Conditions: Intracerebral Hemorrhage
Keywords: Brain hemorrhage Cerebral hemorrhage Bleeding in the brain Deferoxamine iDEF trial
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 294
Healthy volunteers: no

Brief summary:
The investigators hypothesize that treatment with the iron chelator, Deferoxamine Mesylate, improves the outcome of patients with brain hemorrhage.

The purpose of this study is to determine whether treatment with Deferoxamine Mesylate is of sufficient promise to improve outcome before pursuing a larger clinical trial to examine its effectiveness as a treatment for intracerebral hemorrhage.

Detailed description:
This is a prospective, multi-center, double-blind, randomized, placebo-controlled, phase-II clinical trial.

Subjects will be randomized to either deferoxamine mesylate (DFO) at 32 mg/kg/day (up to a maximum daily dose of 6000 mg/day), or saline placebo, given by IV infusion for 3 consecutive days.

Treatment will be initiated within 24 hours after ICH symptom onset. Randomization will control baseline imbalances associated with baseline ICH score, ICH onset-to-treatment time (OTT), ICH volume, baseline NIHSS score, and warfarin use.

All subjects will be followed for 6 months and will receive standard of care therapy while participating in the study.

Throughout the study, we will continue to assess the safety of DFO. At the conclusion of the study, the proportion of DFO-treated subjects with a good clinical outcome at 3 months (defined as modified Rankin Scale (mRS) score of 0-2) will be compared to the placebo proportion in a futility analysis to determine if it is futile to move DFO forward to Phase III efficacy evaluation.

Arms:
arm 1: Deferoxamine Mesylate (32 mg/kg/day) given by an intravenous infusion for 3 consecutive days arm 2: Normal saline (0.9% sodium chloride) given by intravenous infusion for 3 consecutive days

Interventions: intervention 1: Deferoxamine Mesylate intervention 2: Placebo (for Deferoxamine Mesylate)
Intervention descriptions: intervention 1: None intervention 2: None

Protocol document:
Prot_000.pdf: 
FULL PROTOCOL TITLE 
FUTILITY STUDY OF DEFEROXAMINE MESYLATE IN INTRACEREBRAL HEMORRHAGE 
SHORT TITLE 
INTRACEREBRAL HEMORRHAGE DEFEROXAMINE TRIAL 
(iDEF TRIAL) 
Study Chair: 
Magdy H Selim, MD, PhD 
Harvard Medical School 
Beth Israel Deaconess Medical Center 
Department of Neurology 
Division of Stroke and Cerebrovascular Diseases 
Boston, MA 
Supported by: 
The National Institute of Neurological 
Disorders and Stroke (NINDS) 
(U01 NS074425) 
Sponsor of IND: 
Magdy H Selim, MD, PhD 
IND# 77,306 


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1 
TABLE OF CONTENTS 
  PAGE 
STUDY ADMINISTRATIVE ORGANIZATION 
4-5
STUDY TEAM ROSTER 
6 
STUDY SYNOPSIS  
7-9
1.
STUDY OBJECTIVES
10-11
1.1 
Primary Objective  
1.2 
Secondary Objectives 
1.3 
Exploratory Analyses  
2.
BACKGROUND
11-20
2.1 
Rationale   
11-14
2.1.1 
ICH is a major public health problem 
11
2.1.2 
Role of hemoglobin and iron in neuronal injury after ICH 
11-12
2.1.3 
Deferoxamine Mesylate (DFO)  
12-14
2.1.3.1 Pharmacokinetics  
12
2.1.3.2 Safety Considerations 
13-14
2.1.3.3 Neuroprotective Effects 
14
2.2 
Supporting Evidence  
14-20
2.2.1 
Supporting evidence that DFO attenuates neuronal injury after ICH 
14-16
2.2.2 
Safety & tolerability of DFO in patients with ICH 
16-18
2.2.3 
High dose DEFeroxamine in ICH (HI-DEF) 
18-19
2.3 
Summary of background and rationale 
19
2.4 
Significance 
19-20
3.
STUDY DESIGN
20-24
 3.1 Study Objectives 
20 
 3.2 Overview 
20 
 3.3 Rationale 
21-24
4.
SELECTION AND ENROLLMENT OF SUBJECTS
25-28
4.1 
Inclusion Criteria 
 
25 
4.2 
Exclusion Criteria   
25-26
4.3 
Study Enrollment Procedures 
26-28
 4.3.1 
Screening for potential subjects 
26
 4.3.2 
Screening evaluations 
26-27
 4.3.3 
Informed consent 
27
 4.3.4 
Enrollment 
27


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2 
 4.3.5 
Determination of subject’s body weight 
27-28
 4.3.6 
Treatment allocation/randomization 
28
5.
STUDY INTERVENTIONS
28-31
5.1 
Interventions, Administration, and Duration 
28 
5.2 
Safety monitoring during study drug administration 
29 
5.3 
Handling of Study Interventions  
30 
 5.3.1 
Supply, storage, preparation, and disposition of study drug 
30 
 5.3.2    Study drug tracking and accountability 
30 
5.4 
Concomitant Interventions 
30-31
6.
CLINICAL AND LABORATORY EVALUATIONS
31-35
6.1 
Patients’ evaluation and data collection schedule 
31 
 6.1.1 
Clinical and laboratory assessments 
31-33
 6.1.2 
Radiological assessments 
33
6.2 
Schedule of Evaluations 
33-34
6.3 
Blood Banking Repository Sub-Study 
34-35
7.
ADVERSE EVENTS
35-39
7.1 
Definition of adverse events 
35
7.2 
Potential complications of study interventions 
35-36
7.3 
Prevention and management of adverse events 
36-37
7.4 
Criteria for intervention discontinuation 
37-38
7.5 
Reporting of adverse events 
38
7.6 
Monitoring of adverse events 
38-39
7.7 
Stopping rule 
39
8.
STATISTICAL CONSIDERATIONS
39-42
8.1 
Randomization Procedures 
39 
8.2 
Study Endpoints and Data Analysis 
40-41
 8.2.1 
Clinical Endpoints 
40
 8.2.2 
Safety Endpoints 
40-41
8.3 
Study Samples 
41
8.4 
Sample size calculation 
41
8.5 
Loss to follow up and missing data 
41-42
9.
STUDY MONITORING
42-44
9.1 
Monitoring for study progress and recruitment 
42 
9.2 
Quality management and data control 
42-43


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9.3 
Ongoing Safety Monitoring During the Study 
43-44
10.
HUMAN SUBJECTS
44-45
10.1 
Ethical conduct of the study 
44 
10.2 
Institutional Review Board (IRB) Review and Informed Consent 
44 
10.3 
Subject Confidentiality 
44-45
10.4 
Data and Safety Monitoring Plan 
 
45
10.5 
Study Modification/Discontinuation 
45
11.
PUBLICATION OF RESEARCH FINDINGS
45-46
12.
ANCILLARY STUDIES POLICY
46 
13.
RESOURCE AND DATA SHARING POLICY
46 
14.
REFERENCES
46-52
15.
APPENDICES
52- 
I.
DFO in ICH (Phase I) Manuscript
II.
RECOMMENDED PROTOCOL FOR MANAGEMENT OF PATIENTS WITH ICH
III.
ASA/AHA ICH MANAGEMENT GUIDELINES
IV.
DETERMINATION OF ICH SCORE AND HEMATOMA VOLUME (ABC METHOD)
V.
ASSESSMENT SCALES (NIHSS – GCS - mRS – MoCA – SIS-16) AND INSTRUCTIONS
VI.
ARDSNet RECOMMENDATIONS FOR MANAGEMENT OF INTUBATED PATIENTS
VII 
BERLIN CRITERIA FOR DIAGNOSIS OF ACUTE RESPIRATORY DISTRESS SYNDROME 
VIII.
DECLARATION OF HELSINKI


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4 
STUDY ADMINISTRATIVE ORGANIZATION 
EXECUTIVE COMMITTEE  
The Executive Committee consists of Magdy Selim, MD, PhD (the study Principal Investigator and chair); Sharon 
Yeatts, PhD (Biostatistician and Principal Investigator of the Statistics and Data Management Center (SDMC)); Yuko 
Palesch, PhD (Biostatistician); Aaron Perlmutter, MPH, MSW (Project Manager), Catherine Dillon, CCRP (the Superviso-
ry Data Manager); Andre Thornhill (the Data Manager); and Claudia Moy, PhD (NINDS-appointed liaison).  
The Advisory Committee members, Drs. Daniel Hanley, Steven Greenberg, Lewis Morgenstern, and Guohua Xi are also 
expected to participate in the Executive Committee teleconferences, as needed.   
The Committee will be responsible for the development and amendment of the study documents (including the proto-
col, case report forms (CRF), and manual of operations); collection, review and oversight of the dissemination of serious 
adverse event (SAE) occurrences and other important events pertinent to the study; and communication among all 
components of the study administrative organizations.   
THE STUDY CHAIR OFFICE 
The Study Chair Office, housed in the Department of Neurology – Stroke Division at the Beth Israel Deaconess Medical 
Center (BIDMC) in Boston, provides overall scientific coordination and fiscal management of the trial and is responsible 
for preparing progress reports for the NINDS and FDA. The office is comprised of the trial's Principal Investigator (Mag-
dy Selim, MD, PhD), his Study Coordinator, and a senior research administrator from BIDMC. The Principal Investigator 
provides overall leadership to the entire Trial to ensure its successful implementation. He will visit all clinical sites on a 
periodic basis and collaborate with the SDMC in organizing all necessary meetings and conference calls. As the Sponsor 
of the Investigational New Drug (IND) application, he ensures that the trial is conducted according to FDA's Good Clini-
cal Practice (GCP) guidelines and regulations. The Study Coordinator assists the PI in day-to-day implementation of the 
trial and serves as a major contact person for investigators and study coordinators at the clinical sites. The senior re-
search administrator, together with the PI, is responsible for the budgetary management of the grant. These responsi-
bilities include preparation of consortium agreements and subcontracts, handling of invoices, and directing disburse-
ment of funds.  
THE STATISTICS AND DATA MANAGEMENT CENTER (SDMC)  
The Data Coordination Unit (DCU), located in the Department of Public Health Sciences - College of Medicine at the 
Medical University of South Carolina, will serve as the SDMC for the trial.  The SDMC is responsible for statistical design 
and analysis, database development and maintenance, data and project management activities, as well as interim safe-
ty monitoring and report generation. 
DATA AND SAFETY MONITORING BOARD (DSMB)  
The DSMB is appointed by the Director of NINDS and managed by the NINDS Clinical Trials group.  It is comprised of 
Neurologists with special expertise in stroke and ICH, a hematologist, a statistician, and an ad hoc expert in pulmonary 
diseases and critical care medicine. Its responsibility will be the oversight of participant safety, review of the safety re-
ports, requesting additional data/information (if necessary), and advising the NINDS regarding continuation/ discontin-
uation of the study. Peter Gilbert, Sc.M., serves as the NINDS-appointed liaison for the DSMB.      


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INDEPENDENT MEDICAL SAFETY MONITORS (MSM)  
Two experts (a Neurocritical care specialist and a pulmonary critical care specialist with special expertise in Adult Res-
piratory Distress Syndrome) serve as the Independent Medical Safety Monitors (MSMs) for the study. They will be re-
sponsible for monitoring the study with regard to safety on an ongoing basis and reviewing all serious adverse events 
quickly to identify any safety concerns.  The MSMs’ task is to review all adverse events and to adjudicate all serious ad-
verse events as recruitment progresses. The MSMs will communicate with the investigators for any questions or 
clarifications regarding an event, in order to determine whether events are unexpected and related to study drug 
administration. They will report to the Executive Committee their findings that any SAE is unexpected and suspected to 
be related to the study drug. They will also review and adjudicate all cases of respiratory compromise to ascertain the 
underlying cause and to determine whether  the event is related to the study drug. 


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6 
STUDY TEAM ROSTER 
PRINCIPAL INVESTIGATOR AND CHAIR 
Magdy Selim, MD, PhD 
Beth Israel Deaconess Medical Center 
Dept. of Neurology – Stroke Division 
330 Brookline Avenue – Palmer 127 
Boston, MA 02215 
Tel: 617-632-8913 
Fax: 617-632-8920 
E-mail: mselim@bidmc.harvard.edu
STUDY STATISTICIAN, PRINCIPAL INVESTIGATOR OF THE SDMC 
Sharon Yeatts, PhD 
Data Coordination Unit  
Department of Public Health Sciences 
College of Medicine 
Medical University of South Carolina 
Tel: 843-513-9085 
E-mail: yeatts@musc.edu
PROJECT MANAGER, SDMC 
Aaron Perlmutter, MPH, MSW 
Data Coordination Unit 
Department of Public Health Sciences 
College of Medicine 
Medical University of South Carolina 
Tel: 843-876-1261 
Fax: 843-876-1923 
E-mail: perlmutt@musc.edu
DATA MANAGER, SDMC 
Andre Thornhill 
Data Coordination Unit 
Department of Public Health Sciences 
College of Medicine 
Medical University of South Carolina 
Tel: 843-876-1914 
Fax: 843-876-1923 
E-mail: thornhil@musc.edu


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STUDY PROTOCOL SYNOPSIS 
Protocol Title 
Intracerebral hemorrhage DEFeroxamine (iDEF) trial 
Clinical Trial Phase 
Phase II 
Clinical Sites 
>20 North American clinical sites
Statistics and Data  
Management Center 
Sponsor Institution 
Data Coordination Unit, Department of Public Health Sciences, College of Medicine, Med-
ical University of South Carolina, Charleston, SC 
Beth Israel Deaconess Medical Center, Boston, MA 
Study Period 
Planned enrollment duration – 36 to 40 months 
Planned duration of study for each participant – 180 ± 7 days 
Planned duration of the study – 36-48 months 
Study Population 
Patients with spontaneous intracerebral hemorrhage (ICH) 
Study Design 
A prospective, multi-center, double-blind, randomized, placebo-controlled, phase-II clini-
cal trial. Subjects will be randomized to either deferoxamine mesylate (DFO) at 32 
mg/kg/day (up to a maximum daily dose of 6000 mg/day), or saline placebo, given by IV 
infusion for 3 consecutive days. Treatment will be initiated within 24 hours after ICH symp-
tom onset. Randomization will control baseline imbalances associated with baseline ICH 
score, ICH onset-to-treatment time (OTT), ICH volume, baseline NIHSS score, and warfa-
rin use. All subjects will be followed for 6 months and will receive standard of care therapy 
while participating in the study. Throughout the study, we will continue to assess the safe-
ty of DFO. At the conclusion of the study, the proportion of DFO-treated subjects with a 
good clinical outcome at 3 months (defined as modified Rankin Scale (mRS) score of 0-2) 
will be compared to the placebo proportion in a futility analysis to determine if it is futile 
to move DFO forward to Phase III efficacy evaluation. 
Primary Objectives 
1- TO assess whether it is futile to move DFO into Phase III evaluation as a therapeutic in-
tervention for ICH, by comparing the outcome of DFO-treated subjects to placebo-treated
subjects with respect to good outcome (defined as mRS of 0-2 at 90 days), in a futility
analysis. The futility hypothesis specifies that if the difference in good outcome propor-
tions is less than 12% in favor of DFO, then it would be futile to move DFO forward to
Phase III evaluation.
2- TO assess the safety of DFO infusions (at a dose of 32 mg/kg/day, up to a maximum dai-
ly dose of 6000 mg/day), given for 3 consecutive days, in a large cohort of ICH patients. We
specifically wish to collect more data on treatment-related adverse events in order to as-
certain that patients with ICH can complete this dose given over 3-day duration of infusion
without experiencing unreasonable neurological complications, mortality, or other serious
adverse events related to DFO use, in particular adult respiratory distress syndrome
(ARDS).
Secondary Objectives 
1- TO explore the differences between early (≤12h) and late (>12-24h) time windows in
DFO treatment effect on functional outcome.
2- TO determine the overall ordinal distribution of scores on mRS at 3 months in DFO- and
placebo-treated subjects; and to perform a dichotomized analysis considering the propor-
tion of DFO- and placebo-treated subjects with mRS 0-3
3- TO perform dichotomized analyses considering the proportion of DFO- and placebo-
treated subjects with mRS 0-2 and 0-3; and to determine the overall ordinal distribution of
scores on mRS at 6 months.
4- TO determine mortality during the 90- and 180-day follow-up periods (all causes and


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8 
ICH-related). 
5- TO obtain data on the changes in NIHSS between presentation and day-90, and Mon-
treal Cognitive Assessment (MoCA) and Stroke Impact Scale (SIS)-16 scores at 3 months
to explore the effects of DFO on neurological, functional, and cognitive functions.
Exploratory Objectives 
1- TO explore the effects of treatment with DFO on relative perihematoma edema (PHE)
volume progression between baseline and post-treatment CT scans in DFO-treated pa-
tients compared to placebo as potential markers of DFO’s biological activity on brain tis-
sue.
2- TO explore whether the effect of DFO on outcome is dependent on initial ICH volume,
after adjusting for other confounding variables that can affect outcome, to determine if
specific limits for ICH volume should be specified as exclusion/inclusion criteria for future
studies.
3- TO explore the effects of DFO on the size of ventricular enlargement in patients with
intraventricular extension of ICH, not requiring an external ventricular drain, as a potential
marker of treatment utility in intraventricular hemorrhage.
4- TO explore the effect of DFO on the incidence of symptomatic cerebral edema (unex-
plained increase in NIHSS >4 points or decrease in GCS >2 points) during hospitalization,
up to day 7 or discharge, whichever is earlier.
5- TO explore whether progression of PHE can be a radiological/biological marker of activi-
ty that can be correlated with clinical outcomes and treatment effect of DFO.
Sample Size 
Approximately 294 subjects 
Inclusion Criteria 
1)
Age ≥ 18 and ≤ 80 years; 2) The diagnosis of ICH is confirmed by brain CT
scan; 3) NIHSS score ≥6 and GCS >6 upon presentation; 4) The first dose of the study drug
is expected to be administered within 24h of ICH symptom onset; 5) Functional independ-
ence prior to ICH, defined as pre-ICH mRS ≤1; and 6) Signed and dated informed consent is
obtained.
Exclusion Criteria 
1)
Previous chelation therapy or known hypersensitivity to DFO products; 2)
Known severe iron deficiency anemia (defined as hemoglobin concentration < 7g/dL or
requiring blood transfusions); 3) Abnormal renal function, defined as serum creatinine >2
mg/dL; 4) Planned surgical evacuation of ICH prior to administration of study drug (place-
ment of a catheter for ventricular drainage is not a contraindication to enrollment); 5)
SUSPECTED secondary ICH related to tumour, ruptured aneurysm or arteriovenous mal-
formation, hemorrhagic transformation of an ischemic infarct, or venous sinus throm-
bosis; 6) Infratentorial hemorrhage; 7) Irreversibly impaired brainstem function (bilateral
fixed and dilated pupils and extensor motor posturing); 8) Complete unconsciousness, de-
fined as a score of 3 on item 1a of the NIHSS (Responds only with reflex motor or auto-
nomic effects or totally unresponsive, and flaccid); 9) Pre-existing disability, defined as
pre-ICH mRS ≥2; 10) Coagulopathy - defined as elevated aPTT or INR >1.3 upon presenta-
tion; concurrent use of direct thrombin inhibitors (such as dabigatran), direct factor Xa
inhibitors (such as rivaroxaban or apixaban), or low-molecular-weight heparin; 11) Patients
with confirmed aspiration, pneumonia, or evident bilateral pulmonary infiltrates on chest
x-ray or CT scan prior to enrollment; 12) Patients with significant respiratory disease such
as chronic obstructive pulmonary disease, pulmonary fibrosis, or any use (chronic or in-
termittent) of inhaled O2 at home; 13) FiO2 >0.35 (>4 L/min) prior to enrollment; 14) Sepsis
(present source of infection ± lactic acidosis), Systemic Inflammatory Response Syndrome
(Temp >100.4F or <96.8F; Heart rate >90; Respiratory rate >20 or PaCo2 <32 mmHg; WBC
>12, <4, or bands >10%), or shock (SBP <90 mmHg) at presentation; 15) The presence of 4
or more of the following risk modifiers for ARDS prior to enrollment: a) Tachypnea (respir-


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9 
atory rate >30); b) SpO2 <95%; c) Obesity (BMI >30); d) Acidosis (pH <7.35); e) Hypoalbu-
minemia (albumin <3.5 g/dL); or f) Concurrent use of chemotherapy; 16) Taking iron sup-
plements containing ≥ 325 mg of ferrous iron, or prochlorperazine; 17) Patients with heart 
failure taking > 500 mg of vitamin C daily; 18) Known severe hearing loss; 19) Known preg-
nancy, or positive pregnancy test, or breastfeeding; 20) Positive drug screen for cocaine 
upon presentation; 21) Patients known or suspected of not being able to comply with the 
study protocol due to alcoholism, drug dependency, noncompliance, living in another 
state or any other cause; 22) Any condition which, in the judgement of the investigator, 
might increase the risk to the patient; 23) Life expectancy of less than 90 days due to co-
morbid conditions; 24) Concurrent participation in another research protocol for investiga-
tion of another experimental therapy; and 25) Indication that a new DNR or Comfort 
Measures Only (CMO) order will be implemented within the first 72 hours of hospitaliza-
tion. 
Study Intervention and 
Follow-up 
Each subject will receive an IV infusion of DFO (32 mg/kg/day) or a matching saline vehicle 
(placebo) for 3 consecutive days. The maximum daily dose will not exceed 6000 mg per 24 
hours, regardless of body weight, and the infusion rate will not exceed 7.5 mg/kg/hour. 
Subjects will be monitored closely and evaluated daily for the first 3 days (during study 
drug infusion while in hospital) and on day 7 or discharge from the hospital, whichever oc-
curs first. Clinic visits will take place on days 30±7 and 90±7. A telephone interview will be 
conducted on days 60±7 and 180±7. 
Randomization 
A combination of minimization and biased coin methodologies will be used to randomize 
participants to either DFO or placebo in 1:1 ratio. Randomization will control baseline im-
balances associated with baseline ICH score, ICH onset-to-treatment time (OTT), ICH vol-
ume, baseline NIHSS score, and concurrent use of anticoagulants at the time of ICH onset, 
as well as clinical site. 
Efficacy Outcome 
Measures 
The primary outcome measure is the mRS, dichotomized to define good functional out-
come as mRS 0-2 at 90 days. 
Safety Outcome 
Measures 
All adverse events (serious and non-serious) will be assessed until day-7 or discharge 
(whichever is earlier), and serious adverse events (SAEs) until day-90. Safety endpoints 
will include all DFO-related adverse events until day-7 or discharge (whichever is earlier), 
and SAEs through day-90. Mortality (all cause and ICH-related) will be assessed through 
day 180.   
The following adverse events will be defined as EVENTS OF SPECIAL INTEREST for safety sur-
veillance during this study: 1- Anaphylaxis (at any time point during study drug infusion); 
2- Hypotension (defined as a decrease in blood pressure requiring medical intervention at
any time point during drug infusion that cannot be explained by other causes); 3- Respira-
tory compromise of any cause during the in-hospital phase; and 4- Development of new
and unexplained visual or auditory changes after initiating treatment with the study drug.
Analyses of safety data will be carried out on an ongoing basis throughout the trial.


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10
STUDY OBJECTIVES 
1.1 
PRIMARY OBJECTIVES 

TO assess whether it is futile to move deferoxamine mesylate (DFO) into Phase III evaluation as a therapeutic inter-
vention for spontaneous intracerebral hemorrhage (ICH), by comparing the outcome of DFO-treated subjects to
placebo-treated subjects with respect to good outcome (defined as modified Rankin Scale [mRS] score of 0-2 at 90
days), in a futility analysis. The futility hypothesis specifies that if the difference in good outcome proportions is less
than 12% in favor of DFO, then it would be futile to move DFO forward to Phase III evaluation.

TO assess the safety of DFO infusions (32 mg/kg/day, up to a maximum daily dose of 6000 mg/day), given for 3 con-
secutive days, in a large cohort of ICH patients. We specifically wish to collect more data on treatment-related ad-
verse events in order to ascertain that patients with ICH can complete this dose given over 3-day duration of infu-
sion without experiencing unreasonable neurological complications, increased mortality, or other serious adverse
events related to DFO use, in particular adult respiratory distress syndrome (ARDS).
1.2 
SECONDARY OBJECTIVES 
As secondary analyses of the primary outcome, we also plan to:  

Explore the differences between early (≤12h) and late (>12-24h) time windows in DFO treatment effect on func-
tional outcome.

Determine the overall ordinal distribution of scores on mRS at 3 months in DFO- and placebo-treated subjects, and
to perform a dichotomized analysis considering the proportion of DFO- and placebo-treated subjects with mRS 0-3.
Although mRS 0-3 is less favorable than the primary outcome of mRS 0-2, it would still be a desirable effect in pa-
tients with ICH given that no treatments exist to reduce disability. The trial is adequately powered to assess the fu-
tility hypothesis using mRS 0-3 as the outcome based on an absolute difference in treatment effect <13% in favor of
DFO.
In addition, we will: 

Perform dichotomized analyses considering the proportion of DFO- and placebo-treated subjects with mRS 0-2 and
0-3; and determine the overall ordinal distribution of scores on mRS at 6 months.

Determine mortality during the 90- and 180-day follow-up periods (all causes and ICH-related).

Obtain data on the changes in NIHSS between presentation and day-90, and Montreal Cognitive Assessment
(MoCA) and Stroke Impact Scale (SIS)-16 scores at 3 months to explore the effects of DFO on neurological, func-
tional, and cognitive functions.
1.3 
EXPLORATORY OBJECTIVES 
Additional planned exploratory analyses include assessments of:  

The effects of treatment with DFO on relative perihematoma edema (PHE) volume progression between baseline
and post-treatment CT scans in DFO-treated patients compared to placebo as potential markers of DFO’s biologi-
cal activity on brain tissue.

Whether the effect of DFO on outcome is dependent on initial ICH volume, after adjusting for other confounding
variables that can affect outcome, to determine if specific limits for ICH volume should be specified as exclu-
sion/inclusion criteria for future studies.


 IDEF  
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11

The effects of DFO on the size of ventricular enlargement in patients with intraventricular extension of ICH, not re-
quiring an external ventricular drain (EVD), as a potential marker of treatment utility in intraventricular hemorrhage
(IVH).

The effect of DFO on the incidence of symptomatic cerebral edema (unexplained increase in NIHSS >4 points or
decrease in GCS >2 points) during hospitalization, up to day 7 or discharge, whichever is earlier.

Whether progression of PHE can be a radiological/biological marker of activity that can be correlated with clinical
outcomes and treatment effect of DFO.
2 
BACKGROUND 
2.1 
RATIONALE 
2.1.1 
INTRACEREBRAL HEMORRHAGE IS A MAJOR PUBLIC HEALTH PROBLEM  
Intracerebral hemorrhage (ICH) is a major public health problem. Approximately 70,000 patients are diagnosed with 
ICH in the United States (US), and up to 400,000 in the Far East, each year [Qureshi 2009; Zhang 2003], and the number 
of patients with ICH is rising with the aging of the population. ICH is a frequent cause of disability and mortality and 
confers a substantial burden on the healthcare system and society [Russell 2006]; approximately 40% of ICH patients 
die within one month, and more than 70% of the survivors are left with serious and permanent disability. The financial 
burden of ICH is enormous; it is estimated that the overall annual costs for ICH patients in the US alone exceed seven 
billion US dollars. 
At present, there is no specific treatment for ICH beyond supportive general medical care. Attention has been focused 
on medical, endoscopic, and surgical treatments targeting hematoma and its expansion [Mendelow 2005; Mayer 2008]. 
However, the utility of these approaches alone is likely to be limited to only a selected subset of ICH patients, since ICH 
expansion often occurs early within the first few hours of onset [Brott 1997; Flaherty 2005]. Although the hematoma 
gradually resolves after ICH, restoration of function is usually incomplete, indicating that neuronal injury after ICH is not 
only related to direct tissue damage and hematoma expansion [Brown 2005]. Other processes including apoptosis, ne-
crosis, iron-mediated oxidative stress, inflammation, autophagy, and edema formation contribute to secondary neu-
ronal injury and disability after ICH [Regan 1993-1996; Castillo 2002; Goldstein 2003; Huang 2002; Wu 2003; Wagner 
2003; Leira 2004; He 2008].  
There is an unmet need for safe and effective neuroprotective strategies to target the secondary effects of ICH in order 
to limit brain injury, facilitate neuronal repair, and improve functional outcome. The iron chelator, deferoxamine me-
sylate (DFO), is potentially promising as a candidate therapeutic intervention to improve the overall outcome of ICH 
patients.  
2.1.2 
THE ROLE OF HEMOGLOBIN DEGRADATION PRODUCTS AND IRON IN SECONDARY NEURONAL INJURY 
AFTER ICH 
Hemolysed red blood cells release their hemoglobin into the brain parenchyma after ICH. The time course for hemo-
globin hemolysis is approximately 2-3 days [Macdonald 2004]. Hemoglobin is a potent neurotoxin, and its toxicity is 
largely iron-mediated [Regan 1993-2003; Hua 2002; Goldstein 2003; Xi 1998; Huang 2002]. Hemoglobin degradation 
products include the iron-containing heme, which is metabolized by heme oxygenase to yield ferric iron. The released 
iron is implicated in neuronal injury and delayed brain edema formation after ICH via several mechanisms including: 
activation of lipid peroxidation, exacerbation of excitotoxicity, inhibition of Na+/K+ ATPase activity, and catalysis of Ha-
ber-Weiss/Fenton reaction {Fe+++ H2O2  Fe++++ OH◦+ OHо}, in which superoxide and hydrogen peroxide (H2O2) are  


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converted into highly reactive toxic hydroxyl radicals (OH◦) leading to oxidative stress and cell death [Regan 1993-1996; 
Winterboum 1995; Goldstein 2003; Huang 2002; Wu 2003; Wagner 2003; Winterbourn1995; Nakamura 2005].  
Experimental studies examining the time course of iron’s role in brain injury after ICH detected an increase in iron-
positive cells, heme oxygenase protein, and markers of DNA damage in the peri-hematoma area within the first day 
after ICH, peaking by day 3 [Nakamura, 2003-2005; Wu 2003]. Emerging evidence also links iron to neuronal injury in 
ICH patients. Serum ferritin upon admission correlates with the relative PHE on day-3 (which coincides with the timing 
for hemoglobin hemolysis), but not at baseline [Mehdiratta 2008], and functional outcome at 3 months [Perez 2010]; 
and the iron content within the hematoma, estimated by MRI, correlates with the relative PHE volume [Lou 2009]. 
These studies indicate that iron accumulates in the brain after ICH, and that its toxicity contributes to neuronal injury 
over many days following ICH. This delayed time window can have important therapeutic implications.   
2.1.3 
DEFEROXAMINE MESYLATE 
2.1.3.1 PHARMACOKINETICS –  
Deferoxamine mesylate (DFO) [N-(5-C3-L (5 aminopentyl) hydroxycarbamoyl)-propionamido)pentyl)-3(5-(N-
hydroxyacetoamido)-pentyl)carbamoyl)-proprionhydroxamic acid] is a hydrophilic, water-soluble, chelator. It complex-
es almost exclusively with ferric iron, at physiologic pH values, chelating iron from ferritin and hemosiderin; and forms a 
stable complex that prevents iron from entering into further chemical reactions. The drug’s serum protein-binding rate 
is less than 10%, and it is distributed throughout all body fluids. It has a volume of distribution of 0.8-1.35 L/kg [Lee 
1993]. The initial distribution half-life of DFO is approximately 5-10 minutes in humans [Perrone 2002]. Peak plasma 
concentrations between 80 and 130 µmol/L are measured 3 minutes after IV injection of DFO at a dose of 10 mg/kg [Al-
lain 1987]. During IV infusion of 50 mg/kg/day, steady state concentrations are generally obtained between 6-12 hours 
with a mean concentration of 7.4 ± 2.73 µM. The drug has a biphasic elimination pattern; the half-life is 1h in the first 
rapid phase and 6h in the second slow phase [Allain 1987]. Despite a molecular weight of 560.7 daltons (656.8 as mesyl-
ate) and low lipid solubility, the drug can diffuse into the brain down a concentration gradient, and there appears to be 
specific mechanism(s) of uptake by neuronal cells. DFO accumulates in the brain at a significant concentration quickly 
after injection [Keberle 1964; Yokel 1991; Palmer 1994; Hershko 1988; Brancaccio 1991] and reduces cerebrospinal fluid 
free iron level in rats subjected to experimental ICH, indicating that it crosses the blood brain barrier after ICH [Wan 
2006-2009].  
Figure 2.1.3.1: Chemical Structure of DFO 
2.1.3.2 SAFETY CONSIDERATIONS – 
Deferoxamine mesylate is approved by the Federal Drug Administration (FDA) for the treatment of acute iron intoxica-
tion and chronic iron overload and has been extensively used in clinical practice for more than 40 years. Safety studies 
indicate that it is relatively well tolerated. The following adverse reactions have been observed, but there are not 


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enough data to support an estimate of their frequency:  At the injection site: localized irritation, pain, burning sensa-
tion, swelling, induration, infiltration, itching, erythema, or wheal formation. These injection site reactions may be as-
sociated with arthralgia, fever, headache, myalgia, nausea, or abdominal pain. Systemic allergic and hypersensitivity 
reactions: rash, urticaria, anaphylactic reaction with or without shock, or angioedema. Cardiovascular system: tachy-
cardia, hypotension, and shock.  Impairment of cardiac function has also been reported in patients with severe chronic 
iron overload following concomitant treatment with DFO and vitamin C in excess of 500 mg per day. Digestive system: 
abdominal discomfort, nausea, vomiting, or diarrhea. Hematologic system: thrombocytopenia and leucopenia.  Muscu-
loskeletal system: muscle cramps. Nervous system: dizziness, paresthesias, tinnitus, high-frequency sensorineural hear-
ing loss, or visual disturbances, such as blurred vision, decreased visual acuity, pigmentary retinopathy, visual field de-
fects, and cataracts. The ocular and auditory disturbances were reported when DFO was administered over prolonged 
periods of time, at high doses, in patients with low ferritin levels. These disturbances were reversible upon immediate 
cessation of treatment, in most cases. Respiratory system: acute respiratory distress syndrome has been reported fol-
lowing treatment with excessively high doses of intravenous DFO in patients with acute iron intoxication or thalasse-
mia. Urinary system: dysuria and impaired renal functions. In addition, rare infections, such as Yersinia and Mu-
cormycosis have been reported in patients with iron-overload. These are thought to be attributed to iron-induced in-
creased susceptibility to infections particularly in patients with low ferritin levels, and are unlikely to occur in patients 
without iron overload. In a recent study, the most frequent adverse events of DFO (40-50 mg/kg/day, 5 days/week, for 
48 weeks) in adult patients with transfusion related iron overload were abdominal pain (34%), diarrhea (26%), fever 
(26%), joint pain (13%), vertigo (13%), and dyspepsia (8.7%) [Piga 2006]. Other less frequent adverse effects of DFO 
were local reactions at the site of injection, such as pain, swelling, or erythema. There were no serious adverse events 
related to DFO use, and the gastrointestinal symptoms were mild and resolved spontaneously within a few days with-
out drug interruption.  
Serious adverse effects are uncommon and are seen primarily with IV doses higher than 125 mg/kg/day, daily doses ex-
ceeding 6000 mg, and chronic long-term use [Porter, 1989]. Hypotension and shock have been reported in up to 2% of 
patients using IV DFO [Westlin 1971]. They were mostly seen with rapid intravenous infusions, and rarely reported when 
the drug is given at a rate ≤1 mg/kg/minute, and a dose ≤125 mg/kg/day [Westlin 1971; Porter 1989]. We will use a slow-
er infusion rate ≤7.5 mg/kg/hour and limit the maximal dose that any subject can receive to 6000 mg per day in the cur-
rent study. 
Small studies have investigated the use of DFO in patients without systemic iron overload who have similar co-
morbidity profile to that of stroke patients, such as patients with coronary artery disease, diabetes mellitus, and elderly 
subjects with Alzheimer’s disease [Duffy, 2001; Hattori, 2002-2003; Crapper, 1991], and healthy volunteers [Allain; 
1987]. The doses used varied from 10 to 80 mg/kg body-weight-adjusted or 250 to 500mg fixed-dose regimens, and the 
duration of treatment varied from a single treatment to repeated daily dosing up to one year.  Previous studies in cardi-
opulmonary bypass patients and in thalassaemia patients with cardiac disease used IV DFO doses of 50 to 100 mg/kg 
[Tamary 1994; Davis, 2000; Ioannis, 2004]. Gordeuk et al. also used DFO infusions (100 mg/kg per day for 72 hours) in 
non-iron overloaded volunteers with asymptomatic Plasmodium falciparum parasitemia and patients with cerebral ma-
laria [Gordeuk, 1992].  These various dose regimens resulted in no serious adverse events to treatment in these studies.  
Because DFO is not approved by the FDA for the treatment of patients with ICH, the primary investigator was granted a 
research (non-commercial) Investigational New Drug (IND) by the FDA to investigate the use of DFO in this patient  
population (IND #77,306). The supporting data section below (section 2.2.4) details our experience with DFO use in ICH 
patients. Our Phase I study indicated that repeated IV infusions of DFO in doses up to 62 mg/kg/day with a maximum 
daily dose of 6000 mg/day in patients with ICH were largely safe and tolerable. The most observed adverse events were 
injection site reaction (irritation, pain, or erythema; 15%), IV infiltration (20%) and a modest decrease in blood pressure 
which did not require any medical intervention (40%). While hypotension may be an undesirable side effect in patients 
with ischemic stroke, a modest reduction in blood pressure may prove to be beneficial, and is often advocated, in  


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patients with ICH. One subject developed visual hallucination during IV infusion of DFO. Almost all of these adverse 
events were mild, self-limited, did not require specific treatment, and resolved spontaneously. There were no deaths or 
serious adverse events related to DFO use.  
In the first part of phase II investigation of DFO in ICH (HI-DEF trial, where subjects were treated with a continuous infu-
sion of DFO at 62 mg/kg/day for 5 consecutive days), however, we observed 7 cases of ARDS among the first 42 partici-
pants; 6 of these cases were in the DFO treatment arm, and 2 of these were fatal. An expert blinded to treatment as-
signment reviewed all these cases and concluded that a plausible cause for ARDS, other than the study drug or ICH it-
self, was identified in 4 cases, while no other explanation, other than the study drug or ICH, was apparent for the re-
maining 3 cases. HI-DEF was terminated due to safety concerns, given the imbalance in the frequency of ARDS be-
tween the DFO- and placebo-treated groups.  This prompted further review of 2 cases of respiratory failure, reported as 
the result of aspiration pneumonia, in Phase I study; both in the 62 mg/kg/day dose-tier. An expert review concluded 
that one of these cases was an undiagnosed case of ARDS, and that aspiration was a plausible explanation for it. We 
have undertaken considerable precautions to minimize the potential pulmonary toxicity of DFO and ARDS risk and to 
enhance the safety of future participants in the current modified protocol by reducing the daily dose of DFO from 62 
mg/kg/day to 32mg/kg/day, decreasing the duration of treatment from 5 to 3 days, and excluding subjects at high risk 
for ARDS. 
The effects of DFO on the fetus are unknown. It is also not known if DFO is excreted in human milk.  Therefore, women 
who are pregnant and those who are breast-feeding will be excluded from this study. 
2.1.3.3 NEUROPROTECTIVE EFFECTS OF DFO –  
By forming a stable complex with ferric iron, DFO decreases free iron’s availability for the production of hydroxyl radi-
cals. DFO also alters iron regulatory genes and proteins binding activity, thereby reducing cellular vulnerability to iron 
[Chen 2010; Messer 2010]. However, DFO has multiple and diverse neuroprotective properties, which may be only part-
ly related to its iron chelating abilities. DFO also prevents apoptosis induced by glutathione depletion and oxidative 
stress in embryonic cortical neuronal cultures by activating a signal transduction pathway leading to activation tran-
scription factor 1/cAMP response element-binding protein (ATF-1/CREB) and hypoxia inducible factor (HIF-1), and ex-
pression of genes known to compensate for oxidative stress [Zaman 1999]. It inhibits prolyl 4-hydroxylase activity, 
which may lead to protection from oxidative stress-induced cell death [Siddiq 2005; Ratan 2008]; induces transcription 
of heme oxygenase-1; suppresses the upregulation of activated c-Jun N-terminus kinase (JNK) seen after ICH [Wan 
2009]; exerts anti-inflammatory effects by stimulating cyclooxygenase [Tanji 2001]; blocks the neurotoxic effects of 
hemoglobin via inhibition of glutamate-mediated excitotoxicity [Regan 1996]; and exerts anti-phagocytic effects in an-
imal models of ICH [He 2008]. Our phase I study also suggests that DFO has a modest blood pressure lowering effect 
when administered by IV infusion, which might be beneficial in patients with ICH [Anderson 2008; Suri 2008].  
2.2 
SUPPORTING EVIDENCE 
2.2.1 
SUPPORTING EVIDENCE THAT DFO ATTENUATES NEURONAL INJURY AFTER ICH 
2.2.1.1 IN VITRO STUDIES  
Several studies have shown that DFO can reduce hemoglobin-induced neurotoxicity in experimental models of ICH. 
Regan and Rogers [Regan, 2003] showed that delayed treatment with DFO markedly attenuates the production of re-
active oxygen species and neuronal death induced by adding hemoglobin to mixed neuronal/astrocyte cell cultures. In 
another study, Regan and Panter [Regan, 1993] showed that DFO completely blocked hemoglobin-induced neuronal 
death in neocortical cultures derived from fetal mice. Regan and Panter [Regan, 1996] also showed that hemoglobin 
potentiates the neurotoxicity of glutamate agonists in primary murine cortical cultures, and that this effect was  


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attenuated by DFO. Goldstein et al [Goldstein, 2003] showed that treatment with DFO significantly reduces the pro-
duction of reactive oxygen species and cell death induced by hemin in human neuron-like cells. Similarly, Levy et al 
[Levy, 2002] showed that DFO diminishes hemin-induced cell death in pheochromocytoma (PC12) and neuroblastoma 
(SH-SY5Y) cell lines.       
2.2.1.2 IN VIVO STUDIES  
Bilgihan et al [Bilgihan, 1994] studied the effects of DFO on lipid peroxidation and Na-K ATPase activity after experi-
mental ICH in guinea pigs, and found that DFO treatment reduces brain malondialdehyde content and induces recovery 
of Na-K ATPase activity, suggesting that DFO can exert potential neuroprotective effects to counteract the deleterious 
effects of ICH. Pelit et al [Pelit, 2003] examined the effects of systemic treatment with DFO on the pathological chang-
es in the optic nerve after experimental retrobulbar hematoma in rabbits, and were able to detect ultrastructural 
changes and abundant iron pigment accumulation in the orbital fat tissue following induction of hematoma. These mi-
croscopic changes were significantly less pronounced in DFO-treated rabbits. Huang et al [Huang, 2002] examined the 
effects of intraperitoneal administration of DFO (500 mg/kg) on brain edema in rats pre-treated with hemoglobin deg-
radation products via stereotactic infusion into the brain; they found that DFO significantly attenuated the brain edema 
induced by hemoglobin and its breakdown products. In similar studies, Nakamura et al [Nakamura, 2004] investigated 
the effects of repetitive administration of DFO (100 mg/kg intraperitoneally every 12 hours), starting 2, 6, and 24 hours 
after induction of ICH in rats, on markers of DNA oxidative damage and repair. They found that treatment with DFO 
ameliorated ICH-induced changes in 8-hydroxyl-2'-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage, and 
increased levels of apurinic/apyrimidinic endonuclease/redox effector-factor 1 (APE/Ref-1), a protein involved in DNA 
repair following oxidative damage, indicating that DFO may be a potential therapeutic agent for ICH by reducing the 
oxidative stress caused by the release of iron from the hematoma. 
Several experimental studies investigated whether DFO can reduce ICH-induced brain injury, by examining its effects 
on brain edema, severity of neurological deficits, and performance on sensorimotor behavioral tests [Nakamura, 2003-
2004; Wan 2006-2009; Gu, 2009; Okauchi 2009]. In a piglet ICH model, ICH resulted in development of a reddish peri-
hematoma zone, and iron accumulation, ferritin upregulation, and neuronal death within that zone, which were all re-
duced by intramuscular administration of DFO at 50 mg/kg bid for 3 or 7 days after ICH [Okauchi 2009] (Figure 2.2.1.2- 
A-C).
Figure 2.2.1.2- A: Deferoxamine (DFX) reduces the 
reddish zone around the hematoma after 3 and 7 
days of treatment in a pig model of ICH.  


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 Figure 2.2.1.2- B 
 Figure 2.2.1.2- C 
Figures 2.2.1.2- (B-C):  B shows iron histochemistry (Perls staining) and ferritin immunoreactivity in the brain af-
ter ICH (* in D & E indicates the hematoma; inserts in C & F indicates hematoxylin and eosin staining).  C shows 
Fluoro-Jade C-positive cells, which indicates neuronal degeneration, in the perihematoma area [A-C]; D shows 
4 sampled field for Fluoro-Jade C cell counting); and Luxol fast blue staining of the white matter [E & F].  Cour-
tesy of Dr. Guohua Xi.  
2.2.2 
SAFETY AND TOLERABILITY OF DEFEROXAMINE IN PATIENTS WITH ACUTE CEREBRAL HEMORRHAGE 
(R01 NS 057127) 
OVERVIEW AND OBJECTIVES - This was a prospective, open-label, multiple-tier, dose-finding, multi-center, preliminary 
clinical study to evaluate the safety and tolerability of repeated treatments with DFO in patients with ICH. The study 
was planned to evaluate DFO dose-tiers ranging from 7 to 125 mg/kg/day (up to a maximum dose of 6000 mg/day) us-
ing the Continual Reassessment Method (CRM). The initial dose of the drug was administered within 18h after stroke 
symptom onset by IV infusion at a rate of 7.5 mg/kg/hour and repeated daily for 3 consecutive days. Four clinical sites 
participated in the study (Beth Israel Deaconess Medical Center, Massachusetts General Hospital, Medical College of 
Wisconsin, and Hartford Hospital). The Division of Biostatistics and Epidemiology at the Medical University of South 
Carolina served as the Statistics and Data Management Center for the study.  Subject recruitment began in July 2008.  
THE PRIMARY OBJECTIVES were: 1) To evaluate the safety and tolerability of repeated IV infusions of DFO in patients with 
spontaneous ICH; and 2) To determine the maximum tolerated dose (MTD) of DFO (highest dose regimen ≤ 125 mg/kg 
that can be safely administered and tolerated) in patients with ICH to be adopted in subsequent studies to test the effi-
cacy of DFO in ICH.  
Enrollment into every dose tier, starting with 7 mg/kg daily for 3 days, was completed before enrollment into the next 
tier began. Each subsequent dose level was determined using the Piantadosi-modified CRM (Piantadosi 1998). The 
dose-toxicity curve was re-estimated based on safety data generated throughout the study. At least 3 patients were 
enrolled in each dose-tier. The safety information, guiding transition from one dose tier to the next, was based on the 
number of subjects in a cohort who experienced pre-defined dose-limiting toxicities (DLT) during the first 7-day period 
or until discharge, whichever occurred first, following initiation of DFO treatment. The acceptable probability of DLT  


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was set at 0.40. The dose-limiting toxicity (DLT) was pre-defined as any of the following adverse events, fatal or non-
fatal, occurring within 7 days or until discharge, whichever occurs first, of treatment initiation: 1) Anaphylaxis (at any 
time point during DFO infusion); 2) Hypotension (defined as a decrease in SBP > 20 mm Hg or DBP > 10 mm Hg, or a 
SBP ≤ 85 mm Hg, confirmed by 3 consecutive readings, requiring medical treatment at any time point during DFO infu-
sion that cannot be explained by other causes); 3) Worsening neurological status (defined as an increase ≥ 4 points on 
NIHSS, or a decrease of ≥ 2 points on GCS, that cannot be explained by other causes, compared to baseline values oc-
curring at any point during DFO infusions); 4) Mortality within 7 days of hospitalization; and 5) Any adverse event pro-
longing hospital stay, resulting in emergent medical therapy, or resulting in death. All of these events were considered 
SAEs for the purpose of this study. Patients underwent repeated clinical (neurological, visual and auditory, and general 
physical examinations) and laboratory evaluations at regular intervals throughout their participation in the study, from 
the day of enrollment until day 90, for adverse events. The 30-day neurological and functional status was determined 
by NIHSS, extended Glasgow outcome scale (eGOS), Barthel index (BI), and mRS. The patients or their surrogates were 
contacted by phone on day 90±7 days to compute mRS, eGOS, and BI, and to assess 3-month mortality.  
STUDY STATUS AND RESULTS: Enrollment was completed in January 2010; the last subject completed the 90-day as-
sessment in April 2010. A total of 20 subjects were enrolled into 5 DFO dose tiers, ranging from 7 mg/kg/day to 62 
mg/kg/day. Because the recommended subsequent dose was within 5% (a pre-specified convergence criterion) of the 
62 mg/kg/day dose, the study was concluded with the 62 mg/kg/day dose as the recommended MTD.  
Number of subjects enrolled by dose-tier: 7 mg/kg = 4; 32 mg/kg = 3; 47 mg/kg = 3; 57 mg/kg = 4; 62 mg/kg = 6. An addi-
tional cohort of 3 subjects was treated at 62 mg/kg once it was determined to be the MTD to ascertain the safety and 
tolerability of this dose before proposing its use in the current study). The detailed results are included in Appendix I. 
The main results are summarized below.  
SAFETY DATA: Sixteen non-serious adverse events were possibly or probably related to the study drug. The most 
common were injection site irritation and IV infiltration. Six subjects experienced 12 SAEs and 3 subjects had 4 DLTs. 
Three subjects (15%) died during the 90-day follow-up period: one subject died in hospital within 7 days of ICH onset 
and two between 7-30 days after ICH onset. None of the SAEs, DLTs, or mortalities was adjudicated to be related to the 
study drug. There were 2 cases of respiratory failure; both in the 62 mg/kg/day dose-tier and were thought to be related 
to aspiration pneumonia and adjudicated as being unrelated to the study drug. However, a recent expert review found 
that one of these cases was an undiagnosed ARDS, and agreed that aspiration was a plausible explanation for it.       
PHYSIOLOGICAL DATA: Laboratory data - There were no differences in routine laboratory values and in the inci-
dence of abnormalities and change from baseline in EKG parameters. Overall, daily DFO infusions at a rate of 7.5 
mg/kg/hour up to 62 mg/kg/day (up to a maximum of 6000 mg/day) were well tolerated without significant alterations 
of hemoglobin or hematocrit, hematological parameters, and renal or hepatic functions. Treatment with DFO for 3 con-
secutive days at all 5 dose-tiers did not result in iron deficiency. Overall, the median values of the change in iron param-
eters from baseline to after the 3rd DFO infusion were as follows: serum iron (3.5 ug/dl; 95% CI -23, 45); transferrin (-18 
ng/ml; 95% CI -43, 5); total iron binding capacity (8.5 ug/dl; 95% CI -47, 30); and ferritin (32.5 ng/ml; 95% CI 6, 74). We 
found no relationship between DFO dose and changes in serum iron studies. Vital signs (heart rate; respiratory rate; 
oxygen saturation; and temperature) - Overall, DFO administration did not result in important alterations in these vari-
ables. Blood pressure: DFO had a moderate BP-lowering effect. Overall, the median mean BP at baseline was 95.8 
mmHg (95% CI 84, 100.3) vs. 93.2 mmHg (95% CI 87.6, 98) during the infusions. A total of 8 patients (40%) experienced 
a maximal drop in mean BP >20% (median 0.29; 95%CI 0.27, 0.39) at some point during the infusions compared to 
baseline values. One subject required vasopressors; his hypotension was thought to be related to intubation and anes-
thesia. None of the remaining 7 subjects required any medical treatment.       
RADIOLOGICAL DATA: We found no radiological evidence to suggest that treatment with DFO exacerbates the 
natural enlargement of ICH or PHE over 72h after treatment. The overall median change in relative PHE volume from 
screening to post-3rd DFO infusion was 0.48 (95% CI 0.10, 0.76) and from screening to day-7 or discharge (whichever  


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occurred first) was 0.87 (95% CI 0.51, 1.28). These exploratory results contrast with previous studies of the natural histo-
ry of relative PHE evolution [Mehdiratta 2008; Gebel 2002], which showed that the relative PHE volume almost doubles 
by 72h.       
FUNCTIONAL OUTCOME DATA: Two subjects withdrew consent, one after the 7-day visit (mRS =5), and one after 
the 30-day visit (mRS =1). Among the remaining 18 subjects, 9 subjects (50%) had mRS scores of 0-2; 2 (11%) had a 
score of 3; and 7 (39%) had scores of 4-6.The 90-day mortality rate was 15%.   
CONCLUSIONS: Repeated daily infusions of DFO at doses up to 62 mg/kg/day (up to a maximum of 6000 mg/day) in pa-
tients with acute spontaneous ICH for 3 consecutive days after ICH onset are feasible, well tolerated and do not increase 
serious adverse events or mortality.    
2.2.3 
HIGH DOSE DEFEROXAMINE IN INTRACEREBRAL HEMORRHAGE (HI-DEF IN ICH)  
(U01 NS074425) 
OVERVIEW AND OBJECTIVES - Part 1 of the current study was a prospective, multi-center, double-blind, randomized, pla-
cebo-controlled, phase-II clinical trial. Subjects were randomized to either DFO at 62 mg/kg/day (up to a maximum dai-
ly dose of 6000 mg/day), or saline placebo, given by continuous IV infusion for 5 consecutive days. Treatment was initi-
ated within 24 hours after ICH symptom onset. Randomization controlled for baseline imbalances associated with base-
line ICH score (0-2 vs. ≥3), ICH onset-to-treatment time (OTT) window (≤12h vs. >12-24h), and concurrent warfarin use. 
All subjects were followed for 3 months and received standard of care therapy while participating in the study. 
THE MAIN OBJECTIVES WERE: 1- TO assess whether it is futile to move DFO forward as a therapeutic intervention for ICH 
into Phase III evaluation, by comparing the outcome of DFO-treated subjects to placebo-treated subjects with respect 
to good outcome (defined as mRS of 0-2 at 90 days), in a futility analysis; and 2- TO assess the safety of DFO infusions 
(at a dose of 62 mg/kg/day, up to a maximum daily dose of 6000 mg/day), given for 5 consecutive days, in a large cohort 
of ICH patients. We specifically wished to collect more data on treatment-related adverse events in order to ascertain 
that patients with ICH could complete this dose given over 5-day duration of infusion without experiencing unreasona-
ble neurological complications, mortality, or other serious adverse events related to DFO use. 
STUDY STATUS AND SAFETY RESULTS - Throughout the study, we continued to assess the safety of DFO. Enrollment was 
placed on hold after enrollment of 42 subjects to investigate a potential safety concern – Adult Respiratory Distress 
Syndrome (ARDS). ARDS is reported as a potential side effect of prolonged intravenous infusions of high dose DFO in 
the product’s package insert. Five cases of ARDS were reported. An expert in ARDS, blinded to treatment assignments, 
reviewed all 5 cases as well as 3 cases reported as respiratory failure and 3 cases reported as pulmonary edema. The ex-
pert concluded that 2/3 cases reported as pulmonary edema were possibly/probably ARDS, suggesting a total of 7 
ARDS cases. The expert review identified a plausible cause for ARDS, other than the study drug or ICH itself, in 4 cases, 
while no other explanation was apparent for the remaining 3 cases. After careful review of the data, the DSMB unblind-
ed the investigators to the treatment assignment. Six of the 7 cases of ARDS occurred in the DFO-treated group. Three 
of the 42 enrolled subjects died (7%); all had ARDS, and the cause of death was attributed to ARDS in 2 subjects. No 
other safety concerns emerged.  A total of 36 SAEs were reported in 15 subjects; 22 of which occurred in 9 DFO-treated 
subjects. One MedWatch report was submitted for hypophosphatemia as it was judged to be severe, unexpected, and 
possibly related to the study drug. Overall, hypophosphatemia was reported as an AE in 3 subjects (2 in the DFO- and 1 
in the placebo group).  Other analyses are yet to be performed. 
CONCLUSIONS - The NINDS-appointed DSMB concluded that: “although the ARDS cases in the treatment group were in 
the ballpark frequency of at least one paper in the literature on ARDS in patients with ICH [Elmer 2013], the imbalance 
in the frequency of ARDS cases between the treatment and placebo groups suggested that pulmonary toxicity of the  


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drug was highly likely”. HI-DEF was therefore terminated due to safety concerns, and the initial protocol has been mod-
ified to minimize the potential for pulmonary toxicity of DFO and to enhance the safety of future participants. 
2.3 
SUMMARY OF BACKGROUND AND RATIONALE 
Collectively, the above data indicate that: 1) extensive preclinical investigations, in vitro and in vivo, by different inves-
tigators and in different species, show that treatment with DFO confers neuroprotection after ICH; 2) although DFO 
may work directly by chelating iron, it also has several other neuroprotective properties which can be beneficial after 
ICH;  3) repeated daily administrations of IV infusions of DFO to ICH patients is feasible and largely well-tolerated; and 
4) the potential for pulmonary toxicity and ARDS is higher in ICH patients treated with continuous IV infusions of DFO
at  62 mg/kg/day (up to a maximum total dose of 6000 mg/day), for 5 consecutive days; a lower dose and shorter dura-
tion of treatment might help to ameliorate this toxicity to improve the benefit/risk ratio.
These findings indicate that DFO is a potentially promising candidate intervention to target the secondary effects of 
ICH, and provide a rationale for its therapeutic use to improve the overall outcome in patients with ICH, particularly if 
the safety concerns encountered in part 1 (HI-DEF) of this study can be ameliorated by lowering the daily and total dos-
age of DFO and improved selection of subjects to exclude those at high risk for ARDS.  Because hemoglobin degrada-
tion starts hours-days after ICH onset, the potentially delayed and slow pace of hemoglobin- and iron-mediated injury 
may facilitate treatment at a delayed time window after ICH onset. This could extend the potential utility of DFO as a 
therapeutic intervention to a large proportion of ICH patients. DFO is relatively inexpensive and is likely to be a highly 
cost-effective therapy for ICH and complementary to ongoing efforts targeting hematoma and its expansion. We gen-
erally hypothesize that treatment with DFO would minimize ongoing neuronal injury after ICH, via several diverse 
mechanisms, and would improve the outcome in these patients. As a prelude to test this hypothesis, this Phase II study 
will assess the futility of DFO as a therapeutic intervention in ICH before embarking on a large Phase III trial. In addition 
to allowing us to assess the futility of moving DFO forward to phase III efficacy testing as a potential therapeutic inter-
vention in ICH, this study will allow us to gain experience with logistical aspects of trial conduct, and the data and re-
sults from this study will be used to guide our planning for a possible future Phase III study.  
2.4 
SIGNIFICANCE 
This study will provide a crucial “go/no-go” signal to determine if embarking on a large-scale, costly phase III trial to in-
vestigate the efficacy of DFO as a treatment for ICH is worthwhile. This study will also provide important information to 
guide the planning and conduct of a future phase-III trial, if it is determined that the treatment with DFO is not futile. It 
will allow us to gain experience with logistical aspects of trial conduct, such as blinding and randomization, prior to 
phase-III testing. Furthermore, results from this study can provide valuable information regarding the dichotomized 
mRS outcome rate among control subjects, the potential for a differential treatment effect in the ≤12 vs. >12-24 hour 
time windows, and appropriate inclusion and exclusion criteria (HI-DEF has been already informative in this regard); 
information which can guide the design of a potential future Phase III trial. In addition, we plan to collect data on 
various outcome scales (mRS, NIHSS, SIS-16 [Duncan 2003], and MoCA [Pendlebury 2010]), which will allow us to 
explore the utility of these various outcome measures in phase-III.  
If successful, this study can potentially result in new means to improve the outcome of patients with ICH. A successful 
study demonstrating the efficacy of DFO in ICH would be of considerable significance to the field and the society. A 
wealth of data, generated from various planned exploratory analyses in this study, can still help to advance our 
knowledge and understanding of the pathophysiology of ICH, such as the relationship between PHE volumes and out-
come and the relationship between ICH and cognitive function, even if DFO is found to be futile.  These additional anal-
yses can generate novel hypotheses for future investigations in ICH. The potential advantage of novel treatments for 
ICH and better understanding of its pathophysiology and relationship to outcome with regard to patients' welfare, pub-
lic health and cost containment is noteworthy. 


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3 
STUDY DESIGN 
3.1 STUDY OBJECTIVES 
THE STUDY HAS TWO PRIMARY AIMS: 1- To determine if it is futile to move DFO forward to Phase III evaluation, using mRS 
0-2 at 90 days as the outcome based on an absolute difference in treatment effect ≥12% in favor of DFO; and 2- To fur-
ther assess the safety of DFO infusions, at a dose of 32 mg/kg/day, given over a consecutive 3-day period, in particular
serious adverse events including ARDS.
THE SECONDARY AIMS are to: 1- Explore the differences between early (≤12h) and late (>12-24h) time windows in DFO 
treatment effect on functional outcome; 2- Determine the overall distribution of ordinal scores on mRS at 3 months in 
DFO- and placebo-treated subjects, and to perform a dichotomized analysis considering the proportion of DFO- and 
placebo-treated subjects with mRS 0-3; 3- Perform dichotomized analyses considering the proportion of DFO- and pla-
cebo-treated subjects with mRS 0-2 and 0-3; and to determine the overall ordinal distribution of scores on mRS at 6 
months; 4- Determine mortality during the 180-day follow-up period (all causes and ICH-related); and 5- Obtain data on 
the changes in NIHSS between presentation and day-90, and MoCA and SIS-16 scores at 3 months to explore the ef-
fects of DFO on neurological, functional, and cognitive functions.   
ADDITIONAL PLANNED EXPLORATORY ANALYSES include assessments of: 1- The effects of treatment with DFO on relative 
PHE volume progression between admission (i.e. screening) and post-treatment CT scans in DFO-treated patients as 
potential markers of DFO’s biological activity on brain tissue [Gebel 2002; Mehdiratta 2008; Leira 2004; Okauchi 2009-
2010]; 2- Whether the effect of DFO on outcome is dependent on initial ICH volume, after adjusting for other prognos-
tic variables, to determine if specific limits for ICH volume should be specified as exclusion/inclusion criteria for future 
studies; 3- The effects of DFO on the size of ventricular enlargement in patients with intraventricular extension of ICH , 
not requiring EVD, as a potential marker of treatment utility in IVH [Chen 2011]; 4- The effect of treatment with DFO on 
incidence of symptomatic cerebral edema (unexplained increase in NIHSS >4 points or decrease in GCS >2 points) dur-
ing hospitalization, up to day 7 or discharge whichever is earlier; and 5- Whether progression of PHE can be a radiologi-
cal/biological marker of activity which can be correlated with clinical outcomes and treatment effect of DFO. 
3.2 OVERVIEW OF STUDY DESIGN 
This is a prospective, multi-center, double-blinded, randomized, placebo-controlled, phase-II clinical trial. The total 
sample size required is 294 subjects with ICH. Subjects will be randomized in a 1:1 ratio to receive either DFO at 32 
mg/kg/day (up to a maximum daily dose of 6000 mg/day), or a matching saline vehicle (placebo), given by IV infusion 
for 3 consecutive days, in a blinded manner. Treatment will be initiated within 24 hours after ICH symptom onset. Sub-
jects will undergo repeated clinical, imaging, and laboratory evaluations at regular intervals throughout their participa-
tion in the study, from the day of randomization until day 180. All adverse events will be assessed until day-7 or dis-
charge (whichever is earlier), and new SAEs until day-90. Mortality and continuing SAEs will be assessed until day 180. 
Functional status will be determined by mRS scores, in person at 30±7 and 90±7 days; the patients or their surrogates 
will be contacted by phone on days 60±7 and 180±7 to assess mRS [Merino, 2005]. 
3.3 RATIONALE FOR STUDY DESIGN AND PROTOCOL MODIFICATIONS 
The rationale for the proposed dose regimen, therapeutic time window, duration of treatment, study design, inclusion 
of a placebo group, and surrogate measures of DFO efficacy is as follows: 


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Dose Reduction and New Dose Selection: The 62 mg/kg/day dose was identified in our Phase I study as the MTD in 
ICH subjects and was the dose used for the 42 participants enrolled in HI-DEF. This was based on safety data from the 
phase I study, where DFO did not appear to increase the overall rate of SAEs or mortality compared with placebo-
treated patients in previous ICH trials [Mayer 2005; Mayer 2008; Lyden 2007; Haley 2005], and the notion that the effi-
cacy of a drug often has a monotonically non-decreasing dose-response relationship. However, the observation of 6 
cases of ARDS among DFO-treated participants in HI-DEF, and the reported association of prolonged high-dose intra-
venous infusions of DFO with the development of ARDS [Tenenbein 1992] prompted us to reduce the dose to 32 
mg/kg/day (up to a maximum daily dose of 6000 mg per day) in the current protocol to maximize safety and minimize 
harm. Pre-clinical studies in aged rats compared 3 dose regimens of DFO; 10 mg/kg, 50 mg/kg, and 100 mg/kg every 12 
hours [Okauchi 2009]. Based on mass constant conversion factors and the FDA guidance for the industry for estimating 
the human equivalent dose (HED) in clinical trials, the calculated HEDs are approximately 3, 16 and 32 mg/kg/day, re-
spectively [http://www.fda.gov/downloads/Drugs/GuidanceCompliance RegulatoryInfmation/Guidances/ ucm078932. 
pdf]. The latter dose regimens (HEDs 16 and 32 mg/kg/day) were superior to the first dose regimen in improving per-
formance on sensorimotor behavioral tests at days 28 and 58 following induction of experimental hemorrhage. We now 
opt for a dose of 32 mg/kg/day based on the following considerations: 
I.
This dose, while assuring increased tolerability and safety of the drug, represents the upper limit of effective doses in
animal models - Data from rat models of ICH indicate that HEDs ranging from 16 to 32 mg/kg/day are effective in
reducing brain edema formation and brain tissue atrophy, and improving neurological and functional recovery
[Okauchi 2009].
II.
This dose provides a better safety margin - No cases of respiratory failure or distress were reported in any of the 10
participants who received DFO in doses ranging from 32 to 57 mg/kg/day in our Phase I study (32 mg/kg/day, n=3; 47
mg/kg/day, n=3; 57 mg/kg/day, n=4).
III.
Equivalent dose in piglet model of ICH exerts biological activity on the brain tissue. It reduced the perihematomal
reddish zone, white matter injury, iron accumulation, ferritin upregulation, and neuronal death within that zone [Gu
2009].
The 6000 mg/day limit, irrespective of body weight, is based on FDA recommendations and the manufacturer’s bro-
chure to avoid the risk of serious adverse effects of higher dose-regimens.  
TIME-TO-TREATMENT WINDOW: The decision to initiate DFO treatment up to 24h after ICH symptom onset and not be-
yond, and to balance randomization based on OTT, is based on data from animal studies. Okauchi et al [Okauchi 2010] 
examined the effects of DFO when administered within 2h, 4h, 12h, 24h, and 48h after the induction of ICH on brain 
edema formation, forelimb placing and corner turn scores, and brain atrophy in aged rats. They found that: 1) Regard-
less of the time-to-treatment window, DFO improved forelimb placing and corner turn scores compared to vehicle 
treatment, and the differences in rats’ performance of these tests between these various time points were not signifi-
cant; 2) DFO effects on functional recovery, however, were seen faster when the treatment was begun within 24h as  
opposed to 48h (28 days vs. 56 days); and 3) DFO reduced brain tissue loss and atrophy when administered within 24h 
after induction of ICH compared to 48h delayed treatment. The faster improvement of function noted in these studies 
is suggestive of greater efficacy for earlier treatment and might also translate into potentially significant cost savings 
from reduction in healthcare related expenses and earlier ability of the patients or their caregivers to return to work and 
avoid lost wages. In addition, very few patients in the phase I study presented more than 24h after ICH onset, indicating 
that only few additional patients are likely to be enrolled by extending the OTT beyond 24h at this stage.  


 IDEF  
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22
Conversely, animal data do not strongly support limiting treatment to an earlier time window, such as within 12h. Alt-
hough Okauchi et al [Okauchi 2010] reported that the optimal therapeutic window of DFO to reduce PHE formation 
was about 12h, the correlation between PHE and deficits on sensorimotor behavioral tests weeks-to-months after ICH 
was weak, which they attributed to the sensitivity of their assays. Previous studies have shown that PHE formation cor-
relates with forelimb placing during the acute phase of ICH [Hua 2002]. A serial MRI study of the natural history of PHE 
in patients with ICH reported similar results; PHE correlated with worsening neurological status at 48h, but not 3-month 
functional outcome [Venkatasubramanian 2011]. Indeed, the influence of PHE on long-term recovery of ICH patients is 
debatable [Inaji 2003; McCarron 1999; Sansing 2011; Sykora 2009; Zazulia 1999]. Therefore, restricting the time-to-
treatment to 12h or even less, for the sole purpose of its potential effect on PHE, seems unnecessary. In addition, very 
few patients presented within 12h in the phase-I study. Attempting to restrict the OTT window to less than 12h could 
limit recruitment into the study as well as the generalizability of the results to the wider ICH population. Therefore, OTT 
time is incorporated into the randomization algorithm in order to achieve an acceptable treatment balance. Similar to 
animal studies, this would also allow for further exploratory analysis to assess the effect of early vs. delayed treatment 
with DFO on PHE and outcome.    
DURATION OF TREATMENT:  Pre-clinical studies that examined the optimal duration of treatment with DFO following ICH 
produced variable results. In one study, treatment with DFO (HED ~ 16 mg/kg/day) for 2, 5, 7, or 14 days improved per-
formance compared with placebo on forelimb placing test at days 28 and 56 [Okauchi 2010]. However, residual neuro-
logical deficit was present on corner turn test in animals treated for less than 7 days, and the 7-day treatment was asso-
ciated with less adverse reactions than the 14 days, leading the investigators to conclude that the optimal duration of 
treatment in this model was 7 days. In other studies, treatment with DFO at HEDs of 16 and 32 mg/kg/day for 3 days 
resulted in significant improvement in rats’ performance on both forelimb placing and corner turn tests and reduced 
brain atrophy at 56 weeks compared with placebo [Okauchi 2009]. In piglets, treatment with DFO for 3 or 7 days had 
similar protective effects on perihematomal reddish zone, white matter injury, iron accumulation, ferritin upregulation, 
and neuronal death within that zone [Gu 2009]. In other experiments, treatment with DFO at 200 mg/kg/day for 3 or 7 
days after ICH also improved forelimb placing and corner turn scores and reduced brain edema [Nakamura 2003-2004]. 
Collectively, these results suggest that treatment with DFO for any duration ≥2 days results in improved functional per-
formance, compared with vehicle treatment.   
The phase I study assessed varying doses of DFO administered over a 3-day infusion period.  The 3-day infusion dura-
tion was chosen for the following reasons: 1- Edema growth is fastest in the first 2-3 days after ICH onset and this initial 
growth (and not the later one) is associated with neurological deterioration [Venkatasubramanian 2011; Inaji 2003; 
Zazulia1999]; 2- The time course for hemoglobin hemolysis and release of its degradation products including iron is ap-
proximately 2-3 days [Macdonald 2004]; and 3- Iron in the perihematoma tissue peaks at day 3 after experimental ICH 
[Wu 2003]. Extending the duration of treatment to 5 days in HI-DEF was based on the above data in aged rats [Okauchi 
2010]. The pulmonary toxicity of DFO is believed to be related to prolonged intravenous infusions of high doses of the 
drug, and a review of the 6 cases of ARDS in the DFO arm in HI-DEF revealed that the mean time from treatment to 
ARDS onset was ~ 78 hours, median ~ 76 hours. The longer duration of treatment might have contributed to increased 
pulmonary toxicity of the drug observed in HI-DEF, where 6/21 (28.6%) subjects in the treatment arm developed ARDS  
compared with 1/6 (16.7%) of patients who received the 62 mg/kg/day dose in phase I. Lowering the duration of treat-
ment would decrease the total dose of the drug and might help to minimize the pulmonary toxicity of the drug without 
compromising the potential effectiveness of the drug. Therefore, we chose to decrease the duration of treatment to 3 
days in this revised protocol.  


 IDEF  
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23
Overall, a 3-day treatment provides the potential for improved drug safety and tolerability without significantly jeop-
ardizing its efficacy.  The 3-day treatment with DFO was superior to saline vehicle in improving functional outcome in 
rat models of ICH [Okauchi 2010]; although iron release from the hematoma continues for days-to-weeks, the time  
course of iron release from the hematoma may not directly correlate with the timing of iron toxicity. With time, there is 
an up-regulation in endogenous iron chelators after ICH which may limit iron toxicity and the need for more extended 
treatment with DFO; and the 32 mg/kg/day dose proposed in part 2 of this study exceeds the animal dose (100 
mg/kg/day; HED 16 mg/kg/day) used in studies examining the optimal duration of DFO therapy after experimental ICH, 
and it is possible that the higher dosage and route of administration (IV as opposed to intramuscular in animal studies) 
might be as effective within a shorter treatment period.  
STUDY DESIGN AND PLACEBO GROUP: The futility design directly addresses the question of whether or not DFO at a dose 
shown to be effective in animal models of ICH given over a clinically feasible duration of 3 days is of sufficient promise in 
improving functional outcome after ICH to consider a large-scale and costly phase-III trial. The proposed futility design 
is logically consistent with the aims of Phase II trials, which are to weed out ineffective interventions. It is not a substi-
tute for phase-III and cannot by itself provide sufficient evidence to indicate that treatment with DFO is efficacious, 
since it is not designed or intended to test the efficacy hypothesis. Furthermore, non-futility in this phase-II study does 
not guarantee a positive phase-III result, which is important in preserving clinical equipoise for Phase III. Lastly, the futil-
ity hypothesis differs from hypotheses specified in traditional phase III efficacy trials and the probabilities of type-I and -
II errors are interpreted differently. In futility analysis, we are less concerned about falsely concluding an ineffective 
treatment is possibly effective.  
The advantage of the proposed two-arm design over a single-arm phase-II futility design is that it avoids the pitfalls as-
sociated with the use of historical data (temporal changes in other aspects of patient management, variations in data 
quality and protocol adherence, and differences in the eligibility criteria and specification of primary outcome measure 
between the various studies). These confounding variables can distort estimates of the historical reference proportion 
and are difficult to account for in a single arm study. Besides addressing these concerns, the inclusion of a placebo arm 
allows us to conduct secondary exploratory imaging analyses to determine the natural history of PHE progression and 
the effects of DFO on PHE as a surrogate marker of its biological activity on brain tissue. Furthermore, the proposed 
two-arm design allows us to configure and evaluate the logistics of randomization and blinding processes.  
CHOICE OF FUTILITY THRESHOLD: The Minimum Clinically Important Difference (MCID) in ICH is to some extent arbitrary 
and is largely derived from Traumatic Brain Injury literature, where demonstration of effectiveness was set at a 10% 
increase in the percentage of patients with favorable outcome in many clinical trials of head injury. Indeed, previous 
and ongoing Phase III trials in ICH, such as ATACH-2, used MCID of 10% or greater in the absence of data supporting 
that lower magnitude of difference will change practice patterns. For example, in the GAIN International and GAIN 
America Studies of the glycine antagonist, Gavestinel, the rate of mRS 0-2 was approximately 4%-to-5% higher in 
Gavestinel-treated patients, compared with placebo, but this difference was not considered encouraging to further de-
velop the drug as a therapy for ICH [Haley 2005]. We relied on data from previous ICH studies, the NINDS rt-PA trials in 
ischemic stroke, and outcome data from the phase-I study to derive a threshold for futility, based on mRS score of 0-2 
at 3 months, in the current study. In phase-II study of factor VIIa in ICH, the proportion of treated patients with mRS 0-2 
at 3 months was 12.8% to 17% (depending on the dose) greater than the placebo patients [Mayer 2005]. In the NINDS 
rt-PA trials, the proportion of rt-PA-treated patients with mRS scores of 0-2 at 3 months was 12% greater than their  
placebo-treated counterparts. Taking these numbers into consideration, the fact that no specific treatments exist to 
prevent disability after ICH, and the frequent observation of “winner’s curse” in previous stroke trials (a phenomenon by 
which effect size tends to be overestimated in pre-phase III trials, only to become smaller in larger Phase III trials which 
involve more sites and greater heterogeneity), a 12% difference in the proportion of DFO-treated subjects who achieve  


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24
favorable outcome compared with placebo was chosen as the threshold for futility in the current study. Although set-
ting the bar lower in this futility study may result in a more positive outcome favoring moving deferoxamine forward for 
Phase III evaluation, at the expense of a much larger sample size, the general consensus was that an effect size of 12% 
for the current proposal provides a balance between setting the bar too high (leading to rejection of a potentially effec-
tive therapy) or too low (leading to further testing of a marginally-effective mediocre therapy with its associated ex-
pense and resources, which could hinder the development of truly effective treatments for ICH). 
CHOICE OF EFFICACY OUTCOME MEASURE: The mRS was chosen for the primary outcome assessment because of its high 
inter-rater reliability and to be consistent with previous ICH trials. The decision to use a dichotomous outcome aims to 
increase the sensitivity of detecting meaningful differences by reducing the rate of misclassification or score assign-
ment. Favorable outcome, as opposed to poor outcome, was specified based on careful review of the putative benefi-
cial mechanism(s) of DFO and expected lack of an effect from treatment on reducing hematoma growth, a major pre-
dictor of mortality and significant disability after ICH [Davis 2006], as well as the phase-I outcome data. However, it is 
possible that DFO might result in an increase in the proportion of patients with mRS 0-3 (and hence a decrease in the 
proportion of patients with mRS 4-6) instead of mRS 0-2. Although mRS 0-3 is less favorable than the primary outcome 
of mRS 0-2, it would still be a desirable effect in patients with ICH given that no treatments exist to reduce disability. 
Therefore, the proportion of DFO- and placebo-treated subjects with mRS 0-3 at 90 days will also be evaluated. The 
trial is adequately powered to assess the alternative futility hypothesis using mRS 0-3 as the outcome based on an ab-
solute difference in treatment effect ≥13% in favor of DFO. Given increased acceptance of ordinal analysis of mRS in the 
stroke field and recognition that ICH patients may take longer to recover, we added ordinal analysis across all mRS 
scores and extended the follow up period to include assessment of mRS by phone at 6 months as secondary outcome 
measures. 
SURROGATE MEASURES OF DFO BIOLOGICAL ACTIVITY: We plan to examine the effect of treatment with DFO on relative 
PHE volume and progression at days 3-4 as a surrogate marker of biological activity on brain tissue. Delayed brain ede-
ma formation following the first 24h after ICH is related to red blood cells’ lysis and hemoglobin- and iron-mediated 
toxicity [Xi 1998/2002; Lee 1996; Qing 2009]. Treatment with DFO reduces brain edema, CSF free iron levels, iron dep-
osition in the perihematoma region, and residual cavity volume after experimental ICH [Nakamura 2003-2004; Wan 
2006-2009; Gu 2009; Okauchi 2009]. Human studies suggest that PHE continues to grow for weeks after ICH, and that 
edema progression is fastest during the first few days [Venkatasubramanian 2011]. Although the influence of PHE on 
long-term recovery after ICH is debatable [Hua 2002; Inaji 2003; Zazulia1999; McCarron 1999; Sykora 2009; Sansing 
2011], there is evidence that early edema progression correlates with early neurological deterioration [Venkatasubra-
manian 2011; Inaji 2003]. Therefore, secondary analyses will examine the effects of treatment on PHE immediately fol-
lowing completion of the infusion and not weeks later, since PHE growth during the early time period seems to be more 
clinically relevant. Regardless of the clinical impact of PHE, demonstrating an effect of DFO on this variable would at 
least provide secondary evidence of the agent’s biological activity under the chosen dosing regimen. The relative PHE 
volume (the absolute PHE volume divided by the hematoma volume) was chosen as it has been used previously to de-
tect serial changes in edema volume while adjusting for subsequent hematoma expansion or retraction [Gebel 2002; 
Mehdiratta 2008].    


 IDEF  
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25 
4 SELECTION AND ENROLLMENT OF SUBJECTS 
Study subjects will be recruited from the emergency departments or inpatient services at > 20 hospitals in North Ameri-
ca under the responsibility of the site investigators. Patients with diagnosis of ICH, as confirmed by CT scan, in whom 
the first dose of the study drug can be administered within 24h from symptom-onset (determined from the time the 
patient was last known to be without presenting deficits), will be prospectively enrolled, according to the eligibility cri-
teria. To minimize selection bias, consecutive patients who meet all eligibility criteria will be considered for enrollment. 
4.1 Inclusion Criteria 
1) Age ≥ 18 and ≤ 80 years
2) The diagnosis of ICH is confirmed by brain CT scan
3) NIHSS score ≥ 6 and GCS >6 upon presentation.
4) The first dose of the study drug is expected to be administered within 24h of ICH symptom onset (as described
above).
5) Functional independence prior to ICH, defined as pre-ICH mRS ≤1.
6) Signed and dated informed consent is obtained.
4.2 Exclusion Criteria 
1) Previous chelation therapy or known hypersensitivity to DFO products
2) Known severe iron deficiency anemia (defined as hemoglobin concentration < 7 g/dL or requiring blood transfusions
3) Abnormal renal functions, defined as serum creatinine greater than 2 mg/dl
4) Planned surgical evacuation of ICH prior to administration of study drug (placement of a catheter for ventricular
drainage is not a contraindication to enrollment)
5) Suspected secondary ICH related to tumour, ruptured aneurysm or arteriovenous malformation, hemorrhagic trans-
formation of an ischemic infarct, or venous sinus thrombosis
6) Infratentorial hemorrhage
7) Irreversibly impaired brainstem function (bilateral fixed and dilated pupils and extensor motor posturing)
8) Complete unconsciousness, defined as a score of 3 on item 1a of the NIHSS (Responds only with reflex motor or au-
tonomic effects or totally unresponsive, and flaccid)
9) Pre-existing disability, defined as pre-ICH mRS ≥2
10) Coagulopathy - defined as elevated aPTT or INR >1.3 upon presentation; concurrent use of direct thrombin inhibi-
tors (such as dabigatran), direct factor Xa inhibitors (such as rivaroxaban or abixapan), or low-molecular-weight heparin
11) Patients with confirmed aspiration, pneumonia, or evident bilateral pulmonary infiltrates on chest x-ray or CT scan
prior to enrollment
12) Patients with significant respiratory disease such as chronic obstructive pulmonary disease, pulmonary fibrosis, or
any use (chronic or intermittent) of inhaled O2 at home
13) FiO2 >0.35 (>4 L/min) prior to enrollment
14) Sepsis (present source of infection ± lactic acidosis defined as serum lactate >5 mmol/L and pH <7.35); Systemic In-
flammatory Response Syndrome (defined as Temp >100.4F or <96.8F; Heart rate >90; Respiratory rate >20 or PaCo2
<32 mmHg; WBC >12, <4 K/uL, or  >10% bands); or shock (SBP <90 mmHg) at presentation
15) The presence of 4 or more of the following risk modifiers for ARDS prior to enrollment:
a) Tachypnea (respiratory rate >30)
b) SpO2 <95%
c) Obesity, defined as Body Mass Index (BMI) >30
d) Acidosis (pH <7.35)
e) Hypoalbuminemia (albumin <3.5 g/dL)
f) Concurrent use of chemotherapy
16) Taking iron supplements containing ≥ 325 mg of ferrous iron, or prochlorperazine


 IDEF  
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26 
17) Patients with heart failure taking > 500 mg of vitamin C daily
18) Known severe hearing loss
19) Known pregnancy, or positive pregnancy test, or breastfeeding
20) Positive drug screen for cocaine upon presentation
21) Patients known or suspected of not being able to comply with the study protocol due to alcoholism, drug depend-
ency, noncompliance, living in another state or any other cause
22) Any condition which, in the judgement of the investigator, might increase the risk to the patient
23) Life expectancy of less than 90 days due to co-morbid conditions
24) Concurrent participation in another research protocol for investigation of another experimental therapy
25) Indication that a new DNR or Comfort Measures Only (CMO) order will be implemented within the first 72 hours of
hospitalization.
4.3 
STUDY ENROLLMENT PROCEDURES 
4.3.1 
SCREENING FOR POTENTIAL SUBJECTS  
The majority of patients is expected to be recruited upon initial evaluation in the Emergency Department (ED).  There-
fore, the ED staff, members of the stroke team, and Neurology/Neurosurgery residents should be made aware of this 
study and in-serviced about its protocol to facilitate recruitment.  It is expected that the stroke team and study coordi-
nator (or other designated members of the study staff) will be informed about all patients who present to the ED with 
ICH, as confirmed by CT; and, that these patients will be reviewed by appropriate members of the study staff as quickly 
as possible to determine eligibility for participation in the study.  All participating sites have an acute stroke response 
team, and it is expected that potential eligible candidates will be examined and interviewed within 60-90 minutes of 
their arrival to the ED.   
The investigators at each site will be required to maintain a screening log for ICH patients who are found ineligible to 
participate in the study or those who are enrolled into another research study, documenting the patients’ age, de-
mographics, and the reason(s) for exclusion from the current study. The study coordinator at each site is required to 
enter the screening log data into the WebDCUTM study database on a monthly basis.
4.3.2 
SCREENING EVALUATIONS 
The following clinical, laboratory, and radiological assessments will be carried out upon screening of potential study 
subjects: 1) Demographic data; 2) Medical history; 3) Review of medications; 4) General physical examination and vital 
signs; 5) Neurological examination, including visual auditory assessment, NIHSS, and GCS; 6) Review of head CT scans 
and chest x-rays; 7) Review of all inclusion/exclusion criteria; 8) Determination of ICH score and pre-ICH mRS score; 9) 
Laboratory tests (hematology, serum chemistries, serum albumin, coagulation parameters, renal and hepatic function 
tests and urine analysis).   
A baseline pre-treatment plain (non-enhanced) brain CT confirming the presence of ICH is required to establish the di-
agnosis of ICH.  The head CT should be reviewed by a Radiologist or a stroke-trained Neurologist experienced in the 
interpretation of CT scans. The need for additional diagnostic tests, such as CTA, conventional angiogram, or MRI/MRA 
to rule out secondary causes of ICH in suspected cases should be based on the judgment of the investigators according 
to the standards of clinical practice at each participating institution and the guidelines from the Stroke Council of the 
American Heart Association [Morgenstern, 2010; please refer to appendix II].  


 IDEF  
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27 
A baseline chest x-ray is required before enrollment to exclude the presence of bilateral pulmonary infiltrates or pulmo-
nary edema. 
A negative serum pregnancy test is required for all women of childbearing potential prior to enrollment into the study. 
4.3.3 
INFORMED CONSENT 
Upon confirmation of patient’s eligibility based on the initial screening evaluation above, an informed consent will be 
obtained.  In accordance with US FDA regulations (21 CFR 50) and ICH-GCP Consolidated Guidelines, a witnessed, IRB-
approved, informed consent is required from all subjects, their legal representative, or family member (as defined in 
21CFR50.3(m)) (legal representative and family members are also referred to as surrogates in this protocol) prior to par-
ticipating in this study.  At the initial contact with a potential candidate, the investigator(s) should provide a compre-
hensive explanation of the purpose, procedures, possible risks/benefits of the study in language that is understandable 
to a non-medically trained person; as well as participant responsibilities and the fact that his/her participation is volun-
tary, that he/she may withdraw from the study at any time, and that the decision not to participate or to withdraw will 
not affect subject’s care in any way.  Potential participants or their surrogates should be given ample opportunity to ask 
questions and to consider their decision.  If the subject or the surrogate on behalf of the subject expresses a sustained 
interest, a signed and dated written informed consent will be obtained.  Patients with a known history of dementia 
should be excluded from self-consent, thereby minimizing the possibility of invalid informed consent. A copy of the 
consent form must be given to the participant or the surrogate, and another copy placed in the subject’s medical rec-
ord. The informed consent must be obtained by either the clinical site PI or other members of the study team who are 
qualified to perform this task and whose names are listed on the Delegation of Authority Log.  
4.3.4 
ENROLLMENT 
Clinically and radiologically eligible patients for whom informed consent is obtained (from the subject or a legal repre-
sentative or family member) will be randomized. Calculation of the ICH score [Hemphill 2001; please refer to appendix 
III] is required prior to randomization. Administration of the study drug to eligible candidates will only be permitted af-
ter completion of liver function tests and urine analysis, if not done upon presentation.
Participants should be reassessed immediately prior to the study drug administration to obtain a pre-treatment GCS 
and baseline NIHSS. Patients who exhibit significant clinical and neurological deterioration (i.e. develop fixed and dilat-
ed pupils or GCS decreases to ≤ 6) prior to administration of the first dose of the study drug should not receive the study 
drug, and their participation in the study should be terminated.   
To ensure that all eligible subjects presenting within 12h of ICH onset are enrolled in a timely fashion without delay: 1) 
enrollment and time from door (arrival-to-emergency room)-to-infusion will be monitored on an ongoing basis, and 
every attempt will be made to ensure that subjects are treated as soon as possible from symptom-onset; and 2) if en-
rollment into the early (≤12h) window is disproportionately low, we will implement a strategy of forced recruitment 
whereby each site will be required to enroll one subject into the early time window for each subject enrolled into the 
later (>12-24h) window (or maintain its overall ratio of ≤12h to >12-24h to at least 1:1) before subsequent enrollment 
into the later time window is permitted at this site.  
4.3.5 
DETERMINATION OF SUBJECT’S BODY WEIGHT 
The subject’s body weight will be obtained by self-report from the subject or his/her accompanying person to the ED, or 
estimated by the treating physician, if actual body weight cannot be determined during the acute phase of evaluation in 
order to prevent delays in administering the study drug.  However, it is expected that all subjects will have their actual  


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28
body weight determined and recorded within 24 hours of admission, and that subsequent dosing will be based on actu-
al body weight.  
4.3.6 
TREATMENT ALLOCATION AND RANDOMIZATION 
Once it is determined that a patient meets all the eligibility criteria, the investigator(s) will log on to the secure, study-
dedicated electronic database (WebDCUTM) to enter demographics and required randomization data. The database will 
generate a randomization code. The investigator will then provide the site’s pharmacy with the randomization code 
and subject weight. The pharmacist will retrieve the label with the corresponding randomization code from the Study 
Randomization Binder maintained separately in the pharmacy.  The pharmacist will prepare the study drug for blinded 
administration. The pharmacists will be specifically instructed not to reveal treatment assignment to the investigators 
and not to break the blind. In the pilot phase I study, the reconstituted solution of DFO was colorless, and there were no 
specific treatment-related changes in laboratory tests including hematology, urine color, or adverse events to suggest 
that the active drug can be identified from placebo. The following additional measures will be taken to assure that the 
integrity of the blinding will remain intact: 1) An independent, unblinded statistician from the DCU, not involved in the 
operations of the study, will create the randomization algorithm; 2) Randomization codes will be numeric and will not 
contain any reference to the type or dose of treatment to be administered; and 3) The randomization binders will be 
kept in secured and locked cabinets in each pharmacy. There is no specific antidote to DFO. Therefore, unblinding is 
unnecessary in most cases. In cases of extreme emergency when the treating physicians request unblinding of treat-
ment assignments for therapeutic purposes, the unblinding will only be revealed to the treating physicians but not the 
investigators. The treating physicians must be instructed not to reveal the blind to the subjects or study investigators. 
The study personnel (pharmacist) will be required to inform the Principal Investigator and the Project Manager within 
24h in the event of unblinding. In cases where the treating physician is one of the study investigators, he/she also will be 
required not to reveal the identity of the study drug to other members of the study team, and not to perform subse-
quent study-related outcome assessments.  
5 
STUDY INTERVENTIONS 
5.1 
INTERVENTIONS, ADMINISTRATION, AND DURATION 
The active study drug (DFO) will be supplied in vials containing 2 gm of sterile, lyophilized, powdered deferoxamine 
mesylate. The drug will be reconstituted for injection, by dissolving it in 20 ml of sterile water, then the volume of the 
reconstituted drug required for a dosing of 32mg/kg/day (up to a maximum of 6000 mg/day) will be calculated based on 
the subject’s body weight and added to normal saline (0.9% sodium chloride) for administration by IV infusion. The 
drug will be reconstituted immediately prior to use to ensure microbiological safety. The matching placebo will be an 
isotonic saline solution (0.9% sodium chloride). Subjects will receive weight-adjusted intravenous infusions of the study 
drug. The maximum daily dose will not exceed 6000 mg per day regardless of subject’s weight. The study drug will be 
administered using an intravenous cannula, dedicated to drug infusion, inserted into an antecubital vein preferably in a 
non-paralyzed limb, and a variable speed infusion pump. Alternatively, a central line, with a port dedicated to the drug 
infusion, may be used. It is important to maintain a dedicated line for the study drug infusion because it may be incom-
patible with other drugs. Applying warm compresses to the site of IV injection throughout each infusion period is 
strongly encouraged as a precaution to minimize the potential for local injection site reactions. The initial dose of the 
study drug must be administered within 12-24 hours (as appropriate) of ICH symptom onset.  Every effort should be 
made to initiate drug administration in consenting subjects as soon as possible after their arrival to the ED. The infusion 
of the study drug must be administered daily during hospitalization for 3 consecutive days by IV infusion at a rate not to 
exceed 7.5 mg/kg/hour. The date and time of drug preparation and administration, as well as rate of infusion, will be 
recorded. Ideally, the IV infusions of the study drug should occur without any interruptions. However, it is possible that  


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interruptions will occur, for example to replace the IV access site. In such cases, the occurrence and duration of these 
interruptions will be documented with an explanation as to why they occurred. 
5.2 
SAFETY MONITORING DURING STUDY DRUG ADMINISTRATION 
All patients will be closely monitored for safety and neurological function during the administration of the study drug, 
and for at least 24 hours after completion of the infusions, in Neurological Intensive or Stroke Care Unit(s), staffed by 
stroke-trained neurologists and nursing staff. Patients will be monitored for all adverse events from the time of study 
drug initiation until day-7 or discharge, whichever occurs first; and, all new SAEs will be monitored until day-90 or reso-
lution of the SAE. Continuing SAEs beyond day-90 will be monitored until day-180.   
The patients will be closely observed during the initial 30 minutes of the infusion for possible adverse effects, such as an 
allergic/anaphylactic reaction, symptomatic bradycardia or hypotension. If there are no adverse effects, the infusion 
will continue. If a patient’s neurological status deteriorates during the infusion, the NIHSS score and GCS will be reas-
sessed by a member of the stroke team/study staff. Significant neurological worsening will be defined as an increase in 
NIHSS score of ≥ 4 points or a decrease of ≥ 2 points in GCS, compared to pre-treatment baseline values, that cannot be 
explained by other causes. A change of this nature lasting >4 hours could lead to premature discontinuation of the drug 
infusion based on the judgment of the local primary investigator(s). Vital signs (blood pressure and pulse) and neurolog-
ical functions will be assessed at least once every 4h during intravenous drug infusion. Daily urinary output checks will 
be carried out every day until the day following the last infusion. An unexplained drop of >20% in mean arterial pressure 
will trigger review by the treating physician to determine its cause and if medical intervention is required. Fluid support 
will be used to prevent or treat hypotension. In more severe cases, use of inotropic agents may be required.  
The skin at the infusion site will be examined at the same time points to assess for the presence and extent of local site 
reactions/irritation. If evidence of mild erythema/irritation or swelling emerges during the infusion, more frequent mon-
itoring will be instituted depending on the severity and observed rate of change to ensure that the reaction does not 
progress further. The use of local anti-inflammatory agents may also be considered. In the event that local irritation 
continues to progress along with worsening edema, and development of pain or papular/vesicular eruption, the infusion 
will be terminated at that site and restarted in a different location. Close monitoring of the new infusion site, as well as 
the original site, will continue. If irritation develops at the new site or progresses at the original site, the infusion will be 
terminated and will not be re-started in this patient. In cases of an allergic reaction or anaphylaxis, the use of antihista-
mines and steroids is often sufficient; ACLS airway management may be required in severe cases. Appropriate treat-
ment and monitoring will proceed according to the standard clinical practice at each institution until the reaction has 
resolved. Routine physical and neurological examinations will be carried out on a daily basis during the infusions, and 
the NIHSS and GCS scores will be determined. Safety laboratories (Hematology; Coagulation; Serum chemistry; Kidney 
and liver functions; and urine analysis) will be performed on the day following the last infusion of the study drug. Labor-
atory studies that are abnormal or worsen from baseline will be repeated. The frequency of subsequent evaluation of 
safety laboratories will be determined by the treating physician on a patient-by-patient case. 
All patients must be closely monitored for any signs or symptoms of respiratory compromise, including ARDS (based on 
Berlin criteria – see appendix VII). A diagnosis of ARDS will lead to premature discontinuation of the drug infusion. Any 
evidence of respiratory compromise should trigger the investigators to complete the AE CRF on Web-DCUTM within 24 
hours of onset for immediate safety review by the Independent MSMs. In addition, the following data must be checked 
at least once daily for all intubated patients (or more frequently in patients with changes in respiratory status requiring 
changes in ventilator settings and increased oxygen requirement) throughout day-7 or discharge and entered into Web-
DCUTM: 1) PaO2/FiO2 ratio; 2) Plateau and peak pressures; and 3) Chest X-ray results (when available). A CHEST X-RAY 
IS REQUIRED IF THE PAO2/FIO2 RATIO IS <300.  


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In order to minimize the potential pulmonary complications of the study drug, the investigators MUST follow the guide-
lines by ARDSNet for management of intubated patients [http://www.ardsnet.org/system/ 
files/Ventilator%20Protocol%20Card.pdf] – please refer to appendix VI and section 7.3 for more details. 
5.3 
HANDLING OF STUDY INTERVENTIONS 
5.3.1 
SUPPLY, STORAGE, PREPARATION, AND DISPOSITION OF STUDY DRUG 
The active drug (DFO) will be purchased by the Department of Health and Human Services (DHHS) from the manufac-
turer and shipped to the Program Support Center, Supply Service Center, Department of Health and Human Services 
(DHHS-SSC), in Perry Point, MD. The DHHS-SSC will serve as the drug distribution center, purchase the study drug pe-
riodically during the trial, and resupply the sites as recruitment progresses. The research pharmacist at each clinical site 
will receive the study drug in shipments.    
The active study drug (DFO) will be supplied in vials containing 2 gm of sterile, lyophilized, powdered deferoxamine 
mesylate. The drug will be reconstituted for injection by dissolving it in 20 ml of sterile water, then the volume of the 
reconstituted drug required for a dosing of 32 mg/kg/day (up to a maximum of 6000 mg/day) will be calculated based on 
the subject’s body weight and added to normal saline (0.9% sodium chloride) in an IV bag to achieve a final concentra-
tion of 7.5 mg per ml for IV administration. The infusion rate will not exceed 7.5 mg/kg/hour. The matching placebo will 
be an isotonic saline solution (0.9% sodium chloride). The drug will be reconstituted immediately prior to use to ensure 
microbiological safety. However, the reconstituted solution may be stored at room temperature for approximately 24h 
before use. In the phase I study, the reconstituted solution of DFO was colorless, and there were no specific treatment-
related changes in laboratory tests including iron, hematology, urine color, or adverse events to suggest that the active 
drug can be identified from placebo. 
The shelf half-life of the drug is 2 years, when stored in a cool place (15ºC-25ºC) away from direct light and heat. The 
drug should be stored in a secured and locked location.  The storage room temperature should be recorded at least dai-
ly, and a temperature log should be maintained.  Deviations from the recommended storage conditions should be im-
mediately communicated to the Project Manager and the PI.  The study drug affected by the deviations should not be 
administered to subjects until the monitoring team has assessed the impact of the deviation and determined whether 
the study drug can be used.  Any materials remaining after each infusion should be discarded by the site in accordance 
with standard clinical practices. 
5.3.2 
STUDY DRUG TRACKING AND ACCOUNTABILITY 
The PI at each site has the overall responsibility for drug accountability at his/her site, which will be carried out in ac-
cordance with ICH/GCP and individual clinical site’s Standard Operating Procedures.  Upon receipt of the study drug, 
the research pharmacist will inspect and count the study drug supply and confirm receipt of the study drug in the Web-
DCUTM study database.  The pharmacy at each clinical site also must maintain records of the amount of study drug re-
ceived, dispensed to study subjects, and destroyed or returned at the end of the study. Drug accountability records and 
storage temperature logs will be inspected by the study monitor and may be subjected to inspection by relevant au-
thorities as well.  Study drug supplies will be counted and reconciled at each site during monitoring visits and at the end 
of the study.    
5.4    
CONCOMITANT MEDICATIONS AND PRECAUTIONS 
Concomitant medications will be recorded during the study period from the time of study drug initiation until day-7 or 
discharge (whichever is earlier). There are few restrictions on the use of concomitant medications for study  


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31
participants.  The use of prochloroperazine (compazine), a phenothiazine derivative, is not allowed before treatment, 
during treatment, or up to 72 hours after completion of the study drug infusion, since the combination of DFO and 
compazine can lead to impairment of consciousness [Blake, 1985].  Concurrent use of other experimental therapy is not 
allowed.  Vitamin C supplements will not be allowed in patients with heart failure during treatment with DFO.   
All standard therapies used in the management of ICH patients will be allowed under close monitoring and supervision. 
These include volume expansion with normal saline or crystalloids, vasopressors, antiedema agents such as steroids or 
mannitol, anti-hypertensives including diuretics, anti-convulsants, anti-arrhythmic agents and anti-emetics, except for 
phenothiazine derivatives.   
In order to minimize the variability in care and to assure consistency in management across sites, the general care of ICH 
patients should conform to the guidelines from the Stroke Council of the American Heart Association [Morgenstern, 2010], 
and the European Stroke Initiative Guidelines [Steiner, 2006].  Pertinent issues are detailed in Appendix II and III. 
6 
CLINICAL AND LABORATORY EVALUATIONS 
6.1 
PATIENT EVALUATION AND DATA COLLECTION SCHEDULE 
All study data will be collected using Case Report Forms (CRFs) designed specifically for the study. Patient confidentiali-
ty will be maintained throughout, as participants will only be identified on the CRFs by a unique study ID number.  Pa-
tients’ identifying information and source documents for the CRF data should be kept by the primary investigator at 
each participating site.  The data obtained will include: 1) Demographics; 2) Previous medical history; 3) Concomitant 
medications; 4) Vital signs; 5) Results of laboratory tests; 6) NIHSS and GCS scores; 7) ICH score; 8) mRS, SIS-16, and 
MoCA scores; 9) Adverse events; 10) Surgical and non-surgical treatments; and 11) Imaging findings, including chest x-
rays, and CT and MRI scans identifying the location of ICH, presence of IVH or hydrocephalus, and etiology of ICH (after 
completing diagnostic work-up).  
6.1.1 
CLINICAL AND LABORATORY ASSESSMENTS 
The following assessments will be carried out throughout the 90-day study period: 
Upon screening of potential study subjects: 1) Review of head CT and determination of the time of ICH onset or the 
time the patient was last known to be without presenting deficits; 2) Demographic data; 3) Medical history; 4) Review 
of medications; 5) General physical examination; 6) Neurological examination, including NIHSS and GCS; 7) Visual and 
auditory assessment (which includes assessments for cataracts, visual loss or field cut, disturbed color vision, hearing 
loss, and presence of tinnitus), whenever possible in awake patients; 8) Determination of pre-morbid mRS; 9); Review 
of admission laboratory tests - Hematology [hemoglobin, hematocrit, red blood cell count and platelet count]; Coagu-
lation [partial thromboplastin time and international normalized ratio]; and serum albumin; and 10) Review of all inclu-
sion/exclusion criteria. A baseline chest x-ray is required to rule out the presence of bilateral pulmonary infiltrates or 
pulmonary edema. A pregnancy test is required for all women of childbearing potential prior to enrollment into the 
study. Subjects in whom all eligibility criteria are met should be considered for enrollment, and those who agree to par-
ticipate via signed and dated informed consent should be enrolled.  
Participants should be reassessed immediately prior to the study drug administration to obtain a pre-treatment base-
line. The Pre-treatment baseline reassessments include: 1) Vital signs, including blood pressure and heart rate; 2)  


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32
Neurological examination, including NIHSS and GCS to confirm stability of their neurological status and to obtain pre-
treatment baseline scores; and 3) Assessment for new adverse events since screening.  Patients who exhibit significant 
clinical or neurological deterioration (i.e. develop fixed dilated pupils or GCS decreases to ≤6) prior to administration of 
the first dose of the study drug should not receive the study drug, and their participation will be terminated.  
Administration of the study drug should only be permitted after obtaining pre-treatment blood sample for serum 
chemistry and glucose, hepatic function tests, and urine analysis (if not already done upon arrival to the ED). 
Subjects who receive the study drug(s) will be evaluated daily for the first 3 days (during study drug infusion while in 
hospital) and on day-7 or discharge from the hospital, whichever occurs first. Clinic visits will take place on days 30±7 
and 90±7. Telephone interviews will be conducted on day 60±7 and 180±7.   
The following assessments should be performed after each daily infusion (every 24±6h) and through the day following 
completion of the last infusion: 1) General physical examination; 2) Visual and auditory assessments; 3) Neurological 
examination, including NIHSS and GCS; and 4) Vital signs, including blood pressure and heart rate. A repeat head CT 
scan and blood samples for hematology, serum chemistry, renal and hepatic function tests, coagulation studies, and 
urine analysis should be obtained within 24±6h following the last infusion of the study drug. Vital signs (blood pressure 
and pulse) and neurological functions should be assessed at least once every 4h during intravenous drug infusions. Daily 
urinary output checks should be carried out every day until the day following the last infusion. In addition, the following 
data must be checked at least once daily for all intubated patients (or more frequently in patients with changes in res-
piratory status requiring changes in ventilator settings and increased oxygen requirement) throughout day-7 or dis-
charge and entered into Web-DCUTM: 1) PaO2/FiO2 ratio; 2) Plateau and peak pressures; and 3) Chest X-ray results 
(when available). A chest x-ray is required if the PaO2/FiO2 ratio is <300. 
On day-7 or Discharge (whichever comes first), the following assessments are done: mRS, NIHSS, GCS, MoCA and 
visual and auditory assessments.  
During hospitalization, the NIHSS and GCS should be done any time a subject’s neurological status deteriorates, or when-
ever the investigator believes it is prudent to do an assessment. Laboratory assessments beyond those required for the 
study should be done whenever clinically indicated.    
The Day-30 (±7) will be an in-person visit comprised of a general physical examination, visual and auditory assess-
ments, review of SAEs, and assessments of NIHSS, SIS-16, MoCA and mRS.  
The Day-60 (±7) assessments, which will be completed via telephone, will be comprised of a functional assessment to 
obtain a score on mRS and query for SAEs.  
The Day-90 (±7) visit will be an in-person visit comprised of a general physical examination, visual and auditory assess-
ments, and assessments of NIHSS, MoCA, SIS-16, and mRS. However, a telephone interview to obtain a score on mRS 
(the primary outcome measure) may be allowed on a case-by-case basis, after consultation with the PI, if the subject is 
unable to return for an in-person evaluation. 
The Day-180 (±7) assessments, which will be completed via telephone, will be comprised of a functional assessment to 
obtain a score on mRS.  
A study staff member certified in NIHSS and mRS and experienced in administering GCS, SIS-16, and MoCA should perform 
the assessments at all time points. We require that all assessments at all time points be performed by the same blinded  


 IDEF  
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33 
investigator, whenever possible, to minimize inter-rater variability. Data on the subject’s functional status (mRS and SIS-
16) can be obtained from a proxy if the subject is unable to provide the information. Please refer to Appendix V for details of
NIHSS, GCS, SIS-16, and MoCA scales.
Concomitant medications and non-drug therapies should be assessed at all points of contact with the subject after 
study drug initiation. All adverse events (serious and non-serious) should be assessed until day-7 or discharge, whichev-
er occurs first. All new SAEs will be monitored until day-90, resolution of the SAE, or withdrawal of consent, whichever 
is earlier. Continuing SAEs beyond day-90 will be monitored until day-180.  
6.1.2 
RADIOLOGICAL ASSESSMENTS 
A baseline pre-treatment non-enhanced brain CT confirming the presence of ICH is required to establish the diagnosis 
of ICH. The need for additional diagnostic tests, such as CTA, MRI/MRA, or a conventional angiogram to rule out sec-
ondary causes of ICH in suspected cases should be based on the judgment of the investigators according to the stand-
ards of clinical practice at each participating institution and the guidelines from the ASA [Morgenstern 2010; refer to 
Appendix II]. The volume of ICH on admission scan must be determined by the local investigators using the ABC/2 
method (please refer to Appendix IV for instructions) before randomizing the subject in WebDCUTM. A repeat non-
enhanced brain CT scan should be obtained within 24±6 hours after completion of the last infusion, with the same 
standard imaging protocols and scanner at a given hospital. Additional CT scans, beyond those required for the study, 
should be done whenever clinically indicated. 
A baseline chest x-ray confirming the absence of bilateral pulmonary infiltrates or pulmonary edema is required before 
considering the subject for enrollment into the study. A repeat chest x-ray is required in intubated patients whenever 
the PaO2/FiO2 ratio is <300. 
6.2 
Schedule of Evaluations 
Table 6.2 below summarizes the timing and type of assessments/evaluations throughout the study. 
Schedule of Assessments and Data Collection 
Screening 
Baseline 
0h 
24h 
48
h 
72h 
24h post-last 
infusion (± 
6h) and as 
needed 
Day 7 or  
Discharge* 
Day  
30 
Day  
60 -
Phone 
Day 
90 
Day 
180 -
Phone 
End of 
Study 
Prior to 
study 
drug 
start 
± 6h 
± 
6h 
± 6h 
± 6h 
± 7 days 
± 7 days 
± 7 
days 
± 7 
days 
Screen Failure Log 
X 
Inclusion/Exclusion Cri-
teria 
X 
Demographics & Medi-
cal History 
X 
Informed Consent 
X 
Subject Enrolment / 
Randomization 
X 
Study Drug Administra-
tion 
X 
X 
X 
X 
Physical Examination 
X 
X 
X 
X 
XX 
X 
X 
Visual & Auditory As-
sessment€ 
X 
X 
X 
X 
XX 
X 
X 
NIHSS 
X 
X 
X 
X 
X 
XX 
X 
X 
X 


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34 
GCS 
X 
X 
X 
X 
X 
XX 
X 
ICH Score 
X 
mRS 
X 
X 
X 
X 
X¥ 
X 
MoCA 
X 
X 
X 
SIS-16 
X 
X 
Vital Signs (BP & pulse)$ 
X 
X 
X 
X 
XX 
X 
Safety Monitoring 
X 
X 
X 
X 
Urinary Output 
X 
X 
X 
Hematology 
X 
XX 
Serum Chemistry 
X 
XX 
Coagulation Parameters 
X 
XX 
Urine Analysis 
X 
XX 
Liver Function Tests 
X 
XX 
Kidney Function Tests 
X 
XX 
Serum albumin 
X 
Pregnancy Test (women 
of childbearing poten-
tial) 
X 
Blood sample for future 
research 
X 
XX 
CT scan 
X 
XX 
Chest x-ray%
X
X
Prior Medications 
X 
Concomitant Medica-
tions 
X 
X 
X 
X 
X 
X 
X 
X 
Concomitant Non-Drug 
Therapies 
X 
X 
X 
X 
X 
X 
X 
X 
Adverse Events 
X 
X 
X 
X 
X 
X 
X 
X 
X 
End of Study 
X 
€ Includes assessments for cataracts, visual loss or field cut, disturbed color of tinnitus – Should be performed, whenever possible in awake patients 
* Day 7 or Discharge– whichever comes first 
$ Vital signs (blood pressure and pulse) and neurological functions will be assessed at least once every 4h during intravenous drug infusion Laboratory tests may be 
performed outside the ±6h window, if it is a standard site procedure to perform laboratory tests6 at a specific time each day. 
XX These assessments must be performed within 24±6h following completion of the last infusion of the study drug  
We plan to collect (bank) additional blood samples at baseline and after the last infusion to be stored and analyzed in the future from subjects providing informed 
consent. 
 Post-treatment scan should be performed within 24±6 hours of the last infusion even if it is terminated before day-5 of treatment 
¥ A telephone interview to obtain a score on mRS (the primary outcome measure) may be allowed on a case-by-case basis if the subject is unable to return for an in-
person evaluation on day 90. 
% Chest x-ray must be performed in any intubated patient if PaO2/FiO2 ratio is <300. 
6.3         Blood Banking Repository Sub-Study 
The main study provides an opportunity to “bank” blood samples from the participants for future innovative ICH re-
search, particularly if the   efforts to develop DFO as a therapy for ICH are successful. Future pharmacogenetic studies 
may be considered to help define other therapeutic targets and responders vs. non-responders to DFO therapy. We, 
therefore, plan to collect additional blood samples at baseline and after the last DFO infusion (approximately 30 ml) to 
be stored and analyzed in the future. Subjects’ participation in this blood-banking repository will be optional; they do 
not have to participate in the repository sub-study in order to participate initially or to continue their participation in the 
main study. The blood samples from all participating sites will be sent to the Beth Israel Deaconess Medical Center (The 
Coordinating Center) by overnight mail. All samples will be stripped of identifiers and randomly assigned a unique code 
number. The key linking code numbers and identifying information will be kept in a secure location accessible only to 
the principal investigator of the study or his designee, on a password protected network drive. All samples will be  


 IDEF  
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35 
stored at -80 degrees Celsius low-temperature, locked, freezer until final planned analyses are formulated and carried 
out. The samples will be stored indefinitely or until no more remains for genomic and protein research that can be per-
formed at future dates.   
The exact questions to be asked and tests to be done in the future are not fully identified at this stage. We tentatively 
plan to investigate the relationship between polymorphisms from a panel of genes encoding iron-handling proteins 
(which includes genes involved in both intra- and extra-cellular iron metabolism, such as ceruloplasmin, haptoglobin, 
hemopexin, transferrin receptor, ferritin heavy- and light-chain, and heme-oxygenase 1 and 2 genes) and PHE, out-
come, and response to DFO therapy. Genotyping will be performed using high-throughput genome-wide SNP genotyp-
ing methods. Genotype results from DFO- and placebo-treated subjects will be examined for differences in allele fre-
quency that may be associated with risk of malignant PHE progression or response to DFO therapy. Extracted DNA 
samples will be genotyped without knowledge of the treatment arm by chip-based methods utilizing mass spectrome-
try. The exact location of genotyping is undetermined at this point in time, although it is likely to be performed at the 
Broad Institute, given our ongoing collaboration in ICH Genome Wide Association Studies.  
We plan to seek ancillary funding to support these additional analyses. Once funding is secured and the final analyses 
are formulated, we plan to seek an IRB approval for the specified analyses and use of the blood banking repository data. 
7 
ADVERSE EVENTS 
7.1 
DEFINITION OF ADVERSE EVENTS 
Adverse events are any untoward medical occurrence whether or not they are considered related to the study drug. An 
adverse event can be any unfavorable and unintended symptom, sign, disease or clinically significant abnormal test 
result occurring during the study which were either not present at baseline, or if present, worsened during the study in 
terms of either severity or frequency.  When evaluating possible adverse events, an assessment should be made regard-
ing its seriousness, severity and temporal relationship to the administration of the study drug.  An adverse event is con-
sidered serious if it is life-threatening, prolongs hospitalization, requires re-hospitalization, results in significant disabil-
ity, leads to death, or requires medical or surgical intervention to prevent one of the other listed outcomes.  An assess-
ment should be made regarding the seriousness, severity and relationship to the administration of the study medica-
tion as well as to the ICH.  The following factors should be considered when evaluating possible adverse events: 1) the 
temporal sequence from drug administration; 2) patient's response after drug discontinuation or re-introduction; and 3) 
severity of the event.  The investigators, on the basis of their clinical judgment and guided by the following definitions, 
should determine the relationship of an adverse event to the study drug(s) as: definitely related, i.e. following in a rea-
sonable temporal sequence, known to be a complication of DFO, and having no other explanation; probably related, i.e. 
following in a reasonable temporal sequence and not reasonably explained by the patient’s clinical state or other thera-
pies; possibly related, i.e. could have been explained by other therapies or patient’s clinical state; or unlikely or  not re-
lated.  The severity of adverse events should be graded as mild, moderate, severe, life-threatening, or fatal.  
All adverse events or complications will be entered into WebDCUTM and assigned to a system-organ class and preferred 
term using the MedDRA coding dictionary. Each subject must be monitored closely throughout his/her hospitalization 
for serious adverse events (SAEs). All adverse events (serious and non-serious) should be assessed until day-7 or dis-
charge, whichever occurs earlier, and serious adverse events until day-90 or withdrawal of consent, whichever is earlier. 
7.2     POTENTIAL COMPLICATIONS OF THE STUDY INTERVENTION 


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36
Aspects of the study that have potential for risks are blood withdrawals, CT scan imaging studies, and injections of the 
study medication(s). There are no additional risks for any of these procedures other than those present in any routine 
clinical situation, especially for blood draws and CT scans. Patients may experience some temporary discomfort, bruis-
ing, or, rarely, infection or the formation of a small clot or swelling at the site of the needle puncture in the process of 
drawing blood or starting the intravenous drip. The condition of ICH, in itself, carries a high risk for secondary complica-
tions including serious life-threatening adverse events, and even death, whether or not the active study drug (DFO) is 
used. Recent studies indicate that approximately 40% of patients with ICH have a serious adverse event during hospi-
talization, of which approximately 26% can be fatal [Haley, 2005; Mayer 2005; Mayer 2006; Lyden 2007].  
DFO has been extensively used in clinical practice for more than 40 years, and is approved by the Federal Drug Admin-
istration for the treatment of acute iron intoxication and of chronic iron overload due to transfusion-dependent anemia.  
Safety studies indicate that it is relatively well tolerated.  The adverse reactions observed with DFO have been previous-
ly detailed in section 2.2.2.  
The most observed adverse events in our Phase I study in ICH patients were injection site reaction (irritation, pain, ery-
thema, infiltration) and a modest decrease in blood pressure, which did not require any medical intervention. One sub-
ject developed visual hallucination during IV infusion of DFO. Almost all of these adverse events were mild, self-limited, 
did not require specific treatment, and resolved spontaneously. There were no deaths or serious adverse events related 
to DFO use. There were 2 cases of respiratory failure, both of which occurred in the 62mg/kg/day dose-tier. They were 
thought to be related to aspiration pneumonia, and unrelated to the study drug. However, a recent expert review found 
that one of these cases was an undiagnosed case of ARDS, and concurred that aspiration was a plausible explanation 
for it.   
In the first part of phase II investigation of DFO in ICH (HI-DEF trial, where subjects were treated with a continuous infu-
sion of DFO at 62 mg/kg/day for 5 consecutive days), 5 cases of ARDS were reported among the first 42 participants. A 
detailed expert review of reported cases of respiratory failure and pulmonary edema revealed 2 more cases of undiag-
nosed ARDS.  Overall, 6/7 of the ARDS cases in HI-DEF were in the DFO treatment arm; 3 were fatal and the cause of 
death was ARDS-related in 2/3 cases. The expert who was blinded to treatment assignment identified a plausible cause 
for ARDS, other than the study drug or ICH itself, in 4/7 ARDS cases, while no other explanation, other than the study 
drug or ICH, was apparent for the remaining 3 cases. Although the overall rate of ARDS was in line with published re-
ports in ICH patients [Elmer 2013], the imbalance in the frequency of ARDS between the DFO- and placebo-treated 
groups raised concerns that the pulmonary toxicity of the drug was likely. There were no other safety concerns. A 
MedWatch report was filed with the FDA and Health Canada for an AE of hypophosphatemia that was judged to be se-
rious (potentially life-threatening), unexpected, and possibly related to the study drug. Overall, hypophosphatemia of 
varying severity was reported in 3 subjects in HI-DEF; 2 in DFO- and 1 in placebo-treated patients. 
The effects of DFO on the fetus are unknown. It is also not known if DFO is excreted in human milk.  Therefore, women 
who are pregnant and those who are breast-feeding will be excluded from this study. Those with child-bearing poten-
tial must have a negative pregnancy test before participating in the study.  
7.3   PREVENTION AND MANAGEMENT OF ADVERSE EVENTS 
Aseptic and sterile techniques should be used during blood draws, which should be performed by experienced phlebot-
omists. Application of warm compresses to the site of IV infusion throughout the infusion period is advised to minimize 
the potential for injection site irritation. In order to maximize safety during this study, we are using a slow infusion rate 
of < 7.5 mg/kg/hour and are limiting the maximal dose that any subject can receive to 6000 mg per day. The subjects 
must be carefully monitored throughout their participation in the study as detailed above. Any medical complications  


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should be managed as appropriate, according to the standard clinical practice at participating institutions. There is no 
specific antidote to DFO. General symptomatic and supportive measures should be applied. 
Subjects should be admitted to the Neurological Intensive Care or Stroke Unit, undergo repeated clinical (neurological 
and general physical examinations) and laboratory evaluations at regular intervals throughout their participation in the 
study for safety monitoring, and receive standard medical management for ICH based on the American Stroke Associa-
tion Guidelines [Morgenstern 2010].  Pertinent issues, including blood pressure management, are detailed in the ap-
pendix II and III. Laboratory studies that are abnormal or worsen from baseline should be repeated.   
It is important that investigators ensure maintenance of intravascular volume prior to administration of the drug infu-
sions, especially in hypovolemic patients, to minimize DFO-induced hypotension; and to carefully observe the subjects 
during the initial 30 minutes of the intravenous infusion for possible adverse effects, such as an allergic/anaphylactic 
reaction, arrhythmias, or hypotension.  If there are no adverse effects, the infusion should continue.  All patients should 
be kept hydrated and, if necessary, fluid support should be used to prevent or treat hypotension. In more severe cases, 
use of inotropic agents may be required.  
The skin at the site of IV infusion must be carefully inspected periodically for signs of irritation, induration, or inflamma-
tion during physical examinations, coincident with the times of all assessments of vital signs, during the infusion of the 
drug and for 24 hours afterwards. Section 5.2 above details appropriate measures for management of local injection 
site reactions. 
In order to minimize the potential pulmonary complications of the study drug, the investigators MUST follow the fol-
lowing guidelines by ARDSNet for management of intubated patients [http://www.ardsnet.org/system/ 
files/Ventilator%20Protocol%20Card.pdf]– please refer to appendix VI: Ventilator settings – 1) Tidal volume (VT)= ≤8 
cc/kg PBW (predicted body weight); 2) Plateau Pressure (Pplat) <30 cm H2O; 3) Peak Pressure (Ppeak) <50 cm H2O; and 4) 
Positive End-Expiratory Pressure (PEEP): at least 5 cm H2O. Investigators MUST also implement a Ventilator Associat-
ed Pneumonia prevention protocol – 1) Head of bed elevation at least 30 degrees or greater; and 2) Regular clorhexi-
dine mouth wash and oral care. In addition, a conservative fluid management strategy that aims to minimize or elimi-
nate positive fluid balance and cautions against indiscriminate use of blood transfusion unless hemoglobin concentra-
tion drops below 7 g/dL is recommended. All patients must be closely monitored for any signs or symptoms of respira-
tory compromise, including ARDS (based on Berlin criteria – see appendix VII). A confirmed diagnosis of ARDS should 
lead to premature discontinuation of the drug infusion. Appropriate treatment and monitoring, including the use of in-
vasive mechanical ventilation, low tidal volume, permissive hypercapnea, or high PEEP, should proceed according to 
the standard clinical practice at each institution. In patients with respiratory failure, strategies that decrease oxygen 
utilization, such as antipyretics to control fever and sedatives to control agitation; and appropriate use of neuromuscu-
lar blockade when asynchrony with the ventilator persists despite adequate sedation are recommended.  
Infections with Yersinia enterocolitica and Y. pseudotubeculosis have been reported in DFO-treated patients. Although 
these are unlikely to occur in patients without significant systemic iron overload, appropriate bacteriological tests 
should be performed, and suitable antibiotic coverage for Yersinia should be instituted if a patient in the study develops 
fever accompanied by acute enteritis, abdominal pain, or pharyngitis. Appropriate treatment and monitoring should 
proceed according to the standard clinical practice at each institution. 
7.4 CRITERIA FOR INTERVENTION DISCONTINUATION 


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The investigators may terminate the study drug infusion if, in their judgment, its continued administration poses harm 
to the patient’s medical condition. The only pre-established criteria for premature discontinuation of the study drug 
are: 1) severe allergic reaction or anaphylaxis; 2) worsening of renal functions, defined as serum creatinine >2 mg/dl on 
2 repeated measures 8 hours apart; 3) development of ARDS based on Berlin criteria/definition (see appendix VII); or 4) 
if the patient or his proxy voluntarily withdraws consent. Subjects whose study drug is discontinued but who do not 
withdraw consent should still be followed for 180 days. 
7.5 REPORTING OF ADVERSE EVENTS 
In order to ensure prompt reporting of adverse events, all adverse events (as well as all related study data) must be en-
tered into the WebDCUTM within five working days following the completion of the Baseline, Treatment, and Day-7 or 
discharge (whichever occurs first) trial phases. All serious adverse events (SAEs) must be reported on the WebDCUTM 
within 24 hours of the study site staff first being made aware of its occurrence. The 24-hour reporting requirement for 
SAEs applies to all study phases.  The investigators are required to provide relevant information, including description 
of the adverse event, date/time of onset and resolution, severity and seriousness, action taken, and suspected relation-
ship to the study drug. Similarly, all adverse events of respiratory compromise, whether serious or not, must be report-
ed in WebDCUTM and the relevant CRF completed within 24 hours of the study site staff first being made aware of its 
occurrence. 
Reporting of respiratory compromise, as well as any serious or life-threatening adverse event, will trigger notification of 
the event to the Project Manager (PM), the independent MSMs, and appropriate members of the Executive Committee 
(EC). The MSMs will conduct an independent review of each of these events to determine its relatedness to the study 
drug along with other elements, and to confirm or exclude the diagnosis of ARDS in cases of respiratory compromise. 
Within 72 hours of receipt of these events for review, the MSMs will enter into the trial’s database their opinion regard-
ing whether the adverse event is, in fact, serious, and if it is unexpected and related to the study drug. If the MSMs be-
lieve all three elements are present in the SAE, an expedited safety report (MedWatch) will be filed with the FDA within 
the stipulated regulatory guidelines: for fatal or life-threatening SAEs, no later than 7 calendar days after the sponsor’s 
initial receipt of information, and for SAEs outside those categories, no later than 15 calendar days after the sponsor 
determines that the event requires expedited reporting (21 CFR 312.32(c)(1)). If the study site investigator(s) and the 
MSMs are not in agreement on these issues and fail to resolve their disagreement after direct communication, the opin-
ion of the MSMs will be considered as the final adjudication. When a MedWatch Report is filed with the FDA, a copy of 
the MedWatch Report (or a link to a secure file in the trial database) will be sent to the DSMB through the NINDS liai-
son. 
Each clinical site PI and primary Study Coordinator will receive an email notification that a MedWatch Report has been 
filed with FDA. The email notification will contain instructions for accessing the report. It is the responsibility of each 
clinical site PI to file these reports with his/her IRB in compliance with their institution’s local requirements. After the 
submission of the initial MedWatch Report, the principal investigator at the corresponding clinical site will be responsi-
ble for obtaining follow-up information about the event and reporting it in the WebDCUTM. 
7.6 MONITORING OF ADVERSE EVENTS 
Two independent Medical Safety Monitor (MSMs), appointed in consultation with the NINDS program director, will 
monitor the study with regard to safety on an ongoing basis to identify any safety concerns. The MSMs will review all 
cases of respiratory compromise and all SAEs to determine whether they are related to study drug administration, and 
to ascertain the diagnosis of ARDS in cases of respiratory compromise. They will communicate with the investigators  


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for any questions or clarifications regarding an event. Periodically throughout the study, the EC and the MSMs will 
review reports on the incidence rates of all reported adverse events, whether serious or not, with particular attention to 
SAEs, respiratory compromise including ARDS, and mortality during the first 7 days of hospitalization (or until dis-
charge, whichever is earlier).  Should such monitoring uncover issues that may threaten subject safety (e.g. 
unexpectedly high rate of adverse events), the study statistician and principal investigator will prepare a report to be 
submitted to the DSMB for their review and further actions to be taken, if any.  
Two statistical reports will be generated semiannually (unless requested at a more frequent interval by the MSMs or 
DSMB) – an open report to be distributed to the Executive Committee and MSMs, and a closed report to be distributed 
only to the NINDS-appointed DSMB.  Each semi-annual report will provide cumulative summary statistics on enroll-
ment, subject status in the study, baseline characteristics, protocol violations, safety data (including a summary of the 
most frequent and most serious adverse events, a summary of all MedWatch Reports, and a listing of all subjects who 
were terminated from the study and the reason for termination), and data management/quality information. The sta-
tistics will be provided for the overall study. For the closed report only, the statistics will also be provided by partially 
blinded treatment group (A vs. B). If the DSMB wishes to be completely unblinded for these reports, a sealed identifica-
tion envelope will be provided to the NINDS DSMB liaison; this envelope can be opened at the discretion of the DSMB. 
An annual report will be submitted to the FDA and Health Canada. 
7.7 STOPPING RULE 
The occurrence of ARDS will be continuously monitored during the course of the trial, in order to facilitate thorough 
review of the data by the DSMB and to stop enrollment into the trial if sufficient evidence of imbalance in the rate of 
ARDS exists. The NINDS-appointed DSMB recommended that recruitment be stopped if the difference in the number 
of confirmed ARDS cases between the groups is 5 at any time during the recruitment of the first 40 subjects; 10 at any 
time during the recruitment of subjects 41-80; 12 at any time during the recruitment of patients 81-120; or if the differ-
ence in the number of confirmed ARDS cases between the groups is statistically significant after 40, 80, or 120 subjects 
have completed the in-hospital phase based on a Pocock-adjusted, one-sided, 0.05 alpha level. This one-sided hypoth-
esis is consistent with our concern for patient safety, wherein early termination is considered only if ARDS incidence is 
higher on the DFO arm than on the control arm.  
These analyses will be based on the occurrence of confirmed ARDS cases (according to the Berlin criteria) thought to be 
at least possibly related to study drug. The ARDS cases will be reviewed on an ongoing basis throughout the trial by the 
MSMs (who will adjudicate all cases together to provide a consensus opinion about the diagnosis, severity, and possible 
plausible causes other than the study drug). . The DSMB will be notified of each individual ARDS occurrence upon con-
firmation by the MSMs, per their request.  Given that a common definition for the diagnosis of ARDS will be used, we 
do not anticipate much discrepancy between the site’s assessment and the MSM’s assessment. However, in the event 
of disagreement, the MSM’s assessment will be used. 
8 
STATISTICAL CONSIDERATIONS 
8.1 
RANDOMIZATION PROCEDURES  
A combination of minimization and biased coin methodologies will be used to randomize participants to either DFO or 
placebo [Pocock 1975; Taves 1974]. The minimization method aims to control imbalance in the treatment groups with 
respect to pre-specified baseline characteristics; the biased coin approach is adopted to avoid deterministic assign-
ment. Throughout the trial, the probability of treatment assignment will take into account the imbalance associated  


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with clinical site, baseline ICH score, OTT, baseline ICH volume, baseline NIHSS score, and concurrent use of anticoagu-
lants at the time of ICH onset.  
8.2 
STUDY ENDPOINTS AND DATA ANALYSIS 
8.2.1 
CLINICAL ENDPOINTS  
The primary efficacy outcome measure is the mRS, dichotomized to define good functional outcome as mRS score of 0-
2 at 90 days. At the conclusion of the study, the proportion of DFO-treated subjects with a good outcome will be com-
pared to the placebo proportion in a futility analysis. The primary futility hypothesis is tested via generalized linear 
model relating the probability of a good outcome to the treatment. Adjustment for OTT, ICH score, baseline ICH 
volume, NIHSS score, and anticoagulant use are included in the model to obtain proper significance due to the inclusion 
of these variables in the randomization scheme. The binomial distribution for Y, with the identity link, is used to derive 
an estimate of the adjusted risk difference for good outcome. The primary futility hypothesis, H0: (πDFO-πplacebo) ≥0.12, 
will be tested at one-sided alpha (the probability that an effective intervention will be called ineffective, or futile) 0.10. 
The corresponding alternative hypothesis,  HA: (πDFO-πplacebo) <0.12, defines futility as a treatment effect less than 
absolute 12% in favor of DFO. The futility analysis will be conducted using a one-sided 90% upper confidence bound on 
the risk difference, which is consistent with the one-sided alternative hypothesis and stated level of significance. To 
declare futility, the entire interval must lie below the value 0.12, indicating that the true difference in risk of good 
outcome is less than 0.12 with 90% confidence. Under this design, a significant result would suggest that it would be 
futile to move DFO forward to Phase III testing.  
As secondary analyses of the primary outcome, the presence of a differential treatment effect in the OTT windows will 
be explored. The generalized linear model described above for the primary analysis will be expanded to include an 
interaction between treatment and OTT window. While the Trial will be underpowered to definitively address this 
question, the magnitude of the treatment effect, and corresponding confidence interval, will be estimated for each 
time window. A dichotomized analysis considering the proportion of DFO- and placebo-treated subjects with mRS 0-3 
will also be performed. Although mRS 0-3 is less favorable than the primary outcome of mRS 0-2, it would still be a de-
sirable effect in patients with ICH given that no treatments exist to reduce disability. The trial is adequately powered to 
assess the futility hypothesis using mRS 0-3 as the outcome based on an absolute difference in treatment effect <13% 
in favor of DFO. We will also perform similar analyses at 180 days and ordinal analysis across all mRS scores as second-
ary outcome measures. 
8.2.2 
SAFETY ENDPOINTS: 
All adverse events will be assessed until day-7 or discharge (whichever is earlier), and SAEs until day-90. Mortality (all 
causes and ICH-related) will be assessed until the end of the study (day-180). Safety endpoints of particular interest in-
clude all DFO-related adverse events until day-7 or discharge (whichever is earlier), all SAEs through day-90, and deaths 
(all causes and ICH-related) through day-180.  
The following events will be defined as ADVERSE EVENTS OF SPECIAL INTEREST (AEOSI) for safety surveillance during this 
study: 1) Anaphylaxis (at any time point during study drug infusion); 2) Hypotension (defined as a decrease in blood 
pressure requiring medical intervention at any time point during drug infusion that cannot be explained by other caus-
es); 3) Respiratory compromise of any cause during the in-hospital phase (day-7 or discharge, whichever is earlier); and 
4) Development of new and unexplained visual or auditory changes after initiating treatment with the study drug.


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All adverse events or complications will be submitted electronically via the WebDCUTM system and assigned to a 
system-organ class and preferred term using MedDRA coding dictionary. The number and percent of all adverse events 
will be summarized for each treatment arm by event type. Tables will be generated to detect any clinically significant 
laboratory abnormalities in change of values from baseline to end of the last infusion. We anticipate no missing safety 
data regarding adverse events, since patients will remain hospitalized and closely monitored until day-7 or discharge 
(whichever is earlier). The cumulative incidences of each AEOSI, as well as mortality, will be compared via 95% confi-
dence intervals.  Mortality will also be assessed via the log rank test for comparing survival curves. 
Analysis of the safety data regarding the occurrence of ARDS will be carried out continuously throughout the trial by 
the unblinded study statistician, who will notify the EC and DSMB if recruitment must be stopped.   The DSMB will be 
notified of each individual ARDS occurrence upon confirmation by the MSMs, per their request.Analysis of other safety 
data by the DSMB will occur semiannually (or more frequently if requested by the DSMB). If any of these analyses re-
veal serious emerging concerns, the EC, in consultation with the DSMB, may implement modifications to assure safety 
as necessary. Apparent, consistent and persistent evidence of net harm that tends to overwhelm any benefit may allow 
for premature termination of the study. 
8.3 
STUDY SAMPLES 
ALL analyses will involve data from two samples: 
1-
Modified Intent-to-Treat (mITT): Outcome data from all subjects who are randomized and in whom the study
infusion begins, even if it is discontinued prematurely, will be analyzed. Classically, ITT analysis is used in Phase III trials
and includes all subjects randomized, regardless of whether they receive the treatment or not. However, in order to
evaluate the efficacy of DFO as administered, randomized subjects in whom the study infusion is never initiated will be
excluded from the primary analysis. As a sensitivity analysis, the futility hypothesis will also be assessed according to
the ITT principle. All subjects randomized will be included and considered in the treatment group to which he/she was
randomized, regardless of the treatment actually received. The outcomes for subjects in whom treatment is never initi-
ated will be imputed based on available baseline data.
Safety analysis will involve data from all patients in whom study infusion begins, even if it is discontinued prematurely 
(i.e. the mITT sample). 
2- Per-protocol sample, i.e. all subjects who have at least one post-treatment assessment and no major protocol viola-
tions that affect the analysis. The Executive Committee (EC) will adjudicate major protocol violations and patients’ in-
clusion in the per-protocol population before unblinding. Analyses based on the per-protocol sample will be supportive
in nature.
8.4 
SAMPLE SIZE CALCULATION 
The sample size is calculated to achieve 80% power for the futility analysis described above using a one-sided alpha 
level of 0.10. It is anticipated that approximately 28% of control subjects will have mRS 0-2 at 3 months; this is based on 
the weighted average of the good outcome proportions reported in the placebo-treated subjects of the Factor VII, 
FAST, GAIN, and CHANT trials [Haley 2005; Lyden 2007; Mayer 2005-2008]. If the true good outcome proportions in 
the DFO and placebo arms are identical (πtx=0.28, πctrl=0.28, a truly futile situation), 254 subjects (127 in each arm) are 
required to test the futility hypothesis with 80% power. This calculation takes into account the 0.12 null value specified 
in the futility hypothesis [Chow, 2003]. The primary analysis will be conducted according to a modified Intent-to-Treat 
(ITT) principle, wherein subjects in whom the study drug infusion is not initiated will be excluded from the analysis.  
Therefore, the final sample size was inflated by a factor of 1.11 (Friedman, Furberg, and DeMets) to account for dilution 
of the treatment effect associated with a conservative drop-out rate of 5% (due to loss-to-follow-up (LTFU) and  


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withdrawal of consent), and an additional factor of 1.04 to account for randomized subjects in whom the study drug is 
not initiated.  Therefore, the final sample size is 294 subjects. 
8.5 
LOSS-TO-FOLLOW-UP AND MISSING DATA 
Extensive efforts will be made to keep missing data, particularly the primary outcome (mRS at 90 days), to a minimum, 
and to ensure near complete follow-up. Missing primary outcome data will be imputed via standard multiple  
imputation (MI) methods (i.e. via logistic regression model predicting outcome based on pertinent baseline and treat-
ment data). As a sensitivity assessment, we will use the Last Observation Carried Forward (LOCF) approach, wherein 
the last available score will be carried forward for missing day 90 assessments. If the treatment effect is robust, we ex-
pect to reach similar conclusions via these imputation methods, particularly for minimal missing data (<5%). 
9 
STUDY MONITORING 
9.1  
MONITORING FOR STUDY PROGRESS AND RECRUITMENT  
The Executive Committee will monitor recruitment progress at each site on a monthly basis to address individual site 
issues and concerns. To minimize unnecessary delays in enrolling eligible subjects into the early time window (≤12h 
from ICH onset), we will monitor the time-from-door-to-infusion on an ongoing basis and make every effort to ensure 
eligible subjects are enrolled as quickly as possible after ED arrival. If we observe that enrollment into the early (≤12h) 
time window is disproportionately low compared to the >12-24h window, we will implement a strategy of forced re-
cruitment whereby each site will be required to enroll one subject into the early time window for each subject enrolled 
into the later (>12-24h) window (or maintain its overall ratio of ≤12h to >12-24h to at least 1:1) before subsequent en-
rollment into the later time window is permitted by the site. If frequent delays are observed at a particular site, the 
Principal Investigator will be asked to take the necessary corrective steps. The Executive Committee additionally may 
require that the infusion of the study drug must be started within a certain time window following the subject’s arrival 
to the emergency room, at a particular site or across all sites, if necessary.  
The Executive Committee will also monitor accrual of subjects on an ongoing basis throughout the study, and will con-
duct formal reviews of the overall recruitment across all sites and site-by-site on a yearly basis (or at more frequent in-
tervals, if needed).  For sites whose recruitment is less than 75% of their expected rate (approximately 4-6 subjects per 
year), the local principal investigator will be asked to submit his/her own analysis of the barriers to recruitment at 
his/her site and corrective actions and plans. The “low” enrolling sites will be monitored closely at 3-month intervals to 
determine if further corrective actions or dismissal from the trial are warranted. 
       9.2  
QUALITY MANAGEMENT AND DATA CONTROL 
The Data Coordination Unit (DCU) in the Department of Public Health Sciences at the Medical University of South 
Carolina (MUSC) serves as the Statistics and Data Management Center (SDMC) for the trial, coordinating all statistical, 
data and project management responsibilities. The study data will be managed (including data queries) by the SDMC 
using the WebDCU™ system. This user-friendly web-based database system, developed and validated by the SDMC, 
will be used for regulatory document management, subject randomization, data entry, data validation, project progress 
monitoring, subject tracking, user customizable report generation and secure data transfer. In addition to the study da-
tabase, the SDMC will provide the clinical site staff access (via password) to a standard set of web-enabled tools, includ-
ing subject visit calendar, subject accrual status, case report form completion status, and outstanding DCR status per-
taining to their respective clinical sites. Furthermore, all approved study materials, such as the protocol, informed con-
sent template and manual of procedures, will be housed on the website to ensure that the clinical sites always have ac-
cess to the most current trial documents.  


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Data should be independently entered by the designated personnel at each clinical site into WebDCU within five work-
ing days following the completion of any trial phase – Baseline, Treatment, day-7 or discharge (whichever occurs first), 
Month 1, Month 2, Month 3, and Month 6. Enrollment data, however, must be entered into the WebDCU database be-
fore the subject can be randomized. The SAE and adverse events of respiratory compromise data must be entered into 
the WebDCU within 24 hours of the site staff’s first awareness of the event.  It is critically important to the effective and  
efficient conduct of the study that ALL data be entered in a timely manner. An electronic copy of the CRFs will be made 
available to the clinical sites prior to initiation of the study to be used as worksheets to capture the required data for the 
study. The SDMC staff will perform range verification, consistency checks, and quality assurance on the data. The staff 
at the SDMC will contact the sites regarding missing data or queries on CRF data. They will maintain direct contact with 
the staff at the participating sites to ensure the study is conducted according to the Good Clinical Practice Guidelines 
and FDA and all applicable regulations. 
Experienced clinical research monitors will be contracted through the SDMC to perform on site source data verification 
(SDV) during the study. The first visit will take place after enrollment of the first subject by the site and will involve 
source document verification of 100% of the data. For subsequent subjects, a checklist of key outcome and safety data 
variables requiring SDV will be developed based on the trial’s endpoints.  The checklist ensures that a target of no less 
than 40% of the clinical data submitted to the Hi-Def database are verified against source documents at the perfor-
mance sites prior to finalization of the database. Of the data on the checklist, the safety and efficacy variables repre-
sent approximately half of the data to be verified. The remaining half of source monitored data include: 100% of deaths 
and 100% of serious adverse events and source data reviews based on the per-patient evaluation of safety parameters 
defined in the protocol. All informed consent and HIPAA documents will be verified by the clinical research monitor. 
Subsequent visits to each site will depend on the number of patients recruited at the site as well as other issues. Moni-
toring will also involve, as appropriate, correspondence and telephone contacts. In addition to data verification, the 
monitor will evaluate drug accountability, site facilities, and regulatory documents. The CRFs and corresponding source 
documents should be made available to the study monitor at each site visit.  It is also expected that the PI, or a desig-
nated member of the research staff, will be available during the monitoring visit to review the data and resolve any que-
ries.  The close out monitoring visit will take place after the study is completed. 
The SDMC will prepare, at selected intervals, a summary report of screened and enrolled patients, completeness and 
quality of CRF data, status of enrolled patients, a listing of SAEs, and a table of event-specific cumulative rates. The Ex-
ecutive Committee and the independent Medical Safety Monitors will review these reports and monitor the study per-
formance (recruitment, compliance, protocol violations, and follow up) and safety data on an ongoing basis. The study 
statistician will also generate a comprehensive statistical report bi-annually to the DSMB, unless requested at more fre-
quent intervals.   
       9.3  
ONGOING SAFETY MONITORING DURING THE STUDY  
Two independent Medical Safety Monitors (MSMs), appointed in consultation with the NINDS program director, will 
monitor the study with regard to safety on an ongoing basis to identify any safety concerns. As mentioned above in 
section 7.6, the MSMs will review all SAEs and cases of respiratory compromise and determine whether they are related 
to study drug administration, and will communicate with the investigators for any questions or clarifications regarding 
an event. Periodically throughout the study, the EC and the MSMs will review reports on the incidence rates of all 
reported adverse events, whether serious or not, with particular attention to SAEs, ARDS, and mortality during the first 
7 days of hospitalization (or until discharge, whichever is earlier).  Should such monitoring uncover issues that may 
threaten subject safety (e.g. an unexpectedly high rate of adverse events), the study statistician and principal 
investigator will prepare a report to be submitted to the DSMB for their review and further actions to be taken, if any.  


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Analysis of the safety data regarding the occurrence of ARDS will carried out continuously throughout the trial by the 
unblinded study statistician, who will notify the EC and DSMB if recruitment must be stopped (please refer to section 
7.7 for the stopping rule).   The DSMB will be notified of each individual ARDS occurrence upon confirmation by the 
MSMs, per their request.Analysis of the overall safety data by the DSMB will take place periodically throughout the tri-
al. The first analysis is planned after the first 40 subjects have completed the in-hospital phase of the study. The num-
ber and timing of subsequent analyses will be determined in consultation with the DSMB as the study progresses.  
Safety analyses will be carried out at least semiannually (unless requested at more frequent intervals by the DSMB or 
MSMs). Two statistical reports will be generated – an open report to be distributed to the Executive Committee and 
MSM, and a closed report to be distributed only to the NINDS-appointed DSMB.  Each semi-annual report will provide 
cumulative summary statistics on enrollment, subject status in the study, baseline characteristics, protocol violations, 
safety data (including a summary of the most frequent and most serious adverse events, a summary of all MedWatch 
reports, and a listing of all subjects who were terminated from the study due to adverse events, whether or not study 
drug-related), and data management/quality information. The statistics will be provided for the overall study. For the 
closed report only, the statistics will also be provided by partially blinded treatment group (A vs. B). If the DSMB wishes 
to be completely unblinded for these reports, a sealed identification envelope will be provided to the NINDS DSMB liai-
son; this envelope can be opened at the discretion of the DSMB. An annual report will be submitted to the FDA. 
10 
HUMAN SUBJECTS 
10.1 
 ETHICAL CONDUCT OF THE STUDY 
This study will be conducted in accordance with Declaration of Helsinki, and in compliance with the protocol, applicable 
regulatory requirements, and GCP/ICH guidelines (please refer to appendix VIII).  The PI at each site is responsible for 
the care and medical follow-up of the patients throughout their participation in the study.  If the PI is not present in the 
clinical site, he/she will leave instructions for other members of the study staff and a telephone number where he/she 
can be reached, if needed.   
10.2 
INSTITUTIONAL REVIEW BOARD (IRB) REVIEW AND INFORMED CONSENT 
This protocol, the ICF, and any subsequent modifications must be reviewed and approved by the local IRB at each of the 
participating institutions. A signed and dated ICF must be obtained from the subject, his/her legal representative, or 
family member as defined in 21CFR50.3(m).  The ICF must also be signed and dated by a member of the study staff 
qualified to be delegated the authority to obtain informed consent, and a witness (if required by the local IRB).  A copy 
of the ICF must be given to the subject, his/her legal representative or family member and the consent process must be 
documented in the subject’s medical record.  The PI or delegated sub-Investigator is responsible for ensuring that in-
formed consent is obtained from each patient, his/her legal representative, or family member prior to conducting any 
study-related activities.  
No deviations from or changes to the study protocol should be initiated except when necessary to eliminate immediate 
hazard to the patient.  However, the IRB and PI must be informed of this as soon as possible thereafter.  It is the study 
site PI’s responsibility to report SAEs occurring during the study and MedWatch/Safety Report to their IRB, as required 
and as soon as possible.       
10.3 
SUBJECT CONFIDENTIALITY 
All participating study investigators must ensure that the confidentiality of personal identity and all personal medical 
information of study participants will be maintained at all times. Additionally, the clinical sites are to follow privacy ob-
ligations to study participants under the Health Insurance Portability and Accountability Act (HIPAA). All records re 


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45 
garding this study must be securely stored in a locked cabinet. Subject confidentiality will be strictly maintained by the 
use of a subject ID number. The study database and any study documents submitted to the SDMC will only identify 
study subjects by this unique study identification code. All data will be stored in a manner that is HIPAA compliant, 
without the ability to track the information back to a specific subject except through a password protected system. Per-
sonal medical information may be reviewed for the purpose of verifying data recorded in the CRF by site monitors. 
Other properly authorized persons, such as the regulatory authorities, also may have access to these records. Personal 
medical information, however, always will be treated as strictly confidential. All SDMC personnel are certified by the  
NIH Office of Human Subjects Research in the Protection (OHRP) of Human Research Subjects course, or other training 
courses acceptable to the NIH OHRP. 
In order to maintain the confidentiality of subjects who elect to participate in the blood bank repository, as well: 1) the 
results of any genetic analyses performed on samples collected from this repository will not appear in the medical 
records and will not be released to the subjects or their healthcare providers; 2) the samples will be coded and the key 
will be kept in a separate locked file; and 3) in the event of future second or third party use of the samples, all codes will 
be removed so that there will be no method by which the sample can be tracked back to the subject. 
10.4 
DATA AND SAFETY MONITORING PLAN 
The DSMB, appointed by the NINDS and managed by the NINDS Clinical Trials group, will be responsible for the 
oversight of safety of trial participants, review of the safety reports, requesting additional data/information (if 
necessary), and advising the NINDS regarding continuation/ discontinuation of the study. The study may be modified or 
discontinued at any time by the NINDS, the FDA, or the Executive Committee to ensure that research subjects are 
protected. All ARDS cases will monitored continuously throughout the trial by the unblinded study statistician, who will 
notify the EC and DSMB if recruitment must be stopped (please refer to section 7.7 for the stopping rule).  The DSMB 
will be notified of each individual ARDS occurrence upon confirmation by the MSMs, per their request. Analysis of the 
overall safety data by the DSMB will take place periodically throughout the trial. The first analysis is planned after the 
first 40 subjects have completed the in-hospital phase of the study (day-7 or discharge, whichever is earlier). The DSMB 
will meet to review the accumulated safety data, with particular attention to mortality, SAEs, and adverse events of 
special interest (AEOSI) including ARDS, from initiation of treatment to day-7 or discharge (whichever occurs first) to 
ensure that there are no emerging safety concerns. Unblinding of treatment assignments will only be allowed by the 
Chair of the DSMB, if felt necessary, but the unblinded data will be confidentially maintained within the DSMB. Enroll-
ment will continue with the recommendation of the DSMB, including necessary modifications, if needed, to address 
any safety concerns early on.  
After this meeting, the DSMB will regularly meet at least once approximately every 6 months or at more frequent 
intervals if needed to review recruitment and safety data. Analyses of the safety data will be carried out on an ongoing 
basis throughout the trial, as mentioned above. All AEOSIs, all SAEs, deaths, and adverse events will be reviewed by 
the DSMB during their meetings. There will be no limit on the number and timing of analyses aimed at guaranteeing 
the safety of the patients.  An apparent, consistent and persistent evidence of net harm that tends to overwhelm any 
benefit may allow for premature termination of the study. Please refer to section 7.7 from the pre-specified stopping 
rule based on the frequency of ARDS cases. 
10.5 
STUDY MODIFICATION/DISCONTINUATION 
The study may be modified or discontinued at any time by the NINDS, the OHRP, the FDA, or a study site IRB (but only 
for that individual study site) as part of their duties to ensure that research subjects are protected. 
11 
PUBLICATION OF RESEARCH FINDINGS 


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Publication and presentations of the results of this trial will be governed by the policies and procedures developed by 
the Executive Committee in conjunction with the NINDS.  The Publication Policy will be fully compliant with the vol-
untary NIH Public Access Policy mandated by the Consolidated Appropriations Act of 2008 (Division G, Title 
II, Section 218 of PL 110-161). The Executive Committee will follow NIH policies on data sharing (as described at the 
site: [http://grants2.nih.gov/grants/policy/data_sharing/data_sharing_ guidance.htm] and any updates thereto).  Any 
manuscript(s) will be made available for review by the sponsor and the NINDS prior to submission.  
A Publications Subcommittee of the Executive Committee, consisting of Drs. Magdy Selim, Sharon Yeatts, Yuko Palesch,  
and Claudia Moy will establish a writing group headed by the Study Chair to oversee the writing of the main study-related 
manuscripts. Any of the Trial’s investigators (clinicians as well as study coordinators) wishing to write a manuscript must  
first submit to the Publications Subcommittee a brief description/proposal of the manuscript content. The Subcommittee  
will review the proposal in a timely manner, and if the proposal is approved, it will be disseminated to all study investigators 
to encourage those who have similar interests in the topic to participate in the writing group. The Publications Subcom- 
mittee will appoint the writing group members. The Publication Subcommittee must review and approve all writing and 
presentation activities of the study prior to submission to a journal or a meeting for scientific presentation. 
12 
ANCILLARY STUDIES POLICY  
Any of the study investigator(s) wishing to conduct ancillary studies must submit in writing to the Executive  
Committee a 2-3 page outline of the proposal. The Executive Committee will discuss the proposal, and each  
member of the Executive Committee will vote to approve or disapprove the proposal. The key criteria for  
evaluation are scientific merit, relevance to the major goals of the main study, and the ancillary study’s impact 
on the conduct and feasibility of the main trial. If the proposal is approved by the Executive Committee, it will  
be forwarded to the DSMB members and the NINDS Administrative Officer who will also vote for approval or  
disapproval. Upon approval by the Executive Committee, the DSMB and the NINDS, the investigator(s) may  
then submit the proposal to potential funding source(s), if needed.  
13 
RESOURCE- AND DATA-SHARING PLAN  
The Executive Committee will follow the NIH policies on data sharing [http://grants2.nih.gov/grants/policy/ da-
ta_sharing/data_sharing_guidance.htm], and any updates thereto. Sharing of the data generated by this study will be 
carried out in several different ways to make our results available to the scientific community interested in stroke and 
ICH and to avoid unintentional duplication of research. Our plan for data sharing will include: 1) Presentations at na-
tional and international scientific meetings, such as the International Stroke Conference, the Annual meeting of the 
American Academy of Neurology, the European Stroke Conference, and the International Cerebral Hemorrhage con-
ference; 2) Posting information about stroke and ICH written primarily for a general audience on the study’s website; 3) 
Making a description of this clinical trial and summary of the results available on www.ClinicalTrials.gov.   
We would also welcome collaboration with others who could make use of the data generated by the study. Upon com-
pletion of the study, the public use database will be prepared by stripping any and all personal identifiers. The public 
use database, consisting of several data files, will contain: (1) baseline and demographic characteristics; (2) outcomes 
assessments; (3) CT data; (4) concomitant medications and procedures; and (5) adverse events. Each data file will be 
made available as a formatted SAS dataset or other electronic format. The data files will be distributed along with the 
data dictionary and a brief instruction file. Anyone wishing to access the data may do so by completing a data-sharing 
agreement and data request form and submitting those forms to the SDMC or subsequently to an external data archiv-
ing unit chosen by the NINDS.  
14 
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77. Suri MF, Suarez JI, Rodrigue TC, Zaidat OO, Vazquez G, Wensel A, Selman WR. Effect of treatment of elevated
blood pressure on neurological deterioration in patients with acute intracerebral hemorrhage. Neurocrit Care.
2008;9(2):177-182.
78. Steiner T, Kaste M, Forsting M, Mendelow D, Kwiecinski H, Szikora I, Juvela S, Marchel A, Chapot R, Cognard C, Un-
terberg A, Hacke W.  Recommendations for the management of intracranial haemorrhage - part I: spontaneous in-
tracerebral haemorrhage. The European Stroke Initiative Writing Committee and the Writing Committee for the
EUSI Executive Committee. Cerebrovasc Dis. 2006;22(4):294-316.
79. Sykora M, Diedler J, Turcani P, Rupp A, Steiner T. Subacute perihematomal edema in ICH is associated with im-
paired blood pressure regulation. J Neurol Sci. 2009;284(1-2):108-112
80. Tanji K, Imaizumi T, Matsumiya T, Itaya H, Fujimoto K, Cui X, Toki T, Ito E, Yoshida H, Wakabayashi K, Satoh K.
Desferrioxamine, an iron chelator, upregulates cyclooxygenase-2 expression and prostaglandin production in a
human macrophage cell line. Biochim Biophys Acta. 2001;1530(2-3):227-235.
80. Tenenbein M, Kowalski S, Sienko A, Bowden DH, Adamson IY. Pulmonary toxic effects of continuous desferriox-
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82. Taves, D.R., Minimization: a new method of assigning patients to treatment and control groups. Clin Pharmacol
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83. Venkatasubramanian C, Mlynash M, Finley-Caulfield A, Eyngorn I, Kalimuthu R, Snider RW, Wijman CA. Natural
history of perihematomal edema after intracerebral hemorrhage measured by serial magnetic resonance imaging.
Stroke. 2011;42:73-80.
84. Wagner KR, Sharp FR, Ardizzone TD, Lu A, Clark JF. Heme and iron metabolism: role in cerebral hemorrhage. J
Cereb Blood Flow Metab. 2003;23(6):629-652.
85. Wan S, Hua Y, Keep RF, Hoff JT, Xi G. Deferoxamine reduces CSF free iron levels following intracerebral hemor-
rhage. Acta Neurochir Suppl. 2006;96:199-202.
86. Wan S, Zhan R, Zheng S, Hua Y, Xi G. Activation of c-Jun-N-terminal kinase in a rat model of intracerebral hemor-
rhage: the role of iron. Neurosci Res. 2009;63:100-105.


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52 
87. Westlin W. Desferrioxamine in the treatment of acute iron poisoning. Clin Toxicol. 1971;4:597-602.
88. Winterbourn CC. Toxicity of iron and hydrogen peroxide: the Fenton reaction. Toxicol Lett. 1995;82-83:969-974.
Wu J,
89. Hua Y, Keep RF, Schallert T, Hoff JT, Xi G. Oxidative brain injury from extravasated erythrocytes after intracerebral
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90. Wu J, Hua Y, Keep RF, Nakamura T, Hoff JT, Xi G. Iron and iron-handling proteins in the brain after intracerebral
hemorrhage.  Stroke. 2003;34(12):2964-2969.
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brain edema. Acta Neurochir. 2006 (supplement) 96;78-80.
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blood, liver and brain. Toxicology. 1991;66(3):313-24.
93. Xi G, Wagner KR, Keep RF, Hua Y, de Courten-Myers GM, Broderick JP, Brott TG, Hoff JT, Muizelaar JP. Role of
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94. Xi G, Keep RF, Hoff JT. Erythrocytes and delayed brain edema formation following intracerebral hemorrhage in
rats.  J Neurosurg. 1998;89(6):991-996.
95. Xi G, Keep RF, Hoff JT. Pathophysiology of brain edema formation.  Neurosurg Clin N Am. 2002;13(3):371-383.
96. Xi G, Keep RF, Hoff JT. The role of thrombin and thrombin receptors in ischemic, hemorrhagic and traumatic brain
injury: deleterious or protective?  J Neurochem. 2003;84(1):3-9.
97. Zaman K, Ryu H, Hall D, O'Donovan K, Lin KI, Miller MP, Marquis JC, Baraban JM, Semenza GL, Ratan RR.  Protec-
tion from oxidative stress-induced apoptosis in cortical neuronal cultures by iron chelators is associated with en-
hanced DNA binding of hypoxia-inducible factor-1 and ATF-1/CREB and increased expression of glycolytic en-
zymes, p21(waf1/cip1), and erythropoietin. J Neurosci. 1999;19(22):9821-9830.
98. Zazulia AR, Diringer MN, Derdeyn CP, Powers WJ.  Progression of mass effect after intracerebral hemorrhage.
Stroke. 1999;30(6):1167-1173.
99. Zhang LF, Yang J, Hong Z, Yuan GG, Zhou BF, Zhao LC, Huang YN, Chen J, Wu YF; Collaborative Group of China
Multicenter Study of Cardiovascular Epidemiology.  Proportion of different subtypes of stroke in China. Stroke.
2003;34(9):2091-2096.
15 
APPENDIX 
I.
DFO in ICH (Phase I) Manuscript
II.
RECOMMENDED PROTOCOL FOR MANAGEMENT OF PATIENTS WITH ICH
III.
ASA/AHA ICH MANAGEMENT GUIDELINES
IV.
DETERMINATION OF ICH SCORE AND HEMATOMA VOLUME (ABC METHOD)
V.
ASSESSMENT SCALES (NIHSS – GCS - mRS – MoCA – SIS-16) AND INSTRUCTIONS
VI.
ARDSNet RECOMMENDATIONS FOR MANAGEMENT OF INTUBATED PATIENTS
VII 
BERLIN CRITERIA FOR DIAGNOSIS OF ACUTE RESPIRATORY DISTRESS SYNDROME 
VIII.
DECLARATION OF HELSINKI

SAP_001.pdf: 
iDEF Trial Statistical Analysis Plan 
Version 1.1 (07/11/18) 
Intracerebral Hemorrhage Deferoxamine Trial 
(iDEF Trial)  
STATISTICAL ANALYSIS PLAN 
Prepared by 
Sharon D. Yeatts, Ph.D. 
Yuko Y. Palesch, Ph.D. 
Lydia D. Foster, MS 
Data Coordination Unit 
Department of Public Health Sciences 
Medical University of South Carolina 
Charleston, SC 29425 
Version 1.1 


iDEF Trial Statistical Analysis Plan 
Version 1.1 (07/11/18) 
TABLE OF CONTENTS 
1.
LIST OF ABBREVIATIONS .................................................................................. 1 
2.
STATISTICAL ANALYSIS PLAN AND STATISTICAL REPORTS ....................... 1 
3.
OBJECTIVES OF THE STUDY ............................................................................ 1 
3.1. 
Efficacy................................................................................................................... 1 
3.2. 
Safety ..................................................................................................................... 2 
4.
STUDY DESIGN................................................................................................... 2 
5.
SAMPLE SIZE DETERMINATION FOR THE PRIMARY EFFICACY ANALYSIS 2
6.
DEFINITION OF TARGET POPULATION AND STUDY SAMPLES .................... 2 
6.1. 
Target Population ................................................................................................. 2 
6.2. 
Modified Intent-to-Treat Sample ........................................................................ 2 
7.
RANDOMIZATION ............................................................................................... 2 
8.
BLINDING ............................................................................................................ 3 
9.
MULTIPLICITY ..................................................................................................... 3 
10.
MISSING DATA .................................................................................................... 4 
11.
PRIMARY ANALYSIS ........................................................................................... 4 
11.1. 
Primary Outcome Variable Analysis .................................................................. 4 
11.1.1.  Statistical Hypothesis........................................................................................... 5 
11.1.2.  Primary Analysis ................................................................................................... 5 
11.1.3.  Additional Analyses of the Primary Outcome: Exploring a Differential 
Treatment Effect According to OTT Window ................................................... 5 
11.1.4.  Additional Analyses of the Primary Outcome: Adjusting for Covariates ...... 6 
11.1.5.  Additional Analyses of the Primary Outcome: Subgroup Analyses ............. 6 
11.1.6.  Additional Analyses of the Primary Outcome: mRS dichotomized0-3 v 4-6
................................................................................................................................. 6 
11.1.7.  Ordinal Analysis of the mRS at 90 days ............................................................7
11.1.8.  Additional Analyses of the Primary Outcome: Per Protocol Analysis .......... 7 
11.1.9.  Additional Analyses of the Primary Outcome: ITT Analysis .......................... 7 
11.2. 
Secondary Outcomes Analyses ......................................................................... 8 
11.2.1.  Mortality at 90 days .............................................................................................. 8 
11.2.2.  Symptomatic Cerebral Edema through 7 days or discharge ........................ 8 
11.2.3.  Functional and Cognitive Measures at 90 days .............................................. 8 
11.2.4.  Analysis of the mRS at 180 days ....................................................................... 9
11.3. 
Exploratory Analyses ........................................................................................... 9 
11.3.1.  Treatment Effect on PHE Volume Progression ............................................... 9 
11.3.2.  Treatment Effect on Ventricular Enlargement ................................................. 9 
11.3.3.  Progression of PHE as a Biological Marker of Activity...................................... 9
12.
SAFETY ANALYSES ............................................................................................ 9 
12.1. 
Safety Monitoring ................................................................................................. 9 
12.2. 
Summary of Adverse Events and Serious Adverse Events ........................ 10 
13.
REFERENCES ................................................................................................... 10 


iDEF Trial Statistical Analysis Plan 
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1.
LIST OF ABBREVIATIONS
AE  
Adverse Event 
AEOSI 
Adverse Events of Special Interest 
ARDS 
Acute Respiratory Distress Syndrome 
DCR 
Data Clarification Request 
DFO 
Deferoxamine Mesylate 
SDMC 
Statistical and Data Management Center 
DSMB 
Data and Safety Monitoring Board 
GCP 
Good Clinical Practice 
HR  
Hazard Ratio 
ICH  
Intracerebral Hemorrhage 
ITT  
Intent-to-Treat 
MoCA 
Montreal Cognitive Assessment 
NIHSS 
National Institute of Health Stroke Scale 
SAE 
Serious Adverse Event 
SIS-16 
Stroke Impact Scale-16 
2.
STATISTICAL ANALYSIS PLAN AND STATISTICAL REPORTS
This document provides the details of statistical analyses planned for the iDEF Trial.  In 
addition, it discusses the statistical issues relevant to these analyses (e.g., sample data to be 
used, missing data, adjustments for multiplicity, etc.) 
The partially unblinded statistician at the Statistics and Data Management Center (SDMC) 
generates closed and open DSMB Reports semiannually. Each semiannual report provides 
cumulative summary statistics on enrollment; subject status in the study (e.g., number 
completed 90 day assessments); baseline characteristics; protocol violations; safety data, 
including AEs and SAEs by AE code, severity, and relatedness to the study medication; and 
data management/quality information (e.g., timeliness and completeness of data entry by the 
clinical centers via the iDEF Trial Website; number of DCRs generated and resolved).  The 
statistics for the closed DSMB Reports are provided by partially unblinded treatment group 
(A/B).  The open report contains aggregate statistics only, i.e., not by treatment group.  
3.
OBJECTIVES OF THE STUDY
3.1. Efficacy
The primary objective of the iDEF Trial is to assess the futility of deferoxamine mesylate
(DFO), when initiated within 24 hours of symptom onset and administered for 3 days, versus
placebo in subjects with ICH.  The primary futility hypothesis is that DFO will increase the
proportion of subjects with a good functional outcome, as defined by the modified Rankin
Scale (mRS) 0-2 at 90 days, by less than absolute 12% when compared to placebo.
In addition, the Trial plans to evaluate the effect of DFO as compared to placebo based on
the proportion of subjects with mRS 0-3.  For supportive evidence, other clinical outcome
measures at 3 months, including the National Institutes of Health Stroke Scale (NIHSS),
Montreal Cognitive Assessment (MoCA), and the Stroke Impact Scale-16 (SIS-16), will also
be assessed.


iDEF Trial Statistical Analysis Plan 
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3.2. Safety 
The safety of DFO when compared to placebo is evaluated by mortality, SAEs, and Adverse 
Events of Special Interest (anaphylaxis, unexplained hypotension requiring medical 
intervention, development of new and unexplained visual or auditory changes, and ARDS).  
4.
STUDY DESIGN
The study is of a two-arm parallel futility design whereby eligible subjects are randomized in a 
1:1 ratio to either the DFO group or to the placebo group.  Each subject is followed for 180 days 
from randomization. 
5.
SAMPLE SIZE DETERMINATION FOR THE PRIMARY EFFICACY ANALYSIS
The total sample size for the futility threshold of 12% (the absolute increase of DFO over the 
placebo arm in the proportion of subjects with good outcome), assuming a placebo proportion of 
28%, and Type I and Type II error probabilities of 0.10 and 0.80, is 254 subjects.   
The primary analysis will be conducted according to a modified intent-to-treat (ITT) principle, 
wherein subjects in whom the study drug infusion is not initiated will be excluded from the 
analysis.  Therefore, the final sample size was inflated by a factor of 1.11 (Friedman, Furberg, 
and DeMets) to account for dilution of the treatment effect associated with a conservative drop-
out rate of 5% (due to loss-to-follow-up (LTFU) and withdrawal of consent), as well as an 
anticipated <4% of randomized subjects in whom the study drug is not initiated.  Therefore, the 
final sample size is 294 subjects.      
6.
DEFINITION OF TARGET POPULATION AND STUDY SAMPLES
6.1. Target Population
The target population to which the DFO treatment regimen may be applied are patients with
ICH in whom study drug can be initiated within 24 hours of symptom onset.
6.2. Modified Intent-to-Treat Sample
As the primary analysis, all efficacy and safety outcome measures are analyzed under the
modified ITT principle. Under this principle, the evaluable sample includes all randomized
subjects in whom the study infusion is initiated, regardless of whether or not it was
prematurely discontinued.  Missing outcome data for subjects included in the modified ITT
sample are handled as specified in Section 10.
7.
RANDOMIZATION
The randomization takes place centrally via the iDEF Trial Website via a combination of 
minimization and biased coin methodologies. Subjects are randomized in a 1:1 ratio (DFO: 
placebo), controlling for ICH score, onset-to-treatment (OTT) time, ICH volume, NIHSS score, 
concurrent use of warfarin at the time of ICH onset and clinical site.  The computer program 


 
iDEF Trial Statistical Analysis Plan 
 
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developed at the SDMC makes the treatment assignment based on the current status of 
treatment group distribution and study drug availability.  The detailed randomization scheme 
and source codes are provided in the Randomization Plan document. 
 
The randomization is never deterministic.  Although a randomization scheme with any 
constraints would yield some bias in the inferences from using standard analytic methods, Efron 
(1971) shows the appropriateness of standard statistical tests under the biased coin 
randomization in large studies.  Friedman et al (1998 p. 72) note that the variance terms under 
the biased coin design tend to be larger than under simple randomization.  The consequence is 
that it would be more difficult to reject the null hypothesis, and therefore, we would be more 
conservative in determining the futility of the treatment. 
 
A “Real-Time” randomization procedure is implemented via the iDEF Trial Website on the 
WebDCUTM System, where the clinical center staff enters the basic baseline (e.g., ICH score, 
anticipated time to treatment, etc) and eligibility information of a subject prior to enrollment.  If 
the subject’s eligibility status is confirmed, the computer program on the WebDCUTM server 
evaluates the treatment arm distribution and selects a randomization number based on the 
randomization scheme.  The randomization number corresponds to a specific label in the 
Randomization Binder maintained at the site pharmacy, indicating whether the subject should 
receive DFO or placebo.     
 
 
8. BLINDING  
 
In the phase I study, the reconstituted solution of DFO was colorless, and there were no specific 
treatment-related changes in laboratory tests or adverse events to suggest that the active drug 
can be identified from placebo.  The study subject and all site staff, with the exception of the 
pharmacist, are blinded to the treatment assignment of all subjects.  The pharmacists are 
specifically instructed not to reveal treatment assignment to the investigators.  The SDMC staff 
(with the exception of the partially unblinded statistician, biostatistical programmer, and the 
senior programmer) are blinded to the treatment assignment of all subjects.  
 
There is no specific antidote to DFO; therefore, unblinding is unnecessary in most cases. In 
cases of extreme emergency when the treating physicians request unblinding of treatment 
assignments for therapeutic purposes, the unblinding will only be revealed to the treating 
physicians but not the investigators. The treating physicians must be instructed not to reveal the 
blind to the subjects or study investigators. The study personnel (pharmacist) will be required to 
inform the Principal Investigator and the Project Manager within 24h in the event of unblinding. 
In cases where the treating physician is one of the study investigators, he/she also will be 
required not to reveal the identity of the study drug to other members of the study team, and not 
to perform subsequent study-related outcome assessments. 
 
 
9. MULTIPLICITY 
 
The primary futility hypothesis is tested via generalized linear model relating the probability of a 
favorable outcome to the treatment, as described in Section 11.  The hypothesis is tested at the 
one-sided alpha level of 0.10.   
 


 
iDEF Trial Statistical Analysis Plan 
 
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Secondary outcomes analyses are considered exploratory.  We present descriptive statistics 
and confidence intervals rather than p-values.  The results of these analyses are interpreted 
with caution.  Because the tests are not necessarily powered to detect a pre-specified effect 
size, a negative test result may be due to lack of power.   
 
For safety outcomes, we will not account for multiplicity.   
 
 
10. MISSING DATA 
 
Based on previous experiences with clinical trials of acute stroke (including HI-DEF), it is 
anticipated that there would be minimal missing data for the 90-day assessment of the primary 
outcome. Only 1 of 42 HI-DEF subjects (2.38%) was missing the primary outcome.  All efforts 
should be put forth to ensure near complete follow-up, in particular with the assessment of the 
primary outcome and occurrence of death.  Nevertheless, minimal missing data may be 
inevitable.     
 
If the missing data are minimal (<5%), we plan to perform a complete case analysis, wherein 
any subjects missing the primary outcome are excluded from the analysis.  Otherwise, standard 
multiple imputation methods are used to account for missing primary outcome data in the 
analyses. A distribution for the primary outcome is derived from a logistic regression model that 
accounts for baseline covariates (the individual components of the ICH score (age, size and 
location of hemorrhage, Glasgow Coma Scale score, and presence of intraventricular 
hemorrhage), continuous time from onset to treatment initiation, serum glucose, statin use at 
onset), treatment, and post-treatment data (the last mRS obtained prior to end of study, initiation 
of anti-edema therapy), and a random sample from this distribution is used to impute values for 
missing primary outcomes.  Multiple sample data sets with complete 90-day outcome are 
generated through PROC MI, and each of the data sets are analyzed according to the method 
described in Section 11.1.2, and the results (regression parameter and covariance matrix 
estimates) for each sample are combined and analyzed with PROC MIANALYZE to derive a 
valid statistical inference about the treatment effect.   
 
As a sensitivity analysis, we plan to impute the missing primary outcome data via the Last 
Observation Carried Forward (LOCF) approach as well as the best/worst imputation. If the 
treatment effect is robust, we expect analyses using these imputation methods would yield 
similar inferences, particularly if the missing data are minimal (<5%).  
 
 
11.  PRIMARY ANALYSIS 
 
11.1. Primary Outcome Variable Analysis 
 
The primary outcome measure is the mRS score, dichotomized to define good functional 
outcome as a score of 0, 1, or 2 on the mRS at 90 days post randomization.  For the primary 
analysis, mRS data collected no sooner than 60 days and no later than 120 days from 
randomization are considered for the primary outcome.   
 
The primary futility hypothesis is tested via generalized linear model relating the probability 
of a good outcome to the treatment, after adjustments as described below.   
 


 
iDEF Trial Statistical Analysis Plan 
 
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The primary analysis is conducted under the modified ITT principle; all randomized subjects 
in whom the study infusion is initiated are included in the analysis and analyzed according to 
the randomly assigned treatment arm.  Covariate misclassifications, however, will be 
corrected in the analysis in order to maintain the intended power (Fan L et al, 2015).  OTT 
times greater than 24 hours will be included in the 12-24 hour category. 
 
11.1.1. Statistical Hypothesis 
 
The analysis model for the primary futility analysis is: 
 
݃ሼܲሺܻ௜ൌ1|ܠሻሽൌߙ൅ߚଵݔଵ൅ߚଶݔଶ൅ߚଷݔଷ൅ߚସݔସ൅ߚହݔହ൅ߚ଺ݔ଺ ,, 
 
where Y is the primary outcome variable (1=favorable, 0=unfavorable), x  is a 
specified vector of covariates, and g is the link function.  Here we consider 
1
0
x 
  if 
treatment=placebo and 
1
1
x 
 if treatment=DFO; ݔଶ, ݔଷ, ݔସ, ݔହ, and ݔ଺  are indicator 
variables, respectively defining ICH score (0-2 vs 3-5), OTT time (≤12 vs 12-24 
hours), baseline ICH volume (≤10 vs >10), screening NIHSS score (≤10 vs >10), and 
warfarin use (1=Yes, 0=No).  Using this model, the primary statistical hypothesis is 
0
1
:
0.12
H

 versus 
1
1
:
0.12
H

. 
 
11.1.2. Primary Analysis 
 
The primary futility hypothesis is tested via generalized linear model relating the 
probability of a good outcome to the treatment, after adjustments as described 
above.  This model is fit using the binomial distribution for Y and the identity link to 
derive an estimate of β1, which yields an estimate of the risk difference for good 
outcome for DFO versus placebo.  The PROC GENMOD procedure of SAS is used 
to obtain the test statistics and the results.  If the model will not converge, the log link 
(to derive an estimate of the relative risk) and/or the logit link (to derive an estimate 
of the odds ratio) will be considered as alternative approaches.   
 
If insufficient distribution of subjects in each level of anticoagulant use or ICH score 
results in quasi-complete separation of data points, these covariates will be excluded 
from the model.  The ICH score is derived by assigning a point value to each of 5 
components: the Glasgow Coma Scale score, ICH volume, presence of 
intraventricular hemorrhage, infratentorial origin, and age of subject.  We do not 
anticipate significant collinearity between the ICH score and any individual 
component.  However, if significant collinearity exists between the ICH score and 
ICH volume, the component adjustment will be excluded from the model.      
 
The futility analysis will be conducted using a one-sided 90% upper confidence 
bound on the risk difference (the β1 parameter), which is consistent with the one-
sided alternative hypothesis and stated level of significance.  To declare futility, the 
entire interval must lie below the value 0.12, indicating that the true difference in risk 
of good outcome is less than 0.12 with 90% confidence.   
 
11.1.3. Exploring a Differential Treatment Effect According to OTT Window 
 


 
iDEF Trial Statistical Analysis Plan 
 
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The generalized linear model described above will be expanded to include an 
interaction between treatment and OTT window.  While the trial will be underpowered 
to definitively address this question, the magnitude of the treatment effect, and 
corresponding confidence interval, will be estimated for each time window.  The 
magnitude of the estimated differential effect can be used in determining whether 
future Phase III trials should maintain the 0-24 hour treatment window or consider 
limiting treatment to the ≤12 hour window.  If the model does not converge, the 
simplified model described in Section 11.1.5 will instead be applied. 
 
11.1.4. Adjusting for Covariates 
 
The mRS is additionally assessed for treatment differences adjusting for a variety of 
pre-randomization covariates deemed clinically or prognostically important, such as 
age (continuous), location of hemorrhage (lobar vs. deep thalamic vs deep non-
thalamic), screening Glasgow Coma Scale score (<8 vs 9-12 vs 13-15), ICH volume, 
IVH volume, serum glucose.    Each covariate is evaluated individually first for a 
relationship with outcome.  Each covariate which is found to be significantly 
associated with outcome will be added to the primary model in order to evaluate the 
impact of its inclusion on the estimated treatment effect. Covariates which cause 
more than a 20% change in the treatment effect estimate will be considered potential 
confounders, and a multivariable model that includes these potential confounders 
may then be constructed. If any components of the ICH score are identified as 
treatment modifiers, the ICH score will be removed from the multivariable model in 
favor of its individual components. 
 
11.1.5. Subgroup Analyses 
 
The primary outcome variable is assessed for treatment differences in the covariate 
categories listed below, assuming sufficient numbers of subjects (at least 60) are 
enrolled in each category. Each covariate is evaluated individually with a model that 
includes treatment, subgroup, and an interaction effect between the treatment (DFO 
or placebo).  This model will be used to derive subgroup specific treatment effect 
estimates and the corresponding 95% confidence intervals, and the significance of 
the interaction presented to reflect heterogeneity across the subgroups. 
 
 
OTT time (≤12 vs >12 hours)  
 
ICH score: 0-2 vs 3-5 
 
Sex 
 
Race  
 
Ethnicity 
 
Age (≥60 vs <60) 
 
ICH volume (<30 vs ≥30 cm3) 
 
Intraventricular hemorrhage (present vs absent) 
 
 
11.1.6. mRS Dichotomized 0-3 v 4-6 
 
Although mRS 0-3 is less favorable than the primary outcome of mRS 0-2, it would 
still be a desirable effect in patients with ICH given that no treatments exist to reduce 
disability. The trial is adequately powered to assess the futility hypothesis using mRS 


 
iDEF Trial Statistical Analysis Plan 
 
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0-3 as the outcome based on an absolute difference in treatment effect ≥13% in 
favor of DFO.  This outcome will be analyzed as described above. 
 
11.1.7. Ordinal Analysis of the mRS  
 
As a strictly supportive analysis, a shift analysis of the full distribution of the mRS will 
be analyzed via proportional odds model, if and only if the proportional odds 
assumption holds.  Given the total sample size, it may be the case that the cell count is 
insufficient to fit the full model with treatment and all covariates outlined in Section 
11.1.1.  If this is the case, a sequential approach will be explored, wherein ICH score 
and OTT window strata are individually added to a model that contains only treatment.  
The proportionality assumption will be assessed; the partial proportional odds model 
and/or the adjacent categories logit model may be considered depending on the type 
and degree of violation.   
 
11.1.8.  Per Protocol Analysis 
 
Due to the nature of the study, variability in patient management is, to some extent, 
unavoidable.  To account for this variability, a complete case analysis of the primary 
outcome variable is also conducted on a per protocol basis.  The evaluable sample 
for this analysis includes all subjects who have at least one post-treatment 
assessment and no major protocol violations that affect the analysis.  Subjects will 
be analyzed in the treatment arm representing the treatment actually received.  
Details for defining the per protocol sample were outlined by the Executive 
Committee (EC) prior to unblinding.   
 
The per protocol analysis is based on the modified ITT population and excludes 
subjects according to the below criteria. 
 
Subjects in whom the qualifying ICH was due to a secondary cause will be 
excluded. 
 
Subjects in whom a major pre-specified eligibility criterion was violated. 
o Screening NIHSS <6 or screening GCS <6  
o Bilateral pulmonary infiltrates on screening CXR or other modifiers of 
ARDS risk specified in the protocol 
 
Subjects who did not receive all 3 doses (in full) of the study drug infusion 
 
Subjects in whom surgery (evacuation of the hematoma or hemicraniectomy) 
was performed before completing all 3 doses of the study drug infusion 
 
Subjects in whom withdrawal of care was instituted within 72 hours of 
enrollment. 
 
 
11.1.9. ITT Analysis 
 
Classically, ITT analysis is used in Phase III trials and includes all subjects 
randomized, regardless of whether they received the treatment or not.  As a 
sensitivity analysis, the futility hypothesis will be assessed according to the full ITT 
principle.  All subjects randomized will be included and considered in the treatment 
group to which he/she was randomized, regardless of the treatment actually 
received.  The subjects in whom treatment is never initiated will be assigned an 
unfavorable outcome (mRS>2), based on the assumption that they did not receive 


 
iDEF Trial Statistical Analysis Plan 
 
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treatment due to clinical decline.  Subjects in whom study drug was initiated but for 
whom the primary outcome is missing will be included via Last Observation Carried 
Forward.      
 
11.1.10. 
As Treated Analysis 
 
In the primary analysis, subjects are analyzed in the treatment arm to which they 
were randomly assigned, regardless of whether the correct treatment was 
administered.  As a sensitivity analysis, the futility hypothesis will also be assessed in 
an as treated analysis.  Subjects will be analyzed in the treatment arm representing 
the treatment actually received. 
 
11.2. Secondary Outcomes Analyses  
 
A series of secondary efficacy outcomes are evaluated. The list below outlines the planned 
analysis methods for the pre-specified secondary efficacy outcomes which are conducted 
with the SAS Software System.   
 
.   
The usual verification of variable and model assumptions and goodness of fit assessments 
accompany each analysis.  Some outcome measures may demonstrate a substantial 
departure from the normal distribution, even after transformation, in which case 
nonparametric methods may be considered.   
 
These analyses are used to confirm or support the findings based on the primary outcome 
analysis.  If most of the secondary outcomes show a change in the opposite direction from 
the primary or no change, we might have less confidence in the primary outcome.   
 
 
11.2.1. Mortality at 90 days and at 180 days 
 
The log-rank test is used to compare the survival curves for each treatment group 
(DFO versus placebo).   
 
11.2.2.  Symptomatic Cerebral Edema through 7 days or discharge 
 
Chi-square test, or Fisher’s exact test if the cell size is not sufficient, will be used to 
examine the effect of treatment on the incidence of symptomatic cerebral edema 
(neurological worsening/deterioration adverse events attributed to cerebral edema) 
up to day 7 or discharge, whichever is earlier.   
 
11.2.3. Functional and Cognitive Measures at 90 days 
 
Generalized linear modeling techniques are used to examine the effect of treatment 
on the dependent variables (NIHSS, MoCA, SIS-16) adjusting for previously 
specified covariates.  As in the analysis of the primary outcome, data collected no 
sooner than 60 days and no later than 120 days from randomization are included. 
The NIHSS is a 42 point scale reflecting neurologic deficit.  The MoCA is a 30 point 
scale assessing various domains of cognitive function. The SIS-16 is a 16 point 
scale, with total scores ranging from 16 to 80.  These will be treated as continuous 
measures.        


 
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11.2.4. Analysis of the mRS at 180 days 
 
The analyses specified in Section 11.1 will also be performed on the mRS at 180 
days.  Data collected no sooner than 150 days and no later than 210 days from 
randomization are included. 
 
11.3. Exploratory Analyses  
 
A series of exploratory analyses are also planned. The list below outlines the planned 
analysis methods for these pre-specified exploratory analyses which are conducted with the 
SAS Software System.   
 
The usual verification of variable and model assumptions and goodness of fit assessments 
accompany each analysis.   
 
11.3.1. Treatment Effect on PHE Volume Progression 
 
The effect of DFO treatment on PHE volume progression will be explored as a 
potential marker of biological activity on brain tissue.  Measures of PHE volume 
progression will include absolute PHE volume, relative PHE, edema extension 
distance (EED) and the rate of PHE growth, derived from pre and post treatment 
scans by the central reader.  These outcomes will be analyzed via general linear 
model which includes the baseline ICH volume and screening glucose as covariates.   
 
11.3.2. Effect of PHE Volume Progression on Clinical Outcome 
 
Progression of PHE is a post-randomization characteristic and can therefore be 
considered an outcome in its own right.  The effect of PHE progression on outcome 
will be evaluated separately in the treatment and control arms via generalized linear 
model.  The model will include adjustments for screening glucose, anti-edema 
agents, and hemicraniectomy. 
 
11.3.3.  Treatment Effect on Ventricular Enlargement 
 
The effect of DFO on the size of ventricular enlargement post infusion in patients 
with intraventricular extension of ICH, not requiring an external ventricular drain, as a 
potential marker of treatment utility in intraventricular hemorrhage, will be explored 
via general linear model which includes the baseline size as a covariate. 
 
 
 
12. SAFETY ANALYSES 
 
12.1. 
Safety Monitoring 
 
The detailed guidelines for monitoring for safety by the Executive Committee, the Medical 
Monitor, and the DSMB are included in the protocol.  
 
The review of safety data will focus on the following Adverse Events of Special Interest 
(AEOSI): 


 
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 
Anaphylaxis (at any time point during study drug infusion) 
 
Hypotension (defined as a decrease in blood pressure requiring medical 
intervention at any time point during drug infusion that cannot be explained by 
other causes) 
 
Development of new and unexplained visual or auditory changes after initiating 
treatment with the study drug  
 
Respiratory compromise 
 
 
12.2. 
Summary of Adverse Events and Serious Adverse Events 
 
All AEs and SAEs are summarized by “preferred term” and associated system-organ class 
according to the MedDRA adverse reaction dictionary and by treatment group in terms of 
frequency of the event, number of subjects having the event, timing relative to 
randomization, severity, and relatedness to the study drug.  
 
For the AEOSIs previously defined, the relative risks and their 95% confidence intervals are 
provided.  The DSMB is alerted if the lower confidence limit on the relative risk approaches 1 
from below. 
 
At the end of the study, treatment differences in the cumulative incidences of mortality, as 
well as the AEOSIs, are evaluated via the relative risk and corresponding 95% confidence 
interval.  
 
 
13.  REFERENCES 
 
Efron B. Forcing a sequential experiment to be balanced.  Biometrika 1971; 58: 403-417. 
Friedman LM, Furberg CD, DeMets DL. Fundamentals of Clinical Trials, 3rd ed.  1998; New 
York: Springer-Verlag. 
Fan L, Yeatts SD, Wolf BJ, McClure LA, Selim M, Palesch YY. The impact of covariate 
misclassification using generalized linear regression under covariate-adaptive randomization. 
Stat Methods Med Res. 2015 Nov 23. pii: 0962280215616405. [Epub ahead of print]