Assessing Combination Hydroxyurea and Exogenous Erythropoietin in Sickle Cell Disease
Phase: PHASE1
Conditions: Anemia, Sickle Cell, Sickle Cell Disease
Sponsor: Julia Xu

 
 
Page 1 of 33 
 
 
Study Protocol Title: Assessing Combination Hydroxyurea and Exogenous Erythropoietin in 
Sickle Cell Disease (ACHiEvE-SCD) 
 
 
Principal Investigator 
Julia Xu, MD 
Department of Medicine 
University of Pittsburgh 
Biomedical Science Tower  
200 Lothrop Street, Suite E1254 
Pittsburgh, PA 15213 
E-mail xujz2@upmc.edu 
 
Sub-Investigators 
Titilope Adeyemo, MSc, FMCPath  
Vincent Osunkalu, PhD, FMCPath 
Enrico Novelli, MD, MS 
Laura De Castro, MD 
Busola Oluwole, MD, MS 
 
Institutions Involved in Conduct of Study (Study Sites): 
1) UPMC Adult Sickle Cell Clinic / University of Pittsburgh 
2) Lagos University Teaching Hospital / University of Lagos 
 
 
Protocol Version: 
Modification: MOD21120027-019 
IRB Approval Date: 7/10/24

 
 
Page 2 of 33 
 
1. Introduction 
 
 
1.1 
Background 
 
Chronic anemia is a hallmark complication of sickle cell disease (SCD). SCD is a major global 
health problem, with over 300,000 SCD births annually.1 Polymerization of deoxygenated 
hemoglobin (Hb) S and consequent sickling of erythrocytes lead to the hallmark manifestations 
of chronic hemolytic anemia and acute vaso-occlusive crises (VOCs) that result in multiorgan 
damage and decreased life expectancy.2,3 The pathophysiology of anemia in SCD is multifactorial, 
with the predominant mechanisms of red blood cell (RBC) destruction being intravascular and 
extravascular hemolysis resulting from damage to RBC membrane and contents through 
repeated cycles of HbS polymerization and depolymerization, phosphatidylserine exposure (PS) 
facilitating complement-mediated lysis, loss of RBC deformability, depletion of adenosine 
triphosphate (ATP) and RBC dehydration, oxidative stress, and clearance of deformed sickle 
erythrocytes by the reticuloendothelial system.4  
 
Anemia plays a key role in the pathogenesis of the chronic multi-organ damage in SCD. 
Cardiopulmonary disease, SCD nephropathy, and cerebrovascular disease are the leading 
causes of morbidity and mortality in SCD and are all exacerbated by the chronic anemic state and 
hypercirculatory physiology in SCD.2,3,5 Compensatory increased cardiac output, lowered 
systemic vascular resistance, and neovascularization result in cardiomegaly, left ventricular 
hypertrophy, high output heart failure, pulmonary hypertension, proliferative retinopathy, and 
cerebral vasculopathy, among other complications.5 Anemia activates the renin-angiotensin-
aldosterone system and sympathetic nervous system, contributing to renal and vascular 
dysfunction, chronic inflammation, and increased oxidative stress.6,7 Furthermore, chronic anemia 
affects the quality of life and physical functioning of individuals with SCD. Fatigue and weakness 
associated with anemia may greatly limit patients’ physical activity, daily functioning, level of 
disability, and overall health-related quality of life.8-11 Additionally, neurocognitive impairment has 
been reported in a large proportion of children and adults with SCD, even in the absence of overt 
or silent strokes suggesting that other factors may contribute to its pathogenesis.12 Two cross-
sectional studies have shown better cognitive performance of individuals taking therapies that 
improve anemia when compared to participants not on these therapies.13 As oxygen delivery is 
crucial for brain function, it is feasible that an increase in hemoglobin level may improve 
neurocognitive outcomes [6].  
 
There are limited therapeutic options for anemia in SCD. Blood transfusions are the mainstay 
therapy for acute anemic episodes but carry risks of hemolytic transfusion reactions, 
alloimmunization, and iron overload. Additionally, anecdotal case reports suggest that rapid 
correction of anemia through simple transfusions may increase the risk of viscosity-related 
complications, likely related to the decreased deformability and increased adhesiveness of sickle 
RBCs.14 As such, the risk of increasing blood viscosity must be weighed against the benefits of 
correcting anemia. Hydroxyurea has a salutary anti-sickling effect, but it has only a modest effect 
on Hb levels, particularly in the adult population.15,16 Chronic anemia is not an indication for 
initiating hydroxyurea therapy, and patients who remain anemic despite hydroxyurea need 
additional therapy. Voxelotor (Oxbryta®, GBT-440) received recent FDA approval on the basis of 
raising hemoglobin levels in SCD,17 though its mechanism of action of increasing Hb oxygen 
affinity has raised concerns about its effects on tissue oxygen delivery.18-21 Indeed, the effects of 
increasing Hb level and blood viscosity may be tissue-specific, demonstrating the need for better 
laboratory biomarkers and tissue-specific measures of blood flow, oxygen saturation, and 
vascular tone.  

 
 
Page 3 of 33 
 
 
Erythropoiesis-stimulating agents (ESAs) may be an effective therapy for anemia in SCD patients 
who remain anemic despite hydroxyurea. ESAs are the standard of care for anemia in chronic 
kidney disease (CKD) and may be ideal for use in SCD due to the high prevalence of subclinical 
renal dysfunction in SCD. In fact, ESAs have long been used in clinical practice for SCD patients 
who have severe or symptomatic anemia and cannot tolerate or accept blood transfusions, have 
severe renal dysfunction, or have an acute aplastic or hemolytic crisis (e.g., acute parvovirus B19 
infection or hyperhemolysis associated with delayed hemolytic transfusion reactions). 
Furthermore, ESAs have been shown to have potential neuroprotective and neurogenerative 
effects22 and may improve neurocognitive functioning in animal models23 and in patients with 
neuropsychiatric disorders.24,25 However, the use of ESA therapy in combination with hydroxyurea 
is still not considered standard-of-care for the treatment of anemia in SCD. Older case reports 
and small studies,26-37 as well as recent retrospective studies have shown that ESAs may increase 
Hb level and Hb F %,33 as well as decrease transfusion burden in patients with SCD.30,32 
Moreover, these studies have not identified an increased risk of VOC or venous thromboembolism 
(VTE) with ESA therapy. Additionally, patients stabilized on hydroxyurea are potentially protected 
from developing vaso-occlusive and viscosity-related complications due to the inhibitory effect of 
HbF on HbS polymerization, and ESAs may synergistically potentiate the Hb F-inducing effect of 
hydroxyurea by increasing stress erythropoiesis.35 Despite the abundance of clinical reports of 
ESA utilization in SCD in the literature,26-37 anecdotal evidence of increased pain or VOCs after 
ESA treatment in SCD patients31,33 and a lack of practice guidelines for how to manage ESA 
therapy in combination with hydroxyurea have led ESAs to be prescribed ad hoc by SCD providers 
and to be potentially underutilized as a therapeutic option. Therefore, there is a need for 
prospective evaluation of the efficacy and safety of ESAs in combination with hydroxyurea as a 
therapeutic strategy for anemia in the SCD population. 
 
1.2 Rationale 
 
Recombinant human erythropoietin (rHuEPO) is commonly used for the treatment of anemia of 
CKD. Epoetins (and its biosimilars) are short-acting rHuEPO drugs that are widely available 
globally. The use of EPO (which henceforth refers to epoetins) in combination with hydroxyurea 
may alleviate the detrimental effects of chronic anemia without increasing vaso-occlusive and 
viscosity-associated complications. In addition, this study will allow for the identification of 
predictors of response to EPO and evaluation of potential biomarkers for monitoring the safety 
and effect of increasing hemoglobin levels on cardiopulmonary status, health-related quality of 
life (HRQoL), red cell health, and tissue hemodynamics.  
 
If successful, our study will advance SCD care and research in multiple settings:  
1) This pilot study will provide prospective evidence of the efficacy and safety of EPO in patients 
with SCD who remain anemic despite hydroxyurea therapy, potentially broadening the menu of 
standard therapeutic options for anemia in SCD; 
 
2) Evaluation of baseline biomarkers (e.g., markers of erythropoiesis, hemolysis, renal function, 
iron homeostasis, and inflammation) in prospectively treated patients will allow for identification 
of predictors of response to EPO in the SCD population;  
 

 
 
Page 4 of 33 
 
3) Evaluation of cardiopulmonary status, neurocognitive function, and HRQoL at baseline and 
after treatment with EPO will prospectively demonstrate the effect of raising Hb levels on 
important clinical and HQoL outcomes; 
 
4) Evaluation of in vivo biomarkers of tissue hemodynamics using DSM colorimeter, and diffuse 
optical technologies (i.e., near-infrared spectroscopy (NIRS)), as well as evaluation of neural 
activity using electroencephalography (EEG), will provide novel insights into tissue-specific 
physiological effects of raising Hb level in patients with SCD; and finally,  
 
5) Evaluation of biomarkers of hemorheology and cardiovascular and thrombotic risk (e.g., 
blood viscosity, markers of myocardial strain and hemostatic activation) will help identify 
potential harmful effects of raising Hb level in patients with SCD and will provide insight into an 
optimal Hb target range that balances clinical benefit and safety in patients with SCD. 
 
 
2. Clinical Study Objectives 
 
2.1 Primary objectives 
 
Prospectively evaluate the efficacy and safety of EPO in combination with hydroxyurea in SCD. 
In SCD patients stabilized on hydroxyurea, we will dose escalate EPO to reach a Hb target of 
10-11 g/dL and measure the rates of Hb response during treatment. 
  
2.2    Secondary objectives 
 
Prospectively evaluate the effect of EPO in combination with hydroxyurea on transfusion burden 
in SCD. We will measure the annualized number of simple red blood cell transfusions received 
before and during treatment. 
 
 
2.3 Exploratory Objectives 
 
a) Evaluate the effect of raising Hb level on clinical outcomes. We will evaluate changes in 
cardiopulmonary status, neurocognitive function, and HRQoL after treatment compared to 
baseline using echocardiography, exercise testing (six-minute walk test), and validated HRQoL 
questionnaires.  
 
b) Identify predictors of response to EPO. We will assess the correlation between Hb response 
to EPO therapy and a variety of biomarkers at baseline and end of study, including markers of 
renal function, iron homeostasis, inflammation, erythropoiesis, and hemolysis.  
 
 
c) Identify biomarkers of the risk and benefit of raising Hb level using EPO therapy. We will 
evaluate changes in a variety of biomarkers after treatment compared to baseline, including 
laboratory biomarkers of red cell health, blood viscosity, and cardiovascular or thrombogenic 
risk, as well as in vivo biomarkers of blood flow, tissue oxygen saturation, and vascular tone 
using near-infrared (NIR) technologies, specifically frequency-domain near-infrared 
spectroscopy (FD-NIRS) and diffuse correlation spectroscopy (DCS). We will be using DSM III 
colorimeter, a highly sensitive device for skin color detection to help enhance the accuracy of 

 
 
Page 5 of 33 
 
data analysis. We will perform these assessments in collaboration with research team members 
from Carnegie Mellon University. In addition, we will evaluate neural activity after treatment 
compared to baseline using electroencephalography (EEG).  
These measurements will be performed only at study sites where the equipment and expertise 
are available. For instance, all endpoints will be obtained at Pitt, but only some measurements 
with NIR technologies will be performed at the Nigeria site due to lack of equipment to perform 
all measurements (e.g., DCS will not be available in Nigeria). EEG also will not be performed on 
participants in Nigeria due to lack of the equipment and expertise.   
 
 
3. Study Design 
 
 
This is a Phase 1b/2 prospective, single-arm, open-label pilot study of EPO in patients with 
SCD. Eligible individuals will be consented and undergo baseline testing. EPO dosing will be 
consistent with the routine clinical practice of the investigators, and as informed by published 
literature and expert opinion. EPO treatment will be initiated at a standard dose of 10,000 units 
three times weekly (TIW), with biweekly clinic visits and phlebotomy for clinical monitoring, dose 
titration, and research laboratory testing (Figure 1 and Table 1). EPO dose will be escalated by 
10,000 units per dose every 2 weeks until reaching a maximum dose of 40,000 units TIW or the 
minimal effective dose for maintaining a Hb level of 10-11 g/dL (Table 2). Dose escalation will 
not be performed if there are any safety concerns, such as adverse events or clinically 
significant laboratory abnormalities related to treatment. A dose reduction or temporary holding 
of therapy may be performed per the PI’s discretion and clinical judgment if any safety concerns 
arise that are assessed by the PI as related to EPO therapy or to increases in hemoglobin level, 
including the following criteria: Hb increase to > 11.0 g/dL, rapid rise in Hb (defined as a > 2 
g/dL increase within 4 weeks), development of increased pain or vaso-occlusive crisis, or any 
Grade ≥ 3 adverse events assessed as related to treatment (Table 3).  
 
A 12-week treatment period was selected based on guidance from the drug label of EPO stating 
that a hemoglobin response is expected within 12 weeks. Following this 12-week treatment 
period, participants who benefit from the therapy may elect to continue EPO treatment clinically 
in discussion with their primary SCD provider. After the 12-week timepoint (end of treatment 
(EOT)), all ongoing treatment decisions will be made clinically, and we will collect endpoints at 
one additional time point at 24 weeks, considered the end of study (EOS) testing. Participants 
may also elect to stop EPO therapy after completion of the 12-week treatment period. 
Participants who permanently discontinue EPO therapy prior to the end of the 12-week period 
will not be considered as completing the study and will complete the EOT testing, after which 
they will be withdrawn from the study. Participants in whom EPO treatment is temporarily held 
for any safety reasons will remain on study and be considered completers so long as the drug is 
not held for more than 4 out of the 12 weeks and treatment is restarted at least 2 weeks prior to 
the EOT visit and testing. Hydroxyurea and L-glutamine dose titration and holding are not 
permitted on study, unless otherwise clinically indicated to ensure the participant’s safety.  
 
 
3.1 Study design schematic 
 
Figure 1: Schematic of treatment period and follow-up visits. 
 

 
 
Page 6 of 33 
 
 
 
 
 
Table 1: Schedule of Events 
 
Study Epoch 
Screening 
Treatment 
Visit Number 
1 
2 
3 
4 
5 
6 
7 
8 
9 
Week on Study Intervention 
 
Day -60 to 
Day 0 
Day 0 
Baseline 
and Drug 
Initiation 
 
Wk 2 
 
Wk 4 
 
Wk 6 
 
Wk 8 
 
Wk 10 
 
Wk 12 
EOT 
 
Wk 24 
EOS 
                           Assessment Window  
Study Procedure 
-7 d 
±3 d 
±3 d 
±3 d 
± 3 d 
± 3 d 
± 7 d 
± 7 d 
Informed consent 
x 
Demographics and Review of 
Eligibility 
X 
Review of Inclusion & 
Exclusion criteria 
X 
 
Pregnancy test** 
X 
X 
 
 
 
 
 
 
 
Medical History*** 
 
X 
 
 
 
 
 
X 
X 
Vital signs  
X 
X 
x 
x 
x 
x 
x 
X 
X 
Physical assessment (including weight) 
 
X 
 
x 
 
x 
 
X 
X 
Height 
 
X 
 
 
 
 
 
 
 
Dosing 
X 
x 
x 
x 
x 
x 
 
 
Clinical Labs# 
x 
X 
x 
x 
x 
x 
x 
X 
X 
Biomarkers 
 
X 
x 
x 
x 
x 
x 
X 
X 
Transthoracic echocardiography (TTE)* 
X 
 
 
 
 
 
X 
X 
6MWD Test with Borg Dyspnea Scores 
(performed by research staff) 
X 
 
 
 
 
 
X 
X 
Quality of Life Questionnaires 
X 
 
 
 
 
 
X 
X 
Neurocognitive assessment 
 
X 
 
 
 
 
 
X 
X 
DSM Colorimeter, NIRS and EEG 
X 
 
x 
 
x 
 
X 
X 
Painimation Assessments 
 
X 
x 
x 
x 
x 
x 
X 
X 
AE and SAE assessments 
 
X 
x 
x 
x 
x 
x 
X 
 
Acute care utilization† 
 
X 
x 
x 
x 
x 
x 
X 
X 
 
* Clinical TTE obtained within 180 days of the screening date (if obtained at baseline state of health, without transfusion in 60 
days prior, and within 1 g/dL of screening hemoglobin level) can be used for the baseline TTE results if all research parameters 
were captured (i.e., TTE will not be repeated at baseline). 
 
** For women of childbearing potential, a serum pregnancy test will be performed at the Screening Visit, and a urine pregnancy 
test will be performed at the Baseline visit. 

 
 
Page 7 of 33 
 
 
*** Medical history includes the number of acute vaso-occlusive events, thrombotic complications, and transfusions in the 12 
months prior to baseline, at 12 week (EOT) and at 24 week (EOS) (Refer to Table 5). General medical history will be captured 
at baseline and updated as needed.  
 
# Safety labs checked at every visit include CBC/Diff, reticulocyte count, CMP, direct bilirubin, and LDH. Other clinical labs may 
only be checked at some visits, e.g., at baseline, 12 weeks (EOT), and 24 weeks (EOS) (refer to Table 4). 
 
 Biomarkers include the research labs listed in Table 4 (note that some are only drawn at baseline, EOT, and EOS). 
 
† Frequency of and reason for acute care visits, defined as any non-routine visit to an urgent care, infusion center, emergency 
room, or hospital to seek medical attention. This will be assessed at every study visit, as well as once per month by phone call 
by a member of the study team in between weeks 12 and 24.  
 
Table 2: Dose Modification Schedule 
 
 
Dose Modification Schedule 
 
 
Dose Level 
Dose of EPO 
If Hb > 11.0 (confirmed on 
repeat Hb measurement), 
reduce to: 
If second reduction needed: 
Level 3 
40,000 units TIW  30,000 units TIW (Level 2) 
 20,000 units TIW (Level 1) 
Level 2 
30,000 units TIW  20,000 units TIW (Level 1) 
10,000 units TIW (Level 0) 
Level 1 
20,000 units TIW 10,000 units TIW (Level 0) 
10,000 units BIW (Level -1) 
Level 0 
10,000 units TIW  10,000 units BIW (Level -1) 
 10,000 units weekly (Level -2) 
 
 
 
 
Table 3: Dose Reduction Criteria 
 
Dose Reduction Criteria 
Hb increase to > 11.0 g/dL (confirmed on 
repeat Hb measurement) 
> 2 g/dL Hb increase in 4 weeks 
Increased pain/VOC symptoms 
Grade 3 hypertension (defined as systolic 
blood pressure ≥160 mmHg or diastolic blood 
pressure ≥100 mmHg; medical intervention 
indicated; more than one drug or more 
intensive therapy than previously used 
indicated) on two consecutive measurements 
Grade ≥ 3 treatment related adverse events 
 
 
 
3.2 Participant Selection: 
 
Participants:   
 

 
 
Page 8 of 33 
 
This prospective interventional study proposes to enroll up to 25 participants with SCD, of HbSS 
and HbS/β0-thalassemia genotypes, from USA and Nigeria. Participants will be either from the 
University of Pittsburgh Medical Center (UPMC) Adult Sickle Cell Disease program and UPMC 
hospitals that serves adult participants with SCD from the Pittsburgh community, or from Lagos 
University Teaching Hospital and the surrounding community. The goal is to get a combined 
total of 15 enrolled participants, from both sites, to complete the study. The study will conclude 
once 15 participants have completed the study. Other genotypes, including HbSC and S/β+-
thalassemia, will not be eligible since these genotypes tend to have milder anemia and 
potentially different pathophysiology and would be too heterogeneous of a group to include in 
this pilot study. Male and female adult participants will be recruited in steady state, defined as 
absence of VOC or acute illness as determined by their treating hematologist, and be on a 
stable dose of hydroxyurea, defined as no dose adjustments within the 60 days prior to 
screening and no planned future dose adjustments. The study will conclude once 15 
participants have completed the study; a completer is defined as a participant that has 
completed the 12-week treatment period and testing.  
 
Lagos University Teaching Hospital site: English is the primary language spoken in Nigeria. 
Lagos is a cosmopolitan metropolis, and most people speak English, therefore, we do not 
anticipate any language barrier. In an effort to avoid exclusion of disadvantaged populations in 
Nigeria, we will not exclude individuals who cannot speak, read, or write English. For individuals 
recruited in Nigeria who do not speak or read English, there will be interpreters available and 
consent forms will be translated into Yoruba, which is the major local language spoken in the 
area where the study recruiting will be carried out. In the event that participants cannot read any 
language, the research nurse will read the ICF out loud to the participant. If the participant 
cannot write, whenever required, a thumb print will be used in place of a signature. 
 
3.3 Participant inclusion criteria : 
 
SCD Participants : 
Inclusion Criteria:  
 
• 
Aged ≥ 18 years 
• 
Confirmed SCD (HbSS or HbS/β0-thalassemia) by hemoglobin electrophoresis, high-
performance liquid chromatography, DNA genotyping, or other comparable 
hemoglobin diagnostic testing 
• 
Screening Hb ≤ 9.0 g/dL   
• 
Screening transferrin saturation ≥ 20% and ferritin ≥ 50 ng/mL 
• 
Must be on stable-dose hydroxyurea treatment (i.e., no changes in dose within 60 days 
prior to screening) and plan to continue taking hydroxyurea at the same dose and 
schedule during the study 
• 
If receiving L-glutamine or crizanlizumab, must have been receiving the drug at a 
stable dose for at least 60 days prior to screening and plan to continue taking the drug 
at the same dose and schedule during the study. 
 
 
3.4 Participant exclusion criteria : 
 
SCD Participants : 
Exclusion Criteria:  
 

 
 
Page 9 of 33 
 
• 
Participating in a chronic transfusion program (pre-planned series of transfusions for 
prophylactic purposes) and/or planning on undergoing an exchange transfusion 
during the duration of the study; episodic transfusion in response to worsened 
anemia or VOC is permitted, but participant should not have received a blood 
transfusion within 60 days of screening 
• 
Received voxelotor or EPO within 60 days of screening 
• 
Untreated iron deficiency, or had initiation or change in dose of supplemental iron 
within 30 days of screening 
• 
Ongoing acute illness, infection, or VOC within 2 weeks of screening 
• 
Arterial or venous thrombosis within 180 days of screening 
• 
Grade 3 hypertension (defined as systolic blood pressure ≥160 mmHg or diastolic 
blood pressure ≥100 mmHg; medical intervention indicated; more than one drug or 
more intensive therapy than previously used indicated) on two consecutive 
measurements 
• 
Unstable angina, uncontrolled seizure disorder, or active malignancy 
• 
End-stage renal disease requiring hemodialysis  
• 
Current pregnancy or breast feeding 
• 
Received active treatment on another investigational trial within 30 days (or 5 half-
lives of that agent, whichever is greater) prior to screening visit or plans to participate 
in another investigational drug trial 
 
 
Participant Recruitment: 
 
Participants in the US will be recruited from the UPMC Adult Sickle Cell Program and other 
UPMC hospitals while those in Nigeria will be recruited from Lagos University teaching hospital 
through method(s) approved by the Lagos Teaching University Hospital (LUTH) Health 
Research Ethics Committee (HREC), an equivalent body to the IRB.  
 
In the US, the physician investigators or a research coordinator will initiate contact with the 
participants at the time they present for their routine follow up appointments at their respective 
clinics, by phone, or by flyers distributed in print or electronically. The study team has usual 
clinical access to the medical records of subjects via Dr. Xu. If the study team identifies an 
eligible subject who is not known to the study team from prior clinical care, a clinician known to 
the patient will obtain their permission to approach for research before initiating recruitment 
discussions. The physician or his/her delegate will then review the participants’ records to 
identify if they meet the inclusion criteria.  
 
In Lagos, participants will be identified and recruited by the study team physicians in the clinics 
of the Lagos Teaching Hospital study site with direct 1:1 contact with subjects. No phone scripts 
or flyers will be used for recruitment. 
 
A total of 50 participants will undergo screening across the 2 sites. A total of up to 25 
participants will be enrolled, and a combined total of 15 participants, from both sites, will 
complete study procedures. US Participants will be compensated a total of $ 675 for 
participation in the study, which translates to $75 for each completed study visit. Participants in 
Nigeria will be compensated a total of ₦112,500 Naira (an equivalent of about $270) for 
participation in the study, which is ₦12,500 Naira (about $30) for each visit. This compensation 
accounts for approximately 3-4 hours’ wage, the cost of travel, inflation, and other out-of-pocket 
expenses incurred by participating in the study. 
 

 
 
Page 10 of 33 
 
Consent Process: 
 
The consent process will be conducted by a physician investigator or his/her delegate. Consent 
agreement will be obtained by the Principal Investigator (PI) or a study co-investigator after a 
potential participant has had the opportunity to ask the investigator all the questions they have, 
and have those questions answered to their satisfaction. Prior to performing any of the research 
study procedures or interventions, participants must provide informed consent. The PI will make 
certain that an appropriate informed consent process is in place to ensure that potential 
research participants, or their authorized representatives, are fully informed about the nature 
and objectives of the clinical study, the potential risks and benefits of study participation, and 
their rights as research participants. The investigator will obtain the written, signed informed 
consent of each participant prior to performing any study-specific procedures on the participant. 
The date and time that the participant signs the informed consent form and a narrative of the 
issues discussed during the informed consent process will be documented in the participant’s 
case history. The investigator will retain the original copy of the signed informed consent form, 
and a copy will be provided to the participant. 
Participants in Nigeria will be consented through method(s) approved by the LUTH HREC. 
Consent will be obtained by a physician investigator at the LUTH site. 
 
Screen failures: 
After signing the informed consent document, participants will undergo screening tests to 
ensure they meet all eligibility criteria. Those individuals who are eligible by criteria and have a 
clinical indication to start treatment with EPO will be provided with a clinical prescription for the 
drug, which they will need to fill prior to returning for the Day 0/drug initiation study visit. 
Individuals who do not fill the clinical prescription within the 60-day screening period or whose 
clinical status changes to meet exclusion criteria during the screening period will be considered 
screen failures. These screen failures may be consented and rescreened, up to a maximum of 3 
total screening attempts. They will be assigned the same study participant number upon 
rescreening. 
 
Discontinuation of Individual Research Participants: 
We will make our best effort to retain participants and to complete their participation in the 12-
week treatment period. However, if a participant does not comply with the study procedure/s or 
treatment with study drug, the study team will communicate with the participant and try to 
understand the reason. If the participant wishes to discontinue participation or the study team 
feels that it would be unsafe to continue the participant on the treatment protocol (for instance, 
for participants who become pregnant while on study and require discontinuation of 
hydroxyurea), the PI will inform the participant about the decision and will withdraw the 
participant. All the data and biospecimen collected up to that point will be retained for future 
analyses.  
 
PI discontinuation of the clinical research study: 
The University of Pittsburgh Institutional Review Board (IRB) will be notified promptly of 
discontinuation of the entire clinical study. All protocol modifications will be submitted 
prospectively to the University of Pittsburgh IRB for discontinuation of parts of the clinical 
research study.      
 
Cost to Participant:  
 
It will not cost anything above the standard costs of clinical care for participants to participate in 
this study. All non-clinical charges for study related activities will be paid for by the study. All 

 
 
Page 11 of 33 
 
Standard of Care procedure deemed necessary and independent of study activities will be 
incurred by the participants.  
 
Currently, EPO is used off-label in clinical practice for treatment of SCD patients with persistent 
anemia despite optimal treatment with hydroxyurea in the US. The cost of the study drug, EPO, 
will be paid for by the study. EPO will be dispensed to the participants through the UPMC 
Investigational Drug Services (IDS) pharmacy. 
 
Participants may also qualify for a limited amount of patient assistance while on study, in line 
with what is offered routinely to patients in the UPMC Adult Sickle Cell Program clinic who 
express financial need.  
 
EPO is available in Nigeria but is not affordable for the vast majority of the patient population. 
Therefore, the cost of EPO drug will also be covered by the study for participants at the Nigerian 
site.   
 
4. Study Interventions 
 
Participants will be treated with escalating doses of EPO, administered subcutaneously three 
times weekly (TIW) for 12 weeks. EPO dose will be initiated at 10,000 units TIW and escalated 
as tolerated by 10,000 units per dose every 2 weeks until reaching a Hb target of 10-11 g/dL, up 
to a maximum dose of 40,000 units TIW (Table 2). 
 
4.1  Drug supplies 
 
 
4.1.1 Preparing and dispensing 
 
In the US, EPO will be supplied free of cost to the participants by the study team through the 
UPMC IDS pharmacy. In Nigeria, EPO will be prescribed clinically and dispensed by the study 
team, because the needed dosages of EPO are not available in the hospital and community 
pharmacies. It is common practice in Nigeria for patients to source their EPO drug directly from 
suppliers who are licensed pharmaceutical companies with importation permits rather than a 
pharmacy. The LUTH study team will therefore regularly source the required doses of EPO 
directly from a licensed supplier and store the study drug safely in a refrigerator with locks and 
temperature monitor and alarms. The refrigerator will be located in a limited-access locked 
research suite accessible to the study team only. The LUTH team will dispense the correct 
amount and dose of drug to participants at each study visit as indicated by the protocol. This is 
standard practice in interventional clinical trials conducted in Lagos. The study team’s research 
nurses are trained in study drug storage, dispensing, and record keeping, and only licensed 
pharmacists are permitted to be drug suppliers/vendors. 
 
4.1.2 Drug administration 
 
After enrollment onto the study and filling of the clinical prescription for EPO, participants will be 
scheduled for their Day 0/drug initiation study visit. Each study site will use the epoetins 
available to them at their site and will carry out their visits in designated clinics. For participants 
in the US, all study activities will be done in the UPMC Adult Sickle Cell Program clinic. At the 
Day 0 visit, participants will be educated on how to administer subcutaneous injections of EPO 
by the research or clinical team, as well as provided with a recorded instruction video to review 
at home. After the participant and the research team are comfortable with the participant’s skills 
for self-administration, they will return home and self-administer doses of EPO at home. If a 
participant cannot physically or does not feel comfortable self-administering the injections, a 

 
 
Page 12 of 33 
 
caretaker may be identified and trained to administer the subcutaneous injections at home. The 
research or clinical team may help the patient with drug administration when the patient is 
present in the UPMC Adult Sickle Cell Program clinic if needed. The study drug should not be 
administered via an intravenous catheter. 
 
4.2  Concomitant Medications 
 
A list of concomitant medication will be collected at screening and updated prior to starting study 
drug for all participants. 
 
5. Study Procedures: 
 
 
5.1 Assessment of adverse events 
 
Adverse events (AEs) will be graded by severity using CTCAE version 5.0. Assessment of 
relationship to the study procedure or treatment will be performed by either the PI or a delegate 
on the clinical research team that is overseen by the PI. All AEs will be collected and compiled 
in a research database by a member of the research team. Serious AEs (SAEs) will be collected 
and reported according to reporting protocol in Section 7.  
 
 
5.2 Blood and Urine Biomarkers 
 
Blood sampling for hematological parameters and circulating biomarkers of hemolysis, renal 
function, iron status, inflammation, red cell rheology, adhesion, and thrombogenesis will be 
performed at baseline and throughout the study according to Table 4. Fresh blood will be used 
for some assays and will be performed depending on availability at each research site. The 
remainder of the blood will be processed, and frozen plasma or serum will be stored and then 
batch-tested for the biomarkers outlined in Table 4. Urine will also be collected for baseline and 
end of study tests including measurements of biomarkers of kidney function as indicated in 
Table 4. 
The study collaborators in Nigeria will perform all common clinical laboratory assays available to 
them at their site in Nigeria. The remainder of the laboratory assays will be stored and shipped 
from Nigeria to University of Pittsburgh for research purposes only. An MTA will be signed. 
 
Table 4. Summary of Blood and Urine Biomarkers 
Sample 
Type 
Volume of 
blood 
(Collection 
tube) 
Frequency 
Clinical 
Complete blood count, absolute 
reticulocyte count 
Fresh 
blood 
3 mL (EDTA) 
Screening, 
Baseline, Q2 
weeks, EOT, EOS 
Lactate dehydrogenase, basic 
metabolic panel, hepatic function panel 
Fresh 
blood 
4.5mL (LtGreen) 
 
Screening, 
Baseline, Q2 
weeks, EOT, EOS 
Iron homeostasis (TIBC, ferritin, 
haptoglobin)  
Fresh 
blood 
Screening, EOT, 
EOS 
Erythropoietin level 
Fresh or 
frozen 
serum 
5 mL (GoldSST)  
Baseline 
Serum and/or Urine Pregnancy Test 
Fresh 
blood or 
urine 
5 mL (GoldSST)  
or 10 mL (Clean 
catch up) 
Screening (serum), 
Baseline (urine) 

 
 
Page 13 of 33 
 
 
 
 
 
5.3 Transthoracic echocardiography  
 
Evaluation of cardiac anatomical structure and function using standard echocardiographic 
techniques and equipment will be performed and interpreted by qualified personnel. Specifically, 
2D and Doppler echocardiographic assessment of left ventricular systolic/diastolic function, right 
ventricular systolic function, and right ventricular systolic pressure (tricuspid regurgitant jet 
velocity): Standard echocardiographic views will be used, with particular attention to specific 
measurements of interest for this study and depending on the ability to perform specific 
measurements at each research site: Pulse wave of mitral inflow in 4 chamber views; 
continuous wave Doppler of TRV in 4 chamber and parasternal views; tissue Doppler of septal 
and lateral mitral annulus and TV annulus at the RV free wall; left atrial (end-systolic) and left 
ventricular (end systolic and end diastolic) volume measurements in the apical 2- and 4-
chamber views; tricuspid annular plane systolic excursion (TAPSE) by M-mode; and cardiac 
index estimated by Doppler. Right and left ventricular ejection fraction will be derived as indices 
HPLC 
Fresh 
Blood 
3 mL (EDTA) 
Baseline, EOT, 
EOS 
Urine albumin-to-creatinine ratio 
(random urine creatinine and urine 
albumin) 
Urine 
10 mL (Clean 
catch cup) 
 
Baseline, EOT, 
EOS 
Exploratory/ 
Research 
(Optional) 
Markers of iron homeostasis (e.g., 
hepcidin, soluble transferrin receptor, 
hemopexin, erythroferrone), B-type 
natriuretic peptide and IL-6 
Frozen 
plasma 
6 mL (EDTA) 
Baseline, EOT, 
EOS 
Markers of thrombosis (e.g., D-dimer, 
prothrombin fragment 1.2, thrombin-
antithrombin complexes, soluble P-
selectin) 
Frozen 
plasma 
3 mL (Blue) 
Baseline, EOT, 
EOS 
Cystatin C, Troponin, C-reactive 
protein 
Frozen 
Serum 
5 mL (GoldSST) 
Baseline, EOT, 
EOS 
Blood samples for future genetic 
studies 
Frozen 
DNA 
Included with 
other biomarkers 
(EDTA) 
Baseline 
Whole blood viscosity, red cell 
deformability and point of sickling 
(Lorrca ektacytometry), F-cell fraction 
Fresh 
blood 
6 mL (EDTA) 
Baseline, Q4 
weeks, EOT, EOS 
Microfluidic flow assays 
Fresh 
blood 
6 mL (EDTA) 
Baseline, Q4 
weeks, EOT, EOS 
Phosphatidylserine exposure, markers 
of platelet activation (e.g., surface P-
selectin/CD62, activated GPIIb/IIIa) 
using flow cytometry, and platelet 
bioenergetics (e.g., mitochondrial 
measures and ROS generation) 
Fresh 
blood 
8 mL (ACD) 
Baseline, Q4 
weeks, EOT, EOS 
Urine Biomarkers ( Kidney Injury 
Molecule-1 (KIM-1), IGFBP7, TIMP-2, 
Nephrin, Alpha-1-microglobulin (A1M), 
IL-18, L-Fatty acid binding protein (L-
FABP), Heme/Hemoglobin 
 
 
Urine 
Included with the 
clinical urine 
sample 
collected. 
(Clean catch 
cup) 
Baseline, EOT, 
EOS 

 
 
Page 14 of 33 
 
of systolic ventricular function. TAPSE and RV annular velocity will give additional measures of 
RV systolic function, presence of LV diastolic dysfunction (Grades I-III) and elevated left atrial 
pressures will be determined according to updated guidelines established by the American 
Society of Echocardiography. RV systolic pressure will be derived from TRV using the modified 
Bernoulli equation. A cardiologist will be consulted if necessary to review the transthoracic 
echocardiograms if there are results of clinical concern. 
 
 
5.4 Six-minute walk test with Modified Borg Dyspnea Scale 
 
The 6MW Test will be carried out indoors, along a long, flat, straight, enclosed corridor with hard 
surface that is seldom traveled. The walking course must be 30 meters in length, but not less 
than 25 meters. The length of the corridor and turnaround points should be marked. Patients will 
be instructed to walk alone, not run, from one end to the other end of the walking course, at their 
own pace, while attempting to cover as much ground as possible in 6 min. The Modified Borg 
Dyspnea Scale will be measured in conjunction with the 6MW Test to measure the level of 
severity of breathlessness and fatigue perceived by the patient before and after 6MW test. The 
severity is measured on a 10-point scale with 0=nothing at all and 10=maximum severity of 
breathlessness or fatigue. Vital signs will be taken before and after the 6MW test.  
 
 
5.5 Assessment of health-related quality of life  
 
Participants will be asked to complete a combination of survey items from the Adult Sickle Cell 
Quality of Life Measurement System (ASCQ-Me), the Patient Reported Outcome Measurement 
Information System (PROMIS) short form (SF), and the 36-Item Short Form Health Survey (SF-
36) questionnaires at baseline (prior to starting study treatment) and at the end of treatment 
(Figure 1 and Table 1). Survey items will be selected from following questionnaires to cover the 
primary functional domains affected in adults with SCD, as well as domains hypothesized to be 
affected by anemia: ASCQ-Me Pain Impact, Stiffness Impact, Pain Episodes, Emotional Impact, 
and Sleep Impact; PROMIS Pain Interference, Fatigue, Dyspnea, and Cognitive Function. All 
listed SF questionnaires have been validated in an SCD population, except for the PROMIS 
Pain Interference and Cognitive Function, which were designed to be applicable across chronic 
diseases. In addition, the SF-36 questionnaire has been used in and found culturally appropriate 
for Nigerians with SCD, and this will be administered in its entirety. Questionnaires will be 
created in an online database (REDCap) built by the study team and administered using a 
portable electronic device. Questionnaires should be completed by the subject without help from 
anyone else (with the exception of help with logistical or technological issues that may arise). 
During the survey, research staff may help to define a term but cannot define a concept where 
the respondent’s subjective interpretation is the goal of the question. Subjects completing the 
survey in clinic will be provided with sufficient space and time to complete the survey. 
 
 
5.6 Assessment of Pain using Visual Analog Scale and Painimation 
 
In addition to using the standard Visual Analog Scale for pain, pain will be assessed at baseline 
and at each clinic visit using a novel pain assessment tool called Painimation. This tool 
addresses the documented communication challenges associated with SCD pain and allows 
participants to express their pain quality, intensity, and location using animations and graphical 
images instead of words and numerical scales. Painimation was validated in 170 participants 
with a mix of non-SCD pain types and was acceptable, easy to use, and useful for 
communicating pain among adults with SCD.38 The assessment will be administered using a 
portable electronic device. This participant-centered technology-based approach to pain 
measurement will complement and support data obtained through the questionnaires. 

 
 
Page 15 of 33 
 
 
 
5.7 Neurocognitive Assessment  
 
 
For a comprehensive assessment of neurocognitive function, subtests of the National Institute 
of Health (NIH) toolbox, a widely-used and validated computer-based cognitive test will be 
administered.39 The NIH toolbox Cognitive Function Battery (CFB) is a brief battery consisting of 
7 tests to assess 8 cognitive abilities. These tests include Dimensional Change Card Sort 
(DCCS, Executive Function-Cognitive Flexibility); Flanker Inhibitory Control and Attention Test 
(Executive Function-Inhibitory Control and Sustained Attention); Picture Sequence Memory Test 
(Episodic Memory-Visual); the Picture Vocabulary Test (Language-Vocabulary 
Comprehension), Oral Reading Recognition Test (Language-Reading Decoding); List Sorting 
Working Memory Test (Working Memory); and Pattern Comparison Processing Speed Test 
(Processing speed). These tests will assess domains that have been shown to be affected in 
SCD. Assessments will be administered using a portable electronic device and will take ~30 
minutes. This participant-centered technology-based method of assessing neurocognitive 
function will complement and support data obtained through the questionnaires and the testing 
in Section 5.8. 
 
 
 
5.8  Near-infrared (NIR) Technologies and electroencephalography (EEG) 
 
 
Several NIR technologies have been validated in SCD and non-SCD populations.40-42 In this 
study, the NIR technologies of diffuse correlation spectroscopy (DCS) and frequency-domain 
NIRS (FD-NIRS) will be used to assess the impact of raising hemoglobin level on microvascular 
hemodynamics in SCD. DCS measures temporal fluctuations in light intensity in deep tissues 
and is sensitive to changes in blood flow.43 FD-NIRS measures absolute tissue oxy- and 
deoxyhemoglobin concentrations and tissue oxygen saturation,41 which allows for both intra-
individual and inter-individual comparisons. DCS and FD-NIRS will be performed at baseline 
and every month thereafter until the end of 12 weeks of treatment. Various non-invasive probes 
will be used to acquire spatially and temporally co-registered measurements from superficial 
forearm muscles and the cerebral cortex to obtain steady-state integrated data on tissue 
hemodynamics. Melanin content in skin has been shown to have an effect on near-infrared 
spectroscopy (NIRS) recordings. 54,55 For this reason, a DSM III Skin Colorimeter (Cortex 
Technology) will be used to record the melanin content in participants' skin. This device uses 2 
high intensity white LEDs to provide recordings of both erythema and melanin content. The 
colorimeter is not FDA approved as it is marketed as a cosmetic research device. In addition, 
electroencephalography (EEG) measurements will be obtained to simultaneously evaluate 
neuronal activity. These measures will be performed, depending on the availability of these 
technologies at each research site. In the US site, the FD-NIRS, DCS, and EEG measurements 
will be performed in collaboration with study team members from the Carnegie Mellon University 
(CMU). In the Lagos site, some measurements with NIR technologies will be performed as well, 
but DCS and EEG will not be performed due to lack of the equipment and/or expertise. The 
CMU or Pitt team will provide the required equipment and material for these non-invasive 
measurements. For the remainder of the protocol, the terms NIR technologies and NIRS will be 
used interchangeably and will refer to all non-invasive imaging technologies using NIR. 
 
Prior to performing the NIRS assessment, the DSM Colorimeter device will be used to check the 
melanin index and erythema value of the participant. It will be placed on the subject's skin for 
about 5 seconds to record a measurement from each location where NIRS will be recorded (left 
forehead, right forehead, and left forearm). Once the NIRS and EEG probes are placed and 
steady-state measurements are obtained, we will have the participant undergo non-invasive 
experimental tasks that allow for evaluation of the muscle and cerebral vascular response to 

 
 
Page 16 of 33 
 
perturbations. For instance, participants will undergo a vascular occlusion challenge, in which a 
standard automated blood pressure cuff will be placed on the same forearm as the NIRS probes 
and inflated to temporarily occlude arterial blood flow into the extremity, for a maximum of 3-5 
minutes per measurement, while participants are seated and at rest. The cuff will then be 
released, and NIRS will be used to measure the magnitude of hyperemic response and other 
parameters related to vascular function.  
 
Other tasks include paced breathing and breath holding after exhalation, as well as brief 
cognitive tests such as the digit symbol substitution test (DSST). Simultaneous measurement of 
cerebral blood flow, tissue oxygenation, and neuronal activity via EEG will allow for evaluation of 
cerebral autoregulation, oxygen extraction, and neurovascular coupling.  Each task is designed 
to induce changes in the blood pressure or physiochemical changes in the brain within normal 
physiologic variation and would therefore be of minimal risk to healthy or at-risk volunteers. 
Blood pressure oscillations will be induced at a well-defined frequency, according to protocols 
that have been already proposed, performed, and reported in the literature.44-47 Paced breathing 
involves following the cue of a metronome to breath at a well-defined frequency within the range 
0.03-0.50Hz. In all cases, the protocol will involve no more than 5 minutes of each breathing 
task at each frequency considered (within the range 0.03-0.5Hz) or 5 minutes of cognitive tests 
for a total measurement time not to exceed 60 minutes. Descriptions and procedures of each 
task will be provided to participants before they are conducted. 
 
DSM colorimeter, EEG and NIRS measurements will be obtained at baseline and repeated at 
monthly clinic visits during the 12-week treatment period, as well as at EOS (24 weeks). The 
information obtained through these methodologies will be purely experimental and will not be 
used to track participant response to drug therapy in real-time. The data collected will be shared 
between the Pitt and the CMU research teams. The data will be analyzed retrospectively and 
will not be used in any way to provide feedback to the study team, treating clinician, or 
participant for the purpose of making clinical or research decisions.  
 
 
 
5.9 Assessment of compliance 
 
Participants will self-report doses of EPO taken and any symptoms observed during the course 
of participation in the study. They will be filling out a daily drug diary for EPO and their 
compliance will be reviewed at each visit. The level of compliance will be calculated as the 
percentage of the total number of doses administered out of the total number of doses 
prescribed during the treatment period. Symptoms reported through the application will be 
reviewed by the study team and will be used to facilitate AE tracking and reporting.  
 
 
6. Efficacy and Safety Assessments: 
 
 
6.1 Safety assessments 
 
 
The PI will review participant safety data as it is generated and ensure that the IRB is 
immediately notified of any serious adverse event. The PI will routinely be notified of any 
medical concerns during the conduct of the study. Expected and unexpected serious (including 
fatal) adverse reactions and major unresolved disputes between the research investigator(s) 
and the research participant or between research investigator(s) will be expeditiously reported 
to the IRB as per Section 3.4 of the University of Pittsburgh IRB reference manual. The PI will 
promptly report the unanticipated problems that occur during the conduct of the study according 
to the reporting criteria and timelines under the current IRB policies. At the time of renewal, the 

 
 
Page 17 of 33 
 
IRB will be provided with a summary of the cumulative adverse event data, information 
regarding participant safety or ethics changes, confidentiality issues, benefit-to-risk changes and 
recommendations on continuing, changing or terminating the study. 
 
Risk/Benefit Assessment: 
 
Risks with Study Treatment. Epoetin (EPO) is a 165 amino acid glycoprotein manufactured by 
recombinant DNA technology with the same biological effect as endogenous erythropoietin. 
Initially approved in 1989, epoetins have a long safety record and are the standard of care for 
anemia in the chronic kidney disease (CKD) population. EPO is also indicated for anemia in 
zidovudine-treated HIV-infected patients, cancer patients on chemotherapy, and surgery 
patients. In studies of EPO treatment in these patient populations, patients have experienced 
increases in blood pressure, greater risk of seizures, increased clotting of the vascular access 
(A-V shunt), and other thrombotic events (e.g., myocardial infarction, cerebrovascular accident, 
transient ischemic attack), especially when targeting higher Hb levels. In controlled trials, 
patients with CKD have experienced greater risk of death, serious adverse cardiovascular 
reactions, and stroke when administering EPO to target a Hb level of > 11 g/dL. Furthermore, 
increased mortality and/or increased risk of tumor progression or recurrence has been found in 
patients with cancer. For these reasons, participants with uncontrolled hypertension, 
uncontrolled seizure disorder, unstable cardiac disorder, active malignancy, or recent 
thrombosis will be excluded from the study. This study will also avoid targeting a Hb level > 11 
g/dL.  
 
In terms of SCD-specific risks, there have been a handful of anecdotal reports of pain and 
VOCs associated with EPO therapy, but of note, the largest and most recent retrospective 
cohort studies did not identify any increased risk of VOC or thrombosis,30,32 suggesting that EPO  
therapy is safe and efficacious in the SCD population. The typical dose range of EPO used to 
achieve a Hb response in the SCD population is higher than the range used in the CKD 
population. Doses of erythropoietin in previous clinical studies have ranged between 100 and 
3000 U/kg per dose (i.e., 7000-210000 U per dose for a 70-kg individual), with dosing 1-3 times 
per week.26-37 The dose range and rate of dose escalation is largely determined by institutional 
and provider practices, but the starting treatment dose used in routine clinical practice is 
typically 10000-20000 U per dose. As higher doses of EPO are associated with increased risk of 
cardiovascular and thrombotic complications in the CKD population and may also increase risk 
of VOC, we have chosen to use low- to mid-range dosing of EPO (10000-40000 U per dose, 3 
times per week) to further reduce the risk of complications.  
 
Additional rare risks of EPO therapy include pure red cell aplasia, serious allergic reactions, and 
severe cutaneous reactions. If any of these reactions occur, EPO therapy should be 
discontinued immediately. Subcutaneous injections of EPO may also be commonly associated 
with slight pain or bruising at the injection site, and less commonly, a chance of bleeding or 
hematoma formation. There is a rare chance of infection at the injection site. Common adverse 
reactions reported in clinical trials for the following non-SCD patient populations are listed in the 
drug label insert for epoetins (e.g. Epogen): 
 
• 
Patients with CKD: Adverse reactions in ≥ 5% of Epogen-treated patients in clinical 
studies were hypertension, arthralgia, muscle spasm, pyrexia, dizziness, medical device 
malfunction, vascular occlusion, and upper respiratory tract infection. 
 
• 
Patients on Zidovudine due to HIV-infection: Adverse reactions in ≥ 5% of Epogen-
treated patients in clinical studies were pyrexia, cough, rash, and injection site irritation. 
 

 
 
Page 18 of 33 
 
• 
Patients with Cancer on Chemotherapy: Adverse reactions in ≥ 5% of Epogen-treated 
patients in clinical studies were nausea, vomiting, myalgia, arthralgia, stomatitis, cough, 
weight decrease, leukopenia, bone pain, rash, hyperglycemia, insomnia, headache, 
depression, dysphagia, hypokalemia, and thrombosis. 
 
• 
Surgery Patients: Adverse reactions in ≥ 5% of Epogen-treated patients in clinical 
studies were nausea, vomiting, pruritus, headache, injection site pain, chills, deep vein 
thrombosis, cough, and hypertension. 
 
Risks with Increasing Hemoglobin Level. Higher hemoglobin levels have been associated 
with higher VOC frequency in SCD in a handful of retrospective studies, but no prospective 
studies have been performed evaluating changes in pain symptoms and VOC frequency with 
EPO therapy. We anticipate an increase in hemoglobin with study treatment and will monitor the 
absolute as well as rate of hemoglobin increase very carefully to avoid rapid or excessive 
increases in hemoglobin that could lead to increased pain or vaso-occlusion in patients with 
SCD. We will not correct the hemoglobin to normal levels but will instead target a hemoglobin 
goal of 10-11 g/dL to minimize potential increases in blood viscosity at near-normal hemoglobin 
levels. As the response to the study drug and tolerance to the level of increased hemoglobin 
likely varies between individuals, we will monitor patient symptoms and wellness through 
adverse event (AE) collection, a Visual Analog Scale for pain, the Painimation tool, and quality 
of life metrics, and will consider dose reduction or discontinuation as indicated for hemoglobin 
levels above target or rapid increases in hemoglobin (Table 3). 
 
Risks with Blood Sampling. As part of this study, up to 50 mL (or just over 3 tablespoons) of 
blood will be collected at the time of each research clinic visit, in addition to routine lab work. 
The risks of collecting blood may include fainting, pain, and/or bruising. Blood will be drawn by 
trained certified personal. After blood draw participant will be monitored for stability. 
 
Risks with DSM Colorimeter. There are no risks associated with the use of the DSM 
Colorimeter since it only measures the wavelength absorbance in the visible range of light. No 
adverse reactions associated with the device have been reported.  
 
Risks with Near-infrared Spectroscopy (NIRS). There are no risks associated with the use of 
near infrared light as it has been described in this protocol. The amount of optical energy is too 
low to cause appreciable tissue heating and no damage will occur to the skin. Light sources are 
used in NIRS technologies and may cause retinal damage upon looking directly into the light 
source. To prevent such instances from occurring and minimizing this type of risk, the lasers will 
be “off” until placed on the skin, and participants will be asked to not look into the light. 
Temporary pain or paresthesias may occur during the approximately 3–5-minute blood pressure 
cuff inflation. The vascular occlusion challenge may also result in redness or minor bruising of 
the limb due to cuff inflation. This occlusion is a standard test that is performed in many 
hemodynamic assessments, such as in the case of peripheral vascular disease. Patients will be 
monitored closely for any concerning signs and symptoms during the occlusion challenge, and if 
there are any concerns for safety or if participants experience significant discomfort, the 
occlusions will be released immediately. Participants may also experience discomforts during 
the breathing maneuvers, such as lightheadedness. Participants will be monitored closely for 
symptoms and signs of discomfort during and after measurements, and participants can ask to 
take breaks between sessions or to stop the maneuver at any time if the discomfort they 
experience is intolerable. 
 
All non-invasive instruments used in this study are research devices used for experimental 
studies only; they are not medical devices based on the criteria: (a) these specific devices are 

 
 
Page 19 of 33 
 
not part of the US Pharmacopeia, (b) these devices are not collecting clinical information for a 
medical diagnosis – all information is purely for research purposes, and (c) these devices are 
non-invasive and are not intended to alter the tissue structure or function in any capacity. For 
these reasons, the devices used in our study are not medical devices and an IDE application is 
not required. 
 
Risks with EEG: There are no risks of physical injury associated with EEG. Adhesive from the 
EEG probes might cause skin irritation. Prior to its removal, the conductive gel for the EEG 
probes may leave residue on the scalp. The conductive gel can easily be removed from the 
scalp. 
 
 
Risk of Inconveniences. Participants may not feel comfortable answering some of the 
questions during questionnaires, pain assessment, or neurocognitive assessment, however this 
information is important to understand. There are no right or wrong answers to questions, and 
all answers will remain confidential. There may also be discomfort with checking vital signs and 
doing physical exams. Although physical tests and checking vital signs do not involve any 
known risks, taking blood pressure may cause discomfort or bruising to upper arm. To maximize 
the patients’ convenience, the NIRS machine will be brought to a research facility or clinic on 
the Pitt campus, and all the tests will be performed in a private room by the collaborating CMU 
research team members. 
 
 
Confidentiality Risk. All records with identifying information will be stored in a password-
protected server. All future research samples (such as blood samples stored for future genetic 
or biomarker testing) will be deidentified by labeling with a study code and stored in a securely 
locked laboratory. Although it is highly unlikely, there is still the possibility that personal 
information could be linked back to research information. For this reason, additional protections 
will be taken to protect information linking the codes with identity (i.e., name or other information 
that could be used to link with the information about the participation in this study), and that 
information will be kept separate from the research records. 
 
 
Importance of the Knowledge to be Gained: 
 
The PI will be reviewing the data from this research throughout the study.  We will promptly tell the 
participants about new information from this or other studies or changes in the study that may 
affect their health, welfare, or willingness to continue in the study.  
 
6.1.1 Data Safety Monitoring Plan (DSMP) 
 
Study sites' local data safety and monitoring plan 
 
A local data and safety monitoring plan will be implemented by each site as per their institutional 
requirements. Oversight of the data and safety monitoring at all sites is the primary 
responsibility of the study site PI. The PI at each participating site will meet with the participant 
when they are first enrolled in the study, and then periodically, every four weeks, the PI will look 
at the individual subject data, as it is generated throughout the study, for any trends in adverse 
events or other events that may pose a concern. Any adverse events meeting reporting 
requirements will be reported as they occur per IRB policy guiding each site. Summary 
information and reportable information required for continuing reviews, will be reported to the 
IRB annually.  

 
 
Page 20 of 33 
 
 
The study team members will monitor participants' recruitment and retention, occurrence of 
adverse events, and confidentiality of the participants’ information at their respective study sites. 
The AEs will be reported as per CTCAE version 5.0 and will include whether the participant 
discontinues to take the drug, start and stop dates, any corrective measures that will be taken, 
and their outcome. All subjects who receive at least one dose of study treatment will be included 
in the safety analysis. All observations pertinent to the safety of the study treatment will be 
recorded and included in a final analytical report. All AEs whether considered drug-related or 
not, will be reported. For all events, the relationship to treatment and the intensity of the event 
will be determined by the investigator. All subjects who have AEs, whether considered 
associated with the use of the study product or not, will be monitored during the treatment 
period of 12 weeks to determine the outcome. They will be referred back to their care provider 
for continued follow up and management in the event they need more medical attention. Our AE 
recording and observation period will stop at the end of treatment (at 12 weeks). AEs will not be 
tracked between end of treatment and end of study, as patient will not be receiving any 
intervention per protocol during this period.  
 
All local site(s) will participate in monthly meetings with the Coordinating Center, Pitt site, to re-
evaluate study goals, subject recruitment, data coding and retention, documentation and 
identification of adverse events, complaints, and confidentiality of subjects.  
 
For the collaborating study site in Nigeria, the PI for the coordinating center, who is the Chief 
Investigator, will initially visit the study site for a study initiation visit. That visit may be done 
remotely if travel restrictions prohibit it. The coordinating center PI will receive updates on the 
study progress during the monthly PI meetings. In the event the PI needs a detailed look into a 
local site participants’ study record, the study site PI or their delegates will send de-identified 
copies of participants’ information to the coordinating center PI. The local sites will also upload 
their data into coordinating center database project hosted on Pitt CTSI REDCap which can be 
accessed. 
 
DATA AND SAFETY MONITORING BOARD  
A Data and Safety Monitoring Board (DSMB) will be created to review this study.  After initial 
approval and at periodic intervals (to be determined by the committee) during the course of the 
study, the DSMB responsibilities are to: 
 
1. Review the research protocol, informed consent documents and plans for data and safety 
monitoring; 
2. Evaluate the progress of the study, including periodic assessments of data quality and 
timeliness, participant recruitment, accrual and retention, participant risk versus benefit, 
adverse events, unanticipated problems, performance of the trial sites, and other factors 
that can affect study outcome; 
3. Consider factors external to the study when relevant information becomes available, such 
as scientific or therapeutic developments that may have an impact on the safety of the 
participants or the ethics of the study; 
4. Review clinical center performance, make recommendations and assist in the resolution 
of problems reported by the PI; 
5. Protect the safety of the study participants; 
6. Report on the safety and progress of the study; 
7. Make recommendations to the PI, and if required, to the funding agency concerning 
continuation, termination or other modifications of the study based on the observed 
beneficial or adverse effects of the treatment under study; 
8. Monitor the confidentiality of the study data and the results of monitoring;  

 
 
Page 21 of 33 
 
9. Assist the PI by commenting on any problems with study conduct, enrollment, sample size 
and/or data collection. 
 
The DSMB will include experts in hematology or vascular medicine and clinical trials/biostatistics.  
Members will consist of persons independent of the investigators who have no financial, scientific, 
or other conflict of interest with the study.  Written documentation attesting to absence of conflict 
of interest will be required. 
 
The University of Pittsburgh Office of Clinical Research, Health Sciences / CTSI will provide the 
logistical management and support of the DSMB.  A safety officer (chairperson) will be identified 
at the first meeting.  This person will be the contact person for serious adverse event reporting.  
Procedures for this will be discussed at the first meeting. 
 
The first meeting will take place before initiation of the study to discuss the protocol, approve the 
commencement of the study, and to establish guidelines to monitor the study.  The follow-up 
meeting frequency of the DSMB will be determined during the first meeting.  An emergency 
meeting of the DSMB will be called at any time by the Chairperson should questions of patient 
safety arise.   
 
 
6.1.2. Data safety monitoring parameters 
 
Adverse events (AE's) will be collected periodically from start of study drug treatment period 
(baseline visit) until end of treatment (EOT), after which only acute care utilization will be 
collected until the end of study (EOS). AE reporting procedure is described in Section 8.2.1. 
 
 
6.1.3 Physical Sign Monitoring 
 
Body temperature (orally), heart rate, and blood pressure will be measured at baseline before 
initiation of EPO administration as well as at each clinic visit.  
 
6.2 Privacy 
 
Privacy of participants will be maintained through a set of procedures that has worked well at 
the University of Pittsburgh.  All University of Pittsburgh faculty participating in this study have 
had HIPAA training. Confidentiality will be ensured by assigning each participant a study code 
and number that will not allow participant name identification; and these will be used to identify 
the participant's data.  Also, all data will be kept in locked file cabinets in a locked and secured 
area or digitally on a server like REDCap with access by research personnel only.   
 
7. Adverse Event Reporting: 
 
7.1 Adverse event definitions  
 
Adverse event. Any untoward medical occurrence in a clinical study; regardless of the causal 
relationship of the event with the investigational drug or study treatment(s). 
 
Associated with the use of the investigational drug or study treatment(s).  There is a reasonable 
possibility that the adverse event may have been caused by the investigational drug or study 
treatment(s). 
 

 
 
Page 22 of 33 
 
Disability. A substantial disruption of a person’s ability to conduct normal life functions. 
 
Life-threatening adverse event. Any adverse event that places the participant or participant, in 
the view of the investigator, at immediate risk of death from the event as it occurred (i.e., does 
not include an adverse event that, had it actually occurred in a more severe form, might have 
caused death). 
 
Serious adverse event. Any adverse event occurring at any dose that results in any of the 
following outcomes: death, a life-threatening adverse event, inpatient hospitalization or 
prolongation of existing hospitalization, a persistent or significant disability/incapacity, or a 
congenital anomaly/birth defect. 
 
Hospitalization. Hospitalization shall include any initial admission (even if less than 24 hours) to 
a healthcare facility as a result of a precipitating clinical adverse event; to include transfer within 
the hospital to an intensive care unit.  Hospitalization or prolongation of hospitalization in the 
absence of a precipitating, clinical adverse event (e.g., for a preexisting condition not associated 
with a new adverse event or with a worsening of the preexisting condition; admission for a 
protocol-specified procedure) is not, in itself, a serious adverse event.   
 
Unexpected adverse event. Any adverse event, the frequency, specificity or severity of which is 
not consistent with the risk information described in the clinical protocol(s). 
 
8. Recording/Reporting requirements  
 
 
8.1 Safety Assessment and Reporting 
 
8.1.1. Eliciting adverse event information 
 
Clinical study participants will be routinely questioned about adverse events at study visits. 
 
8.1.2. Recording requirements 
 
All observed or volunteered adverse drug events (serious or non-serious) and abnormal test 
findings, regardless of treatment group or suspected causal relationship to the investigational 
drug or study treatment(s) will be recorded in the participants’ case histories. For all adverse 
events, sufficient information will be pursued and/or obtained so as to permit 1) an adequate 
determination of the outcome of the event (i.e., whether the event should be classified as a 
serious adverse event); and 2) an assessment of the casual relationship between the adverse 
event and the study treatment(s).    
 
Adverse events or abnormal test findings felt to be associated with the study drug will be 
followed until the event (or its sequelae) or the abnormal test finding resolves or stabilizes at a 
level acceptable to the investigator-sponsor. 
 
8.1.3 Abnormal test findings 
 
An abnormal test finding will be classified as an adverse event if one or more of the following 
criteria are met: 
 
• 
The test finding is accompanied by clinical symptoms. 

 
 
Page 23 of 33 
 
• 
The test finding necessitates additional diagnostic evaluation(s) or medical/surgical 
intervention, including significant additional concomitant drug treatment or other 
therapy. 
• 
Note: simply repeating a test finding, in the absence of any of the other listed criteria, 
does not constitute an adverse event.   
• 
The test finding leads to a change in study dosing or discontinuation of participant 
participation in the clinical study. 
• 
The test finding is considered an adverse event by the investigator-sponsor. 
Serious Adverse Events will be reported to the IRB according to their guidelines.  
 
8.1.4. Causality and severity assessment 
 
The investigator-sponsor will promptly review documented adverse events and abnormal test 
findings to determine 1) if the abnormal test finding should be classified as an adverse event; 2) 
if there is a reasonable possibility that the adverse event was caused by the investigational drug 
or study treatment(s); and 3) if the adverse event meets the criteria for a serious adverse event. 
 
If the investigator-sponsor’s final determination of causality is “unknown and of questionable 
relationship to the investigational drug or study treatment(s)”, the adverse event will be 
classified as associated with the use of the investigational drug or study treatment(s) for 
reporting purposes. If the investigator-sponsor’s final determination of causality is “unknown but 
not related to the investigational drug or study treatment(s)”, this determination and the rationale 
for the determination will be documented in the respective participant’s case history.  
 
8.2. Reporting of adverse events  
  
8.2.1 Reporting adverse events to the responsible IRB 
 
In accordance with applicable policies of the University of Pittsburgh Institutional Review Board 
(IRB), the investigator-sponsor will report, to the IRB, any observed or volunteered adverse 
event that is determined to be 1) associated with the investigational drug or study treatment(s); 
2) serious; and 3) unexpected.  Adverse event reports will be submitted to the IRB in 
accordance with the respective IRB procedures.  
 
Applicable adverse events will be reported to the IRB as soon as possible and, in no event, later 
than 10 calendar days following the investigator-sponsor’s receipt of the respective information.  
Adverse events which are 1) associated with the investigational drug or study treatment(s); 2) 
fatal or life-threatening; and 3) unexpected will be reported to the IRB within 24 hours of the 
investigator-sponsor’s receipt of the respective information.  
 
Follow-up information to reported adverse event will be submitted to the IRB as soon as the 
relevant information is available.  If the results of the sponsor-investigator’s follow-up 
investigation show that an adverse event that was initially determined to not require reporting to 
the IRB does, in fact, meet the requirements for reporting; the investigator-sponsor will report 
the adverse event to the IRB as soon as possible, but in no event later than 10 calendar days, 
after the determination was made. 
 
8.3 Withdrawal of participants due to adverse events 
 

 
 
Page 24 of 33 
 
Participants may be withdrawn from participation in the interventional study if they suffer a 
serious adverse event felt to be related to the study treatment. The decision to withdraw will be 
made by the PI.  
 
9.  Statistical Methods/Data Analysis:  
 
Descriptive statistics will be presented for all baseline and post-treatment characteristics. 
Graphs showing intra-individual change will be presented for all laboratory parameters. Baseline 
clinical and laboratory factors, including age, gender and Hb level, will be correlated with Hb 
response to identify predictors of individual response. Changes in clinical and laboratory factors 
from baseline to the end of study will be correlated with Hb response as well. Laboratory 
measures are listed in Table 4. 
 
For quality-of-life measures, individual item raw scores will be calculated for all items, and 
scores at the end of treatment will be compared to baseline. If an entire instrument is used (e.g., 
the PROMIS Fatigue short form), then the individual item raw scores will be combined to obtain 
a total raw score for each subscale, which will then be converted to standardized t scores (with 
50 as the mean and 10 as standard deviation of the reference population). Descriptive statistics, 
correlation, and regression models will be used to present changes in clinical measures and 
their relationship to changes in Hb level.  
 
9.1 Study endpoints 
 
Primary Endpoint: The primary endpoint of Hb response, defined as a Hb increase of ≥ 1.0 g/dL 
at the end of treatment compared to baseline, was chosen based on the endpoint definition used 
in the HOPE trial (Phase 3 study of voxelotor for patients with SCD).17 This endpoint will serve to 
define responders to EPO, identify laboratory and clinical predictors or modifiers of response, as 
well as meaningful biological and clinical correlates of the surrogate marker of Hb increase. All 
enrolled participants who have completed baseline and end of treatment testing, regardless of 
duration of treatment with EPO, will be included in the analysis. Participants completing the full 
12 weeks of EPO treatment per protocol will be evaluated in a planned sub-analysis. Frequency 
of adverse events (AEs), including treatment-related AEs and SAEs will also be reported. AEs 
will be tabulated by type, grade, and relatedness to treatment. 
 
Secondary Endpoint: The secondary endpoint will be the annualized number of units of simple 
red blood cell transfusions received in the 12 months before treatment initiation compared to 
during active treatment. 
 
Exploratory Endpoints (Optional):  
Changes in hematological, hemolytic, and other clinical laboratory parameters will be assessed 
at EOT and EOS compared to baseline (Table 4). As renal dysfunction can affect endogenous 
EPO production and may also improve with increased Hb level and tissue perfusion, a pre/post 
serum creatinine, eGFR (using CKD-EPI and Cystatin C), urine albumin-to-creatinine ratio, and 
serum EPO level will be assessed at baseline and correlated with Hb response. Iron mobilization 
and inflammation are other factors that contribute to erythropoietic drive and may predict 
responsiveness to EPO, and therefore will be assessed at baseline and correlated with Hb 
response. All laboratory parameters assessed at EOS will be compared to baseline and EOT to 
evaluate longer-term treatment effects of EPO. Exploratory endpoints of Hb response will include 
a Hb increase of ≥ 1.0 g/dL above baseline at any time between 4 and 12 weeks, a Hb increase 
of ≥ 1.0 g/dL at 24 weeks, and sustained Hb response between 4 and 12 weeks (defined as the 

 
 
Page 25 of 33 
 
percentage of Hb values ≥ 1.0 g/dL above baseline). Modeling of the trajectory and heterogeneity 
of the Hb response over time will be performed. 
Clinical and imaging exploratory endpoints are listed in Table 5. Baseline acute vaso-occlusive, 
thrombotic events, and transfusions (within past 12 months) will be captured at the baseline visit 
and compared to the number of events at EOT and EOS. Cardiopulmonary function will be 
assessed by 6-minute walk test with oximetry and 2-dimensional Doppler echocardiogram. 
Neurocognitive assessments will also be administered, and a composite global score will be 
generated from the battery of tests. Quality of life items will be scored as mentioned above. 
Participants will record Visual Analog Scale and Painimation scores to assess pain level. These 
clinical endpoints will be assessed at EOT and EOS, compared to baseline and correlated with 
Hb response, as well as laboratory markers of hemolysis, cardiovascular risk, blood viscosity, and 
imaging biomarkers (Tables 4 and 5).  
Whole blood viscosity (Table 4) will be measured using an automated and/or cone-plate 
viscometer and used to calculate the hematocrit-to-viscosity ratio (HVR), which has been widely 
used as a surrogate measure of oxygen transport effectiveness in the microvasculature and 
appears to correlate with non-invasive measures of tissue oxygen saturation.48 Therefore, 
measurements of HVR will be correlated to Hb level and NIRS measures and may help to identify 
safe target Hb levels at which oxygen delivery and tissue perfusion are maximized in SCD. Whole 
blood viscosity will also be correlated with measurements obtained using microfluidic flow assays 
performed by Dr. Umut Gurkan at Case Western Reserve University and/or BioChip Labs, LLC. 
A number of other measures obtained from fresh whole blood that potentially influence blood 
rheology will be correlated with Hb response and NIRS measures, including the following: red cell 
deformability and point of sickling (PoS) (by Lorrca ektacytometer) measured in the laboratory of 
the PI (Dr. Julia Xu), red cell phosphatidylserine exposure and platelet activation (by flow 
cytometry) and/or bioenergetics measured in the laboratory of co-investigator Dr. Sruti Shiva, F-
cell fraction measured in the laboratory of co-investigator Dr. Adam Straub. Known markers of 
myocardial stress, thrombogenesis, and vascular injury49-51 will be measured using frozen plasma 
or serum samples collected during the study and batched for analysis to further investigate the 
safety of EPO therapy and increasing Hb level (Table 4). DNA will also be stored and potentially 
used to identify genetic polymorphisms that may influence response to EPO. DSM colorimeter 
and NIRS measurements will be correlated with EEG measurements to evaluate neurovascular 
coupling.  
 
Table 5. Summary of clinical and imaging endpoints 
Frequency 
Secondary 
 
Transfusions: number of units of simple transfusions of packed red 
blood cells  
Baseline, EOT, 
EOS 
Exploratory 
(Optional) 
Acute vaso-occlusive events: acute episodes of pain not related 
to other etiologies and requiring acute pain treatment during a 
medical encounter (i.e., VOC), acute chest syndrome, hepatic or 
splenic sequestration, or priapism 
Thrombotic events: Stroke, myocardial infarction, or venous 
thromboembolism 
Baseline, EOT, 
EOS 
Cardiopulmonary function: 6-minute walk distance and oxygen 
saturation, TRV, cardiac index, left ventricular (LV) size as 
measured by LV dimensions and end-diastolic volume  
Baseline, EOT, 
EOS 
Neurocognitive Functioning: composite global score from the NIH 
Toolbox, a computerized neurocognitive assessment tool 
Baseline, EOT, 
EOS 
Select survey items: ASCQ-Me and PROMIS; SF-36 
Baseline, EOT, 
EOS 

 
 
Page 26 of 33 
 
 
 
9.2 Sample size determination 
 
This study aims to have 15 participants completing the study (defined as completion of 12 
weeks of EPO treatment and end of treatment testing) across both study sites. We will enroll up 
to 25 participants across both sites (Pitt and LUTH), with the expectation that no more than 10 
participants will be lost to follow-up or withdraw from the study prior to completion of the 12 
weeks. The goal is to determine an estimate with 95% confidence intervals for the population 
probability that a randomly chosen individual from the eligible SCD population would show a Hb 
response (i.e. Hb increase of ≥ 1.0 g/dL from baseline after 12 weeks of EPO therapy). Prior 
studies of EPO in SCD report a Hb response rate of between 50-60%.30,32,33 In comparison, a 
low rate (7%) of Hb response over 6 months in the placebo arm of the voxelotor study was 
observed.17 Therefore, rejecting a null hypothesis that the true probability of Hb response is  
20% may suggest a relatively robust response. Using these assumptions, if the true probability 
of a Hb response is 0.5, then for a sample size of 25, there is 94% power to reject the null 
hypothesis that this probability is 20%, based on an exact one-sided 95% binomial confidence 
interval.  
 
 
10.  Data Handling and Record-Keeping:   
 
10.1 Data recording/Case Report Forms   
 
Case report forms (CRFs) will be completed for each participant enrolled into the clinical study.  
The principal investigator of each site will review, approve and sign/date each study visit to 
certify that all CRFs have been completed accurately; the principal investigator’s signature 
serving as attestation of the principal investigator’s responsibility for ensuring that all clinical and 
laboratory data entered on the CRF are complete, accurate and authentic.   
 
When applicable, information recorded on the CRF shall match the Source Data recorded on 
the Source Documents.   
 
10.2 Record maintenance and retention   
 
The investigator-sponsor will maintain records in accordance with Good Clinical Practice 
guidelines. 
 
At the University of Pittsburgh, the records will be kept in a locked area at all times, to be 
accessed only by personnel involved in the study. Computer-based files will only be made 
available to personnel involved in the study through the use of access privileges and 
passwords. Prior to access to any study-related information, personnel will be required to sign 
statements agreeing to protect the security and confidentiality of identifiable information, 
whenever feasible, identifiers will be removed from study-related information, precautions are in 
place to ensure the data is secure by using passwords and encryption.  
Pain: Scores from Visual analog scale and Painimation 
Baseline, Q2 
weeks, EOT, 
EOS 
NIRS and EEG testing: tissue oxygen saturation, blood flow 
velocity, metabolic rate of oxygen extraction, magnitude of 
hyperemic response, time to peak hyperemia, EEG frequency band 
power, evoked potentials 
Baseline, Q4 
weeks, EOT, 
EOS 

 
 
Page 27 of 33 
 
 
At the LUTH site, there is a challenge with the medical record system, which is solely on paper 
and can be lost with time. Therefore, investigators at LUTH will make a photocopy of all source 
documentation, and the site Principal Investigator will certify these documents with a wet 
signature. The LUTH study team will maintain these copies in a separate file in order to make it 
easier to retrieve source documents, and these records will be kept in a locked area at all times, 
to be accessed only by personnel involved in the study. 
 
 
 
10.3 Future Use of Stored Specimens and Data 
 
Data collected through NIRS, and EEG methodologies will be stored by the CMU team and will 
be shared with the Pitt team for purposes of comparison and analysis during and at the end of 
the study. If the participant consents to participate in a separate Pitt IRB-approved study 
(STUDY22050118), which has overlapping endpoints and testing, data collected through this 
study may be shared with STUDY22050118 to avoid asking participants to undergo the same 
testing twice. Coded information from the measurement results will be shared between Pitt, 
Lagos, and CMU team. Data may be stored without identifiers indefinitely by both the CMU and 
Pitt teams. 
 
A portion of the blood samples will be stored indefinitely at the University of Pittsburgh’s Core 
Biospecimen Repository for current and future research that might include whole genome 
sequencing. The individual genetic results or incidental findings will not be shared with subjects 
as these tests are research-related and these initial finding will need additional research before 
their clinical significance is understood, and appropriate actions are determined. It is possible 
that some of the genomics research conducted either by the study investigators or secondary 
researchers could eventually lead to the development of new diagnostic tests, new drugs or 
other commercial products. If this should occur, there is no plan for research participants to 
receive any part of the profits generated from such products. 
 
Samples will be stored without identifiers and may be shared with secondary researchers within 
or outside the participating centers. We will ship up to 6 mL of whole blood  from consented 
patients without any identifying information to our collaborator Dr. Umut Gurkan at Case 
Western Reserve University (CWRU) and/or BioChip Labs, LLC which have licensed intellectual 
property from CWRU and are working with Dr. Gurkan at CWRU to translate these 
technologies. With these blood samples, the CWRU and/or BioChip Labs team will perform 
blood cell adhesion, occlusion, and blood rheology experiments, and communicate results of 
that testing back to the site of sample origination. Any residual blood samples may be used for 
additional assay development and validation by CWRU and/or BioChip Labs. We may also send 
a limited data set with the coded blood samples (including clinical, demographic, and 
experimental data) to CWRU and/or BioChip Labs in order to aid in their analysis of samples 
and validation of their technology. There will be no other analyses, such as DNA or RNA 
isolation, performed on the transferred samples. 
 
 
 
 
 
 
11. Ethics: 
 
11.1. Institutional Review Board (IRB) approval   

 
 
Page 28 of 33 
 
 
The investigator-sponsor will obtain, from the University of Pittsburgh Institutional Review Board 
(IRB), prospective approval of the clinical protocol and corresponding informed consent form(s); 
modifications to the clinical protocol and corresponding informed consent forms, and 
advertisements (i.e., directed at potential research participants) for study recruitment.    
 
The only circumstance in which a deviation from the current IRB-approved clinical 
protocol/consent form(s) may be initiated in the absence of prospective IRB approval is to 
eliminate an apparent immediate hazard to the research participant(s).  In such circumstances, 
the investigator-sponsor will promptly notify the University of Pittsburgh IRB of the deviation.  
 
 
 
11.2. Ethical and scientific conduct of the clinical study 
 
The clinical study will be conducted in accordance with the current IRB-approved clinical 
protocol; ICH Guidelines on GCP; and relevant policies, requirements, and regulations of 
the University of Pittsburgh IRB, University of Pittsburgh and UPMC, Commonwealth of 
Pennsylvania, and applicable federal agencies. 
 
 
12. Investigator-Sponsor Discontinuation Criteria: 
 
 
The University of Pittsburgh Institutional Review Board (IRB) will be notified promptly of 
discontinuation of the entire clinical study.  All protocol modifications will be submitted 
prospectively to the University of Pittsburgh IRB for discontinuation of parts of the clinical 
research study.  
 
 
13. Stopping Rules 
 
13.1. Stopping Rule for Qualifying Adverse Events 
 
The stopping rule is designed to halt enrollment for further evaluation of study data by the 
safety committee and determination whether enrollment can continue, or the study should 
be modified or terminated. Qualifying AEs/SAEs include any of the following that are 
possibly, probably, or definitely related to treatment: 
 
 • Arterial or venous thrombosis (including myocardial infarction, cerebrovascular accident, 
and transient ischemic attack)  
• Seizures  
• Pure red cell aplasia 
• Serious allergic reactions  
• Severe cutaneous reactions.  
 
If any of these reactions occur, EPO therapy should be discontinued immediately in the 
patient.  
 
Based on drug package inserts for epoetin alfa and literature review52,53, we anticipate that 
the rate of qualifying adverse events during the study period is no greater than 10%.  

 
 
Page 29 of 33 
 
• 
Our stopping rule is determined by a Bayesian approach. The stopping boundary for an 
experiment is reached if the Bayesian posterior probability that the true probability of 
developing an AE exceeds this benchmark rate of 20% is at least 70%. We take our 
prior distribution to be a beta distribution with parameters (α, β) = (1, 4). The parameters 
are chosen so that the mean α / (α + β) = 0.20 as the maximum acceptable proportion of 
AE and the sum α + β = 5 as the “worth” we place on our prior clinical opinion. This 
indicates that the relative weight we place on our prior opinion is 5/25 =20% of the 
weight we will place on the results if n=25. The following table summarizes the stopping 
rule: 
 
Number of subjects in the study 
Halt if the number of subjects who developed 
a qualifying adverse event during study  
5 
2 
6-9 
3 
10-14 
4 
15-18 
5 
19-23 
6 
24-25 
7 
 
 
 
 
13.2. Stopping Rule for Mortality  
 
A separate stopping rule is proposed for deaths that are deemed to be possibly, probably, or 
definitely related to study intervention. Should such an incident occur, both entry of other 
patients into the study and dose escalation for patients on the study will be suspended, and 
patients receiving study medication will be closely monitored for occurrences of the same 
event whilst assessment of the event occurs. 
 
The IRB/DSMB and the investigators will evaluate the full circumstances of the event and 
determine whether to terminate the study. Should it be determined the study be terminated, 
enrollment in the study will be halted permanently, and subjects already on study drug will 
be withdrawn from treatment. 
 
 
 
 
 
 
 
 
14. REFERENCES:  
 
1. 
Piel FB, Patil AP, Howes RE, et al. Global epidemiology of sickle haemoglobin in 
neonates: a contemporary geostatistical model-based map and population estimates. 
Lancet 2013;381(9861):142-51. DOI: 10.1016/S0140-6736(12)61229-X. 

 
 
Page 30 of 33 
 
2. 
Elmariah H, Garrett ME, De Castro LM, et al. Factors associated with survival in a 
contemporary adult sickle cell disease cohort. Am J Hematol 2014;89(5):530-5. DOI: 
10.1002/ajh.23683. 
3. 
Fitzhugh CD, Lauder N, Jonassaint JC, et al. Cardiopulmonary complications leading to 
premature deaths in adult patients with sickle cell disease. Am J Hematol 2010;85(1):36-
40. DOI: 10.1002/ajh.21569. 
4. 
Steinberg MH. Sickle Cell Anemia: From Basic Science to Clinical Practice. 1 ed: 
Springer International Publishing, 2016. 
5. 
Pecker L, Ackerman H. Cardiovascular adaptations to anemia and the vascular 
endothelium in sickle cell disease pathophysiology. In: Costa FF, Conran N, eds. Sickle 
Cell Anemia: From Basic Science to Clinical Practice Springer International Publishing; 
2016:129-176. 
6. 
Metivier F, Marchais SJ, Guerin AP, Pannier B, London GM. Pathophysiology of 
anaemia: focus on the heart and blood vessels. Nephrol Dial Transplant 2000;15 Suppl 
3:14-8. DOI: 10.1093/oxfordjournals.ndt.a027970. 
7. 
Mozos I. Mechanisms linking red blood cell disorders and cardiovascular diseases. 
Biomed Res Int 2015;2015:682054. DOI: 10.1155/2015/682054. 
8. 
Swanson ME, Grosse SD, Kulkarni R. Disability among individuals with sickle cell 
disease: literature review from a public health perspective. Am J Prev Med 2011;41(6 
Suppl 4):S390-7. DOI: 10.1016/j.amepre.2011.09.006. 
9. 
Anderson LM, Allen TM, Thornburg CD, Bonner MJ. Fatigue in Children With Sickle 
Cell Disease: Association With Neurocognitive and Social-Emotional Functioning and 
Quality of Life. J Pediatr Hematol Oncol 2015;37(8):584-9. DOI: 
10.1097/MPH.0000000000000431. 
10. 
Panepinto JA, Torres S, Bendo CB, et al. PedsQL Multidimensional Fatigue Scale in 
sickle cell disease: feasibility, reliability, and validity. Pediatr Blood Cancer 
2014;61(1):171-7. DOI: 10.1002/pbc.24776. 
11. 
Dampier C, Jaeger B, Gross HE, et al. Responsiveness of PROMIS(R) Pediatric 
Measures to Hospitalizations for Sickle Pain and Subsequent Recovery. Pediatr Blood 
Cancer 2016;63(6):1038-45. DOI: 10.1002/pbc.25931. 
12. 
Prussien KV, Jordan LC, DeBaun MR, Compas BE. Cognitive Function in Sickle Cell 
Disease Across Domains, Cerebral Infarct Status, and the Lifespan: A Meta-Analysis. J 
Pediatr Psychol 2019;44(8):948-958. DOI: 10.1093/jpepsy/jsz031. 
13. 
Partanen M, Kang G, Wang WC, et al. Association between hydroxycarbamide exposure 
and neurocognitive function in adolescents with sickle cell disease. Br J Haematol 
2020;189(6):1192-1203. DOI: 10.1111/bjh.16519. 
14. 
Kato GJ, Gladwin MT, Steinberg MH. Deconstructing sickle cell disease: reappraisal of 
the role of hemolysis in the development of clinical subphenotypes. Blood Rev 
2007;21(1):37-47. DOI: 10.1016/j.blre.2006.07.001. 
15. 
Voskaridou E, Christoulas D, Bilalis A, et al. The effect of prolonged administration of 
hydroxyurea on morbidity and mortality in adult patients with sickle cell syndromes: 
results of a 17-year, single-center trial (LaSHS). Blood 2010;115(12):2354-63. DOI: 
10.1182/blood-2009-05-221333. 
16. 
Steinberg MH, McCarthy WF, Castro O, et al. The risks and benefits of long-term use of 
hydroxyurea in sickle cell anemia: A 17.5 year follow-up. Am J Hematol 
2010;85(6):403-8. DOI: 10.1002/ajh.21699. 

 
 
Page 31 of 33 
 
17. 
Vichinsky E, Hoppe CC, Ataga KI, et al. A Phase 3 Randomized Trial of Voxelotor in 
Sickle Cell Disease. N Engl J Med 2019;381(6):509-519. DOI: 
10.1056/NEJMoa1903212. 
18. 
Hebbel RP, Hedlund BE. Sickle hemoglobin oxygen affinity-shifting strategies have 
unequal cerebrovascular risks. Am J Hematol 2018;93(3):321-325. DOI: 
10.1002/ajh.24975. 
19. 
Henry ER, Metaferia B, Li Q, et al. Treatment of sickle cell disease by increasing oxygen 
affinity of hemoglobin. Blood 2021. DOI: 10.1182/blood.2021012070. 
20. 
Stewart GM, Cross TJ, Joyner MJ, et al. Impact of Pharmacologically Left Shifting the 
Oxygen-Hemoglobin Dissociation Curve on Arterial Blood Gases and Pulmonary Gas 
Exchange During Maximal Exercise in Hypoxia. High Alt Med Biol 2021. DOI: 
10.1089/ham.2020.0159. 
21. 
Ballas SK, Kuypers FA, Gordeuk VR, Hankins JS, Thompson AA, Elliott V. Time to 
rethink haemoglobin threshold guidelines in sickle cell disease. British Journal of 
Haematology 2021. 
22. 
Newton SS, Sathyanesan M. Erythropoietin and Non-Erythropoietic Derivatives in 
Cognition. Front Pharmacol 2021;12:728725. DOI: 10.3389/fphar.2021.728725. 
23. 
El-Kordi A, Radyushkin K, Ehrenreich H. Erythropoietin improves operant conditioning 
and stability of cognitive performance in mice. BMC Biol 2009;7:37. DOI: 
10.1186/1741-7007-7-37. 
24. 
Petersen JZ, Schmidt LS, Vinberg M, et al. Effects of recombinant human erythropoietin 
on cognition and neural activity in remitted patients with mood disorders and first-degree 
relatives of patients with psychiatric disorders: a study protocol for a randomized 
controlled trial. Trials 2018;19(1):611. DOI: 10.1186/s13063-018-2995-7. 
25. 
Miskowiak KW, Vinberg M, Macoveanu J, et al. Effects of Erythropoietin on 
Hippocampal Volume and Memory in Mood Disorders. Biol Psychiatry 2015;78(4):270-
7. DOI: 10.1016/j.biopsych.2014.12.013. 
26. 
al-Momen AK. Recombinant human erythropoietin induced rapid healing of a chronic leg 
ulcer in a patient with sickle cell disease. Acta Haematol 1991;86(1):46-8. DOI: 
10.1159/000204799. 
27. 
Bourantas K, Makrydimas G, Georgiou J, Tsiara S, Lolis D. Preliminary results with 
administration of recombinant human erythropoietin in sickle cell/beta-thalassemia 
patients during pregnancy. Eur J Haematol 1996;56(5):326-8. DOI: 10.1111/j.1600-
0609.1996.tb00725.x. 
28. 
Brooks BJ, Jr., Hanson DS, Cryer PA, Hubbard WJ, Hooper D. Erythropoietin therapy 
for sickle cell anemia in Jehovah's Witnesses. South Med J 1991;84(11):1416-7. DOI: 
10.1097/00007611-199111000-00042. 
29. 
el-Hazmi MA, al-Momen A, Kandaswamy S, et al. On the use of 
hydroxyurea/erythropoietin combination therapy for sickle cell disease. Acta Haematol 
1995;94(3):128-34. DOI: 10.1159/000203994. 
30. 
Ferreira FA, Benites BD, Costa FF, Gilli S, Olalla-Saad ST. Recombinant erythropoietin 
as alternative to red cell transfusion in sickle cell disease. Vox Sang 2019;114(2):178-
181. DOI: 10.1111/vox.12750. 
31. 
Goldberg MA, Brugnara C, Dover GJ, Schapira L, Charache S, Bunn HF. Treatment of 
sickle cell anemia with hydroxyurea and erythropoietin. N Engl J Med 1990;323(6):366-
72. DOI: 10.1056/NEJM199008093230602. 
32. 
Han J, Zhou J, Kondragunta V, et al. Erythropoiesis-stimulating agents in sickle cell 
anaemia. Br J Haematol 2018;182(4):602-605. DOI: 10.1111/bjh.14846. 

 
 
Page 32 of 33 
 
33. 
Little JA, McGowan VR, Kato GJ, et al. Combination erythropoietin-hydroxyurea 
therapy in sickle cell disease: experience from the National Institutes of Health and a 
literature review. Haematologica 2006;91(8):1076-83. 
34. 
Nagel RL, Vichinsky E, Shah M, et al. F reticulocyte response in sickle cell anemia 
treated with recombinant human erythropoietin: a double-blind study. Blood 
1993;81(1):9-14. (https://www.ncbi.nlm.nih.gov/pubmed/8417806). 
35. 
Rodgers GP, Dover GJ, Uyesaka N, Noguchi CT, Schechter AN, Nienhuis AW. 
Augmentation by erythropoietin of the fetal-hemoglobin response to hydroxyurea in 
sickle cell disease. N Engl J Med 1993;328(2):73-80. DOI: 
10.1056/NEJM199301143280201. 
36. 
Steinberg MH. Erythropoietin for anemia of renal failure in sickle cell disease. N Engl J 
Med 1991;324(19):1369-70. DOI: 10.1056/NEJM199105093241916. 
37. 
Tomson CR, Edmunds ME, Chambers K, Bricknell S, Feehally J, Walls J. Effect of 
recombinant human erythropoietin on erythropoiesis in homozygous sickle-cell anaemia 
and renal failure. Nephrol Dial Transplant 1992;7(8):817-21. 
(https://www.ncbi.nlm.nih.gov/pubmed/1325614). 
38. 
Jonassaint CR, Rao N, Sciuto A, et al. Abstract Animations for the Communication and 
Assessment of Pain in Adults: Cross-Sectional Feasibility Study. J Med Internet Res 
2018;20(8):e10056. DOI: 10.2196/10056. 
39. 
Ott LR, Schantell M, Willett MP, et al. Construct validity of the NIH toolbox cognitive 
domains: A comparison with conventional neuropsychological assessments. 
Neuropsychology 2022;36(5):468-481. DOI: 10.1037/neu0000813. 
40. 
Mesquita RC, Durduran T, Yu G, et al. Direct measurement of tissue blood flow and 
metabolism with diffuse optics. Philos Trans A Math Phys Eng Sci 
2011;369(1955):4390-406. DOI: 10.1098/rsta.2011.0232. 
41. 
Yazdi HS, O'Sullivan TD, Leproux A, et al. Mapping breast cancer blood flow index, 
composition, and metabolism in a human subject using combined diffuse optical 
spectroscopic imaging and diffuse correlation spectroscopy. J Biomed Opt 
2017;22(4):45003. DOI: 10.1117/1.JBO.22.4.045003. 
42. 
Lee SY, Cowdrick KR, Sanders B, et al. Noninvasive optical assessment of resting-state 
cerebral blood flow in children with sickle cell disease. Neurophotonics 
2019;6(3):035006. DOI: 10.1117/1.NPh.6.3.035006. 
43. 
Durduran T, Yodh AG. Diffuse correlation spectroscopy for non-invasive, micro-vascular 
cerebral blood flow measurement. Neuroimage 2014;85 Pt 1:51-63. DOI: 
10.1016/j.neuroimage.2013.06.017. 
44. 
JANA M. KAINERSTORFER, ANGELO SASSAROLI, MICHELE L. PIERRO, 
BERTAN HALLACOGLU, FANTINI S. COHERENT HEMODYNAMICS 
SPECTROSCOPY BASED ON A PACED BREATHING PARADIGM — REVISITED. 
Journal of Innovative Optical Health Sciences 2014;7(1). 
45. 
Kainerstorfer JM, Sassaroli A, Hallacoglu B, Pierro ML, Fantini S. Practical steps for 
applying a new dynamic model to near-infrared spectroscopy measurements of 
hemodynamic oscillations and transient changes: implications for cerebrovascular and 
functional brain studies. Acad Radiol 2014;21(2):185-96. DOI: 
10.1016/j.acra.2013.10.012. 
46. 
Kainerstorfer JM, Sassaroli A, Tgavalekos KT, Fantini S. Cerebral autoregulation in the 
microvasculature measured with near-infrared spectroscopy. J Cereb Blood Flow Metab 
2015;35(6):959-66. DOI: 10.1038/jcbfm.2015.5. 

 
 
Page 33 of 33 
 
47. 
Wood S, Fanta A, Dosunmu-Ogunbi A, et al. Assessing Dynamic Cerebral 
Autoregulation in Patients with Sickle Cell Disease Using Near-Infrared Spectroscopy 
and Paced Breathing. Blood 2020(136):57-58. 
48. 
Waltz X, Hardy-Dessources MD, Lemonne N, et al. Is there a relationship between the 
hematocrit-to-viscosity ratio and microvascular oxygenation in brain and muscle? Clin 
Hemorheol Microcirc 2015;59(1):37-43. DOI: 10.3233/CH-131742. 
49. 
Byrnes JR, Wolberg AS. Red blood cells in thrombosis. Blood 2017;130(16):1795-1799. 
DOI: 10.1182/blood-2017-03-745349. 
50. 
Shet AS, Wun T. How I diagnose and treat venous thromboembolism in sickle cell 
disease. Blood 2018;132(17):1761-1769. DOI: 10.1182/blood-2018-03-822593. 
51. 
Merten M, Thiagarajan P. P-selectin in arterial thrombosis. Z Kardiol 2004;93(11):855-
63. DOI: 10.1007/s00392-004-0146-5. 
52. 
Srisuwananukorn A, Raslan R, Zhang X, et al. Clinical, laboratory, and genetic risk 
factors for thrombosis in sickle cell disease. Blood Adv 2020;4(9):1978-1986. DOI: 
10.1182/bloodadvances.2019001384. 
53. 
Naik RP, Streiff MB, Lanzkron S. Sickle cell disease and venous thromboembolism: 
what the anticoagulation expert needs to know. J Thromb Thrombolysis 2013;35(3):352-
8. DOI: 10.1007/s11239-013-0895-y. 
54. 
Wassenaar, E.B. and Van den Brand, J.G. (2005) Reliability of Near-Infrared 
Spectroscopy in People with Dark Skin Pigmentation. Journal of Clinical Monitoring and 
Computing, 19, 195-199. http://dx.doi.org/10.1007/s10877-005-1655-0 
55. 
Couch, L., Roskosky, M., Freedman, B. A., & Shuler, M. S. (2015). Effect of Skin 
Pigmentation on Near Infrared Spectroscopy. American Journal of Analytical Chemistry, 
6(12), 911. https://doi.org/10.4236/ajac.2015.612086