NEOLEV3: A PHASE IIB DOSE ESCALATION STUDY OF

LEVETIRACETAM IN THE TREATMENT OF NEONATAL

SEIZURES OF MILD TO MODERATE SEVERITY
National Clinical Trial (NCT) Identified Number: <Number, if available>
Principal Investigators: Sonya, Wang, MD, Jeff Gold, MD, PhD, Cynthia
Sharpe MBChB and Richard Haas MBBChir
<IND/IDE> Sponsor: Richard Haas, #109622

Funded by:
Version Number: 6.6
27 Sept 2022

Summary of Changes from Previous Version:

Affected Summary of Revisions Made Rationale
Section(s)
1.1, 1.2, Changed criteria for maximum In response to FDA advice
2.1, 2.2, seizure burden to <30 minutes
2.3.3, seizure/ hour in phase 3 of the study
5.2,5.5,
6.1.2, 7.1,
8.1, 8.2
2.2 Emphasized that persyst algorithm In response to FDA query
used as research tool only
1.2, 6.1.2, Reverted back to 3 step dose In response to FDA advice
8.1, 10.4 escalation for phase III. Revised
section 8.1 language about Persyst to
be consistent throughout protocol.
6.1.2 Fixed the missed line to revert back In response to FDA comment
to 3 step dose escalation for phase III
1.2 Schema A corrected
1.3 Schedule of activities corrected to In response to UCSD IRB comment
show timing of LEV3 and PHB
2.3.3 Statement added in minimization of
risk to confidentiality to the effect
that study records may be shared
with UCSD IRB or FDA
 6.1.2 Corrected to show more clearly time
duration of observation in the
scenario of continued seizures that
have not worsened ( 2 hours)
Edited to clarify that additional 15%
6.1.2 efficacy is expected to be above
baseline efficacy of 40% as in
NEOLEV2
1.2 Schema C and D edited to reduce In response to USCD IRB
confusion regarding criteria for comment.
maintenance therapy
Schema C and D changed to Schema
phase 1, Schema phase 2, Schema
6.1.2 phase 3.
Maintenance therapy indication
clarified as 50% seizure reduction

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 Table of Contents
STATEMENT OF COMPLIANCE 1
1 PROTOCOL SUMMARY 1
1.1 Synopsis 1
1.2 Schema 2
1.3 Schedule of Activities (SoA) 6
2 INTRODUCTION 6
2.1 Study Rationale 6
2.2 Background 7
2.3 Risk/Benefit Assessment 11
2.3.1 Known Potential Risks 11
2.3.2 Known Potential Benefits 12
2.3.3 Assessment of Potential Risks and Benefits 12
3 OBJECTIVES AND ENDPOINTS 14
4 STUDY DESIGN 15
4.1 Overall Design 15
4.2 Scientific Rationale for Study Design 16
4.3 Justification for Dose 16
4.4 End of Study Definition 16
5 STUDY POPULATION 16
5.1 Inclusion Criteria 16
5.2 Exclusion Criteria 16
5.3 Lifestyle Considerations 17
5.4 Screen Failures 17
5.5 Strategies for Recruitment and Retention 17
6 STUDY INTERVENTION 19
6.1 Study Intervention(s) Administration 19
6.1.1 Study Intervention Description 19
6.1.2 Dosing and Administration 19
6.2 Preparation/Handling/Storage/Accountability 21
6.2.1 Acquisition and accountability 21
6.2.2 Formulation, Appearance, Packaging, and Labeling 21
6.2.3 Product Storage and Stability 21
6.2.4 Preparation 21
6.3 Measures to Minimize Bias: Randomization and Blinding 22
6.4 Study Intervention Compliance 22
6.5 Concomitant Therapy 22
6.5.1 Rescue Medicine 22
7 STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT DISCONTINUATION/WITHDRAWAL 22
7.1 Discontinuation of Study Intervention 22
7.2 Participant Discontinuation/Withdrawal from the Study 23
7.3 Lost to Follow-Up 23
8 STUDY ASSESSMENTS AND PROCEDURES 23
8.1 Efficacy Assessments 23
8.2 Safety and Other Assessments 26
8.3 Adverse Events and Serious Adverse Events 27

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 8.3.1 Definition of Adverse Events (AE) 27
8.3.2 Definition of Serious Adverse Events (SAE) 27
8.3.3 Classification of an Adverse Event 27
8.3.4 Time Period and Frequency for Event Assessment and Follow-Up 30
8.3.5 Adverse Event Reporting 31
8.3.6 Serious Adverse Event Reporting 35
8.3.7 Reporting Events to Participants 35
8.3.8 Events of Special Interest 35
8.3.9 Reporting of Pregnancy 35
8.4 Unanticipated Problems 35
8.4.1 Definition of Unanticipated Problems (UP) 35
8.4.2 Unanticipated Problem Reporting 36
8.4.3 Reporting Unanticipated Problems to Participants 36
9 STATISTICAL CONSIDERATIONS 37
9.1 Statistical Hypotheses 37
9.2 Sample Size Determination 37
9.3 Populations for Analyses 39
9.4 Statistical Analyses 39
9.4.1 General Approach 39
9.4.2 Analysis of the Primary Efficacy Endpoint(s) 40
9.4.3 Analysis of the Secondary Endpoint(s) 41
9.4.4 Safety Analyses 42
9.4.5 Baseline Descriptive Statistics 42
9.4.6 Planned Interim Analyses 42
9.4.7 Sub-Group Analyses 42
9.4.8 Tabulation of Individual participant Data 42
9.4.9 Exploratory Analyses 43
10 SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS 43
10.1 Regulatory, Ethical, and Study Oversight Considerations 43
10.1.1 Informed Consent Process 43
10.1.2 Study Discontinuation and Closure 44
10.1.3 Confidentiality and Privacy 44
10.1.4 Future Use of Stored Specimens and Data 45
10.1.5 Key Roles and Study Governance 45
10.1.6 Safety Oversight 45
10.1.7 Clinical Monitoring 46
10.1.8 Quality Assurance and Quality Control 46
10.1.9 Data Handling and Record Keeping 46
10.1.10 Protocol Deviations 47
10.1.11 Publication and Data Sharing Policy 47
10.1.12 Conflict of Interest Policy 48
10.2 Additional Considerations 48
10.3 Abbreviations 49
10.4 Protocol Amendment History 50
11 REFERENCES 51

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1. STATEMENT OF COMPLIANCE

The trial will be conducted in accordance with International Conference on

Harmonisation Good Clinical Practice (ICH GCP), applicable United States (US) Code of
Federal Regulations (CFR), and the FDA’s Terms and Conditions of Award. The
Principal Investigators will assure that no deviation from, or changes to the protocol will
take place without prior agreement from the Investigational New Drug (IND) or
Investigational Device Exemption (IDE) sponsor, funding agency and documented
approval from the Institutional Review Board (IRB), except where necessary to eliminate
an immediate hazard(s) to the trial participants. All personnel involved in the conduct of
this study have completed Human Subjects Protection and ICH GCP Training.

The protocol, informed consent form(s), recruitment materials, and all participant
materials will be submitted to the IRB for review and approval. Approval of both the
protocol and the consent form must be obtained before any participant is enrolled. Any
amendment to the protocol will require review and approval by the IRB before the
changes are implemented to the study. All changes to the consent form will be IRB
approved; a determination will be made regarding whether a new consent needs to be
obtained from participants who provided consent, using a previously approved consent
form.

1 PROTOCOL SUMMARY

1.1 SYNOPSIS
Title: NEOLEV3:A Phase IIb Dose escalation study of Levetiracetam
in the Treatment of Neonatal Seizures

Study Description: This study is an open label dose-escalation, preliminary safety and
efficacy study with a randomized control treatment component. The
main purpose of this study is to determine the maximum tolerated
dose of LEV in the treatment of neonatal seizures . Our hypothesis is
that optimal dosing of Levetiracetam (LEV) to treat neonatal seizures
is significantly greater than 60mg/kg.

Objectives: Aim 1: To determine the recommended maximal safe dose of LEV in

the setting of neonatal seizures .

Aim 2: To study the pharmacokinetics of high dose LEV in

neonates.

Aim 3: To estimate the additional efficacy of higher doses of LEV

in neonates with seizures .

Aim 4: To improve technologies for the prompt detection of neonatal

seizures

Endpoints: The primary endpoint of this study is the maximum tolerated dose of
LEV in the treatment of neonatal seizures, that is, the maximal

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dose studied where dose limiting toxicity is not observed.

Secondary endpoints will include:

● LEV population pharmacokinetic parameters;

● Estimate of additional efficacy of LEV at higher doses;

● Rates of adverse events seen with high dose LEV treatment.

● Sensitivity and specificity of the latest Persyst neonatal

seizure detector algorithm as compared with the markings of
expert neurophysiologists;

Study Population: 133 Term neonates (corrected gestational age between 35 and 44
weeks, postnatal age less than 28 days), with weight >2200g, with
confirmed electrographic seizures. In phases 1 and 2 we will recruit
only patients with seizure burden less than 8 minutes/hr. Once
phases 1 and 2 have been completed, we will move to phase 3 to
recruit patients with seizure burden less than 30 minutes/hr.

Phase: 2b

Description of The study will enroll in 6 neonatal intensive care units, selected
Sites/Facilities because of their capacity to perform cEEG monitoring with real time
Enrolling response. 4 sites are in the US and 2 in New Zealand. The four sites
Participants: in the US include: UC San Diego, Rady Children’s Hospital, Sharp
Mary Birch, University of Minnesota. The two New Zealand sites
include: Starship Hospital Auckland and Middlemore Hospital
Auckland

Description of Study Patients with confirmed electrographic seizures will be treated with
Intervention: intravenous LEV 60mg/kg as first line and if seizures persist will
receive additional dose escalation doses of LEV. Dose escalation
will increase over the course of the study. If seizures are controlled
by high dose LEV, higher dosing will be maintained for 5 days. If
LEV does not control seizures, standard of care PHB will be
administered.

Study Duration: 54 months; including 6 months after study completion for data
analysis
Participant Duration: 5 days of active treatment. Planned neurodevelopmental follow up at
24 months.

1.2 SCHEMA

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Schema A: Study Recruitment Flow Chart: Study Intervention and Control

Schema B: Phases of LEV Dose Escalation over 4 years
Schema C: Study Intervention Arm Timeline Flow Diagram for Phases 1 and 2
Schema D: Study Intervention Arm Timeline Flow Diagram for Phase 23
Schema E: Study intervention Arm Timeline Flow Diagram for Phase 3

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Schema A: Study Recruitment Flow Chart: Study Intervention and Control

Intervention Control
Arm Arm

Schema B: Phases of LEV Dose Escalation over 4 years

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Schema C: Study Intervention Arm Timeline Flow Diagram Phases 1 and 2

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Schema D: Study Intervention Arm Timeline Flow Diagram Phase 2

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Schema ED: Study Intervention Arm Timeline Flow Diagram Phase 3

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1.3 SCHEDULE OF ACTIVITIES (SOA)

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1 1 1 I 7
5 h 5 f
E h o d
l m o u d P a
e i u 1 r a H y
c n r 5 p y B s
t m o s
r p p i s g a
Sei Sei
o o o n t m i f
zur zur Ne
g Seiz s s p L a v t
1 es es B uro
r ures t t o E 2 i e e
5 per per e dev
a pers s V 4 n n r
E sist sist f elo
p ist L L t 3 t
nr m or or o pm
h or E E L h e a s
ol i rec rec r ent
i recu V V E o n t t
l n ur ur e al
c r 15 2 2 V u a a u
m 15 15 test
min- 3 r n n d
en p mi mi d ing
S 24 - - s c y y
t o n- n- i at
z hour 1 p e
D s 24 24 s 24
V s 1 h o s d
ay t ho ho c mo
i post o s L t r
-4 urs urs h nth
s LE h u t E a u
to L pos pos a s
i V1 o r L V g g
1 E t t r +/-
t u p E e
V LE LE g 1
r o V h c
1 V2 V3 e mo
1 s i o
nth
, p t g m
o L h p
D s E l
a t V d e
y 3 o t
L s e
1 E e
V
Procedures 2
Informed consent X
Demographics X
Medical history X
cEEG x x
Administer LEV 60 mg/kg
IV (LEV1) over 15 X
minutes
Randomization to higher
LEV intervention or PHB X X
control if seizures persist
Administer escalation dose
x
LEV2 over 15 minutes
Administer escalation dose
LEV3 over 15 minutes x
**Only in Phase 2 and 3
PHB 20mg/kg IV over 15 x
minutes- PHB load timing
dependant on x x x
randomization arm and
response to LEV
Physical exam (including
weight, head x x x
circumference, length)
Hammersmith exam x
Vital signs X x x x x x X x
Hematology X X X
serum chemistry a X X X
EKG (as indicated) x x x
LEV PK specimen trough X X X X
LEV PK specimen peak x x
Daily adverse event x x X
review and evaluation, X
CRF if AE

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1 1 1 I 7
5 h 5 f
E h o d
l m o u d P a
e i u 1 r a H y
c n r 5 p y B s
t m o s
r p p i s g a
Sei Sei
o o o n t m i f
zur zur Ne
g Seiz s s p L a v t
1 es es B uro
r ures t t o E 2 i e e
5 per per e dev
a pers s V 4 n n r
E sist sist f elo
p ist L L t 3 t
nr m or or o pm
h or E E L h e a s
ol i rec rec r ent
i recu V V E o n t t
l n ur ur e al
c r 15 2 2 V u a a u
m 15 15 test
min- 3 r n n d
en p mi mi d ing
S 24 - - s c y y
t o n- n- i at
z hour 1 p e
D s 24 24 s 24
V s 1 h o s d
ay t ho ho c mo
i post o s L t r
-4 urs urs h nth
s LE h u t E a u
to L pos pos a s
i V1 o r L V g g
1 E t t r +/-
t u p E e
V LE LE g 1
r o V h c
1 V2 V3 e mo
1 s i o
nth
, p t g m
o L h p
D s E l
a t V d e
y 3 o t
L s e
1 E e
V
Procedures 2
Neurodevelopmental
x
testing
Stakeholder interview x x

2 INTRODUCTION
2.1 STUDY RATIONALE

There are no FDA approved treatments for neonatal seizures and few data to help guide their
management. Standard of care treatments are Phenobarbital and phenytoin. NEOLEV2 showed
that 20 to 40mg/kg of Phenobarbital was much more effective than 40 to 60mg/kg of LEV. In
settings of high seizure burden we suggest that PHB should be used as first line treatment given
the mounting evidence that ongoing seizure activity can cause brain injury, particularly in the
setting of hypoxic ischemic encephalopathy (HIE).

However, in settings of lower seizure burden it is less clear that the benefits of seizure
elimination outweigh the risk of the acute and chronic side effects of PHB.

In NEOLEV2 adverse events occurred in 30% of patients randomized to PHB including

hypotension requiring inotropic support, respiratory suppression requiring intervention, and
sedation with impact on feeding and duration of hospital stay. Concerns persist that chronic
exposure to PHB may be associated with decreased cognitive ability. In animal models PHB
causes accelerated neuronal apoptosis in the immature brain.

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LEV, although much less effective, had a preferable side effect profile. There are promising
data to suggest that higher doses of LEV may have greater efficacy. If higher dose LEV has
increased efficacy in neonatal seizures, the excellent safety profile of LEV should be exploited
to realize this potential.

Neonates in NEOLEV2 had wide ranging seizure severity, and single brief seizures were
treated in the same way as neonatal status epilepticus.

In NEOLEV3 we will target treatment according to seizure severity. In the first two phases of
the study we will recruit patients with mild to moderate seizure burden only, since in these
patients we expect a 40% or greater response to LEV 60 mg/kg to 120 mg/kg, and there is a
lower risk of harm from ongoing seizures. The stopping rules for lack of efficacy indicate that
we will only proceed to the third and final phase of the study if there has been increased
efficacy demonstrated at 120mg/kg. Therefore, in the third and final phase we will recruit
patients with a wider range of seizure severity, up to a maximum of 30 min seizure burden/
hour. Widening our recruitment in phase 3 in this manner will result in a more generalizable
estimate of the efficacy when using high dose LEV. This recruitment modification also allows
for a more useful efficacy comparison with other ASMs when planning for a definitive phase III
randomized clinical trials.

2.2 BACKGROUND
Existing knowledge:. Neonatal seizures are an important clinical problem, associated with
poor outcomes; 7-33% of infants with neonatal seizures die, and of survivors 40-60% have
permanent disabilities of cerebral palsy and/or global developmental delay. 10-20% develop
post neonatal epilepsy1. Better treatments could improve neurodevelopmental outcomes. Much
of the mortality and morbidity in neonates with seizures can be attributed to the underlying
condition. However, there is mounting evidence that seizures themselves are harmful,
especially in the asphyxiated neonatal brain. Animal studies clearly show that seizures can
damage the developing brain2,3. In humans a strong correlation has been demonstrated
between the amount of electrographic seizure activity and subsequent morbidity and mortality4.
Preliminary data also demonstrates an improved outcome with prompt and complete cessation
of seizures5. In a controlled trial, treatment of subclinical seizures showed a trend for reduction
in seizure duration which was associated with less brain injury on MRI6. Brain MR spectroscopy
in neonates suffering seizures secondary to perinatal asphyxia shows brain injury as a
consequence of the seizures7. Neonatal seizures have been shown to independently contribute
to poor neurodevelopmental outcome at 4 years of age in neonatal hypoxic-ischemic injury8.
Rare Disease Prevalence: Reported incidence of neonatal seizures varies from 1/1000 live
births to 5/1000 live births9. The most recent US study reported an incidence of 1.8/1000 live
births 2, Assuming an average 3-day duration of seizures, 63 neonates experience clinical
seizures on any given day in the US. The true incidence and prevalence figures are likely
double these numbers as most neonatal seizures are electrographic only without clinical
manifestations and so are underdiagnosed.4, 5
Current treatments remain inadequate: The only randomized clinical trial of first line
treatment of neonatal seizures prior to NEOLEV2, conducted in 1999, showed that the two
standard of care medications, Phenobarbital and Phenytoin were both effective in approximately
45% of cases10. In the NEOLEV2 trial, conducted in the hypothermia era, and with rapid
detection and treatment of seizures, PHB had a higher efficacy than expected (80%). However
adverse events occurred in 30% of patients randomized to PHB including hypotension requiring
inotropic support, suppression of respiratory drive requiring intervention, and sedation with

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impact on feeding and duration of hospital stay. Concerns persist that chronic exposure to PHB
may be associated with decreased cognitive ability11-13. In rat and ape models these agents
cause accelerated neuronal apoptosis in the immature brain14,15. With few exceptions16 neonatal
seizure trials to date have evaluated seizure cessation as the primary outcome. However, the
end point of greatest concern is neurodevelopmental outcome. A drug that is less effective in
achieving seizure cessation but leads to better neurodevelopmental outcome through a
neuroprotective effect or lack of neurotoxicity may be the preferred first-line treatment.
Existing data regarding Levetiracetam for neonatal seizures: LEV is not approved for use
in children less than 1 month of age. The neonatal response to AEDs is fundamentally
different to that of the adult brain17-20. Specific study of AED’s within the neonatal population is
essential since drugs that are effective in terminating seizures in older patients may be less
effective and have more toxicity in neonates. In older patients LEV has established efficacy
and an excellent safety profile21-28 Multiple recent clinical trials have demonstrated the efficacy
of intravenous LEV for status epilepticus in older children29-31 Ideal pharmacokinetics: LEV
has many pharmacokinetic (PK) characteristics that are considered “ideal” for an antiepileptic
drug. It has low protein binding and linear pharmacokinetics over a dosage range of 500 to
5000 mg. Its metabolites are inactive; it causes no enzyme induction and has few drug
interactions. There has been extensive study of the pharmacokinetics of LEV in older
children32-35 Safety and toxicity: LEV toxicity data show a very wide safety margin, with a
TD50/ED50 ratio of >148 in rodents36 .No deaths, organ failure or other irreversible toxicity
were seen after long-term oral treatment up to doses of 1,800 mg/kg/d in the rat, 960 mg/kg/d
in the mouse and 1,200 mg/kg/d in the dog 37Clinical experience with LEV use in adults and
in children has found it to be well tolerated and safe35 . Rapid intravenous infusion over 5
minutes has been shown to be safe38. LEV is a low risk anticonvulsant in pregnancy39.
Animal fetal toxicity data are reassuring. Data from the UK Epilepsy and pregnancy register
documents that no fetal malformations occurred in 39 pregnancies where levetiracetam was
used as monotherapy. Where levetiracetam was used with other anticonvulsants 3 major
malformations occurred in 78 pregnancies. Reports or significant hematological, hepatic or
renal toxicities are very rare. In children and adults, the most common side effects, occurring
in 20 to 30% of patients, are somnolence and behavioral side effects including agitation,
anxiety, apathy, depersonalization, depression, emotional lability and hostility.

Neuroprotection: Three of four studies in animals suggest that LEV may be

neuroprotective40. In contrast to other AEDs LEV does not cause accelerated neuronal
apoptosis in the immature rat model41,42.These studies dosed up to 1000mg/kg42.

Existing neonatal data for LEV: NEOLEV2 provides the first prospective randomized
controlled data on the safety and efficacy of LEV used as a first line treatment in neonates. The
efficacy of LEV seen in NEOLEV2 (28%) was lower than previously reported efficacy from case
series (30% and 84%)43,44. Safety data from NEOLEV2 aligns with previous neonatal data and
continues to show LEV to be a remarkably safe medication, associated with fewer adverse
events than PHB.
Neonates with congenital heart disease undergoing cardiac surgery have a high incidence
(up to 20%) of seizures. Aggravation of hypotension as an antiseizure medication side effect is
particularly problematic in this group. A retrospective study in 51 such neonates demonstrated
significantly better side effect profile of LEV.45
The pharmacokinetics of LEV in neonates has been studied34,46,47. Data from NEOLEV2 was in
agreement with pharmacokinetic modelling based on our prior 7-day pharmacokinetic study,
confirming that with 40mg/kg and 60mg/kg loading dosing LEV continues to show linear
pharmacokinetics in neonates.

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Importance of dose optimisation in neonates: If higher dose LEV has increased efficacy in
neonatal seizures, the excellent safety profile of this drug should be exploited to realize this
potential. There is reason to hope higher dose of LEV will have greater efficacy. In NEOLEV2
7.5% increased efficacy was seen with the small dose increment from 40 to 60 mg/kg. Two case
series in older children with medically refractory epilepsy have demonstrated that high doses of
Levetiracetam can achieve seizure freedom where standard dosing has not48,49. In Obeid’s case
series up to 275mg/kg/day of LEV achieved a 50% reduction or greater reduction in seizures for
14/32 subjects, 5 achieved seizure freedom. Behavioural side effects occurred in 4 children,
there were no other adverse side effects. In Depositario-Cabacar’s case series 8/9 subjects
treated with up to 280mg/kg/day of LEV had resolution of acute repetitive seizures, 2 were
entirely seizure free, >80% reduction of seizures was seen in additional 4 patients, and an
additional 1 patient had > 50% reduction in seizures.
Neonatal seizures vary in severity: treatment strategies should reflect this. In treating
neonatal seizures physicians must weigh the known acute side effects and the risk of
neurocognitive effects of ASM against the risk of brain injury from ongoing seizure activity. The
appropriate treatment varies with seizure severity. The risk of brain injury from seizures is
highest for neonates in status epilepticus. In these patients PHB should be used as first line
treatment. In the situation of brief infrequent seizures, the risk of brain injury from seizures is low
and may not warrant the risks of treatment PHB both acute and chronic. Furthermore, treatment
response varies with seizure severity.
Figure 1: Drug Efficacy by Pre-treatment severity of seizures
Posthoc analysis of the
NEOLEV2 data has shown
that while efficacy of LEV was
inferior to PHB in all seizure
severity groups, LEV was
more effective in patients with
low pretreatment seizure
severity than in patients with
high seizure burden. In
neonates with pre-treatment
seizure severity less than 8
minutes per hour LEV had
41% efficacy.

Importance of continued improvements in technologies for rapid detection of neonatal

seizures: Recent research demonstrates that early detection and treatment of seizures leads
to better response to treatment 50 and increased seizure burden is associated with worse
neurological outcome 8,51,52. However, there are major logistical difficulties involved in achieving
rapid detection and treatment of neonatal seizures. Because neonatal seizures are very
frequently entirely subclinical it is accepted that cEEG monitoring is the only way to adequately
detect neonatal seizures and the only objective means to judge the response to AED
treatment17,53,54 55 56.Despite this, cEEG monitoring is not yet standard of care for neonates at risk
of seizures in many NICUs. Even in well-resourced NICU’s where cEEG is available, EEG
monitoring may be reviewed every 12 hours or even less frequently, thus seizures may go
untreated for hours. Even within the research setting of the NEOLEV2 study we struggled with
resourcing the real time specialist review of suspect seizures.
A reliable automated seizure detection algorithm would be invaluable, but such algorithms are
not yet in widespread use for real time neonatal seizure detection. Automated detection of

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electrographic seizures in the newborn requires different techniques from those employed in
adults and must be developed and tested specifically in neonates. Existing algorithms have
been shown to have sensitivities of between 43 % to 63% and specificities between 56% to
90%, not high enough for use in a clinical environment 56-58 In NEOLEV2 we incorporated
validation of the Persyst neonatal seizure detection algorithm within our study design and used
it for real time seizure detection. All study neurologists found the algorithm a useful tool for
seizure detection; however, not a practical tool due to its limitations in identifying artifact. Many
commented that the algorithm demonstrated fairly high sensitivity but that false positives were
common, often related to patting artifact. The low accuracy of the algorithm ultimately required
review by an electroencephalographer to be useful; however, most found that the algorithm
improved their efficiency in identifying seizures.
We believe that facilitating early detection and fast treatment of neonatal seizures is of equal
importance to developing better drugs in improving outcomes for neonates with seizures. In
NEOLEV2 we used remote centralised reviewing of cEEG monitoring via the internet and
evaluated the Persyst neonatal automated seizure detector in real time. In the proposed study
we will continue to facilitate innovation and improvement of automated seizure detection
technologies, through our continued collaboration with the Persyst company. Using cEEG data
from NEOLEV2, Persyst has refined and improved the neonatal seizure detector. Included in
those innovations is a new patting artifact detection algorithm. The new version of the Persyst
seizure detector will runin real time in NEOLEV3 and its performance evaluated against expert
neurophysiologists. At our request Persyst have developed a simple trend that will allow study
investigators to quickly assess seizure burden over the preceding 2 hours. This will assist in
identifying neonates with mild-moderate seizure burden, appropriate for study with LEV, and
escalate quickly where appropriate to treatment with PHB if seizure burden increases during the
study. The seizure detection algorithm and the unvalidated new trend will be used as resource
tools and adjuncts in monitoring only. All treatment decisions will be made by the neurologist
based on review of the raw EEG. Site neurologists will regularly review the cEEG to detect
seizures and ensure ongoing subject suitability for study inclusion.

2.3 RISK/BENEFIT ASSESSMENT

2.3.1 KNOWN POTENTIAL RISKS

The potential risks to the subjects include 1) adverse drug reaction risk secondary to high
dose LEV, 2) risk from delay in receiving PHB 3) risks of blood drawing, 4) risks of skin
irritation from EEG, and 5) risks regarding confidentiality.

Potential risks related to High Dose LEV: There is potential risk to patients of adverse
effects from off label high dose LEV. As described in section 2.2 safety and toxicity data from
standard as labeled dosing is reassuring and data from NEOLEV2 are reassuring. Data from
small case series of high dose LEV and from cases series of overdose with LEV are also
reassuring. LEV toxicity data show a very wide safety margin, with a TD50/ED50 ratio of
>148 in rodents.2 No deaths, organ failure or other irreversible toxicity were seen after long-
term oral treatment up to doses of 1,800 mg/kg/d in the rat, 960 mg/kg/d in the mouse and
1,200 mg/kg/d in the dog.

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High dose LEV case series: In Obeid’s pediatric case series levetiracetam up to 275
mg/kg/day caused reversible behavioural side effects in 4/32 patients, no other adverse side
effects were noted. In Depositario-Cabacar’s case series using high dose LEV up to 280
mg/kg/day no adverse side effects were reported.

The highest known dose of oral levetiracetam received in the clinical development
program was 6000 mg/day. Other than drowsiness, there were no adverse reactions in the few
known cases of overdose in clinical trials.

In case series of LEV overdose doses between 15,000 mg and 50,000 caused no adverse
effects or mild side effects were reported in 14/15 patients, moderate side effects occurred in
one patient. There is once case report where overdose with 30,000 mg of Levetiracetam caused
respiratory depression with decreased muscle tone requiring respiratory support (Barrueta). In
this case the patient made a full recovery the following day. There are reports of levetiracetam
causing pruritus and decreased muscle tone in pediatric overdoses.3

Safety monitoring within the study will be vigilant to guard against these risks. Please see Data
Safety Monitoring Plan for details.

Potential risks from delay in PHB administration:

There is mounting evidence that neonatal seizures add to brain injury. In this study we will be
giving a drug known to be less effective than the standard of care treatment PHB, and there is
therefore potential risk of brain injury from ongoing seizures.

Potential risks of blood drawing: blood loss, pain, dizziness, fainting, and infection
Blood draws will be required or measuring the pharmacokinetics of LEV and for safety
monitoring, although in most patients these laboratory tests would be obtained as standard
care because of their underlying illness.

Potential risk of skin irritation from EEG:

to record the EEG it is necessary to rub gently the scalp underneath each electrode. This can cause slight redness of
the skin

Potential risks regarding privacy and confidentiality:

The research will involve collection of health information from mothers and infants. This carries
a risk to the confidentiality of that private information.

2.3.2 KNOWN POTENTIAL BENEFITS

There may be no benefit to the individual participant. A potential benefit of the study to the
participant is that LEV may be a safer drug associated with fewer side effects than the
standard treatments. The participant may benefit from more frequent review of the cEEG
monitoring than would be standard of care, resulting in earlier detection and treatment of
seizures.

The main benefit of the study will be to society and future neonates with seizures, in the
knowledge that will be gained from the study. If LEV efficacy, safety and pharmacokinetics can
be established in neonates this will be of great benefit to all neonates with seizures.

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Developing better algorithms for the automated early detection of neonatal seizures will also
significantly improve the management of this condition.

2.3.3 ASSESSMENT OF POTENTIAL RISKS AND

BENEFITS

There is an ethical dilemma inherent in conducting research in vulnerable neonates. However,

treatments cannot be tested in an older, less vulnerable population. The results of studies in
older patients do not extrapolate reliably to neonates, it is imperative that better treatments for
seizures are found and tested in neonates. A comprehensive data safety monitoring plan will be
in place to protect subjects.

Minimization of risks:

Risk of adverse effect from high dose LEV will be minimized by careful clinical and laboratory
monitoring and stepwise escalation of doses only if and when each phase of the study proves
safe. This is a major focus of our data safety monitoring plan and stopping rules.

Risk of brain injury due to ongoing seizure activity and delay to treatment with PHB will be
minimized as follows:
To guard against this risk, in phases 1 and 2 we will only recruit neonates with a mild to
moderate seizure burden. We have used a conservative definition for this of < 8 minutes/ hour
seizure burden. If, at any point in the study, a patient has a seizure burden over this threshold
they will exit the LEV treatment protocol and receive treatment with PHB. Because our study
involved more intensive cEEG monitoring and review of monitoring than is standard the risk of
untreated significant seizure burden is reduced for recruited patients. Phase 3 of the study will
only take place if higher efficacy of LEV is seen at 120mg/kg. In phase 3 we will recruit
neonates with seizure burden up to 30 minutes/ hour.

Risk of harm from blood draws will be minimized as follows:

The required volume of each blood sample for LEV levels will be 0.5 mL. Our inclusion criteria include a weight requirem

Risks to patient privacy will be minimized as follows:

The treating neonatology staff will first approach a newborn’s parents to ask permission for the
study investigator to discuss the study with the family. Only if and after parents agree to this
step will a study investigator then talk with the parents and review the subject’s medical record.

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Once informed consent is obtained, the parents may need to answer a few questions about the
maternal and family health history, but study staff will obtain most of the data from the subjects’
medical records.

Risks to Confidentiality of private health information will be minimized as follows: The

physicians and nurses caring for each newborn will need to know that the newborn is enrolled
in this study but will not have access to data collected only for research purposes. Medical
information collected during this study will become part of the subject’s hospital record, if the
information is determined to be pertinent to the subject’s medical care or is a usual part of the
subject’s care (e.g., results of tests ordered by treating clinicians that are also recorded for
research purposes). Medical records are available to other health care professionals at the
hospital and may be reviewed by hospital staff in their course of carrying out their
responsibilities. However, they are required to maintain confidentiality in accordance with
applicable laws and hospital policies. Information contained in a medical record may not be
given to anyone unaffiliated with the hospital in a way that could identify the subject or parent
without written consent, except as required or permitted by law. Information collected during
the study that does not become part of a subject’s medical record will be stored in separate
research files maintained by the investigators. These research records will not be made
available to any individuals who are not part of the research team except upon a parent’s
request or as required by law. If a subject is withdrawn from the research study, information
that has already been collected will become part of the research database.

Only the research coordinator, neurology fellow and site PI at each NICU will have access to
individually identifiable information about newborns enrolled at that site. Data will be collected
by the study investigators and research coordinator and recorded onto case report forms
(CRFs) by the research coordinator. The CRFs and the electronic database containing the
research data will contain only de-identified information to preserve confidentiality of patients’
protected health information. Only the study identification number will identify the subjects on
the CRFs and in the electronic database. Files that link the study identification number for
each subject to the subject’s personal information (such as name, medical record number,
parents’ names) will be kept at each study site and accessible only by the site PI and
research coordinator. The research database containing the subjects' study ID numbers and
research data will be stored on a single computer, protected with a password, and will be
backed up on the network in a private folder accessible only to study staff.

The research records may be made available to the UCSD IRB and to the FDA, since this is a
federally funded study.

Overall the value of the information to be gained outweighs the risks of participation in
the study:
Several potential benefits to enrollment in this study compare favorably with the risks
associated with participation in the study. Possible benefits of LEV include: (1) lower risk of
hypotension, (2) lower risk of pulmonary compromise, (3) lower risk of drug-drug interactions
especially with medications digested by the liver, (4) less sedation leading to improved feeding
and earlier discharge, (5) potentially avoiding phenobarbital-induced neuronal toxicity although
this concern is based on the animal and not human literature. Although there are potential
risks associated with administration of LEV, the overall risk of adverse events are low based
on the cited experiences of groups with high-dose LEV in older children and adults. All
subjects will be carefully monitored with respect to clinical, laboratory and EEG data in a more
systematic fashion than is typical in the care of such newborns, thus any adverse event will be
recognized quickly. Our study design ensures if LEV is ineffective at the study dose, the child

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may quickly move to another treatment, which also mitigates risks from the trial. No drug study
in neonates is without risk, however IV LEV appears to be a very safe drug. There is a dire
need for better anti-epileptic agents for neonates and there are substantial risks associated
with the use of standard therapies. The potential benefit to all neonates with seizures
outweighs the risks that would be involved in this study. There is considerable potential that
the risk/benefit ratio is favorable in the study subjects themselves.

3 OBJECTIVES AND ENDPOINTS

OBJECTIVES ENDPOINTS JUSTIFICATION FOR

ENDPOINTS
Primary
To determine the The primary endpoint of this This study is a dose
recommended maximal safe study is the maximum safe and escalation study, as
and tolerable dose of LEV in tolerated dose of LEV in the such the endpoint is the
the setting of neonatal seizures treatment of neonatal seizures, maximum safe and
of mild to moderate severity. that is, the maximal dose tolerated dose.
studied where dose limiting
toxicity is not observed.
We will use a continual
reassessment method to
determine the maximal safe and
tolerated dose.
Secondary
To study the pharmacokinetics LEV population pharmacokinetic It is important in
of high dose LEV in neonates parameters vulnerable neonates to
with seizures of mild to determine whether PK
moderate severity. parameters remain
predictable at high dose
To estimate the additional Estimate of additional efficacy of This allows for
efficacy of higher doses of LEV LEV at higher doses comparison between
in neonates with seizures of efficacy of high dose
mild to moderate severity. LEV to 60mg/kg LEV.
Tertiary/Exploratory
Rates of adverse events seen A trial of this size cannot
To study the safety of higher with high dose LEV treatment compare rates of
doses of LEV in neonates with will be reported and compared to adverse events between
seizures of mild to moderate rates seen in PHB control arm. treatment arms with
severity statistical significance.
However, safety is a key
concern in treatment
decisions so it important
to study safety to the
degree that we are able

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OBJECTIVES ENDPOINTS JUSTIFICATION FOR

ENDPOINTS
Sensitivity and specificity of the Assessing the function
To improve technologies for Persyst neonatal seizure of the latest version of
the prompt detection of detector algorithm as compared the algorithm will aid
neonatal seizures with the markings of expert resource allocation in
neurophysiologists. neonatal seizure
monitoring.

4 STUDY DESIGN

4.1 OVERALL DESIGN

• Hypothesis The main purpose of this study is to determine the maximum safe and
tolerated dose of LEV in the treatment of neonatal seizures of mild to moderate severity.
Our hypothesis is that optimal dosing of Levetiracetam (LEV) to treat neonatal seizures
is significantly greater than 60mg/kg.
• Phase of the trial 2b

• Study Design: Open label dose-escalation, preliminary safety and efficacy study. There
will be a randomized control treatment component.
• Diagnosis of seizures and treatment efficacy will be validated by independent
neurophysiologist review of the EEG record to minimize bias.
• Dose escalation or dose-ranging details should be contained in Section 6.1.2, Dosing
and Administration
• The number of study groups/arms and study intervention duration
There will be 2 study arms, the experimental high dose LEV arm and an active treatment
control arm where patients will receive the standard of care PHB.
The study intervention will last for 5 days of active treatment.
• Under the proposed continual reassessment method, the first group of 10 patients will
receive 90mg/kg of Levetiracetam and the decision to escalate dose to the next
120mg/kg is informed and guided by the number of infants observed with adverse
events. We will repeat the above process until we (a) reach the proposed maximum
dose 150mg/kg or (b) stop the study before reaching this targeted maximum dose
because of evidence of dose-limiting toxicity exceeding our set limits.
• Indicate if single site or multi-site: multi-site

• Name of study intervention(s) Levetiracetam

• Note if interim analysis is planned: Under the proposed CONTINUAL REASSESSMENT

METHOD with our stopping rules there will effectively be an interim analysis prior to
each phased escalation of dose, looking for dose limiting toxicity, and after 10 subjects

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have been studied at 120mg/kg looking for lack of efficacy. Refer to details in Section
9.4.6, Planned Interim Analysis
• No stratification is planned.

4.2 SCIENTIFIC RATIONALE FOR STUDY DESIGN

The study design is open label dose-escalation, preliminary safety and efficacy study. An active
drug treatment control arm (PB group) is included in the study design. This study is not
designed or powered to compare high dose LEV and PB groups, however the randomized
control group will help with interpretation of adverse events and seizure cessation efficacy seen
in the high dose escalation group. This is particularly important because of the high rates of
morbidity in neonates with seizures.

4.3 JUSTIFICATION FOR DOSE

60 mg/kg is the maximal dose for which LEV is licensed in any age group, and therefore as high
as was ethical in NEOLEV2, the first controlled study in vulnerable neonates.

Having established safety but inadequate efficacy at that dose we will now study higher doses.
Case series in children have demonstrated that high doses of LEV up to 280 mg/kg/day can
achieve seizure freedom when standard dosing has failed.
In vulnerable neonates we have selected 200mg/kg/day as the maximum that we will increase
to in phase 3 of the study;150mg/kg load followed by 25 mg/kg maintenance doses every 8
hours. The 200mg/kg/day maximum is the summation of 150mg/kg load followed by the two
25mg/kg maintenance doses given within a 24 hours period.
To ensure safety we will use the Continual Reassessment Method only increasing to higher
dose escalation once 90mg/kg and 120 mg/kg dosing loads have been studied in at least 10
subjects.

4.4 END OF STUDY DEFINITION

A participant is considered to have completed the study if he or she has completed all phases of
the study including the last visit or the last scheduled procedure shown in the Schedule of
Activities (SoA), Section 1.3.

The end of the study is defined as completion of the last visit or procedure shown in the SoA in
the trial globally.

5 STUDY POPULATION

5.1 INCLUSION CRITERIA

● At risk for having seizures or suspected to be having seizures

● All seizure etiologies except correctable metabolic abnormalities such as hypoglycemia,

and hypocalcemia

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● Term neonates (corrected gestational age between 35 and 44 weeks, postnatal age less
than 28 days

● > 2200g in weight

5.2 EXCLUSION CRITERIA

● Cumulative seizure burden of 8 minutes/ hour or more in phases 1 and 2; Cumulative

seizure burden of 30 minutes/hour or more in phase 3.
● Renal failure: defined as anuria in the first 24 hours of life

● Patients in whom death seems imminent.

● Seizures caused by correctable metabolic abnormality, such as hypocalcemia,

hypoglycemia

Pre-treatment with other anticonvulsants will not preclude recruitment and enrollment.
A minimum seizure burden of 30 seconds will be required to receive ASM and to receive
further dose escalation.
5.3 LIFESTYLE CONSIDERATIONS

● Not Applicable

5.4 SCREEN FAILURES

All consented subjects who do not receive treatment within the study will still have the following
minimum information set collected: demographic data, screen failure details, eligibility criteria,
and any serious adverse events (SAE)

5.5 STRATEGIES FOR RECRUITMENT AND RETENTION

The target study sample size is 133 patients with neonatal seizures.
From NEOLEV2 data, we expect to screen 2.8 patients at risk for neonatal seizures for each
patient that prove to have confirmed electrographic seizures on cEEG monitoring.
From NEOLEV2, we expect 56% of patients with electrographic seizures to meet the mild to
moderate seizure burden criteria required for recruitment into phases 1 and 2 of the study, and
88% of patients with electrographic seizures to meet the seizure burden criteria set for phase 3..

Anticipated annual accrual rate is 35 patients per year.

Anticipated number of sites and participants to be enrolled from the U.S. and outside the
U.S. We will recruit at 6 NICU’s, 4 in the US and 2 in New Zealand. From NEOLEV2
data, we expect that each site will recruit between 5-9 patients annually.

Source of participants inpatient hospital setting, neonatal intensive care units

How potential participants will be identified and approached

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The treating neonatology staff will identify infants at risk of developing seizures or suspected to
be having seizures. Neonatology staff will first approach a newborn’s parents to ask permission
for the study investigator to discuss the study with the family. Only if and after parents agree to
this step will a study investigator then talk with the parents and review the subject’s medical
record.

Eligibility will be confirmed and consent will be obtained. In phases 1 and 2 study neurologists
will identify neonates with mild-moderate seizure burden (less than 8 minutes cumulative
seizure activity per hour), appropriate for study with LEV, and exclude patients with higher
seizure burden where treatment with PHB is more appropriate. In phase 3 the inclusion criterion
will be less than 30 minutes cumulative seizure burden/ hour.

Types of recruitment strategies

Prior to study commencement in-servicing sessions will be held at all study sites to increase
awareness of the study within each neonatal unit. Study coordinators and neonatologist co-
investigators will maintain a high level of awareness of the study within the units.

Procedures that will be used to enhance participant retention

This study has its endpoint within the neonatal period and so retention is unlikely to be a
problem. However, in line with INC recommendations we will endeavor to track long-term
neurodevelopmental outcomes in this cohort, data that is part of routine clinical care for these
high- risk newborns. Study team members will conduct an exit interview with families at the
conclusion of the active phase of the study and discuss the importance of ongoing
neurodevelopmental follow up. Study coordinators will assist with ensuring study subjects are
not lost to this planned clinical follow up.

Specific strategies that will be used to recruit and retain historically under-represented
populations We expect the ethnic and racial make-up of NEOLEV3 to be similar to that of
NEOLEV2.The study will recruit equal numbers of male and female patients.

Baseline characteristics of patients in NEOLEV2

Total
Gender
Boy 55 (51.89%)
Girl 51 (48.11%)
Total 106 (100%)
Race
American Indian/Alaska Native 1 (0.95%)
Asian 5 (4.76%)
Native Hawaiian or other Pacific Islander 9 (8.57%)
Black or African American 5 (4.76%)
White 59 (56.19%)
Mixed Race 4 (3.81%)
Unknown 10 (9.52%)
Other 12 (11.43%)
Total 105
(99.99%)
Ethnicity
Hispanic or Latino 28 (26.67%)
NOT Hispanic or Latino 64 (60.95%)
Not Reported 12 (11.43%)
Unknown 1 (0.95%)

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Total 105 (100%)

Justification for inclusion of vulnerable newborns

There is an ethical dilemma inherent in conducting research in vulnerable neonates.
However, treatments cannot be tested in an older, less vulnerable population. The
results of studies in older patients do not extrapolate reliably to neonates, it is imperative
that better treatments for seizures are found and tested in neonates. A comprehensive
data safety monitoring plan will be in place to protect subjects

No Compensation or incentives will be offered to participants or their families.

6 STUDY INTERVENTION

6.1 STUDY INTERVENTION(S) ADMINISTRATION

6.1.1 STUDY INTERVENTION DESCRIPTION

Patients will receive off-label treatment with IV Levetiracetam. IV Levetiracetam is not licensed
for use below 1 month of age. Furthermore, this dose escalation study will involve much higher
doses than the doses for which Levetiracetam is currently licensed.
Patients in the control arm of the study will receive IV phenobarbital. There are no FDA
approved treatments for neonatal seizures, however phenobarbital and phenytoin are the
standard of care treatments.

6.1.2 DOSING AND ADMINISTRATION

Phases 1 and 2
If seizures of mild to moderate severity are confirmed, (minimum seizure burden for treatment
30 seconds, maximal under 8 minutes/ hour) enrolled subjects will receive 60mg/kg of LEV.
Initial 60mg/kg LEV load will be given by IV infusion over 15 minutes and an additional 15
minutes allowed for the anticonvulsant to reach peak effect.

If there are no further seizures, the subject will be continuously monitored for 24-hours and if
seizures recur at any point, they will be treated with standard of care (starting with PHB). During
this 24-hour period and if no further seizures are detected, the subject will receive maintenance
doses of LEV for 5 days. Monitoring may also continue for 1-3 days following the 24-hour initial
monitoring period, depending on a variety of factors related to the subject’s standard care. For
example, babies who receive hypothermia treatment are kept cool for 72 hours and EEG
monitoring must be continued until the baby has been rewarmed, since this is a period of risk for
seizure recurrence If any seizure activity is detected at any time while the subject is undergoing
EEG monitoring, they will be treated with standard of care.

Subjects whose seizures persist or recur following treatment with 60mg/kg of LEV will be
randomized in the dose escalation study. Patients in the dose escalation study will be randomly

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assigned to receive either higher dose LEV or treatment with the control drug PHB in a 3:1
allocation ratio, stratified by site.
Dose-escalation LEV loads or randomized control drug PHB load will be given over 15 minutes
with an additional 15 minutes allowed for the anticonvulsant to reach peak effect.

Rolling cumulative seizure burden over the preceding 2 hours will be continuously monitored.
If seizure burden escalates at any stage above the 8 minute/hour threshold, the patient will exit
the study and receive PHB.

Initial 60mg/kg LEV load will be given by IV infusion over 15 minutes and an additional 15
minutes allowed for the anticonvulsant to reach peak effect.
If seizures persist or recur dose-escalation of LEV will occur.
Further dose-escalation LEV loads will again be given over 15 minutes with an additional 15
minutes allowed for the anticonvulsant to reach peak effect.
Efficacy of the final LEV dose will be assessed over 12 hours.
The rolling cumulative seizure burden will be continuously monitored and if seizure burden
escalates at any stage above the 8 minute/hour threshold, the patient will exit the study and
receive PHB, but seizure burden below the 8 minute/hour threshold will not result in further
medication during the 1-hour observation period.

After this 1-hour efficacy assessment period continuous video EEG monitoring will continue
within the study for 24 hours. In the monitoring period from the end of the efficacy assessment
period after the final LEV dose to the end of the 24-hour time point, if seizures totaling more
than 30 seconds persist or recur the patient will receive PHB
If seizures totaling at least > 30 seconds persist or recur 2 hours after the final LEV dose the
patient will receive PHB.

CEEG monitoring will continue for 24 hours seizure freedom.

Maintenance dosing: Subjects in whom high dose LEV controls seizures or reduces seizures by
at least 50% will receive maintenance high dose LEV for 5 days, IV or oral depending on
whether the patient can be fed. Subjects whose seizures have been reduced by 50% but who
are still experiencing some seizure activity (defined as > 30 seconds total seizure activity over
the EEG monitoring period) will receive maintenance LEV and PHB load and PHB maintenance.
Receiving 2 medicines for treatment of neonatal seizures in these situations is within usual
standard of care parameters. After the 5-day high dose active study period, LEV will be reduced
to standard dosing and continued treatment decisions will be at the discretion of the treating
neurologist.

Rationale for consideration of 50% reduction in seizure burden: 50% seizure reduction will be
defined based on EEG confirmed seizure burden in the 690 minutes after LEV administration
compared to EEG confirmed seizure burden in the time period prior to administration of
treatment. (LEV is administered over 15 minutes, with a further 15 minutes allowed for the
medication to take effect, therefore 90 minutes will remain in the 2-hour assessment window).
The 50% reduction in seizure severity is clinically useful and meaningful, maintaining a level of
LEV is clinically advisable in this situation. Both measures of efficacy; complete cessation of
seizures and 50% reduction in seizure burden in time period after treatment compared with
baseline pre-treatment are valid and commonly used metrics in epilepsy medication trials.

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If there are no further seizures, the subject will be continuously monitored for 24-hours and if
seizures recur at any point, they will be treated with standard of care (starting with PHB). During
this 24-hour period and if no further seizures are detected, the subject will receive maintenance
doses of LEV for 5 days.
Monitoring may also continue for 1-3 days following the study 24-hour initial monitoring period,
depending on a variety of factors related to the subject’s standard care. For example, babies
who receive hypothermia treatment are kept cool for 72 hours and EEG monitoring must be
continued until the baby has been rewarmed, since this is a period of risk for seizure recurrence
If any seizure activity is detected at any time while the subject is undergoing EEG monitoring,
they will be treated with standard of care.

Phase 3

If seizures are confirmed, (minimum seizure burden for treatment 30 seconds, maximal under
30 minutes/ hour) enrolled subjects will receive 60mg/kg of LEV. Initial 60mg/kg LEV load will be
given by IV infusion over 15 minutes and an additional 15 minutes allowed for the
anticonvulsant to reach peak effect.

Subjects whose seizures persist or recur following treatment with 60mg/kg of LEV will be
randomized in the dose escalation study. Patients in the dose escalation study will be randomly
assigned to receive either higher dose LEV or treatment with the control drug PHB in a 3:1
allocation ratio, stratified by site.

Dose-escalation LEV load of 60mg/kg or randomized control drug PHB load will be given over
15 minutes with an additional 15 minutes allowed for the anticonvulsant to reach peak effect.

Rolling cumulative seizure burden over the preceding 2 hours will be continuously monitored. If
seizure burden escalates at any stage above the 30 minute/hour threshold, the patient will exit
the study and receive PHB.

Initial 60mg/kg LEV load will be given by IV infusion over 15 minutes and an additional 15
minutes allowed for the anticonvulsant to reach peak effect.
If seizures persist or recur, dose-escalation of LEV will occur. A dose-escalation LEV load of
60mg/kg will again be given over 15 minutes with an additional 15 minutes allowed for the
anticonvulsant to reach peak effect.
If seizures persist or recur, a final dose-escalation of LEV will occur. Tthe final dose-escalation
LEV load of 30mg/kg will again be given over 15 minutes with an additional 15 minutes allowed
for the anticonvulsant to reach peak effect.

Efficacy of the third and final LEV dose will be assessed over 1 hour if the seizure burden is less
than 8 min/hr. The efficacy assessment period will be shortened to 30 minutes if the seizure
burden remains between 8 and 30 min/hr. 2 hours.
Rolling cumulative seizure burden will be continuously monitored. If seizure burden escalates at
any stage above the 30 minute/hour threshold, the patient will exit the study and receive PHB.

After the 30-to-60-minute efficacy assessment period continuous video EEG monitoring will
continue within the study for 24 hours. In the monitoring period from the end of the efficacy

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assessment period to the end of the 24-hour time point, if seizures totaling more than 30
seconds persist or recur the patient will receive PHB.
If seizures > 30 seconds persist or recur 2 hours after the final LEV dose the patient will receive
PHB.

CEEG monitoring will continue for 24 hours seizure freedom.

Maintenance dosing: Subjects in whom high dose LEV reduces seizures by 50% will receive
maintenance high dose LEV for 5 days, IV or oral depending on whether the patient can be fed.
Subjects whose seizures have been reduced by 50% but who are still experiencing some
seizure activity (defined as > 30 seconds total seizure activity over the EEG monitoring period)
will receive maintenance LEV and PHB load and PHB maintenance. Receiving 2 medicines for
treatment of neonatal seizures in these situations is within usual standard of care parameters.
After the 5-day high dose active study period, LEV will be reduced to standard dosing and
continued treatment decisions will be at the discretion of the treating neurologist.

As in earlier phases of the study EEG monitoring may also continue for 1-3 days following the
study 24-hour monitoring period, depending on a variety of factors related to the subject’s
standard care. For example, babies who receive hypothermia treatment are kept cool for 72
hours and EEG monitoring must be continued until the baby has been rewarmed, since this is a
period of risk for seizure recurrence If any seizure activity is detected at any time while the
subject is undergoing EEG monitoring, they will be treated with standard of care.

Phases of dose escalation:

As illustrated below the maximum Levetiracetam dose to be used will increase over the course
of the study in three phases. The study will be terminated early if dose limiting toxicity is
observed or if additional efficacy of high dose LEV is less than 15%. We expect the efficacy of
standard dose LEV to be 40% based on NEOLEV2 efficacy in patients with seizures of mild to
moderate severity, therefore the study will be stopped if 120mg/kg LEV is not effective in
stopping seizures in at least 55% of these subjects. Detailed definition of dose limiting
toxicity, dose escalation rules and study stopping rules are in the data safety monitoring
plan.

In phase 1 a cohort of 10 subjects will receive a dose escalation increment of 30mg/kg, total
dose 90mg/kg. Maintenance dosing with 15mg/kg/dose q8 hourly will be continued for 5 days. If
no dose limiting toxicity is observed at this dose level the study will enter phase 2.

In phase 2 a cohort of at least 10 subjects will receive, if needed to control their seizures 2
incremental doses of 30mg/kg, total loading dose of 120mg/kg. Maintenance dosing with
20mg/kg/dose q 8 hourly will be continued for 5 days. If no dose limiting toxicity is observed at
this dose level the study will enter phase 3.

In phase 3 subjects will receive, if needed to control their seizures, 2 incremental doses: one of
60mg/kg and one of 30mg/kg, total dose 150mg/kg. (This fasttrack regimen escalating in two
additional load will be used rather than three 30mg/kg loads to reduce the delay in progressing
to alternative anti-seizure medications if higher dose LEV is not effective.) Maintenance dosing
with 25mg/kg/dose q8 hourly will be continued for 5 days.
If study continues to completion, 40 subjects will be studied in this phase, enabling adequate
assessment of the safety and efficacy of these higher doses.

Study Intervention Timeline Flow Diagram

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6.2 PREPARATION/HANDLING/STORAGE/ACCOUNTABILITY

1. 6.2.1 ACQUISITION AND ACCOUNTABILITY

The study is an open label study. At the US sites research pharmacies will distribute to the
neonatal units study LEV doses for infusion. Phenobarbital will be given at standard doses and
is held in NICU repository.

At the NZ sites, both LEV and Phenobarbital will be held in the NICU repositories, and NICU
nurses will draw up and administer non-standard doses of the IV medication according to
protocolled instructions and orders.

2. 6.2.2 FORMULATION, APPEARANCE, PACKAGING, AND LABELING

Levetiracetam in Sodium Chloride Injection is a clear, colorless, sterile solution that is
available as a generic medication in several forms.

Injectable phenobarbital is supplied by West-ward pharmaceuticals as a clear liquid in 1ml vials

in two strengths, the most appropriate for neonatal 20mg/kg dosing is the 130mg/ml strength.

6.2.3 PRODUCT STORAGE AND STABILITY

Levetiracetam should be stored at 20° to 25°C (68° to 77°F). The product is for single dose
use. Phenobarbital should be stored at 20°-25°C (68°-77°F), excursions permitted to 15°-30°C
(59°-86°F). The product is for single dose use.

6.2.4 PREPARATION
Research pharmacies will provide doses of study LEV to US neonatal units in prepared
syringes. At the NZ study sites, registered neonatal nurses will draw up nonstandard doses from
Levetiracetam bags on a ml/kg basis as per study protocol.

6.3 MEASURES TO MINIMIZE BIAS: RANDOMIZATION AND BLINDING

For the randomized control component we will randomize subjects in a 3:1 ratio to high dose
LEV or PHB using block randomization, with a block size of 4, stratified by each of the 6 study
sites. The block randomization will ensure balancing of 3 high dose LEV subjects for each PHD
within each site to the extent possible. Analyses of efficacy for Aim 3 will control for potential site
differences (see Section 9, Statistical considerations for details).

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Justification of non-blinded study

NEOLEV2 was a blinded study designed just prior to the catastrophic September 2012 outbreak
of fungal infections due to contamination of medicines prepared at a sterile compounding
facility, which has led to the permanent cessation of external sterile compounding. To provide
blinded study medications it was therefore necessary in NEOLEV2 to perform sterile dilutions
every 2 weeks at each of the 5 hospitals. This was prohibitively time consuming and expensive.
In addition, our experience in NEOLEV2 was that because of the obvious immediate side effects
seen in any subject receiving phenobarbital (EEG suppression, sedation, respiratory
suppression, and lowered blood pressure) true blinding was impossible. For these reasons we
propose an unblinded study.

To minimize bias EEG data will be verified by neurophysiologists who will be blinded to
treatment to the degree this is possible.
6.4 STUDY INTERVENTION COMPLIANCE
Details of each dose of study drug given as recorded on patient drug chart will be entered into
Redcap, including total dose, dose time and date. Serum levels of LEV done for
pharmacokinetic testing will provide additional assurance regarding protocol compliance.

6.5 CONCOMITANT THERAPY

Patients in this study are sick neonates and are frequently on multiple medications. All
concomitant medications will be recorded at enrollment and daily throughout the study.
Administration of oxygen or respiratory support and hypothermia treatment will also be
specifically recorded

6.5.1 RESCUE MEDICINE

Not applicable

7 STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT

DISCONTINUATION/WITHDRAWAL
7.3 DISCONTINUATION OF STUDY INTERVENTION

In phases 1 and 2 of the study if seizure burden increases to high seizure burden, the patient
will exit the study and receive PHB. Site neurologists will regularly review the raw trace EEG to
detect seizures and ensure ongoing subject suitability for study inclusion.

Discontinuation from the study intervention does not mean discontinuation from the study, and
remaining study procedures should be completed as indicated by the study protocol. If a
clinically significant finding is identified (including, but not limited to changes from baseline) after
enrollment, the investigator or qualified designee will determine if any change in participant
management is needed. Any new clinically relevant finding will be reported as an adverse event
(AE).

The data to be collected at the time of study intervention discontinuation will include the
following:
Reason for study discontinuation, alternative treatment for seizures given, (Phenobarbital), vital
signs before and 15 minutes after Phenobarbital infusion, any adverse events noted in the
following 5 days. Total seizure burden prior to administration of Phenobarbital, Total time from

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first indication for study discontinuation to administration of Phenobarbital, Total seizure burden
over period of cEEG monitoring.

7.4 PARTICIPANT DISCONTINUATION/WITHDRAWAL FROM THE STUDY

Participants are free to withdraw from participation in the study at any time upon request.
An investigator may discontinue or withdraw a participant from the study for the following
reasons:

If any clinical adverse event (AE), laboratory abnormality, or other medical condition or situation
occurs such that continued participation in the study would not be in the best interest of the
participant.
If the participant meets an exclusion criterion (either newly developed or not previously
recognized) that precludes further study participation.

The reason for participant discontinuation or withdrawal from the study will be recorded on the
Case Report Form (CRF).

Subjects who sign the informed consent form and are randomized but do not receive the study
intervention may be replaced. Subjects who sign the informed consent form, and are
randomized and receive the study intervention, and subsequently withdraw, or are withdrawn or
discontinued from the study, will not be replaced

7.5 LOST TO FOLLOW-UP

This study has its endpoint within the neonatal period and so loss to follow up within the study
period will not be a problem. However, in line with INC recommendations we will endeavor to
track long-term neurodevelopmental outcomes in this cohort, data that is part of routine clinical
care for these high- risk newborns. Study team members will conduct an exit interview with
families at the conclusion of the active phase of the study and discuss the importance of
ongoing neurodevelopmental follow up. Study coordinators will assist with ensuring study
subjects are not lost to this planned clinical follow up.

8 STUDY ASSESSMENTS AND PROCEDURES

8.3 EFFICACY ASSESSMENTS

Study staff will collect minimal deidentified data set for all screened patients not
consented to the study: date of screen; indication for cEEG monitoring; gestational age;
reason not enrolled.

Procedures/evaluations that will take place following Consent

Study staff will record demographic data and medical history data for all consented subjects
including birth weight, gestational age at birth, postnatal age, gender, pregnancy history,
mode of delivery, general anaesthesia for delivery, Apgar scores, cord blood gas and Sarnat
stage if applicable.

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All consented patients, by definition are considered at risk for seizures and will be monitored
with cEEG monitoring for a minimum of 24 hours, except in the circumstance of benign
neonatal myoclonus, where the monitoring can be stopped as soon as the abnormal
movement of concern has been captured on video EEG and confirmed not to be seizure
related. Patients with hypoxic ischemic encephalopathy undergoing hypothermia treatment
will remain on cEEG monitoring throughout the 72 hours of cooling and through the
rewarming period, as this is a period of risk for seizure occurrence.

The study investigator will review the first 15 minutes of cEEG monitoring to review seizure
burden immediately. In phases 1 and 2 patients with seizure burden > 8 minutes/ hour will not
be eligible for the study. Patients commenced on LEV treatment in the study during phase 1 and 2,
who subsequently develop seizure burden > 8 minutes/ hour will exit the study and receive PHB.

Consented patients will only be enrolled in the study and receive study medication if
electrographic seizures occur of at least 30 seconds cumulative duration

Procedures that will take place following enrollment in the study and the
administration of study medication

Study staff will record additional demographic and medical history data for enrolled patients
including: family medical history, etiology of seizures, results of neuroimaging, investigations to
determine seizure etiology including genetic test results if applicable.

All concomitant medications will be recorded.

Height, weight and head circumference will be recorded

Vital signs: At the beginning of any infusion of study drug and 15 minutes following any
infusion of study drug HR and BP, Respiratory status will be recorded, with respect to
whether any respiratory support is being provided. Vasopressor support will be noted

For all enrolled subjects, study staff will do daily review for adverse event monitoring see
7.1.2

Baseline and monitoring laboratory tests: at the time of administration of study drug, if the
patient has not already had full blood count and chemistry including Creatinine and ALT
tested within the previous 24 hours, those tests will be sent. The same tests will be
rechecked after 24 hours and 5 days of treatment.

1 minute of ECG rhythm strip will be printed before dose escalation and at highest dose to
monitor for arrhythmia, PR interval or QTc abnormality.

MRI imaging: All patients with neonatal seizures have MRI brain as part of their routine care.
Deidentified copies of MRI imaging will be collected for this study

EEG data monitoring and automated seizure detection: Continuous video-EEG data will be
collected for 24 hours after study drug administration. Each site will use already available video
EEG equipment with Persyst software installed. Neurologists at each study site will review
monitoring and make all treatment decisions. The seizure detection algorithm and the
unvalidated new trend will be used as resource tools and adjuncts in monitoring only. The video
EEG stripped of personally identifiable health information will be archived on the UCSD server

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for analysis by neurophysiologist. Data transfer will be achieved encrypted password protected
external hard drives.

Blood specimens for LEV pharmacokinetic studies. LEV levels will be drawn at time points
indicated on SOP. Each specimen should be 0.6ml collected in yellow or red top microtainers.
Time and date of specimen should be written on the specimen label. The blood sample should
be centrifuged and frozen within 30 minutes. Centrifuge should spin at 3200 rpm for 8 minutes
(no temperature control). Serum collected in this manner should be frozen and stored at each
site. Batch frozen shipments to Quest laboratory every 18 months for analysis.

Parent/ stakeholder exit questionnaire will be developed and approved by IRB. It will be
conducted by study staff on day 5 as active study period ends.

At hospital discharge a Hammersmith Infant Neurological Examination will be recorded.

Feeding method at discharge , and whether breastmilk fed, time to establish full sucking feeds,
respiratory support required, results of hearing and vision screening, all discharge diagnoses
and discharge medications.

8.4 SAFETY AND OTHER ASSESSMENTS

Safety component of screening procedure

Our inclusion and exclusion criteria are designed to maximise study safety. Preterm neonates
are still more vulnerable than term neonates and are therefore not included in this study. Weight
criteria ensure that blood volumes drawn for safety monitoring and pK levels are not injurious to
the patient. Anuric patients are excluded from the study since LEV is renally excreted.
Exclusion of patients with cumulative seizure burden > 8 minutes/hour in phases 1 and 2, and
>30 minutes/hour in phase 3 ensure that those patients receive timely treatment with the
standard of care antiseizure medication PHB, since high seizure burden may cause brain injury.

Safety data: Continuous monitoring of vital signs will occur as part of routine neonatal intensive
care. Vital signs will be recorded at baseline, and at 15 minutes after each drug infusion. Daily
review for adverse event monitoring will be systematically recorded for the 5 days of study drug
treatment. Blood tests of haematological indices, electrolytes, glucose, renal function and liver
enzymes will be tested and recorded at baseline, at 24 hours and at 5 days. 1 minute of ECG
rhythm strip will be printed before dose escalation and at highest dose to monitor for arrhythmia,
PR interval or QTc abnormality.

8.5 ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS

8.5.1 DEFINITION OF ADVERSE EVENTS (AE)

Adverse event means any untoward medical occurrence associated with the use of an
intervention in humans, whether or not considered intervention-related (21 CFR 312.32 (a))

8.5.2 DEFINITION OF SERIOUS ADVERSE EVENTS (SAE)

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An adverse event (AE) or suspected adverse reaction is considered "serious" if, in the view of
either the investigator or sponsor, it results in any of the following outcomes: death, a life-
threatening adverse event, inpatient hospitalization or prolongation of existing hospitalization, a
persistent or significant incapacity or substantial disruption of the ability to conduct normal life
functions, or a congenital anomaly/birth defect. Important medical events that may not result in
death, be life-threatening, or require hospitalization may be considered serious when, based
upon appropriate medical judgment, they may jeopardize the participant and may require
medical or surgical intervention to prevent one of the outcomes listed in this definition.
Examples of such medical events include allergic bronchospasm requiring intensive treatment in
an emergency room or at home, blood dyscrasias or convulsions that do not result in inpatient
hospitalization, or the development of drug dependency or drug abuse (21 CFR 312.32 (a)).

8.5.3 CLASSIFICATION OF AN ADVERSE EVENT

Relationship to Study Intervention

All adverse events (AEs) must have their relationship to study intervention assessed by the
clinician who examines and evaluates the participant based on temporal relationship and his/her
clinical judgment. The degree of certainty about causality will be graded using the categories
below. In a clinical trial, the study product must always be suspect.
In NEOLEV3 we will use the International Neonatal Consortium (INC) Neonatal Adverse
event scale59 . This resource provides a descriptive severity scale for the most common
neonatal AE’s . For grading of adverse laboratory results we refer to the DIADS scale and that
scale is included in a table below, since the INC scale does not provide an adverse event scale
for laboratory results.

SEVERITY GRADES
As defined in the INC Neonatal adverse event scale. If there are no severity scales
specified for an AE, this generic scale should be used.

Grade 1 Grade 2 Grade 3 Grade 4 Grade 5

Mild Moderate Severe Life Death
threatening
Mild; Moderate; Severe; Life- Death related
asymptomatic resulting in resulting in threatenin to AE
or mild minor changes major g; Resulting
symptoms; of baseline changes of in life-
clinical or age- baseline age- threatenin
diagnostic appropriate appropriate g changes
observations behavior*; behavior* or in basal
only; no requiring non- life physiologic
change in minor changes threatening al
baseline age- in baseline changes in processes*
appropriate care or basal *; requiring
behavior*; no monitoring*** physiological urgent
change in processes**; major

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baseline care requiring change in

or monitoring major change baseline
indicated in baseline care
care or
monitoring**
**

NEOLEV3 GRADING ABNORMAL LABORATORY VALUES

General. An asymptomatic, abnormal laboratory finding without an accompanying AE should not
be reported in an expedited timeframe unless it meets protocol-specific reporting requirements.
Values below Grade 1. Any laboratory value that is between the ULN and grade 1 (for high
values) or the LLN and grade 1 (for low values) should not be graded or reported as an AE.
Sites should consult the Manual for Expedited Reporting of Adverse Events to DAIDS, Version
2.0 and their protocol when making an assessment of the need to report an AE.
Overlap of Local Laboratory Normal Values with Grading Table Ranges. When local laboratory
normal values fall within grading table laboratory ranges, the severity grading is based on the
ranges in the grading table unless there is a protocol-specific grading criterion for the laboratory
value. For example, “Magnesium, Low" has a grade 1 range of 1.2 to < 1.4 mEq/L, while a
particular laboratory’s normal range for magnesium may be 1.3 to 2.8 mEq/L. If a study
participant’s magnesium laboratory value is 1.3 mEq/L, the laboratory value should be graded
as grade 1.

PARAMETER GRADE 1 MILD GRADE 2 GRADE 3 GRADE 4

MODERA SEVERE POTENTIALL
TE Y
LIFE-
THREATENIN
G
ALT or SGPT, 1.25 to < 2.5 x 2.5 to < 5.0 x ULN 5.0 to < 10.0 x 10.0 x ULN
High ULN ULN
Report only one

Bicarbonate, Low 16.0 to < LLN 11.0 to < 16.0 8.0 to < 11.0 8.0 < 8.0 < 8.0
(mEq/L; mmol/L) 16.0 to < LLN 11.0 to < 11.0
to < 16.0
Calcium, High 10.6 to < 11.5 11.5 to < 12.5 12.5 to < 13.5 13.5 3.38
(mg/dL; mmol/L) 2.65 2.88 3.13 to < 3.38
7 days of age to < 2.88 to < 3.13
< 7 days of age 12.9 to < 13.5 3.38
3.23 13.5
11.5 to < 12.4 12.4 to < 12.9
2.88 3.10
to < 3.10 to < 3.23 to < 3.38
Calcium > ULN to < 6.0 > 6.0 to < 6.4 1.5 to 6.4 to < 7.2 1.6 to 7.2 1.8
(Ionized), < <
High (mg/dL; ULN to < 1.5 1.6 1.8
mmol/L)
Calcium, Low 7.8 to < 8.4 1.95 7.0 to < 7.8 1.75 6.1 to < 7.0 1.53 to < 6.1 < 1.53
(mg/dL; mmol/L) to < 2.10 to < 1.95 < 1.75
7 days of age
< 7 days of age 6.5 to < 7.5 1.63 to 6.0 to < 6.5 1.50 to 5.50 to < 6.0 1.38 < 5.50 < 1.38
< 1.88 < 1.63 to < 1.50

Creatinine, High 1.1 to 1.3 x ULN , > 1.3 to 1.8 x ULN > 1.8 to < 3.5 x 3.5 x ULN
*Report only one 1 to <1.2 umolLl 1.2 to <1.6 OR ULN >3.2 OR

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Increase to 1.3 1.6to <3.2 OR

to < Increase to 1.5 to Increase of 2.0 x participant’s baseline
<
1.5 x
participant’s 2.0 x
Glucose 116 to 160 6.44 to baseline participant’s 500 27.75
< baseline
> 250 to 500
> 160 to 250 8.89 13.89
to
Nonfasting, High 8.89 < 13.89 to < 27.75

Glucose, 50 to 54 2.78 to < 40 to < 50 2.22 to 30 to < 40 1.67 to < 30 < 1.67
Low< 1 month 3.00 < <
of age 2.78 2.22

PARAMETER GRADE 1 MILD GRADE 2 GRADE 3 GRADE 4

MODERA SEVERE POTENTIALL
TE Y LIFE-
THREATENIN
G
Potassium, High 3.50.6toto<<3.64.0 2.65.0toto<<3.60.5 2.60.5toto<<2.75.0 <2.70.0 < 27.0.0
(mEq/L; mmol/L) 3.50.6toto 2.65.0toto 2.60.5toto
<<3.64.0 <<3.60.5 <<2.75.0
Potassium, Low
(mEq/L; mmol/L)
Sodium, High 146 to < 150 146 150 to < 154 150 154 to < 160 154 160 160
(mEq/L; mmol/L) to < 150 to < 154 to < 160
Sodium, Low 130 to < 135 130 125 to < 130 125 121 to < 125 121 120 120
(mEq/L; mmol/L) to < 135 to < 130 to < 125

Absolute 800 to 1,000 600 to 799 400 to 599 < 400

Neutrophil Count 0.800 x 109 to 0.600 x 109 to 0.400 x 109 to < 0.400 x
(ANC), Low 1.000 0.799 0.599
x 109 x 109 x 109 109
(cells/mm3;
cells/L)
> 7 days of age
2 to 7 days of age 1,250 to 1,500 1,000 to 1,249 750 to 999 < 750
1.250 x 109 to 1.000 x 109 to 0.750 x 109 to < 0.750 x
1.500 1.249 0.999
x 109 x 109 x 109 109
1 day of age 4,000 to 5,000 3,000 to 3,999 1,500 to 2,999 < 1,500
4.000 x 109 to 3.000 x 109 to 1.500 x 109 to < 1.500 x
5.000 3.999 2.999
x 109 x 109 x 109 109
Hemoglobin, Low 11.0 to 13.0 9.0 to < 11.0 8.0 to < 9.0 < 8.0
8 to 21 days of 6.81 to 8.10 5.57 to < 6.81 4.96 to < 5.57 < 4.96
age (male and
female)
7 days of age 13.0 to 14.0 10.0 to < 13.0 9.0 to < 10.0 < 9.0
(male and female) 8.05 to 8.72 6.19 to < 8.05 5.59 to < 6.19 < 5.59
WBC, Decreased
(cells/mm3; 2,000 to 2,499 1,500 to 1,999 1,000 to 1,499 < 1,000
cells/L) 2.000 x 109 to 1.500 x 109 to 1.000 x 109 to
> 7 days of age 2.499 1.999 1.499 < 1.000 x
x 109 x 109 x 109 109
7 days of age 5,500 to 6,999 4,000 to 5,499 2,500 to 3,999 < 2,500
5.500 x 109 to 4.000 x 109 to 2.500 x 109 to < 2.500 x
6.999 5.499 3.999

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x 109 x 109 x 109 109

Platelet 100,000-125,000 50,000-100,000 <25,000-50,000 <25,000

s,
Decreas
ed
cells/m
m3
>150

ADVERSE EVENT EXPECTEDNESS

The patient’s neonatologist will be responsible for determining whether an adverse event (AE) is
expected or unexpected, based on the patients underlying clinical condition.
Unexpected treatment emergent adverse clinical events or laboratory abnormalities in the high
dose LEV group with severity grading 3, 4 or 5 will guide dose escalation decisions and may
trigger early termination of the study. (See study stopping rules joint probability chart)
Expected adverse clinical events will also be closely monitored. Study PI and study
biostatistician will report summary of the observed expected adverse clinical events in study
subjects. This will allow comparison of rates of expected adverse events with historical control
data from adverse events rates reported for neonates with hypoxic ischemic encephalopathy
and seizures in head cooling trials .60 and in NEOLEV2. See section 7.1.3.

8.5.4 TIME PERIOD AND FREQUENCY FOR EVENT

ASSESSMENT AND FOLLOW-UP

The occurrence of an adverse event (AE) or serious adverse event (SAE) may come to the
attention of study personnel during daily systematic review, or be reported to study personnel by
neonatal staff at any time.

Solicited AE: Daily review will include systematic daily monitoring for hypotension, heart rate or
ECG abnormality, respiratory abnormality, sedation, irritability, poor feeding, infection, need for
oxygen, ventilation or vasopressor treatment. Complete blood count and a comprehensive
metabolic panel will be measured before after 48 hours of treatment. Unsolicited AE Daily
review will also include review of the clinical notes for the past 24 hours and direct enquiry with
neonatal staff as to whether any new problems have arisen. To avoid double capture the
Redcap data base will only allow single entry of any adverse event each day.

All AEs including local and systemic reactions not meeting the criteria for SAEs will be captured
on the appropriate case report form (CRF). Information to be collected includes event
description, time of onset, clinician’s assessment of severity, relationship to study product
(assessed only by those with the training and authority to make a diagnosis), and time of
resolution/stabilization of the event. All AEs occurring while on study must be documented
appropriately regardless of relationship. All AEs will be followed to adequate resolution.
Study coordinators at each site will enter daily safety monitoring and laboratory test monitoring
data as well as any adverse event data directly into centralized Redcap database.

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Any medical condition that is present at the time that the participant is screened will be
considered as baseline and not reported as an AE. However, if the study participant’s condition
deteriorates at any time during the study, it will be recorded as an AE.

Changes in the severity of an AE will be documented to allow an assessment of the duration of

the event at each level of severity to be performed. AEs characterized as intermittent require
documentation of onset and duration of each episode.

Study coordinator or investigator will record all reportable events with start dates occurring any
time after informed consent is obtained until 7 (for non-serious AEs) or 30 days (for SAEs) after
the last day of study participation. At each study visit, the investigator will review the clinical
notes and speak with neonatology staff about the occurrence of AE/SAEs since the last visit.
Events will be followed for outcome information until resolution or stabilization.

Accrual report, eligibility verification, and data completion will be monitored by the clinical trials
coordinator and the PI’s. Monthly conference calls will be held with site study coordinators and
investigators.

1.1.1 ADVERSE EVENT REPORTING

SAEs that are unanticipated, serious, and possibly related to the study intervention will be
reported to the DSMB, Independent Monitor, IRB or record, local IRB and to the FDA in an IND
safety report.

The Principle investigators and clinical trials coordinator will review with site study investigators
any unexpected adverse events with a grade of 3,4, or 5 and possibly related to the study
intervention within 72 hours.
The principle investigators are responsible for reporting SAE’s to overseeing bodies.

● Unexpected fatal or life-threatening AEs related or possibly related to the intervention

will be reported to the FDA Program Officer, DSMB, and Independent Monitor and IRBs
within 7 days of the investigator becoming aware of the event.
● Other serious and unexpected AEs related to the intervention will be reported within 15
days.
● Anticipated or unrelated SAEs will be handled in a less urgent manner but will be
reported to the DSMB, Independent Monitor, IRB, and other oversight organizations in
accordance with their requirements. These will be reported to FDA on an annual basis.
● All other AEs documented during the course of the trial will be reported to FDA on an
annual basis by way of inclusion in the annual report and in the annual AE summary
which will be provided to FDA, the DSMB, and to the Independent Monitor. The DSMB
Report will state that all AEs have been reviewed.
● The DSMB will review all AE and safety data prior to each phased escalation stage.

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Process for reporting disease-related events (DREs)/ expected adverse events

Any adverse events that are expected, not related to high-dose LEV, and not severe in nature,
will be recorded in the database, and reviewed at regular meetings of the DSMB and prior to
progression in the phases of the study. Study PI and study biostatistician will report summary of
the observed expected adverse clinical events. This will allow comparison of rates of expected
adverse events with historical control data. After review of the data, the DSMB will report the
risks of the study to sponsor.

The study will be conducted in a sick neonatal population, with a high rate of morbidity, clinical
adverse events, abnormal laboratory values and mortality expected due to the underlying
causes of neonatal seizures. Risk information from adverse events rates reported for neonates
with hypoxic ischemic encephalopathy and hypoxic ischemia in head cooling trials .60 and in
NEOLEV2 are presented below.

Head cooling trial data

Adverse events occurring in Infants with hypoxic ischemic encephalopathy Percentage

receiving hypothermia treatment

Death 33 %
Cause of death (more than one possible)
Encephalopathy 29%
Persistent pulmonary hypertension 3.5%
Sepsis 1.8%
Renal failure 3.5%
Intractable hypotension 3.5%

Severe hypotension despite full support 3%

Unanticipated serious adverse event 1%

Minor cardiac arrhythmia 9%

Hypotension 55%
Coagulopathy 19%
Prolonged coagulation times 19%
Abnormal renal function 65%
Hyponatremia 44%
Hypokalemia 63%
Platelet count <100,000 per microliter 32%
Raised liver enzymes concentrations 38%
Metabolic acidosis 20%
Respiratory distress 84%
Systemic infection 3%
Haemoconcentration 3%
Hypoglycaemia 13%
Hypocalcaemia 44%
Difficulties in temperature control 32%

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Clinical Adverse Events and Treatment Emergent Laboratory Abnormalities seen in

neonates with seizures and HIE from NEOLEV2 data.
Event

Death 6/ 56

Total Maximal Severity Grading

Grade 1 Grade 2 Grade 3 Grade 4
Respiratory depression 52 3 12 2 35
Hypotension 28 3 5 20

Glucose high 5 2 1 2
Glucose low 1 1
Sodium high 2 2
Sodium low 4 2 1 1
Potassium high 0
Potassium low 4 4
Calcium high 1 1
Calcium low 0
Creatinine high 2 2
ALT high 4 1 1 2

Hemoglobin low 6 6
White cell count low 5 3 2
Platelet count low 13 5 8

Clinical Adverse events and treatment emergent laboratory abnormality seen in

NEOLEV2 subjects treated for seizures who did not have HIE.
Event

Death 0/50

Total Maximal Severity Grading

Grade 1 Grade 2 Grade 3 Grade 4
Respiratory depression 29 7 14 1 7
Hypotension 4 1 3
Cardiac arrhythmia ( PVC’s) 1 1
Bloody stools 1 1

Glucose high 2 1 1
Glucose low 0
Sodium high 1 1
Sodium low 0
Potassium high 0
Potassium low 3 1 2
Calcium high 0
Calcium low 0
Creatinine high 0
ALT high 1 1

Hemoglobin low 0
White cell count low 0
Platelet count low 0

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1.1.2 SERIOUS ADVERSE EVENT REPORTING

Any serious adverse event, whether or not considered study intervention related, will be
reported and will include an assessment of whether there is a reasonable possibility that the
study intervention caused the event. Any death will be reported.

The study principal investigators will be responsible for notifying the Food and Drug
Administration (FDA) of any unexpected fatal or life-threatening suspected adverse reaction as
soon as possible, but in no case later than 7 calendar days after the sponsor's initial receipt of
the information. In addition, the sponsor must notify FDA and all participating investigators in an
Investigational New Drug (IND) safety report of potential serious risks, from clinical trials or any
other source, as soon as possible, but in no case later than 15 calendar days after the sponsor
determines that the information qualifies for reporting.

The DSMB will determine if any adverse events change the known risks to study subjects. If the
known risk to subjects changes, the DSMB’s report regarding the change in risk will be released
to all participating investigators via the sponsor. The DSMB may request changes to the DSMP.

All serious adverse events (SAEs) will be followed until satisfactory resolution or until the site
investigator deems the event to be chronic or the participant is stable.

1.1.3 REPORTING EVENTS TO PARTICIPANTS

Parents of participating patients will be informed of any AE or SAE as soon as they are
recognized.

A summary of the safety data and AE monitoring from each phase of the CONTINUAL
REASSESSMENT METHOD will be shared with all investigators and will inform informed
consent process in later phases.

1.1.4 EVENTS OF SPECIAL INTEREST

Not applicable
1.1.5 REPORTING OF PREGNANCY
Not applicable

1.2 UNANTICIPATED PROBLEMS

1.2.1 DEFINITION OF UNANTICIPATED PROBLEMS (UP)

The Office for Human Research Protections (OHRP) considers unanticipated problems
involving risks to participants or others to include, in general, any incident, experience, or
outcome that meets all of the following criteria:

• Unexpected in terms of nature, severity, or frequency given (a) the research procedures
that are described in the protocol-related documents, such as the Institutional Review
Board (IRB)-approved research protocol and informed consent document; and (b) the
characteristics of the participant population being studied;

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• Related or possibly related to participation in the research (“possibly related” means

there is a reasonable possibility that the incident, experience, or outcome may have
been caused by the procedures involved in the research); and
• Suggests that the research places participants or others at a greater risk of harm
(including physical, psychological, economic, or social harm) than was previously known
or recognized.

1.2.2 UNANTICIPATED PROBLEM REPORTING

Reporting of unanticipated problems meeting the definition above will be report in accordance
with local IRB regulations, along with reporting to the DSMB chair within 24 hours of receiving
the report.

This will include the following information:

• Protocol identifying information: protocol title and number, PI’s name, and the IRB
project number;
• A detailed description of the event, incident, experience, or outcome;
• An explanation of the basis for determining that the event, incident, experience, or
outcome represents an UP;
• A description of any changes to the protocol or other corrective actions that have been
taken or are proposed in response to the UP

The DSMB will determine if any adverse events change the known risks to study subjects. If the
known risk to subjects changes, the DSMB’s report regarding the change in risk will be released
to all participating investigators via the sponsor. The DSMB may request changes to the DSMP
or changes as outlined below.

• Modification of inclusion or exclusion criteria to mitigate the newly identified risks

• Implementation of additional safety monitoring procedures (change in DSMP)
• Suspension of enrollment of new participants or halting of study procedures for enrolled
participants
• Modification of informed consent documents to include a description of newly recognized
risks
• Provision of additional information about newly recognized risks to previously enrolled
participants.

1.2.3 REPORTING UNANTICIPATED PROBLEMS TO PARTICIPANTS

Parents of participating patients will be informed of any unanticipated problem as soon as they
are recognized. Informed consent documents will be modified to include a description of newly
recognized risks.

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2 STATISTICAL CONSIDERATIONS

There will be a formal Statistical analysis plan (SAP).

2.1 STATISTICAL HYPOTHESES

Aim 1: Hypothesis 1.
The optimal dosing of Levetiracetam (LEV) to treat neonatal seizures is significantly greater
than 60mg/kg.

Aim 2: Hypothesis 2.
LEV will display the same pharmacokinetic parameters of CL and V at high dosing that have
been documented at standard dosing.

Aim 3: Hypothesis 3.
Treatment response rate improves as dose escalates from 60mg/kg LEV.

Aim 4: Hypothesis 4.
The new version of the Persyst neonatal seizure detector algorithm will show excellent
sensitivity and specificity in the detection of neonatal seizures.

2.2 SAMPLE SIZE DETERMINATION

The proposed study needs to enroll 133 patients with seizures.
Data from NEOLEV2 predict we will need to recruit and screen 2.8 patients at risk for seizures
or suspected of having seizures for every patient who will actually have seizures.
Data from NEOLEV2 predict that of patient with seizures, 56% will have seizures < 8 minutes
cumulative seizure burden/ hour as required for recruitment into phases 1 and 2 of the study
( 20/60 subjects); and 88% of patients will have cumulative seizure burden < 30 minutes/hour as
required for recruitment into phase 3 of the study (40/60 subjects).
We are confident that we if will be feasible to enroll the necessary number of participants based
on recruited numbers in the NEOLEV2 trial, and considering the inclusion of further sites and
our widened inclusion criteria (gestational age, and pretreatment allowed). (See table below)
Site NEOLEV2 Average 77%<set Number
seizures/ seizures/year sz needed/
year burden* year
Rady 7.5 39 30 14
UCSD 3 11 8.5 4
SMB 5 11 8.5 4
Auckland 8 12 9 5
Middlemor NA 10 8 4
e
U. Minn NA 11 8.5 4
Totals 94 72.5 35
*(56%*20/60+88%*40/60 =77%)
Of the 133 patients with seizures of mild to moderated severity, we expect initial treatment with
60mg/kg LEV will adequately control 40% of these subjects. The remaining 80 subjects with
seizures refractory to 60mg/kg of LEV will enter in the randomized dose escalation study. 60 of

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these subjects will be randomized to receive higher dose LEV, 20 will receive control treatment.
Using the estimate that each dose escalation step will have a 5% additional efficacy, it would be
expected that 35 subjects would continue to seize and reach the final dose escalation step of
150mg/kg total loading dose, allowing adequate study of this dosing step. If each dose
escalation step had 20% additional efficacy (a high estimate), it would still be expected that 23
subjects would reach the 150mg/kg total loading dose step.

● Outcome measure used for calculations

o Maximum safe and tolerated dose
● Test Statistic
o Aim 1: Comparison of dose limiting toxicity events during dose escalation
o Aim 3: Estimates of response rates in response to dose escalation from 60mg/kg
LEV (secondary outcome)
● Null and Alternative Hypotheses
o Aim 1: Null hypothesis: DLT is the same between the standard 60 mg/kg and
max tolerated dose:
▪ Alternative hypothesis: DLT is higher in the max tolerated dose than the
standard 60 mg/kg
o Aim 3: Null hypothesis: Treatment response rate is the same between each
escalated dose level and the standard 60mg/kg LEV
▪ Alternative hypothesis: Treatment response rate improves as dose
escalates from the standard 60mg/kg LEV
● Type I error rate (alpha)
o Two sided alpha = 0.05
● Power level
o Power is at least 80%
● Assumed event rate
o Response rate is estimated at 28% for the standard 60mg/kg LEV and will be
improved by an estimated 15% for the max tolerated dose
● Sample size calculation
o We estimated sample size for logistic regression using the approach by Hsieh,
FY, Bloch, DA, and Larsen, MD. A simple method of sample size calculation for
linear and logistic regression. Statistics in Medicine. 1998; 17:1623- 1634 and the
"powerMediation" package in R.
● Anticipated impact of dropout rates
o We anticipate a low dropout rate such as 5% and will recruit addiotnal subjects to
reach the proposed sample size
● Interim analyses
o No formal interim analysis is planned; however, we will use a continual
reassessment method (CRM) to assess safety in dose escalation.

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● Power of secondary endpoints

o The study is powered only for the primary outcome. However, if effect size for
the secondary outcome in Aim 3 is large, the proposed sample size may show
statistically significance for this outcome after adjustment for multiple
comparisons.

Sample size: Estimating additional efficacy of higher dose LEV

Assuming no dose limiting toxicity is demonstrated and the study proceeds to completion, 50
subjects will be treated at 120mg/kg or higher dosing level. This sample size will give
satisfactory power for estimating additional efficacy of higher doses. For example, if we
document an additional response rate of 15%, this sample size will provide a 95% confidence
interval (0.051, 0.248) around that estimate.

Sample size: Pharmacokinetic study

Our prior infant population pharmacokinetic study was able to determine key parameters with
149 concentrations from 18 subjects. In that prior study the precision of pharmacokinetic
parameters was good for CL and V estimates with 95% CIs < + 20% of their mean values. The
pharmacokinetic data expected from the proposed study has larger number of subjects n=60
but few samples per subject (> 3) than the first infant study and is expected to generate similar
pharmacokinetic parameter confidence intervals. The data from this study will be nested with
raw existing infant LEV concentrations data from our prior studies to formally assess the
potential impact of higher doses being used and better characterize changes in
pharmacokinetics during the first week of life.

The sample size will also provide sufficient power for planned exploratory analysis assessing
the sensitivity and specificity of the neonatal seizures detection algorithm. The performance of
the algorithm will be able to be assessed in 133 patients with seizures and several hundred
patients without seizures.
The sample size is too small to assess differences in rates of adverse events between the
treatment arms with statistical significance. Much larger trials will be required to study this
adequately.

2.3 POPULATIONS FOR ANALYSES

Populations for analysis will include
● Intention-to-Treat (ITT) Analysis Dataset (all randomized participants)

● Modified Intention-to-Treat Analysis Dataset (all randomized participants in whom EEG

can be validated by independent neurophysiologist .)
● Safety Analysis Dataset: (participants who took at least one dose of high dose lev)

● Per-Protocol Analysis Dataset: ITT population who remain on high dose LEV for 5 days.

2.4 STATISTICAL ANALYSES

2.4.1 GENERAL APPROACH

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Demographic and baseline characteristics of the 2 treatment groups will be reported as means
with standard deviations, medians and range for the continuous data and percentages for the
categorical data. Treatment groups will be compared using Fisher’s exact test for categorical
variables and Wilcoxon-rank sum test for continuous variables.
All analyses will incorporate the intention-to-treat principle; all randomized participants will be
included in the analysis. Analyses for all efficacy outcomes will be guided by descriptive and
exploratory analyses. All results will be reported as point estimates (proportions or mean
differences across groups, as appropriate) and interval estimates (95% confidence intervals)
with two-sided p-values denoting statistical significance. Since this is a phase II preliminary
efficacy and safety study, no adjustments for multiple comparisons will be made and a p-value
of 0.05 will be considered statistically significant.
2.4.2 ANALYSIS OF THE PRIMARY EFFICACY ENDPOINT(S)

Analysis Plan for Aim 1

The study will determine the maximum safe and tolerated dose of LEV in the treatment of
neonatal seizures. The maximum safe and tolerated dose of LEV will be the maximal dose
studied where dose limiting toxicity is not observed. We will use a continual reassessment
method (CRM) to assess safety in dose escalation with a two-tier approach, monitoring for two
different severities of adverse events. Dose limiting toxicity (DLT) will be defined as either a 5%
incidence of grade 4 or 5 unexpected treatment emergent adverse events OR a 15% incidence
of grade 3 unexpected treatment emergent adverse events.

The first group of 10 patients will receive 90mg/kg of Levetiracetam and the decision to escalate
dose to the next 120mg/kg will be informed and guided by the number of infants observed with
adverse events. We will repeat the above process until we (a) reach the proposed maximum
dose 150mg/kg or (b) stop the study before reaching this targeted maximum dose because of
evidence of DLT exceeding our set limits.
Stopping rule for dose limiting toxicity
Joint probability distribution: grade 4-5 (X1) and grade 3 (X2) adverse events calculated
under 5% DLT for X1 and 15% for X2.
X1
0 1 2 3
0 0.999 0.401 0.086 0.011
X2
1 0.803 0.322 0.069 <0.01
2 0.456 0.183 0.039 <0.01
3 0.179 0.072 0.015 <0.01
4 0.049 0.020 <0.01 <0.01
The entry in the table for a given entry X1 = i and X2 = j indicates the probability of observing at
least i grade 4-5 and at least j grade 3 adverse events. The distribution shows that there is
strong evidence of DLT exceeding our set limits if the probability for the observed grade 4-5
and/or grade 3 events is less than 0.05. All such probabilities are bolded in the table. For
example, if we observe X1 = 1 grade 4-5 and X2 = 0 grade 3, there is no indication of DLT
exceeding 5% for grade 4-5 or DLT exceed 15% for grade 3 and dose escalation can safely
continue. But, if we observe X1 = 2 grade 4-5 and X2 = 2 grade 3 adverse events, there is
strong evidence of DLT exceeding 5% for grade 4-5 or DLT exceed 15% for grade 3 or both
and we will stop the study.

Stopping rule for lack of efficacy.

Probability Table: P(X<=x) if actual additional efficacy response rate is 15%. P=0.10
Number of responding subjects

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0 1 2 3 4 5
Number 10 0.197 0.544 0.820 0.950 0.990 0.999
Of 20 0.039 0.176 0.405 0.648 0.830 0.933
Observed 30 0.008 0.048 0.151 0.322 0.524 0.711
Subjects 40 0.002 0.012 0.049 0.130 0.263 0.432
The study will be terminated early if less than 15% additional efficacy is evident at 120mg/kg or greater
dosing. Conditions for stopping early are bolded in the table.

Missing data there are no imputation techniques that are able to be used within the CRM. As
such every effort will be made to minimize missing data. Building on lessons learned during the
NEOLEV2 we believe that rapid transfer and neurophysiologist review of cEEG data and regular
site PI review of data completeness will reduce this problem.

2.4.3 ANALYSIS OF THE SECONDARY ENDPOINT(S)

Analysis Plan for Aim 2: Pharmacokinetic analysis: The actual individual concentration data,
collection times and dosing histories will be used to create graphs of plasma LEV concentration
vs. time for each infant. Median plasma drug concentration vs. time curves will also be
generated. Summary statistics (i.e., n, mean, standard deviation, median, minimum, maximum,
and coefficient of variation) will be calculated for plasma concentrations for each time point and
each dose level. LEV population pharmacokinetic parameters will be determined using the
computer program NONMEM (ver. 7.2 or later). The pharmacokinetics will be modeled
recognizing the two stages of a nonlinear hierarchical model development. The first stage
introduces the structural model (e.g. one compartment open model), the population parameters,
individual effects and the within-patient variation. The second portion of the model development
recognizes that variation between patients in the pharmacokinetic parameters exists and will
attempt to determine covariates that may identify different pharmacokinetic subpopulations. A
one-compartment model with first order elimination and linear pharmacokinetics will be utilized
consistent with prior pediatric pharmacokinetic studies including infant studies. The impact of
clinical and demographic factors (including weight, sex, age, race, serum creatinine, postnatal
age and dosing regimen) will be assessed as fixed effects in the model. Nested models will be
compared graphically and with a likelihood ratio test.

Our prior infant population pharmacokinetic study was able to determine key parameters with
149 concentrations from 18 subjects. In that prior study the precision of pharmacokinetic
parameters was good for CL and V estimates with 95% CIs < + 20% of their mean values. The
pharmacokinetic data expected from the proposed study has larger number of subjects n=60
but few samples per subject (> 3) than the first infant study and is expected to generate similar
pharmacokinetic parameter confidence intervals. The data from this study will be nested with
raw existing infant LEV concentrations data from our prior studies to formally assess the
potential impact of higher doses being used and better characterize changes in
pharmacokinetics during the first week of life.

Analysis Plan for Aim 3: Analysis of efficacy of higher doses of LEV in neonates with
seizures. Logistic regression will be used to model the relationship between dose and response
(seizure cessation), in which dose will be the independent and seizure cessation will be
dependent variable. As response rates may be low, especially in the lower dose range such as
90mg/kg, we may not observe any infant who responds to the intervention, given the relatively
small group size in this study. In this case we will not be able to estimate model parameters, a
phenomenon known as data separation (Tan et al., 2012). If this occurs, we will use the exact

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conditional logistic regression for deriving model estimates and inference (Tan et al., 2012).
Assuming no dose limiting toxicity is demonstrated and the study proceeds to completion, 50
subjects will be treated at 120mg/kg or higher dosing level. This sample size will give
satisfactory power for estimating additional efficacy of higher doses. For example, if we
document an additional response rate of 15%, this sample size will provide a 95% confidence
interval (0.051, 0.248) around that estimate.

As described in section 9.4.2 every effort will be made to reduce missing data. If missing data is
over 5% best-worst case imputation analysis will be performed as additional analyses.

Analysis Plan for Aim 4: To improve technologies for the prompt detection of neonatal
seizures: Sensitivity and specificity of the Persyst neonatal seizure detector algorithm will be
compared against the markings of expert neurophysiologists. The utility of the rollingcumulative
seizure burden will be assessed qualitatively by study neurologists.

2.4.4 SAFETY ANALYSES

Safety endpoints to be analyzed include clinical AE’s coded as per INC adverse event rating
tables and laboratory AE’s coded as per DIADS rating tables as presented above in section 8.
Each AE will be counted only once per patient. Summary statistics comparing treatment arm
groups will be presented. Severity, frequency, and relationship of AEs to study intervention will
be presented by System Organ Class (SOC). Each AE will be described in detail including (e.g.,
start date, stop date, severity, relationship, expectedness, outcome, and duration). Adverse
events leading to premature discontinuation from the study intervention and serious treatment-
emergent AEs will be presented either in a table or a listing.

2.4.5 BASELINE DESCRIPTIVE STATISTICS

Dose escalation and PHB active control groups will be compared on baseline characteristics ,
including seizure etiology, hypothermia treatment, gender, cord pH, 5 minute Apgar, gestational
age, birth weight and pretreatment seizure severity.

2.4.6 PLANNED INTERIM ANALYSES

There will be interim analyses after every 10 patients under the proposed Continual
reassessment method. Please see section 9.4.2.

2.4.7 SUB-GROUP ANALYSES

No subgroup analyses for the primary endpoint are planned.

Analysis of LEV population pharmacokinetic parameters will include assessment of the impact
of clinical and demographic factors (including weight, sex, age, race, serum creatinine,
postnatal age and dosing regimen) as fixed effects in the model. Nested models will be
compared graphically and with a likelihood ratio test.

A sub-group analysis is planned for the estimate of additional efficacy of LEV at higher doses
within the sub-group of patients with seizures of acute symptomatic etiology. This sub-group is
expected to constitute around 70% of the total cohort.

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2.4.8 TABULATION OF INDIVIDUAL PARTICIPANT DATA

Not applicable

2.4.9 EXPLORATORY ANALYSES

Not applicable

3 SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS

3.1 REGULATORY, ETHICAL, AND STUDY OVERSIGHT CONSIDERATIONS

3.1.1 INFORMED CONSENT PROCESS

3.1.1.1 CONSENT/ASSENT AND OTHER INFORMATIONAL DOCUMENTS PROVIDED TO

PARTICIPANTS
Parental permission forms describing in detail the study intervention, study procedures, and
risks are given to the participant’s parent/guardian and written documentation of informed
consent is required prior to starting intervention/administering study intervention.

3.1.1.2 CONSENT PROCEDURES AND DOCUMENTATION

Parental or guardian permission must be obtained in accordance with the requirements for
informed consent (21 CFR 50.55(e)) and be documented in accordance with 21 CFR 50.27. (21
CFR 50.55(f)).)

The study will follow the OHRP regulations that define “permission” under 46.402(c) as the
“agreement of parent(s) or guardian to the participation of their child or ward in research.” The
term “parent” means a “child's biological or adoptive parent.” The term “guardian” means “an
individual who is authorized under applicable State or local law to consent on behalf of a child to
general medical care.”

Informed consent must meet the requirements of 21 CFR 50.20, and must include the basic
information required by 21 CFR 50.25(a).

Parental/guardian permission for the child’s participation in research will be initiated in English
or Spanish prior to the study and will continue throughout the child’s study participation. A
parental/guardian permission form will be Institutional Review Board (IRB)-approved and the
parent/guardians will be asked to read and review the document. The investigator will explain
the research study to the parent/guardians and answer any questions that may arise. A verbal
explanation will be provided in terms suited to the parent/guardian’s comprehension of the
purposes, procedures, and potential risks of the study and of their and their child’s rights as
research participants. The parent/guardians will have the opportunity to carefully review the
written form and ask questions prior to signing. The parent/guardians should have the
opportunity to discuss the study with their family or surrogates or think about it prior to agreeing
to participate. The parental permission form will be signed by at least one parent/guardian prior
to any procedures being done specifically for the study. The parent/guardians will be informed
that participation is voluntary and that they may withdraw their child from the study at any time,
without prejudice. A copy of the document will be given to the parent/guardians for their records.
The informed consent process will be conducted and documented in the source document

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(including the date), and the form signed, before the child participant undergoes any study-
specific procedures. The rights and welfare of the child will be protected by emphasizing to them
that the quality of their medical care will not be adversely affected if the parent/guardians
decline to participate in this study.

Under 45 CFR 46.406, the above outline for obtaining permission will be applied.

3.1.2 STUDY DISCONTINUATION AND CLOSURE

This study may be temporarily suspended or prematurely terminated if there is sufficient
reasonable cause. Written notification, documenting the reason for study suspension or
termination, will be provided by the suspending or terminating party to <study participants,
investigator, funding agency, the Investigational New Drug (IND), sponsor and regulatory
authorities>. If the study is prematurely terminated or suspended, the Principal Investigator (PI)
will promptly inform study participants, the Institutional Review Board (IRB) and will provide the
reason(s) for the termination or suspension. Study participants will be contacted, as applicable,
and be informed of changes to study visit schedule.

Circumstances that may warrant termination or suspension include, but are not limited to:
● Determination of unexpected, significant, or unacceptable risk to participants

● Demonstration of efficacy that would warrant stopping

● Insufficient compliance to protocol requirements

● Data that are not sufficiently complete and/or evaluable

● Determination that the primary endpoint has been met

● Determination of futility

Study may resume once concerns about safety, protocol compliance, and data quality are
addressed, and satisfy the sponsor, IRB and/or Food and Drug Administration (FDA).

3.1.3 CONFIDENTIALITY AND PRIVACY

Participant confidentiality and privacy is strictly held in trust by the participating investigators,
their staff, and the sponsor(s) and their interventions. This confidentiality is extended to cover
the clinical information relating to participants. Therefore, the study protocol, documentation,
data, and all other information generated will be held in strict confidence. Research records may
be made available to UCSD IRB and FDA as this clinical trial is federally funded.No information
concerning the study or the data will be released to any unauthorized third party without prior
written approval of the sponsor.

All research activities will be conducted in as private a setting as possible.

The study monitor, other authorized representatives of the sponsor, representatives of the
Institutional Review Board (IRB), regulatory agencies or pharmaceutical company supplying
study product may inspect all documents and records required to be maintained by the
investigator, including but not limited to, medical records (office, clinic, or hospital) and
pharmacy records for the participants in this study. The clinical study site will permit access to
such records.

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The study participant’s contact information will be securely stored at each clinical site for internal
use during the study. At the end of the study, all records will continue to be kept in a secure
location for as long a period as dictated by the reviewing IRB, Institutional policies, or sponsor
requirements.

Study participant research data, which is for purposes of statistical analysis and scientific
reporting, will be transmitted to and stored at the University of Minnesota Data Coordinating
Center. This will not include the participant’s contact or identifying information. Rather, individual
participants and their research data will be identified by a unique study identification number.
The study data entry and study management systems used by clinical sites and by the
University of Minnesota Data Coordinating Center research staff will be secured and password
protected. At the end of the study, all study databases will be de-identified and archived at the
University of Minnesota Data Coordinating Center.

3.1.4 FUTURE USE OF STORED SPECIMENS AND DATA

Data collected for this study will be analyzed and stored at the University of Minnesota Data
Coordinating Center. After the study is completed, the de-identified, archived data will be
transmitted to and stored at the University of Minnesota Data Repository, for use by other
researchers including those outside of the study. Permission to transmit data to the University of
Minnesota Data Repository will be included in the informed consent. Specifically, after EEG data
has been completed, analyzed and de-identified, the archived data will be transmitted and
stored at the University of California San Diego Data Repository.

During the conduct of the study, an individual participant can choose to withdraw consent to
have data stored for future research.

When the study is completed, access to study data and/or samples will be provided through the
University of Minnesota Data Repository.

3.1.5 KEY ROLES AND STUDY GOVERNANCE

Principal Investigator Medical Monitor
Multi-PI Study Leadership: The Independent Steering
Sonya Wang, MD Committee and Independent
Jeffrey Gold, MD PhD Data Safety Monitoring Board
Richard Haas, MB, BChir, will oversee the study.
Cynthia Sharpe, MBChB
University of Minnesota
420 Delaware Street S.E.
612-301-1454
sgwang@umn.edu
Study leadership committee consists of the four multi-principal investigators who will provide
oversight of the entire program; this includes the development and implantation of all policies,
and procedures and processes.
An Independent Steering Committee comprised of clinical experts in neonatology, neurology
and pharmacology will be responsible for the scientific integrity of the research by reviewing the
scientific validity of the study protocol and assessing the study quality.
New Zealand sites have the same administrative requirements and functions as the USA sites.
3.1.6 SAFETY OVERSIGHT

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There will be a formal independent DSMB. The board will be composed of an experienced
neonatologist, a pediatric neurologist, an independent biostatistician, a research
pharmacologist, and a representative of the FDA orphan drug research program.

Members of the DSMB will be independent from the study conduct and free of conflict of
interest, or measures should be in place to minimize perceived conflict of interest. The DSMB
will meet at least semiannually to assess safety and efficacy data on each arm of the study. The
DMSB will operate under the rules of an approved charter that will be written and reviewed at
the organizational meeting of the DSMB. At this time, each data element that the DSMB needs
to assess will be clearly defined.

3.1.7 CLINICAL MONITORING

Clinical site monitoring is conducted to ensure that the rights and well-being of trial participants
are protected, that the reported trial data are accurate, complete, and verifiable, and that the
conduct of the trial is in compliance with the currently approved protocol/amendment(s), with
International Conference on Harmonisation Good Clinical Practice (ICH GCP), and with
applicable regulatory requirement(s).

• Monitoring for this study will be performed by two federally funded Clinical and
Translational Science/Research Institutes (University of Minnesota and UC San Diego)
and a contracted Clinical Research Associate for the other sites involved.
• Monitoring will consist of random review of sampled data with targeted data verification
of endpoint, safety, and other key data variables. These monitoring visit will occur every
6 months throughout the study remotely or on-site. All data will be centralized in RedCap
at University of Minnesota.
• Site PIs will be provided copies of monitoring reports within 5 days of visit.
• Details of clinical site monitoring are documented in a Clinical Monitoring Plan (CMP).
The CMP describes in detail who will conduct the monitoring, at what frequency
monitoring will be done, at what level of detail monitoring will be performed, and the
distribution of monitoring reports.

3.1.8 QUALITY ASSURANCE AND QUALITY CONTROL

Each clinical site will perform internal quality management of study conduct, data collection,
documentation and completion. An individualized quality management plan will be developed to
describe a site’s quality management.

Quality control (QC) procedures will be implemented beginning with the data entry system and
data QC checks that will be run on the database will be generated. Any missing data or data
anomalies will be communicated to the site(s) for clarification/resolution.

Following written Standard Operating Procedures (SOPs), the monitors will verify that the
clinical trial is conducted and data are generated, collected, documented (recorded), and
reported in compliance with the protocol, International Conference on Harmonisation Good
Clinical Practice (ICH GCP), and applicable regulatory requirements (e.g., Good Laboratory
Practices (GLP), Good Manufacturing Practices (GMP)).

The investigational site will provide direct access to all trial related sites, source
data/documents, and reports for the purpose of monitoring and auditing by the sponsor, and
inspection by local and regulatory authorities.

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3.1.9 DATA HANDLING AND RECORD KEEPING

3.1.9.1 DATA COLLECTION AND MANAGEMENT RESPONSIBILITIES

Data collection is the responsibility of the clinical trial staff at the site under the supervision of
the site investigator. The investigator is responsible for ensuring the accuracy, completeness,
legibility, and timeliness of the data reported.
All source documents should be completed in a neat, legible manner to ensure accurate
interpretation of data.

Hardcopies of the study visit worksheets will be provided for use as source document
worksheets for recording data for each participant enrolled in the study. Data recorded in the
electronic case report form (eCRF) derived from source documents should be consistent with
the data recorded on the source documents.

Clinical data (including adverse events (AEs), concomitant medications, and expected adverse
reactions data) and clinical laboratory data will be entered into REDCap, a 21 CFR Part 11-
compliant data capture system provided by the University of Minnesota. The data system
includes password protection and internal quality checks, such as automatic range checks, to
identify data that appear inconsistent, incomplete, or inaccurate. Clinical data will be entered
directly from the source documents.

3.1.9.2 STUDY RECORDS RETENTION

Study documents should be retained for a minimum of 2 years after the last approval of a
marketing application in an International Conference on Harminosation (ICH) region and until
there are no pending or contemplated marketing applications in an ICH region or until at least 2
years have elapsed since the formal discontinuation of clinical development of the study
intervention. These documents should be retained for a longer period, however, if required by
local regulations. No records will be destroyed without the written consent of the sponsor, if
applicable. It is the responsibility of the sponsor to inform the investigator when these
documents no longer need to be retained.

3.1.10 PROTOCOL DEVIATIONS

A protocol deviation is any noncompliance with the clinical trial protocol, International
Conference on Harmonisation Good Clinical Practice (ICH GCP), or Manual of Procedures
(MOP) requirements. The noncompliance may be either on the part of the participant, the
investigator, or the study site staff. As a result of deviations, corrective actions are to be
developed by the site and implemented promptly.

These practices are consistent with ICH GCP:

• 4.5 Compliance with Protocol, sections 4.5.1, 4.5.2, and 4.5.3
• 5.1 Quality Assurance and Quality Control, section 5.1.1
• 5.20 Noncompliance, sections 5.20.1, and 5.20.2.

It is the responsibility of the site investigator to use continuous vigilance to identify and report
deviations within 5 working days of identification of the protocol deviation, or within 5 working
days of the scheduled protocol-required activity. All deviations must be addressed in study
source documents, reported to the University of Minnesota data coordinating center. Protocol
deviations must be sent to the reviewing Institutional Review Board (IRB) per their policies. The

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site investigator is responsible for knowing and adhering to the reviewing IRB requirements.
Further details about the handling of protocol deviations will be included in the MOP.

3.1.11 PUBLICATION AND DATA SHARING POLICY

Multiple investigators are involved in this study. The study leadership (Multi-PIs) will be
responsible for developing publication procedures and resolving authorship issues.

This study will be conducted in accordance with the following publication and data sharing
policies and regulations:
● The NIH Public Access Policy, the NIH Policy on the Dissemination of NIH-Funded
Clinical Trial Information, The Food and Drug Administration Amendments Act of 2007
(FDAAA), Clinical Trials Registration and Results Information Submission rule,
● The NIH Data Sharing Policy

National Institutes of Health (NIH) Public Access Policy, which ensures that the public has
access to the published results of NIH funded research. It requires scientists to submit final
peer-reviewed journal manuscripts that arise from NIH funds to the digital archive PubMed
Central upon acceptance for publication.

This study will comply with the NIH Data Sharing Policy and Policy on the Dissemination of NIH-
Funded Clinical Trial Information and the Clinical Trials Registration and Results Information
Submission rule. As such, this trial will be registered at ClinicalTrials.gov, and results
information from this trial will be submitted to ClinicalTrials.gov. In addition, every attempt will be
made to publish results in peer-reviewed journals. Data from this study may be requested from
other researchers up 10 years after the completion of the primary endpoint by contacting Sonya
Wang, MD at University of Minnesota.

3.1.12 CONFLICT OF INTEREST POLICY

The independence of this study from any actual or perceived influence, such as by the
pharmaceutical industry, is critical. Therefore, any actual conflict of interest of persons who
have a role in the design, conduct, analysis, publication, or any aspect of this trial will be
disclosed and managed. Furthermore, persons who have a perceived conflict of interest will be
required to have such conflicts managed in a way that is appropriate to their participation in the
design and conduct of this trial. The study leadership in conjunction with the FDA has
established policies and procedures for all study group members to disclose all conflicts of
interest and will establish a mechanism for the management of all reported dualities of interest.

3.2 ADDITIONAL CONSIDERATIONS

A designated IRB of Record will be identified and will be registered with the OHRP. The IRB of
Record will undertake the responsibilities of oversight of the whole study/application and will
communicate with, inform, and update all other participating IRBs of issues from other centers.

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3.3 ABBREVIATIONS
The list below includes abbreviations utilized in this template. However, this list should be customized
for each protocol (i.e., abbreviations not used should be removed and new abbreviations used should be
added to this list).

AE Adverse Event
ANCOVA Analysis of Covariance
cEEG Continuous Electroencephalography
CFR Code of Federal Regulations
CMP Clinical Monitoring Plan
CONSORT Consolidated Standards of Reporting Trials
CRF Case Report Form
DCC Data Coordinating Center
DHHS Department of Health and Human Services
DSMB Data Safety Monitoring Board
eCRF Electronic Case Report Forms
FDA Food and Drug Administration
FDAAA Food and Drug Administration Amendments Act of 2007
FFR Federal Financial Report
GCP Good Clinical Practice
GLP Good Laboratory Practices
HIPAA Health Insurance Portability and Accountability Act
ICH International Conference on Harmonisation
IND Investigational New Drug Application
IRB Institutional Review Board
ISM Independent Safety Monitor
ISO International Organization for Standardization
ITT Intention-To-Treat
LEV Levetiracetam
MOP Manual of Procedures
MSDS Material Safety Data Sheet
NCT National Clinical Trial
NIH National Institutes of Health
OHRP Office for Human Research Protections
PHB Phenobarbital
PI Principal Investigator
QA Quality Assurance
QC Quality Control
SAE Serious Adverse Event
SAP Statistical Analysis Plan
SOA Schedule of Activities
SOP Standard Operating Procedure
UP Unanticipated Problem
US United States

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3.4 PROTOCOL AMENDMENT HISTORY

Version Date Description of Change Brief Rationale
2 November 4 Changed exclusion criteria in phase 3 Change made in response to
2020 of the study to a higher maximum suggestion from FDA and in
seizure burden of <30 minutes/hour order to make estimate of the
efficacy of higher dose LEV
more generalizable to all
neonates with seizures
3 December 6 Changed text to consistently Changes made in response to
2020 emphasize that Persyst algorithm will suggestion from FDA
be used as a research tool only.
3 December 6 Reverted back to protocol for dose Changes made in response to
2020 escalation in phase III in 3 doses, suggestion from FDA
60mg/kg then 60mg/kg/ then
30mg/kg

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