Phase I/II Safety and Dose Escalation Trial of the Omega-3 Fatty Acids Docosahexaenoic Acid (DHA) and Eicosapentaenoic Acid (EPA) in Children and Young Adults With Sickle Cell Disease (SCD)
Phase: PHASE1
Conditions: Sickle Cell Disease
Sponsor: Robin E. Miller

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PHASE I/II SAFETY AND DOSE ESCALATION TRIAL OF THE OMEGA-3 
FATTY ACIDS DOCOSAHEXAENOIC ACID (DHA) and EICOSAPENTAENOIC ACID 
(EPA) IN 
CHILDREN AND YOUNG ADULTS WITH SICKLE CELL DISEASE (SCD) 
NCT02947100 
 
 
Version 5, date April 18, 2018 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

2 
 
PHASE I/II SAFETY AND DOSE ESCALATION TRIAL OF THE OMEGA-3 
FATTY ACIDS DOCOSAHEXAENOIC ACID (DHA) and EICOSAPENTAENOIC ACID (EPA) IN 
CHILDREN AND YOUNG ADULTS WITH SICKLE CELL DISEASE (SCD) 
 
 
STUDY DRUG:      
SCD-OmegatexTM  (Enteric Fish Oil 250 DHA/27 EPA Soft Gelatin Capsule, 450 
mg) 
 
SPONSOR: 
National Institute of General Medical Sciences of the National Institutes of Health 
(NIH/NIGMS) under Award Number P20GM109021  
 
IND 133143 
Clinicaltrials.gov ID NCT02947100 
IRB# 1019241 
 
SITE 
 Nemours/Alfred I duPont Hospital for Children 
 
     1600 Rockland Rd. 
 
 Wilmington, DE 19899 
 
PRINCIPAL 
INVESTIGATOR (PI): 
 
Robin Miller, MD 
                                            
Director Sickle Cell Disease Program 
 
     Nemours Center for Cancer and Blood Disorders 
Nemours/Alfred I duPont Hospital for Children 
 
    1600 Rockland Rd. 
 
     Wilmington, DE 19899 
 
 
 
 
 
 
Assistant Clinical Professor 
                                              Thomas Jefferson University 
                                            
1025 Walnut Street 
                                            
Philadelphia, PA 19107 
 
     
 
 
Office telephone: 302-651-5500 
                                             Fax: 302-651-5510 
 
 
Cell: 302-690-0363 
                                             e-mail: rmiller@nemours.org 
 
 
CO-INVESTIGATORS: 
 Corinna Schultz, MD, MSHP  
 
 
 
 
 Nemours Center for Cancer and Blood Disorders 
 
 
 
 
 Nemours/Alfred I duPont Hospital for Children 
 
 
 
 
 Wilmington, DE 
 
     Telephone: 302-651-5500 
                                            
 Fax: 302-651-5510 
 
 
 
 
 e-mail: corinna.schultz@nemours.org 
 
 
 
 
 
 

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E. Anders Kolb, MD 
Associate Professor 
Vice Chairman for Research 
Department of Pediatrics 
Sidney Kimmel Medical College at Thomas Jefferson University 
Director, Nemours Center for Cancer and Blood Disorders 
Nemours/Alfred I. duPont Hospital for Children 
1600 Rockland Road 
Wilmington, DE 19803 
 
MEDICAL MONITOR: 
Carlton Dampier, MD, CPI 
 
 
 
 
Professor of Pediatrics 
 
 
 
Emory University School of Medicine    
 
 
 
  
Aflac Cancer and Blood Disorders Center 
2015 Uppergate Dr. Rm 464 
Atlanta, GA 30322 
 
STATISTICIAN 
 
Scott W. Keith, PhD 
 
 
 
 
Thomas Jefferson University 
 
 
 
 
Department of Pharmacology and Experimental Therapeutics 
 
 
 
 
Division of Biostatistics 
 
 
 
 
Philadelphia, PA 19107 
STUDY 
COORDINATOR: 
 
Dawn S. Brown, PCNS, FNP, CCRC 
Sickle Cell Comprehensive Care and Research Program  ( SCCRP) 
NIH Center of Biomedical Research Excellence   
Nemours Center for Cancer and Blood Disorders  
Nemours/Alfred I duPont Hospital for Children 
1600 Rockland Rd. 
Wilmington, DE 19803 
P-302-298-7411 
C-302-494-7377 
 
 
BACK UP STUDY 
COORDINATOR:  
 
Lynn Marrs, RN, CCRC 
 
 
 
 
Nemours Biomedical Research 
 
 
 
 
Nemours/Alfred I duPont Hospital for Children 
 
 
 
 
1600 Rockland Rd 
 
 
 
 
Wilmington, DE 19803 
 
 
 
 
P 302-651-5394 
 
 
 
 
C 302-494-6192 
 
 
RESEARCH 
PHARMACIST:                  
Lisa M. Brown PharmD, RPh, Research Pharmacist 
Nemours/Alfred I duPont Hospital for Children 
3rd Floor Pharmacy 3C-168  
1600 Rockland Road 
Wilmington DE 19803 
Ph 302-651-4909 
Fax 302-651-6020 
 

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EXPERT  
CONSULTANTS: 
 
Marie Stuart, MD 
 
 
 
 
Charles Serhan, PhD 
 
 
 
Version 5, April 2018 
 

 
5 
 
TRIAL SUMMARY 
Full Title 
Phase I/II Safety and Dose Escalation Trial of the Omega-3 Fatty Acids (n-3 
FAs) Docosahexaenoic Acid (DHA) and  Eicosapentaenoic Acid (EPA)  in 
Children and Young Adults with Sickle Cell Disease (SCD)  
Abbreviated Title 
Omega-3 Fatty Acids in SCD 
Primary 
Objectives 
1) To determine the clinical safety of DHA/EPA supplementation with SCD-
Omegatex™ in children with Sickle Cell SS Disease (HbSS), Sickle C 
Disease (HbSC) and Sickle β0 Thalassemia (HbSthal) as evidenced by 
an absence of adverse events (AEs) related to the fatty acid (FA) 
supplementation 
2) To determine whether 6 months of supplementation with SCD-
Omegatex™ will reduce thermal  sensitivity  as measured by Quantitative 
Sensory Testing (QST) to below pre-treatment levels  
Secondary 
Objectives 
1) To determine whether 6 months of supplementation with SCD-
Omegatex™ will increase health-associated Quality of Life (QoL) 
responses using the Varni Pediatric QoL InventoryTM SCD Module. 
2) To determine the effect of treatment by assessing pain days using an 
iPad daily report pain diary. 
3) To measure the effect of treatment on individual thermal sensitivity 
parameters by QST 
Exploratory 
Objectives 
1) To measure the effect of treatment on select biomarkers of inflammation 
including pro and anti-inflammatory cytokines, adhesion molecules,  
hsCRP.and  plasma lipid mediators derived from DHA, EPA and 
Arachidonic Acid (AA).  
2) To assess the effect of SCD-Omegatex™   treatment on thrombin 
generation as assessed by Computerized Automated Thrombogram 
(CAT). 
Study Drug 
SCD-OmegatexTM (the Drug Product, DP) consists of a fish-oil derived FA 
concentrate, containing mainly omega-3 fatty acid ethyl esters (EE), 
predominantly docosahexaenoic acid (DHA-EE). Drug Substance (DS) also 
contains eicosapentaenoic acid (EPA-EE) but at a concentration lower than 
DHA-EE. DHA and EPA ratio is approximately 9:1 Alpha-tocopherol is added 
during manufacturing the drug substance at a concentration of 2 mg/g as an 
antioxidant. 
  
Study Design 
Phase I/II Safety and Dose Escalation Trial 
Research Setting Single Site – Nemours/AI duPont Hospital for Children (NAIDHC), Wilmington, 
Delaware 
Trial Blinding 
Open Label  

 
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Treatment Groups  
 
Phase I Portion: The Dose Limiting Toxicity (DLT) portion of the study 
will follow a “3+3” design. Three subjects will be enrolled at the starting 
dose of 25 mg/kg body weight of omega-3 FAs (combined DHA and 
EPA).  If none of the subjects has a DLT after 2 months of treatment, the 
dose is escalated for the next 3 subjects to 37.5 mg/kg. The original 3 
enrollees will remain on the 25 mg/kg dose for the remainder of the trial.  
If one DLT is observed at 25 mg/kg, the next 3 subjects are enrolled at 25 
mg/kg.  If one out of 6 subjects has a DLT at 25 mg/kg, the dose is 
escalated to 37.5 mg/kg for the next group of subjects.  If 2 or more of 6 
subjects develops a DLT at 25 mg/kg, escalation is stopped and no 
Maximum Tolerated Dose (MTD) is found.  If the dose is escalated to 
37.5 mg/kg and 1 or fewer DLT is observed in the first 3 subjects after 2 
months, 3 more subjects will be enrolled at the 37.5 mg/kg. If in 2 more 
months, fewer than 2 out of 6 subjects show toxicities at 37.5 mg/kg, 37.5 
mg/kg is the recommended Phase II dose.  If 2 or more DLTs are noted, 
25 mg/kg of DHA is the MTD.  The length of treatment at each dose level 
in Phase I will be 2 months, with carryover to Phase II of subjects 
receiving the MTD.  The maximum sample size for this portion of the 
study is 12 subjects.   
 
Phase II Portion:  A sample size of 30 is needed to have 80% power to 
detect a moderate 1.5 degree (0.5 standard deviation) mean thermal 
sensitivity reduction at 6 months  following treatment using a 2-sided 
paired-t test of size 0.05.  Since 6 of the subjects for this phase will come 
from the Phase I portion, the additional sample size needed for this 
Phase is 24 subjects.  Allowing for approximately 20% attrition we will 
recruit 30 additional subjects in this phase with a total maximum sample 
size for both Phases I and II combined being 42 subjects (Phase I=12,  
Phase II=30). 
 
Number of trial 
subjects 
42 
Estimated 
duration of trial 
3  years 
Duration of 
Participation of 
each subject 
 
9 months 
 
 
 
 
 
 
 
 
 
 

 
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ABBREVIATIONS 
 
AA 
 
arachidonic acid 
AACC  
American Association for Clinical Chemistry 
ACS 
 
acute chest syndrome 
AE 
 
Adverse Event 
ALT 
 
alanine aminotransferase 
AST 
 
aspartate aminotransferase 
BHT 
 
butylated hydroxyl-toluene 
CAP 
 
College of American Pathologists 
CAT 
 
Computerized Automated Thrombogram 
CBC 
 
complete blood count 
CiC 
 
Child in Care 
CL 
 
calibrator 
CLIA               Clinical Laboratory Improvement Ammendments 
CP 
 
cold pain 
CRF   
case report form 
CS 
 
cold sensitivity 
CTCAE 
Common Terminology Criteria for Adverse Events 
DHA 
 
Docosahexaenoic Acid 
dL 
 
deciliter 
DLT 
 
dose limiting toxicity 
EDTA  
ethylenediaminetetraacetic acid 
EE 
 
ethyl ester 
ELISA  
enzyme-linked immunosorbent assay 
EMR  
electronic medical record 
EPA 
 
eicosapentaenoic acid 
ER 
 
emergency room 
ET-1 
 
endothelin-1 
ETP 
 
endogenous thrombin potential 
FA 
 
fatty acid 
FAMES 
fatty acid methyl esters 
F1.2 
 
prothrombin fragment 1.2 
FDA 
 
Food and Drug Administration 
fmol 
 
femtomole 
g  
 
gram 
GC 
 
gas chromatography 
GCP   
Good Clinical Practice 
GI 
 
gastrointestinal 
GLP 
 
Good Laboratory Practice 
GMP  
Good Manufacturing Practice 
GMR  
Geometric Mean Ratio 
Hb  
 
hemoglobin 
HbF 
 
fetal hemoglobin 
HbS 
 
sickle hemoglobin 
 
HbSβthal 
Sickle β Thalassemia 
HbSC  
Sickle C Disease 
HbSS   
Sickle Cell SS Disease 
17-HDHA 
17-hydroxydocosahexaenoic acid 
18-HEPE 
18-hydroxyeicosapentaenoic acid 
12-HETE 
12-hydroxyeicosatetraenoic acid 
 
HP 
 
heat pain 

 
8 
 
HRQoL 
health related quality of life 
HS 
 
heat sensitivity 
hsCRP  
high sensitivity C reactive protein 
HU 
 
hydroxyurea 
IB 
 
Investigator’s Brochure 
IL 
 
interleukin 
IND  
 
Investigational New Drug 
IRB  
 
Institutional Review Board 
ISO 
 
International Organization for Standardization 
iu 
 
international units 
kg 
 
kilogram 
l 
 
liter 
LC-MS  
liquid chromatography-mass spectrometry 
LDH 
 
lactate dehydrogenase 
LFT 
 
liver function tests 
LT 
 
leucotriene 
MaR1  
Maresin 1 
µg 
 
microgram 
mg  
 
milligram 
µl 
 
microliter 
ml  
 
milliliter 
min 
 
minutes 
µM 
 
micromolar 
MTD 
 
maximum tolerated dose 
n3-FAs 
omega-3 fatty acids 
n6-FAs 
omega-6 fatty acids 
NAIDHC 
Nemours/Alfred I duPont Hospital for Children 
NCLLS 
National Committee for Clinical Laboratory Standards 
NFB   
nuclear factor kappa B 
ng 
 
nanogram 
NHANES 
National Health and Nutrition Examination Survey 
NIH/NIGMS    National Institute of General Medical Sciences of the National Institutes of Health 
nM 
 
nanomolar 
NSAID  
non-steroidal anti-inflammatory Drug 
O3I 
 
omega-3 index 
PC 
 
phosphatidylcholine 
PD1 
 
Protectin D1 
PE 
 
phosphatidylethanolamine 
pg 
 
picogram 
PI  
 
Principal Investigator 
PLPs  
phospholipids 
pM 
 
picomolar 
PPT 
 
pressure pain threshold 
PRBCs 
packed red blood cells 
PS 
 
phosphatidylserine 
PT 
 
prothrombin time 
PTT 
 
partial thromboplastin time 
PUFA  
polyunsaturated fatty acid 
QoL 
 
quality of life 
QST 
 
Quantitative Sensory Testing 
RBC   
red blood cell 
Retic  
reticulocyte count 
Rv 
 
resolvin  

 
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SAE   
Serious Adverse Event 
SCD   
Sickle Cell Disease 
SD 
 
standard deviation 
SL selectin 
soluble L selectin 
SPM 
 
specialized pro-resolving mediators 
SP selectin 
soluble P selectin 
TCD 
 
transcranial doppler 
TG 
 
thrombin generation 
TxB2  
thomboxane B2 
TLC 
 
thin layer chromatography 
TNFα   
tumor necrosis factor α 
TRPV1 
transient receptor potential vanilloid 1 
TRPA1 
transient receptor potential ankryn 1 
Tt peak 
time to peak 
UA 
 
urinalysis 
ULN 
 
upper limit of normal 
USA 
 
United States 
VAS 
 
visual analog scale 
VCAM-1  
vascular cell adhesion molecule-1 
VOC   
vaso-occlusive Crisis 
VS 
 
vital signs 
WBC   
white blood cell 
xg  
 
x gravity 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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TABLE OF CONTENTS 
 
1.0 Background and Significance……………………………………………………………………………… 12-22 
1.1  Pain in SCD…12 
1.2  Quantitative Sensory Testing…14 
1.3  The Omega-3 Fatty Acids in SCD…14 
1.4  Research Studies From Our Group:  Preliminary Data…19 
1.4.1 Deficiency of omega-3 FAs EPA and DHA in Children with SCD is Associated  
    with Increased Levels of Inflammatory Biomarker hs-CRP…19 
1.4.2 Quantitative Sensory Testing in SCD….20 
1.4.3 Thrombin  Generation in SCD: Insights  from Computerized Automated 
    Thrombography (CAT)….21 
1.4.4     hsCRP…22 
1.4.5 Pain Diary…22 
 
2.0 Study Objectives and Hypotheses…………………………………………………………………………  22-23 
 
3.0 Rationale for Dose Selection………………………………………………………………………………..  23-24 
 
4.0 Trial Design……………………………………………………………………………………………………    24-30 
4.1 Dose Escalation…24 
4.1.1 Phase I Portion….24 
4.1.2 Phase II Portion...24 
4.2 Primary and Secondary Endpoints and Additional Markers of Interest…27 
4.2.1 Primary Endpoints….27 
4.2.2 Secondary/Exploratory Endpoints…27 
4.3 Inclusion/Exclusion Criteria…27 
4.3.1 Inclusion Criteria…27 
4.3.2 Exclusion Criteria…27 
4.4 Safety Assessments…28 
4.5 Compliance Assessments…28 
4.6 Selection of Subjects and Screening...28 
4.7 Monitoring for Toxicity and Safety…29 
4.7.1 Dose Limiting Toxicities…29 
4.7.2 Criteria for Subject Discontinuation from Study…29 
4.7.3 Criteria for Re-Initiation of Drug…30 
4.7.3.1 Criteria for Re-initiation of Drug following Interruption for Toxicity…30 
4.7.3.2 Criteria for Re-initiation of Drug following Interruption for Minor 
Surgery, Dental Procedures or Minor Trauma…30 
4.7.3.3 Special Considerations Related to Continued Study Participation…30 
 
5.0 Treatment of Subjects………………………………………………………………………………………..  31-36 
5.1 Description of Study Drug and Dosing Regimen….31 
5.1.1 Description of Study Drug…31 
5.1.2 Dosing Regimen….32 
5.2 Packaging, Labeling and Return of Study Drug.…32 
5.2.1 Drug Accountability…32 
5.2.2 Medication Storage…33 
5.2.3 Destruction…33 

 
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5.3 Prior and Concomitant Therapy…33 
5.4 Subject Compliance...33 
5.5 Study Procedures….33 
5.5.1 Recruitment…33 
5.5.2 Visit One (Study Entry:  Month-2)…34 
5.5.3 Visit Two, Initiation of Study Drug (Study Day 0, Month 0)…34 
5.5.4 Visit Three (Month 1)....34 
5.5.5 Visit Four (Month 2)…34 
5.5.6 Visit Five (Month 3)…35 
5.5.7 Visit Six (Month 4)…35 
5.5.8 Visit Seven (Month 5)…36 
5.5.9 Visit Eight (Month 6)…36 
5.5.10 Visit Nine (Month 7)….36 
 
6.0 Clinical and Laboratory Evaluations………………………………………………………………………   36-40 
6.1 Efficacy and Safety Laboratory Evaluations….36 
6.2 Standard Laboratory Evaluations…38 
6.2.1 CBC…38 
6.2.2 Chemistry Panel…39 
6.2.3 PT and PTT…39 
6.2.4 Urinalysis…39 
6.2.5 Bleeding Assessment…39 
6.2.6 Pain Diary…39 
6.2.7 Varni Pediatric Quality of Life InventoryTM SCD Module…39 
6.2.8 Quantitative Sensory Testing…..40 
6.2.9 Research Laboratory Studies…40 
 
7.0 Safety Evaluations and Reporting Plan…………………………………………………………………..   40-47 
7.1 Safety Assessments Overview and by Study Visit…40 
7.2 Adverse Events (AEs)…40 
7.3 Serious Adverse Events (SAEs)….41 
7.4 Assessment of AE Severity, Relationship to Treatment and Expectedness…43 
7.5 Monitoring and Outcome of AEs….44 
7.6 Reporting of Unexpected SAEs…45 
7.6.1 Expected SAEs…44 
7.6.2 Review of SAEs…44 
7.6.3 Subject Discontinuation due to AE(s)…45 
7.7 Reporting Requirements…46 
7.7.1 Reporting of Serious Safety Issues; Suspension Guidelines…46 
7.8 Pregnancy Reporting…46 
7.9 Monitoring if Subject Accrual and Compliance....46 
7.10 
Data collection and Monitoring…46 
7.10.1 CRF and Source Documentation…46 
7.10.2 Data Management…47 
7.10.3 Staff Training and Data Monitoring...47 
 
8.0 Statistical Considerations...................................................................................................................  47-48 
8.1 Data Analysis…47 
8.1.1 General Considerations…47 
8.1.2 Primary Efficacy End Point…47 
8.1.3 Secondary End Points…48 
8.1.4 Exploratory Analyses…48 
8.1.5 Interim Analysis….48 

 
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9.0 Human Subjects Protection………………………………………………………………………………..  48-50 
9.1 Clinical Trial Monitoring…...48 
9.2 Ethics…49 
9.2.1 Good Clinical Practice and IRB Review…49 
9.2.2 Informed Consent…49 
9.2.3 Data Safety and Confidentiality….49 
9.2.4 Disclosure of Data…49 
9.2.5 Publication of Research Findings…..49 
9.3  Discontinuation of Study….49 
 
10.0 Subject Compensation………………………………………………………………………………………….. 50 
 
Appendix 1  Pain Diary……………………………………………………………………………………………. 51-52 
 
Appendix 2  Bleeding Assessment…………………………………………………………………………….. 53-54 
 
Appendix 3  Dietary Assessment……………………………………………………………………………….  55-63 
 
Appendix 4  QST Methods……………………………………………………………………………………….   64-65 
 
Appendix 5  Research Laboratory Methods…………………………………………………………………..  66-71 
 
Appendix 6  Dose Calculator …………………………………………………………………………………………72 
 
References…………………………………………………………………………………………………………    73-81 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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1.0  Background and Significance: 
Sickle Cell Disease (SCD) is a chronic disorder caused by a mutant hemoglobin. The substitution of the amino 
acid valine by glutamic acid in the sixth position of the beta subunit of hemoglobin produces sickle hemoglobin 
(Hb S). This mutation promotes the hydrophobic interaction and polymerization of deoxygenated Hb S 
molecules, producing rigid, elongated red blood cells (RBCs), or sickle cells, that obstruct the microcirculation. 
The gene for Hb S arose in historically malarious regions of the world because the heterozygous state offered 
protection against the malaria parasite, and the gene is now found in individuals of African, Arabic, Hispanic, 
Greek, and Italian descent. The gene frequency of Hgb S is particularly high in individuals of African ancestry in 
the United States (USA), among whom approximately 1 in 12 are heterozygotes. SCD occurs in individuals who 
are homozygous for Hb S or are compound heterozygotes for Hb S and certain other abnormal hemoglobins. 
Approximately 1 in 500 African-Americans has SCD and 90-100,000 individuals are affected in the US (CDC 
Data and Statistics 2015).  Globally, the numbers of affected individuals are much larger with more than 275,000 
individuals born annually (Modell and Darlison 2008). 
 
1.1 Pain in SCD 
The painful vasocclusive crisis (VOC) is the hallmark of microvessel occlusion in SCD and its most common and 
debilitating feature (Dampier, Setty, et al. 2004; Platt et al. 1991; Smith et al. 2008; Stuart and Nagel 2004). 
While the initiating event is the polymerization of Hg S,  investigations into its pathophysiology have led to the 
delineation of a complex process, encompassing interactions between sickle RBCs, white cells (WBCs), 
endothelium, plasma proteins and other factors (Frenette and Atweh 2007).   
 
There is very strong evidence suggesting that SCD is a chronic inflammatory state (Belcher et al. 2003; Platt 
2000). Hebbel and Kaul (Kaul and Hebbel 2000) have linked the reperfusion injury paradigm to the VOC event. 
These investigators and the work of Frenette (Frenette 2002; Turhan et al. 2002) have focused on activated 
leucocytes and their released inflammatory mediators as playing a critical role in VOC pathobiology. Enhanced 
production of inflammatory cytokines including interleukin (IL)-1ß and tumor necrosis factor (TNF)α and 
leukotrienes (LTs) such as LTB4 in SCD promote cell-cell adhesion, endothelial activation, up regulation of 
endothelial adhesion molecules, activation of transcription factors including nuclear factor ( NF) kB, and further 
leukocyte recruitment (Belcher et al. 2003; Hebbel, Osarogiagbon, and Kaul 2004; Platt 2000).  These 
inflammatory cytokines and leukotrienes also sensitize nociceptors acting as agonists for the transient receptor 
potential vanilloid 1 (TRPV1) and ankyrin (TRPA1) which together with other TRP channels play a role in neural 
and nociceptive pain (Bautista, Pellegrino, and Tsunozaki 2013; Fernandes, Fernandes, and Keeble 2012; 
Geffeney and Goodman 2012)  via both peripheral and spinal cord mechanisms. Of note, increased levels of the 
biomarker high sensitivity C-Reactive Protein (hsCRP), produced in the liver in response to a variety of these 
inflammatory cytokines, have been shown to correlate positively with pain hospitalizations and vasoocclusive 
events (Krishnan et al. 2010).  
 
SCD is a chronic hypercoagulable state. Evidence for this includes elevated plasma levels of multiple markers 
of thrombin and fibrin generation, increased tissue factor expression, and decreased levels of circulating 
anticoagulants. Platelets are activated at baseline in SCD patients, with further activation during VOC (Heeney 
et al. 2016).  SCD patients are at significantly increased risk for thrombotic complications such as pulmonary 
embolism and stroke. Inflammation, ischemia-reperfusion injury and hemolysis are all likely contributors to 
coagulation activation (Ataga et al. 2012; Noubouossie, Key, and Ataga 2015; Tomer et al. 2001). We have used 
the Calibrated  Automated Thrombogram (CAT),a semiautomated high throughput method as the closest 
relevant representation of in  vivo clotting  (Baglin 2005).  Using this assay in children and adults with SCD we 
have noted striking  alterations  in the initial phase of thrombin generation ( a shorter lag time, and lesser time to 
peak ) although overall endogenous thrombin generation  was not significantly different (Betal et al. 2009). 
 
For SCD patients, the experience of recurrent acute VOCs has a devastating impact on quality of life (Dampier 
et al. 2010; Dampier et al. 2011) and is a risk factor for early mortality (Platt et al. 1991).   In addition, VOCs 
place a heavy burden on the USA health care system, accounting for a large number of Emergency Room (ER) 
visits and hospital admissions annually at a cost of approximately $500 million (Weisberg et al. 2013).  Despite 

 
14 
 
an extensive literature addressing pain in SCD, the nature of vasoocclusive pain, factors underlying the transition 
from acute to chronic pain and the wide variability in the pain experience between patients with SCD remain 
poorly understood. Optimal management strategies for this complex pain syndrome remain highly problematic 
despite over three decades of basic and clinical research in the field (Ballas, Gupta, and Adams-Graves 2012). 
There are major overlaps between the areas of neuropathic pain, ie pain initiated by dysfunction of the peripheral 
or central nervous system and inflammatory or nociceptive pain, where pain is the result of tissue injury.  Such 
clear differentiation between these types of pain in any given subject with SCD may not be practically feasible.  
SCD subjects use neuropathic pain descriptors, and may have the hypersensitivity to tactile stimuli usually 
observed in neuropathic pain (hyperalgensia and allodynia) and a recent study by Brandow and coworkers found 
that a third of the SCD patient group (median age 20 years) had evidence of neuropathic pain when screened 
with a validated neuropathic screening tool (Brandow, Farley, and Panepinto 2014). 
 
To preserve survival, the human body has evolved complex protective mechanisms to detect and interpret 
noxious stimuli. Tissue injury from a variety of mechanical, thermal or chemical stimuli produce biochemical 
mediators, stimulating afferent peripheral nerve fibers (nociceptors)  that synapse in the dorsal horn of the spinal 
cord, transmitting signals through ascending pathways to the brain (figure 1). Descending neural pathways 
arising in multiple areas of the brain also regulate transmission of pain signals in both a positive and negative 
fashion at the level of the spinal cord (Ossipov, Dussor, and Porreca 2010). 
 
Figure 1 
 
However, alterations in this system may occur which can result in the pathologic experience of pain. Tissue 
damage with associated accumulation of inflammatory factors released from activated nociceptors and other 
infiltrating cells can enhance the excitability of the nerve fiber resulting in an augmentation in sensitivity to painful 
stimuli. This process is known as peripheral sensitization (Basbaum et al. 2009). Repetitive exposure to 

 
15 
 
noxious stimuli can result in amplification in the pain response through changes in signaling pathways in the 
spinal cord and cortex, known as central sensitization (Kuner 2010).   Mounting data supports the idea that 
peripheral and/or central sensitization may play a key role in the experience of sickle cell vasoocclusive pain.  A 
recent study has implicated mast cell activation with neurogenic inflammation and hyperalgesia in SCD mice 
(Vincent et al. 2013). 
 
1.2 Quantitative Sensory Testing (QST) 
QST refers to a set of noninvasive psychophysical procedures performed to measure subjective somatosensory 
responses to various physical stimuli in order to evaluate sensory function of the peripheral nervous system.  
QST has been successfully performed and shown to be feasible in children as young as three years (Hilz et al. 
1996) with good reproducibility over short intervals of retesting (Meier et al. 2001).  In prior studies, pediatric 
subjects have not reported severe pain or distress and have not withdrawn from study enrollment (Meier et al. 
2001; Zohsel et al. 2006).  Reference values for thermal and mechanical detection and pain thresholds are 
available for children and adolescents aged 6 and up (Blankenburg et al. 2011; Blankenburg et al. 2010). Thermal 
testing for these and many other recent studies, including  prior studies where QST testing was done in children 
(Brandow et al. 2013; O'Leary et al. 2014; Jacob et al. 2015) and adults with SCD (Campbell et al. 2016; Ezenwa 
et al. 2016), used the Medoc TSA II analyzer (Medoc Advanced Medical Sciences) and subjects tolerated the 
testing well, with no  reports of significant pain or distress.  
 
There is strong evidence that sickle mice exhibit marked hypersensitivity to thermal and mechanical stimuli 
(Garrison et al. 2012; Hillery et al. 2011; Kohli et al. 2010).  Brandow et al have documented significantly lower 
median cold and heat detection and pain thresholds in SCD subjects (all genotypes; median age 14 years) 
evaluated by QST (Brandow et al. 2013). Studies by O’Leary et al and Jacob et al have also demonstrated 
altered thermal sensitivity in children with SCD, though with somewhat variable results (Jacob et al. 2015; 
O'Leary et al. 2014).  
 
Although experimental pain does not replicate many features of clinical pain such as emotional overlay and 
actual tissue injury, it can provide relevant and valuable insights.  Clinical relevance of QST in assessing patients 
with a variety of pain syndromes, including fibromyalgia, temporomandibular disorders, headache disorders and 
pelvic pain syndromes has been well documented in the literature.  Suprathreshold experimental pain responses 
can predict pain intensity following surgical procedures.  Of particular relevance to the current protocol, QST has 
been shown to be a sensitive index of treatment outcomes (Edwards et al. 2005). This technology will be a 
valuable tool to aid in assessing the efficacy of interventions, such as omega-3 fatty acid (n-3 FA) 
supplementation, to ameliorate VOC in children with SCD.  
 
1.3  The Omega-3 Fatty Acids in SCD 
While current treatment strategies can ameliorate some of the suffering experienced by SCD patients with VOC, 
the development of effective and minimally toxic preventative strategies is clearly the most desirable approach. 
The ideal drug for the efficacious treatment of SCD should be non-toxic and orally administered so that it can be 
used daily to prevent VOC, rather than as an acute intervention after VOC has occurred. It should also be 
inexpensive to facilitate use in underdeveloped countries where SCD is so prevalent. Hydroxyurea (HU) is the 
only known drug with proven efficacy in preventing VOC and its efficacy rests on its pleiotropic erythrocyte and 
extra-erythrocytic effects (Charache et al. 1995; Stuart and Nagel 2004; Wang et al. 2011).   However, there are 
many SCD patients who do not respond adequately, or at all, to this medication and safe administration requires 
burdensome and expensive laboratory monitoring. 
 
Interest in marine oils has passed through many phases from the 19th C lighting of New England lamps to the 
“Eskimo Paradox” where despite their high fat intake the Inuit experience little cardiovascular disease.  The 
essential n-3 FAs Docosahexaenoic Acid or DHA (c22:6n-3) and Eicosapentaenoic Acid or EPA (C20:5n-3), 
depicted in figure 2 and characterized by a double bond (3 carbons from the methyl terminal of the molecule), 
have profound biologic effects (Table 1).  A multitude of clinical trials and studies have documented their role in 
dyslipidemias and cardiovascular health (Bradberry and Hilleman 2013; Davidson 2013; Davidson et al. 2012), 
immune function and inflammatory disorders (Calder 2015; Levy et al. 2001; Serhan and Savill 2005) , brain 

 
16 
 
development and neurologic disorders (Das 2006; Vesco et al. 2015; Yassine et al. 2016) and pain (Serhan 
2014). 
 
 
 
Eicosapentaenoic Acid (C20:5n-3) 
 
 
 
 
 
 
      Docosahexaenoic Acid (C22:6n-3) 
 
 
 
Figure 2 
 
To emulate the success of HU, we need to identify drugs with effects both on the RBC and the other circulating 
cellular elements of blood, and the endothelium which serves as the template on which cell-cell interactions 
occur.  The n-3FAs DHA and EPA are ideal candidates to fulfill this role since (a) Following incorporation into 
cell membranes DHA and EPA give rise to potent anti-inflammatory mediators the Resolvins and Protectins of 
the D (from DHA) and E (from EPA) series which “stop the clock” on the inflammatory process (Ji et al. 2011; 
Serhan 2007).  (b) Resolvins modulate pain hypersensitivity by inhibiting TRPV1 and TRPA1 channels with DHA 
metabolites having much greater potency than those from EPA (Park et al. 2011; Xu et al. 2010; Xu and Ji 2011) 
(c) DHA supplementation reduces the increase in thermal pain sensitivity exhibited by the SCD mouse 
(Wandersee 2012; Wandersee et al. 2015). (d) Preliminary data shows that children with HbSS have a cell 
membrane content that favors the pro-inflammatory FA Arachidonic Acid (AA) over the anti-inflammatory FAs, 
DHA and EPA, and that DHA/EPA supplementation can favorably modulate this (Daak, Ghebremeskel, Hassan, 
et al. 2013; Setty et al. 2015). (f) In addition, DHA/EPA possess many other salutary effects on RBCs, platelets, 
and endothelium that can modulate SCD pathology (Table 1).  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
17 
 
Table 1: Pleiotropic effects of omega-3 fatty acids on circulating blood cells and endothelium 
 
RBCs 
● Improves membrane deformability and fluidity (Lund et al. 1999; Mills, 
Galey, and Dixon 1993; Popp-Snijders et al. 1986; Terano et al. 1983) 
●Decreases viscosity of RBC suspensions (Popp-Snijders et al. 1986; 
Terano et al. 1983) 
●Increases enzyme levels including glutathione peroxidase (De Caterina, 
Liao, and Libby 2000)  
● Effects on Fetal Hb via TR4-mediated CD36 transactivation (Campbell et 
al. 2011; Xie et al. 2009) 
 
 
WBCs 
●Suppresses production of inflammatory cytokines (Calder 2013; Ji et al. 
2011; Li et al. 2014; Serhan 2007; Song et al. 2016) 
●Protects against ischemia-reperfusion injury in vital organs by modulating 
leukocyte-mediated inflammation  (Chen et al. 2003; Lehr et al. 1991; 
Marcheselli et al. 2003; McGuinness et al. 2006; Serhan 2007) 
●Induces the potent lipid mediators Resolvins, Protectins and Docosatrienes 
(Levy et al. 2001; Serhan and Savill 2005; Serhan 2007, 2014) 
●Decreases monocyte activation and gene expression of IL-1β and TNF-α 
(Calder 2013; Hudert et al. 2006; Mayer et al. 2002; Saku, Kobayashi, and 
Kitamura 1999; Taccone-Gallucci et al. 2006) 
●Inhibits the 5-lipoxygenase pathway in neutrophils and monocytes with 
decreased LTB4 levels(Goodnight, Harris, and Connor 1981; Saku, 
Kobayashi, and Kitamura 1999; Taccone-Gallucci et al. 2006) 
 
 
 
Platelets and Hemostasis 
●Decreases platelet aggregation, adhesion and release of intracellular 
constituents (Goodnight, Harris, and Connor 1981; Terano et al. 1983; 
Weiner et al. 1986) 
●Decreases monocyte procoagulant activity (Hansen et al. 1989)  
●Decreases fibrinogen, vWF and plasma viscosity (Piatti et al. 1993; Saynor 
and Gillott 1992) 
●Decreases production of proaggregatory Thromboxane A2 (Rao, Radha, 
and White 1983; Weiner et al. 1986) 
●Prevents vascular thromboses and re-stenosis after angioplasty (Dehmer et 
al. 1988) 
 
 
 
Endothelium 
●Inhibits cytokine-induced adhesion molecule expression and transcription 
(VCAM-1, ICAM-1 and E-selectin) (Collie-Duguid and Wahle 1996; Dehmer 
et al. 1988; Nohe, Johannes, and Dieterich 2003) 
●Inhibits PAF generation (Hudert et al. 2006)  
● NFkB inhibited by DHA (Marsella and Borgna-Pignatti 2014; Serhan 2007) 
●Increases PGI3 levels  (Hishinuma, Yamazaki, and Mizugaki 1999) 
● DHA causes predominant endothelial-independent vasodilation (Mori et al. 
2000) 
● Inhibits sickle red cell-endothelial adhesion (Stuart preliminary data) 
 
 

 
18 
 
Based on what is known about the pathophysiology of SCD, these demonstrated effects of the n-3 FAs on blood 
cells, the endothelium, inflammatory mediators and the coagulation system have great potential, by way of 
multiple mechanisms, to favorably impact the vasoocclusive complications in SCD.  A wealth of scientific data 
as well as some small clinical trials  (Okpala et al. 2011; Tomer et al. 2001; Wandersee 2012) have indicated 
that the n-3 FAs EPA and DHA, with their pleiotropic effects on circulating blood cells and endothelium, represent 
a promising approach to ameliorate the experience of pain in SCD, with essentially no toxicity (Table 2).  A 2001 
study by Tomer demonstrated a reduced frequency of VOC with reduction in some prothrombotic mediators 
(Tomer et al. 2001).  In a small Nigerian study, a decrease in the number of VOC was seen in 16 SCD patients 
treated with oral DHA and EPA for 6 months (Okpala et al. 2011).  In Khartoum (Sudan), Daak and coworkers, 
in a recent placebo controlled evaluation, treated 128 subjects with Hb SS with an n-3 FA supplement (using a 
capsule with a DHA to EPA ratio of 7:1) daily for 1 year  (Daak, Ghebremeskel, Hassan, et al. 2013). Their study 
demonstrated a reduction in VOC events, and a modulation of severe anemia in the treated group with no side 
effects or bleeding.  Dyspepsia occurred in 4% of both treated and control groups.  In a follow up study, the same 
group demonstrated lower expression of NFB gene expression in the patients treated with their n-3 FA 
supplement compared with untreated subjects (Daak et al. 2015).  NFB is a transcription factor which plays a 
key role in the up-regulation of inflammatory cytokines and adhesive molecules (Hoesel and Schmid 2013; Min 
et al. 2005), providing further evidence that n-3 FA supplementation may have a beneficial effect on inflammation 
and adhesion in SCD. Additional support comes from recent studies in SCD transgenic mice. Wandersee et al. 
demonstrated that DHA supplementation improved RBC flexibility and reduced irreversibly sickled cells in SCD 
mice (Wandersee et al. 2015). In addition, DHA treated SCD mice demonstrated reduced cold hypersensitivity, 
whereas no change was seen in mice who remained on a normal diet (Wandersee 2012).  Kalish treated SCD 
mice with a diet rich in n-3 FAs for 6 weeks. The investigators were able to clearly demonstrate a reduction in 
vascular activation, inflammatory response and SCD related end organ damage in SCD mice on the n-3 FA diet 
(Kalish et al. 2015). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
19 
 
Table 2: Studies related to n-3 FAs in SCD 
Table 2a: Human Studies 
 
 
(Tomer et al. 
2001) 
Prospective, double-blind trial comparing treating adults with SCD and frequent VOC 
with menhaden fish oil (n=5) vs olive oil control (n=5). Observed decreased frequency 
of VOC in n-3 FA treated subjects (p<0.01). Pretreatment, significant decreases in 
platelet activation markers, platelet secreted proteins, thrombin generation and 
fibrinolysis (approaching control values) and decrease in prothrombin fragment 1.2 
(F1.2), d-dimer and plasmin-antiplasmin complexes (PAP) were also noted in n-3FA 
treated subjects. The dosages of DHA and EPA used in this study were approximately 
30 mg/kg/day EPA and 45 mg/kg/day DHA 
There were no significant adverse events. 
(Okpala et al. 
2011) 
16 subjects with SCD in Nigeria treated with EPA 15 mg/kg/day and DHA 10 mg/kg/day 
for 6 months showed a drop in median number of VOC (p<0.0001) 
Here were no significant adverse events. 
 
(Daak, 
Ghebremeskel, 
Hassan, et al. 
2013) 
Double blind, placebo controlled trial in which 128 subjects with SCD in Sudan (mean 
age 8.1 years) were randomized to treatment with DHA/EPA (7:1 ratio, approx 25 
mg/kg/day n-3 FA) vs placebo for one year. N-3 FA treated group demonstrated 
significant reduction in VOC, severe anemia, blood transfusions, WBC and missed 
school days as compared to controls.  
There were no significant adverse events. 
(Daak, 
Ghebremeskel, 
Mariniello, et 
al. 2013) 
80 children with SCD in Sudan treated with DHA/EPA (7:1 ratio, approx 25 mg/kg/day n-
3 FA) vs placebo for 6 mos. N-3 FA treated children showed significant decrease in 
CU/Zn superoxide dismutase activity and cytosolic glutathione peroxidase compared to 
pretreatment baseline suggestive of antioxidant protection. 
(Setty et al. 
2015) 
Ratio of pro-inflammatory AA to anti-inflammatory DHA and EPA was significantly 
increased in RBC membranes of children with SCD as compared with control. This 
correlated positively with hs-CRP levels.  
(Daak et al. 
2015) 
Follow up to Daak 2013 study. Compared children with SCD treated with DHA/EPA 7:1 
(n=24), HU treated (n=18) and placebo (n=21) for minimum 1 year with healthy controls 
(n=25). N-3FA treated group showed lower NFB gene expression compared with HU 
and control groups (p<0.05) 
 
Table 2b:  Animal Studies 
(Wandersee 
2012; 
Wandersee et 
al. 2015) 
SCD mice whose diet was supplemented with DHA for 8 weeks were compared with 
control SCD mice. RBCs from SCD mice on DHA diet showed deceased stiffness, 
decreased osmotic fragility and fewer irreversibly sickled cells compared to control mice. 
In addition, DHA treated SCD mice demonstrated reduced cold hypersensitivity, whereas 
no change was seen in mice who remained on normal diet (p =0.007). 
 
 (Kalish et al. 
2015) 
 
 
SCD and control mice fed standard diet versus n-3 FA enhanced diet. SCD mice fed n-3 
FA diet demonstrated lower neutrophil counts and diastolic BPS. N-3 FA diet resulted in 
reduced aortic expression of endothelin-1 (ET-1, vasoactive, proinflammatory cytokine), 
VCAM-1 (marker of endothelial activation) and heme-oxygenase-1 (anti-oxidant). 
Pulmonary vascular permeability and ET-1 expression were also reduced in mice fed n-3 
FA diet. In a hypoxia-reoxygenation model of VOC, n-3 FA reduced inflammation and 
protected against SCD related end-organ injury.  
 
Since AA, EPA and DHA are the precursors of lipid mediators with effects on inflammatory/anti-inflammatory 
tone, the content of these FAs in the RBC membranes in children with HbSS disease and age- and race-matched 
controls was evaluated by Setty, et al (Setty et al. 2015).  While AA gives rise to proinflammatory eicosanoid 
mediators, EPA and DHA are the precursors of recently identified potent anti-inflammatory products including 
Resolvins, Protectins and Maresins.  Table 3 shows preliminary data from Drs. Setty and Stuart’s research lab 
(presented at the 9th Annual Sickle Cell Disease Research and Educational Symposium and 38th National 
SCD Scientific Meeting, Hollywood, Fl., April 2015). 

 
20 
 
 
1.4   Research Studies from Our Group:  Preliminary Data 
 
1.4.1 Deficiency of RBC Membrane n-3 FAs EPA and DHA in Children with SCD is Associated with 
Increased Levels of Inflammatory Biomarker hs-CRP 
In a study population included 38 children with HbSS (ages 1.7 to 18.3 years, mean age 10.8 years) and 18 age- 
and race- matched controls (mean age 11.9 years), Setty et al. measured the FA content of membrane 
phospholipids (PLPs) by capillary gas chromatography (GC) (Setty et al. 2015). PLP Recovery was monitored 
using diheptadecanoyl-Phosphatidylcholine (PC) and dipentadecanoyl-Phosphatidylethanolamine (PE), and the 
FA values presented were corrected for recovery. An ELISA assay was used to measure plasma levels of hs-
CRP.  Data Analysis was performed using unpaired t-test, Mann-Whitney Rank-Sum test and/or Spearman test. 
This data demonstrates a marked increase in the AA to EPA+DHA ratio in the HbSS patient group. This abnormal 
ratio is due to a decrease in n-3, and an increase in  omega-6 (n-6) FAs in one or both PLP fractions as presented 
in Table 3.  Since the n-6 FA AA is proinflammatory while the n-3 FAs EPA and DHA are membrane precursors 
of crucial anti-inflammatory effector metabolites, this imbalance should increase proinflammatory tone.  In figure 
3 the data demonstrates a significant correlation between the AA to EPA+DHA ratio and levels of the biomarker 
of inflammation hsCRP in children with SCD.  
 
 
Table 3: Red Cell Membrane Fatty Acid Composition in Controls and HbSS subjects (nMol/109 RBCs) 
 
 
 
 
 
 
 
  
 
Figure 3 
 
 
Fatty Acid Ratio
4
5
6
7
8
9
15
10
hs-CRP in Patients  (mg/L)
0.1
1.0
10.0
Fatty Acid Ratio
2
3
4
5
6
7
8
9 10
hs-CRP in Controls  (mg/L)
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
0.01
0.10
1.00
10.00
r = 0.366
p = 0.024
n = 38
r = 0.044
p = 0.856
n = 18

 
21 
 
1.4.2 Quantitative Sensory Testing in SCD 
 
In a recently completed trial, we tested thermal detection and pain thresholds  in children with SCD compared 
with African American controls. In this study, SCD subjects at baseline health and healthy African American 
controls age 8-20 years were administered heat and cold sensitivity and pain threshold testing using a Medoc 
TSAII in triplicate on the forearm. To exclude outliers, the average of the two closest values was taken, discarding 
the third. Controls were tested at baseline and 3 months; SCD subjects were also tested at 6 months.  Subjects 
were tested for four parameters: Cold sensitivity (CS), cold pain (CP), heat sensitivity (HS) and heat pain (HP).  
Sixty controls and 57 subjects with SCD completed thermal testing. Although data analysis is still underway, we 
have the following preliminary findings:  
 
●Aggregating thermal sensitivity across these parameters into one thermal composite score endpoint (TCS, 
described in section 8.1.2) at baseline and 6-months showed a mean reduction in sensitivity of only 0.75C ± 
2.9oC for SCD patients.   
●There were no significant differences in thermal thresholds between SCD and control subjects.  
●Age did not significantly associate with thermal thresholds except among SCD subjects whose CS increased 
on average by 0.14C per year (p=0.01).  
●In SCD subjects, there was no significant association between thermal thresholds and chronic transfusion 
therapy, but heat pain thresholds were significantly higher by approximately 2C among SCD subjects on 
hydroxyurea therapy (p=0.04). 
●The QST measurements were consistent for both SCD and control subjects for CP, HS and HP (each ICC>0.6), 
but not necessarily for CS (ICC=0.52 for SCD subjects and ICC=0.46 for controls). Although internally consistent, 
there was some variability in mean thermal thresholds with in SCD subjects over time. Results for HS varied by 
a mean of +0.47C (95% CI 0.02, 0.93), CS by a mean of -0.61C (95% CI -1.00, -0.21), and HP by a mean of 
+0.81C (95% CI 0.28, 1.35) from baseline to 6 months, but the only significant difference between the 3- and 
6-month means was for HP (+0.66C; 95% 1.19, 0.14). See Table 4. 
 
Table 4:  Test-retest in SCD only. 
Modality 
Day 
1 
Month 
3 
Month 
6 
Difference (M3-D1) 
Difference (M6-D1) 
Difference (M6-M3) 
Mean 
95% CI 
p-
value Mean 
95% CI 
p-
value Mean 
95% CI 
p-
value 
CS 
29.01 28.43 
28.40 
-0.58 (-0.96,-0.20) 0.003 -0.61 (-1.00,-0.21) 0.003 -0.03 
(0.36,-
0.41) 
0.886 
CP 
22.23 21.62 
21.24 
-0.60 (-1.74,0.54) 0.298 -0.99 (-2.18,0.20) 0.102 -0.39 
(0.77,-
1.54) 
0.510 
WS 
35.73 36.24 
36.21 
0.51 
(0.08,0.95) 
0.022 
0.47 
(0.02,0.93) 
0.040 -0.04 
(0.41,-
0.48) 
0.870 
HP 
39.75 39.90 
40.57 
0.15 
(-0.36,0.66) 0.563 
0.81 
(0.28,1.35) 
0.003 
0.66 (1.19,0.14) 0.013 
TCS 
6.58 
7.08 
7.33 
0.50 
(-0.19,1.19) 0.154 
0.75 
(0.01,1.49) 
0.047 
0.25 
(0.91,-
0.41) 
0.457 
 
 
There were 6 subjects who started hydroxyurea therapy either after their baseline QST testing or immediately 
prior, before one would expect any significant clinical effect. Thus, at the time of baseline QST, they were 
“hydroxyurea naïve” and their 6 month QST testing would be expected to reflect the effect of hydroxyurea 
treatment. This data shows a very significant change (p<0.001) in all 4 thermal thresholds (Table 5).  
 
 
 

 
22 
 
 
 
Table 5:  Subjects who started hydroxyurea within one month of baseline QST testing or just after baseline QST  
testing 
 
 
Day 
1 
Month 
3 
Month 
6 
Change from Baseline to 3 
Months 
Change from Baseline to 6 
Months 
Modality Mean Mean 
Mean 
Mean 
Change 
95% CI 
P 
Mean 
Change 
95% CI 
P 
CS 
29.76 
29.20 
27.77 
-0.56 (-1.34,0.22) 
0.141 
-1.99 (-2.77,-1.21) 
<.001 
CP 
23.43 
22.29 
17.28 
-1.13 (-3.03,0.76) 
0.213 
-6.14 (-8.04,-4.24) 
<.001 
WS 
34.85 
35.31 
37.53 
0.46 (-0.55,1.46) 
0.333 
2.68 (1.68,3.69) 
<.001 
HP 
38.67 
41.87 
43.01 
3.19 (2.11,4.27) 
<.001 
4.33 (3.25,5.42) 
<.001 
 
 
Summary:  
• Thermal testing results tended to be consistent over a 6 month period in subjects with SCD, which is critical to 
inform on the use of QST in evaluating the efficacy of preventative pain therapies.  
 • Results between the second and third testing sessions were highly consistent, suggesting that reliability may 
improve once subjects have some experience with the testing procedures.  
• We did not demonstrate any significant differences in thermal thresholds by QST between sickle cell and control 
subjects. These results differ from the findings of Brandow et al. {Brandow, 2013 #29} in which thermal 
hypersensitivity was seen in SCD subjects.   
•The data showing that SCD patients newly on hydroxyurea exhibited significant decreases in sensitivity across 
all thermal parameters over the six month study period strongly supports the utility of QST as an outcome 
measure in a clinical trial testing a new medication to prevent vasoocclusive pain. 
 
Based on this data which has laid the groundwork and methodologic set up, and supported by 
the literature, we have chosen to use thermal sensitivity by QST as the primary efficacy endpoint 
in this Phase I/II Safety and Dose Escalation Trial of SCD-Omegatex™, a novel formulation of DHA 
and EPA. 
 
 
1.4.3  Thrombin  Generation in SCD: Insights  from Computerized Automated Thrombography (CAT) 
(Betal et al. 2009) 
SCD is considered to be a hypercoagulable state.  The Setty and Stuart Lab evaluated hemostatic perturbations 
in subjects with SCD by employing CAT, a novel thrombin generation assay that provides a global measure of 
coagulation potential and a direct assessment of the coagulation phenotype (Hemker et al. 2006).  A total of 23 
adults with Sickle Cell SS Disease (Hb SS) and Sickle β-thalassemia (Hb Sβthal) 18 to 53 years old were 
studied as well as 6 age matched controls.  Plasmas were obtained during routine visits.  Platelet Poor Plasma 
(with and without corn trypsin inhibitor to minimize variability from activation of the contact pathway during 
sample collection) was analyzed by CAT using 5 picomolar (pM) Tissue Factor with 4 micromolar (µM) 
phospholipid. Five CAT assay parameters were evaluated including Lag Time, Endogenous Thrombin Potential 
(ETP), Peak thrombin (Peak), time to peak (tt peak) and start tail. Student t test was used to compare means of 
CAT parameters between SCD subjects and controls.  Significant differences were noted in  SCD compared to 
the controls with shortened Lag Times of 1.9 +/- 0.39 minutes (min) ( mean +/- standard deviation (SD) )  and tt 
peak 3.72 +/- 0.55 min when compared to controls whose values were 2.39 +/- 0.42 min and 4.77 +/- 0.49 min 
for Lag Time and tt Peak respectively ( p < 0.001).  However there was overall a lower and less sustained 
thrombin generation  with SCD subjects demonstrating an early start tail ( 16.76 +/- 2.29 min ) and less total 
ETP  ( 1377 +/- 360 nanomolar (nM )) when compared to the controls whose values for start tail and ETP  were 
20 .33 +/- 1.71 and 1629 +/- 212 nM  respectively ( p < 0.01).  Peak thrombin levels were similar in both groups 

 
23 
 
at 354 and 336 nM respectively.  Thus the subject with SCD has a significant early upsurge in thrombin 
generation followed by the phenomenon of a rapid attenuation.  
  
1.4.4  hsCRP 
In a study of 70 children with SCD at steady state evaluated by a broad panel of biomarkers representing 
previously examined mechanisms of pathogenicity in SCD, hs-CRP, a marker of low-grade, systemic 
inflammation, emerged as the most significant laboratory correlate of hospitalizations for VOC. While markers of 
increased hemolytic status, endothelial activation and coagulation activation all correlated positively with VOC 
events by univariate analysis, baseline hs-CRP levels provided the most significant contribution to the 
association in multiple regression models (22%), and, hs-CRP, along with age, provided the best fit in negative 
binomial models (Krishnan et al. 2010). These data highlight the clinical relevance of the role of inflammation in 
pediatric VOC, providing both a rationale for future therapeutic strategies targeting inflammation in microvessel 
occlusive complications of SCD, and the potential clinical use of hs-CRP as a biomarker that correlates with 
VOC induced pain.   
 
1.4.5 Pain Diary 
Daily diaries are a well documented approach to provide qualitative and quantitative data on the pain experienced 
by SCD patients in the home setting. The validity and reliability of this type of data has been well described in 
published reports describing the use of pain diaries in both paper (Dampier et al. 2002a; Dampier et al. 2002b; 
Dampier, Ely, et al. 2004; Ely et al. 2002; Smith et al. 2008) and electronic form (Bakshi et al. 2015; Jacob et al. 
2013; McClellan et al. 2009). The content of the diary to be used in this study is included in Appendix 1. We 
have piloted use of this pain diary in our  QST trial. All families have reported access to WiFi. Overall compliance 
has been fair with the iPad version with some subjects using paper version of diary.   Approximately 15% of 
participants declined the iPAD Mini, preferring a paper pain diary or choosing to use their own electronic device 
for diary completion.  Preliminary data reveals that 5862 diary entries were completed. Approximately 30% of 
study participants completed the electronic pain diary greater than or equal to 75% of the time, approximately 
46% of the participants completed the electronic pain daily greater than or equal to 50% of the time and 
approximately 70% of the participants completed the electronic pain daily greater than or equal to 25% of the 
time.  Because paper diaries allow for backfilling of entries which provide a less accurate assessment of the pain 
experience (Stone and Broderick 2007; Stone et al. 2003), we would like to assess pain in real time on a daily 
basis.  Thus, for the current trial we will only offer the diary in iPad format. The diary is designed so that an 
assessment can occur only between 6:30 PM and 3 AM each day, after which that day is no longer accessible 
to the subject.  
 
 
2.0 Study Objectives and Hypotheses 
 
We are undertaking a safety and dose escalation trial of SCD-Omegatex™(the Study Drug or Drug Product), 
a novel formulation of DHA ethyl ester (EE) and EPA EE )(approximately 9:1 ratio), in pediatric and 
young adult patients with Hb SS, Sickle C Disease (Hb SC) and Hb Sβ°thal.  
 
Primary Objective 
1) To determine, in a dose escalation trial, the clinical safety of DHA/EPA supplementation with SCD-
Omegatex™ as evidenced by an absence of AEs related to the FA supplementation. 
2) To determine whether 6 months of supplementation with SCD-Omegatex™  will reduce thermal 
sensitivity as measured by QST to below pre-treatment levels. 
 
Pain Related Secondary Objectives   
1) To determine whether 6 months of supplementation with SCD-Omegatex™  will increase health-
associated Quality of Life responses using the Varni Pediatric Quality of Life (QoL) InventoryTM SCD 
Module. 
2) To determine the effect of treatment by assessing pain days using an iPad daily report pain calendar. 
3) To measure the effect of treatment on individual thermal testing parameters by QST. 

 
24 
 
 
 
 
Exploratory Objectives 
1) To measure the effect of treatment on select biomarkers of inflammation including pro and anti-
inflammatory cytokines, adhesion molecules, hsCRP and plasma lipid mediators derived from DHA, EPA 
and AA..  
2) To assess the effect of SCD-OmegatexTM  treatment on thrombin generation as assessed by CAT. 
 
Primary Hypotheses: 
SCD-Omegatex™  will be safe at both the lower and higher dosages to be administered and will significantly 
reduce thermal sensitivity measured by QST after 6 months of use. 
 
Secondary Hypotheses: 
Following 6 months of supplementation with SCD-Omegatex™  we hypothesize that the following will occur:   
● An increase in the Varni Pediatric QoL InventoryTM SCD Score  
● A decrease in pain days 
● A decrease in the proinflammatory cytokines and chemokines as well as hsCRP and an increase in anti-
inflammatory markers. 
● Normalization of the altered kinetics of thrombin generation  
3.0 Rationale for Dose Selection and Monitoring of Study Drug Intake 
The DHA/EPA EE formulation to be used in this trial is similar to the formulation used in the studies by Daak et 
al (Daak, Ghebremeskel, Hassan, et al. 2013; Daak et al. 2015), with a significantly higher concentration of DHA 
as compared to EPA. Daak’s studies used DHA/EPA EE in a 7:1 ratio while the SCD OmegatexTM formulation 
used in the current study contains an approximate 9:1 ratio DHA/EPA EE. The Resolvin metabolites of DHA 
possess more potent anti-inflammatory and analgesic effects than those from EPA (Park et al. 2011; Xu et al. 
2010; Xu and Ji 2011) as well as a greater effect on membrane fluidity in endothelial cells (Hashimoto et al. 
1999). Thus, the rationale for the 9:1 DHA/EPA ratio is: 
●  to maximize the anti-inflammatory and analgesic properties of the DHA metabolites. 
● to utilize a formulation similar to the n-3 FA product shown in the Daak study to be safe and effective in children 
with SCD.  
● to utilize a starting dosage based on the Daak trial, in which the dose was approximately 25 
milligrams/kilogram/day (mg/kg/day) body weight DHA+EPA.  The resulting EPA dose with this formulation was 
approximately 3 mg/kg/day.  Using a two dose escalation protocol will enable us to evaluate safety and 
preliminary efficacy data at 2 dosages (25 and 37.5 mg/kg/day DHA+EPA) to inform dosing in a later Phase III 
trial.  
 
DHA is the major omega-3 FA in all of the cells and tissues evaluated to date (Arterburn, Hall, and Oken 2006).  
We have chosen to evaluate the packed total blood cell incorporation of the n-3 FAs DHA and EPA 
during therapy as a monitor of subject protocol adherence.  (Note that our preliminary results presented in 
Appendix 5 demonstrate that the FA indices between red cell membranes and packed total blood cells are 
comparable). Incorporation of DHA and EPA into RBC membranes appears to reflect the incorporation of these 
FAs into other tissues (Harris 2008; Neubronner et al. 2011).  Studies performed to date on long-term (6 to 12 
months) n-3 FA supplementation, in general, demonstrate that steady state levels of DHA and EPA in 
circulating cells and tissues are achieved between 2 and 6 months (depending on the nature of FA 
supplement) (Arterburn, Hall, and Oken 2006; Harris 2008; Katan et al. 1997; Marangoni et al. 1993). While 
EPA steady state levels in the RBC are achieved within 2 months, DHA incorporation into erythrocyte 
phospholipids during supplementation continues to increase between 4 to 6 months using either the fish oil 
preparation, ethyl ester or the re-esterified triglyceride form (Arterburn, Hall, and Oken 2006; Harris 2008; 
Katan et al. 1997; Marangoni et al. 1993).      
 

 
25 
 
We have attempted to standardize the mechanics of ingestion as much as possible by requiring that the 
capsules be ingested with the biggest meal of the day (dinner) which in general also will accommodate the 
highest fat content to enhance absorption.  We emphasize that our monitoring of total blood cell incorporation 
of n-3 FAs is not because total blood cell increase in DHA and EPA is our major consideration regarding it’s 
mode of action in SCD therapy.  Rather, the effects of these FAs on pain sensation as assessed by 
standardized Qualitative Sensory Testing (QST) is our primary and critical goal.  We are testing whether the 
effect of the DHA/EPA metabolites on the enhanced pain hypersensitivity that has been documented in SCD 
by other investigators (Brandow et al. 2013) will be rectified by the n-3 FAs, especially by DHA.  Metabolites of 
DHA have markedly greater potency in modulating pain hypersensitivity in the SCD mouse model (Wandersee 
2012; Wandersee et al. 2015) and this  FA is particularly abundant in neural tissue where it is several 
hundredfold more abundant than EPA (Arterburn, Hall, and Oken 2006).   Moreover, since the half life of RBCs 
is markedly decreased in SCD (to a few days) the use of red cell membrane n-3 FA content is fraught  with 
interpretive difficulty as to when RBC membrane saturation is attained such that its main utility for us as 
investigators is to monitor compliance.  When the RBC phospholipid accumulation of DHA and EPA reaches 
saturation point is not our focus.  The effect of the n-3 FAs on pain hypersensitivity, i.e. on nerve tissue, as 
measured by our primary QST endpoint is the focus of this clinical trial, in particular whether thermal sensitivity 
by QST undergoes a significant change from baseline in subjects with SCD post DHA/EPA supplementation. 
Further supporting the effect of n-3 FAs on neural tissue is recent randomized, double blind, placebo controlled 
trial in the Sudan which documented a significant reduction in seizure frequency in patients with drug-resistant 
epilepsy treated with DHA and EPA (Ibrahim, Daak, and Ghebremeskel 2016). 
  
We are using the 37.5 mg/kg n-3 FA dose in a cautious dose escalation phase to a maximum of 4 grams (g) 
DHA and EPA since this level has been shown to be without toxicity in numerous previous studies.  In addition, 
as in the literature, if levels of DHA content of cell lipids continues to increase in cells and tissues till 4 to 6 
months post treatment inception, by using the higher dose we hope to achieve maximal benefit on our primary 
end point during the time frame of our study.  
     
 
4.0 Trial Design    
 
This is a Phase I/II, single site, open-label safety and dose escalation trial in which children with Hb SS, Hb SC 
and Hb Sβ0thal will be given a new n-3 FA formulation (SCD-Omegatex™) containing DHA and EPA at a ratio 
of approximately 9:1 DHA:EPA  at one of two daily oral doses for a period of 6 months. 
 
4.1  Dose Escalation (see also figure 4) 
4.1.1 Phase I Portion:  
The Dose Limiting Toxicity (DLT) portion of the study will follow a “3+3” design. Three subjects will be enrolled 
at the starting dose of 25 mg/kg body weight of SCD OmegatexTM.  If none of the subjects has a DLT after 2 
months of treatment, the dose is escalated for the next 3 subjects to 37.5 mg/kg. The original 3 enrollees will 
remain on the 25 mg/kg dose for the remainder of the trial to complete 6 months of study drug.  If one DLT is 
observed at 25 mg/kg, the next 3 subjects are enrolled at 25 mg/kg.  If one out of 6 subjects has a DLT at 25 
mg/kg, the dose is escalated to 37.5 mg/kg for the next group of subjects. Note that the maximum total daily 
dose for this trial will be 4 g per day. If 2 or more of 6 subjects develops a DLT at 25 mg/kg, escalation is 
stopped and no Maximum Tolerated Dose (MTD) is found.  If the dose is escalated to 37.5 mg/kg and 1 or fewer 
DLT is observed in the first 3 subjects after 2 months, 3 more subjects will be enrolled at the 37.5 mg/kg. If in 2 
more months, fewer than 2 out of 6 subjects show toxicities at 37.5 mg/kg, 37.5 mg/kg is the recommended 
Phase II dose.  If 2 or more DLTs are noted, 25 mg/kg is the MTD.  The length of treatment at each dose level 
in Phase I will be 2 months, with carryover to Phase II of subjects receiving the MTD.  The maximum sample 
size for this portion of the study is 12 subjects.  Efficacy data for all subjects will be analyzed.  DLTs will be 
managed as described in section 7.0. 
 
4.1.2 Phase II Portion:   

 
26 
 
From our unpublished pilot data, the standard deviation (SD) of thermal sensitivity reduction over a 6 month 
period is approximately 2.9 in African American children with SCD as measured by the TCS composite endpoint 
described in section 8.1.2. A sample size of 30 is needed to have 80% power to detect a moderate 1.5 degree 
(~0.5 SD) difference in TCS following treatment using a 2-sided paired-t test of size 0.05.  Since 6 of the subjects 
for this phase will come from the Phase I portion, the additional sample size needed for this Phase is 24 subjects.  
Allowing for approximately 20% attrition we will recruit 30 additional subjects in this phase with a total maximum 
sample size for both Phases I and II combined being 42 subjects (Phase I=12,  Phase II=30). 
 

 
27 
 
 
 

 
28 
 
 
 
 
4.2  Primary and Secondary End Points, and Additional Markers of Interest 
4.2.1 Primary Endpoints 
Safety: Clinical safety will be demonstrated by the absence of AEs related to study drug.  
 
Efficacy: The primary efficacy endpoint will be reduction of thermal sensitivity as measured by QST to below 
pre-treatment levels. 
4.2.2 Secondary/Exploratory End Points 
Change from pretreatment levels in QoL scores and number of pain days will be noted following 6 months of 
EPA and DHA supplementation with SCD-Omegatex™.  Effect of supplementation on individual thermal 
sensitivity thresholds by QST will be measured. Pre and post treatment levels of proinflammatory cytokines, 
adhesion molecules, hsCRP, plasma lipid mediators derived from DHA, EPA and AA as well as CAT parameters 
will be assessed.  
 
4.3 Inclusion/Exclusion Criteria 
 
4.3.1 Inclusion Criteria 
Subjects who meet all of the following criteria are eligible for enrollment into the study:  
 
 
Participant has signed the informed consent/assent with parent signing informed consent as age 
appropriate. 
 
Established diagnosis of SCD including Hg SS, Hb SC and  Hb Sβo Thal genotypes 
 
Regular compliance with comprehensive care. 
 
Aged 8 years or greater and less than 26 years. 
 
At enrollment and at the time of study drug initiation, subject should be in his/her baseline steady state 
and not in the midst of any acute complication due to SCD.  Must be at least 2 weeks from infection or 
VOC requiring inpatient admission at time of initiation of study drug. 
•    If subjects are on a disease modifying agent such as hydroxyurea or l-glutamine, the dose per kg must     
be stable for at least 3 months prior to enrollment with a plan in place to keep dose stable throughout   
     the course of the study unless a change in clinical status requires a dose modification . 
 
4.3.2 Exclusion Criteria   
 Subjects with baseline hemoglobin (Hb) levels <5.5 grams/deciliter (g/dL). 
 Inability to swallow capsules 
 Poor compliance with previous treatment regimens. 
 Hepatic dysfunction (alanine aminotransferase (ALT) >2X upper limit of normal (ULN) or conjugated bilirubin 
>2X the patients baseline within the last 6 weeks).  
 Renal dysfunction (A creatinine level within the past 6 weeks of ≥ 1.0 mg/dL for children and ≥ 1.2mg/dL for 
a subject ≥ 18 years of age).  
 Prothrombin time (PT) and/or partial thromboplastin Time (PTT)  ≥ 20% higher than ULN 
•  Platelet count less than 100,000  
 Allergy to fish, shell fish or soy 
 Pregnancy. 
 Chronic transfusion therapy. 
 Transfusion within the last 30 days. 
 Treatment with any investigational drug or regular fish oil supplementations in last 60 days. 
 Currently receiving another investigational agent, or on such an agent within the last 60 days. 
 Changes in per kilogram dosing of HU or l-glutamine in  preceding 3 months if on these medications  

 
29 
 
 Diagnosed bleeding disorder or patient on concomitant anti-coagulation. 
 Conditional or abnormal result on most recent Transcranial Doppler (TCD) or history of stroke. 
 Other active chronic illness that could adversely affect subjects performance such as HIV, tuberculosis, 
cancer, autoimmune disease requiring immunosuppressive therapy. 
 Children in Care (CiC): A child in care is a child who has been placed under the control or protection of an 
agency, organization, institution or entity by the courts, the government or a government body, acting in 
accordance with powers conferred on them by law or regulation. The definition of a CiC can include a child 
cared for by foster parents or living in a care home or institution, provided that the arrangement falls within 
the definition above.  The determination of whether a child meets the definition of CiC should be made with 
the study center staff in consultation with the responsible Institutional Review Board (IRB). 
 
4.4 Safety Assessments 
The following labs and assessments will be performed at designated intervals (see Table 7) to assess for toxicity 
and safety: 
 
Vital signs (VS) and physical evaluation. 
 
Complete Blood Count (CBC), reticulocyte count (retic)  
 
PT/PTT 
 
Metabolic Panel (Creatinine, blood Glucose and liver function tests (LFTs) including ALT and aspartate 
aminotransferase (AST). 
 
Urinalysis including microscopic (UA) 
 
Fasting lipid panel 
 
Urine pregnancy test (in female subjects ≥ 9 years of age). 
 
Clinical Bleeding Assessment (Appendix 2) 
 
4.5 Compliance Assessments 
 
DHA, EPA and AA levels (% of total FAs), omega-3 index (O3I, %DHA plus % EPA), fatty acid ratios 
(AA to DHA p lus EPA) in the packed total blood cell fraction. This assessment will monitor compliance 
with the therapeutic regimen. 
 
Pill count at Clinic Visit every 4 weeks. 
 
An email message will be sent to the email address provided by the family for study messages at 3 PM 
daily  reminding them to take their study medication and to make entry into their pain diary. Subjects will 
be instructed to reply on daily pain diary that study medication has been taken and responses will be 
directly loaded into REDCapTM database. 
 
4.6 Selection of Subjects and Screening 
Subjects will be enrolled by study personnel in the Sickle Cell Clinic at Nemours/Alfred I duPont Hospital for 
Children (NAIDHC) after parental permission for participation in the study has been obtained by parent or legal 
guardian with appropriate subject assent (8 to 17 years) or informed consent by the study subject themselves 
(18 to <26 year olds). Enrollment is defined as the date and time of signing the informed consent document.  
Subjects will be approached to determine their interest in the study at the time of routine Hematology visits. In 
addition, flyers advertising the study will be distributed at clinical sites in the area where patients with Sickle Cell 
Disease receive medical care and potential subjects, including young adults who were previously followed in the 
NAIDHC Hematology Clinic, may be contacted by phone to determine potential interest in the study. Subjects 
who meet initial eligibility will be scheduled for a protocol discussion informational session via telephone, 
telemedicine or in person visit. Those subjects who meet inclusion criteria and wish to enroll will be scheduled 
for a first study visit at which time appropriate consenting documents will be reviewed and signed. 
 
4.7 Monitoring for Toxicity and Safety 
4.7.1 Dose Limiting Toxicities for Phase I Portion of Study 
DLTs will be assessed using the Common Terminology Criteria for Adverse Events (CTCAE) version 4.03 from 
the U.S. Department of Health and Human Services with modifications for SCD. All CTCAE ≥ Grade III toxicities 

 
30 
 
judged as possibly, probably or definitely related to study medication will be considered as a DLT except for the 
modifications noted below: 
a) Known SCD complications including, but not limited to, VOC, ACS, aplastic crisis, viral/bacterial infections 
and indirect hyperbilirubinemia associated with a hemolytic episode or gallbladder disease will not be 
considered as DLTs. 
b) A DLT related to skin/subcutaneous bleeding will be defined as clinical evidence of hemorrhage of 
CTCAE Grades II and above, or a modified CTCAE of Grade I severity with areas of purpura not 
explainable by trauma,  occurring on ≥4% of body surface (without antecedent injury). 
c) Ophthalmic, gastrointestinal (GI) and respiratory hemorrhage as well as hematuria,  not explained by 
trauma, of ≥ Grade II will be considered a DLT  
d) A rise from baseline in serum creatinine of CTCAE Grade I or higher that persists for more than 2 weeks 
despite treatment discontinuation, or reappears at any time following medication reintroduction (for which 
no other reason can be found to explain the abnormality besides study drug)  will be considered a DLT.   
e) Phase I hepatic DLT are defined below: 
i). ALT ≥ 3x ULN and bilirubin ≥ 2xULN with ≥ 35% direct bilirubin. 
ii). ALT ≥ 5x ULN. 
iii). ALT ≥ 3x ULN if associated with the appearance or worsening of symptoms of hepatitis or 
hypersensitivity such as nausea, vomiting, right upper quadrant pain or tenderness, fever, rash or 
eosinophilia. 
iv). ALT ≥ 3x ULN which does not resolve to ≤ 2x ULN after 2 weeks of treatment discontinuation or 
recurs following resumption of study medication. 
During the Phase I portion of the trial, prior to moving from the initial dose level to the higher dosage level, a 
formal meeting will be held in which the PI, Co-investigators, coordinator and the medical monitor will review 
any DLTs and adverse events and all must agree to proceed with the dose escalation.  A similar meeting will 
take place prior to establishing the MTD to be used in the Phase II portion of the study. 
 
4.7.2 Criteria for Subject Discontinuation from Study 
Subjects may decide to discontinue participation at any time during the study.  Investigators may discontinue 
any subject at their discretion, if in their professional opinion, the subject’s health, safety, and/or well-being is 
threatened by continued participation in the study. In addition, the following circumstances require 
discontinuation of subjects:  
a) Any SCD-related complication (such as ACS) requiring exchange transfusion or multiple transfusions 
such that Hb S% ≤ 40%. 
b) Drop in hemoglobin to a level < 5 g/dL requiring multiple transfusions such that Hb S% ≤ 40%.  
c) Phase II hepatic chemistry stopping criteria are defined below: 
 
i). ALT ≥ 3x ULN and bilirubin ≥ 2x ULN with ≥3.5% direct bilirubin. 
ii). ALT ≥ 5x ULN. 
iii). ALT ≥ 3x ULN if associated with the appearance or worsening of symptoms of hepatitis or 
hypersensitivity such as nausea, vomiting, right upper quadrant pain or tenderness, fever, rash or 
eosinophilia. 
iv). Interim dose discontinuation will occur if ALT exceeds 3x ULN. LFTs will be evaluated 2 weeks 
after interruption of study drug. If ALT returns to ≤ 2x ULN after 2 weeks of treatment discontinuation, 
study drug may be reinstituted at the initial dose. However, following re-challenge with study 
medication, if there is once again an increase in ALT ≥3x ULN, study drug will be discontinued and 
subject will be removed from study. 
d) Renal dysfunction:   
i) A rise in serum creatinine of CTCAE Grade I or higher persisting for 4 weeks. If creatinine returns to 
within 20% of baseline 2 weeks after treatment discontinuation, the therapy may be reinstituted. 

 
31 
 
However, following reintroduction of study medication if the creatinine rises to CTCAE Grade I or 
higher the subject will be removed from the study 
e)  Focal neurological changes or stroke (excluding seizure in subject with known seizure disorder). 
f) Allergic reaction to the study drug 
g) Pregnancy 
h) Hospitalized for >2 weeks while on study for any SCD-related event except for pain 
i) 
Miss more than one study visit 
j) 
Documented non-compliance with study drug 
k) Interim dose discontinuation will occur if platelet count drops below 100,000. If platelet count fails to 
recover above 100,000 within 2 weeks or if platelet count again drops below 100,000 subject will be 
removed from the study. 
 
Note: AEs caused by participation in the study may necessitate modifications to the level of participation of a 
subject or discontinuation of subjects from participation in the study. Subjects who discontinue from the study 
prior to completing month 2 of study drug (4th study visit) will be replaced. Subjects who discontinue 
prematurely from the study for any reason will be encouraged to complete at a minimum, a safety follow-up at 
one month. 
 
4.7.3 Criteria for Interruption and Re-initiation of Drug  
4.7.3.1 Criteria for Interruption and Re-initiation of Drug following 
Interruption for Toxicity in Phase II Portion of Study 
AEs possibly related to the study drug that are of at least moderate grade will lead to study drug 
interruption. Study drug may be resumed when the AE has resolved as detailed below: 
 Liver function Tests: Interim dose discontinuation will occur if  ALT exceeds 3x ULN. LFTs will be evaluated 
2 weeks after interruption of study drug. If ALT returns to ≤ 2x ULN normal after 2 weeks of treatment 
discontinuation, study drug may be reinstituted at the initial dose. However, following re-challenge with study 
medication, if there is once again an increase in ALT ≥ 3x ULN, study drug will be discontinued and subject 
will be removed from study. 
 Renal function:  Interim dose discontinuation of study drug will occur for a rise in serum creatinine of CTCAE 
Grade I or higher.  Serum creatinine levels will be reassessed 2 weeks after interruption of study drug. If 
serum creatinine levels fall to within 20% of pre-study baseline level, study drug will be reinitiated at the same 
dose prior to discontinuation. However, following re-challenge with study medication, if there is once again an 
increase to CTCAE Grade I or higher, study drug will be stopped and subject will be removed from study. 
 Platelet count:  Interim dose discontinuation of study drug will occur if platelet count drops below 100,000. If 
platelet count rises to ≥ 100,000 after 2 weeks, study drug will be reinitiated. If count fails recover to >100,000 
after 2 weeks or if platelet count again drops to below 100,000 after reinitiation of study drug, patient will be 
removed from study. 
 
4.7.3.2 Criteria for Re-initiation of Drug following Interruption for Hospitalization,  Minor Surgery, Dental 
Procedures or Minor Trauma 
● Study drug should be held for at least the first 48 hours of any hospital admission to determine the severity of 
illness and address any concerns that illness may be related to study drug. After 48 hours study drug may be 
restarted at the discretion of study investigator.  
 
● Subjects undergoing minor surgical or dental procedures e.g. tooth extraction, will have the study drug held 
for 48 hours prior to, and 48 hours after the procedure. In the event of minor trauma with bleeding for which 
medical attention is sought by the study subject, study drug will be held for 48 hours (e.g. lacerations requiring 
suture).  
 

 
32 
 
4.7.3.3 Special Considerations Related to Continued Study Participation 
Subjects admitted for any acute, SCD-related event (e.g. fever or infection, ACS, VOC), and subjects who receive 
a packed red blood cell (PRBC) transfusion at any time during the study can remain on study and continue 
receiving study drug.  However, at the study visit immediately following the admission or PRBC transfusion, all 
other study-related measures will be collected EXCEPT for efficacy outcome measures, including QST, QoL and 
research laboratory measures which may need to be delayed depending on timing.  The next efficacy outcome 
measurement will be performed at the next scheduled study visit that is ≥ 42weeks from either the hospitalization 
and ≥ 4 weeks from a PRBC transfusion.  If the patient has not yet started on study drug, initiation of drug must 
be delayed until baseline research studies can be performed and subject will continue to keep pain diary. 
Subjects can continue to take non-steroidal anti-inflammatories (NSAID) and other medications for pain control 
while on study but amount taken will be documented.  Subjects should not take pain medication within 24 hours 
of a QST testing session, and QST testing should not take place within 2 weeks of a VOC severe enough to 
require inpatient hospital admission. If necessary, QST testing should be rescheduled for an appropriate time 
prior to or at the next scheduled study visit. 
 
 
5.0 Treatment of Subjects 
 
5.1 Description of Study Drug and Dosing Regimen 
5.1.1 Description of Study Drug 
The active ingredient (the Drug Substance Solutex0656SCD) of the study drug, SCD-Omegatex™ has been 
produced by Solutex GC, S.L. (Mallen, Spain). Solutex0656SCD (the Drug Substance, DS) is a fish-oil derived 
fatty acid concentrate, containing mainly omega-3 fatty acid ethyl esters (EE), predominantly docosahexaenoic 
acid (DHA-EE). DS also contains eicosapentaenoic acid (EPA-EE) but at a concentration lower than DHA-EE. 
Other constituents include naturally occurring product-related substances (ex. other polyunsaturated FAs). 
Alpha-tocopherol is added during manufacturing the drug substance at a concentration of 2 mg/g as an 
antioxidant.  
 
The drug substance was filled in soft gelatin capsule under Good Manufacturing Practice (GMP) quality 
encapsulation and tested for stability by Olds Softgels, Inc (Alberta, Canada). Each 450 mg soft gel capsule 
contains no less than 250 mg DHA and 27 mg EPA per capsule. Capsules are enteric coated to reduce fishy 
after-taste. Please see Investigator’s Brochure (IB) and Quality Overall Summary (QOS) for detailed information 
on Physical, Chemical and Pharmaceutical Properties and Formulation of study drug. The soft gelatin capsules 
are stored at controlled room temperature for at least  24 months in 200-counts bottles. 
 
While SCD-Omegatex™ is a new formulation of DHA and EPA, the n-3 FAs have been widely used as over the 
counter supplements. There are currently 3 Food and Drug Administration (FDA) approved prescription 
formulations of n-3 FA available on the market, Lovaza®, Vascepa® and Epanova®. Lovaza® and Vascepa® 
are ethyl ester formulations while Epanova® is a free fatty acid formulation. Vascepa® is a high purity EPA 
agent.  Lovaza®, which contains approximately 465 mg EPA and 375 mg DHA in each 1 gram capsule, is the 
product which most closely resembles SCD-Omegatex™ although the EPA/DHA ratio is much higher. There is 
a great deal of safety information on Lovaza®. This medication and the n-3 FAs in general, are very well tolerated 
and have shown minimal toxicity. Since its FDA approval in 2004, Lovaza® has been widely used as a triglyceride 
lowering agent in adults. See IB for detailed prescribing and toxicity data on Lovaza®.  In a pediatric trial at our 
institution, 42 children with hypertriglyceridemia were given Lovaza® at a dose of 4 g/day for 8 weeks. The only 
reported AEs were GI symptoms, fishy taste and more frequent nosebleeds, all of which were mild to moderate 
in nature, did not require discontinuation of study medication and resolved on their own (Gidding et al. 2014).  
Given the composition of Lovaza®, the dosage of DHA that a 50 kg patient received on this study would have 
been approximately 30 mg/kg, which exceeds the Phase I dose in the current protocol. The EPA dose on this 
study far exceeded the per kg dose to be used in the current protocol.  Additionally, there are a growing number 
of published studies treating children with a variety of diagnoses including autism spectrum, psychiatric disorders 
and non-alchoholic liver disease with EPA and DHA containing supplements without significant side effects 
(Janczyk et al. 2015; Mankad et al. 2015; Vesco et al. 2015).  A study from Denmark reported on the 

 
33 
 
administration of a fish oil supplement (55% EPA and 37% DHA) at a dose of 2.4g versus placebo daily to 736 
pregnant women starting at 24 weeks gestation without reported significant side effects, and demonstrated a 
reduction in asthma and lower respiratory tract infections in the offspring of the treated women {Bisgaard, 2016 
#487}. Two recent studies in infants have used significant per kg doses of DHA than is planned for this study 
without significant bleeding concerns or other adverse events. Collins et al randomized 31 infants born less than 
30 weeks gestation to receive either 40, 80 or 120 mg/kg/day of DHA as an emulsion via feeding tube starting 
within 4 days of first enteral feed. They reported that all levels of DHA in the emulsion were well tolerated without 
adverse effects {Collins, 2015 #494}. The same group published a study randomizing 1273 infants <29 weeks 
gestation to receive an enteral emulsion providing DHA at a dose of 60 mg/kg/day beginning within 3 days after 
their first enteral feed until 36 weeks postmenstrual age. They reported no significant difference in adverse events 
between the treatment and control groups, including no increased incidence of intraventricular hemorrhage 
{Collins, 2017 #499}  Of note, as of June 2016 there were 234 trials (excluding those with unknown status) listed 
on ClinicalTrials.gov which aim to treat children with n-3 FAs.  
 
Most relevant to the current protocol however, is the study in the Sudan by Daak (Daak, Ghebremeskel, Hassan, 
et al. 2013; Daak et al. 2015) in which children with SCD were treated with a formulation of EPA/DHA very similar 
in concentration and dosage to the formulation used in this trial, with a significantly higher concentration of DHA 
as compared to EPA. The dosage used in the Daak trial was approximately 25 mg/kg DHA+EPA, which matches 
the initial dose to be used in the current trial. There were no significant AEs reported in these trials and the only 
reported side effect was dyspepsia and increased appetite. 
 
 5.1.2 Dosing Regimen 
The starting dose for the current trial is based on the dose used in the studies by Daak et al for children with 
SCD (Daak, Ghebremeskel, Hassan, et al. 2013; Daak et al. 2015), as previously discussed, which was well 
tolerated and showed evidence of efficacy.   Subjects receiving the starting dose will receive 25 mg/kg n-3 FA 
(DHA plus EPA) given once daily, while subjects treated at dose level 2 will receive 37.5 mg/kg once daily. The 
maximum total daily dose for subjects on this trial will be 4g per day. Doses will be rounded up or down for 
capsule size. Doses that calculate to ≥ 0.5 capsule will be rounded up while doses calculating to < 0.5 will be 
rounded down. Dosing will be once a day, taken after the evening meal, which is the usually the most fatty meal 
of the day, to enhance absorption. If subjects are unable to tolerate taking the capsules as one single daily dose, 
after discussion with the study team, they will be permitted to divide into two doses, one taken after breakfast 
and a second after dinner. Capsules must not be broken, crushed or chewed. Dosing will be based on the actual 
DHA and EPA content of the capsules for the batch of SCD-OmegatexTM to be used in the trial. Based on testing 
done February, 2017 each capsule contains 336 mg EPA + DHA (34 mg EPA and 302 mg DHA). See dosing 
calculator in Appendix 6 for weight based dosing.  
 
 
5.2 Packaging, Labeling, Storage and Return of Study Drug 
The study drug will be dispensed to subjects enrolled in this trial as per the study protocol, in a light resistant 
container. The study drug container will be labeled to indicate that the drug is being used for investigational 
purposes in this trial.  Per FDA regulation, the package of an investigational new drug intended for human use 
shall bear a label with the statement “Caution: New Drug – Limited by Federal (or United States) law to 
investigational use.  The label or labeling of an investigational new drug shall not bear any statement that is false 
or misleading in any particular way and shall not represent that the investigational new drug is safe or effective 
for the purposes for which it is being investigated.  The dispensing label will abide by Federal and State law. 
 
5.2.1 Drug Accountability 
The PI and pharmacy delegate are responsible for maintaining accurate records of study drug receipt, use and 
destruction. The study site will maintain center level and subject level accountability records of all received, 
dispensed, expired, and destroyed study medication. Each study drug accountability log will be uniquely 
numbered. The logs will be kept current at all times and available for inspection. Any measures to resolve a 
discrepancy will be documented appropriately. 
 

 
34 
 
5.2.2 Medication Storage 
The capsules will be stored at room temperature with specific instructions that the capsules should not be stored 
in the freezer or refrigerated.  The pharmacy storage room is locked with badge access to pharmacists only. The 
room is dual temperature monitored with Dickson devices. Store at 25°C (77°F); excursions are permitted 
between 15°C and 30°C (59°F and 86°F); do not freeze. 
 
5.2.3 Destruction 
Once therapy is completed, all returned medication will be adjudicated and destroyed on-site by the study 
pharmacist per standard operating procedures in compliance with Federal and State Laws. Any remaining 
inventory at the end of the study including expired inventory will be destroyed on site. 
 
5.3 Prior and Concomitant Therapy   
Subjects enrolled in this trial will receive all therapy and monitoring that is considered standard of care for patients 
with SCD. Patients on HU or l-glutamine can participate in the study but must have been on a stable per kilogram 
dose for at least 3 months prior to enrollment and may not have per kilogram dose escalated during the study 
period.  If subjects have been taking a vitamin preparation, it should be determined that it does not contain 
EPA/DHA from any source (i.e. from plant or fish source).  Subjects can continue to take non-steroidal anti-
inflammatory medications and opioids for pain control while on study.  Subjects may not take anti-coagulant 
medications while enrolled on study.   
 
5.4 Subject Compliance 
Compliance will be assessed by querying the study patient and through laboratory testing that includes FA 
measurement in total packed blood cells (see also section 3.0). An email message will be sent to the subject at 
3PM each afternoon reminding them to take their study medication and complete their pain diary. Participants  
will be instructed to bring the bottles with all remaining capsules to each study visit. Remaining capsules will be 
counted and number recorded by the study pharmacist or research coordinator to document adherence.. 
Patients are required to stay on drug therapy beyond week 24 until their visit 8 appointment occurs. This is 
essential to accommodate critical end of study data collection requirements.  A six week supply of study drug 
will be dispensed at study visits 2 through 6.  The extra 14 day supply will ensure the subject has an adequate 
supply in the event that a study visit is delayed for unavoidable reasons.  An eight week supply of study drug will 
be dispensed at visit 7 to account for any delay in the scheduled visit 8 appointment. All remaining dispensed 
bottles and unused capsules will be collected at visit 8 for final adjudication.  
 
5.5 Study Procedures 
 
5.5.1  Recruitment 
Prospective study participants will be recruited at the NAIDHC Center for Cancer and Blood Disorders outpatient 
clinic. All prospective participants who are approached about study enrollment will be given consent documents 
to take home and review.  A phone call, telemedicine visit or an in person meeting will be offered to discuss 
study enrollment and study procedures.  Ample time will be given for questions to be solicited and answered.  
Should participants wish to enroll in the study they will meet with a study investigator and study coordinator at 
the time consent documents are signed and study procedures initiated.   
 
Once on study medication, subjects will have study evaluations at one-month intervals. These visits should occur 
within ± 7 days of when they are due and are depicted in Table 6.  However subjects will be given 6 week supply 
of study drug at visits 2 through 6 and an 8 week supply at visit 7 to accommodate any unavoidable delays in 
scheduled study visits (section 5.4). Patients are required to stay on drug therapy beyond week 24 until their visit 
8 appointment and associated studies occur. This is essential to accommodate critical end of study data 
collection requirements which will be performed at that visit. 
 
5.5.2 Visit One (Study Entry: Month - 2) 
If subjects meet initial eligibility requirements, informed consent discussion will take place and appropriate 
consents and assents will be signed. Inclusion and exclusion criteria will be checked to make sure that the patient 

 
35 
 
is eligible for the study. Subject’s hemoglobin electrophoresis and any available globin genotyping results must 
be verified and entered into database. Medical and demographic information about the subject including type of 
SCD, current medications including HU use and dosing, number of health care provider contacts for illness (e.g., 
clinic visits, ER visits, hospitalizations), and history of previous SCD complications (e.g. VOC, ACS, invasive 
infection, splenic sequestration) over the preceding 3 years will be gathered from the electronic medical record 
(EMR). For each subject, the number of pain episodes leading to a visit to the hospital or outpatient clinic over 
the 3 years preceding entry into the study will be extracted from the EMR.  Pain episodes per year will be used 
to provide an assessment of disease severity (Platt et al. 1991). A clinical bleeding assessment will be taken 
(see Appendix 2).   A physical examination will be performed.   
 
Blood and urine will be collected for baseline hematology, chemistry and research labs. These labs will include: 
CBC, retic, PT/PTT, chemistries including creatinine and LFTs, fasting lipid panel, hsCRP, lipidomic analysis, 
UA  and female subjects ≥ 9 years of age will receive a urine pregnancy test. 
 
If subject meets all eligibility criteria for entry into the study, the study coordinator will instruct subject on use of 
the i-Pad pain diary and provide them with an iPad mini (Apple Inc). Subjects will be instructed to begin filling 
out the diary each day immediately. At either visit One or Visit Two, a registered dietician or research coordinator 
will administer a validated questionnaire to assess n-3 FA content in the subject’s diet in the average week (see 
Appendix 3) (Lanigan et al. 2012) and content of any nutritional supplements the subject may be taking. QST 
testing will be performed. QST data obtained at this session will not be used as pretreatment baseline, but rather 
to acquaint subjects with the procedure. Pre-treatment baseline QST data will be obtained at Visit Two. The next 
study visit will be scheduled in two months. 
Total volume of blood drawn = 21 milliliters (ml)  
Minimum urine volume = 2 ml 
Estimated visit duration: 2.5 hours 
 
5.5.3 Visit Two, Initiation of Study Drug (Study Day 0, Month 0) 
At Visit 2, the Baseline/Study Initiation Visit, blood will be drawn for CBC, retic, chemistry panel, and female 
subjects ≥ 9 years of age will receive a urine pregnancy test. Research labs will include a baseline lactate 
dehydrogenase (LDH), hsCRP, fetal hemoglobin (HbF), inflammatory cytokines, LTB4, cell activation and 
adhesion markers including L and P selectins, ET-1, VCAM-1, lipidomic analysis, plasma RvD1 and packed 
blood cell membrane AA, EPA and DHA.   Blood will also be collected for analysis of hemostatic markers of 
thrombin generation (F1.2 and d-dimer) as well as CAT assay. Urine will be collected for RvD1, total protein 
random urine with creatinine and urine microalbumin/creatinine ratio. The patient and parent/guardian (if 
applicable) will complete PedsQL TM SCD Module.  Acute care/hospitalization, home pain, and concomitant 
medication information will be updated. Each subject will have a physical exam. A clinical bleeding assessment 
will be performed. At either visit One or Visit Two, a registered dietician or research coordinator will administer a 
validated questionnaire to assess n-3 FA content in the subject’s diet in the average week (see Appendix 3) 
(Lanigan et al. 2012) and content of any nutritional supplements the subject may be taking. Baseline QST testing 
will be performed. The research coordinator will review study procedures with the subject/parent/guardian as 
well as compliance with pain diary.  A 6 week supply of study drug will be dispensed to the subject. 
 
Total volume of blood drawn = 39.5 ml     
Minimum urine volume = 21 ml 
Estimated visit duration: 2 hours 
 
5.5.4 Visit Three (Month 1) 
At Visit 3 the patient will have been receiving the study drug for one month. Baseline safety labs will include: 
CBC, retic, chemistry panel, UA and pregnancy test for female subjects ≥ 9 years of age. Acute 
care/hospitalization, home pain, concomitant medication, clinical bleeding assessment and AE information will 
be collected. Every subject will have a physical exam. Compliance with pain diary will be reviewed. 
Discontinuation criteria will be checked to make sure the patient is still eligible for the study. All unused study 

 
36 
 
drug that was distributed at previous Visit 2 will be counted, and documented.  An additional 6 week supply of 
study drug will be dispensed to the subject. 
 
 
  Total volume of blood drawn = 4 ml   
  Minimum urine volume = 2 ml 
              Estimated visit duration: 1 hour 
 
5.5.5  Visit Four (Month 2) 
At Visit 4 the patient will have been receiving the study drug for two months. Safety assessment labs will be 
done: CBC and retic, chemistry, and fasting lipid panel. Female subjects ≥ 9 years of age will receive a urine 
pregnancy test. Research labs include LDH and RBC membrane studies. Acute care/hospitalization, home pain, 
concomitant medication, clinical bleeding assessment and AE information will be collected. Each subject will 
have a physical exam. Compliance with pain diary will be reviewed. Discontinuation criteria will be checked to 
make sure the patient is still eligible for the study. All unused study drug that was distributed at previous visit will 
be counted, and documented.  An additional 6 week supply of study drug will be dispensed to the subject. 
     Total volume of blood drawn = 9 ml  
     Minimum urine volume = 1 ml 
     Estimated visit duration: 1 hour 
 
5.5.6 Visit Five (Month 3) 
At Visit 5 the patient will have been receiving the study drug for three months. Blood samples will be drawn and 
distributed for local and research laboratory analyses: Safety labs will include: CBC, retic, chemistry panel, UA 
and female subjects ≥ 9 years of age will receive a urine pregnancy test. A physical exam will be performed. 
Compliance with pain diary will be reviewed. Research labs will include a baseline LDH, hsCRP, HbF, 
inflammatory cytokines, LTB4, cell activation and adhesion markers including L and P selectins, ET-1, VCAM-1, 
lipidomic analysis, plasma RvD1 and packed blood cell membrane AA, EPA and DHA.   Blood will also be 
collected for analysis hemostatic markers of thrombin generation (F1.2 and d-dimer) as well as CAT assay. Urine 
will be collected for RvD1, total protein random urine with creatinine and urine microalbumin/creatinine ratio. The 
patient and parent/guardian will complete PedsQL TM SCD Module. QST testing will be performed. Dietary 
assessment will be performed. Acute care/hospitalization, home pain, concomitant medication, clinical bleeding 
assessment and AE information will be collected. Discontinuation criteria will be checked to confirm the patient 
is still eligible for the study.  All unused study drug that was distributed at previous visit will be counted, and 
documented. An additional 6 week supply of study drug will be dispensed to the subject. 
 
 
 
Total blood volume drawn = 39.5 ml   
 
Minimum urine volume = 22 ml 
                  Estimated visit duration: 2 hours 
 
5.5.7 Visit Six (Month 4) 
At Visit 6 the patient will have been receiving the study drug for four months. Safety assessment labs will be 
done CBC/retic, chemistries and female subjects ≥9 years of age will receive a urine pregnancy test. A physical 
exam will be performed. Compliance with pain diary will be reviewed. Acute care/hospitalization, home pain, 
concomitant medication, clinical bleeding assessment and AE information will be collected. Discontinuation 
criteria will be checked to confirm the patient is still eligible for the study.  All unused study drug that was 
distributed at previous visit will be counted, and documented. An additional 6 week supply of study drug will be 
dispensed to the subject. 
 
 
Total volume of blood drawn = 4 ml    
 Minimum urine volume = 1 ml 
 Estimated visit duration: 1 hour 
 
5.5.8 Visit Seven (Month 5) 

 
37 
 
At Visit 7 the patient will have been receiving study drug for five months. Safety assessment labs will be done 
CBC/retic, chemistries, and female subjects ≥ 9 years of age will receive a urine pregnancy test. A physical exam 
will be performed. Compliance with pain diary will be reviewed. Acute care/hospitalization, home pain, 
concomitant medication, clinical bleeding assessment and AE information will be collected. Discontinuation 
criteria will be checked to confirm the patient is still eligible for the study.  All unused study drug that was 
distributed at previous visit will be counted, and documented. An additional 6 week supply of study drug will be 
dispensed to the subject. 
 
Total volume of blood drawn = 4 ml      
Minimum urine volume = 1 ml 
Estimated visit duration: 1 hour 
 
 
5.5.9 Visit Eight (Month 6) 
Visit 8 will be the last evaluation on study drug.  At Visit 8 the patient will have been receiving the study drug for 
6 months. Blood samples will be drawn and distributed for local and research laboratory analyses: Safety labs 
will include: CBC, retic, chemistry panel, UA and female subjects ≥ 9 years of age will receive a urine pregnancy 
test. A physical exam will be performed. Compliance with pain diary will be reviewed. Research labs will include 
a baseline LDH, hsCRP, HbF, inflammatory cytokines, LTB4, cell activation and adhesion markers including L 
and P selectins, ET-1, VCAM-1, lipidomic analysis, plasma RvD1 and packed blood cell membrane AA, EPA 
and DHA.   Blood will also be collected for analysis hemostatic markers of thrombin generation (F1.2 and d-
dimer) as well as CAT assay. Urine will be collected for RvD1, , total protein random urine with creatinine and 
urine microalbumin/creatinine ratio. The patient and parent/guardian (where applicable) will complete PedsQL 
TM SCD Module. QST testing will be performed. Dietary assessment will be performed. Acute care/hospitalization, 
home pain, concomitant medication, clinical bleeding assessment and AE information will be collected. 
Discontinuation criteria will be checked to confirm the patient is still eligible for the study.  All unused study drug 
that was distributed at previous visit will be counted and documented. Any remaining study drug will be collected 
and no further study drug will be dispensed. 
 
Total blood volume drawn = 39.5 ml       
Minimum urine volume = 22 ml 
Estimated visit duration: 2 hours 
 
5.9.10 Visit Nine (Month 7) 
Visit 9 will be the final follow-up visit. At this visit, safety data regarding AEs within 4 weeks of coming off study 
drug will be noted. A physical exam will be performed. Additionally blood will be drawn for the local and research 
laboratory analyses to include: Safety labs: CBC, chemistry panel and UA.  Information about hospitalization, 
pain and concomitant medications will also be noted. 
 
          Total blood volume drawn = 4 ml       
           Minimum urine volume = 1 ml 
           Estimated visit duration: 1 hour 
 
 
Note:  A reminder phone call will be made midway between visits 2 and 3 and between visits 4 and 5 by a study 
team member to each individual subject, related to medication, pain diary, and any interim history. A 
“reinforcement” discussion related to protocol adherence and date of next scheduled appointment will be 
included during the telephone call   
 
Note: At each study visit, subject’s clinical needs will be assessed and addressed by clinical providers.  Adult 
subjects who do not receive their medical care at Nemours will be directed back to their usual providers for 
clinical care.  

 
38 
 
 
Note: If, due to difficulties with venipuncture or issues with specimen quality, all scheduled research labs are not 
able to be obtained at a given study visit, subject will be permitted to remain on study and samples will be drawn 
at next study visit (or sooner if opportunity permits).  If such difficulties occur at study visit 2 prior to initiation of 
study drug, drug initiation may be delayed at discretion of investigator until the baseline research labs can be 
obtained. A notation of any deviation in timing of specimen collection will be made in subject’s CRF.  
 
 
 
6.0 Clinical and Laboratory Evaluations 
 
6.1 Efficacy and Safety Laboratory Evaluations 
Laboratory evaluations (screening safety) will be performed at month -2, as well as QST.  Both safety and 
research labs will be performed at month 0 just prior to initiation of study drug. Safety and research laboratory 
tests will be performed over the time of study as appropriate.  Specific measurement times for each of the tests 
are outlined in Table 6 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
39 
 
Table 6: Study Schedule and Procedures 
 
 
 
6.2 Standard Laboratory Evaluations 
 
6.2.1 CBC  
CBC differential and retic will be performed in the Clinical Laboratories Improvement Amendments (CLIA) 
certified clinical lab at NAIDHC and will be measured at each visit. These evaluations will be to monitor safety, 
to ensure that the hemoglobin level is above the cut-off for exclusion from study.   
 
 
 
H & P 
Visit 1 
Visit 2 
Visit 3 
Visit 4 
Visit 5 
Visit 6 
Visit 7 
Visit 8 
 
Visit 9 
 
Initial Medical History 
X 
 
 
 
 
 
 
 
 
Update medical history 
 
X 
X 
X 
X 
X 
X 
X 
X 
Physical exam 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
BLOODWORK 
 
 
 
 
 
 
 
 
 
CBC/RETIC 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Chemistry panel 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Fasting Lipid Panel 
X 
 
 
X 
 
 
 
 
 
PT/PTT 
X 
 
 
 
 
 
 
 
 
AA, DHA +EPA in packed blood cells 
 
X 
 
X 
X 
 
 
X 
 
Lipodomic analysis with plasma 
X 
X 
 
 
X 
 
 
X 
 
Lipodomic assays with serum 
X 
X 
 
 
X 
 
 
X 
 
Plasma Resolvin (Rv) D1 
 
X 
 
 
X 
 
 
X 
 
hsCRP 
X 
X 
 
 
X 
 
 
X 
 
Inflammatory cytokines and LTB4 
 
X 
 
 
X 
 
 
X 
 
Cell activation/adhesion molecules 
 
X 
 
 
X 
 
 
X 
 
ET-1 
 
X 
 
 
X 
 
 
X 
 
Hemostatic markers and CAT 
 
X 
 
 
X 
 
 
X 
 
LDH 
 
X 
 
X 
X 
 
 
X 
 
Fetal Hemoglobin 
 
X 
 
 
X 
 
 
X 
 
 
Total Blood Volume (ml) 
21 
39.5 
4 
9 
39.5 
4 
4 
39.5 
4 
Clinical Bleeding 
Assessment 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
URINE 
 
 
 
 
 
 
 
 
 
Urinalysis 
X 
 
X 
 
X 
 
 
X 
X 
Urine βHCG (if applicable) 
X 
X 
X 
X 
X 
X 
X 
X 
 
Urine for RvD1 
 
X 
 
 
X 
 
 
X 
 
Urine protein/creatinine 
 
X 
 
 
X 
 
 
X 
 
Urine microalbumin/creatinine 
 
X 
 
 
X 
 
 
X 
 
 
Total Urine Volume (ml) 
2 
21 
2 
1 
22 
1 
1 
22 
1 
ASSESSMENTS 
 
 
 
 
 
 
 
 
 
Pain Days 
X 
X 
X 
X 
X 
X 
X 
X 
 
Quality of Life Survey 
 
X 
 
 
X 
 
 
X 
 
Sensory Testing 
X 
X 
 
 
X 
 
 
X 
 
Dietary assessment 
*Visit 1 or 2 
X * 
X* 
 
 
X 
 
 
X 
 
Follow-up phone calls 
 
X 
 
 
X 
 
 
 
 
Compensation 
$100 
$100 
$100 
$100 
$100 
$100 
$100 
$100 
$250 

 
40 
 
6.2.2 Chemistry Panel  
Chemistry panel will be performed in the CLIA certified clinical lab at NAIDHC. Included in the chemistry panel 
are creatinine, blood glucose and LFTs. All chemistries will be measured initially at Month -2 prior to 
randomization, and subsequently at each monthly visit while on study drug to monitor for potential toxicities. 
Additionally, chemistries will be re-examined at 4 weeks after coming off study drug, to monitor for delayed 
toxicity. Safety monitoring of fasting lipid panel will be performed as per Table 6.  
 
6.2.3 PT and PTT   
A screening PT/PTT will be performed in the CLIA certified clinical lab at NAIDHC. This testing is one of the 
exclusion criteria for the study to ensure that patients are not at excessive risk for bleeding and will be done at 
Month -2. 
 
6.2.4 Urinalysis, total protein random urine with creatinine, urine microalbumin/creatinine ratio and Urine 
pregnancy testing  
UA, total protein random urine with creatinine, urine microalbumin/creatinine ratio and urine pregnancy test will 
be performed in the CLIA certified clinical lab at NAIDHC. UA will be performed at baseline/study entry and 
subsequently at Months 1, 3, and 6 to monitor safety and at 4 weeks after coming off study drug. Urine pregnancy 
test, when appropriate, will be determined prior to randomization at Month -2, and subsequently at each visit to 
determine eligibility for continuation on study drug. Urine for total protein random urine with creatinine and urine 
microalbumin/creatinine ratio will be performed at visits 2, 5 and 7. 
 
6.2.5 Bleeding Assessment 
As an added safety measure, a questionnaire to assess bleeding symptoms (Appendix 2) will be administered 
at baseline and at Study visits 2-7. Any increase in bleeding symptoms as identified on this questionnaire will be 
reported as AEs. 
 
6.2.6 Pain Diary  
Subjects with SCD will be asked to keep a daily pain diary to be initiated at Visit 1, two months prior to initiation 
of study drug and continued until Visit 7, one month post completion of study drug. Apple iPad minis will be 
distributed to the subjects at the time of enrollment and pain diary entries will be entered into the iPad for the 
entire 9 months. Subjects are instructed to log in each day and indicate if they have pain. If they report pain, then 
they are prompted to answer additional questions including  duration and location of pain symptoms, type of 
medication administered, indicate the maximum severity of pain and answer some questions regarding the 
impact of pain on daily functioning.  They must also indicate whether they have taken their study drug that day. 
See Appendix 1 for content of diary.  The diary is designed so that an assessment can occur only between 6:30 
PM and 3 AM each day, after which that day is no longer accessible to the subject. To improve compliance, 
subjects will be compensated $1 per day for filling out the diary.  In addition, participants who complete their 
diary entries at least 70% of the time will be permitted to keep the iPad after they complete the trial. Estimated 
time to complete a daily diary entry “with pain” is approximately 5 minutes and an entry “without” pain should be 
less than 1 minute. Families will not be held financially responsible for lost or stolen iPads. 
 
6.2.7 Varni Pediatric Quality of Life InventoryTM SCD Module  
The PedsQL TM SCD Module Inventory is a brief questionnaire that can be answered by both subject and parent, 
taking only 5 to 10 minutes to complete.  It has been developed for the evaluation of SCD disease-specific 
HRQoL both in the research and clinical settings.  It is a feasible, reliable, and valid tool to measure HRQoL in 
children with SCD (Panepinto et al. 2008; Panepinto et al. 2013).  In the current trial, this inventory will be used 
as one assessment of baseline disease severity and also to assess for changes over the 6 months of treatment 
with study drug.  For young adult subjects over 18 years, the parent report module will be optional for reasons 
of feasibility. Institutional licenses for use of the Varni PedsQL
scales have been purchased by Nemours.  The 
inventory will be administered and scored according to Scoring Instructions and Administrative Guidelines 
provided on the Varni PedsQL
 website. http://www.pedsql.org/ 

 
41 
 
 
6.2.8 Quantitative Sensory Testing:  
A detailed description of QST methods is included in Appendix 4. QST testing will be performed at study entry 
(Month -2), Month 0, Month 3 and Month 6. 
 
6.2.9 Research Laboratory Studies   
Research laboratory methods are detailed in Appendix 5. These investigations will be carried out as noted in 
Table 6. 
 
 
7.0 SAFETY EVALUATIONS AND REPORTING PLAN 
 
7.1 Safety Assessments Overview and by Study Visits 
Subjects will be evaluated every month for the study duration (see Table 7). At each visit, subjects and/or their 
parents will be queried regarding recent medical events or procedures. Specific events will be documented at 
specific visits to ascertain the nature and treatment of the event, including VOC, episodes of ACS, transfusions, 
hospital admissions and other interim illnesses and investigative procedures. These reportable events and 
diagnoses will be followed up by the study staffwho will review medical records document the medical history, 
physical exam and select lab results of a clinical nature in the EHR and these notes will be printed for the 
subject’s study file.  
 
Table 7: SAFETY MONITORING BY STUDY VISIT 
 
 
7.2 Adverse Events 
An AE is defined for the purposes of this study as any untoward medical occurrence, that does not necessarily 
have a causal relationship with treatment, in a subject who is administered a pharmaceutical product. An AE can 
therefore be any unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or 
disease temporally associated with the use of a medicinal product (investigational drug or placebo), whether or 
not related. AE data are recorded on the case report form (CRF). 
 
Examples of an AE include: 
 
Exacerbation of a chronic or intermittent pre-existing condition including either an increase in frequency 
or intensity of the condition. 
 
Significant or unexpected worsening or exacerbation of the condition under study. 
 
A new condition detected or diagnosed after study drug administration even though it may have been 
present prior to the start of the study. 

 
42 
 
 
Signs, symptoms, or clinical sequelae of a suspected overdose of either study drug or a concurrent 
medication (“overdose” per se, should not be reported as an AE). 
 
Pre- or post-treatment events that occur as a result of protocol-mandated procedures (e.g., invasive 
protocol-defined procedures, modification of a subject’s previous drug treatment regimen).  
 
An AE does not include:  
 
Medical or surgical procedures (e.g. tonsillectomy, endoscopy). The medical condition that leads to the 
procedure is an AE. 
 
Social or convenience hospital admissions where an untoward medical occurrence did not occur. 
 
Day to day fluctuations of pre-existing disease or conditions present or detected at the start of the study 
that do not worsen. 
 
7.3 Serious Adverse Events 
A serious adverse event (SAE) is defined as any untoward medical occurrence that at any dose: 
 
 
Results in death. 
 
Is life-threatening (i.e., an event in which the subject was at risk of death at the time of the event; it does 
not refer to an event which hypothetically might have caused death if it were more severe). 
 
Requires hospitalization or prolongation of existing hospitalization. 
 
Results in persistent or significant disability/incapacity. 
 
Results in a congenital anomaly birth defect. 
 
In the opinion of the investigator, important medical events that may not be immediately life-threatening 
or result in death or hospitalization but may jeopardize the subject or may require intervention to prevent 
one of the other outcomes listed in the definition above may be considered serious. Examples of such 
events are intensive treatment in an ER or at home for allergic bronchospasm, blood dyscrasias or 
convulsions that do not result in hospitalization; or development of drug dependency or drug abuse. 
 
An unexpected AE or SAE refers to an event that is not listed in the investigator’s brochure (IB), or if 
it is listed in the brochure, is not listed at the observed specificity or severity.  Unexpected, as used in this 
definition, also refers to adverse events or suspected adverse reactions that the IB mentions as occurring with 
a class of drugs or is consistent with the pharmacological properties of the drug but is not specifically mentioned 
as occurring with the particular drug under investigation 
 
Anticipated events include known consequences of the underlying disease or condition under investigation 
(e.g., symptoms, disease progression, comorbidities) as well as events unlikely to be related to the underlying 
disease or condition under investigation but common in the study population independent of drug therapy. 
 
Although common side effects associated with other drugs in the same class as SCD-OmegatexTM  (ie other n-
3 FA ethyl ester products) are well known and outlined in the Investigator’s Brochure (IB)  accompanying this 
protocol, SCD-OmegatexTM  has not been used previously in human subjects. Therefore as defined by FDA 
guidelines, all AEs will necessarily be classified as unexpected. As the trial proceeds and experience is gained 
with the investigational product, the IB will be updated as appropriate, at least on an annual basis. However, 
since all subjects enrolled in this study have SCD, certain AEs will occur related to the disease process and this 
should be taken into account when determining whether or not an event is related to the therapy. Table 8 lists 
common SCD related events. Events on this list can still be related to therapy if the PI determines that study 
drug may have triggered the event, made it more severe or occur with unexpectedly high frequency. For painful 
vasoocclusive events, an AE will be defined as a pain episode resulting in an acute/urgent care visit or 
hospitalization, or a pain episode that is managed at home that results in a significant change in daily routine 
such as a missed school or work day. 
 

 
43 
 
Particularly close attention will be given to any AEs (even those known to be associated with SCD) that relate to 
bleeding such as hemorrhagic stroke, retinal hemorrhage or hematuria. These will be carefully examined for 
causation. 
 
 
 
 
Table 8: List of Adverse Events Related to Sickle Cell Disease 
 
Acute chest syndrome 
Anemia 
Aplastic crisis 
Aplastic crisis/anemia 
Arthralgia 
Avascular necrosis of joint 
Bone infarction 
Cardiomegaly 
Cerebrovascular accident 
Cholecystitis,  
Hepatic sequestration 
Cranial nerve palsy 
Decreased kidney function 
Decreased lung function 
Delayed growth/puberty 
Dyspepsia 
Elevated urinary urobilinogen 
Fever 
Hand-foot syndrome 
Hematuria 
Hemiplegia 
Hemolysis 
Hepatosplenomegaly 
Headache 
Hyperplastic bone marrow 
Hyposthenuria 
Hypoxemia (PO2 < 65mm Hg) 
Infection 
Jaundice 
Leukocytosis 
Meningitis 
Pain Crisis 
Pain, joint 
Pain, long bone 
Pain, severe abdominal 
Priapism 
Pulmonary embolism 
Pulmonary hypertension 
Pulmonary parenchymal infiltrates 
on chest x-ray 
Pyelonephritis 
Renal failure 
Renal insufficiency/albuminuria 
Renal papillary necrosis 
Reticulocytosis  
Retinal Disease 
Retinal hemorrhage 
Rhabdomyolysis 
Sepsis 
Skin ulcers 
Splenic sequestration 
Stroke 
Vaso-Occlusive Crisis 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
44 
 
 
 
 
Table 9:  Overview of Definitions of Safety Measures 
 
 
Word or Phrase and (Abbreviation) 
 
 
Any untoward clinical or medical occurrence. 
Adverse event (AE) 
Any untoward clinical or medical occurrence. 
 
 
Serious adverse event (SAE) 
Any adverse event that results in any of the following outcomes: 
Death, a life-threatening adverse event, inpatient hospitalization 
or prolongation of existing hospitalization, a persistent or 
significant disability/incapacity, or a congenital anomaly/birth 
defect. 
 
Anticipated SAE 
An anticipated SAE for subjects in this study is an adverse event 
that is listed in Table 8 and is a SAE, as defined above. 
 
Unanticipated SAE 
An unanticipated SAE for subjects in this study is any AE that is 
not listed in Table 8 and is a serious adverse event, as defined 
above. 
 
Adverse Clinical Laboratory Results 
Any safety laboratory measurement that is measured beyond the 
acceptable limits (either high or low) for the population being 
studied, as described by the investigator. 
 
Suspected Adverse Reaction 
 
Any adverse event for which there is a reasonable possibility 
that the study drug caused the adverse event 
 
Unexpected 
Suspected 
Adverse 
Reaction 
A suspected adverse reaction is considered ‘‘unexpected’’ if 
it is not listed in the investigator brochure or is not listed at 
the specificity or severity that has been observed.  
 
Serious and Unexpected Suspected 
Adverse Reaction (SUSAR) 
A suspected adverse reaction that is both serious and 
unexpected; an unexpected SAE reasonably possibly 
related to study drug 
 
 
7.4 Assessment of AE Severity, Relationship to Treatment, and Expectedness 
The principal investigator or co-investigators should the PI be unavailable will  grade the severity of identified 
AEs, which is based on their impact on the patient and will also determine the relationship of the event to the 
treatment. Where applicable, severity will be assessed on the basis of CTCAE version 4.03. The following scale 
will be used to "grade" the severity of all AEs: 
1. Mild. Awareness of sign, symptom, or event, but easily tolerated; does not interfere with usual daily 
activities or tasks. 
2. Moderate. Discomfort enough to cause interference with usual daily activity; may warrant therapeutic 
intervention. 
3. Severe. Incapacitating; inability to perform usual activities and daily tasks; significantly affects clinical 
status; requires therapeutic intervention. 
4.   Life-threatening. AE is life-threatening. 

 
45 
 
5.   Death. AE causes death. 
The standard nomenclature we will use for defining the causal relationship between an AE and the study drug is 
listed in Table 10. The category that overall best "fits" the relationship between the AE and the study drug will 
be chosen and recorded in the EMR, REDCapTM and MedWatch form as necessary. 
 
Table 10:  Relationship between Treatment and AE 
Unrelated 
• No temporal association to study product. 
• An alternate etiology has been established or event is consistent with sequelae of 
sickle cell disease (see Table 8). 
• The event does not follow the known pattern of response to study product. 
• The event does not reappear or worsen with rechallenge. 
Probably not 
related / remote 
• No temporal association to study product. 
• Event could readily be produced by clinical state, environmental or other 
interventions. 
• The event does not follow the known pattern of response to study product. 
• The event does not reappear or worsen with rechallenge. 
 
Possibly Related 
• Reasonable temporal relationship to study product. 
• The event is not readily produced by clinical state, environmental, or other 
interventions. 
• The event follows a known pattern of response to the study product or as yet 
unknown pattern of response. 
Probably related 
• There is a reasonable temporal association with the study product. 
• The event is not readily produced by clinical state, environmental, or other 
interventions. 
• The event follows a known pattern of response to the study product. 
• The event decreases with de-challenge. 
Definitely related  
 
• There is a reasonable temporal relationship to the study product. 
• The event is not readily produced by clinical state, environmental, or other 
interventions. 
• The event follows a known pattern of response to the study product. 
• The event decreases with de-challenge and recurs with re-challenge. 
 
 
7.5 Monitoring and Outcome of AEs 
Every AE must be followed to a satisfactory outcome or stabilization of the event, even when this requires a time 
period beyond the scope of the study (this is particularly applicable to SAEs). Outcome includes information on 
recovery and any sequelae, as well as specific tests and/or treatments that may have been required and their 
results. All AEs will be collected after the subject signs informed consent and will continue for 30 days after the 
last dose of study drug. For a fatal outcome, cause of death and a comment on its possible relationship to the 
suspected reaction should be provided. 
The terms used to define outcome are as follows (outcome of reaction/event at the time of last observation): 
• Ongoing 
• Resolved without sequelae 
• Resolved with sequelae 

 
46 
 
• Death 
 
Actions taken in response to an AE and follow-up results must be recorded in the subject’s medical record (this 
includes follow-up laboratory results). Any treatment administered for the AE must be recorded in the subject’s 
CRF. When subjects are discontinued from the study due to an AE, relevant clinical assessments and laboratory 
tests will be repeated as necessary until final resolution or stabilization occurs. 
 
SAE reporting will begin with events that arise after the informed consent is signed until 30 days after the last 
dose of study drug. 
 
 
7.6 Unexpected SAEs 
Within 24 hours of the realization that an unexpected SAE has occurred to a study subject, study personnel must 
notify the PI (or physician co-investigator if PI is not readily available), who will give an initial report to the 
Institutional IRB and the Medical Monitor. 
This initial report should describe the event as fully as possible and should include the following minimum 
information: an identifiable subject; study product; an identifiable reporting source; and an event or outcome that 
can be identified as serious. Supporting documentation (e.g., CRF pages, lab reports, summary notes, autopsy 
reports) should accompany the report as appropriate.  The PI, study coordinator and the Medical Monitor will 
collaborate to prepare a detailed report of the unexpected SAE.   
The Nemours IRB must be notified within 24 hours if an SAE involves death of a subject. Otherwise all 
unexpected SAEs judged by the PI or co-investigators if the PI is unavailable as possibly, probably or definitely 
related to treatment will be reported to the Nemours IRB within 5 days. AEs judged as possibly, probably or 
definitely related to treatment occurring with an unexpected increase in severity or frequency that represent an 
increased risk of harm will be similarly reported within 5 days. Unexpected fatal or life-threatening AEs possibly, 
probably or definitely related to the intervention will be reported to the NIH/NIGMS and the FDA within 7 days. 
Other serious and unexpected AEs possibly, probably or definitely related to the intervention will be reported to 
the FDA and NIH/NIGMS within 15 days. The exception will be unexpected SAEs which are anticipated as part 
of the SCD disease process (Table 8) and deemed by the PI to be unlikely related to the study drug.  These will 
be reported to the Medical Monitor and Nemours IRB, but will not be reported to NIH/NIGMS or the FDA in an 
expedited fashion.  The PI is responsible for submitting to the NIH/NIGMS and FDA any recommendation of the 
Medical Monitor to suspend or terminate the study. All SAEs, regardless of expected status, are also recorded 
in the AE section of the study’s CRF. The PI will follow the progress of a subject who experiences an unexpected 
SAE until the SAE is resolved or considered stable. When the unexpected SAE has not resolved by the report 
deadline, the PI will make follow-up reports in accordance with directions from the Medical Monitor and/or the 
Nemours IRB.  Note: The PI for this study functioning as Sponsor/Investigator. 
 
7.6.1 Expected SAEs 
Study personnel will report all expected SAEs within 3 business days to the PI (or physician co-investigator if PI 
is not readily available), and Medical Monitor. The PI and the Medical Monitor will collaborate to prepare a report 
of the expected SAE.  
 
7.6.2 Review of Serious Adverse Events 
Three months after the first subject is enrolled in the study, and at the end of each 3-month period thereafter, if 
any SAEs (expected or unexpected) have been reported in the study during the preceding 3 months, the Medical 
Monitor and the PI will meet by phone to review. The PI in collaboration with the Medical Monitor will prepare a 
semi-annual report to the NIGMS Project Officer.. 
 
7.6.3 Subject Discontinuation due to AE(s) 
The following criteria will be used to determine whether or not subjects exhibit toxicities of the study drug(s) 
sufficient to require discontinuation from the study. 

 
47 
 
We will closely monitor signs, symptoms, and laboratory findings to assess for unexpected toxicities (detailed in 
section 4.7.2). Treatment will be discontinued in any subject who experiences the following: 
1. Acute allergic reaction 
2. Serious bleeding manifestations 
3. Changes in LFTs or creatinine that do not return to baseline on treatment discontinuation or returns       
to abnormal values on re-challenge 
4. Pregnancy 
5. Drop in platelet count below 100,000 that fails to recover within 2 weeks or recurs on rechallenge. 
 
Treatment may also be discontinued at the discretion of the PI in the event of any other serious or unanticipated 
side effects. 
 
7.7 Reporting Requirements:  
Nemours IRB 
The Nemours IRB must be notified within 24 hours of an unforeseen death of a subject. All unexpected SAEs 
judged by the PI, or co-investigators if the PI is unavailable, as possibly, probably, or definitely related to 
treatment will be reported to the Nemours IRB within 5 days as these are assumed to represent an unanticipated 
problem involving risk to participants. AEs judged as possibly, probably, or definitely related to treatment 
occurring with an unexpected increase in severity or frequency that represent an increased risk of harm will be 
similarly reported within 5 days. 
External Agencies 
Unexpected fatal or life-threatening AE/SAEs possibly, probably, or definitely related to the study intervention 
will be reported to the NIH/NIGMS within 7 days. As the study intervention involves a drug these events likely 
also meet the definition of an Unexpected fatal or life-threatening suspected adverse drug reactions, they also 
will be reported to the FDA within 7 days. Other unexpected SAEs possibly, probably, or definitely related to the 
intervention will be reported to NIH/NIGMS within 15 days. As these events also meet the definition of Serious 
unexpected suspected adverse drug reactions, they will also be reported to the FDA within 15 days.   
 
7.7.1 Reporting of Serious Safety Issues; Suspension Guidelines 
Serious safety issues that arise in this study will be brought to the attention of PI and Medical Monitor, who will 
make recommendations to the IRB regarding possible suspension or termination of the study. The PI also will 
notify the NIH/NIGMS (study funder) and the FDA if the study is temporarily suspended or terminated. The NIH 
and FDA will consider the recommendations, determine an appropriate action, and notify the PI who will 
implement the actions directed by NIH and/or FDA.  
 
 7.8 Pregnancy Reporting 
Because of potential unknown effects on the fetus, pregnant subjects will be excluded from the study. Female 
subjects who are pubertal will be regularly assessed throughout the study. In the event of a test indicating the 
study subject is pregnant the subject will be informed of this result and will immediately have study drug 
discontinued.  If female participants acknowledge sexual activity they will be informed that they need to use a 
reliable form of birth control for the duration of the study. 
 
7.9 Monitoring of Subject Accrual and Compliance 
The PI will assess the overall enrollment and compliance with the clinical protocol. The monitoring will focus on 
data. All protocol violations/deviations will be documented, and will include a description of the event and the 
corrective action plan. 
 
7.10 Data Collection and Monitoring 
7.10.1 Case Report Form (CRF) and Source Documentation 
The site study coordinator will complete a CRF or eCRF for each subject. On all other study documents except 
for the study visit exams which will be documented in the EMR, subjects will be identified by a study subject 

 
48 
 
number assigned at enrollment, and subject will not be identified by name. Data will be entered into a REDcapTM 
database.  Some source documents such as QoL and dietary assessment will be directly downloaded into 
REDcapTM   via a secure web access. Otherwise data entry into REDcapTM will be done by study team members 
only. 
 
 
7.10.2 Data Management   
The REDcapTM   database will be structured to simplify data entry with rules for restricting data to valid response 
and ranges (e.g., no negative height values allowed). Random subsets of subjects will be audited periodically 
for data quality and completeness. If data quality and completeness problems are detected, the audit will be 
expanded and the entry process will be revised as necessary to avoid the detected problems.   
Validation rules will be applied at several points in the data management process. An error correction procedure 
will be applied to correct data values that fail validation rules. 
 
 
7.10.3 Staff Training and Data Monitoring 
Prior to the onset of enrollment, study personnel will be trained to ensure adherence to the protocol and assure 
the highest possible data quality. Training will address informed consent procedures, study operations and 
protocol requirements, maintenance of source documentation, CRF completion and review, routine reporting 
requirements, and data management. Coordinators will have the responsibility of monitoring CRFs and source 
documents for accuracy, protocol compliance, subject safety, and adherence to guidelines. As the study 
progresses, informed consents and completed data forms will be reviewed periodically by the PI and compared 
to source documentation (medical records) to confirm accuracy. 
 
8.0 Statistical Considerations  
 
8.1 Data Analyses:  
 
8.1.1 General Considerations  
Discrete variables will be summarized with frequency counts and percentages. Any continuous variables will be 
summarized with means and SDs unless there are variables not approximately normally distributed, in which 
cases we will summarize them with their medians and first and third quartiles. This data will be tabled and plotted 
to guide decisions on the adequacy of statistical modeling and testing assumptions. All efficacy analysis will be 
performed on subjects with pre-treatment and post-treatment data.  That is, subjects who drop out will be 
excluded from efficacy analysis.  The primary analysis of each outcome will be performed without regard to 
patient adherence.  Secondary analysis will exclude non- adherent patients.  Rates of AEs will be estimated for 
all treated patients.  All effect measures estimates for efficacy and safety outcomes will be estimated with 
appropriate confidence limits. SAS version 9.4 (Cary, NC) will be used for all statistical analyses.  
 
8.1.2 Primary Efficacy End Point   
The Phase II portion of the Trial is designed to provide preliminary evidence of efficacy in reducing thermal 
sensitivity by QST. For each subject at each time, two technical replicate measurements (i.e., duplicates) will be 
taken for each QST modality (i.e., CS, CP, HS, and HP). A QST thermal composite score (TCS) measure will 
then be evaluated in these data. It will be based on an aggregation of the absolute values of temperature 
deviations from the instrument’s 32oC baseline level (d’s) across all eight of the QST measurements at a given 
visit (i.e., higher values indicating lower sensitivity). A crude estimate of the TCS for the jth participant at the kth 
visit would be the mean of the measured QST’s at that visit, 
 
TCS𝑗𝑘= 1
8 ∑∑𝑑𝑗𝑘𝑖𝑟
2
𝑟=1
4
𝑖=1
   
 

 
49 
 
where i is an index for the 4 QST modalities, r is an index for the duplicates, and djkir is the thermal sensitivity 
deviate measurement. For instance, suppose that for the fifth participant, at the second visit, their heat sensitivity 
(say, i = 3) first duplicate measurement is 25.3oC. Then the d5231 = | 25.3oC - 32oC | = 4.7oC.   
 
Changes in these d’s over time will be modeled using mixed effects linear regression adjusted for modality.  We 
will treat time as a categorical variable.  Correlation among repeated measurements will be modeled using a 
compound symmetric (i.e., random intercept) or first-order autoregressive within-subject covariance 
structure.  We will examine the distribution of the residuals to assess the normality assumption, and will apply 
appropriate transformations as needed.  Within this model, we will directly estimate parameters for the TCS 
changes from baseline to all follow-up visit times (after 3 and 6 months of treatment). These results will include 
an estimate and a two-sided 0.05-level Wald’s test of the primary hypothesis of reductions in thermal sensitivity 
from baseline to 6 months post treatment.  We will also evaluate, in these models, potential confounders 
identified in summary or exploratory analyses and adjust the primary endpoint model for confounding if it 
becomes a concern.    
 
  
8.1.3 Secondary End Points 
 (i) QoL: For the Varni Pediatric Quality of Life InventoryTM Sickle Cell Disease Module, pre- and post-treatment 
values will be summarized using the median and interquartile range.  Significance of change in QOL scores will 
be evaluated using the Wilcoxon signed rank test.  We will evaluate association of change in QoL with change 
in thermal sensitivity and RVD1 using Spearman rank correlation coefficients. 
(ii) The number of pain days per month will be modeled using Poisson regression within a generalized estimating 
equations framework to account for correlation among the repeated measurements.   
(iii) Reductions in thermal sensitivity by individual QST parameter measures will be treated and modeled, 
respectively, using the same mixed modeling approach described above for the primary endpoint analysis.  
 
8.1.4 Exploratory Analyses 
(i) Changes in  biomarkers, adhesion molecules and lipid metabolites: We will consider changes in biomarkers, 
adhesion molecules and lipid metabolites  using two approaches.  First, we will perform paired t-tests for each 
outcome separately.  Second, a longitudinal mixed effects regression modeling approach will be utilized on these 
biomarker data similarly as for the primary endpoint of thermal sensitivity.     
(ii) hsCRP: Values of hsCRP will be log transformed prior to analysis.  Efficacy will be measured by calculating 
the average change in transformed hsCRP values (post minus pretreatment) and reverse transforming this mean 
change to obtain the Geometric Mean Ratio (GMR).  Statistical significance will be evaluated using a one-sided 
paired t-test with alpha=0.05.  The null hypothesis is that there is no change (GMR=1).  The alternate hypothesis 
is that post treatment hsCRP values are not equal to (lower than) pretreatment values (GMR<1). Further analysis 
of hsCRP will include longitudinal mixed effects regression modeling similarly as for the primary endpoint.  
(iii) CAT: Changes in CAT parameters (endogenous thrombin potential or ETP, Lag Time, Peak Thrombin, Time 
to Peak, and start tail) will be evaluated by paired t-tests separately. Depending on the findings, we may further 
model these data in a manner similar to the latent process modeling described for changes in biomarkers. 
 
8.1.5 Interim Analysis 
 No interim analyses will be performed in this study. 
 
9.0 Human Subjects Protection 
 
9.1 Clinical Trial Monitoring 
Independent from our study team, we will have a clinical monitor, external to the study, to review study for 
adherence to regulatory requirement, Good Clinical Practice (GCPs) and the protocol. The clinical monitor will 
assure that data reporting, including safety reporting, is accurate and complete and that the rights and well-being 
of human subjects are protected. 

 
50 
 
9.2 Ethics 
9.2.1 Good Clinical Practice and IRB Review 
Compliance with GCP guidelines for the conduct and monitoring of this clinical trial will occur through observation 
of the ethical and regulatory requirements presented in ICH E6, Good Clinical Practice: Consolidated Guideline. 
By signing this protocol, the investigator agrees to adhere to these requirements. The study (protocol and 
informed consent) will be reviewed and approved by the IRB at NAIDHC.  All changes to the protocol will be 
approved by the Nemours IRB and will also be reported to the NIH/NIGMS Program Director and the FDA. 
Subjects must sign written informed consent and (when appropriate) assent prior to being screened and before 
undergoing any study procedures.  
 
9.2.2 Informed Consent 
Informed consent is an ongoing process that includes the signing of an informed consent document. The 
protocol, parental permission form and assent forms will be submitted for approval by the Nemours IRB for full 
review and approval obtained prior to initiating this study. If it is necessary to amend the protocol and/or consent 
forms those items will be submitted to the IRB prior to implementation. All staff involved in the study will have 
completed formal coursework in the protection of human subjects. Study staff will obtain written informed 
consent, parental consent and age-appropriate child assent prior to conducting any research procedures in 
accordance with ICH E6; 4.8, “Informed Consent of Trial Subjects.” The informed consent form will adhere to the 
guidelines in ICHE6: it will contain the elements as specified in section 4.8.10 of that guideline which can be 
modified to include local IRB required language. Potential subjects will have ample opportunity to ask questions 
prior to signing consent. They will be informed that they are free to opt out of the study at any time and that 
declining participation will not in any way influence the care they will receive at NIADHC.  One copy of the signed 
consent will be given to the subject and one will be kept in the study files for documentation.  Signed consent 
will also be scanned into the EMR. Subjects will also be provided with the telephone numbers of study personnel 
who can assist with their questions and concerns. 
 
9.2.3 Data Safety and Confidentiality 
Subject confidentiality will be maintained by the investigator, the investigator’s associates and co-workers, and 
by all administrators who are part of the project. Confidentiality will be maintained according to ICH E6; 4.8.10, 
Part O: “Records identifying the subject will be kept confidential and, to the extent permitted by the applicable 
laws and/or regulations, will not be made publicly available. If the results of the trial are published, the subject’s 
identity will remain confidential.” All study participants will be assigned a unique study identification number, 
which will be used on all study documents instead of their name or other identifying information. This number 
will be used in all data collection and tabulation. Study participants’ protected health information will be linked 
with their study identification number in a separate computer database that will be password protected. All study 
computers and databases will be password protected, and study documents will be kept in locked filing cabinets 
or computers in the offices of project staff at NAIDHC.  Study visit exams will be documented in the EMR.  
 
9.2.4 Disclosure of Data 
The PI or her staff and associates, and the appropriate regulatory agencies may use the information included in 
this protocol as necessary for the conduct of the trial and the safety of subjects. Data from the trial are confidential 
and may not be disclosed without the written permission of the NIH. 
 
9.2.5 Publication of Research Findings 
Manuscript(s) and abstract(s) prepared from the data collected during this trial can be prepared by the study 
investigators with the subjects’ identity remaining confidential. 
 
9.3 Discontinuation of Study 
Safety will be monitored by the PI, Medical Monitor and Nemours IRB, who can recommend suspension or 
termination of the trial for safety reasons at any time. The PI, Medical Monitor and IRB will receive expedited 
reports for deaths or unexpected SAEs and regular summaries as described earlier under AEs. Particular 
attention will be paid to specific SAEs such as death of a participant, stroke or cerebral hemorrhage, and any 

 
51 
 
grade 3-4 bleeding events. Subjects can discontinue for any reason at any time during the trial, with no 
consequences to the quality of their treatment.  This study will be stopped prior to its completion if: 1) the 
intervention is associated with AEs that call into question the safety of the study drug, 2) difficulty in study 
recruitment or retention will significant impact the ability to evaluate the study endpoints, 3) any new information 
becomes available during the trial that necessitates stopping the trial; or 4) other situations occur that might 
warrant stopping the trial.  
 
10.0 Subject Compensation 
 
Subjects will be compensated at the time of each study visit as reimbursement for their time and for parking fees. 
For visits 1 through 8, subjects will receive $25 to cover transportation expenses and $75 compensation. At visit 
9, subjects who remain in the study and have completed all study visits will receive $250.  In addition, subjects 
will receive $1 for each daily entry into their pain diary.  Subjects who are compliant with pain diary entries at 
least 70% of the time will be permitted to keep their iPad mini at the conclusion of the study.  
 
Bill S.139 Ensuring Access to Clinical Trials Act of 2015 permanently allows an exclusion under the Supplemental 
Security Income program and the Medicaid program for compensation provided to individuals who participate in 
clinical trials for rare diseases or conditions of which SCD is included.  Therefore, compensation up to $2000.00 
annually, in this clinical trial, will not negatively impact the SSI and Medicaid Income received by study 
participants. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
52 
 
 
Appendix 1: Sickle Cell Pain Survey 
 
Participant ID: 
Today’s Date: ________________ 
 
Did you take your study medication today?     Yes       No 
 
Did you have any pain today:        Yes       No  
If yes, please continue. 
If no, you are finished and don’t have to complete any more questions. 
 
About how many hours did you have pain since the last time you filled out diary?  (Circle a single number on the 
line.) 
 
 
If you circled 0 or one, you are finished and don’t have to complete any more questions 
 
How bad was the pain at it’s very worst? (Circle a single number on the line.) 
 
 
Please color in all of the body parts where you felt pain. 
 
 

 
53 
 
 
Did you take any medicine for your pain?       Yes       No 
 
If you took medicine for your pain, what medications did you take for your pain (check all that you took) 
  Tylenol (acetaminophen) 
  Ibuprofen (motrin) 
  Tylenol with codeine 
  Percocet  
  Hycet 
  Oxycodone 
  Morphine 
  Dilaudid 
 
Was today a school day?          Yes       No 
If yes,  did you miss school today because you had pain?      Yes       No 
 
Did you have trouble sleeping last night?     Yes       No 
 
If yes, was it because of pain?          Yes       No 
 
 
 
 

 
54 
 
Appendix 2: Bleeding Questionnaire   
 
Please fill in Yes or No to each of the following questions: 
 
Symptom 
Yes 
No 
Bruising 
 
 
<1cm 
 
 
 
2-5cm 
 
 
>5cm 
 
 
Spontaneous 
 
 
Minimal Trauma 
 
 
Oral Cavity Bleeding 
 
 
Spontaneous 
 
 
After Brushing 
 
 
Nose Bleeds 
 
 
1-5 min 
 
 
>5 min 
 
 
Medical Attention 
 
 
Bleeding from Minor Cuts 
 
 
Medical Attention 
 
 
GI Bleeding 
 
 
Medical Attention 
 
 
Increase in Average Duration of Menstruation 
 
 
 
 
 
 
 
 
Note:  All areas of bruising are to be marked on body map 
 
 

 
55 
 
Body Map: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
56 
 
Appendix 3: Dietary Questionnaire 
 
 

 
57 
 
 

 
58 
 
 

 
59 
 
 

 
60 
 
 

 
61 
 
 

 
62 
 
 

 
63 
 
 

 
64 
 
 

 
65 
 
Appendix 4: Quantitative Sensory Testing Methods   
 
Testing will take place in a dedicated, quiet room with controlled air temperature (68-72 oC).  Testing will be 
performed without a parent, guardian or observer in the room as presence of mother during testing has been 
shown to influence results (Zohsel et al. 2006). However, in the presence of the subject, parent/guardian will be 
permitted to undergo a test trial of the planned thermal and mechanical stimuli to reduce concern that child will 
experience unacceptable discomfort.  Parent/guardian will be permitted to observe testing procedures via video 
monitoring from an adjacent room.  Subjects will be given 10 minutes to acclimate to the room temperature. 
Testing will be performed by a trained research team member who will provide very clear and concise instructions 
on testing procedures to subjects.  Subjects will be seated in a comfortable chair, and positioned so they cannot 
see monitors.   
 
Thermal sensory equipment has been purchased from, Medoc U.S.A, Compass Medical Technologies, Inc. 
 
Neurosensory Analyzer Model TSA-II 
 
The TSA II is a computerized device designed for both clinical and advanced research applications of the 
quantitative assessment of small nerve fiber dysfunctions. It measures sensory thresholds such as the sensation 
of warmth, cold, heat-induced pain and cold-induced pain. This device has FDA clearance via a 510K.  The TSA 
II measures thresholds for four sensory sub-modalities: 
 
 
Warm sensation (WS), a C fiber mediated sensation. 
 
Cold sensation (CS), mediated by A-delta fibers. 
 
Heat induced pain (HP),  a mostly C fiber mediated sensation, with some involvement of A-delta fibers. 
 
Cold induced pain (CP),  at about 10°C. Mediated by a combination of both C and A-delta fibers. 
 
 
 
Thermal testing: 
Thermal testing will be performed using the TSA II Neurosensory Analyzer TSA-II (Medoc Ltd, Ramat Yishai, 
Israel) at non-tissue damaging temperatures using Peltier-type contact thermal stimulators. Testing sites will be 
the volar surface of the forearm and the thenar eminence bilaterally.  These sites were chosen based on previous 
studies which have consistently shown that lower extremity thresholds are consistently higher than upper (Meier 
et al. 2001).  A 9 cm2  thermode will be used.  The baseline temperature will be 32 oC with stimulus range between 
0-50 oC. Thermal thresholds will be determined using the Method of Limits where subjects are instructed to press 
a button or tell the tester to stop when the sensation in question is first perceived. This method has been used 
in other pediatric QST studies in SCD (Brandow et al. 2013; O'Leary et al. 2014) as well as adult SCD studies 
(Campbell et al. 2016) and requires the shortest testing duration of all methods (desirable for a pediatric study). 
The order of testing will be as follows (Brandow et al. 2013; Heldestad et al. 2010): 
1) Cold detection threshold 
2) Warm detection threshold 
3) Cold pain threshold 
4) Heat pain threshold 
5) Heat pain tolerance 
Cold and Heat Detection Thresholds: 
The probe temperatures will be increased/decreased from baseline at increments of 0.5 oC /sec Subject will be 
asked to press a button or acknowledge verbally when they first perceive a sensation of heat or cold. This will 
be defined as the cold/heat detection threshold.  Temperature will then be returned to baseline.. This will be 
repeated twice at each site with an interstimulus interval designated by the participant to signal when they are 
ready for a following stimulus.  
 
Cold and Heat Pain Thresholds: 

 
66 
 
The probe temperatures will be increased/decreased from baseline at increments of 0.5 oC. Subject will be asked 
to press a button when they first perceive the stimulus to produce pain. This will be defined as the cold/heat pain 
threshold. This will be repeated twice at each site with an interstimulus interval designated by the participant. 
 
Heat Pain Tolerance: 
The probe temperatures will be increased from baseline at increments of 0.5 oC. Subject will be asked to press 
a button when they first perceive the stimulus to produce pain which they find to be intolerable. This will be 
defined as the heat pain tolerance threshold. Temperature will then be returned to baseline.. This will be repeated 
once on each forearm with an interstimulus interval designated by the participant.  Fifteen seconds after they 
press the button to stop the test, subjects will be asked to rate the pain that they still feel at that time on a scale 
of 1 to 10 to test for after sensations. 
 
 
Pain perception and Anxiety 
Unpleasantness and anticipatory anxiety will be assessed using a 10 point scale , with 0 indicating the lowest 
level and 10 indicating the highest level.  Before testing, subjects will be instructed on use of the scale. Prior to 
each testing session, each participant will be asked to identify using the scale “how nervous, afraid or worried” 
they are about the upcoming task to indicate level of anxiety.   For “unpleasantness”, subjects will be asked at 
the end of the testing session how unpleasant the experience was for them.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
67 
 
Appendix 5: Research Laboratory Methods 
 
Assay of Docosahexaenoic Acid (DHA), Eicosapentaenoic Acid (EPA) and Arachidonic Acid (AA) in 
Circulating Blood Cells to Monitor Study Drug Compliance:   
Omega-3 index (O3I), defined as the sum of EPA and DHA as a percentage of total FAs, has been used clinically 
to monitor the beneficial effects of dietary supplements of n-3 FAs or the diets enriched in these FAs (Harris 
2008; Superko et al. 2013). To assess the intake of these FAs, the levels of EPA and DHA have been measured 
in phospholipids or total lipids from several blood fractions and tissues including adipose tissue, plasma, serum, 
whole blood, platelets and erythrocytes (Bell et al. 2010; Katan et al. 1997; Marangoni et al. 1993).  Measurement 
of n-3 FAs in the erythrocyte membranes, however, has been regarded as the gold standard, since erythrocytes 
have a long lifespan of approximately 120 days which could reflect long term intake of n-3 FAs (Harris and Von 
Schacky 2004; Poppitt et al. 2005).  In contrast to erythrocytes, other blood cells are short lived with a half life of 
~10 days for platelets and a few hours for WBCs.  RBCs from subjects with SCD are also short lived with an 
average lifespan of less than 20 days and are fragile (McCurdy and Sherman 1978).  In addition, measurement 
of n-3FAs in the erythrocyte phospholipids involves the isolation of erythrocytes from whole blood and separation 
of phospholipid fraction from other lipid components.  Because of the fragile nature, older sickle RBCs could be 
lost during in vitro manipulation resulting in a non-representative and skewed red cell population.  While washed 
red cell preparation involves multiple washing/centrifugation steps, packed total blood cells can be obtained with 
minimal in vitro manipulation of whole blood with a single centrifugation step.  Moreover, packed total blood cells 
are mainly erythrocytes with minimal number of WBCs and platelets.  To test whether packed total blood cells 
could be used to assess O3I in our clinical trial patients (so that their blood samples would be minimally subjected 
to in vitro manipulation), in preliminary experiments using methods detailed below and blood from control 
volunteers, we measured EPA, DHA and AA levels in packed total blood cells, determined their O3I index values 
and FA ratios (between AA and EPA plus DHA), and compared the values with those generated with erythrocyte 
phospholipids from the same donors.  Preliminary results from a representative control presented in the Table 
below demonstrate that levels of AA, DHA, EPA, O3I index, and fatty acid ratios measured in packed total blood 
cells and erythrocyte phospholipids are similar (Table-1).  These results demonstrate that packed total blood 
cells can be used to monitor fatty acid intake and we will use packed total blood cell assay to determine O3I 
index in our clinical trial patient cohort.  
 
 
 
 
Blood cells from EDTA anticoagulated whole blood (100 microliters (μl)) will be pelleted by centrifugation at 
1500 x gravity (xg) for 10 minutes. Packed cells prepared (in 2-ml microfuge tubes) shortly after blood 
collection (30-45 min) will be saved immediately at -80⁰C until further analysis.  At the time of analysis, cell 
pellets will be thawed on ice, lysed in 0.5 ml water and transferred to extraction tubes.  Samples will be spiked 
with 25 micrograms (μg) 1,2,3-tri-heptadecanoyl-glycerol (a representative synthetic triglyceride) and 25 μg 
1,2-dipentadecanoyl-sn-glycero-3-phosphoethanolamine (a representative synthetic phospho-lipid).  These 
synthetic lipids will be added to monitor extraction recovery of blood lipids and their conversion to Fatty Acid 
Methyl Esters (FAMEs).  Total lipids from lysed blood cells  will be extracted as described by Folch et al (Folch, 
Lees, and Sloane Stanley 1957) using a mixture of chloroform and methanol (2:1) in the presence of 0.01% 
butylated hydroxy-toluene (BHT).  BHT will be provided as an antioxidant to protect polyunsaturated FAs 
Fatty Acid (% of total)
Red blood Cells
Total Blood Cells
Arachidonic Acid (AA)
9.51
9.63
Eicosapentaenoic acid (EPA)
1
1.02
Docosahexaenoic acid (DHA)
4.43
4.2
Omega-3-Index (O3I)
5.43
5.22
Fatty Acid Ratio (AA to (EPA + DHA))
1.75
1.85
Table-1:  Fatty Acid levels and Omega-3-Index values from Erythrocyte Phospholipids and Total Blood Cells

 
68 
 
(PUFAs) from oxidative degradation. BHT will also be present during subsequent lipid analysis procedures 
including thin layer chromatography (TLC). FAMEs will be prepared by heating lipid extracts in 250 µl of 
anhydrous toluene (added to dissolve all neutral lipids including triglyceride and cholesterol esters) and 1.5 ml 
of anhydrous methanolic-HCl (0.5 normal solution, a transmethylating reagent) for 90 minutes at 80C in 
sealed glass tubes (Blau and Halket 1993).  Under these reaction conditions, which were established in 
preliminary experiments, both esterified and non-esterified FAs in lipid extracts (from up to 200 µl whole blood) 
are completely converted to FAMEs.  Following dilution of the reaction mixture with 1.5 ml water, FAMEs will 
be extracted with 2.0 ml hexane, separated from other interfering components (such as free cholesterol, which 
can co-extract with FAMEs) by TLC (Dodge and Phillips 1967; Mangold 1965)  and analyzed using an Agilent 
gas chromatograph (6850 GC System).  Analysis will be performed using a polyethyleneglycol capillary column 
(30m x 250 m x 0.25 m, HP-INNOWax) and a temperature gradient between 150C and 240C (David, 
Sandra, and Wylie 2001).  The injector and detector temperatures will be maintained at 240C and 250C, 
respectively.  Helium will be used as a carrier gas.  FAMEs will be identified by comparing their retention times 
with authentic standards.  The gas chromatography (GC) conditions employed resolve all n-3 and n-6 FAs 
with chain lengths between C18 and C22 with a base-to-base resolution of all FAMEs as shown in Figure A 
below.  Peak areas will be integrated using Agilent ChemStation Software, and quantitated using a standard 
curve set up with methyl heptadecanoate (a C17 synthetic FA methyl ester). Assay conditions 
[transmethylation, and GC parameters including detector response and reproducibility (intra- and inter-assay 
variability)] have been set up in initial experiments employing either synthetic lipid standards or whole blood 
lipid extracts.  The detector response is linear between 0.25 and 6.0 µg of FA with an r2-value of 0.995. The 
variability between injections is <3% and between experiments is <4%.  The recovery of added lipid standards 
through GC was >80%.  
 
 
Figure A: Representative GC profile of standard fatty acid methyl esters. 
Peaks: Myrisitic acid (C14:0), myristoleic acid (C14:1), palmitic acid (C16:0), palmitoleic acid (C16:1), 
Heptadecanoic acid (C17:0 internal standard), Stearic acid (C18:0), Oleic acid (C18:1c), elaidic acid (C18:1t), 
linoleic acid (C18:2 n-6), γ-linolenic acid (C18:3 n-6), α-linolenic acid (C18:3 n-3), arachidic acid (C20:0), 
eicosaenoic acid (C20:1), eicosadienoic acid (20:2 n-6), homo-γ-linolenic acid (20:3 n-6), arachidonic acid 
(C20:4 n-6, AA), eicosapentanoic acid (C20:5 n-3, EPA), behenic acid (C22:0), docosaenoic acid (C22:1), 

 
69 
 
docosatetraenoic acid (C22:4 n-6), docosapentaenoic acid (C22:5 n-3, DPA) and docosahexaenoic acid 
(C22:6 n-3, DHA).  
 
Analysis of Plasma Lipid Mediators including Resolvin (Rv) D1 and Leukotriene B4 (LT) B4 :   
RvD1, LTB4 and other plasma lipid mediators [eicosanoids (oxygenated metabolites of AA and EPA) and 
docosanoids (oxygenated metabolites of DHA)] will be analyzed, in initial experiments,  by Liquid 
chromatography–mass spectrometry (LC-MS)-based targeted lipidomics as described previously (Maddipati and 
Zhou 2011; Maddipati et al. 2014; Markworth et al. 2013)  in collaboration with Dr Maddipati (Director, Lipidomics 
Core Facility, Bioactive Lipids Research Program, Department of Pathology, Wayne State University, Detroit, 
MI).  Lipidomics studies have used either serum or plasmas prepared in the presence of various anticoagulants.  
Previous studies, however, have shown that the levels of eicosanoids and docosanoids measured in serum are 
different when compared to those levels measured in plasma.  For example, serum levels of thromboxane B2 
(TxB2) and 12-hydroxyeicosatetraenoic acid (12-HETE) are increased, while the levels of 18-
hydroxyeicosapentaenoic acid (18-HEPE) and 17-hydroxydocosahexaenoic acid (17-HDHA) are decreased 
(Mas et al. 2012).   These differences in mediator levels could be due to an increased synthesis of some (TxB2 
and 12-HETE) and possibly due to metabolic conversion of others (for example, 18-HEPE and 17-HDHA are the 
upstream precursors for the synthesis of E- and D- series resolvins, respectively).   While plasma levels reflect 
a more accurate measure of circulating eicosanoids and docosanoids, serum levels may indicate maximal 
eicosanoid and docosanoid production by activated cellular elements of blood.  We will therefore assay both 
plasma and serum samples in our lipidomic assays using LC-MS/MS.  Studies have shown that the levels of 
resolvin are not significantly different between plasmas collected into different anticoagulants (Mas et al. 2012).  
LTB4 levels measured in EDTA plasma, however,  are significantly lower compared to those measured in 
plasmas prepared with other anticoagulants (Parameter LTB4 Assay Kit from R&D Systems), which appears to 
be due to prevention by EDTA of ex-vivo conversion of LTA4 to LTB4 during blood collection and processing.  
We will therefore use EDTA plasmas in lipidomic assays and in all our biomarker assays with the exception of 
assays involving hemostatic biomarkers and CAT assays, for which we will use citrate-anticoagulated plasmas.  
While measurable levels of RvD1, RvD2, 17R-RvD1 and RvE1 have been reported in control plasmas, presence 
of other SPMs including Protectin D1 (PD1), Maresin 1 (MaR1), RvE2, RvE3, and 18R-RvE3 have been 
documented to be present in plasmas from both control and inflammatory arthritic patients who were on n-3FA 
therapy (Colas 2014; Mas et al. 2016; Psychogios et al. 2011).  In addition, a significant increase in plasma RvD1 
has been noted in subjects after n-3 FA supplementation (Mas et al. 2016).   Median levels of these plasma 
mediators range between 17 pg/ml and 774 pg/ml (Barden et al. 2016; Mas et al. 2016). In addition, 17-HDHA 
and 18-HEPE, the upstream precursors of D- and E- series resolvins, respectively, are also present in plasma 
at concentrations ranging between 63.6 and 235.3 pg/ml and their levels increase by 2 to 4-fold following n-3FA 
therapy (Mas et al. 2016).  The increase in 17-HDHA following fatty acid therapy may be clinically relevant as it 
can affect several inflammatory responses including promotion of phagocytosis (Kohnke et al. 2013) and 
suppression of the synthesis of pro-inflammatory cytokines including TNF-α (Gonzalez-Periz et al. 2006). In our 
clinical trial patients, we will quantitate all of the specialized pro-resolving mediators (SPMs) and their upstream 
precursors documented to be present in plasmas from individuals pre- and post- n-3 FA therapy.  Analysis will 
also include any novel biologically relevant docosanoid and eicosanoid identified in the future.  Using appropriate 
statistical tests (Pearson and/or Spearman Correlation tests) we will evaluate whether there is any relationship 
exist between the levels of SPMs that are elevated following FA therapy and the levels of pro-inflammatory 
cytokines including IL-1β and TNF-α to assess whether FA therapy provided protection against inflammation and 
also to identify the SPM(s) mediating the anti-inflammatory response.   
If lipidomic assay delivers results comparable to those measured by ELISA for LTB4 and RvD1 with acceptable 
intra- and inter- assay variabilities, the ELISA assays will be used in the Clinical Trial.  ELISA kits for both RvD1 
(Cayman Chemicals) and LTB4 (R&D Systems) are commercially available.    We have previously assessed 
LTB4 levels in plasma using an immunoassay kit following extraction and HPLC separation with appropriate 
correction for recovery (Setty and Stuart 2002).  LTB4 levels in race-matched controls and children with HbSC 
and HbSS disease were 136 ± 32 (mean ± SD), 177 ± 83 and 207 ± 64 femtomole (fmol)/ml, respectively (Setty 
and Stuart 2002). The plasma LTB4 levels measured in our laboratory are comparable to those reported in serum 

 
70 
 
by LC-MS/MS (Markworth et al. 2013) [ranged between 28 and 72 picogram (pg)/ml in the placebo group 
equivalent of 89 to 214 fmol/ml].  Plasma LTB4 levels in our study population will be measured using a Parameter 
LTB4 Assay Kit from R&D Systems.  The R&D kit can detect LTB4  in the range of 3.7 to 10.9 pg/ml with a mean 
minimum detectable level of 8.2 pg/ml.  The assay range is between 10.3 and 2500 pg/ml.  RvD1 levels in 
controls, assayed with LC-MS/MS, ranged between 3.7 and 17.09 pg/ml (Colas 2014; Psychogios et al. 2011). 
The Cayman RvD1 assay kit can detect RvD1 at concentration as low as 10-20 pg/ml and the assay is linear 
between 50 and 100 pg/ml.  Assuming a plasma RvD1 concentration of 10 pg/ml, a 5 ml plasma sample provides 
sufficient amount of RvD1 for quantitation by ELISA. We will therefore employ RvD1 ELISA kit to assess plasma 
RvD1 levels.  The lipid mediators in plasma will be extracted using C18 extraction columns as previously 
described (Maddipati and Zhou 2011),  the column eluate will be dried under a stream of nitrogen, reconstituted 
in ELISA assay buffer and quantitated following Manufacturer’s instructions.  We have previously used 
immunoassays to quantitate multiple eicosanoids in plasma and urine samples from patients with SCD and 
controls following extraction of lipid mediators using C-18 cartridges (Setty, Chen, and Stuart 1995; Setty et al. 
1998; Setty and Stuart 2002).  
Analysis of Urinary Resolvin D1:   
A recent study, using an LC/MS/MS assay, has documented the presence in human urine of docosanoids 
including RvD1 which is present at mean levels of 71.4 ± 53.3 and 72.8 ± 48.3 pg per mg creatinine in non-
smoking men (n=13) and women (n=10), respectively (Sasaki et al. 2015).  Based on the urinary creatinine levels 
(500 to 2000 mg/day) and urine volume (800 to 2000 ml/day), a 5 ml urine sample provides sufficient amount of 
RvD1 for quantitation by ELISA. We will therefore employ RvD1 ELISA kit (Cayman) to assess urinary RvD1 
levels.  Fresh urine samples will be centrifuged at 2900 xg for 5 min at room temperature and supernatant saved 
at -80⁰C until assayed.  At the time of the assay urine samples will be diluted with water to adjust creatinine 
levels to 0.6 mg/ml.  RvD1 will be extracted from 5 ml diluted urine samples using C-18 cartridges, extracts 
evaporated to dryness, re-constituted in ELISA assay buffer and quantitated following Manufacturer’s 
instructions.   
 
General ELISA Quality Control Measures:  
Plasma will be prepared shortly after blood collection (30-45 min), and aliquots (50 to 250 µI) saved 
immediately at -80⁰C until further analysis. Multiple freeze-thaw of frozen plasmas will be avoided.    For 
biomarker analysis, batched plasma samples will be thawed completely on ice, and mixed well prior to 
analysis.  Plasma will be centrifuged if large amount of particulate material is present in the sample, 
and this sample treatment will be recorded. Severely hemolyzed and lipemic plasmas will not be included 
in the analysis when possible.  The bio-markers: CRP, cytokines, soluble cell adhesion molecules, ET-1 
and hemostatic markers will be assayed employing commercial ELISA assay kits. Kit manufacturers’ 
provide all reagents and buffers needed to perform the assay.  These kit reagents and buffers will be stored 
and used according to the manufacturer instructions.  All reagents, buffers and assay strips will be warmed to 
recommended temperature before use.  Reagents from different kits will not be mixed.  Kits will not be used 
after their expiration date.  All standards, controls and samples will be run in duplicate.  In addition to kit 
controls, in-house controls will be included with every assay to cover low-end, mid-range, and high-end of the 
standard curve. If control values fall outside pre-established ranges, the assay will be repeated after 
appropriate instrument maintenance procedure.  Assays with standard curves with an r2 value <0.98 will be 
repeated.  Samples with values outside the pre-set calibration range will be diluted and repeated. Other 
general quality control measures include pipette calibration to meet American Association for Clinical Chemistry 
(AACC), International Organization for Standardization (ISO), Good Laboratory Practice (GLP) / Good 
Manufacturing Practice (GMP), National Committee for Clinical Laboratory Standards (NCLLS) and College 
of American Pathologists (CAP) guidelines with dated logs for each pipette used in the assay, and 
individual biomarker analyses using assay kits from the same lot number when possible. We have used 
age-matched African American controls with an n-value ranging from 10 to 29 subjects for biomarker 
controls in our labs. Mean intra- and inter-assay variability are <5%, and <8%, respectively. Control marker 
levels reported from our laboratories are comparable to those reported in the literature. In addition, our 
research laboratory served as a Core Biomarker Facility between 2005 and 2009 for the National 

 
71 
 
Comprehensive Sickle Cell Centers' Clinical Trial of Dexamethasone in Acute Chest Syndrome (Quinn 
et al. 2011). 
 
Assessment for C Reactive Protein (CRP):  
CRP in plasma will be assayed using ALPCO hs-CRP Immunoassay kit.  The standard curve spans a range of 
1900-150000 pg/ml, with a minimum detection limit of 921pg/ml.  Median (25th and 75th percentile) CRP level in 
our African-American controls is 0.1 (0.05, 0.5) mg/liter (l) (Krishnan et al. 2010) and is similar to National Health 
and Nutrition Examination Survey (NHANES) control values in age and race-matched children.  
 
Analysis of Pro- and Anti-Inflammatory Cytokines:   
Representative pro- (IL-1β, IL-6, IL-8 and TNF-α) and anti- (IL-4 and IL-10) inflammatory cytokines that have 
been documented to be abnormal in patients with SCD (Bowers et al. 2013; Hyacinth et al. 2014; Keikhaei et al. 
2013; Raghupathy et al. 2000; Setty et al. 2012) will be evaluated in EDTA plasma using commercially available 
ELISA kits (R&D Systems).  Assay range, minimum detection level, and reported control levels (from our 
laboratories and others) for the test cytokines are shown in Table 2 below.  No matrix interference was noted 
with plasma/serum at dilution between 1:2 and 1:16.  
 
Table 2: Cytokine assay Parameters 
Cytokine 
Assay Range (Standard 
Curve) 
Minimum Detection Level 
Reported Control Plasma 
Range 
IL-1β 
0.125 to 8 pg/ml 
0.057 pg/ml 
0.00 -  0.452 pg/ml 
IL-4 
7.8 to 500 pg/ml 
<2 pg/ml 
15 pg/ml (mean) 
IL-6 
0.156 to 10 pg/ml 
0.039 pg/ml 
0.435 - 9.57 pg/ml 
IL-8 
1.0 to 64 pg/ml 
0.4 pg/ml 
2.27 – 11.1 pg/ml 
IL-10 
0.78 to 50 pg/ml 
0.09 pg/ml 
<0.78 pg/ml 
TNF-α 
0.5 to 32 pg/ml 
0.106 pg/ml 
0.00 - 1.195 pg/ml 
Soluble Cell Activation Markers:  Soluble VCAM-1 (endothelial activation), soluble P-selectin (platelet 
activation) and soluble L-selectin (neutrophil activation) will be evaluated using ELISA kits from R&D Systems.  
Reported plasma levels in pediatric controls for sVCAM-1, sP-selectin and sL-selectin from our laboratories were 
536 ± 105 nanograms (ng)/ml, 38.1 ± 8.5 ng/ml, and 1321± 418 ng/ml, respectively (Setty and Stuart 2002; Setty 
et al. 2012). These levels were comparable to those reported by other investigators. 
 
Endothelin-1:  Endothelin-1 levels in plasma will be assessed using an ELISA assay kit from R&D Systems.  
The assay is linear between 0.4 and 25 pg/ml with a minimum detection limit of 0.207 pg/ml.  Plasma levels in 
controls range between 0.58 and 1.96 pg/ml with a mean level of 1.17 pg/ml (n=35).  No significant interference 
was noted with big endothelin-1 and -2 or endothelin-2.   
 
Standard Hemostatic Markers: Hemostatic markers, including the prothrombin fragment F1.2 and D-dimer will 
be evaluated by ELISA as previously described (Krishnan et al. 2010; Setty et al. 2012). 
  
Novel Hemostatic Markers: Calibrated Automated Thrombograpy (CAT) is a novel assay of in vitro thrombin 
generation, which is employed to evaluate several CAT parameters including endogenous thrombin potential 
(ETP), peak thrombin, lagtime, time to peak, and start tail (Hemker et al. 2003). CAT parameters will be 
measured on platelet poor plasma in a 96-well plate fluorometer (Fluoroskan Ascent, Thermo Electron Corp.) 
with 390/460 nanometers (nm) excitation/emission filters and a dispenser to dispense the substrate. For each 
experiment, 2 sets of readings will be taken: one from a well in which thrombin generation (TG well) takes place 
and a second from a well in which calibrator (CL well) has been added. In the TG well, 20 μl trigger (consisting 
of desired concentrations of tissue factor and phospholipids) is added followed by 80 μl plasma sample. In the 
CL well, 20 μl of alpha2-Macroglobulin-Thrombin complex solution is added followed by 80 μl plasma. The plate 
will be warmed at 37oC for 10 min in the instrument, 20 μl fluorogenic substrate dispensed to all the wells, zero 
time registered, and reading initiated at a predetermined sampling rate for 45-60 minutes. All experiments will 

 
72 
 
be carried out in triplicate. A dedicated software program (Diagnostica Stago) will generate the thrombogram 
and calculate thrombin concentration and other CAT parameters. 
 
Using the assay described above, we have previously measured CAT parameters in 23 individuals with Sickle 
Cell Disease with SS or Sβthal genotype (18 to 53 years old) and 6 age matched controls (Betal et al. 2009).  
Significant differences were noted in the SCD compared to the controls with shortened Lag Times [1.9 ± 0.39 
(mean ± SD) vs 2.39 ± 0.42 minutes, p<0.001] and time to peak (3.72 ± 0.55 vs 4.77 ± 0.49 min, p<0.001).  
However, there was overall a lower and less sustained thrombin generation  with SCD subjects demonstrating 
an early start tail (16.76 ± 2.29 vs 20.33 ± 1.71 min, p<0.01) and less total ETP (1377 ± 360 nM vs 1629 ± 212 
nM, p<0.01) when compared to the controls.  Peak thrombin levels were similar in both groups at 354 and 336 
nM, respectively.  Thus the subject with SCD has a significant early upsurge in thrombin generation followed by 
the phenomenon of a rapid attenuation.  
 
While the anti-thrombotic mechanism ascribed to n-3 FAs relates to in vitro anti-platelet aggregation effect, 
limited information is available about its effect on the plasmatic or fluid phase of coagulation. This study therefore 
provides an opportunity for some exploratory analyses on the effects of n-3 FA on thrombin generation as a 
novel measure of coagulation activation which we will contrast with standard measures of thrombin generation 
(F1.2) and fibrin dissolution (D-Dimer). These data could provide both a qualitative measure of efficacy 
(suppressing ancillary pathogenicity related to coagulation activation) and potential safety measure (suppression 
of thrombin generation as an indicator of potential additive bleeding risk in these patients).   
Lactate Dehydrogenase (LDH): Plasma LDH levels will be evaluated using an LDH assay kit (TOX07, Sigma).  
The enzyme activity international units (iu)/l) will be read from a calibration curve generated using an LDH 
standard (Sigma) as previously described (Krishnan et al. 2010).   
Fetal Hemoglobin (HbF):  HbF levels will be determined using cation exchange-high performance liquid 
chromatography on 50 to 100 μl of stored frozen red cell pellets.  We will use the services available at the Mayo 
Medical Laboratories, Mayo Clinic for HbF analysis (Krishnan et al. 2010).    
Other Relevant Inflammatory Biomarkers:  Please NOTE that no extra blood samples are collected for future 
studies.  We will save any unthawed remaining plasma and urine aliquots for evaluation of novel and relevant 
biomarkers identified in the future.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
73 
 
Appendix 6 
Dose Calculator for SCD-OmegatexTM 
Dosage:  
Please note: all dosing is based on total omega-3 fatty acid content of capsules (combined DHA plus 
EPA). Dosing will be based on a per capsule combined DHA plus EPA content of 336 mg.  
Doses will be rounded to capsule size. Doses that calculate to ≥ 0.5 capsule will be rounded up while 
doses calculating to < 0.5 capsule will be rounded down for capsule size 
 
Lower dose range is 25 mg/kg/day 
Higher dose range is 37.5 mg/kg/day 
Maximum total daily dose is 4 grams 
 
Dose calculation in number of capsules for 25 mg/kg/day dose level 
Weight in kg 
Number of capsules per day 
15-20 
1 
21-33 
2 
34-47 
3 
48-60 
4 
61-73 
5 
74-87 
6 
88-100 
7 
101-107 
8 
 
Dose calculation in number of capsules per day for 37.5 mg/kg/day dose level 
Weight in kg 
Number of capsules per day 
15-22 
2 
23-31 
3 
32-40 
4 
41-49 
5 
50-58 
6 
59-67 
7 
68-76 
8 
77-85 
9 
86-94 
10 
95-103 
11 
104-111 
12 
 
Sample Calculation: 
For a 35 kg child: 
At 25 mg/kg dose range child will receive 25 mg x 35 kg = 875 mg divided by 336 mg EPA+DHA per capsule = 
2.6 capsules, round up to 3 capsules. 
At 37.5 mg/kg dose range child will receive 37.5 mg x 35 kg = 1312.5 mg divided by 336 mg EPA+DHA per 
capsule = 3.9 capsules, round up to 4 capsules. 
 
Maximum total daily dose is 12 capsules =3624mg DHA plus 408 mg EPA in a 9:1 ratio (4032 mg omega-3 
fatty acid total, combined DHA plus EPA). 
 
 
 

 
74 
 
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