Reporting Research 
Events”.
Reports to the IRB at the time of continuing review:
The PI or designee will refer to HRPP Policy 801 “Reporting Research Events” to determine IRB reporting 
requirements.
Reports to the CD:
The PI or designee will refer to NHLBI DIR guidelines to determine CD reporting requirements.
In addition, the following commonly occurring disease specific Serious Adverse Events (SAEs) will be 
captured in the database but are not required to be reported to the CD per NHLBI guidelines:
•
Neutropenic fever
•
Minimal bleeding (such as nose bleed or menorrhagia)
•
Transfusion reactions to red blood cells and/or platelets
10.2.5.2 NHLBI DSMB Reporting
Reports of serious adverse events that are unexpected and suspected will also be forwarded as soon as 
possible, but no later than seven (7) days in the case of death or life-threatening serious adverse events or 
within fifteen (15) days after the occurrence of all other forms of serious adverse events to the Data and 
Safety Monitoring Board (DSMB).  All events will be included in DSMB reports for review by the DSMB.
10.2.5.3 Sponsor and FDA Reporting
IND: 104877
IND Sponsor Representative:  Cynthia E. Dunbar, MD
The PI will report SAEs to the Sponsor according to the requirements of 21 CFR 312.64(b) and as agreed 
upon with the sponsor.  The Sponsor (or designee) will determine the reportability of the event to the FDA 
and IND safety report will be submitted to the FDA as required.
IND Safety Reports to the FDA (Refer to 21 CFR 312.32)
The sponsor must notify the FDA in an IND safety report of potential serious risks, from clinical trials or 
any other source, as soon as possible, but in no case later than 15 calendar days after the sponsor determines 
that the information qualifies for reporting.  The sponsor must also notify FDA of any unexpected fatal or 
life-threatening suspected adverse reaction as soon as possible but in no case later than 7 calendar days after 
the sponsor's initial receipt of the information.
15-day reporting
The sponsor must report any suspected adverse reaction that is both serious and unexpected.  The sponsor 
must report an adverse event as a suspected adverse reaction only if there is evidence to suggest a causal 
relationship between the drug and the adverse event, such as:

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•
A single occurrence of an event that is uncommon and known to be strongly associated with drug 
exposure (e.g., angioedema, hepatic injury, Stevens-Johnson Syndrome);
•
One or more occurrences of an event that is not commonly associated with drug exposure, but is 
otherwise uncommon in the population exposed to the drug (e.g., tendon rupture);
An aggregate analysis of specific events observed in a clinical trial (such as known consequences of the 
underlying disease or condition under investigation or other events that commonly occur in the study 
population independent of drug therapy) that indicates those events occur more frequently in the drug 
treatment group than in a concurrent or historical control group
The sponsor must submit each IND safety report in a narrative format or on FDA Form 3500A.
FDA contact:
CDER Therapeutic Biological Products Document Room
Center for Drug Evaluation and Research, Food and Drug Administration
5901 B Ammendale Road, Beltsville, MD 20705-1266
(301) 796-0683
A summary of all SAEs, non-serious AEs, and other events will be recorded and submitted to the Sponsor and 
FDA in annual progress reports (21 CFR 312.64(b)).
10.2.5.4 Reporting Serious Adverse Events to CRADA Sponsor
Novartis:
All unexpected and possibly, probably or definitely related SAEs occurring during the study or within 30 
days of the last administration of eltrombopag will be reported to Novartis within 24 hours of the research 
team learning of the event.  A copy of the SAE report will be forwarded as soon as possible, but no later 
than seven (7) days in the case of death or life-threatening serious adverse events or within fifteen (15) days 
after the occurrence of all other forms of serious adverse events.  If the SAE is unexpected and determined 
possibly, probably or definitely related to the study drug the SAE report (in the appropriate format, e.g., 
NIH Reportable Events Form, narrative, and/or MedWatch form, Appendix A) will be forwarded to 
Novartis and FDA within 24 hours of learning of event.  Follow-up reports regarding the subject’s 
subsequent course will be submitted until the SAE has resolved or until the subject’s condition stabilizes 
(in the case of persistent impairment) or the subject dies.  The SAE report will contain full written summary 
detailing relevant aspects of the adverse events in question.  Where applicable, information from relevant 
hospital case records and autopsy reports will be included.  The investigator will always provide an 
assessment of causality at the time of the initial report as described in ‘Assessment of Causality.’
10.3
Reporting of Pregnancy
Subjects who become pregnant during the study should discontinue the study drugs (CsA and eltrombopag) 
immediately.  The investigator, or his/her designee, will collect pregnancy information on any subject who 
becomes pregnant while participating in this study.
The investigator, or his/her designee, will submit pregnancy information to the NHLBI Clinical Director, 
the IRB and Novartis within two weeks of learning of a subject’s pregnancy.  Information on the status of 
the mother and child will be forwarded to Novartis.  Generally, follow-up will be no longer than 6 to 8 
weeks following the estimated delivery date.  Any premature termination of the pregnancy will be reported.  
While pregnancy itself is not considered to be an AE or SAE, any pregnancy complication or elective 
termination of a pregnancy for medical reasons will be recorded and reported to Novartis as an AE or SAE.  

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A spontaneous abortion is always considered to be an SAE and will be reported to Novartis.  Furthermore, 
any SAE occurring as a result of a post-study pregnancy and is considered reasonably related to the 
investigational product by the investigator, will be reported to Novartis.  While the investigator is not 
obligated to actively seek this information in former study participants, he/she may learn of an SAE through 
spontaneous reporting.
10.4
Protocol Monitoring
As per ICH-GCP 5.18 and FDA 21 CFR 312.5 clinical protocols are required to be adequately monitored 
by the study sponsor.  The monitoring of this study will be conducted by Clinical Research Associates 
(CRAs)/Monitors working under an agreement with NHLBI to monitor aspects of the study in accordance 
with the appropriate regulations and the approved protocol.  The objectives of a monitoring visit will be: 1) 
to verify the existence of signed informed consent form (ICF) and documentation of the ICF process for 
each monitored subject; 2) to verify the prompt and accurate recording of all monitored data points, and 
prompt reporting of all SAEs; 3) to compare abstracted information with individual subjects’ records and 
source documents (subject’s charts, laboratory analyses and test results, physicians’ progress notes, nurses’ 
notes, and any other relevant original subject information); and 4) to help ensure investigators are in 
compliance with the protocol.  The monitors also will inspect the clinical site regulatory files to ensure that 
regulatory requirements (Office for Human Research Protections-OHRP), FDA and applicable guidelines 
(ICH-GCP) are being followed.  During the monitoring visits, the investigator (and/or designee) and other 
study personnel will be available to discuss the study progress and monitoring visit.
The investigator (and/or designee) will make study documents (e.g., consent forms and pertinent hospital 
or clinical records) readily available for inspection by the local IRB, the FDA, the site monitors, and the 
NHLBI staff for confirmation of the study data.
11. HUMAN SUBJECT PROTECTION
11.1
Rationale for Subject Selection
The study will be open to all subjects who satisfy the inclusion criteria and provide an informed consent to 
the protocol.  No subjects will be excluded from participation based on gender, race or ethnicity.
This study will be open to all patients who fit the inclusion criteria and provide informed consent to protocol 
participation.  Epidemiologic studies suggest that the gender will be approximately evenly split between 
male and females, and that 90% of the patients will be Caucasian.  However, previous experience at our 
institution suggests that distribution will be:
by gender: 60% males and 40% females;
by race/ethnicity:  approximately 55% White, 15% Black, 6% Asian and 24% Hispanic;
by age:  will range between 2 and 82 (median age of 30) and roughly 20% of patients will 
be under the age of 18.
For subjects of Asian ethnicity:  Plasma eltrombopag area under the curve was approximately 70% higher 
in East and South East Asian (ethnicity self-reported) subjects as compared to non-Asian subjects who were 
predominantly Caucasian.  Therefore, subjects of Asian heritage will be included but they will be initiated 
at a lower dose and monitored closely as described in the treatment plan.
For subjects with renal impairment:  The pharmacokinetics of eltrombopag has been studied in adult 
patients with renal impairment.  Following administration of a single 50 mg dose, there was a trend for 
reduced plasma eltrombopag exposure in patients with renal impairment, but there was substantial 
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volunteers.  Therefore, patients with impaired renal function will be included and given the protocol-defined 
dosages, but participation will be monitored closely.
For subjects with hepatic impairment:  Pharmacokinetics of eltrombopag has been studied in adult 
patients with hepatic impairment. Following the administration of a single 50 mg dose, the AUC0-∞ of 
eltrombopag was increased by 41% in subjects with mild hepatic impairment and by 80% to 90% in subjects 
with moderate or severe hepatic impairment compared with healthy volunteers.  Therefore, patients with 
minimally impaired hepatic function will be included but participation will be monitored closely.  Patients 
with baseline moderate to severe hepatic impairment will be excluded from the study.
Recruitment efforts:  The study will be listed on the clinicaltrials.gov, Clinical Center research studies, 
and may be listed on The Aplastic Anemia Foundation, and the National Heart, Lung and Blood Institute 
patient recruitment websites.  If recruitment goals are not met, a recruitment plan will be developed by the 
Clinical Center Office of Patient Recruitment.  Hematologists and Oncologists throughout the country will 
be informed about the protocol by letter.  Because many aplastic anemia patients may respond to initial 
immunosuppressive treatment with a response that is sufficient to prevent serious infections, but have 
persistent thrombocytopenia, we will also be able to rapidly recruit study patients who have completed 
other trials for aplastic anemia therapy within the Branch.
Reimbursement for protocol travel, food, and lodging will be consistent with NIH guidelines.  In 
determining reimbursement, the following factors are considered applicable to this protocol: the patients 
are diagnosed with a rare disease; the patient population is sick; the protocol offers the potential for direct 
benefit; the protocol regimen is demanding; and in order to complete accrual in a reasonable timeframe a 
geographically dispersed participant population is required.
Payment for participation:  $0. The study participants will not be reimbursed for their time and 
inconvenience.
Competition between Branch Protocols:  There are no competing Branch protocols for this patient 
population.
11.2
Participation of Children
In principle, age is not a consideration.  But in practice, we are limiting the protocol to subjects who are 
age 2 years and older because our clinic does not have the expertise to care for infants.  In addition, per 
Clinical Center guidelines, we are limiting participation to children who weigh >12 kg.
11.3
Exclusion of Pregnant Women and Nursing Mothers
Eltrombopag was not teratogenic when studied in pregnant rats and rabbits but caused a low incidence of 
cervical ribs (a fetal variation) and reduced fetal body weight at doses that were maternally toxic.  There 
are no adequate and well-controlled studies of eltrombopag in pregnant women.  The effect of eltrombopag 
on human pregnancy is unknown.  Therefore, women of childbearing potential must agree to use adequate 
contraception prior to (hormonal or barrier method of birth control; abstinence) and for the duration of study 
participation.  If a woman becomes pregnant or suspects she is pregnant while on study, her treating 
physician should be informed immediately.
There are risks to pregnancy for patients with aplastic anemia or a history of aplastic anemia, and it
is generally advised against, regardless of participation in a research trial.  For this protocol, if
pregnancy occurs when a patient is not actively receiving medical drugs, it will not be cause for
withdrawal from study. In the event of relapse during pregnancy, patient will not be retreated on the protocol

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but recommendations will be provided to the local treating physician.
11.4
Risks and Discomforts
11.4.1 Related to Horse ATG (ATGAM)
Potential Serious Adverse Effects:
Anaphylaxis (less than 1% of patients): Rarely, patients may develop potentially fatal anaphylaxis.  
Production by the patient of antibodies to horse proteins leads to the formation of immune complexes and 
the clinical development of serum sickness, characterized by fever, a characteristic rash, arthralgia, 
myalgia and non-specific gastrointestinal and neurologic symptoms.  Onset is typically at day ten to eleven, 
and the course is self-limited; symptoms may be improved by corticosteroids.  Transient reduction in 
peripheral granulocyte and platelet counts and in the hemoglobin may occur during the period of 
administration of ATG and may lead to a temporary increase in transfusion requirements.
Severe lung injury (less than 1% of patients):  Several cases of a severe lung injury related to Atgam 
treatment have been reported. Although this side effect appears extremely rare, it is serious and can be 
fatal. There is no information about the mechanism or specific treatment for this condition. A few patients 
recovered after intensive medical support including use of a breathing machine.
Cardiac Failure and Pulmonary Edema (less than 5% of patients):  h-ATG is associated with pulmonary 
edema or congestive heart failure
Potential side effects related to h-ATG (ATGAM) include:
Very common side effects (occurring in 10% or more of patients): fever (51%), chills (16%), 
thrombocytopenia (30%), leukopenia (14%), skin rash (27%)
Common side effects (occurring in 5 to 10% of patients): serum sickness like symptoms, dyspnea/apnea, 
arthralgia, headache, chest, back or flank pain, diarrhea and nausea and/or vomiting,
Events reported with frequency of less than 5% of patients in a pre-marketing clinical trial in the treatment 
of aplastic anemia include: diaphoresis, joint stiffness, peri-orbital edema, aches, edema, muscle ache, 
vomiting, agitation, lethargy, listlessness, light-headedness, seizures, diarrhea, bradycardia, myocarditis, 
cardiac irregularity, hepatosplenomegaly, post viral encephalopathy, hypotension, congestive heart failure, 
hypertension, burning soles/palms, foot sole pain, lymphadenopathy, post cervical lymphadenopathy, 
tender lymph nodes, pleural effusion, respiratory distress, and proteinuria.
Related to pregnancy and/or nursing mothers: h-ATG has not been evaluated in either pregnant or lactating 
women therefore administration of h-ATG to pregnant women is not recommended and should be 
considered only under exceptional circumstances.
Post marketing Experience:
During approximately 5 years of post-approval marketing experience, the frequency of adverse reactions in 
voluntarily reported cases is as follows: fever 51%; chills 16%; thrombocytopenia 30%; leukopenia 14%; rashes 
27%; systemic infection 13%.  Events reported in 5% to 10% of reported cases include abnormal renal function 
tests; serum sickness-like symptoms; dyspnea/apnea; arthralgia; chest, back, or flank pain; diarrhea and nausea 
and/or vomiting.  Events reported with a frequency of less than 5% include: hypertension, Herpes Simplex 
infection, pain, swelling or redness at infusion site, eosinophilia, headache, myalgias, or leg pains, hypotension, 
anaphylaxis, tachycardia, edema, localized infection, malaise, seizures, GI bleeding or perforation, deep vein 

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thrombosis, sore mouth/throat, hyperglycemia, acute renal failure, abnormal liver function tests, confusion or 
disorientation, cough, neutropenia or granulocytopenia, anemia, thrombophlebitis, dizziness, epigastric or 
stomach pain, lymphadenopathy, pulmonary edema or congestive heart failure, abdominal pain, nosebleed, 
vasculitis, aplasia or pancytopenia, abnormal involuntary movement or tremor, rigidity, sweating, 
laryngospasm/edema, hemolysis or hemolytic anemia, viral hepatitis, faintness, enlarged or ruptured kidney, 
paresthesias, and renal artery thrombosis.
11.4.2 Related to CsA:
Potential Serious Side Effects Include:
Infection related: Because of low white blood cell counts, patients with aplastic anemia are susceptible 
to infections.  By further blocking the immune system, CsA further increases this risk.  
Cancer related:  When used at high doses in transplant patients, CsA may be associated with an 
increased risk of cancer, especially lymphoma (4 of every 10,000 patients who receive the medication).  
Transplant patients receive higher doses than you will be given and are treated for longer periods than 
the duration of this study.  However, because of the way that CsA acts on the body, there is a chance 
that it may cause effects that may not occur until years after the medicine is used
Blindness: In very rare instances (less than .01%), CsA has been reported to cause blindness
Potential side effects:
Although it is metabolized primarily in the liver, CsA major toxicity is renal.  CsA causes a decrease in 
creatinine clearance, which almost always returns to normal range on cessation of the drug or lowering of 
the dose.  Rare development of a hemolytic-uremic syndrome has been reported in patients with CsA after 
allogeneic bone marrow transplant.  In our patients with SAA, frequent creatinine measurements have 
allowed prompt adjustment of dose and serious renal complications are infrequent.
Evidence of hepatotoxicity is common, usually as transient increases in bilirubin and transaminases.  These 
levels often normalize with continued administration of the drug; reduction of the dose is uniformly 
associated with a return to normal levels.
Additional complications include hypertrichosis, gingival hypertrophy (possibly related to pre-existing 
poor dental hygiene), hyperesthesia, hirsutism, tremors, headaches, nausea and nonspecific gastrointestinal 
complaints.  Hypertension may occur, and be high enough to require treatment.
Neurologic complications include insomnia, dizziness, anxiety, confusion, and vertigo. We have observed 
seizures in patients receiving CsA, when drug levels were within the therapeutic range.  Posterior Reversible 
Encephalopathy Syndrome (PRES) is an increasingly recognized neurologic disorder seen in 1% of patients 
on cyclosporine following solid organ transplantation which manifest with acute to subacute hypertension 
and/or seizures.77  In the event of hypertension, subjects will be prescribed 1 or more medications to control 
blood pressure in an effort to decrease the risk of this complication.71
Hypomagnesemia and hyperkalemia may occur but are asymptomatic.  Increases in uric acid may occur 
and attacks of gout have been rarely reported. Cyclosporine therapy may be associated with a modest 
increase of serum triglycerides or cholesterol.

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Less frequent adverse events include:
Autonomic Nervous System:  dry mouth, increased sweating
Systemic:  allergy, asthenia, hot flushes, malaise, weight decrease, weight increase 
Cardiovascular:  abnormal heart sounds, cardiac failure, myocardial infarction, peripheral ischemia
Central and Peripheral Nervous System:   hypoesthesia, neuropathy, vertigo
Endocrine:  goiter
Gastrointestinal:  constipation, dysphagia, enanthema, eructation, esophagitis, gastric ulcer, gastritis, 
gastroenteritis, gingival bleeding, glossitis, peptic ulcer, salivary gland enlargement, tongue disorder, tooth 
disorder
Infection:  abscess, bacterial infection, cellulitis, folliculitis, fungal infection, herpes simplex, herpes zoster, 
renal abscess, moniliasis, tonsillitis, viral infection
Hematologic:  anemia, epistaxis, leukopenia, lymphadenopathy
Liver and Biliary System:  bilirubinemia
Metabolic and Nutritional:  diabetes mellitus, hyperkalemia, hyperuricemia, hypoglycemia
Musculoskeletal System:  arthralgia, bone fracture, bursitis, joint dislocation, myalgia, stiffness, synovial 
cyst, tendon disorder
Neoplasms:  breast fibroadenosis, carcinoma
Psychiatric:  anxiety, confusion, decreased libido, emotional lability, impaired concentration, increased 
libido, nervousness, paranoia, somnolence
Reproductive (Female):  breast pain, uterine hemorrhage
Respiratory System:  bronchospasm
Skin and Appendages:  abnormal pigmentation, angioedema, dermatitis, dry skin, eczema, nail disorder, 
pruritus, skin disorder, urticaria
Special Senses:   abnormal vision, cataract, conjunctivitis, deafness, eye pain, taste perversion, tinnitus, 
vestibular disorder, blindness
Urinary System:   abnormal urine, hematuria, increased BUN, micturition urgency, nocturia, polyuria, 
pyelonephritis, urinary incontinence
Prolonged low dose administration of CsA:
Patients who undergo solid organ transplantation, such as kidney transplantation, are given cyclosporine 
for life at a high dose (usually 5-10 mg/kg/day) whereas we are proposing to continue at low-dose, 
2mg/kg/day.  Cyclosporine is considered a weak immunosuppressant and few infectious complications are 
observed.  There is ample data on the use of cyclosporine from long-term studies where cyclosporine is 
used for solid organ transplantation.  Nephrotoxicity is the main toxicity, usually reversible, but is typically 
only observed when troughs over 200ng/mL are targeted.  Nephrotoxicity is rarely observed with low-dose 
cyclosporine and monitoring of levels is unnecessary.  The risk of relapse of severe aplastic anemia 
however, can be fatal, and this risk far exceeds the risks long term cyclosporine poses and is why many 
centers have adopted this approach.  We also did not adopt this approach at the start of the protocol because 
we were uncertain whether eltrombopag may actually reduce the risk of relapse and warrant this approach 
unnecessary.  However now we see from our maturing data that this is unlikely to be the case.68,69
11.4.3 Related to Promacta®(eltrombopag)
Potential Serious Adverse Effects:
WARNING: RISK FOR HEPATIC DECOMPENSATION IN PATIENTS WITH CHRONIC HEPATITIS 
C
RISK OF HEPATOTOXICITY
See full prescribing information for complete boxed warning

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In patients with chronic hepatitis C, PROMACTA in combination with interferon and ribavirin may 
increase the risk of hepatic decompensation.
PROMACTA may increase the risk of severe and potentially life-threatening hepatotoxicity. Monitor 
hepatic function and discontinue dosing as recommended.
Warnings and Precautions
Hepatic Decompensation in Patients with Chronic Hepatitis C
In patients with chronic hepatitis C, PROMACTA in combination with interferon and ribavirin may 
increase the risk of hepatic decompensation.  In two controlled clinical trials in patients with chronic 
hepatitis C and thrombocytopenia, ascites and encephalopathy occurred more frequently on the arm 
receiving treatment with PROMACTA plus antivirals (7%) than the placebo plus antivirals arm (4%). 
Patients with low albumin levels (less than 3.5 g/dL) or Model for End-Stage Liver Disease (MELD) score 
greater than or equal to 10 at baseline had a greater risk for hepatic decompensation on the arm receiving 
treatment with PROMACTA plus antivirals.  Discontinue PROMACTA if antiviral therapy is discontinued.
Hepatotoxicity
PROMACTA may increase the risk of severe and potentially life-threatening hepatotoxicity. Measure 
serum ALT, AST, and bilirubin prior to initiation of PROMACTA, every 2 weeks during the dose 
adjustment phase, and monthly following establishment of a stable dose.  PROMACTA inhibits UDP-
glucuronosyltransferase (UGT)1A1 and organic anion-transporting polypeptide (OATP)1B1, which may 
lead to indirect hyperbilirubinemia. If bilirubin is elevated, perform fractionation.  Evaluate abnormal serum 
liver tests with repeat testing within 3 to 5 days. If the abnormalities are confirmed, monitor serum liver 
tests weekly until resolved or stabilized.  Discontinue PROMACTA if ALT levels increase to greater than 
or equal to 3 x ULN in patients with normal liver function or greater than or equal to 3 x baseline (or greater 
than 5 x ULN, whichever is the lower) in patients with pre-treatment elevations in transaminases and are:
•
progressively increasing, or
•
persistent for greater than or equal to 4 weeks, or
•
accompanied by increased direct bilirubin, or
•
accompanied by clinical symptoms of liver injury or evidence for hepatic decompensation.
If the potential benefit for reinitiating treatment with PROMACTA is considered to outweigh the risk for 
hepatotoxicity, then consider cautiously reintroducing PROMACTA and measure serum liver tests weekly 
during the dose adjustment phase.  Hepatotoxicity may reoccur if PROMACTA is reinitiated. If liver test 
abnormalities persist, worsen, or recur, then permanently discontinue PROMACTA.
Isolated cases of severe liver injury were identified in clinical trials. The elevation of liver laboratory values 
occurred approximately three months after initiation of PROMACTA.  In all cases, the event resolved 
following PROMACTA discontinuation.
Thrombotic/Thromboembolic Complications
Thrombotic/thromboembolic complications may result from increases in platelet counts with 
PROMACTA.  Reported thrombotic/thromboembolic complications included both venous and arterial 
events and were observed at low and at normal platelet counts.
Consider the potential for an increased risk of thromboembolism when administering PROMACTA to 
patients with known risk factors for thromboembolism (e.g., Factor V Leiden, ATIII deficiency, 
antiphospholipid syndrome, chronic liver disease).  To minimize the risk for thrombotic/thromboembolic 
complications, do not use PROMACTA in an attempt to normalize platelet counts.  Follow the dose 
adjustment guidelines to achieve and maintain target platelet counts.

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In two controlled clinical trials in patients with chronic hepatitis C and thrombocytopenia, 3% (31/955) 
treated with PROMACTA experienced a thrombotic event compared with 1% (5/484) on placebo.  The 
majority of events were of the portal venous system (1% in patients treated with PROMACTA versus less 
than 1% for placebo).
In a controlled trial in patients with chronic liver disease and thrombocytopenia not related to ITP 
undergoing elective invasive procedures (N = 292), the risk of thrombotic events was increased in patients 
treated with 75 mg of PROMACTA once daily.  Seven thrombotic complications (six patients) were 
reported in the group that received PROMACTA and three thrombotic complications were reported in the 
placebo group (two patients).  All of the thrombotic complications reported in the group that received 
PROMACTA were portal vein thrombosis (PVT).  Symptoms of PVT included abdominal pain, nausea, 
vomiting, and diarrhea. Five of the six patients in the group that received PROMACTA experienced a 
thrombotic complication within 30 days of completing treatment with PROMACTA and at a platelet count 
above 200 x 109/L.  The risk of portal venous thrombosis was increased in thrombocytopenic patients with 
chronic liver disease treated with 75 mg of PROMACTA once daily for 2 weeks in preparation for invasive 
procedures.
Cataracts
In the three controlled clinical trials in adults with chronic ITP, cataracts developed or worsened in 15 (7%) 
patients who received 50 mg of PROMACTA daily and 8 (7%) placebo-group patients.  In the extension 
trial, cataracts developed or worsened in 11% of patients who underwent ocular examination prior to 
therapy with PROMACTA.  In the two controlled clinical trials in patients with chronic hepatitis C and 
thrombocytopenia, cataracts developed or worsened in 8% of patients treated with PROMACTA and 5% 
of patients treated with placebo.
Cataracts were observed in toxicology studies of eltrombopag in rodents.  Perform a baseline ocular 
examination prior to administration of PROMACTA and, during therapy with PROMACTA, regularly 
monitor patients for signs and symptoms of cataracts.
Clinical Experience:
For full information on clinical experience with eltrombopag in for the treatment of all approved 
indications, see PACKAGE INSERT.
Severe Aplastic Anemia:  In the single-arm, open-label trial, 43 patients with severe aplastic anemia received 
PROMACTA. Eleven patients (26%) were treated for greater than 6 months and 7 patients (16%) were treated for 
greater than 1 year.  The most common adverse reactions (greater than or equal to 20%) were nausea, fatigue, 
cough, diarrhea, and headache.
Adverse Reactions (≥10%) from One Open-label Trial in Adults with Severe Aplastic Anemia
Adverse Reaction 
PROMACTA
(n = 43)
(%)
Nausea 
33
Fatigue 
28
Cough 
23
Diarrhea 
21
Headache 
21
Pain in extremity 
19
Dyspnea 
14

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Pyrexia 
14
Dizziness 
14
Oropharyngeal pain 
14
Febrile neutropenia 
14
Abdominal pain 
12
Ecchymosis 
12
Muscle spasms 
12
Transaminases increased 
12
Arthralgia 
12
Rhinorrhea 
12
Rash was reported in 7% of patients.
In this trial, patients had bone marrow aspirates evaluated for cytogenetic abnormalities.  Eight patients had 
a new cytogenetic abnormality reported on therapy, including 5 patients who had complex changes in 
chromosome 7.
USE IN SPECIFIC POPULATIONS
Pregnancy
Pregnancy Category C
There are no adequate and well-controlled studies of eltrombopag use in pregnancy.  In animal reproduction and 
developmental toxicity studies, there was evidence of embryo lethality and reduced fetal weights at maternally 
toxic doses.  PROMACTA should be used in pregnancy only if the potential benefit to the mother justifies the 
potential risk to the fetus.
In an early embryonic development study, female rats received oral eltrombopag at doses of 10, 20, or 60 
mg/kg/day (0.8, 2, and 6 times, respectively, the human clinical exposure based on AUC in patients with ITP at 
75 mg/day and 0.3, 1, and 3 times, respectively, the human clinical exposure based on AUC in patients with 
chronic hepatitis C at 100 mg/day).  Increased pre- and post-implantation loss and reduced fetal weight were 
observed at the highest dose which also caused maternal toxicity.
Eltrombopag was administered orally to pregnant rats at 10, 20, or 60 mg/kg/day (0.8, 2, and 6 times, 
respectively, the human clinical exposure based on AUC in patients with ITP at 75 mg/day and 0.3, 1, and 3 
times, respectively, the human clinical exposure based on AUC in patients with chronic hepatitis C at 100 
mg/day).  Decreased fetal weights (6% to 7%) and a slight increase in the presence of cervical ribs were observed 
at the highest dose which also caused maternal toxicity.  However, no evidence of major structural malformations 
was observed.
Pregnant rabbits were treated with oral eltrombopag doses of 30, 80, or 150 mg/kg/day (0.04, 0.3, and 0.5 times, 
respectively, the human clinical exposure based on AUC in patients with ITP at 75 mg/day and 0.02, 0.1, and 0.3 
times, respectively, the human clinical exposure based on AUC in patients with chronic hepatitis C at 100 
mg/day).  No evidence of fetotoxicity, embryolethality, or teratogenicity was observed.
In a pre- and post-natal developmental toxicity study in pregnant rats (F0), no adverse effects on maternal 
reproductive function or on the development of the offspring (F1) were observed at doses up to 20 mg/kg/day (2 
times the human clinical exposure based on AUC in patients with ITP at 75 mg/day and similar to the human 
clinical exposure based on AUC in patients with chronic hepatitis C at 100 mg/day).  Eltrombopag was detected 
in the plasma of offspring (F1).  The plasma concentrations in pups increased with dose following administration 
of drug to the F0 dams.

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Nursing Mothers
It is not known whether eltrombopag is excreted in human milk.  Because many drugs are excreted in human milk 
and because of the potential for serious adverse reactions in nursing infants from PROMACTA, a decision should 
be made whether to discontinue nursing or to discontinue PROMACTA taking into account the importance of 
PROMACTA to the mother.
Pediatric Use
The safety and efficacy of PROMACTA in pediatric patients 1 year and older with chronic ITP were evaluated in 
two double-blind, placebo-controlled trials.  The pharmacokinetics of eltrombopag have been evaluated in 168 
pediatric patients 1 year and older with ITP dosed once daily.  The safety and efficacy of PROMACTA in 
pediatric patients younger than 1 year with ITP have not yet been established.
The safety and efficacy of PROMACTA in pediatric patients with thrombocytopenia associated with 
chronic hepatitis C and severe aplastic anemia have not been established.
Adverse reactions:
Severe cutaneous reaction
There is a risk that subject may develop a severe cutaneous reaction that may require hospitalization and 
discontinuation of eltrombopag.  As of December 2015, there have been three occurrences of this event in 
subjects enrolled on this protocol.
Investigator Brochure, version 13, dated 4/13/2016 - “Adverse Events considered to be Expected for 
Reporting Purposes”
Below are lists of “Adverse Events considered to be Expected for Reporting Purposes” for each chronic 
ITP and SAA.  This list is based upon evaluation of the available clinical safety information, including data 
from all global clinical trials (phase I-III) and the Novartis safety database, Argus (cut-off date of 29 
February 2016).
Adverse reactions are listed below for each indication by MedDRA body system organ class and by 
frequency.  Frequency category for each adverse drug reaction is based on the following convention 
(CIOMS III).  The frequency categories used are:
Very common: ≥1 in 10 (≥10%)
Common: ≥1 in 100 and <1 in 10 (≥1% and <10%)
Uncommon: ≥1 in 1,000 and <1 in 100 (≥0.1% and <1%)
Rare: ≥1 in 10,000 and <1 in 1,000 (≥0.01% and <0.1%)
Adverse Events considered to be Expected for Reporting Purposes in cITP adults
Infections and infestations
Common:
 Pharyngitis
Urinary tract infection
Gastrointestinal disorders
Very Common: Nausea
Diarrhea
Common:
Dry mouth
Vomiting
Hepatobiliary disorders
Common:
Increased aspartate aminotransferase
Increased alanine aminotransferase
Blood bilirubin unconjugated increased

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Uncommon: Drug-induced liver injury
Skin and subcutaneous tissue disorders
Common:
Alopecia
Rash
Musculoskeletal and connective tissue disorders
Common: 
Back pain
Musculoskeletal chest pain
Musculoskeletal pain
Myalgia
Vascular disorders
Rare: post-marketing cases of Thrombotic microangiopathy with acute renal failure reported 
spontaneously
Additional adverse Events considered to be expected for Reporting Purposes in cITP pediatric 
Patients (Aged 1 to 17 years) in addition to those seen in cITP in adults
Infections and infestations
Very common: Nasopharyngitis, upper respiratory tract infection
Common: Rhinitis
Gastrointestinal disorders
Common: Abdominal pain, toothache
General disorders and administration site conditions
Common: Pyrexia
Respiratory, thoracic and mediastinal disorders
Common: Cough, oropharyngeal pain, rhinorrhea
Vascular disorders
Rare: post-marketing cases of Thrombotic microangiopathy with acute renal failure reported 
spontaneously
Adverse Events considered to be expected for Reporting Purposes in SAA
Blood and lymphatic system disorders
Very common: Anemia
Gastrointestinal disorders
Very common: Abdominal pain, diarrhea, nausea
General disorders and administrative conditions
Very common: Dizziness, fatigue, febrile neutropenia, pyrexia
Hepatobiliary disorders
Very common: Transaminases increased
Musculoskeletal and connective tissue disorders
Very common: Arthralgia, muscle spasms, pain in extremity
Nervous systems disorders
Very common: Headache
Respiratory, thoracic and mediastinal disorders
Common: Cough, dyspnea, oropharyngeal pain, rhinorrhea
Skin and subcutaneous tissue disorders
Very common: Ecchymosis
Vascular disorders
Rare: post-marketing cases of Thrombotic microangiopathy with acute renal failure reported 
spontaneously
Adverse Events considered to be expected for Reporting Purposes in MDS/AML

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Blood and lymphatic system disorders
Very common: Leukocytosis**, white blood cell count increased
Gastrointestinal disorders
Very common: Nausea, diarrhea, vomiting, constipation, abdominal pain
General disorders and administrative conditions
Very common: Fatigue, pyrexia
Hepatobiliary disorders
Uncommon: Drug-induced liver injury
Investigations
Rare: Serum discoloration***
Nervous systems disorders
Very common: Dizziness, Headache
Respiratory, thoracic and mediastinal disorders
Very common: Cough
Skin and subcutaneous tissue disorders
Common: Skin discoloration
Vascular disorders
Very common: Hematoma
** Leukocytosis and white blood cell count increased occur individually with a frequency of common, however the terms were 
grouped as they represent the same medical concept, giving a revised frequency of very common.
*** Serum discoloration has been reported in investigator sponsored studies in MDS/AML, and can lead to analytical interference 
with some colorimetric analytical methods
11.4.4 Related to Corticosteroids
Corticosteroids can make the body retain water and salt, cause diabetes and acne, and worsen high blood 
pressure.  In addition, they will probably increase your appetite and may cause insomnia or mood changes.  
Steroids can also cause stomach ulcers and soften bones, leading to osteoporosis.  The more dangerous 
problems, like bone thinning, only occur with long-term use; the other side effects will stop when the 
medication is discontinued.  A small number of cases of "aseptic necrosis" have been observed when 
steroids have been used in high doses for durations comparable to those we will use in this study.  Aseptic 
necrosis is a thinning of the bone near the joints, which can lead to chronic pain, sometimes requiring joint 
replacement.  Steroids also suppress the immune system and raise your susceptibility to infection; taking 
CsA and steroids together increases this risk still further.  We will ask that you plan to stay in the hospital 
for the first two weeks of your therapy so that infections, if they occur, can be treated promptly.
11.4.5 Related to bone marrow aspirate and biopsy: No major risks are involved with bone marrow 
aspirate and biopsy.  However, a small risk of infections, pain, bleeding, and hematoma formation at the 
site of the aspiration exists with the procedure.
11.4.6 Related to blood draws:  No major risks are involved with blood draws.  Minor complications 
including bleeding, pain, and hematoma formation at the site of blood draws, vasovagal reactions or 
infections may rarely occur.
11.4.7 Related to Cardiac Monitoring
EKG:  An electrocardiogram (EKG) is a test that measures the electrical activity of the heartbeat.  With 
each beat, an electrical impulse (or “wave”) travels through the heart.  This wave causes the muscle to 
squeeze and pump blood from the heart.  A technician will put patches (electrodes) on the chest, arms and 
legs.  The electrodes are soft and don’t cause any discomfort when they’re put on or taken off by the 

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technician.  The machine only records the EKG.  It doesn’t send electricity into the body.  There’s no pain 
or risk associated with having an electrocardiogram.
11.4.8 Related to Central Line Placement
A catheter may be placed in a large vein of the neck, chest, or arm using local anesthetic. Patients will sign 
a separate consent for the placement procedure.  Only trained experienced staff will place the line in order 
to minimize these procedure related risks.
The risks from the procedure are low; they include bleeding, bruising, or infection at the site of insertion.  
Very rarely (less than 1% of the time), the line placement may nick a vein causing one lung to collapse 
during line insertion.  If the lung collapses, a tube may have to be inserted into the chest and remain in place 
until the lung re-expands.  Because of this risk, patients will have a chest x-ray following the procedure to 
make sure the line is in the correct place and that the lung is not collapsed.  Once placed, the line will remain 
in place until drug administration is complete.
11.4.9 Related to Concomitant Medications
•
Pentamidine: cough (31-47%), bronchospasm (10-23%), decreased appetite (53-72%), 
fatigue, metallic taste, shortness of breath, decreased appetite, dizziness, rash, nausea, 
pharyngitis, chest pain/congestion, night sweats, chills, vomiting.
•
Valacyclovir: Nausea and/or vomiting, headache, dizziness, abdominal pain, dysmenorrhea, 
arthralgia, acute hypersensitivity reactions, elevations in liver enzyme laboratory values (e.g. 
AST).  Renal failure and CNS symptoms have been reported in patients with renal impairment 
who received valacyclovir at greater than the recommended dose.
11.5
Risks in Relation to Benefit
For adult subjects: The benefits to the subjects could be reduction or even abolition of transfusion 
requirements and/or improvement of cytopenia, resulting in improved quality of life and also decreased 
morbidity and mortality from transfusion-associated viral agents, iron overload, and/or a susceptibility to 
infections.  Potentially, treatment with other more toxic therapies could also be avoided or postponed.
The benefits of this study and the acquisition of bone marrow and blood samples important for the 
understanding of the pathophysiology of immune-mediated bone marrow failure states have been described 
in the previous paragraphs.
For pediatric subjects:  The inclusion of children satisfies the criteria set forth in 45 Code of Federal 
Regulations 46, Subpart D: 46.405 as follows:
(a) the risk is justified by the anticipated benefit to the subjects: We are offering pediatric subjects with a 
probably lethal hematological disease an alternative to symptomatic therapy.
(b) the relation of the anticipated benefit to the risk is at least as favorable to the subjects as that presented 
by available alternative approaches.  The benefits to the patients could be reduction or even abolition of 
transfusion requirements and/or improvement of low peripheral blood counts, resulting in improved quality 
of life and also decreased morbidity and mortality from transfusion-associated viral agents, iron overload, 
and/or a susceptibility to infections.  Potentially, treatment with other more toxic therapies could also be 
avoided or postponed. 

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(c) adequate provisions are made for soliciting the assent of the children and permission of their parents or 
guardians, as set forth in 46.408.
11.6
Informed Consent Processes and Procedures
Note: Effective January 21, 2019, a witness to the signature of the written long form research consent at an NIH 
site (whether initially approved by an IRB before or after January 21, 2019) is no longer a requirement.
The investigational nature and research objectives of this trial, the procedures and treatments involved and 
their attendant risks and discomforts and benefits, and potential alternative therapies will be carefully 
explained to the patient during the initial clinic evaluation.  The Principal Investigator, Dr. Young, or the 
Associate Investigators on this protocol delegated to obtain informed consents will lead this discussion and 
obtain the informed consent.  The consent form will be signed in the presence of the investigator and a 
witness prior to commencement of the treatment plan.  The treatment plan and risks will be discussed again 
and in detail during their hospital visit for treatment.
If the subject is a minor, the parent who signs the consent for the minor must be a legally recognized parent 
or guardian.  Where deemed appropriate by the clinician, and the child's parent or guardian, the child will 
also be included in all discussions about the trial and a minor's assent will be obtained.  The parent or 
guardian will sign on the designated line on the informed consent attesting to the fact that the child had 
given assent.
In cases where parents share joint legal custody in making medical decisions of their child (e.g. by 
a custody agreement or court order) both parents must give their parental permissions regardless of level of 
risk of the research.  Exceptions may be made if one parent is deceased, becomes incompetent or is not 
reasonably available (e.g. in prison).
If the minor subject is a female of childbearing age, she will be informed about pregnancy testing and will 
be told that if her pregnancy test is positive, we will counsel her and help her tell her parents or we will tell 
her parents. If she does not agree she will be advised not to sign the assent.
If at any time during participation in the protocol, new information becomes available relating to risks, 
adverse events, or toxicities, this information will be provided orally or in writing to each enrolled or 
prospective patient.  Documentation will be provided to the IRB and if necessary the informed consent 
amended to reflect relevant information.
When a pediatric subject reaches age 18, continued participation will require re-consenting of the now adult 
with the standard protocol consent document to ensure legally effective informed consent has been 
obtained.  Should sample or data analysis continue following completion of active participation and the 
subject has reached 18 years of age, we will attempt to contact the subject using the last known contact 
information to obtain consent for continued use of data or samples collected during their prior visit.  Given 
the length of time that may have transpired for some of the subjects since their last visit for this study, we 
request waiver of informed consent for those individuals who after good faith efforts to contact them, we 
are unable to contact.
Requirements for Waiver of Consent consistent with 45 CFR 46.116 (d), each of which must be addressed 
in relation to the protocol:
(1) The research involves no more than minimal risk to the subjects;
a)
Analysis of samples and data from this study involves no additional risks to subjects.
(2) The waiver or alteration will not adversely affect the rights and welfare of the subjects;

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a)
Samples and data will be kept in secure locations in the laboratory of Dr. Young. Retention of 
samples or data does not affect the welfare of subjects.
(3) The research could not practicably be carried out without the waiver or alteration; and
a)
Considering the length of time between a minor’s enrollment and their age of majority, it is possible 
that more than a few subjects may be lost to follow up.  A significant reduction in the number of 
samples analyzed could impact the quality of the research.
(4) Whenever appropriate, the subjects will be provided with additional pertinent information after 
participation.
a)
We only plan to request a waiver of reconsent for those subjects who have been lost to follow-up.
Informed Consent of Non-English-Speaking Research Participants.
We anticipate the enrollment of non-English speaking research participants into our study.  The IRB 
approved full consent document will be translated into that language in accordance with the Clinical MAS 
Policy M77-2.  If there is an unexpected enrollment of a research participant for which there is no translated 
extant IRB approved consent document, the Principal Investigator and or those authorized to obtain 
informed consent will use the Short Form Oral Consent Process as described in MAS Policy M77-2, 45 
CFR 46.117 (b) (2) and 21 CFR50.27 (b) (a).  The summary that will be used is the English version of the 
extant IRB approved consent document.
Informed Consent for adult research participants unable to provide consent:
If there is an unexpected enrollment of a research participant unable to provide informed consent, the 
following justification and procedures per NIH HRPP SOP 14E will be used to enrolled participants in this 
protocol.
Justification for inclusion:  This research provides the prospect of direct benefit; therefore, inclusion is 
justified.  The benefits to the participants could be improvement of cytopenias resulting in improved quality 
of life and also decreased morbidity and mortality from transfusion-associated viral agents, iron overload, 
and/or a susceptibility to infections.  Potentially, treatment with other more toxic therapies could also be 
avoided or postponed.  Not allowing participants who cannot provide consent would deny them the potential 
benefits this protocol offers for their AA.  There are no plans to include institutionalized participants.
Consent and Assent:
Procedures to determine capacity:  If documentation of decision making capacity is not present in the 
medical record or the investigator questions the decision-making capacity of the individual, then the Ability 
to Consent Assessment Team (ACAT) (301-496-9675 or 301-496-2429) will be contacted to make the 
determination.
Procedures for obtaining consent for legally authorized representative (LAR) (Category B per SOP 14E).  
See Appendix F for details.
11.7
Conflict of interest
The Principal Investigator assured that each associate investigator listed on the protocol title page received 
a copy of the NIH’s Guide to preventing conflict of interest.  Investigators added subsequent to the initial 
circulation were provided a copy of the document when they were added. Copies of the Conflict of Interest 
Statement were forwarded to the Clinical Director.  No initial or subsequent members of the research team 
reported a potential conflict of interest.
This protocol has an associated CRADA with Novartis.

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11.8
FWA Coverage Agreement
Dr. Townsley is currently working at Medimmune and will be analyzing identifiable data as an Non-NIH, 
Non-Enrolling Engaged Investigator in this protocol.  Dr. Townsley’s role in the research will be limited to 
data analysis.  An FWA coverage agreement to cover this activity has been executed by Dr. Townsley and 
Dr. Young.
11.9
Collaboration(s)
Phillip Scheinberg, M.D. will continue to collaborate on trial with protocol related questions and 
interpretation of study data.  Dr. Scheinberg has no access to PII and is no longer participating in direct 
patient care.
Between NHLBI (Neal S. Young, MD) and Satu Mustjoki, M.D., Ph.D. Professor of Translational 
Hematology in University of Helsinki (Medicum) and Helsinki University Hospital. Upon execution of the 
MTA, de-identified (coded) peripheral blood and plasma samples of Severe Aplastic Anemia patients will 
be sent for sequencing of DNA using Next Generation Sequencing (NGS), analyze results, seeking somatic 
mutations in lymphoid cells, T-cell receptor (TCR) usage, and autoantibody identification by imputation.  
Collaborator will return the results of the analysis to NHLBI for further analysis and correlation of results 
with NHLBI’s clinical data, especially clinical outcome.  Both parties intend to jointly design experiments 
and analyze the results and data, and publish the results in co-authored articles.
Between NHLBI (Neal S. Young, M.D.) and Shahram Kordasti, M.D., Ph.D. at Kings College London. 
Upon execution of the MTA, de-identified (coded) bone marrow samples of Severe Aplastic Anemia 
patients will be sent to investigate freshly stained bone marrow cells, HSPCs, and supporting cells (stromal 
cells, macrophages, myeloid-derived suppressor cells, and their inflammation cytokine production). 
Additionally, the samples collected after the treatment will be analyzed similarly to explore mechanisms 
by which eltrombopag stimulates hematopoiesis and modulates the immune system. Collaborator will 
return the results of the analysis to NHLBI for further analysis and correlation of results with NHLBI’s 
clinical data.
12. PHARMACEUTICALS
12.1
ELTROMBOPAG (PROMACTA®)
Supply:  The drug Novartis is providing for this study may be either investigational or commercial material, 
based on their supply, and is available in tablets and as a powder for oral suspension.  Each sachet contains 
eltrombopag olamine equivalent to 25 mg of eltrombopag. The tablets are available as 12.5, 25, 50, and 75 
mg tablets.
Preparation:  There is no parental dose for eltrombopag.
Storage and Stability:  Store at 25°C (77°F); excursions permitted to 15º to 30°C (59º to 86°F) [see USP 
Controlled Room Temperature].
Administration:  Eltrombopag may only be given orally.
Toxicities:  see section 11.4.3 Risks related to eltrombopag.
Tablets:

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White, round, film-coated tablets without debossing are provided, containing eltrombopag olamine equivalent to 
12.5 mg, 25 mg, 50 mg, or 75 mg of eltrombopag free acid.  Tablets are packaged in white HDPE bottles with 
white plastic, induction-seal, child-resistant caps.
Commercial image actives (12.5 mg - white, 25 mg- orange or white, 50 mg blue or brown, 75 mg – pink,) which 
are equivalent to the clinical forms with the exception of the film coated color may also be provided for clinical 
use.  These forms are also packed in white HDPE bottles with white plastic, induction -seal, child-resistant caps.  
Desiccant may be included.  Additionally, the commercial image tablets may be provided in aluminum foil blister 
packages.
Eltrombopag Powder for Oral Suspension:
Eltrombopag powder for oral suspension (Eltrombopag PfOS) is a reddish-brown to yellow powder contained 
inside an elongated sachet.  A 25 mg PfOS strength is available, containing eltrombopag olamine equivalent to 25 
mg of eltrombopag free acid.  The powder blend composition contains eltrombopag olamine equivalent to 20 mg 
of eltrombopag free acid.  The powder fill weight is 1.25 gram for PfOS 25 mg.
PfOS sachets will be packaged in a carton. PfOS may be supplied as bulk stickpacks (sachets) or as commercial 
kit.  If bulk stickpacks are supplied, the clinical site will also receive reconstitution bottle, syringe-adapta cap, and 
20mL oral dosing syringes. If commercial kit is provided, each carton pack will hold 30 sachets along with a 
plastic reconstitution container, a syringe-adapt cap and 30 single-use 20mL oral dosing syringes.  The sachet 
should not be opened until ready to use. Add 9.5 mL of water drawn using a 20mL syringe into the provided 
plastic container.  Cut open the sachet and add the entire content of the sachet into the container with water.  The 
container is capped and shaken for 10-20 seconds.  The resulting suspension contains 2 mg/mL of eltrombopag 
dose.  The prescribed volume (dose) is drawn through the syringe port on the cap with a syringe.  Upon dosing, 
the rest of the remaining suspension in the container is discarded.  The container and cap are rinsed with water 
and dried.  If the prescribed dose is > 29 mg, which will require that part or all of a second sachet be used, then 
the suspension can be prepared by adding the contents of the two sachets to 19.0 mL of water, and then following 
the steps outlined above.  The water has to be drawn by using the 20cc syringe, and similarly dosing has to occur 
by using the same 20cc syringe.  If the dosage requires more than two sachets, then for each sachet used 9.5 mL 
of water will be added, and then following the steps outlined above.  The water has to be drawn by using the same 
20cc syringe for each sachet used, and similarly dosing has to occur by using the same 20cc syringe.  A fresh dose 
is prepared everyday just prior to the dosing and no storage of the reconstituted suspension is allowed.
Handling and Storage of Study Treatment:  Eltrombopag PfOS sachets will be stored at a controlled room 
temperature of 20-25°C (68-77°F); excursions between 15-30°C (59-86°F) are permissible.
Shipping: The NIH Investigational Drug Management and Research Section (IMDRS) will be responsible 
for receiving, storing, dispensing and accounting for drug product.  The shipping address for Novartis 
supplied investigational agent is:
National Institutes of Health/CC/PHARM/IMDRS
10 Center Drive, MSC 1196, Building 10, Room 1C230
Bethesda, Maryland 20892-1196
Shipping Designee Name:  Jihyun Esther Jeon
Shipping Designee Phone No: (301) 496-4363
Shipping Designee FAX No: (301) 402-3268
Shipping Designee e-mail: jihyunesther.jeon@nih.gov

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Neal S. Young, M.D.  3/18/2020 (Amendment UU/47)
12.2
CYCLOSPORINE (Gengraf, Sandimmune, Neoral)
Supply:  Cyclosporine will be obtained by the NIH Clinical Center Pharmacy Department from commercial 
sources and is available in capsules (25 mg and 100 mg), USP [MODIFIED], oral solution (100 mg/ml), 
USP [MODIFIED], and as a parenteral concentrate for injection (50 mg/ml).  When oral capsules are 
prescribed for this protocol, the cyclosporine capsules, USP [NON-MODIFIED] should NOT be used.
Preparation:  For parenteral doses, each milliliter of concentrate (50mg/ml) should be diluted in 20 to 
100ml of dextrose 5% in water or sodium chloride 0.9%.  Parenteral doses of cyclosporine will be prepared 
in non-PVC containers and infused with non-PVC administration sets/tubing.  The recommended liquids 
for dilution of the oral solution to improve palatability include milk, chocolate milk or orange juice, 
preferable at room temperature.
Storage and Stability:  Capsules, oral solution, and ampules of parenteral concentrate bear expiration dates 
and are stored at room temperature and protected from light.  Cyclosporine concentrate for injection that 
has been diluted to a final concentration of approximately 2mg/ml is stable for 24 hours in 5% dextrose or 
0.9% sodium chloride injection in glass, PVC or non-PVC plastic containers.  To minimize the potential 
for sorption to PVC plastic bags and tubing as well the leaching of phthalate plasticizer (DEHP) into the 
solution, only non-PVC plastic bags and intravenous administration sets should be utilized.
Administration: Cyclosporine may be given intravenously or orally.
Toxicities:  see section 11.4.2 Risks related to CsA
12.3
ANTI-THYMOCYTE GLOBULIN (equine) sterile solution (ATGAM®)
Other: 
Antithymocyte 
Gammaglobulin, 
Antithymocyte 
Globulin, 
ATGAM, 
Antithymocyte 
Immunoglobulin, lymphocyte immune globulin and h-ATG
Supply / availability:  commercially available (Pharmacia & Upjohn Company)
Product description:  Anti-thymocyte globulin (equine) sterile solution (ATGAM®) is available in 5 ml 
ampoules containing 50 mg of horse gamma globulin/mL (250 mg per ampoule).
Preparation:  The calculated dose of anti-thymocyte globulin should be diluted in 0.9% sodium chloride 
injection to a concentration not to exceed 4 mg/mL.
Storage / stability:  Anti-thymocyte globulin (equine) ampoules should be stored in a refrigerator at 2o to 
8o C. Once diluted, anti-thymocyte globulin (equine) is physically and chemically stable for up to 24 hours 
at concentrations of up to 4 mg/mL in the recommended diluents.  It is recommended that diluted anti-
thymocyte globulin (equine) be stored in a refrigerator if it is prepared prior to the time of infusion. 
Administration:  Anti-thymocyte globulin (equine) should be administered into a high-flow central vein 
through an in-line filter with a pore size of 0.2 to 1 micron.  The dose should be infused over no less than 4 
hours. Infusion times may be extended to up to 24 hours for intolerance.  Patients should be closely 
monitored for infusion / allergic reactions.
Compound:  Principally monomeric IgG, prepared from plasma or serum of healthy horses 
hyperimmunized with human thymus lymphocytes.
Action:  Immunosuppressive agent. Exact mechanism of immunosuppression of ATGAM has not been fully 

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Neal S. Young, M.D.  3/18/2020 (Amendment UU/47)
elucidated but may involve elimination of antigen-reactive T-cells in peripheral blood and/or alteration of 
T-cell function
Side effects:  see section 11.4.1 Risks related to h-ATG
12.4
PENTAMIDINE
Supply:  Commercially available (NebuPent®, American Pharmaceutical Partners, Inc.)
Product description:  Pentamidineisethionate is available as a 300 mg single dose vial containing 300 mg 
of lyophilized powder in a 15 mL capacity vial.  The contents of one vial must be dissolved in 6 mL of 
sterile water for injection, USP. It is important to use only sterile water; saline solution will cause the drug 
to precipitate.
Storage and stability:  Store dry product at controlled room temperature 15-30oC (59-86oF).
Route of administration:  Inhalation; Once reconstituted, the entire contents of a vial should be placed into 
the Respigard® II nebulizer (Marquest) reservoir for administration by inhalation.  Do not mix the 
pentamidine solution with any other drugs.
Toxicities:  see section 11.4.9 Risks related to Concomitant medications.
12.5
VALACYCLOVIR
Generic name: valacyclovir
Brand Name: Valtrex
Supply: Commercially available.
Pharmacology:  Valacyclovir is the hydrochloride salt of L-valyl ester of the antiviral drug acyclovir. After 
oral administration, valacyclovir is rapidly absorbed from the GI tract and nearly completely converted to 
acyclovir and L-valine by first-pass intestinal or hepatic metabolism.
Product description: Valacyclovir is available in 500mg tablets and 1gm tablets.  Dose adjustment is 
necessary in patients with significant renal impairment (refer to the manufacturer’s labeling for dose 
adjustment guidelines.
Storage and Stability: Oral tablets should be stored at 15o to 25oC (59o to 77oF).
Route of administration: Oral
Toxicities: see section 11.4.9 risks related to concomitant medications

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Neal S. Young, M.D.  3/18/2020 (Amendment UU/47)
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Barrett AJ, Longhurst P, Rosengurt N, Hobbs JR, Humble JG. Crossreaction of antilymphocyte globulin 
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Greco B, Bielory L, Stephany D, et al. Antithymocyte globulin reacts with many normal human cell types. 
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Shevach EM. The effects of cyclosporin A on the immune system. Annual review of immunology. 
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Nussenblatt RB, Palestine AG. Cyclosporine: immunology, pharmacology and therapeutic uses. Survey of 
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Bacigalupo A, Bruno B, Saracco P, et al. Antilymphocyte globulin, cyclosporine, prednisolone, and 
granulocyte colony-stimulating factor for severe aplastic anemia: an update of the GITMO/EBMT study on 
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27.
Rosenfeld S, Follmann D, Nunez O, Young NS. Antithymocyte globulin and cyclosporine for severe 
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Frickhofen N, Kaltwasser JP, Schrezenmeier H, et al. Treatment of aplastic anemia with antilymphocyte 
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Brodsky RA, Sensenbrenner LL, Smith BD, et al. Durable treatment-free remission after high-dose 
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30.
Tisdale JF, Dunn DE, Geller N, et al. High-dose cyclophosphamide in severe aplastic anaemia: a 
randomised trial. Lancet. Nov 4 2000;356(9241):1554-1559.
31.
Tisdale JF, Maciejewski JP, Nunez O, Rosenfeld SJ, Young NS. Late complications following treatment 
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Blood. Dec 15 2002;100(13):4668-4670.
32.
Scheinberg P, Nunez O, Wu C, Young NS. Treatment of severe aplastic anaemia with combined 
immunosuppression: anti-thymocyte globulin, ciclosporin and mycophenolate mofetil. Br J Haematol. Jun 
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Scheinberg P, Nunez O, Weinstein B, Biancotto A, Wu CO, Young NS. Horse versus rabbit antithymocyte 
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Tichelli A, Schrezenmeier H, Socie G, et al. A randomized controlled study in newly-diagnosed severe 
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Kojima S, Hibi S, Kosaka Y, et al. Immunosuppressive therapy using antithymocyte globulin, cyclosporine, 
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New England journal of medicine. Aug 25 2011;365(8):734-741.
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Nancy L. Geller, Dean Follmann, Eric S. Leifer, and Shelly L. Carter “Design of Early Trials in Stem Cell 
Transplantation: A Hybrid Frequentist-Bayesian Approach” Chapter 2, pp 41-52, Advances in Clinical 
Trial Biostatistics, 2004, Marcel Dekker: New York
67.
Scheinberg P, Rios O, Scheinberg P, Weinstein B, Wu CO, Young NS. Prolonged cyclosporine 
administration after antithymocyte globulin delays but does not prevent relapse in severe aplastic anemia. 
Am J Hematol. 2014 Jun;89(6):571-4.
68.
Novick AC, Hwei HH, Steinmuller D, Streem SB, Cunningham RJ, Steinhilber D, Goormastic M, Buszta 
C.  Detrimental effect of cyclosporine on initial function of cadaver renal allografts following extended 
preservation. Results of a randomized prospective study. Transplantation. 1986;42(2):154.
69.
Tostivint I, du Montcel ST, Jaudon MC, Mallet A, Le Hoang P, Bodaghi B, Deray G, Bagnis CI.  Renal 
outcome after ciclosporin-induced nephrotoxicity.  Nephrol Dial Transplant. 2007;22(3):880.

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APPENDIX A:  MEDWATCH FORM

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APPENDIX B:  NHLBI HEMATOLOGY BRANCH LABORATORY RESEARCH 
STUDIES v. 2.5.2013
DESCRIPTION OF LABORATORY STUDY BY BRANCH SECTION
Does this test 
pose a greater 
than minimal 
risk to 
pediatric 
subjects per 45 
CFR 46.404?
Does this test 
pose a 
greater than 
minimal risk 
to healthy 
pediatric 
donors per 45 
CFR 46.404?
A
Stem Cell Allotransplantation Section (Dr. A. John Barrett) – No longer active 
as of July 1, 2018
A.1
Measurement of lymphocyte function and immune responses directed toward 
allogeneic tissues, malignant cells, and infectious agents.  Assay of a variety of 
antigens, including standard proliferation, cytotoxicity, and intracellular cytokine 
detection including GVHD predictive markers. Measurement of antigen-specific 
responses including employment of tetramers, ELISPOT technique, gene 
amplification-based assays, and flow cytometry.  Selection of cells using 
immunomagnetic beads or flow cytometry.  Culture, expansion, and selection of 
cells. Surface marker analysis of PB MC using flow cytometry. Cytokine/chemokine 
analysis of plasma/serum samples using ELISA and/or Luminex techniques.
No
No
A.2
Generation of cell lines for the study of immune cell interactions with other cells.  
Transformation of B-lymphocytes using Epstein-Barr virus.  Derivation of malignant 
cell lines from patient leukemic or solid tumor samples.
No
No
A.3
Infection of cells and cell lines with recombinant genes to ascertain the effects of 
expressed molecules on immune responses and on growth and development. 
Transfection of cell lines with specific molecules to study antigen-specific responses.
No
No
A.4
Assays of peripheral blood and bone marrow progenitor cells including primitive and 
late erythroid progenitor-derived colonies, myelomonocytic colonies, and primitive 
multi- potential progenitor-derived colonies.
No
No
A.5
Injection of human cells into experimental animals to study the immune system and 
the growth of normal and malignant cells under varying conditions.
No
No
A.6
Testing of selection methods, cell isolation, and cell expansion leading to the 
development of new cell-based therapies requiring scale-up for clinical application.
No
No
A.7
Identification of individual T cell clones by their T cell receptor sequence.
No
No
A.8
Measurement of tumor and tissue specific antigens in cells of subjects and donors by 
mRNA, protein, or peptide expression in cells or fluids.
No
No
A.9
Laser capture micro dissection of cells from biopsies for GVHD to determine 
clonotypes.
No
No
A.10
DNA and RNA typing of genes that control immune responses in lymphocytes.
No
No
A.11
Microassay studies utilizing cellular DNA, cDNA, and RNA for neoplasia and host-
tumor interactions.
No
No
B
Molecular Hematopoiesis Section (Dr. Cynthia Dunbar) 
B.1
Flow cytometric analysis of cell surface and cytoplasmic proteins, including cell 
adhesion molecules, putative retroviral receptors, and markers of differentiation, 
using bone marrow and mobilized peripheral blood cells.
No
No

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B.2
Hematopoietic progenitor-derived colony ascertainment in vitro (as described above), 
and engraftment of immunodeficient mice for detection of human stem cell number 
and function.
No
No
B.3
Testing ability of hematopoietic progenitor cells to be transduced with retroviral, 
lentiviral, and novel gene transfer vectors in vitro.
No
No
B.4
Reprogramming of adult mature cells, including skin fibroblasts and blood cells, into 
induced pluripotent stem cells in vitro.
No
No
C
Cell Biology Section (Dr. Neal Young) 
C.1
Studies of blood and bone marrow hematopoietic progenitor numbers, including early 
and late erythroid progenitors, myelomonocytic progenitors, and multi-potential 
progenitor cells.  In addition, bone marrow may be placed in long-term bone marrow 
culture to assess the function of stroma and stem cells and to assay more primitive 
progenitors, as well as organelle culture.  Whole or selected bone marrow 
populations are cultured short-term for CD34 cell expansion.
No
No
C.2
Assays of apoptosis in hematopoietic cells and their progeny, using flow cytometric 
methods such as annexin and caspase-3 staining, propidium iodide uptake, and 
mitochondrial permeability tests.
No
No
C.3
Separation and functional study of cell populations characteristic of paroxysmal 
nocturnal hemoglobinuria, identified by absence of glycosylphosphatidylinositol 
anchored proteins.
No
No
C.4
Studies of mutation rates in hematopoietic cells and in buccal mucosa cells, using 
conventional hypoxanthine phosphoribosyltransferase activity functional assays, 
sequencing of mitochondrial DNA after specific gene amplification, and 
measurement of GPI-anchored deficient cells in blood and bone marrow.
No
No
C.5
Assays of immune function of T-cells, including intracellular cytokine staining, 
ELISPOT, semiquantitative gene amplification for gamma-interferon, tumor necrosis 
factor, interleukin-2, and other cytokines, and functional assessment in co-culture 
using specific neutralizing monoclonal antibodies.  In addition, peripheral blood 
lymphocytes are subjected to spectratyping for CDR3 size distribution as well as 
nucleotide sequence of CDR3 peaks obtained.
No
No
C.6
Studies of engraftment of human normal and diseased bone marrow and peripheral 
blood in immunodeficient mice in order to determine the presence of hematopoietic 
repopulating stem cells as well as functional differences among selected populations.
No
No
C.7
Flow cytometric analysis of blood and bone marrow for lymphocyte phenotype, 
especially for evidence of activation of lymphocytes, for markers of apoptosis, and 
for antigens associated with primitive and mature hematopoietic cell populations.
No
No
C.8
Flow cytometric analysis of blood and bone marrow for hematopoietic stem cell 
progenitors and CD34 positive cells.
No
No
C.9
Studies of chromosomal instability in myelopdysplastic syndromes including BM cell 
and CD34 cell response to PAS crosslinking and examination of the cytotoxic effect 
of lymphocytes to the abnormal clone of cells.
No
No
C.10
Surface Enhanced Laser/Desorption Ionization (SELDI) time-of-flight mass 
spectrometry (Ciphergen)  (proteomics methodology).
No
No
C.11
Mitochondrial DNA (mtDNA) sequence heterogeneity.
No
No
C.12
Measurement of EBV viral load.
No
No
C.13
Measurement of EBV LMP-1 via RT-PCR for LMP-1 RNA or flow cytometry for 
LMP-1.
No
No
C.14
Outgrowth assay of EBV transformed B cells.
No
No
C.15
Quantification of serum chemokines and cytokines (e.g. SDF-1, IL-10, IL-6, CXCR4, 
CXCL12).
No
No
C.16
Quantification of EBV cytotoxic T cells (tetramerstaining).
No
No

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Neal S. Young, M.D.  3/18/2020 (Amendment UU/47)
C.17
Telomere length measurement by Southern blot, Q-PCR, flow-fish, in situ 
hybridization and STELA
No
No
C.18
Telomere repair complex gene mutations by nucleotide sequencing of some or all of 
the following:  DKC1 ,TERC, TERT, SBDS, NOp10, NHP2.
No
No
C.19
Analysis of inflammatory markers and/or bacterial, viral, fungal or protozoal 
elements in plasma or serum using molecular, colorimetric, enzymatic, flow 
cytometric or other assays in subjects receiving immunosuppressive therapy, 
chemotherapy and/or bone marrow transplantation.
No
No
C.20
Confocal microscopic imaging of bone marrow.
No
No
C.21
Characterization of intracellular signaling proteins by cell permeabilization and flow 
cytometry, and quantitative immunoblots.
No
No
C.22
Assays for chromosomal aneuploidy by florescence in situ hybridization (FISH) and 
other molecular techniques.
No
No
C.23
Conversion of human dermal fibroblasts into hematopoietic progenitors using Oct4 
transfection.
No
No
D
Virus Discovery Section (Dr. Neal Young) THESE ASSAYS WILL NOT BE 
PERFORMED ON SAMPLES FROM HEALTHY PEDIATRIC DONORS
D.1
Assays of serum, blood cells, and bone marrow cells for B19 parvovirus and possible 
B19 variants using gene amplification, cell culture, and hematopoietic colony 
inhibition assays.
No
N/A
D.2
Assays of blood, bone marrow, liver, and other tissues for potentially novel viruses, 
using a variety of techniques including RNA and DNA assays, differential display, 
gene amplification with conserved and random primers, cell culture assays, 
immunohistochemical methods, and inocculation of mice, rabbits, and monkeys, as 
well as antibody measurements.
No
N/A
D.3
Assays of blood, bone marrow, and liver for known viruses, including herpesviruses 
such as cytomegalovirus, human herpesviruses 6, 7, and 8, enteric viruses such as A-
6, circiviruses, and parvoviruses, using assays as in (2).
No
N/A
D.4
Spectra-typing of blood cells to determine response to known or putative viral 
infections.
No
N/A
D.5
HLA typing or subtyping to determine risk factors/determinants for hepatitis-AA 
studies.
No
N/A
D.6
Cytotoxic lymphocyte assays with intracellular cytokine measurement for 
determining anti-viral response and lymphocyte cloning to obtain clones with 
specific antiviral activity.
No
N/A
E
Solid Tumor Section (Dr. Richard Childs)
E.1
Cr51 cytotoxicity assay to evaluating killing of patient tumor cells by patient NK cell 
clones and T-cells.
No
No
E.2
ELISA for IL-12  maturity of DC's made from subjects monocytes.
No
No
E.3
ELISA for IFN ã to evaluate specificity of CTL clones.
No
No
E.4
H thymidine uptake to evaluate proliferation potential of antigen specific T-cells.
No
No
E.5
PCR of STR to assess chimerism status of cellular subsets grown in-vitro or retrieved 
from subjects post-transplant.
No
No
E.6
Flow sorting of PBL and/or tissue samples to evaluate chimerism of different subsets.
No
No
E.7
Surface marker analysis of peripheral blood mononuclear cells using flow cytometry.
No
No
E.8
cDNA expression arrays to evaluate T-cells expression/gene patterns in subjects with 
GVHD and a GVT effect.
No
No
E.9
Geno typing of tumor or tissue samples by high densitycDNA arrays.
No
No
E.10
VHL mutation analysis on kidney cancer tissue.
No
No

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E.11
Transduction of dendritic and tissue cells with tumor antigens using plasmids, viral 
vectors and hybrid fusions.
No
No
E.12
Lasar capture microdisection of cells from tumor biopsies and tissue samples to 
determine origin (donor vs patient).
No
No
E.13
Quantification of polyoma virus BK exposure by serology and PCR in stem cell 
transplant donors and recipients from blood and urine samples.
No
No
E.14
Quantification of polyoma virus BK specific T cells in stem cell transplant donors 
and recipients from peripheral blood samples.
No
No
E.15
Determination of origin of neovasculature endothelial cells in tumor and tissue 
samples obtained from subjects post transplant.
No
No
E.16
Quantification of lymphocyte subsets CD34 progenitors and endovasculator 
progenitors in G-CSF mobilized peripheral cell allografts.
No
No
E.17
Testing for polyoma virus BK latency in CD34 progenitors, B cells and T cells in the 
G-CSF mobilized peripheral cell allografts.
No
No
E.18
Determination of etiology of membraneous nephropathy using serum from subjects.
No
No
E.19
Serum Proteomic patterns analysis to diagnose complications related to allogeneic 
transplantation.
No
No
E.20
Determine cell origin (donor vs patient) of tissue samples using IHC, IF, sorting, and 
FISH.
No
No
F
Lymphoid Malignancies Section (Dr. Adrian Wiestner)
F.1
Culture of cells from research subjects to investigate molecular disease mechanisms, 
model host tumor interactions, and to test effect of drugs on cell survival and cellular 
functions.
No
No
F.2
Generation of stable cell lines for the study of hematologic malignancies.
No
No
F.3
Modifications of cells using standard expression systems or biologic molecules, e.g. 
interfering RNA, to investigate the effects of candidate genes on cellular functions.
F.4
Identification and monitoring of B or T cell populations as identified by flow 
cytometry and by their B cell or T cell receptor expression.
No
No
F.5
Measurement of gene expression in cells or tissues. Techniques frequently used 
include gene expression profiling on microarrays, quantitative RT-PCR, Western 
blotting, flow cytometry and ELISA assays.
No
No
F.6
Analysis of chromosomal abnormalities or mutations in malignant cells and non-
malignant cells including FISH technology and DNA sequencing.
No
No
F.7
Assays of immune function of B-cells and T-cells, including intracellular cytokine 
staining, ELISPOT, quantitative RT-PCR for cytokines or other immune regulatory 
genes.
No
No
F.8
Analysis of antibody specificities in serum and antigen specificity of the B-cell 
receptor on cells. Techniques may include expression of antibodies in phage display 
systems, generation of antibodies in cell culture systems and use of such antibodies to 
screen for cognate antigens.
No
No
F.9
Transplantation of human cells into mice (xenograft model) to study disease biology 
and to investigate the effect of experimental therapy.
No
No
F.10
Measurements of drug concentrations, biologic molecules and disease markers in 
blood, serum, and plasma.
No
No

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APPENDIX C:  SUPPLEMENTAL FIGURES
Figure 1: Study Design
Figure 2: Study results as defined by response criteria at 12 weeks. Achievement of further lineage responses 
during extension phase at greater than 12 weeks are also indicated.
SAA Eltrombopag Study Results
11 responders (44%)
• 9 platelet responses 
• 2 hemoglobin responses 
• additional 4 at > 12 wks
• 4 neutrophil responses 
• additional 3 at > 12 wks
26 patients 
enrolled
25 evaluable
patients 
25 evaluable
patients 
1 patient ineligible,   
not treated
14 non-responders
• 10 stable disease
• 2 died of progression
• 2 clonal evolution to MDS 
• 1 died 
• 1 HSCT
Median follow up 13 months 
(range 4-28 months) 
Censor date 11/1/2011
26 patients 
enrolled
1 patient ineligible 
not treated

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Figure 3: Responses to eltrombopag in responders over time. For each lineage, individual patients reaching 
response criteria are shown (A. Platelets, B. Hemoglobin, C. Neutrophils). Black lines indicate patients remaining 
on drug. 
Gray lines indicate the patient taken off drug at three months due to possible cataract formation.
40 
10 
20 
30 
0                    3                    6                    9                   12 
Time on eltrombopag (months) 
Platelets K/uL)
Platelet Responses 
50 
Eltrombopag
started
Response 
assessment 
Hemoglobin Responses
Eltrombopag
started
15 
14 
12 
10 
9 
8 
7 
Hemoglobin (g/dL)
11
13 
0                    3                    6                    9                   12 
Time on eltrombopag (months) 
16 
B
A

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Neutrophil Responses 
Neutrophil Absolute (K/uL)
Eltrombopag
started
3
2
1
0
0                     3                      6                     9                    12 
Time on eltrombopag (months) 
Figure 4: Bone marrow biopsies stained with hemotoxylin and eosin and shown at 100X magnification pre-
treatment and 12 months following study entry.  
Bone Marrow Cellularity at 1 Year After Study Entry 
Patient 1: Pre-Tx
Patient 1: Post-Tx 
Patient 2: Pre-Tx 
Patient 2: Post-Tx 
Patient 3: Pre-Tx
Patient 3: Post-Tx 
Patient 4: Post-Tx 
Patient 4: Pre-Tx 
C

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APPENDIX D:  PHARMACOKINETIC STUDIES
Collection of samples for PK Assessments
Subjects enrolled in Cohort 1, will have PK assessments at the landmark 3-month study visit. Subjects 
enrolled into the extension cohort, starting with subject # 120, may have their PK assessment between 
day 8 and day 30. An additional trough collection (within 30 min prior to eltrombopag dosing) may also 
be obtained in subjects on the extension cohort at the 3 or 6 month visit. Subjects must have received 
once daily eltrombopag for at least 7 days prior to PK assessment (i.e., be at PK steady-state with no 
recent dose interruptions).  If a subject is not currently receiving eltrombopag at the time of this 
assessment (because of a dose interruption) or eltrombopag has been reinitiated after a dose interruption 
within the 7 days prior to this assessment, PK assessments will be deferred until the landmark 6-month 
study visit for Cohort 1.  The eltrombopag dosing history for the 2 weeks prior to the PK visit will be 
recorded (any dose interruptions, actual dose administered).
Blood samples (2 mL) for PK analysis will be collected in K2EDTA-containing tubes.  One sample will 
be collected at each of the following times: within 30 min prior to eltrombopag dosing (pre-dose sample), 
and at 2, 4, 6, and 8 h after eltrombopag dosing. An optional sample will be collected 24 h post-dose, 
prior to administration of eltrombopag the next day.  
Record the date, time, and amount (in mg) of the dose administered after the pre-dose PK sample.  
Collect each whole blood PK sample as close as possible to the planned time relative to dosing.  Record 
the actual date and time that each sample was collected.
If a cannula is used, the cannula will be inserted into an arm vein within sufficient time prior to dosing, 
will be kept patent with normal saline and will be removed after the last blood sample is collected or 
earlier if the subject requests.  In order to avoid artificial dilution of the PK sample by the saline, 0.5-
1mL of whole blood will be collected and discarded before each PK sample is collected.
7.7.1
PK Sample Processing and Storage
Each PK samples will be gently mixed by inversion 8 to 10 times (do not shake).  Place the samples on 
ice immediately after collection.  Within 1 hour of sample collection, the samples will be centrifuged in 
a refrigerated (2°C to 8°C) centrifuge at 1500 RPM for 10 minutes.  The resulting plasma will be 
transferred into a properly-labeled polypropylene tube.  Immediately, place the plasma samples upright 
in a -20°C freezer and retain the samples in the freezer until they are shipped for analysis.
7.7.2
Shipping Instructions
Samples should be shipped only on Monday, Tuesday, or Wednesday, not less often than every 2 
months.  Samples must be shipped on dry ice via overnight courier to:
LiMajor Pittman
PPD
2246 Dabney Road
Richmond VA, 23230, USA
Tel: (804) 977-8017
e-mail: limajor.pittman@ppdi.com

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APPENDIX E:  PROMIS QUESTIONNAIRE
PROMIS v.1.0/1.1 - Global
© 2009 PROMIS Health Organization and PROMIS Cooperative Group
Page 1 of 2
Global Items
Pleaserespond to each item by marking onebox per row.
Excellent
Very
good
Good
Fair
Poor
Global01
5
4
3
2
1
Global02
In general, would you say your quality of
life is: ................................ ................................ ...
5
4
3
2
1
Global03
In general, how would you rate your physical 
health?............................. .......................................
5
4
3
2
1
Global04
In general, how would you rate your mental 
health, including your mood and your ability to 
think?.............................. .......................................
5
4
3
2
1
Global05
In general, how would you rate your satisfaction 
with your social activities and 
relationships?.............................................. ...........
5
4
3
2
1
Global09
In general, please rate how well you carry out 
your usual social activities and roles. (This 
includes activities at home, at work and in your 
community, and responsibilities as a parent, 
child, spouse, employee, friend, etc.)...................
5
4
3
2
1
Completely
Mostly
Moderately
A little
Not at all
Global06
To what extent are you able to carry out your 
everyday physical activities such as walking, 
climbing stairs, carrying groceries, or moving a
chair?............................... ......................................
5
4
3
2
1

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PROMIS v.1.0/1.1 - Global
© 2009 PROMIS Health Organization and PROMIS Cooperative Group
Page 2 of 2
Never
Rarely
Sometimes
Often
Always
Global10
How often have you been bothered by emotional 
problems such as feeling anxious, depressed or
irritable?.................................................................
1
2
3
4
5
None
Mild
Moderate
Severe
Very
severe
Global08
How would you rate your fatigue on average? ....
1
2
3
4
5
Global07
How would you rate your 
pain on average?..................
0
1
2
3
4
5
6
7
8
9
10
No
pain
Worst
imaginable
pain

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FACT-An (Version 4)
Below is a list of statements that other people with your illness have said are important. Please circle
or mark one number per line to indicate your response as it applies to the past 7 days.
PHYSICAL WELL-BEING
Not
at all
A little
bit
Some-
what
Quite
a bit
Very
much
GP1
I have a lack of energy .......................................................
0
1
2
3
4
GP2
I have nausea ......................................................................
0
1
2
3
4
GP3
Because of my physical condition, I have trouble
meeting the needs of my family.........................................
0
1
2
3
4
GP4
I have pain..........................................................................
0
1
2
3
4
GP5
I am bothered by side effects of treatment .........................
0
1
2
3
4
GP6
I feel ill ...............................................................................
0
1
2
3
4
GP7
I am forced to spend time in bed........................................
0
1
2
3
4
SOCIAL/FAMILY WELL-BEING
Not
at all
A little
bit
Some-
what
Quite
a bit
Very
much
GS1
I feel close to my friends....................................................
0
1
2
3
4
GS2
I get emotional support from my family ............................
0
1
2
3
4
GS3
I get support from my friends.............................................
0
1
2
3
4
GS4
My family has accepted my illness....................................
0
1
2
3
4
GS5
I am satisfied with family communication about my
illness..................................................................................
0
1
2
3
4
GS6
I feel close to my partner (or the person who is my main
support) ..............................................................................
0
1
2
3
4
Q1
Regardless of your current level of sexual activity, please
answer the following question. If you prefer not to answer it,
please mark this box
and go to the next section.
GS7
I am satisfied with my sex life ............................................
0
1
2
3
4

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FACT-An (Version 4)
Please circle or mark one number per line to indicate your response as it applies to the past 7
days.
EMOTIONAL WELL-BEING
Not
at all
A little
bit
Some-
what
Quite
a bit
Very
much
GE1
I feel sad ..............................................................................
0
1
2
3
4
GE2
I am satisfied with how I am coping with my illness..........
0
1
2
3
4
GE3
I am losing hope in the fight against my illness..................
0
1
2
3
4
GE4
I feel nervous.......................................................................
0
1
2
3
4
GE5
I worry about dying.............................................................
0
1
2
3
4
GE6
I worry that my condition will get worse............................
0
1
2
3
4
FUNCTIONAL WELL-BEING
Not
at all
A little
bit
Some-
what
Quite
a bit
Very
much
GF1
I am able to work (include work at home) ..........................
0
1
2
3
4
GF2
My work (include work at home) is fulfilling.....................
0
1
2
3
4
GF3
I am able to enjoy life..........................................................
0
1
2
3
4
GF4
I have accepted my illness...................................................
0
1
2
3
4
GF5
I am sleeping well ...............................................................
0
1
2
3
4
GF6
I am enjoying the things I usually do for fun ......................
0
1
2
3
4
GF7
I am content with the quality of my life right now..............
0
1
2
3
4

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Neal S. Young, M.D.  3/18/2020 (Amendment UU/47)
FACT-An (Version 4)
Please circle or mark one number per line to indicate your response as it applies to the past 7
days.
ADDITIONAL CONCERNS
Not at
all
A little
bit
Some-
what
Quite
a bit
Very
much
HI7
I feel fatigued .....................................................................
0
1
2
3
4
HI12
I feel weak all over.............................................................
0
1
2
3
4
An1
I feel listless ( washed out ) ..............................................
0
1
2
3
4
An2
I feel tired ...........................................................................
0
1
2
3
4
An3
I have trouble starting things because I am tired................
0
1
2
3
4
An4
I have trouble finishing things because I am tired .............
0
1
2
3
4
An5
I have energy......................................................................
0
1
2
3
4
An6
I have trouble walking........................................................
0
1
2
3
4
An7
I am able to do my usual activities.....................................
0
1
2
3
4
An8
I need to sleep during the day ............................................
0
1
2
3
4
An9
I feel lightheaded (dizzy) ...................................................
0
1
2
3
4
An10
I get headaches ...................................................................
0
1
2
3
4
B1
I have been short of breath.................................................
0
1
2
3
4
An11
I have pain in my chest.......................................................
0
1
2
3
4
An12
I am too tired to eat ............................................................
0
1
2
3
4
BL4
I am interested in sex..........................................................
0
1
2
3
4
An13
I am motivated to do my usual activities ...........................
0
1
2
3
4
An14
I need help doing my usual activities.................................
0
1
2
3
4
An15
I am frustrated by being too tired to do the things I want
to do....................................................................................
0
1
2
3
4
An16
I have to limit my social activity because I am tired..........
0
1
2
3
4

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FACT-Th11 (Version 4)
Please circle or mark one number per line to indicate your response as it applies to the past 7
days.
ADDITIONAL CONCERNS
Not
at all
A little
bit
Some-
what
Quite
a bit
Very
much
An5
I have energy......................................................................
0
1
2
3
4
An7
I am able to do my usual activities.....................................
0
1
2
3
4
Th1
I bleed easily ......................................................................
0
1
2
3
4
Th2
I bruise easily .....................................................................
0
1
2
3
4
Th3
I worry about problems with bruising or bleeding.............
0
1
2
3
4
Th5
I am bothered by nosebleeds..............................................
0
1
2
3
4
Th7
I am bothered by pinpoint bruising beneath my skin.........
0
1
2
3
4
Th8
I am bothered by blood in my urine or stool......................
0
1
2
3
4
Th
10
I avoid or limit physical activity (because of concern
with bleeding or bruising) ..................................................
0
1
2
3
4
Th
12
I am frustrated by not being able to do my usual
activities .............................................................................
0
1
2
3
4
Th
13
I worry that my treatment will be delayed (because of
low blood counts)...............................................................
0
1
2
3
4

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FACT-N (Version 4)
Please circle or mark one number per line to indicate your response as it applies to the past 7
days.
ADDITIONAL CONCERNS
None
of the
time
A little
of the
time
Some
of the
time
Most
of the
time
All
of the
time
N1
I worry about getting sick due to low blood counts............
0
1
2
3
4
N2
I avoid public places for fear of getting an infection ..........
0
1
2
3
4
P1
I get aches and pains that bother me ...................................
0
1
2
3
4
An14
I need help doing my usual activities..................................
0
1
2
3
4
N3
I worry about getting infections ..........................................
0
1
2
3
4
N4
I worry my condition will not improve if my treatment
is delayed.............................................................................
0
1
2
3
4
An5
I have energy.......................................................................
0
1
2
3
4
BRM3
I am bothered by fevers (episodes of high body
temperature) ........................................................................
0
1
2
3
4
BRM2
I am bothered by the chills..................................................
0
1
2
3
4
ES 3
I have night sweats..............................................................
0
1
2
3
4
An16
I have to limit my social activity because I am tired...........
0
1
2
3
4
MS10
I need to rest during the day................................................
0
1
2
3
4
An1
I feel listless ( washed out ) ...............................................
0
1
2
3
4
An13
I am motivated to do my usual activities ............................
0
1
2
3
4
N6
I have mouth sores ..............................................................
0
1
2
3
4
N7
My partner worries about me when my blood counts are
low.......................................................................................
0
1
2
3
4
N8
My low blood counts interfere with my intimate
relationships ........................................................................
0
1
2
3
4
An3
I have trouble starting things because I am tired.................
0
1
2
3
4
MS3
I am bothered by headaches ................................................
0
1
2
3
4

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Neal S. Young, M.D.  3/18/2020 (Amendment UU/47)
PROMIS Item Bank v1.0
Emotional Distress-Depression
Short Form 4a 
Investigator Format
© 2009 PROMIS Health Organization and PROMIS Cooperative Group
Page 1 of 1
Emotional Distress-Depression
Short Form 4a
Pleaserespond to each question or statement by marking onebox per row.
In thepast 7 days...
Never
Rarely
Sometimes
Often
Always
EDDEP04
1
I felt worthless................................ ............
1
2
3
4
5
EDDEP06
2
I felt helpless ................................ ..............
1
2
3
4
5
EDDEP29
3
I felt depressed ................................ ...........
1
2
3
4
5
EDDEP41
4
I felt hopeless ................................ .............
1
2
3
4
5

12-H-0150
84
Neal S. Young, M.D.  3/18/2020 (Amendment UU/47)
PROMIS Item Bank v1.0-Emotional Distress-Anxiety
Short Form 4a
Investigator Format © 2009 PROMIS Health Organization and PROMIS Cooperative Group
Page 1 of 1
Emotional Distress-Anxiety
Short Form 4a
Pleaserespond to each question or statement by marking onebox per row.
Never
Rarely
Sometimes
Often
Always
EDANX01
1
I felt fearful................................ ................
1
2
3
4
5
EDANX40
2
I found it hard to focus on anything other
than my anxiety ................................ ..........
1
2
3
4
5
EDANX41
3
My worries overwhelmed me.....................
1
2
3
4
5
EDANX53
4
I felt uneasy ................................ ................
1
2
3
4
5

12-H-0150
85
Neal S. Young, M.D.  3/18/2020 (Amendment UU/47)
PROMIS Item Bank v1.0 Sleep Disturbance
Short Form 4a 
Investigator Format
© 2009 PROMIS Health Organization and PROMIS Cooperative Group
Page 1 of 1
Sleep Disturbance
Short Form 4a
Pleaserespond to each question or statement by marking onebox per row.
Very poor
Poor
Fair
Good
Very good
Sleep109
1
My sleep quality was ................................
5
4
3
2
1
Not at all
A littlebit
Somewhat
Quitea bit
Very much
Sleep116
2
My sleep was refreshing. ...........................
5
4
3
2
1
Sleep20
3
I had a problem with my sleep..................
1
2
3
4
5
Sleep44
4
I had difficulty falling asleep ....................
1
2
3
4
5

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12-H-0150
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Neal S. Young, M.D.  3/18/2020 (Amendment UU/47)
APPENDIX F:  SOP 14E, APPENDIX B, TABLE 1
REQUIREMENTS FOR THE DETERMINATION OF AN LAR’S APPROPRIATENESS TO CONSENT 
TO RESEARCH NOT INVOLVING GREATER THAN MINIMAL RISK (CATEGORY A) AND FOR 
RESEARCH INVOLVING GREATER THAN MINIMAL RISK BUT PRESENTING THE PROSPECT 
OF DIRECT BENEFIT TO THE INDIVIDUAL SUBJECTS (CATEGORY B)
First preference is #1. If not possible, go to option #2. If #2 is not possible, go to option #3.)
Cognitively Impaired Adults and 
Identification of a LAR 
Requirements for Determining 
Appropriateness of LAR to Consent 
to Research at Clinical Center (CC) 
and non- CC sites 
Role of the LAR at all 
sites 
1. Adults who cannot consent and have a 
court-appointed guardian from a state that 
allows iti or a DPAii for healthcare and/or 
research participation. 
2. Adults who cannot consent and who do 
not have a DPA or court-appointed 
guardian, but who are capable of 
understanding the DPA process and can 
assign a DPAv .
3. Adults who cannot consent, who do not 
have a DPA or court-appointed guardian, 
and cannot appoint a DPA: 
At the CC: A person at the highest level of 
the following may consent to research 
participation if found to be appropriate: 1) 
spouse or domestic partner vi, 2) adult child, 
3) parent, 4) sibling, 5) other close relative 
At non-CC sites: Consult with OGC to 
identify applicable state law. 
PI/designeeiii, unless the IRB designates 
an independent person(s) to perform this 
role (e.g., ACATiv if the protocol is 
taking place at the CC), must assess 
appropriateness of LAR to consent to 
research. 
An appropriate LAR is one who at least: 
(a) Understands that the protocol 
involves research; 
(b) Understands the risks, potential 
benefits (if any), and alternatives to the 
study; and 
(c) Has sufficient reason to believe 
participation in the study is consistent 
with the subject’s preferences and 
values. 
LAR may give 
permission for the 
research and sign the 
consent form for the 
protocol on behalf of 
the subject. 
i A court appointed guardian may only consent to enroll a subject in research if the guardian has authority to do so under the 
laws of the state that issued the guardianship order and the terms of the guardianship order. The Office of the General 
Counsel (OGC) should be asked to review guardianship orders to determine if the guardian has legal authority to consent to 
the subject’s participation in the research. PIs are encouraged to seek an OGC consultation in advance of a potential subject 
with a guardianship order coming to an NIH research site to enroll on a study. 
ii DPA means the individual holding the durable power of attorney for healthcare. Consult with OGC if concerned about the 
authority provided in a DPA. 
iii If the protocol is taking place at the CC, the PI’s designee may be someone on the research team or a member of ACAT. If 
not at the CC, the PI’s designee may be someone on the research team or an independent person outside of the research team 
if it is felt that the team does not have the required competencies to undertake the evaluation. 
iv NIH Ability to Consent Team. For definition please see 14E.4.