Clinical trial NCT01566890
Conditions: Sickle Cell Disease Sickle Cell Anemia
Keywords: sickle cell sickle cell crisis sickle cell pain crisis vaso-occlusive crisis vaso-occlusion sickle cell anemia sickle cell disease genetic diseases adenosine 2A Receptor Agonists Lexiscan Regadenoson contrast-enhanced ultrasound Hydroxyurea
Sponsor: Versiti
Start date: 2012-07-01; Completion date: 2020-11-16
Enrollment: 91 participants; Allocation: NON_RANDOMIZED; Intervention model: SEQUENTIAL

Brief summary:
Sickle cell disease (SCD) is an inherited blood disorder that causes the red blood cells to change their shape from a round shape to a half-moon/crescent or sickled shape. Sickle-shaped cells can cause problems by getting stuck in blood vessels, blocking blood flow, and can cause inflammation and injury to important body parts. There are no specific treatments that improve this condition and promote blood flow hindered by sickle cell blockages. Another big challenge in managing sickle cell disease is that there are no good measures to determine changes and improvements in blood flow.

Contrast-enhanced ultrasound is a technique currently used to detect blood flow in the heart, muscles, and other organs. It is extremely sensitive and can detect blood flow in the smallest of blood vessels. It would be very useful in helping healthcare providers know whether treatment strategies are improving blood flow during sickle cell blockages.

The hypothesis is that contrast-enhanced ultrasound will be a feasible tool for determining changes in blood flow of subjects with sickle cell disease.

Detailed description:
Sickle cell disease is an inherited blood disorder that affects one of every 400 African-Americans newborns in the United States. Sickle cell disease causes the red blood cells to change their shape from a round shape to a half-moon sickle shape. Individuals who have sickle cell disease have a different type of protein that carries oxygen in the blood (hemoglobin) than individuals without sickle cell disease. This different type of hemoglobin makes the red blood cell change into a crescent shape under certain conditions. Sickle shaped cells are a problem because the often get stuck on blood vessels blocking the flow of blood, and cause inflammation and injury to important areas of the body. These symptoms can lead to a painful occurrence called a "sickle cell crisis". Many individuals have to be admitted into the hospital because of the pain caused by a sickle cell crisis.

Regadenoson is a drug that may help prevent inflammation and injury caused by the sickle shaped cells. This drug is approved by the FDA to be used as a bolus during a heart stress test in people unable to exercise enough to put stress on the heart by making it beat faster. In a recent Phase I study, a safe dose for regadenoson was determined for adults with sickle cell disease. This dose was given by a slow IV infusion for a 24-48 hour period.

Hydroxyurea is the only FDA approved drug for the treatment of sickle cell disease. Hydroxyurea is a pill taken orally and works well but is not useful during a severe sickle cell crisis.

In this study researchers will use a new method, contrast-enhanced ultrasound (CEU), to visualize tiny blood vessels in cardiac and skeletal muscle. Changes in CEU measurements before, during and after administration of regadenoson will be examined. Contrast-enhanced ultrasounds will also be performed in individuals who are not having a sickle cell crisis. Some of those individuals will also undergo CEU while they are having a sickle cell crisis to compare those measurements . Lastly CEU results will be compared between individuals with sickle cell anemia and healthy African-Americans. These CEU's will be used to determine if there are changes in the blood flow of tiny blood vessels in certain conditions.

This study wants to know if this new method of contrast-enhanced ultrasound will be a useful tool for physicians to use in individuals with sickle cell anemia. The researchers also want to determine if this new method of CEU can be used to reveal if some treatments for sickle cell anemia work better than others.

Arms: arm 1: Adult subjects with sickle cell anemia will receive a regadenoson infusion with contrast-enhanced ultrasound arm 2: Adult subjects with sickle cell anemia will receive contrast-enhanced ultrasound arm 3: Adults subjects with sickle cell anemia will receive contrast-enhanced ultrasound arm 4: Healthy African American control subjects without sickle cell anemia will receive contrast-enhanced ultrasound arm 5: Healthy volunteers will undergo contrast-enhanced ultrasound.
Interventions: intervention 1: regadenoson infusion with contrast-enhanced ultrasound intervention 2: contrast-enhanced ultrasound intervention 3: contrast-enhanced ultrasound intervention 4: contrast-enhanced ultrasound intervention 5: contrast-enhanced ultrasound
Intervention descriptions: intervention 1: Subjects who are not having a pain crisis receive a 24-hour infusion of regadenoson. Contrast-enhanced ultrasound will be performed four times during the 24 hour regadenoson infusion intervention 2: Subjects who are not having a pain crisis will have contrast-enhanced ultrasound performed up to four times over a two-day period. Time points will resemble the time course used for the Regadenoson Arm, although no investigational drug will be given. intervention 3: Contrast-enhanced ultrasound will be performed on adults with sickle cell anemia at baseline who are not having a pain crisis and performed again during a pain crisis intervention 4: Contrast-enhanced ultrasound will be performed on healthy control subjects at baseline, on the first day of the study and 30 days later intervention 5: Contrast-enhanced ultrasound will be performed on healthy volunteers (Technique Optimization Controls)

Protocol documents:
Prot_SAP_000.pdf: 
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Cover page for Clinicaltrials.gov submission 
 
Title: Effect of Adenosine 2A Receptor Agonist Regadenoson on Microvascular Blood 
Flow in Sickle Cell Anemia 
 
NCT Number: 01566890 
 
Document Date: 02MAY2016 
      


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Effect of Adenosine 2A Receptor Agonist Regadenoson on 
Microvascular Blood Flow in Sickle Cell Anemia 
 
Principal Investigators:  
Joshua Field, MD, MS (Medical College of Wisconsin) 
Jonathan Lindner, MD (Oregon Health & Sciences University) 
 
Co-Investigators:  
Joel Linden, PhD (La Jolla Institute of Allergy & Immunology) 
Roberto Machado, MD (University of Illinois – Chicago) 
Michael Widlansky, MD MPH (Medical College of Wisconsin) 
Jason Rubenstein, MD (Medical College of Wisconsin) 
Nancy Wandersee, PhD (Medical College of Wisconsin) 
 
Conducted by: 
The Medical College of Wisconsin, Milwaukee, Wisconsin  
The University of Illinois, Chicago, Illinois 
Oregon Health and Sciences University, Portland, Oregon 
Dana Farber Cancer Institute, Boston, MA 
La Jolla Institute of Allergy and Immunology, La Jolla, CA 
Protocol Version 5.1 
05/02/2016 
IND# 114,085 


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Table of Contents 
Section   
 
 
 
 
 
 
 
 
 
Page 
1. Background and Significance   
 
 
 
 
 
3 
2. Study Objectives  
 
 
 
 
 
 
 
 
5 
3. Study Population 
 
 
 
 
 
 
 
 
6 
4. Study Design 
 
 
 
 
 
 
 
 
12 
5. Screening Procedures 
 
 
 
 
 
 
 
14 
6. On-Study Procedures 
 
 
 
 
 
 
 
15 
 
Table 1: Study Schedule – Regadenoson Subjects  
 
 
20 
 
Table 2: Study Schedule – Sickle Cell CEU Subjects 
 
 
22 
 
Table 3: Study Schedule –Healthy Control Subjects 
                      23 
 
Table 4: Study Schedule – Technique Optimization Controls 
 
24 
           Table 5: Study Schedule – Sickle Cell Control Subjects  
 
24 
7. Investigational agent 
 
 
 
 
 
 
 
25 
 
8. Toxicities 
 
 
 
 
 
 
 
 
 
30 
9. Correlative Studies  
 
 
 
 
 
 
 
32 
 
10. Subject Remuneration 
 
 
 
 
 
 
 
36 
 
11. Adverse Events 
 
 
 
 
 
 
 
 
37 
 
12. Data and Safety Monitoring 
 
 
 
 
 
 
42 
13. Human Subjects Protection 
 
 
 
 
 
 
43 
 
14. Statistical Considerations 
 
 
 
 
 
 
44 
 
References 
 
 
 
 
 
 
 
 
 
45 
 
 
 
 


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1. 
Background and Significance  
Sickle cell disease (SCD) is an autosomal recessive disorder that is symptomatic in 
the homozygous state. One of every 400 African-American newborns in the United 
States has sickle cell disease 1. The basis for sickle cell disease is a point mutation 
in the sixth codon of the β-globin gene. Under hypoxic conditions, the resulting 
hemoglobin S (ααSS) polymerizes to form an intra-erythrocyte viscous gel that 
induces rigid, dense and deformed erythrocytes. Within the vasculature, sickle 
erythrocytes expressing adhesion molecules interact with adhesion molecule 
receptors on endothelial cells, activating the endothelial cells2. The activated 
endothelial cells call forth further adhesion of inflammatory cells and platelets to 
vascular endothelium causing recurrent microvascular occlusion and leading to 
tissue ischemia and ultimately end-organ damage. The activated inflammatory cells 
(particularly iNKT cells) release cytokines that potentiate inflammation, adhesion and 
obstruction in the affected microvessels3,4. The two most common morbidities in 
sickle cell disease, pain episodes5and acute chest syndrome (ACS)6, are secondary 
to this inflammatory vaso-occlusion and there are no specific therapies available to 
acutely treat these complications. 
Acute vaso-occlusive episodes (pain and acute chest syndrome) are the leading 
cause of morbidity and mortality among individuals with sickle cell disease and few 
therapeutic options are available7,8. Pain episodes result from accelerated vaso-
occlusion within bone marrow, joints and soft tissues. Typically characterized by 
bony pain in the legs, arms, chest and back, patients with HbSS will have, on 
average, one pain episode per year that requires hospitalization9. Acute chest 
syndrome is defined as a new infiltrate on chest radiograph and is often 
accompanied by respiratory symptoms, chest pain, fever and cough10. Laboratory 
evidence consistent with acute chest syndrome include increased rate of hemolysis, 
leukocytosis and hypoxemia11.  
Hydroxyurea is the only FDA-approved drug for the treatment of vaso-occlusion. The 
primary effect of hydroxyurea is to increase fetal hemoglobin production thereby 
disrupting hemoglobin polymerization and, ultimately, erythrocyte sickling 
12. 
Although such anti-sickling therapies have an important role in the prevention of pain 
and acute chest syndrome episodes, this strategy is not effective in the acute 
setting. Therapeutic options for pain and acute chest syndrome events are limited in 
the acute setting to supportive care with fluids, antibiotics, opioids and, in the case of 
severe acute chest syndrome episodes, blood transfusion. Although transfusion 
therapy is effective for the treatment of acute chest syndrome, the risk of 
alloimmunization in patients with sickle cell disease is 3% per unit transfused13, 


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greatly limiting the availability of compatible blood in the future. Alternative strategies 
for the treatment of acute exacerbations of vaso-occlusion are needed. Promising 
approaches to decrease the severity of acute vaso-occlusive episodes include 
interrupting endothelial adhesion and/or decreasing inflammation3. Corticosteroids 
are the most rigorously studied anti-inflammatory agents in sickle cell disease and 
have been shown to decrease length of hospital stay14,15. However, patients treated 
with corticosteroids experienced rebound vaso-occlusion and 25% of study 
participants receiving corticosteroids required re-hospitalization14. This study will 
examine the effects of regadenoson, an adenosine2A receptor agonist that has 
demonstrated potent anti-inflammatory activity (Objective 1) and hydroxyurea 
(Objective 2) on microvascular blood flow using contrast-enhanced ultrasound.  
Identifying an outcome measure to assess the clinical efficacy of sickle cell disease 
therapies has been a barrier to drug development. Prior and ongoing studies 
evaluating drugs aimed to decrease the severity of vaso-occlusion have used length 
of hospital stay 14,15, pain scores (clinical trials.gov #NCT00252122) and transfusion 
requirements (clinical trials.gov #NCT01089439) to assess clinical efficacy. Although 
interventions with a very large effect size may demonstrate benefit using these 
outcomes, confounding factors such as differing physician practice, inter-individual 
variability in pain estimates and concomitant medical problems may complicate the 
assessment of drug effect.  
Contrast-enhanced ultrasound has the potential to significantly improve our ability to 
measure the efficacy of drug candidates. It is notable that in multiple previous 
studies, it has been possible to detect < 10% changes in skeletal muscle vascular 
perfusion, indicating that contrast-enhanced ultrasound can detect small changes in 
vascular perfusion16-20. Dr. Lindner’s (PI) laboratory has used contrast-enhanced 
ultrasound to evaluate flow disturbances in the coronary, renal and skeletal muscle 
microcirculation related to inflammatory diseases and rheologic disorders such as 
ischemia-reperfusion injury21, hyperlipidemia22, insulin resistance20 and endothelial 
dysfunction23. Pertinent to sickle cell disease, contrast-enhanced ultrasound is able 
to not only quantify regional perfusion, but is also able to separately assess tissue 
capillary blood volume, capillary RBC velocity, and spatial heterogeneity of capillary 
perfusion. Contrast-enhanced ultrasound is a portable technique that can be 
performed at bedside in about 5 minutes.  
Two separate FDA-approved agents (Definity and Optison) are available to assess 
skeletal muscle in doses that are within the regulatory agency guidelines. There 
have been over 3 million administered doses of the microbubble contrast agents with 
an excellent safety profile, including in very unstable conditions such as myocardial 


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infarction. The reported incidence of pain associated with Definity is 1.2%40; 
although personal communications with practicing cardiologists suggest that the 
incidence is much lower. In our studies of patients with SCD, the incidence of pain 
events per CEU exam was 4.1% (4/97). Dr. Lindner (Co-PI) has performed studies 
in a murine model implicating complement deposition on the microbubbles as the 
mechanism for the pain events associated with Definity41. In the renal vasculature, 
the complement-laden microbubbles adhere to the endothelium and generate 
bradykinin, which alters nociception and causes pain41. We would like to further 
evaluate the mechanism of pain in this patient population. Currently, there is no 
literature to suggest that patients with SCD, or any other pro-inflammatory disorder, 
are more susceptible to pain reactions due to Definity than other groups. To 
determine whether complement deposition contributed to the pain events associated 
with Definity in patients with SCD, we will perform in vivo and flow cytometry 
analyses on plasma samples.  
The ultimate goal in studying regadenoson is to improve vaso-occlusion and 
dampen the severity of vaso-occlusive episodes. Regadenoson’s mechanism in 
sickle cell disease is to inhibit iNKT cell activation thereby decreasing inflammation 
that contributes to white blood cell, platelet and endothelial cell activation. 
Decreasing inflammation and interrupting the multi-cellular interactions of vaso-
occlusion should result in increased blood flow. If blood flow is not increased 
following regadenoson, it is unlikely to provide clinical benefit in reducing the severity 
of pain or acute chest syndrome episodes. For this proposal, we will quantify 
regional perfusion in the skeletal muscle and myocardium in sickle cell disease 
subjects using contrast-enhanced ultrasound.  
 
2.   
Study Objectives  
 2.1 
Primary Objective: To determine changes in microvascular blood flow using 
contrast-enhanced ultrasound (CEU) in 27 adults age 18 years or older with 
sickle cell anemia before, during and after a 24 hour infusion of regadenoson.  
 2.2 
Secondary Objectives:  
• To determine the association between complement deposition on lipid 
microbubbles and pain events associated with Definity in adults with 
SCD. 
• To examine differences in microvascular blood flow using contrast-enhanced 
ultrasound (CEU) in adults with sickle cell anemia age 18 and older at 
baseline compared to healthy, African American controls. 


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• To examine differences in microvascular blood flow using contrast-enhanced 
ultrasound (CEU) in adults with sickle cell anemia age 18 and older at 
baseline state compared to a pain crisis. 
• To evaluate the activation of iNKT cells before, during and after regadenoson  
3.  
Study Population 
The study population with consist of four study groups, and one technique 
optimization control group: 
• Regadenoson treatment group 
• Sickle Cell CEU measurement group 
• Healthy Control subjects group 
• Technique Optimization Control subjects group 
• Sickle Cell Control group 
 
3.1    Regadenoson Eligibility Criteria 
Regadenoson Study Treatment Inclusion Criteria:  
1. Diagnosis of sickle cell disease confirmed by hemoglobin analysis  
2. Ages 18 to 70 years 
3. Laboratory parameters: 
a. Hemoglobin ≥ 6 g/dL 
b. Platelets > 150,000/mcL 
c. ALT < 2.5 X upper limit of normal 
d. Serum creatinine < 1.5 mg/dL 
e. INR < 1.5 
f. PTT < 40 seconds  
4. Reliable IV access as determined by physician 
Regadenoson Study Treatment Exclusion Criteria:  


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1. Subject is not at baseline (see definition of baseline section 3.7) at the time of 
study treatment 
2. Current physician diagnosis of active asthma (within last 12 months) or current 
use of asthma medications (inhaled corticosteroids, leukotriene antagonists). 
Participants using bronchodilators will be allowed to participate 
3. Second or third degree AV block or sinus node dysfunction 
4. Known or suspected right to left sided cardiac shunts  
5. History of a bleeding diathesis  
6. History of clinically overt stroke within the past 3 years  
7. History of severe hypertension not adequately controlled with anti-hypertensive  
medications (SBP ≥ 200 mmHg and/or DBP ≥ 110 mmHg)  
8. Receiving chronic anti-coagulation or anti-platelet therapy  
9. History of metastatic cancer  
10.  Receiving other investigational study agents, or have received a study agent in 
the last 30 days 
11.  Uncontrolled intercurrent illness including, but not limited to:  
a. Ongoing or active infection 
b. Symptomatic congestive heart failure 
c. Unstable angina pectoris 
d. Cardiac arrhythmia 
e. Psychiatric illness/social situations that would limit compliance with study 
requirements  
12.  Pregnancy (must be excluded by a negative urine pregnancy test in all women 
of childbearing potential) or breastfeeding  
13.  Know HIV infection 
14. Participants who have previously undergone a hematopoietic stem cell 
transplant  
15. Participants who are taking medications that may interact with the 
investigational agent, including dipyridamole, aminophylline, and theophylline  


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16. Caffeine and theophylline may not be taken within 12 hours of starting 
regadenoson.  Dipyridamole and aminophylline may not be taken within 48 hours 
of starting regadenoson 
17. Prior hypersensitivity reactions to either regadenoson or ultrasound contrast 
agents  
 
3.2 Sickle Cell CEU Subject Eligibility Criteria: 
Sickle Cell CEU Subject Inclusion Criteria: 
1. Diagnosis of sickle cell disease confirmed by hemoglobin analysis 
2. Males and females age 18-70 years  
Sickle Cell CEU Subject Exclusion Criteria: 
1. Known pregnancy 
2. Known history of HIV 
3. History of stem cell transplant 
4. Current involvement in a therapeutic clinical trial 
5. Known or suspected right to left sided cardiac shunts 
6. Prior hypersensitivity reactions to ultrasound contrast agents 
7. History of severe hypertension not adequately controlled with anti-hypertensive  
medications (SBP ≥ 200 mmHg and/or DBP ≥ 110 mmHg)  
8. Uncontrolled intercurrent illness including, but not limited to: 
a. Ongoing or active infection 
b. Symptomatic congestive heart failure 
c. Unstable angina pectoris 
d. Cardiac arrhythmia 
 
3.3 Healthy Controls Eligibility Criteria 
Enrollment Inclusion Criteria for Healthy Control Subjects: 
1. African American  
2. Ages 18 to 70 years  


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Enrollment Exclusion Criteria for Healthy Controls: 
1. Sickle cell disease or sickle cell trait confirmed by Sickledex 
2. Known or suspected right to left sided cardiac shunts  
3. Diagnosis of type 1 or type 2 diabetes mellitus 
4. Hypertension defined as (SBP ≥ 200 mmHg and/or DBP ≥ 110 mmHg) or 160/90 
on anti-hypertension medication 
5. History or current diagnosis of dyslipidemia or taking lipid lowering drugs 
6. Diagnosis of coronary artery disease or peripheral vascular disease  
7. Body weight greater than 10% of ideal weight which would hinder ability to obtain 
ultrasound images, as assessed by study ultrasound tech 
8. Uncontrolled intercurrent illness including, but not limited to: 
a. Ongoing or active infection 
b. Symptomatic congestive heart failure 
c. Unstable angina pectoris 
d. Cardiac arrhythmia  
9. Pregnancy (must be excluded by a negative urine pregnancy test in all women of 
childbearing potential) or breastfeeding  
10.  Known HIV infection 
11.  Prior hypersensitivity reactions to ultrasound contrast agents 
3.4 Technique Optimization Controls Eligibility Criteria 
Enrollment Inclusion Criteria for Technique Optimization Controls: 
1. Ages 18 to 70 years  
Enrollment Exclusion Criteria for Technique Optimization Controls: 
1. Known sickle cell disease or sickle cell trait 
2. Known or suspected right to left sided cardiac shunts  
3. Uncontrolled intercurrent illness including, but not limited to: 
a. Ongoing or active infection 
b. Symptomatic congestive heart failure 
c. Unstable angina pectoris 


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d. Cardiac arrhythmia  
4. Known pregnancy or breastfeeding 
Prior hypersensitivity reactions to ultrasound contrast agents 
3.5 Sickle Cell Control Subject Eligibility Criteria: 
Sickle Cell Control Subject Inclusion Criteria: 
1. Diagnosis of sickle cell disease confirmed by hemoglobin analysis. 
2. Ages 18 to 70 years 
Sickle Cell Control Subject Exclusion Criteria: 
3. Subject is not at baseline (see definition of baseline section 3.8) at the time of 
study treatment. 
4. Known pregnancy 
5. Known history of HIV 
6. History of stem cell transplant 
7. Receiving other investigational study agents, or have received a study agent in                 
last 30 days. 
8. Known or suspected right to left sided cardiac shunts 
9. Prior hypersensitivity reactions to ultrasound contrast agents 
10. History of severe hypertension not adequately controlled with anti-hypertensive  
medications (SBP ≥ 200 mmHg and/or DBP ≥ 110 mmHg)  
11. Uncontrolled intercurrent illness including, but not limited to: 
a. Ongoing or active infection 
b. Symptomatic congestive heart failure 
c. Unstable angina pectoris 
d. Cardiac arrhythmia 
 
 
3.6 Selection of Subjects 
Subjects will be recruited in Milwaukee and Chicago. Subjects in all study treatment 
arms will be recruited from the Froedtert Adult Sickle Cell Disease Clinic in 


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Milwaukee, which currently serves more than 200 adults with sickle cell disease. 
Regadenoson is currently being administered to patients in Milwaukee and therefore 
the infrastructure is in place to study this drug. Subjects for the Healthy control group 
and technique optimization control group will be recruited in Milwaukee using an 
advertisement in the Adult Sickle Cell Disease Clinic at Froedtert Hospital, the Adult 
Translational Research Unit at the Medical College of Wisconsin, within the 
BloodCenter of Wisconsin Headquarters building, the Blood Research Institute 
building, and BloodCenter of Wisconsin Donor Centers, including the Milwaukee, 
Brown Deer, and Wauwatosa locations.  
Subjects will also be recruited in Milwaukee and Chicago for the Definity 
complement deposition blood sample. The University of Illinois in Chicago, follows 
over 500 adults with sickle cell disease and may also identify healthy controls 
(according to criteria in section 3.3) to give blood samples, using a method 
approved by their local IRB. 
 
3.7  Definitions 
Acute Chest Syndrome (ACS): ACS is an acute illness characterized by fever 
and/or respiratory symptoms, accompanied by a new pulmonary infiltrate on a 
chest X-ray.  Diagnostic criteria include, but are not limited to: a new segmental 
radiographic pulmonary infiltrate, in addition to at least one of the following: 
temperature ≥38.5ºC, >2% decrease in SpO2 from a documented steady-state 
value on room air (FiO2 = 0.21), PaO2 <60 mmHg, tachypnea (per age-adjusted 
normal), intercostal retractions, nasal flaring, or use of accessory muscles of 
respiration, chest pain, cough, wheezing, or rales 
Baseline: The subject is well, reporting no more than baseline pain and is at least 2 
weeks from a hospitalization or emergency department visit for any reason, or 4 
weeks from a hospitalization if the subject was diagnosed with Acute Chest 
Syndrome (ACS) 
Pain Crisis (pain): An episode characterized as pain related to SCD in the 
extremities, back, abdomen, chest or head lasting at least 2 hours and leading to 
a hospitalization or emergency department visit 9 
Red Blood Cell Exchange (RBCX): An exchange blood transfusion via manual 
exchange (phlebotomy plus transfusion of blood products) or erythrocytapheresis 
Study 
Physician: 
In 
this 
protocol, 
“study 
physician” 
refers 
to 
the 
physician/investigator who oversees protocol therapy. The study physician may 
or may not be the same person as the treating physician, who provides the 
patient’s standard care. 


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Treating Physician: In this protocol, “treating physician” refers to the hospital or 
outpatient supervising physician who provides the patient’s standard care.  The 
treating physician may or may not be the same person as the study physician, 
who oversees protocol therapy. 
 
4. 
Study Design 
4.1  Regadenoson Study Treatment (Milwaukee): To examine the effects of 
regadenoson on microvascular blood flow, we will administer regadenoson at 1.44 
mcg/kg/hr, to 27 evaluable subjects with sickle cell disease ages 18 to 70 years, for 
24 hours. Study coordinators will identify participants from rosters of the sickle cell 
disease clinic populations in Milwaukee. Following informed consent and after 
confirming eligibility, Subjects will receive infusional regadenoson when they are at 
baseline  (see definition of baseline section 3.7). Prior to starting the infusion, a 
limited functional 2D echocardiogram and signal-average electrocardiogram will be 
performed. Contrast-enhanced ultrasound, correlative study lab measurements and 
optional “red and white blood cell” exploratory lab measurements will be obtained at 
baseline, approximately 6 hours after the infusion starts, and prior to stopping the 
infusion. A limited functional 2D echocardiogram may also be repeated prior to 
stopping the regadenoson infusion. Subjects will be monitored for 6 hours after the 
infusion is complete and will repeat the contrast-enhanced ultrasound measurement 
at the conclusion of the observation period. Subjects will also be asked to complete 
a cardiac questionnaire. The primary outcome measure will be a 40% increase in 
skeletal muscle microvascular blood flow when the 24 hour measurements are 
compared to the baseline measurements. 
4.2   Sickle Cell CEU subjects (Milwaukee): To determine variability in contrast-
enhanced ultrasound measurements among adults with  sickle cell disease we will 
also obtain skeletal muscle and myocardial contrast-enhanced ultrasound values, 
limited functional 2D echocardiograms, a signal-average electrocardiogram, 
correlative laboratory study blood samples and optional “red and white blood cell” 
exploratory blood samples, from 30 evaluable subjects with sickle cell anemia at 
baseline.  
Due to variability in an individual’s blood flow, an additional baseline contrast-
enhanced ultrasound may be performed at least 7 days later, but within 30 days, 
when the subject is at baseline, if feasible. The same study procedures, with 
exception of the signal-averaged electrocardiogram, will also be performed when the 
subjects are admitted to the hospital for a pain crisis (see definition in Section 3.7). 


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Of note, the initial measurement may be taken when the subject is in pain, followed 
by a baseline measurement, which should be obtained when the subject is 
experiencing their baseline pain (see definition in Section 3.7). Subjects will also 
complete a cardiac questionnaire at baseline. Of these 30 subjects, up to 2 of the 
individuals may have additional 2D Echo and contrast-enhanced ultrasound 
measurements taken prior to, and following a red blood cell exchange transfusion 
(see definition in Section 3.7), if this therapy has been recommended clinically. The 
contrast-enhanced ultrasound measurement should be taken within 72 hours of 
completing the transfusion, or as soon as reasonably possible.  The subjects 
receiving red blood cell exchange transfusions will have additional correlative 
laboratory blood samples and optional “red and white blood cell” exploratory blood 
samples taken at the time of contrast-enhanced ultrasounds. Pain scores will be 
collected at the time of each study blood draw. 
4.3   Healthy Control subjects (Milwaukee): To determine baseline variability in 
contrast-enhanced ultrasound measurements, we will also obtain skeletal muscle 
and myocardial contrast-enhanced ultrasound values, correlative study lab 
measurements and optional “red and white blood cell” exploratory lab 
measurements from 20 evaluable age and gender-matched African American 
controls without sickle cell disease or sickle cell trait at baseline, 1 day and 30 days 
(plus or minus seven days). Controls will be given an option to obtain additional 6 
hour skeletal muscle and myocardial contrast-enhanced ultrasound on each of their 
visits( Day0, Day 1 and Day 30). Subjects may choose to do the optional 6 hr time 
point CEU at one, all or none of their visits. At baseline, a limited functional 2D 
echocardiogram and signal-average electrocardiogram will be performed. 
4.4   Technique Optimization Control subjects (Milwaukee): To refine the 
contrast-enhanced ultrasound measurement technique, we will obtain skeletal 
muscle and myocardial contrast-enhanced ultrasound values from up to 20 healthy 
volunteers, study-wide. Volunteers will not have sickle cell disease or sickle cell trait.  
Participants may also have an additional contrast-enhanced ultrasound performed at 
least one day after the initial measurement. 
 
4.5 Sickle Cell Control subjects (Milwaukee): To learn about the impact of CEU 
technique and definity microbubbles on the rise in microvascular blood flow rate, we 
will obtain skeletal muscle and myocardial contrast-enhanced ultrasound values 
from up to 20 sickle cell subjects. These subjects will follow the study procedure 
similar to regadenoson arm, however they will not receive regadenoson, hence 
acting as sickle cell control subjects. The 24 hr and 30 hr CEU studies will be 
optional for these subjects.  
 


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The subjects who have already completed Regadenoson arm or Sickle Cell CEU 
Arm of the study may also be enrolled in this arm. Likewise, subject may participate 
in the Regadenoson or Sickle Cell CEU Arm after completed participation in the 
‘Sickle Cell Control’ Arm, if they meet study entry criteria for the respective Arm. 
 
Blood Samples for Definity complement deposition (Milwaukee and Chicago): 
To gather more valuable information about the mechanism for pain and other 
adverse events to contrast agents, such as Definity, we would like to collect blood 
samples from up to 14 subjects with sickle cell and up to 10 healthy controls, study-
wide. Subjects with sickle cell and healthy controls who provide their consent will 
have a one-time blood sample while at their baseline state (see definition in Section 
3.7), which will be used to evaluate complement deposition on study contrast 
(Definity) microbubbles. Labs for clinical complement values (C3, C4, CH50) will 
also be obtained.  This may be a standalone blood draw or performed at another 
scheduled study visit. If this sample is drawn, the total volume of blood drawn for the 
study from any subject will not exceed 50ml per 8 week period.   
 
Individuals who meet basic study entry criteria for any of the study arms (excluding 
the Technique Optimization Controls Arm), or subjects who previously participated in 
the study, may be approached for providing this sample. Of note, self-reported 
health status, (including sickle cell, pregnancy state, or other) may be allowed in 
evaluating eligibility for obtaining this blood sample, however should be confirmed 
via applicable screening procedures (Section 5), if the individual should go on to 
participate in other study arms. 
 
 
5. 
Screening Procedures 
 
*Screening evaluations are to be conducted within 30 days prior to start of 
protocol therapy (regadenoson, sickle cell CEU subjects, healthy controls, 
sickle cell controls, or technique optimization control subjects). 
Historical Hemoglobin analysis and sickle dex test are acceptable. 
5.1  Regadenoson Screening Procedures 
✓ Vital signs, height and weight 
✓ Documentation of pain score prior to lab draw 
✓ Blood Tests: 
o Complete blood count with or without differential & platelet count  
o Comprehensive metabolic panel 
o Coagulation tests  
o Study lab draw for Definity complement deposition and values (optional) 
✓ Urine pregnancy test (in women of childbearing potential) 


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✓ Collect demographics, race, ethnicity, medical diagnostic history  
✓ Concomitant medications reviewed 
✓ Determine caffeine and theophylline will not be taken 12 hours prior to receiving 
regadenoson (infusion can be delayed for 12 hours if the subject ingests caffeine 
or theophylline)   
✓ Determine dipyridamole and aminophylline will not be taken in the 48 hours prior 
to regadenoson infusion 
✓ A study physician or designee may read and assess a cardiac EKG tracing to 
rule out 2° or 3° heart block prior to the administration of regadenoson. A recent 
(≤30 days) EKG may also be used for the same purpose, if it is available. 
 
 
5.2 Sickle Cell CEU Subject Screening Procedures 
✓ Vital signs, height and weight 
✓ Urine pregnancy test in all women of childbearing potential 
✓ Collection of demographics, race, ethnicity,  medical diagnostic history and 
medication history  
 
5.3  Healthy Control Subjects Screening Procedures 
✓ Sickledex blood test 
✓ Collection of demographics, race, ethnicity,  medical diagnostic history and 
medication history  
✓ Urine pregnancy test in all women of childbearing potential 
5.4  Technique Optimization Control Subjects Screening Procedures 
✓ Collection of medical diagnostic history and medication history 
✓ Physical exam, if subject’s medical history suggests possible cardiac disorders 
requiring further assessment, or per study physician’s discretion 
 
      5.5  Sickle Cell Control Subjects Screening Procedures 
 
✓ Collection of demographics, race, ethnicity, medical diagnostic history and 
medication history  
✓ Vital signs, height and weight 
✓ Urine pregnancy test in all women of childbearing potential 
✓ Collection of demographics, race, ethnicity, medical diagnostic history and 
medication history  
 
6. 
On-Study Procedures  
 
6.1  Regadenoson (Within 4 hours prior to infusion) 
• Signal Averaged ECG (+/- 30 days from infusion visit date / Baseline) 


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• Reconfirm eligibility and that subject is at baseline (if necessary) 
• Concomitant medication use reviewed prior to infusion:  
o Assess caffeine and theophylline intake for previous 12 hours (delay the 
onset of the infusion for at least 12 hours if the subject recently ingested 
caffeine or theophylline) 
o Assess dipyridamole and aminophylline intake in previous 48 hours. 
• Limited functional 2D echocardiogram prior to infusion 
• Contrast-enhanced ultrasound is performed prior to infusion 
• Documentation of pain score prior to lab draw 
• Correlative laboratory studies and optional “red and white blood cell” 
exploratory study labs (up to 6ml) are drawn prior to infusion 
o *If subject agrees, and gives the Definity complement sample, “red and 
white blood cell” samples may only be collected if at least +/-8 weeks 
from Definity complement sample being drawn (as not to exceed 50ml 
blood collected in 8 week period) 
• Complete cardiac questionnaire (can be completed any time prior to the end 
of observation period) 
 
Regadenoson Administration Procedure: 
Prior to starting the study drug infusion, a baseline heart rate and blood pressure will 
be established with at least 2 measurements, 5-10 minutes apart. If the last two 
consecutive measurements have a difference greater than 5 beats per minute (heart 
rate) or 10 mm Hg (systolic blood pressure), they should be repeated every 5 
minutes until they stabilize. The baseline measurement is the last heart rate and 
blood pressure measurement prior to study drug infusion. Oxygen saturation and 
heart rate will be measured during drug infusion and blood pressure will be taken 
every 30 minutes for 2 hours (+/-10 minutes), hourly for the next 2 hours, then every 
two hours (+/-15 minutes) for the duration of the infusion, and during the observation 
period.  
 
Blood draws will be performed prior to infusion and at the times contrast-enhanced 
ultrasound is performed, for laboratory measurements. Pain will be assessed at the 
same time as blood draws using a standardized pain scale. Blood pressure 
monitoring and blood draws will be performed on a limb or site other than the 
regadenoson infusion.  
 
The infusion will be stopped for any for any toxicity that is designated a serious 
adverse event (SAE). For this study, an SAE will be defined according to the CTEP 
Active Version of the NCI Common Terminology Criteria for Adverse Events 
(CTCAE) with the exception of heart rate and blood pressure. SAEs are CTCAE 
grade 3 events as well as significant changes in heart rate or blood pressure 
(defined in section 11.3). Before initiation of the regadenoson infusion, resuscitation 
equipment and trained staff will be available.   


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After the 24 hour infusion (or if the infusion is stopped early for an SAE), there will be 
a 6 hour observation period during which the subject will be monitored. Oxygen 
saturation and heart rate will be measured, blood pressure will be recorded every 2 
hours (+/-15 minutes) and blood samples will be collected at the end of the 
observation period at 30 hours. Caffeine may not be ingested during the infusion or 
observation period. 
 
Regadenoson (6 hours after infusion start +/- two hours, or earlier if 
necessary, per discretion of study physician) 
• 
Contrast-enhanced ultrasound  
• 
Documentation of pain score prior to lab draw 
• 
Correlative laboratory studies drawn 
 
Regadenoson (24 hours from infusion start within two hours before infusion 
stop, or earlier if necessary, per discretion of study physician) 
• 
Limited functional 2D echocardiogram 
• 
Contrast-enhanced ultrasound  
• 
Documentation of pain score prior to lab draw 
• 
Correlative laboratory studies and optional exploratory study labs (up to 
6ml) ardrawn 
• 
Infusion may over-run up to 1 hour, with PI permission (if difficulty 
completing study procedures before infusion stopped) 
o *If subject agrees, and gives the Definity complement sample, “red and 
white blood cell” samples may only be collected if at least +/-8 weeks 
from Definity complement sample being drawn (as not to exceed 50ml 
blood collected in 8 week period) 
 
Regadenoson (30 hours from infusion start +/- two hours, or earlier if 
necessary, per discretion of study physician) 
• 
Contrast-enhanced ultrasound  
• 
Documentation of pain score and adverse events, if applicable 
• 
Correlative laboratory studies drawn 
 
Post-regadenoson follow-up period (30 days post infusion) 
• 
Subjects will be contacted by phone weekly (plus or minus 3 days) for 4 
weeks following the conclusion of regadenoson therapy to assess for any 
potential toxicities related to study drug  
 
 
6.2 Sickle Cell CEU Subject 
 
Sickle Cell CEU Subject (Baseline) 


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• Review  medical diagnostic history and medication history 
• Vital signs, height/weight 
• Documentation of pain score prior to lab draw 
• Correlative laboratory studies and optional “red and white blood cell” 
exploratory study labs are drawn 
• Study lab draw for Definity complement deposition and values (optional) 
• Signal-average ECG (plus or minus 30 days from baseline) 
• Limited functional 2D echocardiogram 
• Contrast-enhanced ultrasound  
• Complete cardiac questionnaire 
 
Sickle Cell CEU Subject (Second Baseline, ≤30 days if feasible) 
• Review  medical diagnostic history and medication history (if different) 
• Vital signs,  
• Documentation of pain score prior to lab draw 
• Correlative laboratory studies drawn 
• Contrast-enhanced ultrasound  
• Limited functional 2D echocardiogram (optional)  
 
Sickle Cell CEU Subject (Pain Crisis) 
• Review  medical diagnostic history and medication history 
• Vital signs, height/weight 
• Documentation of pain score prior to lab draw 
• Correlative laboratory studies and optional “red and white blood cell” 
exploratory study labs are drawn 
• Contrast-enhanced ultrasound  
• Limited functional 2D echocardiogram (optional) 
 
 
Sickle Cell CEU Subject (before red blood cell exchange) – up to 2 subjects 
**Note – the ‘Before’ red blood cell exchange event may qualify as either a 
Baseline or Pain Crisis event, depending on patient circumstances 
• Review  medical diagnostic history and medication history 
• Vital signs, height/weightDocumentation of pain score prior to lab draw 
• Correlative laboratory studies drawn 
• Contrast-enhanced ultrasound 
• Limited functional 2D echocardiogram (optional) 
 
Sickle Cell CEU Subject (after red blood cell exchange) – up to 2 subjects 
• Review  medical diagnostic history and medication history 
• Vital signs,  
• Documentation of pain score prior to lab draw 
• Correlative laboratory studies and optional “red and white blood cell” 
exploratory study labs are drawn 


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• Contrast-enhanced ultrasound within 72 hours following transfusion 
• Limited functional 2D echocardiogram (optional) 
 
6.3 Healthy Control Subject Procedures 
 
 
Healthy Control Subject (Day 0) 
• Review  medical diagnostic history and medication history, if applicable 
• Vital signs, height and weight collected 
• Correlative laboratory studies and optional “red and white blood cell” 
exploratory study labs are drawn 
• Study lab draw for Definity complement deposition and values (optional) 
• Signal-average ECG (plus or minus 30 days from baseline) 
• Limited functional 2D echocardiogram 
• Contrast-enhanced ultrasound  
• Optional Contrast-enhanced ultrasound at 6 hours 
 
Healthy Control Subject (Day 1) 
• Vital signs 
• Correlative laboratory studies drawn 
• Contrast-enhanced ultrasound  
• Optional Contrast-enhanced ultrasound at 6 hours 
 
 Healthy Control Subject (Day 30 plus or minus 7 days) 
• Review  medical diagnostic history and medication history, if applicable 
• Vital signs 
• Correlative laboratory studies drawn 
• Contrast-enhanced ultrasound  
• Optional Contrast-enhanced ultrasound at 6 hours 
 
For all the study arms, CBC with diff, platelet count, and comprehensive 
metabolic panel are standard of care   if done within 30 days, and need not be 
repeated. 
 
6.4 Technique Optimization Control Subject Procedures 
 
 
Technique Optimization Control Subject 
• Review  medical diagnostic history and medication history, if applicable 
• Vital signs collected 
• Contrast-enhanced ultrasound  
 
Technique Optimization Control Subject (2nd measurement, if applicable) 
• Vital signs collected 
• Contrast-enhanced ultrasound  


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6.5   Sickle Cell Control Subject 
 
Sickle Cell Control Subject (0 hour): 
 
• Signal Averaged ECG (+/- 30 days from infusion visit date) 
• Reconfirm eligibility and that subject is at baseline (if necessary) 
• Concomitant medication use reviewed  
• Vital signs 
• Limited functional 2D echocardiogram  
• Contrast-enhanced ultrasound is performed 
• Documentation of pain score prior to lab draw 
• Correlative laboratory studies and optional “red and white blood cell” 
exploratory study labs are drawn 
• Study lab draw for Definity complement deposition and values (optional) 
• Complete cardiac questionnaire (can be completed any time prior to the 
end of the 24 hr CEU study) 
 
Sickle Cell Control Subject (6 hours +/- 2hr from baseline): 
• Vital signs 
• Contrast-enhanced ultrasound  
• Documentation of pain score prior to lab draw 
• Correlative laboratory studies are drawn 
 
      Sickle Cell Control Subject (24 hours +/- 2hr from baseline –optional):  
• Vital signs 
• Contrast-enhanced ultrasound  
• Documentation of pain score prior to lab draw 
• Correlative laboratory studies and optional “red and white blood cell” 
exploratory study labs are drawn 
 
       Sickle Cell Control Subject (30 hours +/-2hr from baseline - optional): 
• 
Vital signs 
• 
Contrast-enhanced ultrasound  
• 
Documentation of pain score prior to lab draw 
• 
Correlative laboratory studies are drawn 
 


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Table 1. Study Schedule of Procedures for Regadenoson Subjects 
Procedure 
Screening 
Baseline  
6 hours 
(+/-2hr) 
24 hours 
30 hours 
(+/-2hr) 
30 Day 
Follow up 
Documentation- MedHx, 
medications, Tx  & clinical 
data 
X 
 
 
 
 
 
Signal-average ECG 
Xf 
PT/INR, PTT  
X 
 
 
 
 
 
β-HCGc  
X 
 
 
 
 
 
CBC  
X 
 
 
 
 
 
Comprehensive metabolic 
panelb 
Xb 
 
 
 
 
 
VS (including SBP, HR,  
O2 sat) 
X 
X a 
 
Regadenoson infusion 
 
XJ 
 
 
Adverse event evaluation 
X 
Limited functional 2D Echo 
 
X 
 
X 
 
 
Contrast-Enhanced 
Ultrasound 
 
X 
X 
X 
X 
 
iNKT cell measurement 
 
X 
X 
Xd 
X 
 
Inflammatory Markers 
 
X 
 
X 
 
 
Exploratory “red and white 
blood cell” study labsg 
 
       Xh,i 
 
       Xh,i 
 
 
Definity complement 
deposition blood sample 
(optional) 
Xi, k 
Complement (C3, C4, CH50) 
(drawn if optional Definity 
complement sample drawn) 
Xi, k 
Pain Scorese 
X 
X 
X 
X 
X 
 
Cardiac Questionnaire 
 
X 
 
Coordinator  calls subject weekly to assess for toxicities after regadenoson infusion 
a: BP, O2 saturation, and HR will be taken prior to infusion and at the following times after the start of the 
infusion: every 30 min (+/- 10 min) for 2 hrs,. Then hourly for 2 hours, then every 2 hours for the rest of the 
infusion (+/-15 min). After the infusion ends, there will be a 6 hr observation period, during which BP, HR and 
O2 saturation will be taken every 2 hrs (+/- 15 min). b: Albumin, alkaline phosphatase, total bilirubin, serum 
creatinine, BUN, calcium, chloride, glucose, potassium, total  protein, SGOT [AST], SGPT [ALT], sodium 


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c: Urine pregnancy test (women of childbearing potential) 
d: The 24 hour blood draw should occur prior to stopping the drug. 
e: Pain scores will be taken just prior to each blood draw using a visual analog scale 
f: Signal-averaged ECG may be performed within 30 days of the infusion visit 
g: Optional lab testing for blood flow adhesion and leukocyte activation 
h: up to 6ml blood may be collected at each timepoint (optionally) 
i: if subject agrees to Definity complement sample, optional “Red and white blood cell” samples may only be 
collected if it is at least +/-8 weeks of Definity complement sample (as not to exceed 50ml in 8 week period) 
J; Regadenoson infusion may over-run for up to 1 hour, with PI permission 
k: during study visit or standalone blood draw 
 


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Table 2. Study Schedule of Procedures for Sickle Cell CEU Subjects 
Procedure 
Screening 
Baseline  
Baseline 
#2 
Pain 
Crisis 
Pre-RBCX 
RBCX 
Transfusiona 
Documentation- medical hx, 
medications, transfusions  
& clinical data 
X 
X 
X 
X 
X 
X 
Vital Signs ,Height and 
Weight  
X 
X 
X 
Xc 
X c 
X c 
β-HCGe 
X 
 
 
 
 
 
CBC w/diff, plt. Count, 
reticulocyte c 
 
Xg 
 
Xg 
X 
Xg 
Comprehensive metabolic  
panel b, c 
 
Xg 
 
Xg 
X 
Xg 
Adverse event evaluation 
X 
Signal-average ECG 
 
X 
 
 
 
 
Limited functional 2D 
Echo 
 
X 
Xi 
Xi 
Xi 
Xi 
Contrast-enhanced 
ultrasound 
 
X 
X 
X 
X 
Xh 
Cardiac questionnaire 
 
X 
 
 
 
iNKT cell measurement 
 
X 
X 
X 
X 
X 
Inflammatory Markers 
 
X 
X 
X 
X 
X 
Exploratory “red and white 
blood cell” study labsj 
X 
X 
 
 
Definity complement 
deposition blood sample 
(optional) 
Xk 
 
 
 
Complement (C3, C4, CH50) 
(drawn if optional Definity 
complement sample drawn) 
Xk 
 
 
 
Pain Scoresf 
 
X 
X 
X 
X 
X 
a: Red blood cell exchange transfusion 
b: Albumin, alkaline phosphatase, total bilirubin, serum creatinine, BUN, calcium, chloride, glucose, potassium, 
total  protein, SGOT [AST], SGPT [ALT], sodium 
c: Standard of care measurement or intervention 
e: Urine pregnancy test (women of childbearing potential) 
f: Pain scores will be taken just prior to each blood draw using a visual analog scale 
g: Labs (CBC, metabolic panel) are considered standard of care if they have not been measured within 30 days 
h: Contrast-enhanced ultrasound to be performed within 72 hours after red blood cell exchange transfusion 
i: Limited Functional 2D Echocardiogram is optional 
j: Optional lab testing for blood flow adhesion and leukocyte activation 
k: during study visit or standalone blood draw 


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Table 3. Study Schedule of Procedures for Healthy Control Subjects  
Procedure 
Screening 
Day 0 
Day 1 
Day 30 
Documentation- Med Hx, 
demographics, medications & 
data 
X 
X 
 
X 
Sickledex blood test 
X 
 
 
 
Vital signs, height, and weight 
 
X 
X 
X 
β-HCG a 
X 
 
 
 
CBC w/diff, plt. Count, 
reticulocyte 
 
X 
 
 
Comprehensive metabolic  
panel b, 
 
X 
 
 
Reticulocyte count 
 
X 
 
 
Signal-average ECG 
 
X 
 
 
Limited functional 2D Echo 
 
X 
 
 
Contrast-enhanced ultrasound 
(CEU) 
 
X 
X 
X 
Optional 6 hr time point CEU 
 
X 
X 
X 
iNKT cell measurement 
 
X 
X 
X 
Inflammatory Markers 
 
X 
X 
 
Exploratory “red and white 
blood cell”  study labsc 
 
X 
Definity complement deposition 
blood sample (optional) 
Xd 
Complement (C3, C4, CH50) 
(drawn if optional Definity 
complement sample drawn) 
Xd 
Adverse event evaluation 
 
X 
a: Urine pregnancy test (women of childbearing potential) 
b: Albumin, alkaline phosphatase, total bilirubin, serum creatinine, BUN, calcium, chloride, glucose, potassium, 
total  protein, SGOT [AST], SGPT [ALT], sodium 
c: Optional lab testing for blood flow adhesion and leukocyte activation 
d: during study visit or standalone blood draw 
 


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Table 4. Study Schedule of Procedures for Technique Optimization Control Subjects  
Procedure 
Screening 
Initial Measurement 
Second Measurement 
(if applicable) 
Documentation- medical history  & 
concomitant medications 
X 
 
 
Physical Exam (if necessary) 
X 
 
 
Vital signs 
 
X 
X 
Contrast-enhanced ultrasound 
 
X 
X 
Adverse event evaluation 
 
X 
X 
 
 
 
Table 5. Study Schedule of Procedures for Sickle Cell Control Subjects 
 
Procedure 
Screening 
0 hours  
6 hours (+/-
2hr) 
24 hours (+/-
2hr, optional) 
30 hours ( +/- 
2hr, optional) 
Documentation- medical 
history, medications, 
transfusions  & clinical data 
X 
 
 
 
 
 Vital signs, height and weight 
X 
 
Signal-average ECG 
X 
 
β-HCGa  
X 
 
 
 
 
CBC w/diff, plt. Count, 
reticulocyteb 
 
X 
 
 
 
Comprehensive metabolic 
panelb 
 
X 
 
 
 
VS (including SBP, HR,  
O2 sat) 
 
X  
Adverse event evaluation 
X 
Limited functional 2D Echo 
 
X 
 
 
 
Contrast-Enhanced 
Ultrasound 
 
X 
X 
X 
X 
iNKT cell measurement 
 
X 
X 
X 
X 
Inflammatory Markers 
 
X 
 
X 
 
Exploratory “red and white 
blood cell” study labsc 
 
       Xe 
 
Xe 
 


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Definity complement 
deposition sample (optional) 
Xf 
Complement (C3, C4, CH50) 
(drawn if optional Definity 
complement sample drawn) 
Xf 
Pain Scoresd 
 
X 
X 
X 
X 
Cardiac Questionnaire 
 
X 
a: Urine pregnancy test (women of childbearing potential) 
b: Standard of care measurement or intervention if done with 30 days, or per physician discretion 
c: Optional lab testing for blood flow adhesion and leukocyte activation 
d: pain score taken just prior to blood draw 
e: If optional Definity blood sample is also collected, up to 5ml blood may be collected at each timepoint 
(optionally) 
f: during study visit or standalone blood draw 
 
7.  
Investigational agent  
 
7.1 Regadenoson 
Regadenoson is a selective adenosine2A receptor agonist. Activation of adenosine2A 
receptors inhibits inflammation by directly targeting the iNKT cell. We suspect that 
iNKT cells have an important role in sickle cell disease vaso-occlusion and that 
inhibiting iNKT cells through activation of A2ARs may decrease inflammation and 
improve vaso-occlusion.  
Regadenoson has an FDA indication for radionuclide myocardial perfusion imaging 
in patients unable to undergo adequate exercise stress. Regadenoson has the same 
FDA-approved indication as the injectable formulations of adenosine and 
dipyridamole24. 
Adenosine and dipyridamole are nonselective adenosine receptor agonists, targeting 
adenosine A1, A2B, and A3 receptors, as well as the A2AR25,26. Regadenoson has low 
affinity for the A2A receptor, resulting in increased “off rates” from that receptor and a 
short duration of effect.  
Maximal plasma concentrations are achieved within 1 to 4 minutes after 
regadenoson injection. In a 3-compartment model, the initial half-life was 2 to 4 
minutes, the intermediate half-life was 30 minutes, and the terminal half-life was 2 
hours24,26. Body mass index, body weight, age, and height had no influence on 
regadenoson pharmacokinetics24,27,28. Clearance is not dose-dependent; therefore, 
weight-adjusted dosing is not necessary. Renal excretion accounts for approximately 
58% of the total elimination29. Average plasma renal clearance exceeds glomerular 


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filtration rate, suggesting renal tubular secretion plays a role in regadenoson 
elimination24.  
Dosage adjustments are not necessary in patients with renal function impairment24.  
In healthy volunteers, the regadenoson plasma concentration-time profile is multi-
exponential in nature and best characterized by 3-compartment model. The maximal 
plasma concentration of regadenoson is achieved within 1 to 4 minutes after 
injection of regadenoson and parallels the onset of the pharmacodynamic response. 
The half-life of this initial phase is approximately 2 to 4 minutes. An intermediate 
phase follows, with a half-life on average of 30 minutes coinciding with loss of the 
pharmacodynamic effect. The terminal phase consists of a decline in plasma 
concentration with a half-life of approximately 2 hours24,26.  
 
A population pharmacokinetic analysis including data from subjects and patients 
demonstrated that regadenoson clearance decreases in parallel with a reduction in 
creatinine clearance and clearance increases with increased body weight. Age, 
gender, 
and 
race 
have 
minimal 
effects 
on 
the 
pharmacokinetics 
of 
regadenoson26,27,29. 
 
Metabolism  
The metabolism of regadenoson is unknown in humans. Incubation with rat, dog, 
and human liver microsomes as well as human hepatocytes produced no detectable 
metabolites of regadenoson24.  
 
Excretion  
In healthy volunteers, 57% of the regadenoson dose is excreted unchanged in the 
urine (range 19–77%), with an average plasma renal clearance around 450 mL/min, 
i.e., in excess of the glomerular filtration rate. This indicates that renal tubular 
secretion plays a role in regadenoson elimination24. 
 
For a list of toxicities associated with regadenoson, please refer to section 8.1. 
Our group is currently conducting a Phase I Clinical Trial of regadenoson in Children 
and Adults with sickle cell disease.  The results from this safety and dose-seeking 
trial show that an infusional dose of 1.44 mcg/kg/hr is safe in adults with sickle cell 
disease (greater than 18 years of age) at baseline state (i.e., not in pain crisis) when 
given for 24 hours. For this study, we will use the same dose and similar monitoring 
parameters that were used in the phase I trial to ensure patient safety.  


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7.2 Administration  
Doses will be calculated based on the subject’s weight at screening.  
Based on the ongoing phase I dosing and safety study (Safety of Adenosine 2A 
agonist Lexiscan in Children and Adults with Sickle Cell Disease, MCW IRB# 
PRO13470, we will deliver a dose of 1.44 mcg/kg/hr of regadenoson.  
Full details of administration activities can be found in section 6.1. 
 
7.3 Packaging and Preparation 
Regadenoson is available in a prefilled syringe. Each syringe contains 0.4 mg in 5 
ml solution. For continuous infusions, a syringe pump or portable pump will be used. 
Regadenoson should be administered in a peripheral vein with a catheter or needle 
that is 22 gauge or larger. Regadenoson (Lexiscan) is manufactured by Astellas 
Pharma, US. Astellas Pharma US, Inc. Deerfield, IL 60015.  
 
7.4 Storage and Stability 
Store at controlled room temperature, 25°C (77°F); excursions permitted to 15° to 
30°C (59°–86°F). 
 
7.5 Compatibility 
No formal pharmacokinetic drug interaction studies have been conducted with 
Lexiscan. 
Effects of Other Drugs on Regadenoson 
• Methylxanthines (e.g., caffeine and theophylline) are non-specific adenosine 
receptor antagonists and may interfere with the vasodilation activity of 
regadenoson. Patients should avoid consumption of any products containing 
methylxanthines as well as any drugs containing theophylline for at least 12 
hours before regadenoson administration. Aminophylline may be used to 
attenuate severe or persistent adverse reactions to regadenoson. 
• In clinical studies, regadenoson was administered to patients taking other 
cardioactive drugs (i.e., β-blockers, calcium channel blockers, ACE inhibitors, 


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nitrates, cardiac glycosides, and angiotensin receptor blockers) without 
reported adverse reactions or apparent effects on efficacy. 
• Dipyridamole may change the effects of regadenoson. When possible, 
withhold dipyridamole for at least two days prior to regadenoson 
administration. 
Effect of Regadenoson on Other Drugs 
Regadenoson is not known to inhibit the metabolism of substrates for CYP1A2, 
CYP2C8, CYP2C9, CYP2C19, CYP2D6, or CYP3A4 in human liver microsomes, 
indicating that it is unlikely to alter the pharmacokinetics of drugs metabolized by 
these cytochrome P450 enzymes. 
 
7.7 Handling 
No special precautions for handling are required. 
7.8 Availability 
The regadenoson used in this study must be considered an investigational new drug 
and the IND is pending. Regadenoson will be purchased with study funds from the 
manufacturer by the study at no charge to the study subject.  
7.10 Ordering 
Regadenoson will be purchased with study funds from the manufacturer at no 
charge to the study subject.  The supplier of regadenoson is manufactured by 
Astellas Pharma, US. Astellas Pharma US, Inc. Deerfield, IL 60015.  
7.11 Drug Accountability 
Drug accountability logs will be maintained for all study drugs.  At a minimum, these 
logs must contain quantity of drug received and dispensed, date received and 
dispensed, subject number, expiration date, dose, quantity returned, and initials of 
individual dispensing the study drug.  Used medication vials can be discarded 
according to institutional standards. 
 
7.12 Destruction and Return 
At the end of the study, unused supplies of regadenoson should be destroyed 
according to institutional policies. Destruction will be documented in the Drug 
Accountability Record Form. 


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7.13 Discontinuation Criteria 
Subjects may be removed from the study, or treatment stopped by the Investigator 
for any of the following reasons: 
1. Subject experiences an unacceptable adverse event that warrants withdrawal 
from the study, as judged by the investigator. 
2. Occurrence of any CTCAE grade 3 toxicity with the exception of heart rate and 
systolic blood pressure. 
3. The below criteria will be used to determine whether a subject is experiencing a 
drug-related toxicity and treatment should be stopped: 
A baseline heart rate and blood pressure will be established with at least 2 
measurements, 5-10 minutes apart. If the last two consecutive measurements 
have a difference greater than 5 beats per minute (heart rate) or 10 mm Hg 
(systolic blood pressure), they should be repeated every 5 minutes until they 
stabilize. The baseline measurement is the last heart rate and blood pressure 
measurement prior to study drug infusion. 
 
An increase or decrease of > 30 beats per minute from baseline will be 
immediately rechecked along with blood pressure. Participants who are either 
sleeping or relaxing in bed will be awakened or encouraged to move their limbs 
prior to the repeat measurement. If the heart rate is confirmed > 30 beats per 
minute above or below baseline, it will be rechecked again 15 minutes later along 
with blood pressure. Heart rate differences that remain > 30 beats per minute 
from baseline after 15 minutes and are accompanied by 1 of the following will be 
considered a toxicity and will be criteria for stopping: 
 
1) A sustained decrease in blood pressure of > 30 mmHg from baseline,  
2) A sustained systolic blood pressure < 75 mmHg, or  
3) Signs of hypoperfusion (confusion, cool extremities, mottled skin). 
 
Additionally, a heart rate < 40 beats per minute will be rechecked immediately 
and participants who are either sleeping or relaxing in bed will be awakened or 
encouraged to move their limbs prior to the recheck. If the rechecked heart rate 
is confirmed < 40 beats per minute, it will be checked 15 minutes later. Sustained 
values < 40 beats per minute will be considered criteria for stopping. Physicians 
will be notified when heart rate values are: greater than 30 beats per minute 
above or below baseline or < 40 beats per minute (prior to the 15 minute 
recheck).  
 
A decrease in systolic blood pressure of > 30 mmHg from baseline will be 
immediately rechecked. Participants who are either sleeping or relaxing in bed 


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will be awakened or encouraged to move their limbs prior to the repeat 
measurement. If systolic blood pressure is confirmed > 30 mmHg below baseline, 
it will be rechecked again 15 minutes later. Systolic blood pressure decreases 
that remain > 30 mmHg after 15 minutes will be considered a toxicity and criteria 
for stopping. Additionally, a systolic blood pressure < 75 mmHg will be rechecked 
immediately and participants who are either sleeping or relaxing in bed will be 
awakened or encouraged to move their limbs prior the recheck. If the rechecked 
systolic blood pressure is confirmed < 75 mmHg, it will be checked 15 minutes 
later. Sustained values < 75 mmHg will be considered a toxicity and will be 
criteria for stopping. Physicians will be notified when the systolic blood pressure 
is verified as: a decrease of > 30mmHg from baseline or < 75 mmHg. The study 
drug may be stopped at any time per the discretion of the physician and these 
events would be criteria for stopping.  
 
If the infusion is stopped for a toxicity, participants will be observed off therapy for 
an additional 6 hours. 
 
Management of the above adverse events is outlined in section 8.1. 
4. Participant decides to withdraw from the study 
5. General or specific changes in the participant’s condition render the participant 
unacceptable for further treatment in the opinion of the treating investigator 
 
8.        Toxicities 
8.1  Regadenoson 
Common 
Dyspnea, headache, flushing, chest discomfort, dizziness, angina pectoris, chest 
pain, and nausea 
Less Common  
Abdominal discomfort, dysguesia, feeling hot, bronchoconstriction (wheezing), and 
hypotension and tachycardia (as defined by criteria for toxicity in Section 7.13.6)  
Rare 
Regadenoson also has the potential to cause sinus bradycardia or first, second or 
third degree heart block, heart abnormalities (including atrial fibrillation/atrial flutter), 
seizure or stroke 
Rarely, hypersensitivity episodes have also been reported.  
Mild episodes are defined as events that include hives or urticaria, but are not 
characterized by respiratory compromise, angioedema or reduced blood 
pressure. See below for hypersensitivity management. 


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Severe episodes are defined as events that include either respiratory 
compromise 
(brochospasm, 
hypoxemia, 
stridor, 
dyspnea), 
anaphylaxis, 
angioedema, or reduced blood pressure. See below for hypersensitivity 
management. 
 
Regadenoson Toxicity Management: 
Most toxicities associated with regadenoson are self-limiting and resolve within 15 
minutes of discontinuing the drug. If an increase or decrease in heart rate or 
decrease in systolic blood pressure is recorded, according to protocol guidelines, the 
drug will be discontinued. Within minutes these parameters should return to normal. 
If a more significant decrease in blood pressure occurs, aminophylline is an antidote 
to regadenoson. The dose of aminophylline is 50 to 250 mg given as a slow IV 
injection (e.g., 50-100 mg given over 30-60 seconds).  
For mild hypersensitivity episodes, consider the administration of: 
• Methylprednisolone 1 mg/kg IV 
• Diphenhydramine 50 mg IV (1 mg/kg in children) 
 
For severe episodes, consider the administration of: 
• Epinephrine 1 mg per ml (1:1000) IM 0.3 to 0.5 mg every 5-15 minutes 
(children 0.01 mg/kg up to 0.5 mg dose every 5-15 minutes) 
• Methylprednisolone 1 mg/kg IV 
• Diphenhydramine 50 mg IV (children 1 mg/kg) 
• Ranitidine 50 mg IV or PO (children 1 mg/kg) for mild episodes 
 
8.2 Optison (perflutren lipid microsphere) CEU contrast agent 
Rare 
headache, nausea and/or vomiting, flushing, dizziness 
Very Rare 
Pain (which may be severe)Hypersensitivity episodes have been reported.  
Mild episodes are defined as events that include hives or urticaria, but are not 
characterized by respiratory compromise, angioedema or reduced blood 
pressure.  
 
Severe episodes are defined as events that include either respiratory 
compromise 
(brochospasm, 
hypoxemia, 
stridor, 
dyspnea), 
anaphylaxis, 
angioedema, or reduced blood pressure. 
 
Optison Toxicity Management: 


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The subject should be advised to report any sudden onset of symptoms and report it 
to the ultrasound tech.  The contrast agent should be stopped immediately if 
hypersensitivity or other serious AE develops. 
For possible hypersensitivity reactions, please stop administration of the contrast 
agent and use the suggestions provided previously in section 8.1. 
 
9. 
Correlative studies 
 
9.1. CEU methodology 
Low-mechanical index power modulation imaging (IE33, Philips Ultrasound) will be 
performed at a transmission frequency of 1.7 MHz with a phased-array transducer at 
a mechanical index of 0.15.20,30. We will image the deep forearm flexor muscles 
(short-axis), the myocardium (3-apical views) and kidney (optional) after establishing 
a continuous infusion of lipid-shelled octafluoropropane microbubbles (Optison, GE 
Healthcare), which will be prepared in the following way: 1 vial (0.5ml) diluted into 
15-30ml with sterile saline, and infused at a rate of approximately 90-120ml/hr. 
Adjustment may be necessary to achieve desired imaging effect, but will not exceed 
manufacturer’s dosing recommendation.  This dose may be repeated during the 
CEU if necessary, not to exceed manufacturer’s packaging recommendations. 
Images of the forearm may also be acquired 2 minutes after initiating hand-grip 
exercise (12 Hz at 50% maximal tension) to assess flow reserve. Images will be 
acquired for 15 seconds after a destructive (MI 1.0) 5 frame pulse sequence. Time-
intensity data fit to the function: y=A(1-et) where y is intensity at time t, A is the 
plateau intensity, and the rate constant is the microvascular flux rate reflecting RBC 
flux rate 31,32. There may be a 30 minute break during the course of the contrast-
enhanced ultrasound procedure. Data analysis will take place in the laboratory of 
Jonathan Lindner, MD (Co-PI) in Portland, OR. Microvascular blood volume (MBV) 
will be quantified by (A)/(1.06×ILV×F) where 1.06 is tissue density (g/cm3), and F is 
the scaling factor for a different infusion rate for ILV (0.3 ml/min) which will be 
reduced to avoid dynamic range saturation Quantitative perfusion (ml/min/g) will be 
calculated by the product of MBV and. Spatial distribution of microvascular flow in 
skeletal muscle will be determined by using fractal analysis in the spatial domain on 
parametric images as previously described.  
 
9.2 iNKT cell measurements 
iNKT cell measurements will be performed in the lab of J. Linden (Co-I) in La Jolla, 
CA. 4 ml whole blood will be collected per time point into vacutainer EDTA tubes and 
stored at 4ºC until shipping in cold containers. A rapid express system that we have 


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developed will permit all samples to reach Dr. Linden’s laboratory in La Jolla, CA, in 
24 hours.  
Two methods will be used to identify iNKT cells:  
1) CD1d tetramers, loaded with the α-GalCer-like glycolipid PBS57 (defined as 
tetramer+CD3+) 
 2)  6B11 antibodies (defined as CD45+6B11 high) 
Concordance between cells identified by CD1d tetramers and 6B11 antibodies is 
>90%. Further, only cells staining CD45+6B11 high will be called iNKT cells, 
recognizing that our sensitivity for detecting iNKT cells will be lower, but we will have 
fewer false positives. FACS analysis and counting beads are then used to determine 
the absolute numbers of iNKT cells and the percent of iNKT cells among all 
lymphocytes (live, CD45+ low side scatter cells).  
 
9.3 Limited Functional 2D Echocardiogram 
All patients will undergo limited functional rest 2D echocardiography to assess for 
patent foramen ovale (PFO), atrial septal defect (ASD), right-to-left cardiac shunts, 
and aortic arch diameter. Right ventricular (RV) and left ventricular (LV) and valvular 
function will also be assessed. The following views will be used for assessment: 
parasternal long axis (PLAX), parasternal short axis (PSAX), subcostal, and 
suprasternal. Color flow Doppler will be used across all valves to assess 
regurgitation. The LV ejection fraction will be calculated according to the “method of 
discs”. Normal LV function will be defined as an LVEF > 55%. Left atrial area and 
volume and right atrial volume will be measured at end systole using the apical 4-
chamber view, as well as apical 2-chamber, and apical 3-chamber views. RV areas 
at end-diastole and end-systole will be determined in the apical 4-chamber view, as 
well as apical 2-chamber, and apical 3-chamber views. The tricuspid regurgitant jet 
velocity will be recorded as the highest value obtained with a good quality Doppler 
envelope from either modified apical 4-chamber or parasternal views. Tissue 
Doppler imaging (TDI) will also be performed to obtain the MV annulus in the 
medial/lateral view, and the TV annulus in the lateral view. Data analysis will take 
place in the laboratory of Jonathan Lindner, MD (Co-PI) in Portland, OR. 
 
9.4 Signal-average Electrocardiogram 
A signal-averaged electrocardiogram (SAECG) is a more detailed type of 12-lead 
ECG, during which multiple tracings are obtained over a period of approximately 10-


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20 minutes. The subject will be asked to lie as still as possible for the duration of the 
procedure, while the ECG leads are placed, and the cardiac rhythms are recorded. 
Three techniques will be used to analyze the signals, including temporal signal 
averaging, spatial signal averaging, and spectral analysis. Temporal signal 
averaging, averages a number of QRS complexes over time 34. Beats are detected 
by a computer algorithm, based on voltage thresholds or other criteria, then aligned 
by a constant feature of the signal, the "fiducial" 35. Once beats have been aligned, 
their arithmetic mean is taken. This process diminishes random noise that is not 
synchronized to the QRS complex. ECG spatial averaging involves the summation 
and averaging of electrical potentials simultaneously recorded from multiple pairs of 
closely spaced electrodes 36. This process allows a real-time, beat-by-beat analysis 
of individual QRS complexes, but requires electrical shielding of the patient and 
equipment. The advantage of this technique over temporal signal averaging is that it 
allows assessment of irregular rhythms with changing conduction. Spectral analysis 
considers the QRS complex (or P-wave) to be composed of multiple simple 
waveforms, typically sinusoids. Spectral analysis thus decomposes the QRS 
complex or P-wave into these constituent signals, represented by component 
frequencies and corresponding phase and amplitudes 37 38. Spectral decomposition 
will be performed using Fourier analysis 39. 
9.5 Exploratory study of inflammatory markers 
Since inflammation is known to promote vascular occlusion of sickle erythrocytes, 
the hallmark of a sickle cell pain event, measuring markers of inflammation at 
baseline and during a vaso-occlusive crisis may provide insight into the mechanism 
of inflammation, as it relates to sickle cell vaso-occlusion. Additionally, measuring 
inflammatory markers before and after administration of regadenoson may provide 
further evidence about the effects of regadenoson on sickle cell vaso-occlusion. 
Based on mouse studies we expect that an effective concentration of regadenoson 
will decrease plasma levels of IFN-gamma, certain chemokines and various other 
proinflammatory mediators. 
Samples will be collected from participants in Milwaukee, in the Regadenoson Arm, 
Sickle Cell CEU Arm, Healthy Control Arm, and Sickle Cell Control Arm at baseline, 
and 24 hours after baseline, and/or the start of regadenoson infusion, for 
measurement of markers of inflammation. Samples will be collected in 4 mL 
vacutainer tubes with EDTA. They will be spun for plasma, transferred to 
anonymized plastic cryovials and stored at -80ºC or below until ready for analysis. 
The samples will be destroyed after the analysis is completed. 
 


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9.6 Exploratory studies of “red and white blood cell” blood flow adhesion and 
leukocyte activation (optional) 
 
Samples will be collected optionally from participants in Milwaukee in all study arms 
(with exception of the Technique Optimization Controls Arm). Up to 10ml whole 
blood will be collected into anticoagulated vacutainer tubes, spun for serum and 
plasma separation, and the supernatant transferred to anonymized cryovials and 
stored at -80ºC or below until ready for analysis. The pellets (non-plasma or serum 
fraction) of the vacutainer tubes may be used to isolate erythrocyte and leukocytes 
(see description below), for further examination in this study. Any remaining samples 
will be destroyed after the study analysis is completed.   
 
A flow adhesion assay of isolated blood cells (erythrocytes or leukocytes) will be 
performed under conditions of controlled flow, to determine red cell or leukocyte 
rolling and adhesion.  For this assay, we will count how many blood cells become 
adherent or roll along the surface of the flow chamber.  Furthermore, we may 
examine red cell or leukocyte physiology or structural characteristics, such as red 
cell stiffness of membrane or flow cytometry studies.  Sample and data analyses will 
take place at the lab of Cheryl Hillery, MD, at the Blood Research Institute in 
Milwaukee, WI. 
 
9.7 Exploratory Study of complement deposition on Definity microbubbles in 
vivo (optional) 
 
Complement deposition on the surface of lipid microbubbles will be compared 
between samples collected in subjects with SCD who had pain events associated 
with Definity (n=4), patients SCD who did not have a pain event (n=10) and healthy 
African American controls (n=10). We will collect up to 4ml whole blood into 
anticoagulated tubes, and spin for plasma, transfer to anonymized cryovials, and 
store at -80C until ready for analysis.  Samples will be analyzed at the laboratory of 
Jonathan Lindner, MD in Portland, OR.   
 
In Dr. Lindner’s laboratory in Portland, plasma will be incubated with lipid 
microbubbles and then flow cytometry will be performed to measure surface 
complement deposition on the microbubbles. Approximately 4ml blood will also be 
collected for isolation of serum and analyzed for levels of circulating complement 
(C3, C4, CH50) at a clinical laboratory. 
 


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10. 
Subject Remuneration 
 
10.1 Regadenoson Study Subjects  
For their time and efforts in study activities, regadenoson study subjects will receive 
$200.00 for the completion of the: 
• Infusion Visit and related study activities 
• Complete 4 weekly telephone follow-up calls for assessment of AE’s  
When on-study procedures are partially completed, participants will receive $25 
remuneration. 
  
 
10.2 Sickle Cell CEU Subjects 
For their time and efforts in study activities, sickle cell CEU subjects will receive 
$35.00 for the completion of each study visit (baseline, baseline #2,  admission for 
pain crisis, and/or pre- and post- RBCX). 
 
10.3 Healthy Control Study Subjects 
For their time and efforts in study activities, healthy control study subjects will 
receive $100.00 for the completion of the study activities and $35 for any additional 
6 hr time point CEU (optional). 
When testing is partially completed, participants will receive $25 remuneration. 
 
10.4 Technique Optimization Control Study Subjects 
For their time and efforts in study activities, technique optimization controls will 
receive $25 for each contrast-enhanced ultrasound measurement visit. 
 
10.5 Sickle Cell Control Study Subjects 
   For their time and efforts in study activities, sickle cell control subjects will receive 
$35.00 for the completion of each CEU (0hr, 6 hr, 24hr and 30 hr). 
 
Definity Complement Blood Sample 
Subjects who provide consent and agree to give the optional Definity Complement 
blood sample will receive $25 for their time and this additional blood sample. 
Participants in Chicago may be offered a larger compensation amount, per the 
discretion of the local Principal Investigator. The amount will be based on the 
locally established rate of compensation for similar studies and/or procedures and 
must be approved by the local (UIC) IRB. 
 
 
11.  
Adverse events  


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Adverse event collection and reporting is a routine part of every clinical study.  All 
adverse events will use the descriptions and grading scales found in the CTEP Active 
Version of the NCI Common Terminology Criteria for Adverse Events (CTCAE). 
11.1 Adverse Event Monitoring 
 
11.1.1 Study Staff 
Information on all adverse events, whether reported by the subject, directly 
observed, or detected by physical examination, laboratory test or other means, will 
be collected, recorded, followed, and reported as described in the following sections.  
The PI will monitor and review adverse events monthly or more often as needed. 
 
11.2 Definitions 
 
Adverse Event (AE) – Any untoward or unfavorable medical occurrence in a  
human subject, including any abnormal sign (for example, abnormal physical  
exam or laboratory finding), symptom, or disease, temporally associated with the  
subject’s participation in research, whether or not considered related to the  
subject’s participation in research. 
Serious Adverse Event (SAE) – Any adverse event temporally associated with the 
subject’s participation in research that meets any of the following criteria: 
• Results in death; 
• Is life-threatening (places subject at immediate risk of death from the event as 
it occurs); 
• Requires inpatient hospitalization or prolongation of existing hospitalization, 
within 48 hours of a CEU or other research-related activity (*see below); 
• Results in a persistent significant/incapacity; 
• Results in a congenital anomaly/birth defect; or 
• Any other adverse event that, based upon appropriate  medical judgment, 
may jeopardize the subject’s health and may require medical or surgical 
intervention to prevent on the other outcomes listed in this definition 
Note that seriousness and severity are separate concepts. The term “severe” refers 
to the intensity of a specific event; a severe event may be of minor medical 
significance (e.g., a severe leg cramp). The term “serious” is based on an outcome 
or action criteria that are usually associated with events that pose a threat to the 
patient’s life or functioning. An event that is mild in severity is serious if it leads to 
one of the outcomes defined above. 
 
Grade 4 and 5 events will always be considered Serious Adverse Events. Many 
Grade 3 and some Grade 1 and 2 events may meet the definition of a Serious 
Adverse Event. 


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* Individuals with sickle cell disease are not uncommonly hospitalized for sickle cell-
related ailments, such as pain exacerbations or others, which are part of the disease 
process. It is important to note that any adverse event should be assessed with 
regard to activities that are related to research.   
• SAE’s which originate from a study-related intervention, such as CEU, 
are most likely to occur immediately and the PI should acknowledge all 
hospitalizations, perceived prolongation of hospitalizations or ED visits, 
which occur within 48 hours of a study intervention being performed. 
 
 
Unexpected Adverse Event- Any adverse event occurring in one or more subjects in 
a research protocol, the nature, the severity, or frequency of which is not consistent 
with either: 
• The known of foreseeable risk of adverse events associated with the 
procedures involved in the research that are described in; 
o  the protocol related documents or the current IRB-approved informed 
consent document and;  
o other relevant sources of information, such as a product labeling or 
package insert or; 
• The expected natural progression of any underlying disease, disorder, or 
condition of the subject(s) experiencing the adverse event and the subject’s 
predisposing risk factor profile for the adverse event. 
 
Unanticipated problem involving risks to subjects or others (UP) - Any incident, 
experience or outcome that meets all the following criteria: 
• Unexpected (in terms of nature, severity, or frequency) given: 
o The research procedures that are described in the protocol related-
documents, such the IRB-approved research protocol and the informed 
consent form document and, 
o The characteristics of the subject population being studied;  
• Related or possibly related to the subject participation in research; and 
• Suggests that the research places subjects or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) related to the 
research than was previously known or recognized. 
 
Grading of Adverse Events – Events will be graded by using the Common 
Terminology Criteria for Adverse Events (CTCAE) Criteria version 4.03. 
 
Attribution – the determination and documentation of whether an adverse event is 
related to a medical procedure. 
 
Attribution Categories: 


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1. Not Related –Event clearly related to other factors (e.g., clinical state, other 
therapies; concomitant drugs) 
2. Possibly Related – Sequence of events is compatible with study drug, device, 
or procedure, but could have been produced by other factors 
3. Probably Related - Sequence of event is compatible with study drug, or 
procedure and cannot be explained by other factors without much doubt 
4. Definitely Related - Sequence of event is compatible with study drug, or 
procedure and beyond doubt cannot be explained by other factors 
 
A list of the adverse events and potential risks associated with the agents 
administered in this study can be found in sections 8.1-8.3, as well as the Informed 
Consent Form.  
 
11.3  Timeline for Reporting of Adverse events, Serious Adverse Events, 
Unexpected Adverse Events and Unanticipated Problems 
  
  
All serious adverse events, all unexpected adverse events, and all unanticipated 
problems that occur after the initial dose of study treatment, during treatment, or 
during the defined follow-up period will be reported to the overall PI within 24 hours 
of learning of the event’s occurrence by phone, email, or SAE form regardless of 
attribution to the study drug. A follow-up SAE report should be submitted within 24-
48 hours following initial notification.  
11.4  Recording Adverse Events 
 
Participating investigators will assess the occurrence of AEs and SAEs at all 
participant evaluation time points during the study.  All AEs and SAEs whether 
reported by the participant, discovered during questioning, directly observed, or 
detected by physical examination, laboratory test or other means, will be recorded in 
the participant’s study and/or medical record and on the appropriate study-specific 
case report forms (CRFs). 
 
 
11.5 Reporting Adverse Events to Institutional Review Boards 
For multi-site studies where a MCW/FH investigator is serving as the principal 
investigator, each participating investigator is required to abide by the reporting 
requirements set by the MCW/FH Institutional Review Board (IRB). The study must 
be conducted in compliance with FDA regulations, local safety reporting 
requirements, and reporting requirements of the principal investigator. Each 
investigative site will be responsible to report SAEs that occur at that institution to 
their respective IRB, according to local policy. It is the responsibility of each 


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participating investigator to report serious adverse events to the Sponsor/ 
Investigator and/or others as described in Section 11.6 below. 
 
 
A copy of the submitted institutional SAE form should be forwarded to: 
Dr. Joshua Field: Joshua.field@bcw.edu  
Study Coordinator: zachary.williams@bcw.edu  
 
The MCW/FH Principal Investigator will submit SAE reports from outside institutions 
to the MCW/FH IRB according to policies and procedures in reporting adverse 
events. 
 
11.6 Reporting Events to the Sponsor/Investigator 
11.6.1 Serious Adverse Event Reporting Requirements 
All serious adverse events that occur after the initial dose of study treatment, during 
treatment, or during the defined follow-up period must be reported to the MCW/FH 
Overall Principal Investigator on the local institutional SAE form. This includes SAEs 
described by the criteria outlined in section 11.2, as well as the following: 
• Grade 2 (moderate) and Grade 3 (severe) events that are unexpected and at 
least possibly related/associated with the intervention. 
• All Grade 4 (life-threatening or disabling) events that are unexpected or not 
specifically listed in the protocol as not requiring reporting. 
• All Grade 5 (fatal) events while the participant is enrolled and actively 
participating in the trial OR when the event occurs during the defined follow-
up period of the last study intervention. 
 
Participating investigators must report each serious adverse event to the Overall 
Principal Investigator within 24 hours of learning of the occurrence. In the event that 
the participating investigator does not become aware of the serious adverse event 
immediately (e.g., participant sought treatment elsewhere), the participating 
investigator is to report the event within 24 hours after learning of it and document 
the time of his or her first awareness of the adverse event. Report serious adverse 
events by email or telephone to: 
 
Dr. Joshua Field 
Joshua.field@bcw.edu  
414-937-3848 
 
Email is preferred; however, in urgent situations after normal business hours, the 
Principal Investigator can be reached at 414-318-8597 
 
Within 24-48 hours, the participating investigator must provide follow-up information 
on the serious adverse event, utilizing the appropriate SAE Reporting Form. Follow-
up information should describe whether the event has resolved or continues, if and 


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how the event was treated, and whether the participant will continue or discontinue 
study participation. 
 
Follow-up reports should be emailed to Dr. Field at the email address above and to 
the study coordinator at zachary.williams@bcw.edu  
 
11.6.2 Non-Serious Adverse Event Reporting Requirements  
Non-serious adverse events will be reported to the Principal Investigator on the 
toxicity Case Report Forms. 
11.6 Food and Drug Administration (FDA) Notification by Sponsor/ 
Investigator for Investigational Agent 
The Sponsor-Investigator will report to the FDA any adverse event that is serious, 
unexpected and reasonably related (i.e., possible, probable, definite) to the 
investigational study treatment. 
 
Unexpected fatal or life-threatening experiences associated with the use of the 
investigational study treatment will be reported to FDA as soon as possible but in no 
event later than 7 calendar days after initial receipt of the information. 
 
All other serious unexpected experiences associated with the use of the 
investigational study treatment will be reported to FDA as soon as possible but in no 
event later than 15 calendar days after initial receipt of the information. 
 
Events will be reported to the FDA by telephone (1-800-FDA-1088) or by fax (1-800-
FDA-0178) using Form FDA 3500A (Mandatory Reporting Form for investigational 
agents). Forms are available at http://www.fda.gov/medwatch/getforms.htm. 
 


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12.  
Data and Safety Monitoring 
12.1 Data Management and Collection 
Research information, including consent forms and will be maintained secure as 
described in the Confidentiality section 13.3. 
Data will be collected onto study-specific case report forms.  The data gathered will 
be entered into an electronic data set maintained by the investigator. Each 
participant will have a unique identifier, or study ID, assigned. This study ID will be 
used to label all shipped biological specimens. All data and samples will be coded 
with their respective unique identifier.  Only the study coordinator and investigator 
will have access to the study ID key.  The study coordinator will also retain all source 
documentation for each subject, which will be stored in locked cabinets, inside a 
locked secured building. 
12.2 Data Safety Monitoring Board (DSMB) 
The overall study principal investigator will appoint at least three physicians and/or 
scientists with research experience as members of the DSMB group. They will 
monitor accruing data in order to confirm that the patients in the trial are being cared 
for safely, and make recommendations as appropriate. The DSMB will be 
responsible for:  
 
1) Reviewing and analyzing the progress of the study;  
2) Reviewing recruitment and event rates. 
3) Approving amendments to the trial protocol, if warranted;  
4) Monitoring the risk/benefit ratio, safety and response of study treatments and 
diagnostic procedures;  
5) Ensuring complete and responsible reporting and data quality;  
6) Approving amendments to the trial protocol, if warranted;  
7) Consult with the site monitor physician as needed 
8) Provide a summary of their findings in a written report 
 
The DSMB will meet at least annually by phone, or in person, or more often if 
warranted to review serious adverse events. 
 
Local Data Safety Monitoring Plan -The local principal investigator and/or 
members of the research team will monitor the study for regulatory compliance, 
inclusion/exclusion criteria, accrual/withdrawal rates, and breech of confidentially 
annually prior to submission to the IRB. Any adverse events will be reviewed 
immediately with the Principal Investigator and will be reported to the local 
respective IRB per site IRB regulations. All study events will be summarized once a 
year for the IRB, during annual continuing review progress report.   


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The local IRB will determine when it is necessary to inform participants of any new 
findings that reveal additional risk or information that may alter their willingness to 
participate in the trial. 
 
 
13.   Human Subjects Protection 
 
13.1 Informed Consent 
Subjects are required to sign an informed consent prior to screening, and before 
undergoing any study procedures or assessments, in accordance with ICH E6; 4.8, 
“Informed Consent of Trial Subjects.”  When substantial modifications are made to 
the informed consent, the IRB may require that all subjects currently enrolled in the 
study will be re-consented; ICH E6; 4.8 guidelines would still apply.   
 
Subjects will be provided with a copy of the signed informed consent form used in 
the study, procedures, and assessments.  Subjects will also be provided with the 
contact telephone numbers of the investigator and qualified personnel who can 
assist with their questions and concerns.   
 
13.2 Protected Health Information 
 
Protected health information that will be collected as part of this study includes 
name, date of birth, dates for hospitalization, and medical, diagnostic history and 
medications (as mentioned in Sections 5 and 6).  This information will be stored for 
3 years beyond the completion of the study. After blood is used for study testing it 
will be discarded.  
 
13.3 Confidentiality 
Loss of confidentiality is always a potential risk when collecting protected health 
information. Research Records will remain confidential. Volunteer subject’s records 
will be available to the study staff and to each site’s IRB and for audit purposes. Only 
authorized personnel will have access to the protected health information and 
research records.  In order that confidentiality can be maintained, the principal 
investigators will keep records in locked cabinets/room and results of tests will be 
coded to prevent association with volunteers’ names. All electronic data will be 
entered into a secured data set that is password protected. Identifying information 
will not be included on laboratory samples, faxes, or correspondence. Laboratory 
samples will be shipped with only unique study numbers that will be linked to a key 
kept by the site Principal Investigator.   
All study team members are trained in HIPAA privacy regulations and other 
applicable site privacy policies. No information will be released, nor will participation 


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in the research be acknowledged, to any party except where compulsory according 
to law or intuitional policy. The results of the research study may be published, but 
volunteers’ names or identities will not be revealed. 
13.4 Follow-up and Record Retention 
The current proposal involves on-going data collection for the duration of the study 
at each site. The records will be maintained per site policy but at a minimum of two 
years per FDA requirements. 
 14. Statistical Considerations 
14.1    Description of Endpoints: 
14.1.1 Primary Endpoint: Primary outcome measure will be a 40% increase in 
skeletal muscle microvascular blood flow when 24 hour measurements are 
compared to baseline in subjects receiving regadenoson. 
14.1.2 Secondary Endpoints:  
• Secondary outcome measure will be that there will be more complement 
deposited on lipid microbubbles from the plasma from the subjects with SCD 
who suffered pain events compared to the other groups. 
• Secondary outcome measure will be a 40% increase in skeletal muscle 
microvascular blood flow when 12 week measurements are compared to 
baseline in subjects receiving hydroxyurea. 
• Decrease in iNKT cell activation as measured by NFkB when 24 hour 
measurements are compared to baseline in subjects receiving regadenoson. 
14.2  Statistical Analysis 
 
Primary outcome measure: change in skeletal muscle microvascular blood flow 
during regadenoson infusion, obtained at baseline and at end of infusion (24 hours); 
Secondary outcome measures: myocardial microvascular blood flow, functional 
microvascular density, reduction in iNKT cell activity (as measured by phospho-NF-
kB expression).  
We anticipate that at baseline sickle cell disease subjects will have skeletal muscle 
blood flow similar to that of subjects with diabetes who are hyperemic, for whom 
data suggests a mean blood flow of 0.7 (standard deviation 0.3) 30. We seek to 
detect a 30% increase from baseline in skeletal muscle microvascular blood flow, to 
a mean of 1.0, following the administration of regadenoson (Aim 1). With 27 patients 
enrolled on the 24 hour regadenoson study of Aim 1, we will have 90% power to 


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detect this difference, testing at the one-sided 0.05 significance level. For these 
calculations we assume that the unknown correlation of measurements within 
patient is positive, and that therefore the maximum value for the variance of the 
difference will be two times the variance of a single observation, or 0.18 (standard 
deviation 0.42).  Secondary outcome measures will be summarized and differences 
assessed for regadenoson therapy, and tested with the one-sample t-test if 
symmetry holds and the Wilcoxon signed rank test if it does not.  Serial measures of 
iNKT cell number and activation state and markers of inflammation will be obtained 
during therapy. We will construct longitudinal mixed models of these outcomes, 
controlling for demographic factors.  
Since we do not know the magnitude of complement deposition on the lipid 
microbubbles in this setting, power calculations will use the Wilcoxon rank sum test, 
assessing number of standard deviations of difference. We will have 87% power to 
detect a 1.5 standard deviation difference and 80% power to detect a 1 standard 
deviation difference in complement deposition between the subjects who did and did 
not have reactions, testing at the 0.10 one-sided significance level. 
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