Clinical trial NCT02891837
Conditions: Acute Lung Injury
Keywords: L-citrulline pediatric Lung Injury Bypass Heart Defects Clinical Sequelae
Sponsor: Asklepion Pharmaceuticals, LLC
Start date: 2016-08-01; Completion date: 2019-07-01
Enrollment: 189 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The purpose of this study is to determine whether L-citrulline is effective and safe in the prevention of clinical sequelae of Acute Lung Injury in pediatric subjects undergoing surgery for congenital heart defects.

Detailed description:
This is a randomized, double-blind, placebo controlled, multicenter study that will compare the efficacy and safety of L-citrulline versus placebo in subjects undergoing surgery for congenital heart defects.

Eligible subjects undergoing repair of a large unrestrictive ventricular septal defect (VSD), a partial or complete atrioventricular septal defect (AVSD), or an ostium primum atrial septal defect (primum ASD) will be eligible for enrollment in this study.

Each enrolled subject will be randomized to receive either L-citrulline or placebo throughout all administrations in the study. Subjects will receive an L-citrulline bolus of 150 mg/kg or placebo at the initiation of cardiopulmonary bypass, the addition of L-citrulline at a concentration of 200 μmol/L or placebo given as a bolus during bypass. This may be administered as a one-time bolus or multiple administrations to compensate for fluids containing L-citrulline that may be removed from the patient during the course of the operation and thus to maintain the concentration of 200 μmol/L. L-citrulline bolus of 20 mg/kg or placebo 30 minutes after decannulation from cardiopulmonary bypass, followed immediately by a 9 mg/kg/hr continuous L-citrulline infusion or placebo for up to 48 hours.

The study drug or placebo infusion will be discontinued once invasive arterial blood pressure monitoring is discontinued or at 48 hours, whichever comes first. Subjects will be followed until Day 28 or discharge from the hospital, whichever comes first. For subjects discharged prior to Day 28, a final assessment via telephone will be conducted at Day 28.

Arms: arm 1: * Bolus of 150 mg/kg at the initiation of cardiopulmonary bypass, but after removal of any crystalloid base;
* Addition of study medication at a concentration of 200 μmol/L given as a bolus during bypass. This may be administered as a one-time bolus or multiple administrations to compensate for fluids containing L-citrulline that may be removed from the patient during the course of the operation and thus to maintain the concentration of 200 μmol/L;
* Bolus of 20 mg/kg 30 minutes after decannulation from cardiopulmonary bypass;
* 9 mg/kg/hr continuous infusion for up to 48 hours. arm 2: * Bolus of 150 mg/kg at the initiation of cardiopulmonary bypass;
* Addition of placebo matched for volume given as a bolus during bypass. This may be administered as a one-time bolus or multiple administrations during bypass;
* Bolus of 20 mg/kg 30 minutes after decannulation from cardiopulmonary bypass;
* 9 mg/kg/hr continuous infusion for up to 48 hours.
Interventions: intervention 1: L-citrulline intervention 2: Placebo
Intervention descriptions: intervention 1: * Bolus of 150 mg/kg at the initiation of cardiopulmonary bypass, but after removal of any crystalloid base;
* Addition of study medication at a concentration of 200 μmol/L given as a bolus during bypass. This may be administered as a one-time bolus or multiple administrations to compensate for fluids containing L-citrulline that may be removed from the patient during the course of the operation and thus to maintain the concentration of 200 μmol/L;
* Bolus of 20 mg/kg 30 minutes after decannulation from cardiopulmonary bypass;
* 9 mg/kg/hr continuous infusion for up to 48 hours. intervention 2: * Bolus of 150 mg/kg at the initiation of cardiopulmonary bypass;
* Addition of placebo matched for volume given as a bolus during bypass. This may be administered as a one-time bolus or multiple administrations during bypass;
* Bolus of 20 mg/kg 30 minutes after decannulation from cardiopulmonary bypass;
* 9 mg/kg/hr continuous infusion for up to 48 hours.

Protocol documents:
Prot_000.pdf: 
 
 
A Phase III Double-Blind, Randomized, Placebo Controlled, Multi-
Center Clinical Study to Evaluate the Efficacy and Safety of 
Intravenous L-citrulline for the Prevention of Clinical Sequelae of 
Acute Lung Injury induced by Cardiopulmonary Bypass in Pediatric 
Subjects Undergoing Surgery for Congenital Heart Defects  
(Protocol CIT-003-01) 
 
FDA IND 66,261 
 
Protocol Number: 
 
CIT-003-01 
EudraCT Number:  
 
2016-002427-28 
Sponsor:  
 
Asklepion Pharmaceuticals, LLC  
729 E. Pratt Street; Suite 360  
Baltimore, MD 21202 
Previous Version Number: 
 4.2 
Previous Protocol Date: 
 August 31, 2016 
Revised Version Number:  
4.3.1 (Incorporates Version 4.3) 
Revised Protocol Date: 
August 11, 2017 
 
 
 
 
 
CONFIDENTIAL 
The information in this document is privileged and confidential and may not be disclosed unless 
such disclosure is required by federal or state law. The information in this document may be 
disclosed as necessary only to those persons involved in the conduct of this study. These 
restrictions apply as well to all future information supplied in regard to the study. The confidentiality 
of this information is further protected by the terms of the Confidentiality Agreement entered into 
by the Parties. 
 
 




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Study Personnel 
Sponsor Personnel 
Terrance C. Coyne, MD 
Chief Medical Officer 
Office:  410.545.0494, ext. 103 
Cell:  828-302-2678 
terrance.coyne@asklepionpharm.com 
 
 
Cindy Zychowicz, BA 
Director of Clinical Operations 
Office: 410.545.0494, ext. 104 
Cell: 443.467.5047 
cindy.zychowicz@asklepionpharm.com 
 
CRO Personnel 
ResearchPoint Global 
5301 Southwest Parkway 
Suite 100 
Austin, TX  78735 
Phone: 512-343-1092 
www.researchpointglobal.com 
 
Responsibilities:  
US: Clinical Site Monitoring/Management 
Global: Data Management 
 
 
FGK Clinical Research GmbH 
Heimeranstr. 35 
80339 Munich 
Germany 
Phone: + 49 (0) 89 893 119-0 
Fax: + 49 (0) 89 893 119-20  
www.fgk-cro.com 
 
Safety Reporting: 
Toll-free Phone: 1-888-270-3895 
Fax: + 49 (0) 89 893 119-180  
Email: safety@fgk-cro.com 
Responsibilities:  
Europe/Israel: Regulatory Activities, Clinical Site Monitoring/Site 
Management 
Global: Medical Monitoring/Safety Reporting; Biostatistics, Medical 
Writing  
 
 


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Table of Contents 
Protocol Approval ..................................................................................................................... 2 
Study Personnel ........................................................................................................................ 3 
Table of Contents ...................................................................................................................... 4 
List of Abbreviations and Definition of Terms ........................................................................ 8 
Protocol Synopsis ................................................................................................................... 12 
1 
Introduction ................................................................................................................ 24 
1.1 
Condition to be studied ............................................................................................. 24 
1.1.1 
Cardiopulmonary Bypass-Induced Injury ..................................................... 24 
1.1.2 
Pediatric CHD Patients ................................................................................ 26 
1.2 
L-citrulline .................................................................................................................. 28 
1.3 
Non-clinical data ........................................................................................................ 30 
1.4 
Clinical data ............................................................................................................... 31 
1.5 
Safety of L-citrulline .................................................................................................. 40 
1.6 
Rationale for the study .............................................................................................. 41 
2 
Study Objectives........................................................................................................ 42 
2.1 
Primary Objective ...................................................................................................... 42 
2.2 
Secondary Objective(s) ............................................................................................. 42 
3 
Study Endpoints ........................................................................................................ 43 
3.1 
Primary Endpoint ....................................................................................................... 43 
3.2 
Secondary Endpoints ................................................................................................ 43 
3.2.1 
Secondary Efficacy Endpoints ..................................................................... 43 
3.2.2 
Secondary Safety Endpoints ....................................................................... 44 
4 
Investigational Plan ................................................................................................... 44 
4.1 
Overall Study Design and Plan ................................................................................. 44 
4.2 
Discussion of Study Design...................................................................................... 44 


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4.3 
Selection of Study Population .................................................................................. 45 
4.3.1 
Number of Planned Subjects ....................................................................... 45 
4.3.2 
Inclusion Criteria.......................................................................................... 46 
4.3.3 
Exclusion Criteria ........................................................................................ 46 
4.3.4 
Removal of Subjects from Therapy or Assessments ................................... 47 
4.4 
Investigational Medicinal Products .......................................................................... 48 
4.4.1 
Investigational Medicinal Products Administered ......................................... 48 
4.4.2 
Identity of Investigational Medicinal Products .............................................. 49 
4.4.3 
Packaging and Labeling .............................................................................. 50 
4.4.4 
Method of Assigning Subjects to Treatment Groups .................................... 50 
4.4.5 
Selection of Doses in the Study ................................................................... 50 
4.4.6 
Drug Accountability ..................................................................................... 51 
4.4.7 
Blinding ....................................................................................................... 52 
4.4.8 
Prior and Concomitant Therapy ................................................................... 52 
4.4.9 
Treatment Compliance ................................................................................ 53 
5 
Study Procedures ...................................................................................................... 53 
5.1.1 
Pre-treatment .............................................................................................. 53 
5.1.1.1 
Screening Visit (Day -14 to Day 0) .............................................................. 53 
5.1.1.2 
Baseline Visit (Day of Surgery, Pre-operation) ............................................ 54 
5.1.2 
Treatment Period ......................................................................................... 55 
5.1.2.1 
Day 0, Surgery (Intra-operative) .................................................................. 55 
5.1.2.2 
Day 0, post-surgery, 0 hours (30 minutes post CPB decannulation) ............ 56 
5.1.2.3 
Day 0, post-surgery, 6 hours ....................................................................... 57 
5.1.2.4 
Day 0, post-surgery, 12 hours ..................................................................... 58 
5.1.2.5 
Day 1, post-surgery, 24 hours ..................................................................... 59 
5.1.2.6 
Day 2, post-surgery, 48 hours ..................................................................... 61 
5.1.2.7 
Follow-Up Visit [Day 28 (if still in hospital) or upon Hospital 
Discharge] ................................................................................................... 62 
5.1.2.8 
Follow-Up Phone Assessment (only for patients discharged prior to 
Day 28) ....................................................................................................... 62 
5.1.3 
Duration of Treatment ................................................................................. 63 


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6 
Variables Assessed ................................................................................................... 63 
6.1 
Screening, Baseline, and Surgery Assessments .................................................... 63 
6.1.1 
Demographics ............................................................................................. 63 
6.1.2 
Echocardiography ....................................................................................... 63 
6.1.3 
Physical Examination .................................................................................. 63 
6.1.4 
Cardiopulmonary Bypass Assessments ...................................................... 63 
6.2 
Efficacy and Safety Measurements Assessed ......................................................... 63 
6.2.1 
Efficacy Assessments ................................................................................. 63 
6.2.1.1 
Mechanical Ventilation................................................................................. 63 
6.2.1.2 
Inotropes ..................................................................................................... 64 
6.2.1.3 
Hemodynamic Parameters .......................................................................... 64 
6.2.1.4 
Plasma Citrulline Concentration .................................................................. 64 
6.2.1.5 
Thoracotomy Output/Chest Tube Drainage ................................................. 64 
6.2.1.6 
Vasodilators ................................................................................................ 65 
6.2.1.7 
Hospitalization ............................................................................................. 65 
6.2.2 
Safety Assessments .................................................................................... 65 
6.2.2.1 
Adverse Events ........................................................................................... 65 
6.2.2.2 
Clinical Laboratory Variables ....................................................................... 70 
6.3 
Appropriateness of Measurements .......................................................................... 71 
7 
Statistical Methods .................................................................................................... 71 
7.1 
Statistical and Analytical Plans ................................................................................ 71 
7.1.1 
Datasets Analyzed ...................................................................................... 72 
7.1.2 
Handling of Missing Data ............................................................................. 73 
7.1.3 
Demographic and Other Baseline Characteristics ....................................... 73 
7.1.4 
Efficacy Variables ........................................................................................ 73 
7.1.5 
Safety Variables .......................................................................................... 78 
7.2 
Initial Determination of Sample Size Estimate ......................................................... 78 
7.3 
Sample Size Estimation Revision ............................................................................. 79 
7.4 
Interim Analysis ......................................................................................................... 79 
8 
Quality Assurance and Quality Control ................................................................... 80 


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8.1 
Audit and Inspection ................................................................................................. 80 
8.2 
Monitoring .................................................................................................................. 80 
8.3 
Data Management and Coding .................................................................................. 81 
9 
Records and Supplies ............................................................................................... 83 
9.1 
Drug Accountability ................................................................................................... 83 
9.2 
Financing and Insurance ........................................................................................... 83 
10 
Data and Safety Monitoring Board ........................................................................... 83 
11 
Ethics .......................................................................................................................... 84 
11.1 Institutional Review Board / Independent Ethics Committee ................................. 84 
11.2 Ethical Conduct of the Study .................................................................................... 84 
11.3 Subject Information and Consent ............................................................................. 84 
11.4 Subject Confidentiality (US Studies) ........................................................................ 86 
12 
Reporting and Publication, Including Archiving ..................................................... 87 
13 
References ................................................................................................................. 88 
14 
Appendices ................................................................................................................ 90 
14.1 Investigator Signature Page...................................................................................... 91 
14.2 Changes to Protocol .................................................................................................. 92 
14.2.1 
Version 4.0 .................................................................................................. 92 
14.2.2 
Version 4.1 ................................................................................................ 101 
14.2.3 
Version 4.2 ................................................................................................ 102 
14.2.4 
Version 4.3 ................................................................................................ 111 
14.2.5 
Version 4.3.1 ............................................................................................. 124 
 
 


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List of Abbreviations and Definition of Terms 
% 
= 
per cent 
+/- 
= 
plus/minus 
ABG(s) 
= 
arterial blood gas(es)  
ACT 
= 
activated clotting time  
AE(s) 
= 
adverse event(s) 
ALI 
= 
acute lung injury 
ALT 
= 
alanine aminotransferase 
ANOVA 
= 
analysis of variance  
AP 
= 
alkaline phosphatase 
API 
= 
active pharmaceutical ingredient 
ASD 
= 
atrial septal defect  
ASL 
= 
argininosuccinate lyase 
ASS 
= 
argininosuccinate synthetase 
AST 
= 
aspartate aminotransferase 
AV 
= 
atrioventricular 
AVSD  
= 
atrioventricular septal defect  
BPAP  
= 
bilevel positive airway pressure 
BP 
= 
blood pressure 
BDRM 
= 
blind data review meeting 
BUN 
= 
blood urea nitrogen 
°C 
= 
degrees Celsius 
Ca 
= 
calcium 
CBC  
= 
complete blood count 
cc(s) 
= 
cubic centimeter(s) 
CFR 
= 
Code of Federal Regulations  
cGCP 
= 
current Good Clinical Practice 
cGMP 
= 
cyclic guanosine monophosphate 
Cl 
= 
chloride 
 


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CPAP 
= 
continuous positive airway pressure 
CPB 
= 
cardiopulmonary bypass 
CPMP 
= 
Committee for Proprietary Medicinal Products 
CPSI 
= 
carbamoyl phosphate synthetase I  
CRA(s) 
= 
Clinical Research Associate(s) 
CRF 
= 
case report form 
CRO 
= 
contract research organization       
CVP  
= 
central venous pressure  
DSMB 
= 
data safety monitoring board 
eCRF 
= 
electronic case report form 
EDC 
= 
electronic data capture  
EU 
= 
European Union 
°F 
= 
degrees Fahrenheit 
FAS 
= 
full analysis set 
FDA 
= 
Food and Drug Administration 
g/m2 
= 
grams per square meter 
HCO3 
= 
bicarbonate 
hr(s) 
= 
hour(s) 
ICF 
= 
informed consent form 
ICH 
= 
International Conference on Harmonisation 
ICU 
= 
intensive care unit 
IEC 
= 
Independent Ethics Committee 
IMP 
= 
investigational medicinal product 
iNO 
= 
inhaled nitric oxide 
IRB 
= 
Institutional Review Board 
ITT 
= 
intent-to-treat 
i.v.  
= 
intravenous 
IWRS 
= 
interactive web response system 
K 
= 
potassium 
 


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L- 
= 
levorotatory 
LOCF 
= 
last observation carried forward 
LDH 
= 
lactate dehydrogenase 
LFT(s) 
= 
liver function test(s) 
MAP 
= 
mean arterial (blood) pressure 
MedDRA 
= 
Medical Dictionary for Regulatory Activities 
mFAS 
= 
modified full analysis set 
Mg 
= 
magnesium 
mg/kg 
= 
milligram(s) per kilogram(s)  
mg/kg/hr 
= 
milligram(s) per kilogram(s) per hour 
mg/mL 
= 
milligram(s) per milliliter 
mL 
= 
milliliter(s) 
mm 
= 
millimeter(s) 
N 
= 
number of subjects in the dataset 
Na 
= 
sodium 
NO 
= 
nitric oxide 
NOS 
=  
nitric oxide synthase 
O2 
= 
oxygen 
OR 
= 
operating room  
OTC 
= 
ornithine transcarbamylase  
PA 
= 
pulmonary artery 
PaCO2 
= 
arterial carbon dioxide partial pressure 
PaO2 
= 
arterial oxygen pressure 
PAP 
= 
pulmonary artery pressure 
pH 
= 
hydrogen ion concentration 
PICU 
= 
pediatric intensive care unit 
PK 
= 
pharmacokinetics 
PP 
= 
per protocol analysis set 
PT  
=  
preferred term 
 


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PVT 
= 
pulmonary vascular tone 
QA 
= 
quality assurance 
Qhr 
= 
every hour 
RBC 
= 
red blood cell 
SAE 
= 
serious adverse event 
SAF 
= 
safety analysis set 
SaO2 
= 
arterial oxygen percent saturation 
SAP 
= 
statistical analysis plan 
SAS 
= 
Statistical Analysis System 
sem 
= 
standard error of the mean 
SOC  
= 
system organ class  
SOP(s) 
= 
standard operating procedure(s) 
SUSAR(s) = 
suspected unexpected serious adverse reaction(s) 
TPN 
= 
total parenteral nutrition 
µmol/L 
= 
micromole(s) per liter 
US 
= 
United States 
vs 
= 
versus 
VSD 
= 
ventricular septal defect  
WBC 
= 
white blood cell 
WHO 
= 
World Health Organization 
WMA 
= 
World Medical Association 
 
 
 


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Protocol Synopsis 
Protocol Number 
CIT-003-01 
Protocol Title: 
A Phase III Double-Blind, Randomized, Placebo Controlled, 
Multi-Center Clinical Study to Evaluate the Efficacy and Safety 
of Intravenous L-citrulline for the Prevention of Clinical Sequelae 
of Acute Lung Injury induced by Cardiopulmonary Bypass in 
Pediatric Subjects Undergoing Surgery for Congenital Heart 
Defects. 
Investigational 
Medicinal Product 
L-citrulline 
Number of Study 
Sites 
Approximately 28 investigative sites in the U.S., Europe and 
Israel 
Phase 
III 
Time Schedule 
Study Start: First subject signs informed consent 
Study End: Last subject last visit 
Coordinating 
Investigator: 
Frederick E. Barr, MD 
Objectives: 
Primary: 
The primary objective of the study will be to determine if 
intravenous (i.v.) L-citrulline delivery given perioperatively 
reduces clinical sequelae of acute cardiopulmonary bypass-
induced lung injury in pediatric subjects undergoing repair of 
congenital heart defects as evidenced by the reduction of post-
operative need for mechanical ventilation and inotrope therapy. 
Secondary:  
 
To evaluate the safety of L-citrulline compared with 
placebo 
 
To evaluate the effect of L-citrulline on post-operative 
hemodynamic improvement  
 
To evaluate the effect of L-citrulline on the usage and 
length of time on vasodilators 
 
To evaluate the effect of L-citrulline on length of 
hospitalization 
 
To evaluate the effect of L-citrulline on the duration of 


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chest tube placement  
 
To compare the plasma levels of L-citrulline between 
the two treatment groups 
Study Design: 
This is a randomized, double-blind, placebo controlled, multi-
center study that will compare the efficacy and safety of L-
citrulline versus placebo in subjects undergoing surgery for 
congenital heart defects.  
Eligible subjects undergoing repair of a large unrestrictive 
ventricular septal defect (VSD), a partial or complete 
atrioventricular septal defect (AVSD), or an ostium primum atrial 
septal defect (primum ASD) will be eligible for enrollment in this 
study.  
Each enrolled subject will be randomized to receive either L-
citrulline or placebo throughout all administrations in the study. 
Subjects will receive an L-citrulline bolus of 150 mg/kg or 
placebo at the initiation of cardiopulmonary bypass, the addition 
of L-citrulline at a concentration of 200 μmol/L or placebo given 
as a bolus during cardiopulmonary bypass, an L-citrulline bolus 
of 20 mg/kg or placebo 30 minutes after decannulation from 
cardiopulmonary bypass, followed immediately by a 9 mg/kg/hr 
continuous L-citrulline infusion or placebo for up to 48 hours.    
The study drug or placebo infusion will be discontinued once 
invasive arterial blood pressure monitoring is discontinued or at 
48 hours, whichever comes first. Subjects will be followed until 
Day 28 or discharge from the hospital, whichever comes first. 
For subjects discharged prior to Day 28, a final assessment via 
telephone will be conducted at Day 28.  
Number of 
Subjects 
Approximately 450 subjects will be screened, with 190 
randomized to either L-citrulline or placebo in a 1:1 ratio. 
Study Treatments: 
Eligible subjects will be randomized in a 1:1 fashion to either L-
citrulline or placebo and receive the following: 
 
Bolus of 150 mg/kg at the initiation of cardiopulmonary 
bypass, but after removal of any crystalloid base; 
• 
Addition of study medication at a concentration of 200 
µmol/L given as a bolus during bypass.  This may be 
administered as a contemporaneous one-time bolus or 
multiple administrations to compensate for fluids 
containing L-citrulline that may be removed from the 
patient during the course of the operation and thus to 


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maintain the concentration of 200 µmol/L;  
 
Bolus of 20 mg/kg 30 minutes after decannulation from 
cardiopulmonary bypass; 
 
9 mg/kg/hr continuous infusion for up to 48 hours.   
Study Duration 
The study will consist of an initial screening phase, followed by 
enrollment of eligible subjects to the treatment phase on Day 0 
(Day of Surgery) and will end once study medication is 
discontinued: at 48 hours or upon removal of the arterial line. 
Subjects will be followed until Day 28 or discharge from the 
hospital, whichever comes first. For subjects discharged prior to 
Day 28, a final assessment via telephone will be conducted at 
Day 28. 
The study is planned to be conducted over an 11-month 
enrollment period. 
Subject Selection: 
Inclusion Criteria: 
• 
Subjects or parents or legal guardian of the subject who 
are willing and able to sign consent 
• 
Male and female subjects aged ≤18 years of age 
• 
Infants, children and adolescents undergoing 
cardiopulmonary bypass for repair of a large 
unrestrictive VSD, an ostium primum ASD or a partial 
or complete AVSD 
• 
Pre-operative echocardiogram which confirms the 
cardiovascular anatomy and defect to be surgically 
repaired 
Exclusion Criteria: 
• 
Evidence of pulmonary artery or vein abnormalities on 
the pre-operative echocardiogram that will not be 
addressed surgically. Specific abnormalities excluded 
include the following: 
 
Significant pulmonary artery narrowing not 
amenable to surgical correction 
 
Previous pulmonary artery stent placement 
 
Significant left-sided atrioventricular (AV) valve 
regurgitation not amenable to surgical correction 
 
Pulmonary venous return abnormalities not 
amenable to surgical correction 
 
Pulmonary vein stenosis not amenable to 
surgical correction 
• 
Preoperative requirement for mechanical ventilation or 


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intravenous inotrope support 
• 
Presence of fixed or idiopathic pulmonary hypertension 
(i.e. Eisenmenger’s Syndrome) prior to surgical repair 
• 
Pre-operative use of medications to treat pulmonary 
hypertension 
• 
Pregnancy; Sexually active females of child-bearing 
potential must be willing to practice an acceptable 
method of birth control for the duration of study 
participation (e.g. oral contraceptive, hormonal implant, 
intra-uterine device) 
• 
Participation in another clinical trial within 30 days of 
Screening or while participating in the current study, 
including the 28 days of follow-up post study drug 
administration.  
• 
Any condition which, in the opinion of the investigator, 
might interfere with the study objectives 
Endpoints: 
Primary Efficacy Endpoint: 
A composite variable consisting of the longer of either (1) length 
of time on mechanical ventilation or (2) length of inotrope use. 
The definition of mechanical ventilation shall include invasive 
and non-invasive mechanical ventilation including bilevel 
positive airway pressure (BPAP) or continuous positive airway 
pressure (CPAP). 
Secondary Efficacy Endpoints:  
 
Length of time on mechanical ventilation 
 
Length of time on positive pressure ventilation 
 
Length of time of inotrope use 
 
Inotrope score calculated each hour post-operatively 
from the time of separation from bypass until the 
completion of study drug.  Additionally, the total 
inotrope score over time until Day 28 or hospital 
discharge will be derived. 
 
Hemodynamic improvement (heart rate, systemic 
arterial blood pressure, oxygen saturation, and central 
venous pressure)  
 
Length of time of intubation 
 
Length of PICU stay 


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 
Length of time on vasodilators 
 
Length of hospitalization 
 
Thoracotomy output; duration and volume of chest tube 
drainage 
 
Plasma concentrations of L-citrulline 
Safety 
Assessments 
 
Adverse events  
 
Incidence of refractory hypotension 
 
Laboratory values 
Statistical Methods: Continuous variables will be summarized with means, standard 
deviations, medians, lower and upper quartiles, minimums and 
maximums. Frequencies and percentages will be used to 
summarize categorical variables.  
For time-to-event analyses censoring information will be used. 
No other imputation of missing values (e.g., no last observation 
carried forward (LOCF)) will be used, unless stated otherwise.  
Analysis sets: 
Safety Analysis Set (SAF): 
All randomized subjects who received surgery and study 
medication (independent of whether it is L-citrulline or placebo) 
will be valid for the SAF. The SAF will be used for the evaluation 
of the safety assessments. 
Full Analysis Set (FAS): 
The FAS includes all subjects who received surgery. The FAS 
serves as the primary efficacy analysis set. 
Modified Full Analysis Set (mFAS): 
The mFAS includes all subjects who received surgery and study 
medication (independent of whether it is L-citrulline or placebo). 
The mFAS serves as a sensitivity population for the efficacy 
analyses. 
Per-protocol Analysis Set (PP): 
The PP includes all subjects included in the SAF who had no 
significant protocol deviations and completed through Day 28 or 
until discharge from the hospital (end of study follow-up). The 
PP will only be analyzed for main efficacy outcome measures. 
 


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ANALYSIS OF PRIMARY EFFICACY ENDPOINT: 
Hypothesis to be tested: 
H0: Composite endpoint within the L-citrulline group is equal to 
that of the placebo group  
H1: Composite endpoint within the L-citrulline group is unequal 
to that of the placebo group 
H0 will be tested using a Wilcoxon-rank-sum test. The level of 
significance is 1% (two-sided).  
95% confidence intervals using Hodges-Lehman method will be 
additionally calculated. Survival analyses using Kaplan-Meier 
estimates and plots, log-rank test and Cox-regression modeling 
will serve as sensitivity analyses. 
Additional sensitivity analyses will be performed. 
ANALYSIS OF SECONDARY EFFICACY ENDPOINTS: 
No formal statistical hypotheses are specified for the secondary 
endpoint variables. Nonetheless statistical testing methods will 
be applied to compare treatment groups. A significance level of 
5% (two-sided) will be used for such analyses. Secondary 
efficacy endpoints will be analyzed using the FAS, mFAS and 
the PP.  
Length of time on mechanical ventilation and length of time on 
positive pressure ventilation: 
The same analyses as described for the primary endpoint will 
be applied. 
Length of Time of Inotrope Use: 
The same analyses as described for the primary endpoint will 
be applied. 
Inotrope Score: 
The total inotrope score at each hour post-operatively from the 
time of separation from bypass, until the completion of study 
drug will be calculated. A Wilcoxon-rank-sum test, an ANOVA 
and a repeated measures analysis of variance will be applied.  
Additionally, the total inotrope score over time until Day 28 or 
hospital discharge will be derived. 
Hemodynamic Improvement: 
The absolute changes in hemodynamic parameters from 
baseline at hours 1, 2, 4, 12, 24, and 48 will be compared 


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between groups using an analysis of variance (ANOVA) with a 
fixed effect for treatment group and baseline level.  
Length of Time of Intubation: 
For the length of time of intubation, the same analyses as 
described for the primary endpoint will be applied 
Length of PICU stay: 
For the length of PICU stay, the same analyses as described for 
the primary endpoint will be applied. 
Length of Time on Vasodilators: 
For the length of time on vasodilators the same analyses as 
described for the primary endpoint will be applied.  
Length of Hospitalization: 
For the length of hospitalization, the same analyses as 
described for the primary endpoint will be applied. 
Thoracotomy Output: Duration and Volume of Chest Tube 
Drainage: 
For the total postoperative duration of chest tube drainage, the 
same analyses as described for the primary endpoint will be 
applied.  
The total amount of chest tube drainage in milliliters (mL), added 
up during each duration of chest tube placement, will be 
summarized using descriptive measures and compared 
between groups using an ANOVA with a fixed effect for 
treatment group. 
L-citrulline Plasma Levels: 
L-citrulline blood levels will be obtained on each patient at set 
times, and plotted and examined to confirm that they are above 
the threshold level of 100 µmol/L. 
ANALYSIS OF SAFETY ENDPOINTS: 
Safety endpoints will be analyzed using the SAF. 
Adverse Events: 
Adverse events (AEs) will be summarized. No statistical testing 
procedures will be applied to the analysis of AEs. 
Incidence of Refractory Hypotension: 
The number of subjects with any refractory hypotension from 


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end of surgery until 48 hours will be compared between groups 
using Fishers’ exact test. Additionally, a logistic regression 
analysis will be applied including factor treatment and site. 
Laboratory Values:  
The absolute values and the absolute and percentage changes 
from baseline will be tabulated using summary tables describing 
descriptive measurements for all observed time points. No 
statistical testing procedures will be applied to the analysis of 
laboratory values. 
Sample Size 
Estimation 
A sample size of 92 patients in each group will have 90% power 
to detect a difference between L-citrulline and placebo on the 
primary endpoint, using a Wilcoxon-rank-sum test with a 0.01 
two-sided significance level.  
To allow for subjects being enrolled but not assigned to the full 
analysis set, an overall N=95 subjects per group will be enrolled. 
Interim Analysis: 
One interim analysis is planned to be carried out when the first 
95 consecutively randomized full analysis set (FAS)-patients 
(i.e. 50% of the planned patients) will have undertaken their 
Follow up visit (Day 28 or Hospital Discharge). 
 
 


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Schedule of Study Assessments and Procedures 
 
Pre-Treatment 
Treatment 
Follow-Up 
 
Screen 
 
Baseline 
Day of 
Surgery 
Surgical 
Observation 
 
Post-Operative 
Surgical Observation 
Day 28 or 
Hospital 
Discharge 
Day 28 
Phone 
Assessment 
 
Days 
-14 to 0 
Day 0 
Pre-Op 
Day 0 
Intra-Op 
 
Day 0- 0 hrs 
(30 min post 
decannulation) 
Day 0 
6 hrs 
Day 0 
12 hrs 
Day 1 
24 hrs 
 
Day 2 
48 hrs 
 
 
 
Informed Consent/Assent 
x 
 
 
 
 
 
 
 
 
 
Eligibility Assessment 
x 
 
 
 
 
 
 
 
 
 
Demographic Data 
x 
 
 
 
 
 
 
 
 
 
Medical History 
x 
 
 
 
 
 
 
 
 
 
Physical Examination (Pre-op) 
x 
 
 
 
 
 
 
 
 
 
Body Weight and Height  
x 
 x3 
 
 
 
 
 
 
 
 
Inotrope & other concomitant 
medications, incl. vasodilators 
x 
x 
x 
x 
 
Blood draw for serum 
electrolytes, BUN, and creatinine  
 x1 
 
x5 
 
 
 
x15 
x15 
x16 
 
CBC with differential 
 x1 
 
x5 
 
 
 
x15 
x15 
x16 
 
Blood Sampling for Coagulation: 
ACT  
 x1 
 
x5 
 
 
 
x15 
x15 
 
 
LFTs 
 x1 
 
 
 
 
 
 
x15 
x16 
 
Urinalysis  
x2 
 
 
 
 
 
x2 
 
 
 
Urine pregnancy test for females 
of child-bearing potential 
 
x 
 
 
 
 
 
 
 
 
Randomization 
 
x 
 
 
 
 
 
 
 
 


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Pre-Treatment 
Treatment 
Follow-Up 
 
Screen 
 
Baseline 
Day of 
Surgery 
Surgical 
Observation 
 
Post-Operative 
Surgical Observation 
Day 28 or 
Hospital 
Discharge 
Day 28 
Phone 
Assessment 
 
Days 
-14 to 0 
Day 0 
Pre-Op 
Day 0 
Intra-Op 
 
Day 0- 0 hrs 
(30 min post 
decannulation) 
Day 0 
6 hrs 
Day 0 
12 hrs 
Day 1 
24 hrs 
 
Day 2 
48 hrs 
 
 
 
Pharmacy Prep of Study Drug 
 
x 
 
 
 
 
 
 
 
 
Plasma L-citrulline Collection 
 
x4 
x5 
x10; x12 
x14 
x14 
x15 
x15 
x16 
 
Adverse Events 
 
x 
        x 
x 
x 
Cardiopulmonary Bypass Data 
 
 
x6 
 
 
 
 
 
 
 
Hemodynamic Monitoring  
 
 
x7 
x 
 
 
ABGs 
 
 
x8 
x 
 
 
Thoracotomy Output 
 
 
 
x 
 
Administration of Study Drug 
 
 
x9 
x11 
 
 
Hypotension Assessment 
 
 
 
x 
 
 
Ventilator Settings                           
 
 
x13 
 
Phone Assessment 
 
 
 
 
 
 
 
 
 
x17 
1Results of laboratory tests done as standard of care for pre-op testing are acceptable and may be recorded as screening values, 
provided the tests were performed within 14 days of Day 0.  If any of the required tests were not done as part of the pre-op 
testing, these must be completed within 14 days of Day 0.  
2Urinalysis (not required, but results should be recorded if test is performed). 
3 Weight only 
4 Collect 1.0 mL of blood for plasma L-citrulline levels (prior to surgery).  All L-citrulline plasma samples must be processed 
within four hours of being drawn, stored at -20°F (-28.8°C) or below, and sent on dry ice to the central lab. 


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5 Blood collected for intra-op laboratory values and plasma L-citrulline levels (10 minutes after study drug administered); (1.0 
mL for plasma L-citrulline).  All L-citrulline plasma samples must be processed within four hours of being drawn, stored at -20°F 
(-28.8°C) or below, and sent on dry ice to the central lab. 
6 Cardiopulmonary bypass data include: Equipment type, cardioplegia solution type, composition of isovolumetric exchange fluid 
for ultrafiltration and/or modified ultrafiltration, exchange volumes in and out, documentation that L-citrulline or placebo was 
added to the filtration and hemoconcentration fluids, cardiopulmonary bypass (CPB) start and end times and decannulation 
time. 
7 Hemodynamic monitoring includes heart rate, systemic BP, O2 saturation, CVP, and PAP measurements (if data is available). 
Baseline values are first recorded in the operating room (OR) by Anesthesia.  Results will be monitored during surgery 
according to the Institution’s standard of care, but results will only be recorded approximately every 15 minutes. Standard of 
care will continue to be followed from time of post-op admission to the PICU through 48 hours.  Results at 1, 2, 4, 12, 24, and 
48 hours from baseline (in OR) must be obtained and recorded.  
8 Arterial Blood Gas (ABG) includes PaO2, PaCO2, HCO3, and pH.   ABGs will be drawn according to the Institution’s standard 
of care.  Baseline results are first recorded in the OR by Anesthesia.  All results obtained during surgery, and from the time of 
post-op admission to the PICU until removal of the arterial line or through 48 hours (whichever occurs first), will be recorded. 
9 Begin bolus of intravenous (i.v.) L-citrulline at 150 mg/kg or placebo at initiation of CPB & add L-citrulline at a concentration of 
200 μmol/L or placebo given as a bolus during bypass.  The 200 μmol/L dose may be administered as a one-time bolus or 
multiple administrations, to maintain the concentration at 200 µmol/L. 
10 Collect 1.0 mL of blood for plasma L-citrulline levels (30 minutes after decannulation from CPB in OR; prior to bolus). All 
L-citrulline plasma samples must be processed within four hours of being drawn, stored at -20°F (-28.8°C) or below, and sent 
on dry ice to the central lab. 
11 Bolus of 20 mg/kg of L-citrulline or placebo 30 minutes after decannulation from CPB immediately followed by a continuous 
infusion of L-citrulline at 9 mg/kg/hr or placebo  
12 Collect 1.0 mL of blood for plasma L-citrulline levels (5 minutes after bolus). All L-citrulline plasma samples must be 


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processed within four hours of being drawn, stored at -20°F (-28.8°C) or below, and sent on dry ice to the central lab. 
13 Recorded approximately every hour (Qhr) (+/- 10 minutes) from time of separation from CPB until discontinuation of 
mechanical ventilation 
14 Collect 1.0 mL of blood for plasma L-citrulline levels (at 6 hours and 12 hours post-operatively; +/- 1 hr.  If continuous i.v. 
L-citrulline infusion discontinued before 6 or 12 hour timepoint, collect blood within +/- 1 hr of end of infusion.). All 
samples must be processed within four hours of being drawn, stored at -20°F (-28.8°C) or below, and sent on dry ice to the 
central lab.  
15 Blood collected for post-op laboratory values and plasma L-citrulline levels (24 hours and 48 hours post-operatively; +/- 1 
hr.  If continuous i.v. L-citrulline infusion discontinued before 24 or 48 hour timepoint, collect blood within +/- 1 hr of 
end of infusion.); (1.0 mL for plasma L-citrulline).  All samples must be processed within four hours of being drawn, stored at -
20°F (28.8°C) or below, and sent on dry ice to the central lab.  
16 An attempt should be made to obtain final labs on Day 28 or prior to discharge from the hospital. If lab results within 24 hours 
are available, these may be captured. If results are not available within 24 hours, then record the results of the labs obtained 
closest to discharge from the hospital.  These labs must include LFTs.  If no i.v. access is available on Day 28 or the day of 
discharge, venipuncture is not required since results of the labs obtained closest to discharge may be used. 
17 A phone assessment will be performed on Day 28 only for those patients discharged prior to Day 28 


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1 Introduction 
1.1 
Condition to be studied 
Congenital heart disease is the most common of the major congenital anomalies, and 
represents a significant global health problem. Twenty-eight percent of all major congenital 
anomalies consist of heart defects. In a systematic review of the 8 most common congenital 
heart defect subtypes performed by van der Linde et al. (van der Linde, Konings et al. 2011) 
the authors determined ventricular septal defect (VSD) to be the most common heart defect 
(34% of all cases examined), followed by atrial septal defect (ASD) (13%). Prevalence was 
comparable for Europe and North America, with approximately 2.7 and 2.4 children, 
respectively, per 1000 live births presenting with a VSD and approximately 1.6 and 1.7 
children, respectively, presenting with an ASD.  
L-citrulline is being developed for prevention of clinical sequelae of acute lung injury induced 
by cardiopulmonary bypass (CPB) in pediatric patients undergoing surgery for congenital 
heart defects (CHD). It is well recognized that cardiopulmonary bypass causes a systemic 
inflammatory response characterized clinically by acute compromise of cardiovascular and 
pulmonary function (Apostolakis et al. 2010; Huffmyer and Groves 2015).  However, for a 
number of medical and physiological reasons, pediatric patients subjected to CPB during 
surgical repair of congenital heart defects are more susceptible to this cascade and at greater 
medical risk therefrom than adult patients (Kozik and Tweddell 2006; Shure 2010; 
Shekerdemian 2009).  Reduction of key manifestations of acute CPB-induced lung injury, 
namely the post-operative need for mechanical ventilation and for inotrope therapy, will 
provide evidence of the intended clinical effect. 
1.1.1 Cardiopulmonary Bypass-Induced Injury 
A number of factors place the lung at risk for injury during CPB. Chief among these is surface 
activation of neutrophils and other leukocytes, complement, and cytokines (pro- and anti-
inflammatory) inter alia, and an associated systemic inflammatory cascade (Apostolakis et al. 
2010; Shure 2010). The degree to which the lung is damaged by the inflammatory response 
mediated by contact activation of leukocytes during extracorporeal circulation can vary in 
severity from microscopic changes of no clinical consequence to a capillary leak syndrome, 
or, in the worst case, to acute respiratory failure. 
Pulmonary injury manifests in several ways and may involve both parenchymal and vascular 
lung tissues. Parenchymal effects of CPB are reflected in alterations in pulmonary compliance, 
most commonly related to an increase in lung water. The impact of this on the patient is a 


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requirement for increased ventilatory support and a diminished ability of the lungs to perform 
their function in gas exchange. Vascular effects are manifested by changes in pulmonary 
vascular resistance, which in turn affect the function of the right ventricle. This condition 
constitutes, in effect, pulmonary arterial hypertension. The lungs are in a unique position in 
the circulation and may thus be vulnerable to different mechanisms of injury. Circulating 
leukocytes that elaborate inflammatory mediators following contact with surfaces in CPB 
apparatus or by direct damage by CPB equipment account for only part of the inflammatory 
damage that may occur in the lung (Clark 2006). The lung is also an important source of 
inflammatory cells, as well as being a target for damage by those same cells. The 
consequences of the mechanical and inflammatory effects on the lung are decreased 
functional residual capacity, diminished compliance, and impaired gas exchange. These 
changes are ultimately associated with increased pulmonary vascular resistance and 
pulmonary artery pressure. 
 
Inflammation and mechanical factors are not the only factors causing impaired pulmonary 
function related to CPB. When patients are placed on bypass, the lungs undergo a sudden 
and significant decrease in perfusion via the pulmonary artery. During total bypass, the lungs 
receive only nutrient flow from the bronchial arterial circulation. This ischemic effect of CPB is 
added to its inflammatory effect to produce clinical pulmonary dysfunction. It seems that low-
flow CPB produces worse pulmonary injury than does circulatory arrest, which suggests that 
the interaction between the inflammatory and ischemic components is complex. Both the 
inflammatory 
and 
ischemic 
factors 
damage 
the 
pulmonary 
endothelium 
(http://tele.med.ru/book/cardiac_anesthesia/es_home.htm 2001). 
 
Acute CPB-induced lung injury leads to significant cardiopulmonary problems. The 
inflammatory response leads to constriction of the pulmonary and systemic vasculature. The 
constriction leads to increased right ventricular and left ventricular workload. The inflammatory 
response also leads to pulmonary edema and deterioration in lung compliance and 
postoperative lung function. The standard treatments for these postoperative complications 
include mechanical ventilation until the lung function returns to normal and inotropic support 
until pulmonary and systemic vascular tone returns to normal, eventually decreasing the right 
and left ventricular workload. Mechanical ventilation and inotropic support are therapies that 
can thus serve as effective biomarkers of acute CPB-induced lung injury. Additionally, 
prolonged mechanical ventilation can in turn often lead to other morbidities including ventilator 
associated lung injury, ventilator associated pneumonia (VAP), central line associated blood 
stream infections (CLABSI), and even more prolonged intensive care unit stays. Prevention 
of acute CPB-associated lung injury and its sequelae is therefore a desirable therapeutic goal.  
 
 


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1.1.2 Pediatric CHD Patients 
Children undergoing surgery for congenital heart defects are especially susceptible to 
developing CPB-induced acute lung injury due to age dependent differences in the 
inflammatory response, and the elevated sensitivity of their immature organ systems to injury 
as well as distinct differences between pediatric and adult CPB (Kozik and Twedell 2006).  
Neonates and infants are especially affected as the relatively large extracorporeal circuit size, 
the blood prime and the need for increased flow rates result in greater exposure of blood to 
the foreign surface (Schure AY et al. 2010). 
For congenital cardiac surgery, the extracorporeal circuit must be adjusted to a wide range of 
age groups and size variations, from 1.5kg premature infants to >100kg adolescents or adults. 
Infants and children have smaller circulating blood volumes, higher oxygen consumption rates 
and, often, highly reactive pulmonary vascular beds. In addition, neonates and infants have 
labile thermoregulation and immature organ systems with multiple implications for ischemic 
tolerance and inflammatory response. Many complex repairs require a bloodless operative 
field, which can be difficult to achieve in the presence of intra or extra cardiac shunts, 
aortopulmonary collaterals, or otherwise increased pulmonary venous return (Schure AY et 
al. 2010). 
With the possible exception of secundum ASD cases, in which recovery is relatively and 
consistently robust, there is so far, no known biomarker or patient characteristic among 
pediatric CHD patients linked with the probability of clinically significant CPB-induced ALI.   It 
appears to occur in approximately 1/3 of pediatric CHD surgeries (Russell et al. 1998). 
The functional and structural status of the pulmonary vascular bed plays a pivotal role in the 
presentation and outcome of children with congenital cardiovascular disease. However, it is 
in the immediate postoperative period that these pediatric patients are most vulnerable to 
CPB-induced acute lung injury. CPB-induced acute lung injury represents a complex interplay 
between the preoperative condition of the patient (importantly age at repair, type of lesion and 
presence of a syndrome) and the inevitable disruption in the endocrine and vasoactive peptide 
milieu that results from cardiac surgery. Important factors leading to enhanced 
vasoconstriction are cardiopulmonary bypass, hypothermia and circulatory arrest with some 
degree of associated ischemia. Residual cardiac lesions and the sequelae of the stress 
response, hypoxia, metabolic and respiratory acidosis may all contribute additional 
imbalances that favor pulmonary vasoconstriction. Many of the manifestations of acute lung 
injury associated with CPB can be explained wholly or in part by endothelial dysfunction, which 
provides at once a possible unifying hypothesis as well as a potential therapeutic target. Acute 
lung injury associated with CPB may also lead to important adverse cardiac sequelae.  The 


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inflammatory response after surgery for CHD is commonly associated with abnormal 
ventricular– vascular interaction, with systemic vasoconstriction and elevated afterload, as 
well as with myocardial injury with impaired systolic and diastolic function. In a proportion of 
patients, these haemodynamic manifestations can lead to the serious consequence of low 
cardiac output (Shekerdemian 2009). 
The serious pulmonary and cardiac sequelae of acute lung injury are clinically important to 
outcome.  These sequelae are risk factors for prolonged intensive care stay, and death. 
Lability of pulmonary vascular tone is common in neonates and infants after surgery for CHD. 
This can be most problematic after biventricular repairs in patients who had preoperative 
unrestricted pulmonary flow (large septal defects, common arterial trunk) or pulmonary venous 
hypertension (obstructed anomalous pulmonary venous drainage). Instability of the pulmonary 
vascular resistance is also common after palliative surgery in patients with a functionally 
univentricular circulation, including Norwood-type operations, a systemic-to-pulmonary artery 
shunt, or a pulmonary artery band. While many therapeutic interventions optimize systemic 
oxygen delivery through their direct influences on the myocardium and systemic vasculature, 
manipulation of the pulmonary vascular tone can play an important role in optimizing the 
circulation of children undergoing surgery for heart disease (Shekerdemian 2009). 
 
CPB-induced acute lung injury and in its most severe form, a pulmonary hypertensive crisis, 
often requires an aggressive combination of therapies for right ventricular failure and careful 
management of inotropes and vasopressors (e.g., milrinone, epinephrine). Furthermore, fluids 
need to be prudently balanced, and sinus rhythm and atrioventricular synchrony need to be 
supervised and maintained. The challenge is to find the optimal preload to avoid the 
detrimental effects of ventricular interdependence. The general treatment of acidosis and 
hypoxia includes oxygenation, normocapnia, analgesia, sedation, and muscle relaxation, all 
of which require aggressive utilization of mechanical ventilation. If a postoperative pulmonary 
hypertensive crisis is not improved by general therapies mentioned above, inhaled nitric oxide 
(iNO) is often administered. Finally, because either mechanical ventilation or inotrope usage 
or both precludes discharge from intensive care, CPB induced lung injury can result in 
prolonged utilization of intensive care services.  
In summary, the consequences of CPB-induced lung injury lead to: 
1. Extended requirement for mechanical ventilation 
2. Extended requirement for inotrope therapy 
3. Extended requirement for intensive care 
 
 


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1.2 
L-citrulline  
Citrulline is a naturally occurring amino acid and the first intermediate in the urea cycle. Most 
medical personnel think of the urea cycle as a hepatic metabolic pathway that serves to 
convert nitrogen waste from protein breakdown into urea for excretion.  It is not generally 
appreciated that key elements of the urea cycle exist in other tissues where, for example, in 
lung, it is ultimately responsible for the production of NO under precise metabolic control, as 
shown in Figure 1.  The shift in function of the urea cycle comes about because the enzymes 
catalyzing the first two steps of the hepatic urea cycle (carbamoyl phosphate synthetase I 
(CPSI) and ornithine transcarbamylase (OTC), which lead to the production of citrulline, are 
expressed only in the liver and gut. In the liver and gut, newly synthesized citrulline is 
transported from the mitochondria to the cytoplasm. In contrast, other tissues such as lung 
are dependent upon exogenous circulating citrulline since they cannot synthesize it 
endogenously.  Following either endogenous synthesis or transport into a non-hepatic cell, 
argininosuccinate 
synthetase 
(ASS) 
combines 
citrulline 
with 
aspartate 
to 
form 
argininosuccinate. Subsequently, argininosuccinate lyase (ASL) cleaves fumarate off from 
argininosuccinate to form arginine. Arginine is the precursor for NO. Nitric oxide is synthesized 
by several different isoforms of the enzyme NO synthase (NOS), including constitutive and 
inducible forms. Unlike CPSI and OTC, the remaining enzymes of the pathway, including ASS, 
ASL and NOS, are distributed throughout the body, including the pulmonary vascular 
endothelium (Summar 1998). 
Endogenous NO plays a critical role in the regulation of pulmonary vascular tone. It is a potent 
vasodilator and is produced by vascular endothelial cells in response to many different stimuli. 
Nitric oxide diffuses from the vascular endothelial cell to the vascular smooth muscle cell 
where it activates guanylate cyclase, leading to increased intracellular levels of cyclic 
guanosine monophosphate (cGMP). Increased cGMP, in turn, leads to relaxation of the 
vascular smooth muscle cells and increased blood flow. The extremely short half-life of NO 
allows for very tight vasoregulation. The pathophysiology of increased postoperative 
pulmonary artery pressure is thought to involve CPB induced pulmonary vascular endothelial 
cell dysfunction. 
 
 
 


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Figure 1: Schematic description of the urea cycle in lung 
 
 
 
Source:  Personal communication from Marshall Summar, M.D., Ph.D., Children's National 
Medical Center Wash DC). 
Citrulline is thus a precursor to arginine and NO. New substrate supply for NO generation 
comes entirely from the production of citrulline as part of the normal urea cycle function. Once 
citrulline is generated by the hepatic urea cycle, it crosses mitochondrial and cellular 
membranes easily and therefore is also transported to other organs in the body. In the 
pulmonary vascular endothelium, citrulline can then be converted into arginine and 
subsequently into NO. Because the majority of circulating arginine is from urea cycle synthesis 
and not dietary sources, citrulline availability is critical to maintaining adequate arginine supply 
for NO production. 
9
HCO3 + NH4 + 2 ATP
Carbamyl 
Phosphate
CPSI
Citrulline
Ornithine
N-acetyl glutamate
MITOCHONDRIA
Citrulline
CYTOPLASM
Aspartate
Argininosuccinate
ASS
CITRIN
Arginine
Ornithine
ORNT1
Nitric Oxide
Fumarate
ASL
Urea
ARG I
Lung
ARG 2
eNOS
nNOS
iNOS
OTC


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It has been demonstrated in multiple observational and clinical studies that plasma levels of 
citrulline and arginine drop precipitously and do not recover for up to 48 hours after CPB for 
congenital cardiac surgery.  
A study conducted by Barr et al. (Barr, Beverly et al. 2003) revealed that CPB significantly 
decreases several urea cycle intermediates and NO metabolites after repair of unrestrictive 
VSD and atrioventricular septal defect (AVSD). Both citrulline and arginine levels were 
decreased in the postoperative period. Unlike citrulline, arginine was not decreased 
immediately after surgery, but was significantly decreased at 12, 24, and 48 hours after 
surgery. The slight lag in the drop in arginine levels compared with citrulline levels may reflect 
continued synthesis of arginine from precursors available before surgery. Citrulline and 
arginine levels continued to decline at 48 hours after surgery. Also, levels of NO metabolites 
decreased throughout the postoperative period. Patients undergoing these specific cardiac 
procedures are therefore at risk for elevated postoperative pulmonary vascular resistance, 
especially if they are physiologically stressed with hypoxia. An increase in the availability of 
NO and its precursors could decrease the risk for this postoperative complication. 
In a study of 10 infants undergoing CPB for repair of congenital defects, supplementation of 
the NO precursor L-arginine was shown to partially ameliorate pulmonary endothelial 
dysfunction (Schulze-Neick, Penny et al. 1999). However, intravenous (i.v.) L-arginine has 
been shown to reduce both pulmonary and systemic pressures. This global response would 
not be tolerated in patients after cardiac surgery, who are already prone to low cardiac output 
states. The maintenance of high plasma arginine concentrations is also problematic because 
of poor bioavailability and swift metabolism by intestinal and cytosolic arginase. In contrast 
administration of L-citrulline is more effective in maintaining plasma L-arginine concentrations 
than administration of arginine in healthy volunteers (Haeberle, McCandless et al. 2014). L-
citrulline has no recognized toxicity and is used as replacement therapy for children with urea 
cycle defects. 
In summary, due to its intracellular and intercellular transport mechanisms, citrulline is the 
ultimate substrate for endogenous production of NO. Because the majority of circulating 
arginine is from urea cycle synthesis and not dietary sources, citrulline availability is critical to 
maintaining adequate arginine supply for NO production. L-citrulline supplementation is 
therefore a useful method of increasing arginine and NO synthesis and maintaining plasma 
arginine, citrulline, and NO metabolite levels in the postoperative period (Barr, Beverly et al. 
2003; Smith, Canter et al. 2006; Barr, Tirona et al. 2007).  
1.3 
Non-clinical data                                                                                       
(For further information please see the current version of the Investigator Brochure) 


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A study performed in pulmonary hypertension in newborn piglets showed that pulmonary 
arterial pressure and pulmonary vascular resistance were significantly lower in hypoxic 
animals treated with L-citrulline compared with untreated hypoxic animals (p <0.001). The 
authors suggested that L-citrulline may benefit neonates exposed to prolonged periods of 
hypoxia from cardiac or pulmonary causes (Ananthakrishnan, Barr et al. 2009). 
1.4 
Clinical data  
(For further information please see the current version of the Investigator Brochure) 
A study conducted by Barr et al. (Barr, Beverly et al. 2003) investigated the prevalence of 
increased postoperative pulmonary vascular tone in infants and children undergoing cardiac 
surgery for correction of congenital heart defects. Of 169 patients, 56 (33.1%) developed 
clinical evidence of increased postoperative pulmonary vascular tone (PVT+). Many of these 
patients required clinical intervention including sedation, paralysis, and hyperventilation. 
Cardiopulmonary bypass caused a significant decrease in mean citrulline and arginine levels 
at all postoperative time points compared to preoperative levels. Plasma NO metabolite levels 
were also depressed immediately after surgery but showed a partial rebound at 12 and 24 
hours before returning to preoperative levels at 48 hours. 
In patients who subsequently developed increased postoperative PVT (PVT+), a particular 
decrease in plasma arginine levels was noted compared to patients without increased PVT 
(PVT-). Similar observations were not noted for citrulline and NO metabolites (Figure 2). The 
authors concluded that decreased availability of NO precursors may contribute to the 
increased risk of postoperative pulmonary hypertension. 


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Figure 2: Reduced Post-Operative Arginine Levels in PVT+ Group 
 
 
In a pilot study, Smith, Canter et al. 2006 investigated whether oral citrulline supplementation 
could increase plasma citrulline concentrations and decrease the risk of postoperative 
pulmonary hypertension. Forty children, undergoing CPB and at risk for pulmonary 
hypertension, were randomized to receive 5 perioperative doses (1.9 g/m2 per dose) of either 
oral citrulline or placebo. The 1st dose was administered immediately prior to CPB and the 
2nd dose immediately on arrival in the pediatric intensive care unit (PICU) after surgery, 
followed by another 3 doses every 12 hours. 
Median citrulline concentrations were significantly higher in the citrulline group versus the 
placebo group immediately postoperatively (36 µmol/L vs 26 µmol/L, P=0.012) and at 12 hours 
postoperatively (37 µmol/L vs 20 µmol/L, P=0.015). Mean systemic blood pressure was not 
different between the oral citrulline and placebo groups. 
The most significant result was that postoperative pulmonary hypertension developed in 9 
patients, 6 of 20 (30%) in the placebo group and 3 of 20 (15%) in the citrulline group (P=0.451), 
all of whom had plasma citrulline concentrations less than the age-specific norms. In a 
contingency analysis that reached statistical significance, all of the children developing 
postoperative pulmonary hypertension had a plasma citrulline level <37 µmol/L.  Conversely, 
none of the children with citrulline levels >37 µmol/L developed pulmonary hypertension.  This 
result implicated low citrulline levels as a strong risk factor and possible causal element in the 
78.2
55
37
37.8
47
77.9
58.3
29.9
24.5
26
0
10
20
30
40
50
60
70
80
90
100
Preop
Postop
12 Hrs
24 Hrs
48 Hrs
Arginine (umol/L)
Time
PVT-
PVT+
p<.05
p<.05
means + sem


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development of postoperative pulmonary hypertension.  Therefore, more aggressive citrulline 
supplementation, resulting in consistent citrulline concentrations in excess of 37 µmol/L, could 
potentially prevent postoperative pulmonary hypertension. 
In a Phase I/II clinical trial (Barr, Tirona et al. 2007) determined the pharmacokinetics (PK) 
and safety profile of 3 doses of i.v. L-citrulline in children undergoing CPB. The dose escalation 
design used three concentrations of i.v. L-citrulline: 50, 100, & 150 mg/kg. Each patient was 
given two doses of i.v. L-citrulline: the first in the operating room immediately after initiation of 
CPB and the second 4 hours later in the critical care unit. The overall goal was to achieve a 
sustained citrulline level of 80 to 100 µmol/L up to 4 hours after the initial dose. The 4-hour 
time point was selected to allow for the surgical procedure to be completed and the patient to 
return to the PICU postoperatively before further dosing.  
The results showed that 150 mg/kg was the optimal dose, as it resulted in 4 hour levels close 
to the 100 µmol/L target. However, from PK modeling it was also determined that the ½ life of 
the bolus doses of i.v. L-citrulline was approximately 60-90 minutes and that it would require 
at least Q4hr dosing to achieve a consistently increased citrulline level, which is impractical 
even in an intensive care unit (ICU) setting. The PK modelling suggested a sustained citrulline 
level of approximately 100 µmol/L could be achieved by a bolus dose of 150 mg/kg of i.v. L-
citrulline given at the beginning of surgery after initiation of CPB, followed 4 hours later by a 
continuous infusion of 9 mg/kg/hr. In a second phase of the study, 9 patients were enrolled 
and treated with this continuous infusion protocol. Using this regimen, plasma arginine, 
citrulline, and NO metabolite levels were well maintained (Figure 3). 
Figure 3: Mean Citrulline PK in Infants with Continuous Infusion Protocol (Barr, Tirona et al. 
2007) 
 
Mean Citrulline PK in Infants
0
12
24
36
48
60
10
100
1000
10000
Hours
Citrulline (µM)


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Based on the data from the PK study, a pilot randomized placebo-controlled Phase II/III trial 
of i.v. L-citrulline was performed at Vanderbilt (Vanderbilt Pilot Study SA3 [NCT00335244], 
May 2006-July 2008). Based on the previous results, this study utilized a protocol of an i.v. L-
citrulline bolus of 150 mg/kg during CPB followed postoperatively 4 hours later by a 9 mg/kg/hr 
continuous infusion for 48 hours.   
Patients undergoing one of 5 surgical procedures (VSD, ASD, arterial switch procedure, 
bidirectional Glenn procedure, or Fontan procedure) were eligible for enrollment. A total of 77 
patients were enrolled in this study. Serial arterial blood samples were obtained at 7 
perioperative time points. 
Most patients who received i.v. L-citrulline failed to reach the predicted target threshold of 100 
µmol/L. Of the 37 patients who were treated with i.v. L-citrulline, only 1/3 had a sustained 
plasma citrulline level > 100 µmol/L. Upon investigation, it was found that the duration of 
hemofiltration had been significantly extended during CPB to maintain the hematocrit, plasma 
lactate, and plasma potassium within a tightly controlled target range. This modified 
hemofiltration protocol effectively removed a majority of the i.v. L-citrulline delivered at the 
time of the preoperative bolus. Due to this finding, the design of a subsequent efficacy trial 
was revised to take into account L-citrulline removal by hemofiltration during CPB. 
The recent Phase IB single-blind, randomized, placebo-controlled study CIT-002-01 used a 
revised protocol to determine the PK and safety of i.v. L-citrulline in children undergoing CPB 
for surgical repair of congenital heart defects. The primary objective of the study was to obtain 
and maintain a perioperative plasma level of citrulline >100 µmol/L. Twenty-two children (age 
range 3 months to 5.5 years) with an ASD, a VSD, or a partial or complete AVSD were enrolled 
and treated. The study is completed and the full clinical study report is available. 
Subjects were randomized to receive either L-citrulline or placebo during their congenital heart 
defect procedures as follows: An i.v. L-citrulline bolus of 150 mg/kg or placebo at the initiation 
of CPB, followed by the addition of L-citrulline at a concentration of 200 µmol/L or placebo to 
the filtration or hemoconcentration replacement fluid used during CPB. An L-citrulline bolus of 
20 mg/kg or placebo was administered 30 minutes after decannulation from CPB, immediately 
followed by a 9-mg/kg/hr continuous infusion of L-citrulline or placebo for 48 hours. Blood 
samples were collected at 7 perioperative time points: baseline (prior to surgical incision, used 
for comparison to post-surgery time points), 10 minutes after initial bolus (post-bolus #1), after 
separation from CPB just prior to 2nd bolus (pre-bolus #2), 6h, 12h, 24h, and 48h after pre-
bolus #2. 
 


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The revised dosing protocol achieved plasma citrulline levels consistently above the target 
level of 100 μmol/L as shown below in Figure 4. 
 
Figure 4: Median Plasma Citrulline Levels at Indicated Time Points 
 
 
Patients receiving i.v. L-citrulline showed a reduced duration of mechanical ventilation as 
shown in Figure 5 and  Figure 6.  The duration of postoperative invasive mechanical ventilation 
was derived as the time in hours from separation from CPB until endotracheal extubation. The 
mean duration of invasive mechanical ventilation was clearly longer in the placebo group (37 
hours) than in the citrulline group (5 hours).  Kaplan-Meier survival analyses confirmed this 
reduced duration as seen in Figure 5 with re-intubation time included and ventilation time 
censored and a log rank p-value of 0.0498, and in Figure 6 with reintubation time included 
and zero ventilation time not censored and a log rank p-value of 0.0089. 
 
 
 
 
 
 


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igure 5: Kaplan-Meier Survival Analysis of the Duration of Invasive Mechanical Ventilation 
(Including Re-intubation Time): Censored 
 
 
 
 
 
 
 


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Figure 6: Kaplan-Meier Survival Analysis of the Duration of Invasive Mechanical Ventilation 
(Including Re-intubation Time): Not Censored 
 
Some differences were noted in clinical practice among sites with regard to mechanical 
ventilation. One site tended to extubate patients in the operating room without recording a 
time of extubation for such patients. For purposes of analysis, the duration of postoperative 
mechanical ventilation was set at zero for such patients. When these patients were stratified 
by treatment, it was shown that all 6 patients (100%) receiving i.v. L-citrulline had been 
extubated in the operating room, in comparison to only 2 of 6 patients (33%) in the placebo 
group. Although the numbers are small, these data achieved borderline significance.  The 
results suggest that children receiving citrulline appeared better clinically following 
cardiopulmonary bypass than did children receiving placebo.  
As with the length of time of mechanical ventilation, the duration of inotrope therapy 
(dopamine, dobutamine, milrinone, epinephrine, phenylephrine, or norepinephrine) was also 
recorded.  Length of time on inotropes was documented from the time of first use after surgery 
until completion of the study medication at 48 hours.  Patients still receiving inotropes at hour 
48 were censored.  There was a marked difference between the two treatment groups as 
shown in Figure 7 with censored patients still using inotropes at hour 48, with no additional 
censoring with a log rank p-value of 0.0351. 
 


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Figure 7: Kaplan-Meier Survival Analysis of the Length of Time on Inotropes: No additional 
Censoring 
 
 
 
 
A second analysis also censored patients without inotrope use; i.e., inotrope time = 0 (Figure 
8), which demonstrated a log rank p-value of 0.562.   
 
 
 


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Figure 8: Kaplan-Meier Survival Analysis of the Length of Time on Inotropes: Censored 
 
 
 
Cessation of mechanical ventilation and of inotrope therapy are the two principal determinants 
of readiness for discharge from the intensive care unit. A composite variable comprising (for 
each subject) of the longer of the two parameters - duration of mechanical ventilation or of 
inotrope therapy - can serve as an effective and accurate surrogate for the duration of 
intensive care unit stay.  Since inotrope use was only documented until Hour 48 after surgery 
(end of study medication treatment), patients with inotrope use continuing until Hour 48 and 
with mechanical ventilation duration of ≤ 48 hours were censored at this time point.  If 
mechanical ventilation was continued beyond the 48-hour time point, the duration of 
mechanical ventilation was used in the analysis. Kaplan-Meier plots of the composite endpoint 
are presented in Figure 9, with re-intubation times excluded. One (1) patient in the placebo 
group was re-intubated. This patient was excluded from this analysis; however, results 
including this patient were comparable.  In this analysis, patients with inotrope use still ongoing 
at 48 hours, and mechanical ventilation of less than 48 hours, were censored.  Figure 9 shows 
the difference in the composite variable when L-citrulline and placebo groups are compared. 
Citrulline-treated patients required statistically significantly less mechanical ventilation and 
inotropes than placebo-treated patients. Thus, as assessed by the composite surrogate 
marker variable, patients receiving study drug were ready for discharge from the PICU sooner 
than patients receiving placebo. In addition to indicating shorter PICU time, shorter 
mechanical ventilation time lowers the added risk of physical injury. 


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Figure 9: Kaplan-Meier Survival Analysis of the Longer Duration of Duration of Postoperative 
Mechanical Ventilation and the Length of Time on Inotropes: Re-Intubation Time Excluded 
 
 
In summary, the data from study CIT-002-01 show that the revised dosing protocol achieved 
the PK endpoint. Further, despite the extremely small sample size of the study, CIT-002-01 
indicates that intravenous L-citrulline prevents compromised cardiopulmonary function 
caused by CPB in pediatric patients undergoing surgery for ASD, VSD, or AVSD.  The effect 
is robust and is evident even in centers practicing early extubation as shown in the strong 
preferential early extubation of citrulline patients in comparison to the control population.  
Discharge from the ICU requires cessation of both mechanical ventilation and inotrope 
therapy.  Intravenous L-citrulline shortens the duration of each of these variables, resulting in 
earlier readiness of patients for discharge from the ICU. Thus, CIT-002-01 indicates that i.v. 
L-citrulline treatment can play a beneficial role in preventing clinical sequelae of CPB-induced 
lung injury. 
1.5 
Safety of L-citrulline  
L-citrulline is a naturally occurring amino acid. It is produced by, and normally present in, the 
human body. Typical circulating levels of citrulline in adults are 16-55 µmol/L. In children <12 
years of age, the levels are 16-32 µmol/L. Oral citrulline has been used for years as a food 
supplement to enhance energy and prevent muscle fatigue and has been found safe in doses 
of 2-15 g, which is a multiple of the administered dose in the L-citrulline trials conducted to 
date (http://examine.com/supplements/citrulline/ #summary_full) (Moinard, Nicolis et al. 


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2008). 
No SAEs occurred in the oral citrulline study reported by Smith, Canter et al. 2006. Four 
adverse reports were submitted to the Institutional Review Board (IRB) per protocol on 
patients who developed hypotension secondary to surgical complications and required further 
surgical or medical support. These cases were reviewed and determined to not be related to 
study drug administration. 
In the first report of the use of L-citrulline in humans, L-citrulline was found to be both safe and 
well tolerated (Barr, Tirona et al. 2007). One patient required advanced life support following 
a junctional rhythm postoperatively and profound bradycardia consistent with complete heart 
block. The patient recovered fully. The Data Safety Monitoring Board (DSMB) reviewed the 
event and deemed it not likely related to L-citrulline.  
Serious AEs (SAEs) under L-citrulline treatment in the Vanderbilt pilot study included 
postoperative bleeding (2 patients), and obstructive airway disease (1 patient). Although these 
events occurred in 3 out of 35 L-citrulline patients only, these types of adverse events (AEs) 
will be monitored in future studies. 
Safety data from the completed CIT-002-01 study have shown that of the patients randomized 
and treated, all patients (100%) in the i.v. L-citrulline group and 73% of patients in the placebo 
group experienced at least one AE.  AEs that were judged to be related to study treatment 
were reported in the placebo group (2 patients, 18%), but not in the active treatment group.  
No AE leading to death and no other SAE was reported in this study.  No AE leading to 
treatment discontinuation was reported. 
The safety of administering even higher doses of citrulline than those being used in this study 
has been demonstrated in children with urea cycle dysfunction.  A previous study has shown 
that children with carbamyl phosphate synthetase and OTC deficiencies have benefited from 
citrulline supplementation at doses of 100–170 mg/kg/d (Singh, Rhead et al. 2005).  
Additionally, in children who are genetically unable to produce citrulline, bolus administrations 
of citrulline at 400-500 mg/kg are used routinely, again without observed negative 
consequences (personal communication from Marshall Summar, M.D., Ph.D., Children's 
National Medical Center Wash DC). 
Based on the available published, pre-clinical and clinical data, the only potential adverse 
event would be transient systemic hypotension.  This was seen in 2 out of 11 patients in the 
L-citrulline group and 1 out of 11 patients in the placebo group in the CIT-002 trial. 
1.6 
Rationale for the study 


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Asklepion intends to develop L-citrulline for the prevention of clinical sequelae of acute lung 
injury induced by CPB in pediatric subjects undergoing surgery for congenital heart defects. 
Cardiopulmonary bypass causes a transient systemic inflammatory response leading to 
clinical manifestations of acute compromise of cardiovascular and pulmonary function 
requiring extended mechanical ventilation and use of inotropes (Kozik and Twedell 2006).  
Pediatric patients undergoing congenital heart defects surgery are most vulnerable to 
experiencing lung injury sequelae during the immediate postoperative period. CPB-induced 
acute lung injury represents a complex interplay between the preoperative condition of the 
patient (importantly age at repair, type of lesion and clinical manifestations) and the inevitable 
disruption in the endocrine and vasoactive peptide milieu that results from cardiac surgery. L-
citrulline, with its mechanism of action, is intended as a preventive treatment to improve 
restoration and maintenance of normal vasodilation, thus improving clinical status and 
reducing the need for-and exposure to-mechanical ventilation and inotrope treatment.  
The reduction of key manifestations of acute CPB-induced lung injury, namely the post-
operative need for mechanical ventilation and for inotrope therapy will provide evidence for L-
citrulline's intended clinical effect. 
 
2 Study Objectives 
2.1 
Primary Objective 
The primary objective of the study is to determine if L-citrulline delivery given perioperatively 
reduces clinical sequelae of acute CPB-induced lung injury in pediatric subjects undergoing 
repair of congenital heart defects, as evidenced by the reduction of post-operative need for 
mechanical ventilation and inotrope therapy. 
2.2 
Secondary Objective(s) 
Secondary objectives are to evaluate the effect of L-citrulline compared with placebo 
regarding 
• 
safety and tolerability 
• 
hemodynamic improvement post-operatively 
• 
the usage and length of time on vasodilators 
• 
length of hospitalization 


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• 
the duration of chest tube placement  
• 
the plasma concentrations of citrulline 
 
3 Study Endpoints 
3.1 
Primary Endpoint 
A composite variable consisting of the longer of either (1) length of time on mechanical 
ventilation or (2) length of inotrope use. 
The definition of mechanical ventilation shall include invasive mechanical ventilation or non-
invasive mechanical ventilation including bi-level (biphasic) positive airway pressure (BPAP) 
or continuous positive airway pressure (CPAP). 
3.2 
Secondary Endpoints 
3.2.1 Secondary Efficacy Endpoints 
• 
Length of time on mechanical ventilation 
• 
Length of time on positive pressure ventilation 
• 
Length of time of inotrope use 
• 
Inotrope score calculated each hour post-operatively from the time of separation from 
bypass until the completion of study drug; additionally, the total inotrope score over 
time until Day 28 or hospital discharge will be derived 
• 
Hemodynamic improvement (heart rate, systemic arterial blood pressure, oxygen 
saturation, and central venous pressure) 
• 
Thoracotomy output; duration and volume of chest tube drainage 
• 
Length of time of intubation 
• 
Length of PICU stay 
• 
Length of time on vasodilators 
• 
Length of hospitalization 
• 
Plasma concentrations of citrulline 


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3.2.2 Secondary Safety Endpoints 
• 
Occurrence of adverse and serious adverse events 
• 
Incidence of refractory hypotension 
• 
Change from baseline in laboratory values (serum electrolytes, blood urea nitrogen 
[BUN], creatinine, complete blood count [CBC] with differential, activated clotting time 
[ACT] & liver enzymes) 
 
4 Investigational Plan 
4.1 
Overall Study Design and Plan 
This is a multicenter Phase III double-blind, randomized, placebo controlled study to evaluate 
the efficacy and safety of L-citrulline for the prevention of clinical sequelae of acute lung injury 
induced by CPB in pediatric subjects undergoing surgery for congenital heart defects.  
Children undergoing repair of cardiac defects that include a large unrestrictive VSD or a 
partial or complete AVSD or an ostium primum ASD will be eligible for enrollment in this 
study.  
• 
190 subjects will be randomized in a 1:1 fashion to either L-citrulline or placebo. L-
citrulline treatment will include: 1) an L-citrulline bolus of 150 mg/kg or placebo at the 
initiation of CPB, but after removal of any crystalloid base; 2) the addition of L-
citrulline at a concentration of 200 µmol/L or placebo given as a bolus during bypass.  
This may be administered as a one-time bolus or multiple administrations to 
compensate for fluids containing L-citrulline that may be removed from the patient 
during the course of the operation and thus to maintain the concentration of 200 
µmol/L; and 3) an L-citrulline bolus of 20 mg/kg or placebo 30 minutes after 
decannulation from CPB immediately followed by a 9 mg/kg/hr continuous infusion of 
L-citrulline or placebo for 48 hours (see Section 4.4.1 for further details). The study 
drug or placebo infusion will be discontinued once invasive arterial blood pressure 
monitoring is discontinued or at 48 hours, whichever comes first. Subjects will be 
followed until discharge from the hospital, and a final assessment via telephone will 
be conducted at Day 28. 
4.2 
Discussion of Study Design 
This study will examine patients who develop significant sequelae of CPB-induced lung injury 


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in the absence of confounding co-morbidities. Consequently, secundum ASDs are excluded 
because they are often clinically insignificant and patients with secundum ASDs rarely develop 
significant clinical sequelae of CPB-induced lung injury. Inclusion of the very common 
secundum ASDs would thus dilute study results. Similarly, single ventricle defect patients are 
excluded because of their high incidence of co-morbidities that would prolong the use of 
positive pressure ventilation and inotrope usage by confounding mechanisms other than CPB-
induced lung injury. 
A primary concern in designing a study to confirm that perioperative administration of L-
citrulline to CPB patients does indeed provide a substantive clinical benefit is to construct an 
endpoint or endpoints that are reliable and consistent. 
The following factors underlie the determination of the appropriate primary endpoint for the 
proposed trial: there is consistent pathophysiology among pediatric congenital heart defect 
patients receiving CPB: pulmonary endothelial insult and disruption => CPB-induced acute 
lung injury => increased vascular resistance and elevated vascular pressure => various 
morbidities and risks, including congestive heart failure => prolonged ICU stays with continued 
positive pressure ventilation and inotrope therapy. 
Each patient will be randomized to receive either L-citrulline or placebo for the duration of the 
study. Randomization will be performed using a previously generated randomization scheme, 
and stratified by site to ensure, to the extent possible, equal randomization into both treatment 
arms. 
The study is double-blinded for all investigational staff who make determinations related to the 
study, with the sole exception of the hospital or site pharmacist who will be single-blind in 
order to properly prepare the individual subject’s medication (see Section 4.4.7).   
4.3 
Selection of Study Population 
4.3.1 
Number of Planned Subjects  
Approximately 450 subjects will be screened to obtain 190 patients randomized to either 
placebo or L-citrulline in a 1:1 ratio. A total of approximately 190 subjects are anticipated to 
complete the study.  
The estimated enrollment period is 11 months. The total trial period for a patient is up to 42 
days (up to 2 weeks screening, 2 days treatment, up to 28 days follow-up). The trial end is 
defined as "last patient out" (LPO). 


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4.3.2 Inclusion Criteria 
To be eligible for study entry subjects must satisfy all of the following criteria: 
• 
Subjects, parents, or legal guardian of the subject who are willing and able to sign 
informed consent 
• 
Male and female subjects aged ≤18 years of age (females of child-bearing potential 
must be willing to practice an acceptable form of birth control; i.e. oral contraceptive, 
hormonal implant; etc.) 
• 
Infants, children and adolescents undergoing CPB for repair of a large unrestrictive 
VSD, an ostium primum ASD, or a partial or complete AVSD 
• 
Pre-operative echocardiogram which confirms the cardiovascular anatomy and 
defect to be surgically repaired 
4.3.3 Exclusion Criteria 
Subjects will be excluded from the study if one or more of the following statements are 
applicable: 
• 
Evidence of pulmonary artery or vein abnormalities on the pre-operative 
echocardiogram that will not be addressed surgically. Specific abnormalities 
excluded include the following: 
 
Significant pulmonary artery narrowing not amenable to surgical correction 
 
Previous pulmonary artery stent placement 
 
Significant left sided AV valve regurgitation not amenable to surgical 
correction 
 
Pulmonary venous return abnormalities not amenable to surgical correction 
 
Pulmonary vein stenosis not amenable to surgical correction 
• 
Preoperative requirement for mechanical ventilation or i.v. inotrope support 
• 
Presence of fixed or idiopathic pulmonary hypertension (i.e. Eisenmenger’s 
Syndrome) prior to surgical repair 
• 
Pre-operative use of medications to treat pulmonary hypertension 
• 
Pregnancy; Sexually active females of child-bearing potential must be willing to 
practice an acceptable method of birth control for the duration of study participation 


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(e.g. oral contraceptive, hormonal implant, intra-uterine device) 
• 
Participation in another clinical trial within 30 days of Screening or while participating 
in the current study, including the 28 days of follow-up post study drug 
administration.  
• 
Any condition which, in the opinion of the investigator, might interfere with the study 
objectives 
4.3.4 Removal of Subjects from Therapy or Assessments 
The person(s) providing consent is free to withdraw his/her consent regarding participation in 
this study at any time, for any reason, specified or unspecified and without penalty or loss of 
benefits to which the subject is otherwise entitled. 
Subjects may be withdrawn from the study by the Investigator at any time due to an AE or for 
any other reason if it is in the opinion of the Investigator that continued participation in the 
study is no longer in the best interest of the subject. These reasons include, but are not limited 
to the following: 
• 
Adverse drug reaction or AE or any other reason for which, in the opinion of the 
Investigator, subject’s continued participation in the study is not in the best interest of 
the subject 
• 
Serious protocol violation(s), such as determination of fulfilment of exclusion criteria 
during surgery only 
• 
Lack of response of subject or subject’s parents/legal guardian to the attempts of the 
investigational site to contact him/her (lost to follow-up) 
The reason(s) for withdrawal must be documented in the case report form (CRF). 
For subjects withdrawing or being withdrawn from the study, it will be encouraged to have the 
same final evaluations completed as those for subjects completing the study according to this 
protocol, particularly safety evaluations.  
For subjects who are lost to follow-up, reasonable efforts will be made to contact the parents 
or legal guardians of subjects, or the subjects themselves if they provided their own consent. 
These efforts must be documented in the subject’s file.  
Subjects who have been randomized and who withdraw or are withdrawn for any reason will 
not be replaced.   


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The sponsor has the right to terminate the study at any time in case of SAEs or if special 
circumstances concerning the investigational medicinal product (IMP) or the company itself 
occur, making further treatment of subjects impossible. In this event, the investigator(s) will 
be informed of the reason for study termination. 
4.4 
Investigational Medicinal Products 
4.4.1 Investigational Medicinal Products Administered 
The main objective of the dosing schedule, as outlined below, is to achieve a minimum 
systemic target plasma trough level concentration of 100 µmol/L of L-citrulline to provide the 
most beneficial effect as demonstrated in the previous phase 2 study.  As a general guiding 
principal during bypass, once dosing has begun, all fluids entering the circuit must contain 
200 µmol/L of L-citrulline in order to achieve and maintain the trough level of 100 µmol/L of 
L-citrulline. Therefore, if fluids are being added or removed, it is crucial to take this into 
consideration when timing the administration of the required doses.  The following section 
describes the administration of L-citrulline or placebo, while taking into account the above 
objective. 
• 
The first (initial) dose administered will be an L-citrulline bolus of 150 mg/kg or 
placebo, administered at the initiation of CPB, but after removal of any crystalloid 
base.   
o If fluid is added to the priming circuit, but removed prior to initiation of bypass, 
then the initial 150 mg/kg bolus dose should be added after the crystalloid 
based fluids are removed and at the initiation of bypass.  
o If fluid is added to the priming circuit, but not removed, then the initial 150 
mg/kg dose should be added to the circulating prime used for the bypass.  
• 
The second dose administered consists of L-citrulline at a concentration of 200 
µmol/L or placebo, given as a bolus during bypass.  This may be administered as a 
one-time bolus or multiple administrations, to maintain the concentration at 200 
µmol/L. The total dose will be dependent upon the total amount of any volume 
administered and removed during CPB.  This includes the crystalloid portion of the 
cardioplegia so as not to dilute the loading dose; it is significantly important when 
using a hemoconcentrator. 
• 
The third dose administered will be a bolus of 20 mg/kg of L-citrulline or placebo, 
administered 30 minutes after decannulation from CPB.  


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• 
The above third dose is immediately followed by a 9 mg/kg/hr continuous infusion of 
L-citrulline or placebo for up to 48 hours.  The study drug or placebo infusion will be 
discontinued once invasive arterial blood pressure monitoring is discontinued or at 
48 hours, whichever comes first. 
Placebo will be matched for volume using suitable fluid recognized as standard of care for 
such procedure (e.g. normal saline or Plasmalyte without L-citrulline added). 
All study medications will be prepared and provided by each site’s investigational pharmacy. 
A pharmacy manual will be provided to the hospital or institution Investigational Pharmacist. 
The manual will contain detailed instructions for the preparation of the investigational product. 
The manual will include specific directions on randomization procedures, preparation of study 
drug or placebo and labeling of product for administration. The label should contain subject 
information (e.g. subject initials, subject identification number), study number, volume of 
product to be administered, and the date of administration. However, the label may not contain 
any information that identifies the actual product being administered.  
All study medications will be administered by the Investigator or by medically trained personnel 
under his/her supervision (e.g., the anesthesiologist, perfusionist, PICU personnel, etc.).  
Doses will be given by a central i.v. catheter that will be emplaced after induction of anesthesia 
or via the bypass circuit. Separate i.v. access is not required for the study drug. The study 
drug is isotonic and can run through either a peripheral i.v. or a central venous catheter at the 
discretion of the clinical staff. The study drug is an amino acid (or placebo) and thus, for 
compatibility purposes, the drug product is treated like parenteral nutrition. Additionally, it is 
compatible with fluids used for filtration or hemoconcentration during CPB. 
No dose adjustments will be allowed; however, study drug may be discontinued as noted 
above. 
4.4.2 Identity of Investigational Medicinal Products 
The L-citrulline drug product is a sterile, non-pyrogenic injectable clear solution intended for 
intravenous administration. It is supplied as a 10 mL, single use vial containing 500 mg L-
citrulline in 10 mL of acidified water. The product is packaged in a clear glass vial (13 mm 
neck) with a grey stopper and blue flip-off cap/aluminum seal. The L-citrulline drug product is 
manufactured aseptically at a concentration of 50 mg/mL using fully synthetic drug substance 
dissolved in water for injection. The resulting solution pH is adjusted to near pH 3, sterile 
filtered and filled. 


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Study drug should be stored as instructed: Store at 2-8⁰C (36-46⁰F); do not freeze. 
 
The drug product is manufactured and imported according to current Good Manufacturing 
Practice and the relevant regulatory requirements.  
4.4.3 Packaging and Labeling 
L-citrulline study drug comes in a clear glass vial (13 mm neck) with a grey stopper and blue 
flip-off cap/aluminum seal. Boxes of 25 vials are supplied to the study centers. 
The batch number, strength, storage conditions, and federally required language stating that 
the product is “for investigational use only” or “for clinical trial use only” will be printed on the 
outer package. A Material Safety Data Sheet (MSDS) will be provided with the shipment. 
All packaging and labeling operations will be performed according to the current Good 
Manufacturing Practice for Medicinal Products and the relevant regulatory requirements. 
4.4.4 Method of Assigning Subjects to Treatment Groups 
Screened subjects will be assigned a unique 5-digit number, with the first two digits identifying 
the site and the next three digits identifying the subject, e.g. 01-001. At each site, the 
investigator will assign numbers to subjects in ascending order (e.g. 001, 002, 003). 
Eligible subjects will be randomized to a treatment (placebo or L-citrulline) in a 1:1 ratio. 
Randomization will be performed per study site via an interactive web response system 
(IWRS). The randomization schedule will be generated using a validated randomization 
program. A unique randomization number will be assigned to each randomized subject. 
Randomization assignment will be stratified at each site to ensure balance of the number of 
randomized subjects to both treatment groups. 
4.4.5 Selection of Doses in the Study 
Initial supplementation trials utilized oral citrulline at a dose of 1.9 g/m2 per dose, administered 
before CPB, postoperatively upon admission to the intensive care unit and every 12 hours at 
hour 12, 24 and 36. Oral citrulline was well tolerated with no evidence of significant AEs. In 
addition, it was noted that patients who had a 12-hour plasma citrulline level >37 µmol/L (the 
upper range of normal levels) did not develop increased PVT. Unfortunately, not all patients 
receiving oral citrulline reached these levels (Smith, Canter et al. 2006).  


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In a dose escalation study targeting a sustained plasma level of approximately 100 µmol/L, it 
was noted that L-citrulline has a fairly short half-life, thus requiring a classic bolus and 
continuous infusion drug delivery protocol (Barr, Tirona et al. 2007). A subsequent study (CIT-
002-01) confirmed that the following L-citrulline administration would result in sustained 
plasma L-citrulline levels of approximately 100 µmol/L:  
• 
An L-citrulline bolus of 150 mg/kg or placebo at the initiation of CPB  
• 
The addition of L-citrulline at a concentration of 200 µmol/L or placebo to both the 
bypass circuit priming fluid as well as to the hemofiltration fluid utilized during CPB 
• 
 A bolus of L-citrulline of 20 mg/kg or placebo, 30 minutes after decannulation from 
CPB, immediately followed by initiation of a 9 mg/kg/hr continuous infusion of L-
citrulline or placebo for 48 hours  
This treatment regimen, with clarification added for the dose(s) given during bypass, will also 
be used in the current study.  
4.4.6 Drug Accountability 
The Pharmacist at the study site is responsible for keeping accurate drug accountability 
records throughout the study regarding the receipt of study drug, the preparation of study drug 
and dispensation to subjects, and the reconciliation of all used and unused study drug.  
All empty and partially used vials should be retained by the pharmacy in order for study drug 
accountability to be performed.   The total amount of study drug prepared for administration 
to the patient will be calculated by the investigational pharmacist, with verification of the 
calculations performed by a second qualified person (e.g. pharmacy technician).  Total 
volume of study drug administered to the patient will be documented in the source 
documents and in the electronic case report forms (eCRFs). 
A Drug Preparation Log as well as a Drug Dispensing and Accountability Log must be kept 
current and must contain the following information: 
• 
Batch number 
• 
Subject number for whom the drug was prepared 
• 
Date on which drug was dispensed 
• 
Quantity of the drug administered 
 


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4.4.7 Blinding 
Randomization will take place via an IWRS system. The site’s investigational pharmacist will 
be responsible for obtaining the randomization assignment. Therefore, the site pharmacist will 
only be aware of the treatment assigned to each individual subject. The pharmacist has no 
other role in the study other than preparing the study product. Study drug or placebo will be 
prepared and labeled with the appropriate subject identifiers; however, no information that 
would reveal the actual contents of the dose to be administered (active vs. placebo) will be 
included on the label. Study drug will be provided in either syringes or bags and mask-labeled. 
Placebo is also mask-labeled and provided in either syringes or bags. The bags are the same 
size, shape, and fluid clarity, and hence masked to both investigators and staff (if other than 
the investigator) administering the drug. Therefore, the investigator and staff administering the 
product will remain blinded as to its contents. Only the pharmacist and the unblinded monitor 
responsible for performing drug accountability (a different monitor than the person performing 
routine data monitoring) will be aware of the treatment assignment.  
Data may be unblinded at the request of the DSMB to the study Medical Monitor in the event 
that a safety issue arises for which they determine there is a need to review the subject’s 
treatment assignment. If data is unblinded for any reason, the DSMB will not share the 
treatment assignment with the Sponsor, unless there is a medical or ethical reason to do so.  
The randomization assignment should not be revealed before the study has been completed 
and the database has been cleaned and closed. The study will be unblinded using a study 
specific unblinding procedure. 
In rare emergencies, such as the investigator feels adequate treatment of an AE requires 
knowledge of study treatment, unblinding of an individual subject may be necessary. In case 
of an emergency, the treatment administered to the subjects can be revealed using the 
unblinding function of the IWRS. 
In such events, the Investigator must make every attempt to inform the study Medical Monitor 
before breaking the blind or as soon as possible after unblinding has been performed (within 
24 hours after the emergency unblinding). The date, time and reason for breaking the code, 
the treatment administered (placebo or L-citrulline), and the sponsor’s representative who was 
informed of the unblinding, will be documented within the eCRF/IWRS. 
4.4.8 Prior and Concomitant Therapy 
Any medication taken by a subject during their hospitalization and the reason for use will be 
recorded on the case report form. These include medications taken before, during and after 


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surgery. Concomitant therapy includes, but is not limited to, inotropic or vasoactive 
medications, CPB replacement fluids, blood products and perfusion fluids.  
Administration of total parenteral nutrition (TPN) will be at the discretion of the clinical staff in 
the PICU and should not be affected by study participation. Any substance that is incompatible 
with TPN should not be infused with L-citrulline, since TPN also contains amino acids. A “Y” 
site may be utilized for the infusion at the discretion of the Principal Investigator.  
Subjects can be treated with iNO if the therapy is indicated at the discretion of the Principal 
Investigator. However, prophylactic treatment with NO is not permitted. Any use of iNO will be 
tracked.  
Preoperative invasive mechanical ventilation or i.v. inotrope support, as well as concomitant 
use of any pulmonary hypertension medications, is not allowed and renders a subject ineligible 
for treatment (see Section 4.3.3). 
Rescue medications, including use of iNO, after surgery will be permitted. Use of any rescue 
medications will be recorded and their use will be tracked as an outcome.  
4.4.9 Treatment Compliance 
The administration of the study medication will be performed by the Investigator or Sub-
Investigator or medically trained personnel and will be documented accordingly.  
 
5 Study Procedures 
5.1.1 Pre-treatment 
5.1.1.1 Screening Visit (Day -14 to Day 0) 
A series of screening evaluations will be performed in order to determine whether prospective 
study participants meet the selection criteria for the trial. 
• 
The Investigator or designated study personnel will inform each prospective subject’s 
family of the nature of the study, explain the potential risks, and obtain written 
informed consent prior to performing any study related procedures.  
• 
Assessment for eligibility against the inclusion and exclusion criteria 
• 
Demographic data (age, sex, race and ethnicity) 


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• 
Screening evaluations will consist of medical history documentation, including 
documentation of previous echocardiogram confirming cardiovascular anatomy and 
the defect to be surgically repaired, and a physical examination, including body 
weight and height  
• 
Blood draw*: 
− 
serum electrolytes (sodium (Na), potassium (K), calcium (Ca), magnesium 
(Mg) and chloride (Cl)), BUN, and creatinine 
− 
CBC with differential 
− ACT 
− 
liver function tests (LFTs), including bilirubin (total), alkaline phosphatase 
(AP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and 
lactate dehydrogenase (LDH) 
 
* Results of laboratory tests done as standard of care for pre-op testing are 
acceptable and may be recorded as screening values, provided the tests were 
performed within 14 days of Day 0.  If any of the required tests were not done as 
part of the pre-op testing, these must be completed within 14 days of Day 0. 
• 
Urinalysis (not required, but results should be recorded if test is performed) 
− 
Visual: Color, Clarity/Turbidity 
− 
Chemical: pH, Specific gravity, glucose, ketones, nitrites 
− 
Microscopic: RBCs, WBCs, epithelial cells, casts, bacteria 
• 
Concomitant medications 
5.1.1.2 Baseline Visit (Day of Surgery, Pre-operation) 
The following evaluations will be performed on the day of surgery, prior to the operation: 
• 
Body weight for calculating study drug dosage 
• 
Inotrope and other concomitant medication documentation 
• 
Urine pregnancy test for females of child-bearing potential  
• 
Randomization 
• 
Preparation of study drug by the hospital pharmacy 
− 
Blood draw (prior to surgery; in the operating room [OR], after induction of 


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anesthesia); for: plasma L-citrulline (1.0 mL)  
• 
Adverse events documentation (pre-treatment) 
5.1.2 Treatment Period 
5.1.2.1 Day 0, Surgery (Intra-operative) 
The following evaluations will be performed in the OR, during surgery: 
• 
Collection of CPB data, including the following:  
− 
Equipment type 
− 
Cardioplegia solution type 
− 
Composition of isovolumetric exchange fluid for ultrafiltration and/or modified 
ultrafiltration  
− 
Exchange volumes in and out 
− 
Documentation of L-citrulline or placebo administration 
− 
CPB start and end times and decannulation time 
• 
Hemodynamic monitoring, including the following: 
− 
Heart rate 
− 
Systemic blood pressure (BP) 
− 
O2 saturation 
− 
Central Venous Pressure (CVP) measurements 
− Pulmonary Artery Pressure (PAP) measurements (if PA line is placed) 
Baseline values are first recorded in the OR by Anesthesia.  Results will be 
monitored during surgery according to the Institution’s standard of care, but 
results will only be recorded approximately every 15 minutes.  
• 
Arterial Blood Gases (ABGs) 
− 
PaO2, PaCO2, HCO3, and pH measurements 
Baseline values are first recorded in the OR by Anesthesia.   All results 
obtained after induction of anesthesia and during surgery will be recorded.  
• 
Bolus of L-citrulline at 150 mg/kg or placebo at initiation of CPB; addition of L-
citrulline at a concentration of 200 µmol/L or placebo given as a bolus during bypass.  
This may be administered as a one-time bolus or multiple administrations to 
compensate for fluids containing L-citrulline that may be removed from the patient 


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during the course of the operation and thus to maintain the concentration of 200 
µmol/L.  The total dose will be dependent upon the total amount of any volume 
administered and removed during CPB.   
• 
Blood draw (10 minutes after study drug is administered): 
− 
serum electrolytes, BUN, and creatinine  
− 
CBC with differential 
− 
ACT  
− 
plasma L-citrulline (1.0 mL) 
• 
Inotrope and other concomitant medication documentation 
• 
Adverse events documentation 
5.1.2.2 Day 0, post-surgery, 0 hours (30 minutes post CPB decannulation) 
The following evaluations will be performed 30 minutes post decannulation: 
• 
Blood draw for: 
− 
 plasma L-citrulline (prior to bolus); 1.0 mL 
• 
Bolus of L-citrulline at 20 mg/kg or placebo 30 minutes after decannulation from CPB 
immediately followed by a continuous infusion of L-citrulline at 9 mg/kg/hr or placebo 
for up to 48 hours.  The study drug or placebo infusion will be discontinued once 
invasive arterial blood pressure monitoring is discontinued or at 48 hours, whichever 
comes first. 
• 
Hemodynamic monitoring including: 
− 
Heart rate 
− 
Systemic BP 
− 
O2 saturation 
− 
CVP measurements 
− PAP measurements (if PA line is placed) 
Results will be recorded according to the Institution’s standard of care from 
time of post-op admission to the PICU through 48 hours.  Results at 1, 2, 4, 12, 
24, and 48 hours from baseline (in OR) must be obtained and recorded.  
• 
ABGs 
− 
PaO2, PaCO2, HCO3, and pH measurements 
ABGs will be drawn according to the Institution’s standard of care. All results 


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obtained from the time of post-op admission to the PICU until removal of the 
arterial line or through 48 hours (whichever occurs first) will be recorded.  
• 
Thoracotomy output measurements (for the duration of chest tube placement) 
• 
Blood draw for: 
− 
plasma L-citrulline (5 minutes after bolus); 1.0 mL 
• 
Inotrope and other concomitant medication documentation 
• 
Documentation of ventilator settings. Recorded approximately every hour (+/- 10 
minutes) from time of separation from CPB until end of mechanical ventilation or 28 
days (whichever occurs first) 
• 
Hypotension assessment (Refractory hypotension is defined as a 20% drop of mean 
arterial blood pressure [MAP] below specific age-related criteria (Haque and Zaritsky 
(2007) for greater than 30 minutes) (See below) 
− 
Infants:  
MAP of 40 
− 
Age 1 year:       MAP of 40 
− 
Age 2 years:        MAP of 44 
− 
Age 3 years:        MAP of 47 
− 
Age 4 years:        MAP of 50 
− 
Age 5 years: 
MAP of 52 
 
− 
Age 6 years:        MAP of 53 
− 
Age 7 years:        MAP of 52 
− 
Age 8 years:        MAP of 54 
− 
Age 9 years:        MAP of 55 
− 
Age 10 years:      MAP of 56 
− 
Age 11 years:      MAP of 57 
− 
Age 12 years:      MAP of 58 
− 
Age 13 years:      MAP of 59 
− 
Age 14 years:      MAP of 61 
− 
Age 15 years:      MAP of 62 
− 
Age 16 years:      MAP of 63 
− 
Age 17-18 years: MAP of 64 
• 
Adverse events documentation 
5.1.2.3 Day 0, post-surgery, 6 hours  
The following evaluations will be performed: 
• 
Continuous infusion of L-citrulline at 9 mg/kg/hr or placebo for up to 48 hours.  The 


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study drug or placebo infusion will be discontinued once invasive arterial blood 
pressure monitoring is discontinued or at 48 hours, whichever comes first. 
• 
Hemodynamic monitoring including: 
− 
Heart rate 
− 
Systemic BP 
− 
O2 saturation 
− 
CVP measurements 
− PAP measurements 
Results will be recorded according to the Institution’s standard of care from 
time of post-op admission to the PICU through 48 hours.  Results at 1, 2, 4, 
12, 24, and 48 hours from baseline (in OR) must be obtained and recorded.  
• 
ABGs 
− 
PaO2, PaCO2, HCO3, and pH measurements 
• 
ABGs will be drawn according to the Institution’s standard of care. All results 
obtained from the time of post-op admission to the PICU until removal of the arterial 
line or through 48 hours (whichever occurs first) will be recorded. Thoracotomy 
output measurements (for the duration of chest tube placement) 
• 
Blood draw for: 
− 
plasma L-citrulline (6 hrs; +/-1 hour; collect at end of infusion if infusion 
discontinued prior to 6 hours); 1.0 mL 
• 
Inotrope and other concomitant medication documentation 
• 
Documentation of ventilator settings. Recorded approximately every hour (+/- 10 
minutes) from time of separation from CPB until end of mechanical ventilation or 28 
days (whichever occurs first) 
• 
Hypotension assessment (Refractory hypotension is defined as a 20% drop of MAP 
below specific age-related criteria for greater than 30 minutes) (See Section 5.1.2.2) 
• 
Adverse events documentation 
5.1.2.4 Day 0, post-surgery, 12 hours  
The following evaluations will be performed: 
• 
Continuous infusion of L-citrulline at 9 mg/kg/hr or placebo for up to 48 hours.  The 
study drug or placebo infusion will be discontinued once invasive arterial blood 


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pressure monitoring is discontinued or at 48 hours, whichever comes first. 
• 
Hemodynamic monitoring including: 
− 
Heart rate 
− 
Systemic BP 
− 
O2 saturation 
− 
CVP measurements 
− PAP measurements 
Results will be recorded according to the Institution’s standard of care from 
time of post-op admission to the PICU through 48 hours.  Results at 1, 2, 4, 
12, 24, and 48 hours from baseline (in OR) must be obtained and recorded.  
• 
 ABGs 
− 
PaO2, PaCO2, HCO3, and pH measurements 
ABGs will be drawn according to the Institution’s standard of care. All results 
obtained from the time of post-op admission to the PICU until removal of the 
arterial line or through 48 hours (whichever occurs first) will be recorded.  
• 
Thoracotomy output measurements (for the duration of chest tube placement) 
• 
Blood draw for: 
− 
plasma L-citrulline (12 hrs; +/-1 hour; collect at end of infusion if infusion 
discontinued prior to 12 hours); 1.0 mL  
• 
Inotrope and other concomitant medication documentation 
• 
Documentation of ventilator settings. Recorded approximately every hour (+/- 10 
minutes) from time of separation from CPB until end of mechanical ventilation or 28 
days (whichever occurs first) 
• 
Hypotension assessment (Refractory hypotension is defined as a 20% drop of MAP 
below specific age-related criteria for greater than 30 minutes) (See Section 5.1.2.2) 
• 
Adverse events documentation 
5.1.2.5 Day 1, post-surgery, 24 hours  
The following evaluations will be performed: 
• 
Continuous infusion of L-citrulline at 9 mg/kg/hr or placebo for up to 48 hours.  The 
study drug or placebo infusion will be discontinued once invasive arterial blood 


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pressure monitoring is discontinued or at 48 hours, whichever comes first. 
• 
Hemodynamic monitoring including: 
− 
Heart rate 
− 
Systemic BP 
− 
O2 saturation 
− 
CVP measurements 
− PAP measurements 
Results will be recorded according to the Institution’s standard of care from 
time of post-op admission to the PICU through 48 hours.  Results at 1, 2, 4, 12, 
24, and 48 hours from baseline (in OR) must be obtained and recorded.  
• 
 ABGs 
− 
PaO2, PaCO2, HCO3, and pH measurements 
• 
ABGs will be drawn according to the Institution’s standard of care. All results 
obtained from the time of post-op admission to the PICU until removal of the arterial 
line or through 48 hours (whichever occurs first) will be recorded. Thoracotomy 
output measurements (for the duration of chest tube placement) 
• 
Blood draw for: 
− 
serum electrolytes, BUN, and creatinine  
− 
CBC with differential 
− 
ACT  
− 
plasma L-citrulline (24 hrs; +/-1 hour. Collect at end of infusion if infusion 
discontinued prior to 24 hours); 1.0 mL  
• 
Urinalysis (not required, but results should be recorded if test is performed 
• 
Inotrope and other concomitant medication documentation 
• 
Documentation of ventilator settings. Recorded approximately every hour (+/- 10 
minutes) from time of separation from CPB until end of mechanical ventilation or 28 
days (whichever occurs first) 
• 
Hypotension assessment (Refractory hypotension is defined as a 20% drop of MAP 
below specific age-related criteria for greater than 30 minutes) (See Section 5.1.2.2) 
• 
Adverse events documentation 
 


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5.1.2.6 Day 2, post-surgery, 48 hours  
The following evaluations will be performed: 
• 
Continuous infusion of L-citrulline at 9 mg/kg/hr or placebo for up to 48 hours.  The 
study drug or placebo infusion will be discontinued once invasive arterial blood 
pressure monitoring is discontinued or at 48 hours, whichever comes first. 
• 
Hemodynamic monitoring including: 
− 
Heart rate 
− 
Systemic BP 
− 
O2 saturation 
− 
CVP measurements 
− PAP measurements 
Results will be recorded according to the Institution’s standard of care from 
time of post-op admission to the PICU through 48 hours.  Results at 1, 2, 4, 
12, 24, and 48 hours from baseline (in OR) must be obtained and recorded.  
• 
 ABGs 
− 
PaO2, PaCO2, HCO3, and pH measurements 
ABGs will be drawn according to the Institution’s standard of care. All results 
obtained from the time of post-op admission to the PICU until removal of the 
arterial line or through 48 hours (whichever occurs first) will be recorded.  
• 
Thoracotomy output measurements (for the duration of chest tube placement) 
• 
Blood draw for: 
− 
serum electrolytes, BUN, and creatinine  
− 
CBC with differential 
− 
ACT  
− 
LFTs 
− 
plasma L-citrulline (48 hrs; +/-1 hour; collect at end of infusion if infusion 
discontinued prior to 48 hours); 1.0 mL 
• 
Inotrope and other concomitant medication documentation 
• 
Documentation of ventilator settings. Recorded approximately every hour (+/- 10 
minutes) from time of separation from CPB until end of mechanical ventilation or 28 
days (whichever occurs first) 


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• 
Hypotension assessment (Refractory hypotension is defined as a 20% drop of MAP 
below specific age-related criteria for greater than 30 minutes) (See Section 5.1.2.2) 
• 
Adverse events documentation 
5.1.2.7 Follow-Up Visit [Day 28 (if still in hospital) or upon Hospital Discharge]  
The following evaluations will be performed: 
• 
Thoracotomy output measurements (for the duration of chest tube placement) 
• 
Blood draw* for: 
− 
serum electrolytes, BUN, and creatinine 
− 
CBC with differential  
− 
LFTs 
− 
Plasma L-citrulline (1.0 mL)  
* (An attempt should be made to obtain final laboratory samples on Day 28 or 
prior to discharge from hospital. If laboratory results within 24 hours are 
available, these may be captured. If results are not available within 24 hours, 
then record the results of the labs obtained closest to discharge from the 
hospital.  These labs must include LFTs.  If no i.v. access is available on Day 
28 or the day of discharge, venipuncture is not required since results of the 
labs obtained closest to discharge may be used. 
• 
Inotrope and other concomitant medication documentation 
• 
Documentation of ventilator settings. Recorded approximately every hour (+/- 10 
minutes) from time of separation from CPB until end of mechanical ventilation or 28 
days (whichever occurs first) 
• 
Adverse events documentation 
Patients withdrawing from the study prior to Day 28 or hospital discharge will be encouraged 
to complete all follow-up assessments.  
5.1.2.8 Follow-Up Phone Assessment (only for patients discharged prior to Day 28) 
Patients discharged prior to Day 28 will have their follow-up visit procedures completed upon 
discharge from the hospital (see section 5.1.2.7).  Therefore, in order to assess their survival 
status at Day 28, patients (or parents, dependent upon patient age) will be contacted by phone 
on Day 28 to assess their status. 


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5.1.3 Duration of Treatment 
The total duration of treatment will be the treatment time during CPB plus 48 hours from time 
of decannulation after CPB onwards. 
 
6 Variables Assessed 
6.1 
Screening, Baseline, and Surgery Assessments 
6.1.1 Demographics 
Relevant demographic data include age, sex, race and ethnicity.  
6.1.2 Echocardiography 
The results of a transthoracic echocardiogram will be required to document the presence of a 
congenital heart defect and to determine eligibility for the study. The results of the 
echocardiogram must be documented in the patient’s medical history so that eligibility can be 
confirmed. 
6.1.3 Physical Examination 
A complete physical examination, including height and weight, will be performed at screening.  
6.1.4 Cardiopulmonary Bypass Assessments 
The following data on CPB will be documented in the OR: Equipment type, cardioplegia 
solution type, composition of isovolumetric exchange fluid for ultrafiltration and/or modified 
ultrafiltration, exchange volumes in and out, documentation that L-citrulline or placebo was 
added to the filtration and hemoconcentration fluids, CPB start and end times and 
decannulation time. 
6.2 
Efficacy and Safety Measurements Assessed  
6.2.1 Efficacy Assessments 
6.2.1.1 Mechanical Ventilation 
The definition of mechanical ventilation includes invasive mechanical ventilation or non-
invasive mechanical ventilation including BPAP or CPAP. Length of time on mechanical 


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ventilation will be assessed from the time of separation from CPB until mechanical ventilation 
is discontinued. 
6.2.1.2 Inotropes 
Inotropes are defined as dopamine, dobutamine, milrinone, epinephrine, phenylephrine, and 
norepinephrine. Inotrope use will be assessed from the start of CPB until the subject is 
discharged from the hospital or at Day 28. 
Total Inotrope Score = Dopamine + Dobutamine + 10*Milrinone + 100*Epinephrine 
+100*Phenylephrine + 100*Norepinephrine. 
6.2.1.3 Hemodynamic Parameters 
Hemodynamic evaluations include heart rate, systemic arterial blood pressure, oxygen 
saturation, and CVP. If a PA catheter is placed for clinical reasons, PAP will be recorded. 
Hemodynamic parameters will be assessed prior to anesthesia for CPB and repeatedly until 
48 hrs post-operation (Day 2). 
6.2.1.4 Plasma Citrulline Concentration 
L-citrulline treatment in this study is designed to maintain a sustained therapeutic plasma 
citrulline level above the target threshold of 100 µmol/L from the initiation of CPB, throughout 
surgery, and for up to 48 hours after surgery. Plasma citrulline concentrations will be assessed 
in both treatment groups to determine the number of patients who reach the therapeutic 
sustained target plasma citrulline level of ≥100 µmol/L.  
Blood collection for assessment of plasma citrulline concentrations will be taken prior to 
surgery, during surgery, 30 minutes post-decannulation after CPB (prior to bolus and again 5 
minutes after bolus), at the specified post-operative time points (6h, 12h, 24h, 48h), and at 
hospital discharge or Day 28, whichever occurs first.  
All plasma citrulline samples will be sent to a central laboratory for analysis. 
6.2.1.5 Thoracotomy Output/Chest Tube Drainage 
The thoracotomy output is defined as the total volume of chest tube drainage recorded in cc 
prior to discontinuation of chest intubation. 
The duration of chest intubation as well as the volume of chest tube drainage will be recorded 
from 30 minutes post decannulation after CPB until chest tube is removed.  


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6.2.1.6 Vasodilators 
Use of vasodilators and infusion dose will be recorded. Examples of vasodilators include 
nitroprusside, nitroglycerin, and nicardipine 
Use of vasodilators will be recorded from first use following separation from bypass, until the 
subject is discharged from the hospital or at Day 28. 
6.2.1.7 Hospitalization 
Hospitalization will be documented from the time of hospital admission until Day 28 or 
discharge, whichever occurs earlier. Days and hours in the PICU will be indicated. 
6.2.2 Safety Assessments 
6.2.2.1 Adverse Events 
Adverse Event Definition 
An adverse event (AE) is any event, side effect, or other untoward medical occurrence, 
(including dosing errors) that may be present during treatment with a medical product or 
intervention and may or may not be related to that product or intervention.  An AE can, 
therefore, be any unfavorable and unintended sign (e.g., a clinically significant abnormal 
laboratory finding), symptom or disease temporally associated with the use of a medicinal 
product or intervention, whether or not the event could be considered related to use of the 
product or intervention.   
Clinical adverse events are illnesses, signs, or symptoms that have appeared or worsened 
during the study or during the follow-up period.  These may include the following: 1) the 
exacerbation of a pre-existing illness, 2) an increase in the frequency or severity of a pre- 
existing event or condition, 3) any condition detected or diagnosed after study drug 
administration even though it may have been present prior to the start of the study or 4) 
continuous persistent disease or symptoms present at baseline that worsen following the start 
of the study.  Laboratory AEs are clinically significant abnormal values obtained on laboratory 
tests during the acute and follow-up period. 
Any medical condition or clinically significant laboratory abnormality with an onset date before 
the date the Informed Consent is signed is considered to be pre-existing and should be 
documented on the Medical History case report form (CRF.)  All AEs from the time the 
Informed Consent is signed, whether believed to be study-related or not, must be recorded on 
the appropriate CRF. 


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It is the responsibility of the investigator to document all AEs that occur during the study.  
Occurrence of AEs will be assessed through observation by Investigational staff and reported 
on the appropriate page of the CRF. 
Serious Adverse Event Definition 
A Serious Adverse Event (SAE) is any untoward medical occurrence or effect that, at any 
dose, results in any of the following outcomes: 
• 
Death 
• 
Life-threatening (subject at immediate risk of death) 
• 
Requires inpatient hospitalization or prolongation of existing hospitalization 
• 
Results in congenital anomaly/birth defect 
• 
Results in a persistent or significant disability or incapacity 
• 
An important medical event that may not result in death, be life-threatening, or require 
hospitalization may be considered a serious adverse event when, based upon 
appropriate medical judgment, it may jeopardize the subject and may require medical 
or surgical intervention to prevent one of the outcomes listed in this definition. 
Documentation and Assessment of AE’s and SAE’s 
Every adverse event, whether serious or not, must be recorded as soon as is reasonably 
possible in the patient’s CRF.  The following must be included: 
• 
date of onset (or exacerbation in the case of worsening of an existing AE) 
• 
 frequency 
• 
duration of effect 
Additionally, the Principal Investigator (PI) must assess every adverse event in the CRF for 
severity and causality and must define the action taken (defined below). 
Assessment of Severity (not to be confused with “seriousness”) 
Each AE/SAE will be assigned a category by the investigator as follows: 
Mild 
An AE/SAE that is easily tolerated by the subject, causes minimal 
discomfort and does not interfere with everyday activities. 


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Moderate 
An AE/SAE that is sufficiently discomforting to interfere with normal 
everyday activities; intervention may be needed. 
Severe 
An AE/SAE that prevents normal everyday activities; treatment or other 
intervention usually needed. 
If there is a change in severity of an AE/SAE, it must be recorded as a separate event. 
Assessment of Causality 
Every effort will be made by the investigator to assess the relationship of the AE, if any, to the 
IMP. Causality should be assessed using the categories defined below: 
Unrelated 
Clinical event where evidence exists that the symptom is definitely 
related to etiology other than the study treatment (e.g., trauma 
secondary to a motor vehicle accident or a symptom suggestive of 
another illness which is not accepted to be a possible reaction to the 
study treatment). 
Unlikely 
Clinical event with a temporal relationship to IMP administration makes 
a causal connection improbable, but that could reasonably be 
explained by underlying disease or other drugs or chemicals. 
Possible 
Clinical events with a reasonable temporal relationship to IMP 
administration, but that could also be explained by concurrent disease 
or other drugs or chemicals that have a possible temporal relationship 
to the study treatment; however, a potential alternative etiology exists 
which may be responsible for the symptom (e.g., fever or irritability 
when other symptoms are present that suggest another etiology, for 
example: upper respiratory infection). 
Probable 
Clinical events with temporal relation to the study treatment, and a 
potential alternative etiology is not apparent (e.g., fever or irritability 
when no other symptoms suggestive of an illness are present) with a 
reasonable time relationship to IMP administration, and is unlikely to 
be attributed to concurrent disease or other drugs or chemicals. 
Definite 
Clinical events with plausible temporal relationship to IMP 
administration, and that cannot be explained by concurrent disease or 
other drugs or chemicals; events which have a timely relation to the 
study and no alternative etiology is present.  They must have occurred 
within a reasonable temporal sequence of the treatment, must be 
reasonably explained and must follow a known pattern of response. 
 


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Action Taken 
 
The investigator will describe the action taken in the appropriate section of the CRF, as 
follows: 
• None 
• IMP stopped 
• IMP temporarily interrupted 
• Concomitant medication 
• Other, specify 
 
“Expected” Adverse Events 
“Expected” adverse events are those AE’s that are associated with the condition or the 
treatment and are listed as such in the trial protocol and/or the Investigator Brochure, whether 
or not they occur in every case. 
Congenital heart surgery and events associated with the surgery are considered “expected” 
in many participants as part of the underlying disease of study and therefore not necessarily 
reportable to the IRB as adverse events.  A compilation of these AE’s is in Table 1. 
 
 


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Table 1: Adverse Events Associated with Pediatric Congenital Heart Defect Surgery 
 
Based on available data with L-citrulline treatment, there are no anticipated risks or side 
effects. The only potential AE would be transient systemic arterial hypotension.  
Unexpected Adverse Events 
An unexpected adverse event is any adverse event that has not been previously described in 
available risk information (included in the protocol or the investigator’s brochure).  
Reporting of Adverse Events 
All AEs occurring during the study must be documented on the relevant CRF pages.  Non-
serious AE’s will only be recorded in the CRF.  Serious AE’s will be recorded in the CRF and 
 


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also reported to the CRO.  SAEs must be reported by the investigator if it occurs during the 
clinical study or within 30 days of the patient receiving the IMP, whether or not the SAE is 
considered to be related to the IMP.   
SAEs must be reported within 24 hours to the following:   
 
Edgar Fenzl 
FGK Clinical Research GmbH 
Heimeranstr. 35 
80339 Munich 
Germany 
Toll-free phone: 1-888-270-3895 
P: + 49 (0) 89 893 119-0 
F: + 49 (0) 89 893 119-180  
Email: edgar.fenzl@fgk-pv.com 
Cc:      safety@fgk-cro.com 
 
The investigator should not wait to receive additional information to document the event before 
notification of an SAE, although additional information may be requested.  Where applicable, 
information from relevant laboratory results, hospital case records, and autopsy reports should 
be obtained. 
SAEs must be followed until resolution or death, including those still ongoing at the Follow-up 
visit. 
The CRO Medical Monitor must, in turn, forward the SAE report to the Asklepion Chief Medical 
Officer (CMO) as soon as is reasonably possible, but in any case, within 24 hours of receipt 
from the site.  The CRO Medical Monitor or designee will notify the participating Institutional 
Review Board(s) (IRBs)/Ethics Committee(s), and CMO will notify the DSMB as appropriate.   
SUSAR (Suspected Unexpected Serious Adverse Reaction) 
A SUSAR is a serious adverse event that is suspected to be at least possibly related to the 
investigational drug (per the definitions of Causality in this section) and Unexpected (per the 
preceding definition of expectedness).   
6.2.2.2 Clinical Laboratory Variables 
The laboratory analyses will be performed at the local hospital/institution laboratories. 
Reference ranges will be supplied by all appropriate laboratory facilities and used by the 
investigator to assess the laboratory data for clinical significance and pathological changes. 
The following laboratory tests will be performed: 


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Hematology 
Hemoglobin, hematocrit, red blood cell (RBC) count, white blood cell (WBC) count (total and 
differential), platelet count  
Serum Electrolytes, BUN, and creatinine 
Serum electrolytes (Na, K, Ca, Mg and Cl), BUN, and creatinine 
Liver function tests 
Bilirubin (total), AP, AST, ALT, and LDH 
Coagulation parameter 
ACT 
A total amount of 16-19 mL of blood will be taken from each subject during the study for both 
clinical laboratory testing and citrulline plasma concentration determinations.  
Urinalysis 
Visual: Color, Clarity/Turbidity 
Chemical: pH, Specific gravity, glucose, ketones, nitrites 
Microscopic: RBCs, WBCs, epithelial cells, casts, bacteria 
6.3 
Appropriateness of Measurements 
All efficacy and safety assessments are widely used and generally recognized as reliable, 
accurate and relevant to the disease condition. 
 
7 Statistical Methods 
7.1 
Statistical and Analytical Plans 
All aspects of statistical analyses will be detailed within a statistical analysis plan (SAP), which 
will be finalized prior to both data base lock and subsequent unblinding.  
Continuous variables will be summarized by means, standard deviations, medians, lower and 
upper quartiles, minimums and maximums. Frequencies and percentages will be used to 
summarize categorical variables. 
Analyses will be performed by visit/ time point, if not stated otherwise. Baseline will be defined 


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as the most recent non-missing value prior to surgery. 
For time to event analyses censoring information will be used for statistical analyses. No other 
imputation of missing values (e.g., last observation carried forward [LOCF]) will be used, if not 
stated otherwise. 
7.1.1 Datasets Analyzed 
Safety Analysis Set (SAF) 
All randomized subjects who received surgery and study medication (independent of whether 
it is L-citrulline or placebo) will be valid for the SAF. Within the SAF a subject will be considered 
for the treatment actually received and not for the treatment assigned by randomization, if 
different. The SAF will be used for the evaluation of the safety assessments.  
Full Analysis Set (FAS) 
The FAS includes all subjects who underwent surgery. Within the FAS a subject will be 
considered for the treatment assigned by randomization and not for the treatment actually 
received, if different, i.e. following the intent-to-treat (ITT) principle. The FAS will be used for 
the evaluation of the efficacy assessments. The FAS serves as the primary efficacy analysis 
set.  
Modified Full Analysis Set (mFAS) 
The mFAS includes all subjects who underwent surgery and received study medication 
(independent of whether it is L-citrulline or placebo). Within the mFAS, a subject will be 
considered for the treatment assigned by randomization and not for the treatment actually 
received, if different, i.e. following the ITT principle. The mFAS will be used for the evaluation 
of the efficacy assessments. The mFAS serves as a sensitivity population for the efficacy 
analyses.  
Per-protocol Analysis Set (PP) 
The PP includes all subjects included in the SAF who had no significant protocol deviations 
and completed through Day 28 or until discharge from the hospital (end of study follow-up). 
For the PP, all subjects will be assigned to the treatment actually received. The PP will only 
be analyzed for main efficacy outcome measures.  
L-citrulline Plasma Levels 
L-citrulline blood levels will be obtained on each patient at set times, and plotted and examined 
to confirm that they are ≥ the threshold level of 100 µmol/L. 


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Blind data review meeting (BDRM) 
Subjects will be assigned to the SAF, FAS, mFAS, and PP during a blind data review meeting 
(BDRM). Further details on the analysis sets, criteria for the PP, and the DRM will be specified 
within the SAP. 
7.1.2 Handling of Missing Data 
For laboratory data, concentrations below the limit of quantification will be set to zero.   
Missing safety data or safety assessments and missing plasma levels will not be estimated 
nor imputed. All safety summaries and analyses will be based on observed data. 
Censoring procedures will be applied for all time variables as described within the sections 
below. Further specifications on handling of missing data are added below, as applicable. 
7.1.3 Demographic and Other Baseline Characteristics 
Baseline data, including demographic data will be analyzed descriptively. Patient disposition, 
including discontinuation and reasons for discontinuation, and subjects’ exposure will be 
tabulated in detail. Concomitant medication will be coded according to World Health 
Organization (WHO) drug dictionary and tabulated accordingly. 
7.1.4 Efficacy Variables 
Analysis of Primary Efficacy Endpoint 
The primary efficacy endpoint is a composite variable consisting of the longer of either (1) 
length of time on mechanical ventilation or (2) length of time of inotrope use.  
The definition of mechanical ventilation includes invasive mechanical ventilation or non-
invasive mechanical ventilation including BPAP or CPAP. Length of time on mechanical 
ventilation will be measured until the subject is discharged from the hospital or at Day 28. The 
length will be derived as the time in hours from separation from CPB until discontinuation of 
all mechanical ventilation including non-invasive support. If invasive or non-invasive 
mechanical ventilation is stopped but has to be restarted, time will continue to accrue. If a 
subject did not receive any mechanical ventilation the length is set to 0. For subjects who died 
before discharge from the hospital or before Day 28, respectively, the observed length of time 
on mechanical ventilation will be used. As sensitivity analyses subjects who died will (1) be 
excluded from analysis and (2) be assigned a length of time on mechanical ventilation of 28 
days. 


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The definition of inotrope use includes medications which are considered within the derivation 
of the total inotrope score (dopamine, dobutamine, milrinone, epinephrine, phenylephrine, 
norepinephrine). Length of inotrope use will be measured until the subject is discharged from 
the hospital or until Day 28, whichever occurs first. The length will be derived as the time of 
first use until last use (in hours). Any duration of re-use will continue to accrue. If a subject did 
not use any inotropes the length is set to 0. For subjects who died before discharge from the 
hospital or before Day 28, respectively, the observed length on inotropes will be used. As 
sensitivity analyses subjects who died will (1) be excluded from analysis and (2) be assigned 
a length of time on inotropes of 28 days. 
Hypothesis to be tested:  
H0: Composite endpoint within the L-citrulline group is equal to that of the placebo group  
H1: Composite endpoint within the L-citrulline group is unequal to that of the placebo group 
H0 will be tested using a Wilcoxon-rank-sum test.  
The level of significance is 1% (two-sided).  
The FAS will be the primary analysis set for the analysis on the composite endpoint. Analyses 
using the mFAS and the PP serve as sensitivity analyses.  
Two-sided 95% confidence intervals using Hodges-Lehman method will be additionally 
calculated. Survival analyses using Kaplan-Meyer estimates and plots, log-rank test and Cox-
regression modeling will serve as sensitivity analyses.  
For survival analyses, different sets of analyses will be done:  
(1) The composite variable for subjects without any mechanical ventilation and without any 
use of inotropes is set to 0, observations will not be censored 
(2) The composite variable for subjects without any mechanical ventilation and without any 
use of inotropes is set to 0, observations will be censored 
(3) Subjects without any mechanical ventilation and without any use of inotropes will be 
deleted from analysis 
Subjects who receive mechanical ventilation or are still using inotropes on the last day of 
observation (which can be Day 28, day of early discontinuation, or day of death) will be 
censored for survival analyses.  


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Analysis of Secondary Efficacy Endpoints 
No formal statistical hypotheses are specified for the secondary endpoint variables. 
Nonetheless statistical testing methods will be applied to compare treatment groups. A 
significance level of 5% (two-sided) will be used for such analyses. Secondary efficacy 
endpoints will be analyzed using the FAS, mFAS and the PP.  
Length of Time on Mechanical Ventilation  
The same definitions and analyses as described for the primary endpoint will be applied.  
Length of Time on Positive Pressure Ventilation 
The same definitions and analyses as described for the primary endpoint will be applied.  
Length of Time of Inotrope Use 
The same definitions and analyses as described for the primary endpoint will be applied.  
Inotrope Score 
The total inotrope score at each hour post-operatively from the time of separation from bypass, 
until the completion of study drug will be calculated as:  
Total Inotrope Score = Dopamine + Dobutamine + 10*Milrinone + 100*Epinephrine 
+100*Phenylephrine + 100*Norepinephrine.  
The total inotrope score will be summarized at each post-operative hour and analyzed using 
a Wilcoxon-Rank sum test and an analysis of variance (ANOVA) with a fixed effect for 
treatment group. Also, a repeated measures analysis of variance will be used to compare total 
inotrope score between placebo and L-citrulline over time.  
Additionally, the total inotrope score over time until Day 28 or hospital discharge will be derived 
and analyzed using a Wilcoxon-Rank sum test and an ANOVA with a fixed effect for treatment 
group. 
The total inotrope score will be imputed to zero after the bypass pump has been removed. It 
will also be imputed as zero for patients not using inotropes at all. For any missing scores 
between two non-missing scores they will be imputed using the most recent non-missing score 
(the last observation carried forward principle).   
Hemodynamic Improvement 
Hemodynamic evaluations include heart rate, systemic arterial blood pressure, oxygen 


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saturation, and central venous pressure. The absolute changes from baseline at hours 1, 2, 
4, 12, 24, and 48 will be compared between groups using an ANOVA with a fixed effect for 
treatment group and baseline level.  
An ANOVA adding site and site-by-treatment group interaction will be conducted as a 
sensitivity analysis. If the p-value for the site-by-treatment group interaction is lower than 0.1 
the analysis will also be provided by site. Summary tables describing descriptive 
measurements will be generated for absolute values and absolute change from baseline 
values for all observed time points.  
Length of time on intubation  
The length will be derived as the time in hours from separation from CPB until discontinuation 
of intubation. Any duration of re-use of intubation will continue to accrue. If a subject did not 
use any intubation the length is set to 0. As a sensitivity analysis, subjects with no use of 
intubations will be excluded. For subjects who died before discharge from the hospital or 
before Day 28, respectively, the observed length of time on intubation will be used. As 
sensitivity analyses subjects who died will (1) be excluded from analysis and (2) be assigned 
a length of time on intubation of 28 days. 
For the length of time on intubation the same analyses as described for the primary endpoint 
will be applied. 
Length of PICU stay  
The length of PICU stay will be calculated as the total number of days postoperative until 
discharge from PICU. For subjects who died before discharge from PICU or before Day 28, 
respectively, the observed length of PICU stay will be used. As sensitivity analyses subjects 
who died will (1) be excluded from analysis and (2) be assigned a length of PICU stay of 28 
days. 
For the length of PICU stay the same analyses as described for the primary endpoint will be 
applied. 
Length of Time on Vasodilators 
Length of time on vasodilators will be measured from first use following separation from 
bypass, until the subject is discharged from the hospital or at Day 28. The length will be derived 
as the time in hours from separation from CPB until discontinuation of all vasodilators. Any 
duration of re-use of vasodilators will continue to accrue. If a subject did not use any 
vasodilators the length is set to 0. As a sensitivity analysis, subjects with no use of vasodilators 


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will be excluded. For subjects who died before discharge from the hospital or before Day 28, 
respectively, the observed length of time on vasodilators will be used. As sensitivity analyses 
subjects who died will (1) be excluded from analysis and (2) be assigned a length of time on 
vasodilators of 28 days. 
For the length of time on vasodilators the same analyses as described for the primary endpoint 
will be applied. 
Length of Hospitalization 
The length of hospitalization will be calculated as the total number of days postoperative until 
discharge from the hospital. For subjects who died before discharge from the hospital or 
before Day 28, respectively, the observed length of hospitalization will be used. As sensitivity 
analyses subjects who died will (1) be excluded from analysis and (2) be assigned a length of 
hospitalization of 28 days. 
For the length of hospitalization, the same analyses as described for the primary endpoint will 
be applied. 
Thoracotomy Output - Duration and Volume of Chest Tube Drainage: 
The total postoperative duration (in hours) that the chest tube is used will be calculated as the 
time from the end of the surgery to the time the chest tube is removed. If an additional chest 
tube is required or reinserted (until discharge from the hospital or at Day 28) the duration that 
the additional chest tube was used (from time of insertion to time of removal) will be added to 
the time the original chest tube was used for the total postoperative duration. If a subject did 
not use any chest tube the duration is set to 0. As a sensitivity analysis, subjects with no use 
of chest tube will be excluded. For subjects who died before discharge from the hospital or 
before Day 28, respectively, the observed duration of chest tube drainage will be used.  
For the total postoperative duration, the same analyses as described for the primary endpoint 
will be applied. Again, as sensitivity analyses subjects who died will (1) be excluded from 
analysis and (2) be assigned a length of time of chest tube placement of 28 days. 
The total amount of chest tube drainage (mL), added up during each duration of chest tube 
as described above, will be summarized using descriptive measures and compared between 
groups using an ANOVA with a fixed effect for treatment group.  
L-citrulline Plasma Levels 
Summary statistics using descriptive measures will be tabulated for plasma L-citrulline levels.  


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7.1.5 Safety Variables 
Safety endpoints will be analyzed using the SAF.  
Adverse Events 
Pre-treatment adverse events and treatment adverse events will be analyzed separately. AEs 
will be summarized by system organ class (SOC) and preferred term (PT) according to 
Medical Dictionary for Regulatory Activities (MedDRA). The number of events, as well as the 
number and rate of affected subjects will be reported. AEs (SOC and PT) will also be 
summarized by seriousness, as well as by severity and relationship to study medication. No 
statistical testing procedures will be applied to the analysis of AEs. No statistical testing 
procedures will be applied to the analysis of AEs. 
Incidence of Refractory Hypotension 
Refractory hypotension is defined as a 20% drop in mean MAP below specific age-related 
criteria for more than 30 minutes. The number of subjects with any refractory hypotension 
from end of surgery until 48 hours will be compared between groups using Fishers’ exact test. 
Additionally, a logistic regression analysis will be applied including factor treatment and site.  
Laboratory Values (CBC with differential, serum creatinine and electrolytes & liver enzymes) 
The absolute values, absolute and percentage changes from baseline will be tabulated using 
summary tables listing descriptive measurements for all observed time points. No statistical 
testing procedures will be applied to the analysis of laboratory values. 
7.2 
Initial Determination of Sample Size Estimate 
The primary endpoint of this study is a composite variable consisting of the longer of either (1) 
length of time on mechanical ventilation or (2) length of inotrope use. The primary analysis will 
be the based on the full analysis set and will be conducted using a Wilcoxon-rank-sum test. 
As a sensitivity analysis, the primary endpoint variable will also be analyzed using survival 
analysis methods.  
The results for the composite endpoint variable, with re-intubation periods included, from the 
Phase IB study, CIT-002-01, are shown in Table 2: 


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Table 2: Phase IB study (CIT-002-01) Composite endpoint results  
 
Mean (SD) 
Placebo 
(N = 11) 
Citrulline 
(N = 11) 
Total  
(N = 22) 
 
 
 
 
Composite endpoint 
44.4 (63.62) 
14.3 (14.06) 
29.3 (47.54) 
 
 
 
 
SD = standard deviation, N = number of patients.  
Data source: Table 14.2.2.5.1.1, CIT-002-01 Clinical Study Report 
 
The sample size calculation was calculated using nQuery 7.0. The common standard 
deviation (SD) was estimated to be 46.3 using the observed SDs of 64 and 14. Again, using 
an assumed SD of 50 and expected mean values of 44 for placebo and 14 for the L-citrulline 
group, the effect size is estimated as being 0.600, which yields a sample size of N=92 per 
group when applying a two-sided Wilcoxon-rank-sum test with a 0.01 significance level and a 
power of 90%.   
The power is even larger than 90% when applying the Phase IB results of the composite 
endpoint variable with re-intubation periods excluded. Only one patient within the Placebo 
group was re-intubated, resulting in a lower mean composite endpoint value of 32 and a SD 
of 26.26.  
To allow for subjects being enrolled but not assigned to the full analysis set, an overall N=95 
subjects per group will be enrolled.  
7.3 
Sample Size Estimation Revision  
A sample size re-estimation will be done as soon as the first 95 consecutively randomized full 
analysis set (FAS)-patients will have undertaken their Follow up visit (Day 28 or Hospital 
Discharge). (Refer to section 5.1.2.7 for details.) 
7.4 
Interim Analysis 
One interim analysis is planned to be carried out when the first 95 consecutively randomized 
full analysis set (FAS)-patients (i.e. 50% of the planned patients) will have undertaken their 
Follow up visit (Day 28 or Hospital Discharge), data required for the analysis of the composite 
endpoint have been entered into the eCRF, and those data have been cleaned appropriately. 
Only a sample size re-estimation will be carried out during this interim analysis; no other 
statistical analyses will be conducted. The sample size re-estimation will be done by an 
independent, unblinded statistician following the same procedures and criteria as for the initial 


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sample estimate, as described in section 7.2. Only the results of the sample size re-estimation 
will be provided to Asklepion.  
Only the re-estimated variance values will be used for sample re-calculation, while the 
assumed values will not be re-estimated but kept as described in Section 7.2. 
In the event that the sample size re-estimation leads to a reduction of the sample size due to 
lower re-estimated variance compared to the assumed variance values, the sample size as 
currently planned will be kept, and not reduced, to ensure robust estimations for safety and 
efficacy variables. 
If the trial is to be continued with the re-estimated sample size, an appropriate protocol 
amendment will be submitted to the FDA as soon as feasible. 
Final analysis will be based on the population of all randomized patients without taking the 
sample size re-estimation results into consideration. Due to the fact that only the interim 
variance will be assessed, no alpha adjustment of statistical analyses is required. 
 
8 Quality Assurance and Quality Control 
8.1 
Audit and Inspection 
Investigator sites and study documentation may be subject to Quality Assurance (QA) audit 
during the course of the study by the sponsor, CRO or its nominated representative. In 
addition, inspections may be conducted by regulatory authorities at their discretion. It is 
important that the investigator(s) and their relevant personnel are available during audits or 
inspections and that sufficient time is devoted to the process. 
8.2 
Monitoring 
Data for each subject will be recorded on an eCRF. Data collection must be completed for 
each subject for whom there is an informed consent form (ICF) and is administered IMP. 
In accordance with current Good Clinical Practice (cGCP) and International Conference on 
Harmonisation (ICH) guidelines, the study monitor will carry out source document verification 
at regular intervals to ensure that the data collected in the eCRF are accurate and reliable. 
The frequency of monitoring visits will be determined by the rate of subject recruitment.  
The following will be reviewed at these visits: 


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• Compliance with the protocol. 
• Consent procedure. 
• Source documents. 
• AE procedures. 
• Storage and accountability of materials. 
The monitoring visits also provide the sponsor with the opportunity to ensure the investigator’s 
obligations and all applicable ICH or health authority regulation requirements are being 
fulfilled. 
The investigator must permit the monitor, the IRB/IEC, the sponsor’s internal auditors and 
representatives from regulatory authorities direct access to all study-related documents and 
pertinent hospital or medical records for confirmation of data contained within the CRFs. 
Subject confidentiality will be protected at all times. 
An electronic medical record may be the source document; however, the study site must 
provide a standard operating procedure(s) (SOPs) that details review and approval of data 
entries by the principal, investigator(s). 
8.3 
Data Management and Coding 
The sponsor or CRO will be responsible for activities associated with the data management 
of this study. This will include setting up a relevant database and data transfer mechanisms, 
along with appropriate validation of data and resolution of queries. Data generated within this 
clinical study will be handled according to the relevant SOPs of the data management and 
biostatistics departments of sponsor or CRO. 
For Electronic Data Capture (EDC): 
Study sites will enter data directly into an EDC system by completing the eCRF via a secure 
internet connection. Data entered into the eCRF must be verifiable against source documents 
at the study site. Data to be recorded directly on the CRF (without prior written or electronic 
record) will be identified and the CRF will be considered the source document. Any changes 
to the data entered into an EDC system will be recorded in the audit trail and is Food and Drug 
Administration (FDA) Code of Federal Regulations (CFR) Title 21 Part 11 compliant. 
Data entered into the eCRF will be validated as defined in the Data Validation Plan. Validation 
includes, but is not limited to, validity checks (for example range checks), consistency checks 
and customized checks (logical checks between variables to ensure that study data are 
accurately reported). A majority of edit checks will be triggered during data entry and will 


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therefore facilitate efficient “point of entry” data cleaning. CRO Clinical Data Management will 
perform both manual eCRF review and review of additional electronic edit checks to ensure 
that the data are complete, consistent and reasonable. The electronic edit checks will run 
continually throughout the course of the study and the issues will be reviewed manually online 
to determine what action needs to be taken. 
Manual queries may be added to the system by Clinical Data Management or Clinical 
Research Associates (CRAs). Data Managers and CRAs are able to remotely and proactively 
monitor the subject eCRFs to improve data quality. 
CRO or designee will be responsible for: 
• eCRF database creation and validation 
• eCRF review and data validation 
 
Medical coding will use MedDRA for concomitant diseases and adverse events and WHODrug 
for therapies. 
Prior to the creation of a site data archive, it is necessary to be sure the database for the study 
in question has been closed. As an EDC system is being utilized for the study, CRO resources 
will produce the site data archives and deliver these archives to the study sites. Site data 
archive shall include not only an archive of the data entered, but also a record of the audit trail 
and any electronically created queries and comments. The site archival material shall be 
produced separately for each study site and then each set of materials or CDs should then be 
delivered to the study site for archival. 
All study materials will be returned to the sponsor within a pre-agreed time interval from final 
database approval. The CRO clinical database (SAS), along with the SAS programs, will be 
stored on the CRO computer for a period of time to be agreed with the sponsor following 
completion of all activities due to be carried out for sponsor for the study prior to archiving. 
The archived electronic file will be destroyed after a pre-agreed period of time. 
Missing or inconsistent data will be queried for clarification. Subsequent modifications to the 
database will be documented. 
 
 


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9 Records and Supplies 
9.1 
Drug Accountability 
On receipt of the IMP, the investigator (or pharmacist) will conduct an inventory of the supplies 
and verify that IMP supplies are received intact and in the correct amounts prior to completing 
a supplies receipt. The pharmacist will retain a copy of this receipt at the study site and return 
the original receipt to the unblinded study monitor. The inventory of supplies at each study site 
may be checked at any time during the study by the unblinded monitor. 
It is the responsibility of the unblinded study monitor to ensure that the investigator (or 
pharmacist) has correctly documented the amount of IMP received and dispensed on the 
dispensing log that will be provided. A full drug accountability log will be maintained at the 
study site at all times. The study monitor will also perform an inventory of IMP at the close-out 
visit to the site. All discrepancies must be accounted for and documented. 
9.2 
Financing and Insurance 
Financing and insurance of this study will be outlined in a separate agreement between CRO 
and the sponsor. 
10 Data and Safety Monitoring Board 
The Data and Safety Monitoring Board (DSMB) will act in an advisory capacity to the 
Coordinating Investigator and Asklepion Pharmaceuticals, LLC to monitor participant safety.  
The DSMB responsibilities are to:  
• 
Protect the safety of the study participants; 
• 
Review the research protocol and plans for data safety and monitoring;  
• 
Evaluate participant risk versus benefit, and other factors that can affect study safety 
outcome; 
• 
Consider factors external to the study when relevant information becomes available, 
such as scientific or therapeutic developments that may have an impact on the safety 
of the participants or the ethics of the trial; 
• 
Report to the study sponsor, Asklepion Pharmaceuticals, on the safety and progress 
of the trial;  


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• 
Make recommendations concerning continuation, termination or other modifications 
of the trial based on the observed beneficial or adverse effects of the treatment 
under study; and 
• 
If applicable, review interim analyses in accordance with stopping rules, which are 
clearly defined in advance of data analysis and have the approval of the DSMB. 
11 Ethics 
11.1  Institutional Review Board / Independent Ethics Committee 
Before initiation of the study at each investigational site, the protocol, all protocol amendments, 
the ICF, and any other relevant study documentation will be submitted to the appropriate 
IRB/IEC. Written approval of the study and all relevant study information must be obtained 
before the study site can be initiated or the IMP is released to the investigator. Any necessary 
extensions or renewals of IRB/IEC approval must be obtained; in particular, for changes to 
the study such as modification of the protocol, the informed consent form, the written 
information provided to subjects and/or other procedures. 
The investigator will report promptly to the IRB/IEC any new information that may adversely 
affect the safety of the subjects or the conduct of the study. The investigator will submit written 
summaries of the study status to the IRB/IEC annually, or more frequently if requested by the 
IRB/IEC. On completion of the study, the sponsor will notify the IRB/IEC that the study has 
ended. 
11.2 Ethical Conduct of the Study 
The Guidelines of the World Medical Association (WMA) Declaration of Helsinki in its revised 
edition (Seoul, 2008), the ICH guidelines for cGCP (CPMP/ICH/135/35), Directive 2001/20/EC 
for EU studies, and by the FDA CFR (Title 21, § 50, § 54, § 56) for US studies, as well as the 
demands of national drug and data protection laws and other applicable regulatory 
requirements will be followed. 
11.3 Subject Information and Consent 
The informed consent will include all 8 basic required elements (45 CFR 46.1116a) as well as 
6 additional elements (45 CFR 46.116b). A waiver of consent will not be sought for this study. 
The study investigator or coordinator will review all portions of the consent in great detail. Any 
questions will be addressed prior to asking for a signature. Study staff will emphasize that the 


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parent or legal guardian may continue to ask questions at any time during the study and 
he/she may withdraw consent at any time.  
Documented informed consent will be obtained from each potential volunteer’s parent or legal 
guardian. Consent must be documented by the volunteer’s parents or legal guardian’s dated 
signature on a Consent Form along with the dated signature of the person conducting the 
consent discussion.  
If the volunteer’s parent or legal guardian is illiterate, an impartial witness should be present 
during the entire informed consent reading and discussion. Afterward, the volunteer’s parent 
or legal guardian should sign and date the informed consent, if capable. The impartial witness 
should also sign and date the informed consent along with the individual who read and 
discussed the informed consent i.e. study staff personnel. 
A copy of the signed and dated consent and assent forms will be given to the volunteer’s 
parent or legal guardian before participation in the trial begins. The original signed consent 
forms will remain in locked, study files and will be available for review at any time. 
The initial informed consent form and any subsequent revised written informed consent form, 
and written information must receive IRB approval in advance of use. The volunteer’s parent 
or legal guardian will be informed in a timely manner if new information becomes available 
that may be relevant to the volunteer’s parent’s or legal guardian’s willingness to continue 
participation in the trial. The communication of this information will be documented. 
The investigator is responsible for ensuring that no subject undergoes any study related 
examination or activity before that subject and/or legal guardian has given written informed 
consent to participate in the study. The written consent must be given by the subject and/or 
the legal guardian of the subject, after detailed information about the study has been given 
and in accordance with any national provisions on the protection of clinical study subjects. 
The verbal explanation will cover all the elements specified in the written information provided 
for the subject. 
The investigator or designated personnel will inform the subject and/or legal guardian of the 
subject of the objectives, methods, anticipated benefits and potential risks and inconveniences 
of the study. The subject’s legal guardian should be given every opportunity to ask for 
clarification of any points he/she does not understand and, if necessary, ask for more 
information. At the end of the interview, the subject’s legal guardian will be given time to 
consider the study, if this is required, or if the subject’s legal guardian requests more time. 
Subjects and/or legal guardians will be required to sign and date the informed consent form. 


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After signatures are obtained, the informed consent form will be kept and archived by the 
investigator in the investigator’s study file for possible inspection by regulatory authorities, the 
IEC, sponsor, and/or CRO personnel. 
It should be emphasized to the subject’s legal guardian that he/she is at liberty to withdraw 
from the study at any time, without penalty or loss of benefits to which the subject is otherwise 
entitled. Subjects’ legal guardians who refuse to give written informed consent should not be 
included. For those subjects whose legal guardians withdraw consent participation in the study 
should be discontinued.  
11.4 Subject Confidentiality (US Studies) 
All personal data collected and processed for the purposes of this study should be managed 
by the investigator and his/her staff with adequate precautions to ensure confidentiality of 
those data, and in accordance with the Health Insurance Portability and Accountability Act, 
applicable to national and/or local laws and regulations on personal data protection. 
Monitors, auditors, and other authorized agents of the sponsor and/or its designee, the ethics 
committees approving this research, and the United States (US) FDA, as well as that of any 
other applicable agency(ies), will be granted direct access to the study subjects’ original 
medical records for verification of clinical study procedures and/or data, without violating the 
confidentiality of the subjects to the extent permitted by the law and regulations. In any 
presentations of the results of this study or in publications, the subjects’ identity will remain 
confidential. 
Volunteer confidentiality: 
The results of the research study may be published by the sponsor, but volunteers’ names or 
identities will not be revealed. Records will remain confidential. In order that confidentiality can 
be maintained, the principal investigators will keep records in locked cabinets and results of 
tests will be coded to prevent association with volunteers’ names. Volunteers’ records will be 
available to the FDA, study staff, representatives of Asklepion Pharmaceuticals and the IRB. 
Serum samples will be collected during this study and stored with code numbers as the only 
identifiers. Laboratory assays containing data from the study will be labelled only with 
volunteer code numbers. 
 


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12 Reporting and Publication, Including Archiving 
Essential documents are those documents that individually and collectively permit evaluation 
of the study and quality of the data produced. After completion of the study, all documents 
and data relating to the study will be kept in an orderly manner by the investigator in a secure 
study file. This file will be available for inspection by the sponsor or its representatives or FDA. 
Essential documents should be retained for 2 years after the final marketing approval in an 
ICH region or for at least 2 years after the discontinuation of clinical development of the IMP. 
It is the responsibility of the sponsor to inform the study site(s) of when these documents no 
longer need to be retained. The investigator must contact the sponsor before destroying any 
study related documentation. In addition, all subject medical records and other source 
documentation will be kept for the maximum time permitted by the hospital, institution or 
medical practice. 
The sponsor must review and approve all results of the study or abstracts for professional 
meetings prepared by the investigator(s). The sponsor agrees to publish the results of this 
study; however, publishing may be delayed until the formal filings with government regulatory 
agencies for market approval are submitted. Published data must not compromise the 
objectives of the study. Data from individual study sites in multi-center studies may not be 
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13 References 
Ananthakrishnan, M., F. E. Barr, et al. (2009). "L-Citrulline ameliorates chronic hypoxia-
induced pulmonary hypertension in newborn piglets." American Journal of Physiology-
Lung Cellular and Molecular Physiology 297(3): L506-L511. 
Apostolakis E., S. F. Kriton, et al. (2010) “Lung dysfunction following cardiopulmonary bypass” 
Journal of Cardiac Surgery 25: (47-55). 
Barr, F. E., H. Beverley, et al. (2003). "Effect of cardiopulmonary bypass on urea cycle 
intermediates and nitric oxide levels after congenital heart surgery." The Journal of 
Pediatrics 142(1): 26-30. 
 
Barr, F. E., R. G. Tirona, et al. (2007). "Pharmacokinetics and safety of intravenously 
administered citrulline in children undergoing congenital heart surgery: potential therapy 
for postoperative pulmonary hypertension." The Journal of Thoracic and Cardiovascular 
Surgery 134(2): 319-326. 
Clark, S. C. (2006). "Lung injury after cardiopulmonary bypass." Perfusion 21(4): 225-
228.Curis, E., I. Nicolis, et al. (2005). "Almost all about citrulline in mammals." Amino 
acids 29(3): 177-205. 
Department of Health and Human Services. Health Insurance Portability and Accountability 
Act of 1996. Pub. L. 104-191. 
Directive 2001/20/EC of the European Parliament and of the Council of 04 April 2001 on the 
approximation of the laws, regulations and administrative provisions of the Member 
States relating to the implementation of good clinical practice in the conduct of clinical 
trials on medicinal products for human use. Official Journal of the European 
Communities L 121, 1.5.2001. 
Haeberle J., S. E. McCandless (2014). "Orphan drugs in development for urea cycle disorders: 
current perspectives." Orphan Drugs: Research and Reviews 4: 63-70. 
Haque I, Zaritsky A. (2007). “Analysis of the evidence for the lower limit of systolic and mean 
arterial blood pressure in children.” Pediatric Critical Care Medicine 8: 138-144 
Huffmyer, J. L. and D. S. Groves (2015). "Pulmonary Complications of Cardiopulmonary 
Bypass." Best Practice & Research Clinical Anaesthesiology 29(2): 163-75. 
International Conference on Harmonisation. ICH Harmonised Tripartite Guideline. Good 
Clinical Practice. CPMP/ICH/135/35. 17 January 1997. 


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Kozik, D. J. and J. S. Tweddell (2006). "Characterizing the inflammatory response to 
cardiopulmonary bypass in children." The Annals of Thoracic Surgery 81(6): S2347-
S2354. 
Moinard, C., I. Nicolis, et al. (2008). "Dose-ranging effects of citrulline administration on 
plasma amino acids and hormonal patterns in healthy subjects: the Citrudose 
pharmacokinetic study." British Journal of Nutrition 99(04): 855-862. 
Russell, I.A.M., M.S. Zwass, et.al. (1998). “The Effects of Inhaled Nitric Oxide on 
Postoperative Pulmonary Hypertension in Infants and Children Undergoing Surgical 
Repair of Congenital Heart Disease.”  Anesthesia and Analgesia 87: 46-51. 
Schulze-Neick I., D. J. Penny, et al. (1999) "L-arginine and substance P reverse the pulmonary 
and endothelial dysfunction caused by congenital heart surgery." Circulation 100:749-
55. 
Schure, A. Y. (2010) “Cardiopulmonary Bypass in Infants and Children: What’s New?” South 
African Journal of Anesthesia and Analgesia 16(1): 25-27. 
Shekerdemian, L. (2009) “Perioperative manipulation of the circulation in children with 
congenital heart disease.”  Heart 95: 1286-1296. 
Singh, R.H., W.J. Rhead, et al. (2005) “Nutritional Management of Urea Cycle Disorders.”  
Critical Care Clinics 21: S27-S35 
Smith, H. A., J. A. Canter, et al. (2006). "Nitric oxide precursors and congenital heart surgery: 
a randomized controlled trial of oral citrulline." The Journal of Thoracic and 
Cardiovascular Surgery 132(1): 58-65. 
Summar, M. (1998). "Molecular genetic research into carbamoyl-phosphate synthase I: 
molecular defects and linkage markers." Journal of Inherited Metabolic Disease 21(1): 
30-39. 
van der Linde, D., E. E. Konings, et al. (2011). "Birth prevalence of congenital heart disease 
worldwide: a systematic review and meta-analysis." Journal of the American College of 
Cardiology 58(21): 2241-2247. 
World Medical Association, Declaration of Helsinki. Ethical Principles for Medical Research 
Involving Human Subjects. Last amended by the 59th WMA General Assembly, Seoul, 
October 2008. 
 
 
 


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14 Appendices 


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14.1 Investigator Signature Page 
Protocol Title: 
A Phase III Double-Blind, Randomized, Placebo Controlled, Multi-
Center Clinical Study to Evaluate the Efficacy and Safety of 
Intravenous L-citrulline for the Prevention of Clinical Sequelae of 
Acute Lung Injury induced by Cardiopulmonary Bypass in Pediatric 
Subjects Undergoing Surgery for Congenital Heart Defects  
Protocol Number: CIT-003-01; Version 4.3.1 
Confidentiality and cGCP Compliance Statement 
I, the undersigned, have reviewed this protocol, including appendices and I will conduct the 
study as described in compliance with this protocol, GCP, and relevant ICH guidelines. 
Once the protocol has been approved by the IRB/IEC, I will not modify this protocol without 
obtaining prior approval of Asklepion Pharmaceuticals, LLC and of the IRB/IEC. I will submit 
the protocol modifications and/or any ICF modifications to Asklepion Pharmaceuticals, LLC 
and IRB/IEC, and approval will be obtained before any modifications are implemented. 
I understand that all information obtained during the conduct of the study with regard to the 
subjects’ state of health will be regarded as confidential. No subjects’ names will be disclosed. 
All subjects will be identified by assigned numbers on all Case Report Forms, laboratory 
samples or source documents forwarded to the sponsor. Clinical information may be reviewed 
by the sponsor or its agents or regulatory agencies. Agreement must be obtained from the 
subject before disclosure of subject information to a third party. 
Information developed in this clinical study may be disclosed by Asklepion, to other clinical 
investigators, regulatory agencies, or other health authority or government agencies as 
required. 
Investigator Signature 
Date 
Printed Name 
Institution 


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14.2 Changes to Protocol 
14.2.1 Version 4.0 
1. Page 2; Study Personnel; CRO Personnel: Added clarification regarding 
responsibilities. 
 
Previously Stated: 
US: Clinical Site Monitoring and Data Management 
Veristat LLC 
 
Europe/Israel: Regulatory Activities, Clinical Site Monitoring, Medical Monitoring/ Safety 
Reporting, Biostatistics, Medical Writing  
FGK Clinical Research GmbH 
Changed To: 
Veristat LLC 
US: Clinical Site Monitoring/Management 
Global: Data Management 
 
FGK Clinical Research GmbH 
Europe/Israel: Regulatory Activities, Clinical Site Monitoring/Management 
Global: Medical Monitoring/Safety Reporting; Biostatistics, Medical Writing  
2. Page 13; Synopsis; Study Treatments: Added clarification for initial bolus dose. 
  Previously Stated: 
 Bolus of 150 mg/kg at the initiation of cardiopulmonary bypass 
Changed To: 
 Bolus of 150 mg/kg at the initiation of cardiopulmonary bypass, but after removal of any 
crystalloid base; 
3. Pages 13-14; Synopsis; Study Treatments: Added clarification for second dose. 
Previously Stated: 
Addition of study medication at a concentration of 200 µmol/L to the filtration or 
hemoconcentration replacement fluid utilized during cardiopulmonary bypass; 
Changed To: 
Addition of study medication at a concentration of 200 µmol/L given as a bolus during 


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bypass. This may be administered as a one-time bolus or multiple administrations to 
compensate for fluids containing L-citrulline that may be removed from the patient during 
the course of the operation and thus to maintain the concentration of 200 µmol/L;  
4. Page 15; Synopsis; Exclusion Criteria; 2nd Bullet: Removed term “invasive” to be 
consistent with remainder of protocol. 
Previously Stated: 
Preoperative requirement for invasive mechanical ventilation or intravenous inotrope 
support 
Changed To: 
Preoperative requirement for mechanical ventilation or intravenous inotrope support 
5. Pages 21-23; Schedule of Study Assessments and Procedures; Footnotes 2, 4, 9, 
11, 13, and 14: Added clarification regarding storage of plasma L-citrulline. 
Previously Stated: 
All L-citrulline plasma samples must be processed within four hours of being drawn, stored 
at -20°F and sent on dry ice to the central lab. 
Changed To: 
All L-citrulline plasma samples must be processed within four hours of being drawn, stored 
at -20°F (28.8°C) or below, and sent on dry ice to the central lab. 
6. Page 22; Schedule of Study Assessments and Procedures; Footnote 6: Added 
clarification regarding recording of hemodynamic monitoring results. 
Previously Stated: 
Results will be recorded every 5 minutes during surgery, and then every hour from time of 
post-op admission to the PICU through 48 hours.  
Changed To: 
Results will be recorded approximately every 5 minutes during surgery, and then 
approximately every hour from time of post-op admission to the PICU through 48 hours.  
Results at Hours 1, 2, 4, 12, 24, and 48 must be obtained and must be within +/- 10 minutes 
of the required time point. 


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7. Page 22; Schedule of Study Assessments and Procedures; Footnote 7: Added 
clarification regarding recording of arterial blood gas results. 
Previously Stated: 
All results obtained during surgery will be recorded, and then recorded every 2-3 hours 
from time of post-op admission to the PICU until removal of arterial line or through 48 hours, 
whichever occurs first. 
Changed To: 
All results obtained during surgery will be recorded, and then recorded every 2-3 hours (+/- 
15 minutes) from time of post-op admission to the PICU until removal of arterial line or 
through 48 hours, whichever occurs first. 
8. Page 22; Schedule of Study Assessments and Procedures; Footnote 12: Added 
clarification regarding recording of ventilator settings. 
Previously Stated: 
Recorded every hour (Qhr) from time of separation from CPB until discontinuation of 
mechanical ventilation 
Changed To: 
Recorded approximately every hour (Qhr) (+/- 10 minutes) from time of separation from 
CPB until discontinuation of mechanical ventilation 
9. Page 41; Section 4.1; Overall Study Design and Plan; Last Paragraph:  Added 
clarification regarding dosing  
Previously Stated: 
190 subjects will be randomized in a 1:1 fashion to either L-citrulline or placebo. L-citrulline 
treatment will include an L-citrulline bolus of 150 mg/kg or placebo at the initiation of CPB, 
the addition of L-citrulline at a concentration of 200 µmol/L or placebo to the filtration or 
hemoconcentration replacement fluid utilized during CPB, an L-citrulline bolus of 20 mg/kg 
or placebo 30 minutes after decannulation from CPB immediately followed by a 9 mg/kg/hr 
continuous infusion of L-citrulline or placebo for 48 hours. 
Changed To: 
190 subjects will be randomized in a 1:1 fashion to either L-citrulline or placebo. L-citrulline 
treatment will include: 1) an L-citrulline bolus of 150 mg/kg or placebo at the initiation of 
CPB, but after removal of any crystalloid base; 2) the addition of L-citrulline at a 


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concentration of 200 µmol/L or placebo given as a bolus during bypass.  This may be 
administered as a one-time bolus or multiple administrations to compensate for fluids 
containing L-citrulline that may be removed from the patient during the course of the 
operation and thus to maintain the concentration of 200 µmol/L; and 3) an L-citrulline bolus 
of 20 mg/kg or placebo 30 minutes after decannulation from CPB immediately followed by 
a 9 mg/kg/hr continuous infusion of L-citrulline or placebo for 48 hours (see Section 4.4.1 
for further details). 
10. Pages 44-45; Section 4.4.1; Investigational Medicinal Products Administered:  
Added clarification regarding dosing  
Previously Stated: 
An L-citrulline bolus of 150 mg/kg or placebo will be administered at the initiation of CPB; 
L-citrulline at a concentration of 200 µmol/L or placebo will be added to the filtration and 
hemoconcentration fluid utilized during CPB; a bolus of 20 mg/kg of L-citrulline or placebo 
will be administered 30 minutes after decannulation from CPB, immediately followed by a 
9 mg/kg/hr continuous infusion of L-citrulline or placebo for 48 hours.  
Changed To: 
The main objective of the dosing schedule, as outlined below, is to achieve a minimum 
systemic target plasma trough level concentration of 100 µmol/L of L-citrulline to provide 
the most beneficial effect as demonstrated in the previous phase 2 study.  As a general 
guiding principal during bypass, once dosing has begun, all fluids entering the circuit must 
contain 200 µmol/L of L-citrulline in order to achieve and maintain the trough level of 100 
µmol/L of L-citrulline. Therefore, if fluids are being added or removed, it is crucial to take 
this into consideration when timing the administration of the required doses.  The 
following section describes the administration of L-citrulline or placebo, while taking into 
account the above objective. 
• The first (initial) dose administered will be an L-citrulline bolus of 150 mg/kg or 
placebo, administered at the initiation of CPB, but after removal of any crystalloid 
base.   
o If fluid is added to the priming circuit, but removed prior to initiation of bypass, 
then the initial 150 mg/kg bolus dose should be added after the crystalloid 
based fluids are removed and at the initiation of bypass.  
o If fluid is added to the priming circuit, but not removed, then the initial 150 
mg/kg dose should be added to the circulating prime used for the bypass.  


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• The second dose administered consists of L-citrulline at a concentration of 200 
µmol/L or placebo, given as a bolus during bypass.  This may be administered as a 
one-time bolus or multiple administrations, to maintain the concentration of 200 
µmol/L. The total dose will be dependent upon the total amount of any volume 
administered and removed during CPB.  This includes the crystalloid portion of the 
cardioplegia so as not to dilute the loading dose; it is significantly important when 
using a hemoconcentrator. 
• The third dose administered will be a bolus of 20 mg/kg of L-citrulline or placebo, 
administered 30 minutes after decannulation from CPB.  
• The above third dose is immediately followed by a 9 mg/kg/hr continuous infusion of 
L-citrulline or placebo for 48 hours.  
11. Page 46; Section 4.4.2; Identity of Investigational Medicinal Products; 2nd 
Paragraph:  Added clarification regarding storage of investigational product 
Previously Stated: 
Study drug should be stored as indicated on the package label: Store at room temperature, 
at 15°C - 30°C (59-86°F).  Study drug should not be refrigerated or frozen. 
Changed To: 
Study drug should be stored as indicated on the package label: Store at 25°C (77°F) or 
below; do not freeze. 
12. Page 46; Section 4.4.2; Identity of Investigational Medicinal Products; 3rd 
Paragraph:  Removed statement regarding replacement fluid  
Previously Stated: 
The L-citrulline bolus and L-citrulline infusion will be provided by each site’s investigational 
pharmacy at a concentration of 50 mg/mL. The filtration or hemoconcentration replacement 
fluid will be provided as standard fluid (Plasmalyte) with L-citrulline added to achieve an L-
citrulline concentration of 200 µmol/L. 
Changed To: 
The L-citrulline bolus and L-citrulline infusion will be provided by each site’s investigational 
pharmacy at a concentration of 50 mg/mL. 
 


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13. Page 47; Section 4.4.4; Method of Assigning Subjects to Treatment Groups: 
Clarified numbering format 
Previously Stated:  
Screened subjects will be assigned a unique 4-digit number, with the first two digits 
identifying the site and the last two digits identifying the subject, e.g. 01-01. At each site, 
the investigator will assign numbers to subjects in ascending order e.g. 01, 02, 03 etc. 
Changed To: 
Screened subjects will be assigned a unique 5-digit number, with the first two digits 
identifying the site and the next three digits identifying the subject, e.g. 01-001. At each 
site, the investigator will assign numbers to subjects in ascending order (e.g. 001, 002, 
003). 
14. Page 52; Section 5.1.2.1; Day 0, Surgery (Intra-Operative); 4th Bullet: Added 
clarification regarding dosing 
Previously Stated: 
Bolus of L-citrulline at 150 mg/kg or placebo at initiation of CPB; addition of L-citrulline at a 
concentration of 200 µmol/L or placebo to the filtration and hemoconcentration fluids 
utilized during CPB 
Changed To: 
Bolus of L-citrulline at 150 mg/kg or placebo at initiation of CPB; addition of L-citrulline at a 
concentration of 200 µmol/L or placebo given as a bolus during bypass.  This may be 
administered as a one-time bolus or multiple administrations to compensate for fluids 
containing L-citrulline that may be removed from the patient during the course of the 
operation and thus to maintain the concentration of 200 µmol/L.  The total dose will be 
dependent upon the total amount of any volume administered and removed during CPB.   
15. Pages 52-59; Section 5.1.2; Treatment Period; All Visits: Added clarification 
regarding recording of hemodynamic monitoring, arterial blood gases, and 
ventilator settings 
Previously Stated: 
1) Hemodynamic Monitoring (Day 0, Surgery (Intra-Operative)): Results will be recorded 
every 5 minutes after induction of anesthesia and during surgery 
Hemodynamic Monitoring (Day 0, Post-Surgery, 0 Hours through Day 2, Post-Surgery, 
48 Hours): Results will be recorded every hour from time of post-op admission to the 


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PICU through 48 hours.   
2) Arterial Blood Gases (Day 0, Post-Surgery, 0 Hours through Day 2, Post-Surgery, 48 
Hours): 
Results will be recorded every 2-3 hours from time of post-op admission to the PICU 
until removal of arterial line or through 48 hours, whichever occurs first. 
3) Ventilator Settings (Day 0, Post-Surgery, 0 Hours through Follow-Up Visit (Day 28 or 
Hospital Discharge)): Recorded every hour from time of separation from CPB until 
end of mechanical ventilation or 28 days (whichever occurs first) 
Changed To: 
1) Hemodynamic Monitoring (Day 0, Surgery (Intra-Operative)): Results will be recorded 
approximately every 5 minutes after induction of anesthesia and during surgery 
Hemodynamic Monitoring (Day 0, Post-Surgery, 0 Hours through Day 2, Post-Surgery, 
48 Hours):  Results will be recorded approximately every hour from time of post-op 
admission to the PICU through 48 hours.  Results at Hours 1, 2, 4, 12, 24, and 48 must 
be obtained and must be within +/- 10 minutes of the required time point. 
2) Arterial Blood Gases (Day 0, Post-Surgery, 0 Hours through Day 2, Post-Surgery, 48 
Hours):   Results will be recorded every 2-3 hours (+/- 15 minutes) from time of post-
op admission to the PICU until removal of arterial line or through 48 hours, whichever 
occurs first. 
3) Ventilator Settings (Day 0, Post-Surgery, 0 Hours through Follow-Up Visit (Day 28 or 
Hospital Discharge)): Recorded approximately every hour (+/- 10 minutes) from time 
of separation from CPB until end of mechanical ventilation or 28 days (whichever 
occurs first) 
16. Pages 53-58; Section 5.1.2.2 through 5.1.2.6; Treatment Period; Visits Day 0, post-
surgery, 0 hours through Day 2 post-surgery, 48 hours: Added age-related criteria 
for MAP 
Previously Stated (Day 0, post-surgery, 0 hours): 
Hypotension assessment (Refractory hypotension is defined as a 20% drop of mean 
arterial blood pressure [MAP] below specific age-related criteria for greater than 30 
minutes) 
Changed To: 
Hypotension assessment (Refractory hypotension is defined as a 20% drop of mean 
arterial blood pressure [MAP] below specific age-related criteria (Haque and Zaritsky 


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(2007) for greater than 30 minutes) (See below) 
− 
Infants:  
MAP of 40 
− 
Age 1 year:       MAP of 40 
− 
Age 2 years:        MAP of 44 
− 
Age 3 years:        MAP of 47 
− 
Age 4 years:        MAP of 52 
− 
Age 6 years:        MAP of 53 
− 
Age 7 years:        MAP of 52 
− 
Age 8 years:        MAP of 54 
− 
Age 9 years:        MAP of 55 
− 
Age 10 years:      MAP of 56 
− 
Age 11 years:      MAP of 57 
− 
Age 12 years:      MAP of 58 
− 
Age 13 years:      MAP of 59 
− 
Age 14 years:      MAP of 61 
− 
Age 15 years:      MAP of 62 
− 
Age 16 years:      MAP of 63 
− 
Age 17-18 years: MAP of 64 
Previously Stated (Day 0, post-surgery, 6 hours through Day 2, post-surgery, 48 hours): 
Hypotension assessment (Refractory hypotension is defined as a 20% drop of mean 
arterial blood pressure [MAP] below specific age-related criteria for greater than 30 
minutes) 
Changed To: 
Hypotension assessment (Refractory hypotension is defined as a 20% drop of mean 
arterial blood pressure [MAP] below specific age-related criteria for greater than 30 
minutes) (See Section 5.1.2.2) 
17. Page 59; Section 5.1.2.8; Follow-Up Phone Assessment: Added clarification 
regarding assessment 
Previously Stated: 
Patients discharged prior to Day 28 will have their follow-up visit procedures completed 
upon discharge form the hospital (see section 5.1.2.7).  Therefore, in order to assess their 
status at Day 28, patients will be contacted by phone on Day 28 to assess their status. 
 
Changed To: 
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upon discharge form the hospital (see section 5.1.2.7).  Therefore, in order to assess their 
survival status at Day 28, patients (or parents, dependent upon patient age) will be 
contacted by phone on Day 28 to assess their status. 
18. Page 80; Section 10; Data and Safety Monitoring Board: Added section regarding 
responsibilities of DSMB 
Previously Stated: 
N/A 
Changed To: 
Data and Safety Monitoring Board 
The Data and Safety Monitoring Board (DSMB) will act in an advisory capacity to the 
Coordinating Investigator and Asklepion Pharmaceuticals, LLC to monitor participant 
safety.  The DSMB responsibilities are to:  
• 
Protect the safety of the study participants; 
• 
Review the research protocol and plans for data safety and monitoring;  
• 
Evaluate participant risk versus benefit, and other factors that can affect study 
safety outcome; 
• 
Consider factors external to the study when relevant information becomes 
available, such as scientific or therapeutic developments that may have an impact 
on the safety of the participants or the ethics of the trial; 
• 
Report to the study sponsor, Asklepion Pharmaceuticals, on the safety and 
progress of the trial;  
• 
Make recommendations concerning continuation, termination or other 
modifications of the trial based on the observed beneficial or adverse effects of 
the treatment under study; and 
• 
If applicable, review interim analyses in accordance with stopping rules, which are 
clearly defined in advance of data analysis and have the approval of the DSMB. 
 
 
 
 
 


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14.2.2 Version 4.1 
1. Page 22; Schedule of Study Assessments and Procedures; Footnote 8: Removed 
reference to filtration and hemoconcentration fluids for 200 μmol/L or placebo 
dose. 
Previously Stated: 
Begin bolus of intravenous (i.v.) L-citrulline at 150 mg/kg or placebo at initiation of CPB & 
add L-citrulline at a concentration of 200 μmol/L or placebo to the filtration and 
hemoconcentration fluids utilized during CPB. 
Changed To: 
Begin bolus of intravenous (i.v.) L-citrulline at 150 mg/kg or placebo at initiation of CPB & 
add L-citrulline at a concentration of 200 μmol/L or placebo given as a bolus during bypass.  
This may be administered as a one-time bolus or multiple administrations, to maintain the 
concentration at 200 µmol/L. 
2. Page 51; Section 5.1.2.1; Day 0, Surgery (Intra-Operative); Collection of CPB Data; 
Sub-bullet 5: Updated wording regarding documentation of L-citrulline or placebo. 
Previously Stated: 
Documentation that L-citrulline or placebo was added to the filtration and 
hemoconcentration fluids 
 
 
Changed To: 
Documentation of L-citrulline or placebo administration 
 
 
 


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14.2.3 Version 4.2 
1. Cover Page:  Added EudraCT Number. 
2016-002427-28 
2. Page 13; Synopsis; Study Design: Added clarification for second dose. 
Previously Stated: 
…the addition of L-citrulline at a concentration of 200 μmol/L or placebo to the filtration or 
hemoconcentration fluid utilized during cardiopulmonary bypass,… 
Changed To: 
…the addition of L-citrulline at a concentration of 200 μmol/L or placebo given as a bolus 
during cardiopulmonary bypass,… 
3. Page 15; Synopsis; Subject Selection: Added further clarification regarding 
females of child-bearing potential.  Added new exclusion. 
 
Previously Stated: 
• 
Pregnancy 
Changed To: 
• 
Pregnancy; Sexually active females of child-bearing potential must be willing to 
practice an acceptable method of birth control for the duration of study participation 
(e.g. oral contraceptive, hormonal implant, intra-uterine device) 
• 
Participation in another clinical trial within 30 days of Screening or while participating 
in the current study, including the 28 days of follow-up post study drug 
administration.  
4. Page 20; Schedule of Study Assessments and Procedures: Moved “x(s)” for 
laboratory specimens. 
Moved blood draws for serum electrolytes, BUN, creatinine, CBC with differential, 
Coagulation: ACT, and LFTs, and Urinalysis from Day 0, Pre-Op to Screen, Day -14 to 0. 
5. Page 20; Schedule of Study Assessments and Procedures: Deleted “x” for 
laboratory specimens. 
Deleted blood draw for LFTs at Day 1, 24 hours. 


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6. Page 20-21; Schedule of Study Assessments and Procedures: Updated Schedule of 
Study Assessments and Procedures. 
Moved “x(s)” and rearranged order of procedures to be consistent with changes made to 
collection of laboratory specimens. 
7. Page 21; Schedule of Study Assessments and Procedures; Footnotes 1,2,3: 
Changed timing for blood draws for baseline laboratory values.  Added statement 
regarding acceptability of using pre-op laboratory values for Screening.  Also 
changed order of footnotes to match new placement in Schedule of Assessments 
and Procedures. 
Previously Stated: 
1 Weight only 
2 Blood collected for baseline laboratory values and plasma L-citrulline levels (prior to 
surgery); 3 mL total (0.5 for plasma L-citrulline).  All L-citrulline plasma samples must be 
processed within four hours of being drawn, stored at -20°F (28.8°C) or below, and sent on 
dry ice to the central lab. 
3 Urinalysis not required, but results should be recorded if test is performed  
Changed To: 
1 Results of laboratory tests done as standard of care for pre-op testing are acceptable and 
may be recorded as screening values, provided the tests were performed within 14 days 
of Day 0.  If any of the required tests were not done as part of the pre-op testing, these 
must be completed within 14 days of Day 0.  
2Urinalysis not required, but results should be recorded if test was performed. 
3 Weight only 
8. Page 21-23; Schedule of Study Assessments and Procedures; Footnotes 2, 4, 9, 11, 
13, and 14: Changed volume of blood draw for plasma L-citrulline and deleted total 
volume for blood draw. 
Previously Stated: 
2,4,14 Blood collected for … laboratory values and plasma L-citrulline levels (…); 3.0 mL total; 
(0.5 mL for plasma L-citrulline).  
9,11,13 Blood collected for plasma L-citrulline levels (…); 0.5 mL 
Changed To: 


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5,15 Blood collected for … laboratory values and plasma L-citrulline levels (…); (1.0 mL for 
plasma L-citrulline).  
4,10,12,14 Collect 1.0 mL of blood for plasma L-citrulline levels… 
9. Page 43-44; Section 4.3.3; Exclusion Criteria; 3rd, 5th, and 6th bullet: Added 
additional detail to match synopsis.  Added further clarification regarding females 
of child-bearing potential.  Added new exclusion. 
Previously Stated: 
• 
Presence of fixed or idiopathic pulmonary hypertension prior to surgical repair 
• 
Pregnancy 
Changed To: 
• 
Presence of fixed or idiopathic pulmonary hypertension (i.e. Eisenmenger’s 
Syndrome) prior to surgical repair 
• 
Pregnancy; Females of child-bearing potential must be willing to practice an 
acceptable method of birth control for the duration of study participation (e.g. oral 
contraceptive, hormonal implant, intra-uterine device) 
• 
Participation in another clinical trial within 30 days of Screening or while participating 
in the current study, including the 28 days of follow-up post study drug 
administration.  
10. Page 46; Section 4.4.1; Investigational Medicinal Products Administered 3rd bullet: 
Added additional detail to match synopsis. 
Previously Stated: 
• 
The above third dose is immediately followed by a 9 mg/kg/hr continuous infusion of 
L-citrulline or placebo for 48 hours.  
Changed To: 
• 
The above third dose is immediately followed by a 9 mg/kg/hr continuous infusion of 
L-citrulline or placebo for up to 48 hours.  The study drug or placebo infusion will be 
discontinued once invasive arterial blood pressure monitoring is discontinued or at 
48 hours, whichever comes first. 
 
 


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11. Page 48; Section 4.4.5; Selection of Doses in the Study; Last Paragraph: Added 
clarification regarding dosing regimen. 
Previously Stated: 
This treatment regimen will also be used in the current study.  
Changed To: 
This treatment regimen, with clarification added for the dose(s) given during bypass, will 
also be used in the current study.  
12. Page 51; Section 5.1.1.1; Screening Visit (Day -14 to Day 0): Added new bullet to 
allow use of results of laboratory specimens collected for pre-op testing.  
Previously Stated: 
N/A 
Changed To: 
• 
Blood draw*: 
− 
serum electrolytes (sodium (Na), potassium (K), calcium (Ca), magnesium 
(Mg) and chloride (Cl)), BUN, and creatinine 
− 
CBC with differential 
− ACT 
− 
liver function tests (LFTs), including bilirubin (total), alkaline phosphatase 
(AP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and 
lactate dehydrogenase (LDH) 
 
* Results of laboratory tests done as standard of care for pre-op testing are 
acceptable and may be recorded as screening values, provided the tests were 
performed within 14 days of Day 0.  If any of the required tests were not done as 
part of the pre-op testing, these must be completed within 14 days of Day 0. 
• 
Urinalysis (not required, but results should be recorded if test is performed) 
− 
Visual: Color, Clarity/Turbidity 
− 
Chemical: pH, Specific gravity, glucose, ketones, nitrites 
− 
Microscopic: RBCs, WBCs, epithelial cells, casts, bacteria 
 
 


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13. Page 51; Section 5.1.1.2; Baseline Visit (Day of Surgery, Pre-operation); 6th and 7th 
bullets: Deleted total volume for blood draw and deleted most Day of Surgery labs.  
Changed volume for plasma L-citrulline sample. Also deleted Urinalysis (moved to 
Screening; see above).  
Previously Stated: 
• 
Blood draw (prior to surgery; in the operating room [OR], after induction of anesthesia); 
3 mL total for: 
− 
serum electrolytes (sodium (Na), potassium (K), calcium (Ca), magnesium 
(Mg) and chloride (Cl)), BUN, and creatinine 
− 
CBC with differential 
− 
ACT 
− 
liver function tests (LFTs), including bilirubin (total), alkaline phosphatase (AP), 
aspartate aminotransferase (AST), alanine aminotransferase (ALT), and 
lactate dehydrogenase (LDH) 
− 
plasma L-citrulline (0.5 mL)  
• 
Urinalysis (not required, but results should be recorded if test is performed) 
− 
Visual: Color, Clarity/Turbidity 
− 
Chemical: pH, Specific gravity, glucose, ketones, nitrites 
− 
Microscopic: RBCs, WBCs, epithelial cells, casts, bacteria 
Changed To: 
• 
Blood draw (prior to surgery; in the operating room [OR], after induction of anesthesia 
total for: 
− 
plasma L-citrulline (1.0 mL)  
14. Page 53; Section 5.1.2.1; Day 0, Surgery (Intra-operative); 5th bullet: Deleted total 
volume for blood draw and changed volume for plasma L-citrulline sample. 
Previously Stated:  
• 
Blood draw (10 minutes after study drug is administered); 3 mL total for: 
− 
serum electrolytes, BUN, and creatinine  
− 
CBC with differential 
− 
ACT  


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− 
plasma L-citrulline  
Changed To: 
• 
Blood draw (10 minutes after study drug is administered): 
− 
serum electrolytes, BUN, and creatinine  
− 
CBC with differential 
− 
ACT  
− 
plasma L-citrulline (1.0 mL) 
15. Page 53-54; Section 5.1.2.2; Day 0, post-surgery, 0 hours (30 minutes post 
decannulation); 1st, 2nd, 5th, and 6th bullets: Changed volume of blood drawn for 
plasma L-citrulline sample. Added additional detail to match synopsis.  Added 
clarification regarding thoracotomy outputs. 
Previously Stated:  
• 
Blood draw for: 
− 
 plasma L-citrulline (prior to bolus); 0.5 mL 
• 
Bolus of L-citrulline at 20 mg/kg or placebo 30 minutes after decannulation from CPB 
immediately followed by a continuous infusion of L-citrulline at 9 mg/kg/hr or placebo  
• 
Thoracotomy output measurements  
• 
Blood draw for: 
− 
plasma L-citrulline (5 minutes after bolus); 0.5 mL 
Changed To: 
• 
Blood draw for: 
− 
 plasma L-citrulline (prior to bolus); 1.0 mL 
• 
Bolus of L-citrulline at 20 mg/kg or placebo 30 minutes after decannulation from CPB 
immediately followed by a continuous infusion of L-citrulline at 9 mg/kg/hr or placebo 
for up to 48 hours.  The study drug or placebo infusion will be discontinued once 
invasive arterial blood pressure monitoring is discontinued or at 48 hours, whichever 
comes first. 
• 
Thoracotomy output measurements (for the duration of chest tube placement) 
• 
Blood draw for: 
− 
plasma L-citrulline (5 minutes after bolus); 1.0 mL 


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16. Page 54-58; Section 5.1.2.3 through 5.1.2.6; Day 0, post-surgery, 6 and12 hours, 
Day 1, post-surgery, 24 hours, and Day 2, post-surgery, 48 hours; 1st, 4th, and 5th 
bullets: Added additional detail to match synopsis.  Added clarification regarding 
thoracotomy outputs.  Changed volume of blood drawn for plasma L-citrulline 
sample. 
Previously Stated: 
• 
Continuous infusion of L-citrulline at 9 mg/kg/hr or  
• 
Thoracotomy output measurements  
• 
Blood draw for: 
− 
plasma L-citrulline (6 {12, 24, 48} hrs; +/-1 hour; collect at end of infusion if infusion 
discontinued prior to 6 {12,24,48} hours); 0.5 mL 
Changed To:  
• 
Continuous infusion of L-citrulline at 9 mg/kg/hr or placebo for up to 48 hours.  The 
study drug or placebo infusion will be discontinued once invasive arterial blood 
pressure monitoring is discontinued or at 48 hours, whichever comes first. 
• 
Thoracotomy output measurements (for the duration of chest tube placement) 
• 
Blood draw for: 
− 
plasma L-citrulline (6 {12, 24, 48} hrs; +/-1 hour; collect at end of infusion if 
infusion discontinued prior to 6 {12, 24, 48} hours); 1.0 mL 
17. Page 57; Section 5.1.2.5; Day 1, post-surgery, 24 hours: Deleted total blood 
volume.  Deleted LFTs.   
Previously Stated: 
• 
Blood draw (3 mL total) for: 
− 
serum electrolytes, BUN, and creatinine  
− 
CBC with differential 
− 
ACT  
− 
LFTs 
 
Changed To: 
• 
Blood draw for: 


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− 
serum electrolytes, BUN, and creatinine  
− 
CBC with differential 
− 
ACT  
 
18. Page 58; Section 5.1.2.6; Day 2, post-surgery, 48 hours: Deleted total blood 
volume.   
Previously Stated: 
• 
Blood draw (3 mL total) for: 
Changed To: 
• 
Blood draw for: 
19. Page 59; Section 5.1.2.7; Follow-Up Up Visit [Day 28 (if still in hospital) or upon 
Hospital Discharge]: Added thoracotomy output to ensure collection of data if 
patient still in hospital.  Changed volume for plasma L-citrulline sample. 
Previously Stated: 
• 
Blood draw* for: 
− 
serum electrolytes, BUN, and creatinine* 
− 
CBC with differential*  
− 
LFTs* 
* (An attempt should be made to obtain final laboratory samples while i.v. 
access is available, either on Day 28 or prior to discharge from hospital. If 
laboratory results within 24 hours are available, these may be captured. If no 
i.v. access is available, venipuncture is not required unless indicated for 
clinical reasons.) 
− 
Plasma L-citrulline (0.5 mL)  
Changed To: 
• 
Thoracotomy output measurements (for the duration of chest tube placement) 
• 
Blood draw* for: 
− 
serum electrolytes, BUN, and creatinine 
− 
CBC with differential  
− 
LFTs 
− 
Plasma L-citrulline (1.0 mL)  


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* (An attempt should be made to obtain final laboratory samples while i.v. access 
is available, either on Day 28 or prior to discharge from hospital. If laboratory 
results within 24 hours are available, these may be captured. If no i.v. access is 
available, venipuncture is not required unless indicated for clinical reasons.) 
 
20. Page 68; Section 6.2.2.2; Clinical Laboratory Variables: Adjusted total volume of 
blood draw needed for clinical laboratory testing. 
Previously Stated: 
A total amount of 14-17 mL of blood will be taken from each subject during the study for 
both clinical laboratory testing and citrulline plasma concentration determinations.  
Changed To: 
A total amount of 16-19 mL of blood will be taken from each subject during the study for 
both clinical laboratory testing and citrulline plasma concentration determinations.  
 
 


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14.2.4 Version 4.3 
1. Page 3; Study Personnel; CRO Personnel: Deleted Veristat, LLC; Added 
ResearchPoint Global. 
Previously Stated: 
 
Veristat LLC 
118 Turnpike Rd, Suite 200 
Southborough, MA  01772 
P: 508-429-7340 
www.veristat.com 
Changed To: 
 
ResearchPoint Global 
5301 Southwest Parkway 
Suite 100 
Austin, TX  78735 
Phone: 512-343-1092 
www.researchpointglobal.com 
 
2. Page 14; Synopsis; Subject Selection; Exclusion Criteria: Clarified exclusion 
regarding left-sided AV valve regurgitation 
 
Previously Stated: 
 
 
Significant left-sided atrioventricular (AV) valve regurgitation  
Changed To: 
 
Significant left-sided atrioventricular (AV) valve regurgitation not amenable to 
surgical correction 
 
3. Page 22; Schedule of Study Assessments and Procedures; Footnote 7:  Changed 
requirement for obtaining hemodynamics to align with Institution’s standard of 
care. 
Previously Stated: 
 
…Results will be recorded approximately every 5 minutes during surgery, and then 
approximately every hour from time of post-op admission to the PICU through 48 hours.   
 
 
 


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Changed To: 
 
… Results will be recorded, during surgery and from time of post-op admission to the PICU 
through 48 hours, according to the Institution’s standard of care.   
 
4. Page 22; Schedule of Study Assessments and Procedures; Footnote 8:  Changed 
requirement for obtaining ABGs to align with Institution’s standard of care. 
Previously Stated: 
 
…Baseline values are first recorded in the OR by Anesthesia.  All results obtained during 
surgery will be recorded, and then recorded every 2-3 hours (+/- 15 minutes) from time of 
post-op admission to the PICU until removal of arterial line or through 48 hours, whichever 
occurs first. 
 
Changed To: 
…ABGs will be drawn according to the Institutions’ standard of care.  Baseline results are 
first recorded in the OR by Anesthesia.  All results obtained during surgery, and from the 
time of post-op admission to the PICU until removal of the arterial line or through 48 hours 
(whichever occurs first), will be recorded. 
 
5. Page 28; Section 1.2; L-citrulline: Updated section to clarify urea cycle. 
Previously Stated: 
Citrulline is a naturally occurring amino acid and the first intermediate in the urea cycle 
(Figure 1). The first two steps of the hepatic urea cycle, leading to the production of 
citrulline, are carried out by carbamoyl phosphate synthetase I (CPSI) and ornithine 
transcarbamylase (OTC). These two enzymes are limited to the liver and gut. Afterwards, 
citrulline is transported from the mitochondria to the cytoplasm. There, argininosuccinate 
…endothelium (Summar 1998). 
Changed To: 
Citrulline is a naturally occurring amino acid and the first intermediate in the urea cycle. 
Most medical personnel think of the urea cycle as a hepatic metabolic pathway that serves 
to convert nitrogen waste from protein breakdown into urea for excretion.  It is not generally 
appreciated that key elements of the urea cycle exist in other tissues where, for example, 
in lung, it is ultimately responsible for the production of NO under precise metabolic control, 
as shown in Figure 1.  The shift in function of the urea cycle comes about because the 
enzymes catalyzing the first two steps of the hepatic urea cycle (carbamoyl phosphate 
synthetase I (CPSI) and ornithine transcarbamylase (OTC), which lead to the production of 
citrulline, are expressed only in the liver and gut.  In the liver and gut, newly synthesized 


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citrulline is transported from the mitochondria to the cytoplasm. In contrast, other tissues 
such as lung are dependent upon exogenous circulating citrulline since they cannot 
synthesize it endogenously.  Following either endogenous synthesis or transport into a non-
hepatic cell, argininosuccinate…endothelium (Summar 1998). 
 
6. Page 29; Figure 1; Title and Figure: Changed Title and Figure to be consistent with 
new information added to Section 1.2 
Previously Stated: 
Figure 1: Schematic description of the hepatic urea cycle 
[See previous figure] 
Figure 10: Schematic description of the urea cycle in lung 
[See new figure] 
 
7. Page 32-40; Section 1.4; Clinical data: Updated section to reflect results of data 
from CIT-002-01 final Clinical Study Report. 
a. Page 32; 3rd Paragraph: 
Previously Stated: 
Postoperative pulmonary hypertension developed in 9 patients, 6 of 20 (30%) in the 
placebo group and 3 of 20 (15%) in the citrulline group (P=0.451), all of whom had plasma 
citrulline concentrations less than age-specific norms. None of the patients with plasma 
citrulline level >37 µmol//L developed pulmonary hypertension. 
Changed To: 
The most significant result was that postoperative pulmonary hypertension developed in 9 
patients, 6 of 20 (30%) in the placebo group and 3 of 20 (15%) in the citrulline group 
(P=0.451), all of whom had plasma citrulline concentrations less than the age-specific 
norms. In a contingency analysis that reached statistical significance, all of the children 
developing postoperative pulmonary hypertension had a plasma citrulline level <37 µmol/L.  
Conversely, none of the children with citrulline levels >37 µmol/L developed pulmonary 
hypertension.  This result implicated low citrulline levels as a strong risk factor and possible 
causal element in the development of postoperative pulmonary hypertension.  Therefore, 
more aggressive citrulline supplementation, resulting in consistent citrulline concentrations 
in excess of 37 µmol/L, could potentially prevent postoperative pulmonary hypertension. 


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b. Page 33; 4th Paragraph: 
Previously Stated: 
It was concluded that elevations in plasma citrulline concentrations above age-specific 
norms, whether naturally occurring or with citrulline supplementation, were associated with 
a decreased risk of postoperative pulmonary hypertension. Therefore, more aggressive 
citrulline supplementation, resulting in consistent citrulline concentrations in excess of 37 
µmol/L, could prevent postoperative pulmonary hypertension. 
Changed To: 
N/A [Deleted first sentence; moved 2nd sentence to last sentence of previous paragraph] 
c. Page 34; 2nd Paragraph: 
Previously Stated: 
… clinical study report is currently being generated. 
Changed To: 
… full clinical study report is available. 
d. Page 34; 3rd Paragraph: 
Previously Stated: 
…after end of CPB… 
Changed To: 
…after separation from CPB… 
e. Page 35; 1st Paragraph: 
Previously Stated: 
Patients receiving i.v. L-citrulline showed a reduced duration of mechanical ventilation as 
shown in Figure 5. The differences in mechanical ventilation were statistically significant by 
the Wilcoxon rank sum test (p = 0.0222) and by the ANOVA t-test (p = 0.0317). 
Changed To: 
Patients receiving i.v. L-citrulline showed a reduced duration of mechanical ventilation as 
shown in Figure 5 and Figure 6.  The duration of postoperative invasive mechanical 
ventilation was derived as the time in hours from separation from CPB until endotracheal 
extubation. The mean duration of invasive mechanical ventilation was clearly longer in the 


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placebo group (37 hours) than in the citrulline group (5 hours).  Kaplan-Meier survival 
analyses confirmed this reduced duration as seen in Figure 5 with re-intubation time 
included and ventilation time censored and a log rank p-value of 0.0498, and in Figure 6 
with reintubation time included and zero ventilation time not censored and a log rank p-
value of 0.0089. 
f. Page 36; Figure 5; Title and Figure: 
Previously Stated: 
Figure 5: Kaplan-Meier Survival Analysis of the Duration of Invasive Mechanical Ventilation 
from End of Surgery until Last Extubation (Excluding Re-intubation)  
[See previous figure] 
Changed To: 
Figure 5: Kaplan-Meier Survival Analysis of the Duration of Invasive Mechanical Ventilation 
(Including Re-intubation Time): Censored 
[See new figure] 
g. Page 37; Added new Figure 6: 
Previously Stated: 
N/A 
Changed To: 
Figure 6: Kaplan-Meier Survival Analysis of the Duration of Invasive Mechanical Ventilation 
(Including Re-intubation Time): Not Censored 
[See new figure] 
h. Page 37; 1st Paragraph: 
Previously Stated: 
Some differences…such patients (Figure 5).  When these patients were stratified by 
treatment, it was shown that all 6 patients (100%) receiving i.v. L-citrulline had been 
extubated in the operating room, in comparison to only 2 of 6 patients (33%) in the placebo 
group. 
Changed To: 
Some differences…such patients.  When these patients were stratified by treatment, it was 
shown that all 6 patients (100%) receiving i.v. L-citrulline had been extubated in the 
operating room, in comparison to only 2 of 6 patients (33%) in the placebo group. Although 


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the numbers are small, these data achieved borderline significance.  The results suggest 
that children receiving citrulline appeared better clinically following cardiopulmonary bypass 
than did children receiving placebo. 
i. Page 37; Last Paragraph: 
Previously Stated: 
As with the length of time of mechanical ventilation, the duration of inotrope therapy 
(dopamine, dobutamine, milrinone, epinephrine, phenylephrine, or norepinephrine) showed 
marked differences between the two treatment groups as shown in Figure 6. 
Changed To: 
As with the length of time of mechanical ventilation, the duration of inotrope therapy 
(dopamine, dobutamine, milrinone, epinephrine, phenylephrine, or norepinephrine) was 
also recorded.  Length of time on inotropes was documented from the time of first use after 
surgery until completion of the study medication at 48 hours.  Patients still receiving 
inotropes at hour 48 were censored.  There was a marked difference between the two 
treatment groups as shown in Figure 7 with censored patients still using inotropes at hour 
48, with no additional censoring with a log rank p-value of 0.0351. 
j. Page 38; Figure 6; Title and Figure: 
Previously Stated: 
Figure 6: Kaplan-Meier survival analysis of the length of time on inotropes 
[See previous figure] 
Changed To: 
Figure 7: Kaplan-Meier Survival Analysis of the Length of Time on Inotropes: No 
additional Censoring 
 
[See new figure] 
k. Page 38; Last Paragraph; Added new paragraph: 
Previously Stated: 
 
N/A 
Changed To: 
A second analysis also censored patients without inotrope use; i.e., inotrope time = 0 
(Figure 8), which demonstrated a log rank p-value of 0.562.   
 


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l. Page 39; Added new figure 
Previously Stated: 
 
N/A 
Changed To: 
Figure 8: Kaplan-Meier Survival Analysis of the Length of Time on Inotropes: Censored 
[See new figure] 
m. Page 39; 1st Paragraph: 
Previously Stated: 
Cessation of mechanical ventilation and of inotrope therapy are the two principal 
determinants of readiness for discharge from the intensive care unit. A composite variable 
comprising (for each subject) of the longer of the two parameters - duration of mechanical 
ventilation or of inotrope therapy - can serve as an effective and accurate surrogate for the 
duration of intensive care unit stay.  Figure 7 shows the difference in the composite variable 
when L-citrulline and placebo groups are compared. Patients receiving study drug showed 
shorter composite durations of mechanical ventilation and inotrope therapy than did 
patients receiving placebo. Thus…physical injury. 
Changed To: 
Cessation of mechanical…stay.  Since inotrope use was only documented until Hour 48 
after surgery (end of study medication treatment), patients with inotrope use continuing until 
Hour 48 and with mechanical ventilation duration of ≤ 48 hours were censored at this time 
point.  If mechanical ventilation was continued beyond the 48-hour time point, the duration 
of mechanical ventilation was used in the analysis. Kaplan-Meier plots of the composite 
endpoint are presented in Figure 9, with re-intubation times excluded. One (1) patient in 
the placebo group was re-intubated.  This patient was excluded from this analysis; however, 
results including this patient were comparable.  In this analysis, patients with inotrope use 
still ongoing at 48 hours, and mechanical ventilation of less than 48 hours, were censored. 
Figure 9 shows the difference in the composite variable when L-citrulline and placebo 
groups are compared. Citrulline-treated patients required statistically significantly less 
mechanical ventilation and inotropes than placebo-treated patients. Thus…physical injury. 
n. Page 40; Figure 7; Title and Figure: 
Previously Stated: 
Figure 7: Kaplan-Meier survival analysis of the longer duration of duration of 
postoperative mechanical ventilation and the length of time on inotropes. 


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[See previous figure] 
Changed To: 
Figure 9: Kaplan-Meier Survival Analysis of the Longer Duration of Duration of 
Postoperative Mechanical Ventilation and the Length of Time on Inotropes: Re-Intubation 
Time Excluded 
[See new figure] 
o. Page 40; 1st Paragraph  
Previously Stated: 
In summary, the data from study CIT-002-01 show that the revised dosing protocol 
achieved the PK endpoint. Further, despite the extremely small sample size of the study, 
CIT-002-01 demonstrated clear treatment-dependent differences in favor of L-citrulline for 
the duration of mechanical ventilation and inotrope therapy between the treated and control 
groups. Combined in a composite variable, the results show clinically meaningful 
therapeutic efficacy for L-citrulline for the time to discharge from the intensive care unit. 
Thus, CIT-002-01 indicates that i.v. L-citrulline treatment can play a beneficial role in 
preventing clinical sequelae of CPB-induced lung injury. 
Changed To: 
In summary…CIT-002-01 indicates that intravenous L-citrulline prevents compromised 
cardiopulmonary function caused by CPB in pediatric patients undergoing surgery for ASD, 
VSD, or AVSD.  The effect is robust and is evident even in centers practicing early 
extubation as shown in the strong preferential early extubation of citrulline patients in 
comparison to the control population.  Discharge from the ICU requires cessation of both 
mechanical ventilation and inotrope therapy.  Intravenous L-citrulline shortens the duration 
of each of these variables, resulting in earlier readiness of patients for discharge from the 
ICU. Thus…injury. 
 
8. Page 41; Section 1.5; Safety of L-citrulline; 4th Paragraph: Updated section to 
reflect results of data from CIT-002-01 final Clinical Study Report. 
Previously Stated: 
Safety data…have shown that i.v. L-citrulline administration to 11 patients was safe and 
no unexpected AEs and /or SAEs occurred under this treatment. 
 


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Changed To: 
Safety data…have shown that of the patients randomized and treated, all patients (100%) 
in the i.v. L-citrulline group and 73% of patients in the placebo group experienced at least 
one AE.  AEs that were judged to be related to study treatment were reported in the placebo 
group (2 patients, 18%), but not in the active treatment group.  No AE leading to death and 
no other SAE was reported in this study.  No AE leading to treatment discontinuation was 
reported. 
 
9. Page 46; Section 4.3.3; Exclusion Criteria: Clarified exclusion criteria for patients 
with AV valve regurgitation. 
Previously Stated: 
 
Significant left sided AV valve regurgitation  
Changed To: 
 
Significant left sided AV valve regurgitation not amenable to surgical 
correction 
 
10. Page 49; Section 4.4.1; Investigational Medicinal Products Administered; 1st 
Paragraph: Added new language regarding matching placebo. 
Previously Stated: 
N/A 
Changed To: 
Placebo will be matched for volume using suitable fluid recognized as standard of care for 
such procedure (e.g. normal saline or Plasmalyte without L-citrulline added). 
All study medications will be prepared and provided by each site’s investigational 
pharmacy. 
11. Page 49-50; Section 4.4.2; Identity of Investigational Medicinal Products: Updated 
wording regarding Investigational Medicinal Product; Updated storage conditions. 
Previously Stated: 
Citrulline is a naturally occurring amino acid. The L-citrulline drug substance as fully 
synthetic active pharmaceutical ingredient (API) is a white crystalline powder. The L-
citrulline drug product is a sterile, non-pyrogenic injectable clear solution in water. The 


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concentrated solution is supplied to the study sites in heat-sterilized borosilicate glass vials 
labeled “L-citrulline for Injection”. Each vial contains 500 mg sterile L-citrulline in 10 mL of 
sterile water. The containers are closed with 13 mm brominated butyl stoppers and light 
blue, plastic/aluminum flip-off seals.  
Study drug should be stored as indicated on the package label: Store at 25°C (77°F) or 
below; do not freeze. 
The L-citrulline bolus and L-citrulline infusion will be provided by each site’s investigational 
pharmacy at a concentration of 50 mg/mL. 
Placebo will be matched for volume using either normal saline or Plasmalyte without L-
citrulline added.  
The IMP is manufactured and imported according to the current Good Manufacturing 
Practice and the relevant regulatory requirements.  
Changed To: 
The L-citrulline drug product is a sterile, non-pyrogenic injectable clear solution intended 
for intravenous administration. It is supplied as a 10 mL, single use vial containing 500 mg 
L-citrulline in 10 mL of acidified water. The product is packaged in a clear glass vial (13 mm 
neck) with a grey stopper and blue flip-off cap/aluminum seal. The L-citrulline drug product 
is manufactured aseptically at a concentration of 50 mg/mL using fully synthetic drug 
substance dissolved in water for injection. The resulting solution pH is adjusted to near pH 
3, sterile filtered and filled. 
Study drug should be stored as instructed: Store at 2-8⁰C (36-46⁰F); do not freeze. 
The drug product is manufactured and imported according to current Good Manufacturing 
Practice and the relevant regulatory requirements. 
12. Page 50; Section 4.4.3; Packaging and Labeling: Updated description of packaging. 
 
Previously Stated: 
 
L-citrulline study drug is provided as vials for injection and supplied to the study centers. 
The study medication vials will be packed and dispatched in containers.  
The batch number, strength, storage conditions, and federally required language stating 
that the product is for investigational use only will be given on the outer package. A batch 


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release certificate and a Good Manufacturing Practice-conformity statement will be 
provided, in addition to required language noting investigational use only.  
Changed To: 
L-citrulline study drug comes in a clear glass vial (13 mm neck) with a grey stopper and 
blue flip-off cap/aluminum seal. Boxes of 25 vials are supplied to the study centers. 
The batch number, strength, storage conditions, and federally required language stating 
that the product is “for investigational use only” or “for clinical trial use only” will be printed 
on the outer package. A Material Safety Data Sheet (MSDS) will be provided with the 
shipment. 
13. Page 51; Section 4.4.6; Drug Accountability: Added wording regarding drug 
accountability (deleted previous language from Section 5.1.2.6), and updated 
instructions. 
 
Previously Stated: 
 
N/A 
 
Changed To: 
 
All empty and partially used vials should be retained by the pharmacy in order for study 
drug accountability to be performed.   The total amount of study drug prepared for 
administration to the patient will be calculated by the investigational pharmacist, with 
verification of the calculations performed by a second qualified person (e.g. pharmacy 
technician).  Total volume of study drug administered to the patient will be documented in 
the source documents and in the electronic case report forms (eCRFs). 
14. Page 55; Section 5.1.2.1; Day 0, Surgery (Intra-Operative): Changed requirement for 
obtaining hemodynamics to align with Institution’s standard of care. 
Previously Stated: 
…Results will be recorded approximately every 5 minutes after induction of anesthesia and 
during surgery. 
Changed To: 
…Results will be recorded according to the Institution’s standard of care during surgery. 


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15. Page 56-61; Section 5.1.2.2 through 5.1.2.6; Day 0, post-surgery, 0, 6, and 12 hours, 
Day 1, post-surgery, 24 hours, and Day 2, post-surgery, 48 hours: Changed 
requirement for obtaining hemodynamics to align with Institution’s standard of 
care. 
 
Previously Stated: 
Results will be recorded approximately every hour from time of post-op admission to the 
PICU through 48 hours.   
 
Changed To: 
 
Results will be recorded according to the Institution’s standard of care from time of post-op 
admission to the PICU through 48 hours.   
 
16. Page 56-61; Section 5.1.2.2 through 5.1.2.6; Day 0, post-surgery, 0, 6, and12 hours, 
Day 1, post-surgery, 24 hours, and Day 2, post-surgery, 48 hours: Changed 
requirement for obtaining ABGs to align with Institution’s standard of care. 
 
Previously Stated: 
 
Results will be recorded every 2-3 hours (+/- 15 minutes) from time of post-op admission 
to the PICU until removal of arterial line or through 48 hours, whichever occurs first. 
 
Changed To: 
 
ABGs will be drawn according to the Institution’s standard of care. All results obtained from 
the time of post-op admission to the PICU until removal of the arterial line or through 48 
hours (whichever occurs first) will be recorded. 
 
17. Page 57; Section 5.1.2.2; Day 0, post-surgery, 0 hours (30 minutes post CPB 
decannulation): Corrected MAP criteria for Age 4 years, and added MAP criteria for 
Age 5 years (missing from previous list). 
 
Previously Stated: 
− 
Age … 
− 
Age 4 years:        MAP of 52 
− 
Age 6 years:        MAP of 53 
− 
Age … 
 
Changed To:  
 
− 
Age … 
− 
Age 4 years:        MAP of 50 
− 
Age 5 years: 
MAP of 52 
 


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− 
Age 6 years:        MAP of 53 
− 
Age … 
 
18. Page 62; Section 5.1.2.6; Day 2, post-surgery, 48 hours: Removed reference to 
drug accountability here, and moved to Section 4.4.6. (See #11 above). 
 
Previously Stated: 
 
Any remaining IMP, as well as empty or partially used IMP containers (e.g. bags, 
syringes), will be returned to the pharmacy and an accountability check will be 
performed.   The total amount of study drug administered to the patient will be calculated 
by the investigational pharmacist, with verification of the calculations performed by a 
second qualified person (e.g. pharmacy technician).   
Changed To: 
N/A 
19. Page 80; Section 7.4; Interim Analysis; 2nd Paragraph: Added additional wording 
regarding sample size re-estimation; Removed wording which stated study will be 
stopped. 
 
Previously Stated: 
 
In the event that the calculated sample size has already been exceeded, the study will be 
stopped. 
Changed To: 
Only the re-estimated variance values will be used for sample re-calculation, while the 
assumed values will not be re-estimated but kept as described in Section 7.2. 
In the event that the sample size re-estimation leads to a reduction of the sample size due 
to lower re-estimated variance compared to the assumed variance values, the sample size 
as currently planned will be kept, and not reduced, to ensure robust estimations for safety 
and efficacy variables. 
 
 
 


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14.2.5 Version 4.3.1 
1. 
Page 22; Schedule of Study Assessments and Procedures; Footnote 7:  Further 
clarified instructions for obtaining hemodynamics. 
Previously Stated (Version 4.3): 
 
Baseline values are first recorded in the operating room (OR) by Anesthesia.  Results will 
be recorded, during surgery and from time of post-op admission to the PICU through 48 
hours, according to the Institution’s standard of care. Results at Hours 1, 2, 4, 12, 24, and 
48 must be obtained and must be within +/- 10 minutes of the required time point. 
 
Changed To: 
 
Baseline values are first recorded in the operating room (OR) by Anesthesia.  Results 
will be monitored during surgery according to the Institution’s standard of care, but results 
will only be recorded approximately every 15 minutes. Standard of care will continue to be 
followed from time of post-op admission to the PICU through 48 hours.  Results at 1, 2, 4, 
12, 24, and 48 hours from baseline (in OR) must be obtained and recorded. 
2. 
Page 23; Schedule of Study Assessments and Procedures; Footnote 16: Clarified 
instructions for obtaining final laboratory sample. 
Previously Stated: 
 
An attempt should be made to obtain final labs while i.v. access is available, either on 
Day 28 or prior to discharge from hospital. If lab results within 24 hours are available, 
these may be captured. If no i.v. access is available, venipuncture is not required unless 
indicated for clinical reasons. 
 
Changed To: 
 
An attempt should be made to obtain final labs on Day 28 or prior to discharge from the 
hospital. If lab results within 24 hours are available, these may be captured. If results are 
not available within 24 hours, then record the results of the labs obtained closest to 
discharge from the hospital.  These labs must include LFTs.  If no i.v. access is available 
on Day 28 or the day of discharge, venipuncture is not required since results of the labs 
obtained closest to discharge may be used. 
 
 
 


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3. 
Page 55; Section 5.1.2.1; Day 0, Surgery (Intra-Operative): Further clarified 
instructions for obtaining hemodynamics. 
Previously Stated (Version 4.3): 
…Results will be recorded according to the Institution’s standard of care during surgery. 
Changed To: 
Results will be monitored during surgery according to the Institution’s standard of care, 
but results will only be recorded approximately every 15 minutes. 
 
4. 
Page 56-61; Section 5.1.2.2 through 5.1.2.6; Day 0, post-surgery, 0, 6, and 12 hours, 
Day 1, post-surgery, 24 hours, and Day 2, post-surgery, 48 hours: Further clarified 
instructions for obtaining hemodynamics. 
 
Previously Stated (Version 4.3): 
 
…Results at Hours 1, 2, 4, 12, 24, and 48 must be obtained and must be within +/- 10 
minutes of the required time point. 
Changed To: 
 
…Results at 1, 2, 4, 12, 24, and 48 hours from baseline (in OR) must be obtained and 
recorded.  
 
5. 
Page 62; Section 5.1.2.7; Follow-Up Visit [Day 28 (if still in hospital) or upon 
Hospital Discharge]: Clarified instructions for obtaining final laboratory sample. 
 
Previously Stated: 
 
• 
Blood draw* for: 
…. 
 
* (An attempt should be made to obtain final laboratory samples while i.v. 
access is available, either on Day 28 or prior to discharge from hospital. If 
laboratory results within 24 hours are available, these may be captured. If no 
i.v. access is available, venipuncture is not required unless indicated for 
clinical reasons.) 
 
Changed To: 
 
• 
Blood draw* for: 


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…. 
 
* (An attempt should be made to obtain final laboratory samples on Day 28 or 
prior to discharge from hospital. If laboratory results within 24 hours are 
available, these may be captured. If results are not available within 24 hours, 
then record the results of the labs obtained closest to discharge from the 
hospital.  These labs must include LFTs.  If no i.v. access is available on Day 
28 or the day of discharge, venipuncture is not required since results of the 
labs obtained closest to discharge may be used. 
 
 
 
 

SAP_001.pdf: 
Statistical Analysis Plan  
Final Analysis 
1/24 
CIT-003-01 
 
BS-FRM-001_01-2.0 
October 01, 2018 
SAP (version 2.0) 
 
 
Statistical Analysis Plan 
for 
Final Analysis 
 
Version 2.0 
 
 
 
 
 
Study: 
A Phase III Double-Blind, Randomized, Placebo Controlled, 
Multi-Center Clinical Study to Evaluate the Efficacy and Safety 
of Intravenous L-citrulline for the Prevention of Clinical Sequelae 
of Acute Lung Injury induced by Cardiopulmonary Bypass in 
Pediatric Subjects Undergoing Surgery for Congenital Heart 
Defects 
 
 
Study-ID: 
CIT-003-01 
 
 
Sponsor / Contact: 
 
 
Asklepion Pharmaceuticals, LLC 
729 E. Pratt Street; Suite 360 
Baltimore, MD 21202 
 
 
Evaluation: 
FGK Clinical Research GmbH 
 
 
FGK code: 
3359 
 
 
Version: 
Version 2.0 October 01, 2018 (final) 
 
 
Previous versions: 
  
 
 
The content of this Statistical Analysis Plan is confidential and must not be passed to any third 
party without permission of Asklepion Pharmaceuticals. 
 
 
 


Statistical Analysis Plan  
Final Analysis 
2/24 
CIT-003-01 
 
BS-FRM-001_01-2.0 
October 01, 2018 
SAP (version 2.0) 
Revision history 
Version 
Author 
Date 
Reason for Revision 
1.0 
O.Boehm 
17 Nov 2017 
Final version 1.0 
2.0 
V. Voelkl 
01 Oct 2018 
Final version 2.0 
• 
PF ratio and 28-day mortality added as 
exploratory endpoints (according to EU Study 
Protocol) 
• 
Definition of FAS, mFAS, and SAF clarified 
• 
Sensitivity analyses added for the primary 
efficacy variable and for secondary efficacy 
variables ‘Length of time on mechanical 
ventilation’, and ‘Length of time of inotrope 
use’ 
• 
Numbering of TLG corrected in SAP Appendix 
A 
• 
SAP appendix B replaced by external sample 
size reestimation document describing 
procedure 
 


Statistical Analysis Plan  
Final Analysis 
3/24 
CIT-003-01 
 
BS-FRM-001_01-2.0 
October 01, 2018 
SAP (version 2.0) 
Table of Contents 
 
LIST OF ABBREVIATIONS ............................................................................................................................... 4 
1 
GENERAL ................................................................................................................................................... 5 
1.1 
Study Design .................................................................................................................................. 5 
1.2 
Objectives....................................................................................................................................... 5 
1.3 
Sample Size Calculation ................................................................................................................ 6 
1.4 
Interim Analysis .............................................................................................................................. 6 
1.5 
Visit Terminology ............................................................................................................................ 7 
2 
EFFICACY AND SAFETY VARIABLES..................................................................................................... 8 
2.1 
Primary Efficacy Variable ............................................................................................................... 8 
2.2 
Secondary Efficacy Variables ........................................................................................................ 8 
2.3 
Secondary Safety Variables ........................................................................................................... 8 
2.4 
Exploratory Variables ..................................................................................................................... 8 
2.5 
Other Relevant Variables ............................................................................................................... 8 
3 
STATISTICAL ANALYSIS SETS ............................................................................................................... 9 
3.1 
Safety Analysis Set ........................................................................................................................ 9 
3.2 
Full Analysis Set ............................................................................................................................. 9 
3.3 
Modified Full Analysis Set .............................................................................................................. 9 
3.4 
Per-Protocol Set ............................................................................................................................. 9 
3.5 
Assignment of Analysis Sets to Analysis ....................................................................................... 9 
4 
STATISTICAL EVALUATION ................................................................................................................... 10 
4.1 
Dispositions of Subjects and Analysis Sets ................................................................................. 10 
4.2 
Demographics and Other Covariates ........................................................................................... 10 
4.3 
Study Drug Administration and Intra-operative Details ................................................................ 11 
4.4 
Efficacy Analysis .......................................................................................................................... 11 
4.4.1 
Analysis of Primary Efficacy Variable ..................................................................................... 11 
4.4.2 
Analysis of Secondary Efficacy Variables .............................................................................. 12 
4.5 
Safety Analysis ............................................................................................................................. 16 
4.6 
Analysis of Exploratory Variables ................................................................................................ 19 
4.7 
Analysis of Other Relevant Variables .......................................................................................... 19 
4.8 
Missing Values ............................................................................................................................. 20 
4.9 
Data Base Closure and Data Review .......................................................................................... 20 
4.10 
Miscellaneous .............................................................................................................................. 21 
5 
CHANGES FROM PROTOCOL ............................................................................................................... 21 
6 
LITERATURE ............................................................................................................................................ 23 
7 
APPENDICES ........................................................................................................................................... 23 
8 
SIGNATURES ........................................................................................................................................... 24 
 


Statistical Analysis Plan  
Final Analysis 
4/24 
CIT-003-01 
 
BS-FRM-001_01-2.0 
October 01, 2018 
SAP (version 2.0) 
List of Abbreviations 
 
In the following abbreviations are listed as used within this statistical analysis plan or which might 
occur within the tables, listings and graphs outputs: 
ABG 
Arterial blood gases 
ACT  
Activated clotting time 
AE  
Adverse event  
ANOVA 
Analysis of variance 
ASD 
Atrial septal defect 
ATC 
Anatomical therapeutic chemical classification 
AVSD 
Atrioventricular septal defect 
BDR  
Blind data review meeting 
BMI 
Body mass index 
BPAP  
Bilevel (biphasic) positive airway pressure  
CBC  
Complete blood count 
CI 
Confidence interval 
CPAP  
Continuous positive airway pressure  
CPB 
Cardiopulmonary bypass 
CRF 
Case report form 
CS  
Clinically significant  
EC 
Exclusion criteria 
ECG 
Echocardiogram  
ET 
Early termination 
FAS 
Full analysis set 
FDA  
Food and Drug Administration 
H0 
Null hypothesis 
H1 
Alternative hypothesis 
IC 
Inclusion criteria 
ICF 
Informed consent form 
ITT 
Intention-to-treat 
LOCF  
Last observation carried forward 
MedDRA 
Medical dictionary for regulatory activities 
mFAS 
Modified full analysis set 
N 
Number of subjects 
NCS 
Not clinically significant  
OR 
Odds ratio 
PICU  
Pediatric intensive care unit 
PP 
Per-protocol analysis set 
PT 
Preferred term 
PTAE 
Pre-treatment adverse event 
RD 
Risk difference 
RR 
Risk ratio 
SAE  
Serious adverse event 
SAF  
Safety analysis set  
SAP  
Statistical analysis plan  
SD 
Standard deviation 
SOC 
System organ class 
TAE 
Treatment adverse event 
TLG 
Tables, listings, graphs 
VSD 
Ventricular septal defect 
WHO-DD 
World Health Organization drug dictionary 
 
No abbreviations of units and of laboratory and blood gas values are included in this list. 


Statistical Analysis Plan  
Final Analysis 
5/24 
CIT-003-01 
 
BS-FRM-001_01-2.0 
October 01, 2018 
SAP (version 2.0) 
1 
General 
This Statistical Analysis Plan (SAP) was defined by the Sponsor and the responsible Statistician 
without knowledge of the randomization code. It is based upon the global Study Protocol (version 
4.3.1 of August 11, 2017) and the EU Study Protocol (version 2.0 of July 12, 2018) and contains 
detailed description of the statistical methods described therein. 
The SAP describes prospectively the analyses to be performed on study data. It was finalized prior to 
data base lock and subsequent unblinding. 
1.1 
Study Design 
This is a multicenter Phase III double-blind, randomized, placebo controlled study to evaluate the 
efficacy and safety of L-citrulline for the prevention of clinical sequelae of acute lung injury 
induced by cardiopulmonary bypass (CPB) in pediatric subjects undergoing surgery for 
congenital heart defects. Children undergoing repair of cardiac defects that include a large 
unrestrictive ventricular septal defect (VSD) or a partial or complete atrioventricular septal defect 
(AVSD) or an ostium primum atrial septal defect (ASD) will be eligible for enrollment in this 
study.  
190 subjects will be randomized in a 1:1 fashion to either L-citrulline or placebo. Treatment will 
include:  
1) An L-citrulline bolus of 150 mg/kg or placebo at the initiation of CPB, but after removal of any 
crystalloid base;  
2) The addition of L-citrulline at a concentration of 200 μmol/L or placebo given as a bolus during 
bypass. This may be administered as a one-time bolus or multiple administrations to compensate 
for fluids containing L-citrulline that may be removed from the patient during the course of the 
operation and thus to maintain the concentration of 200 μmol/L;  
3) An L-citrulline bolus of 20 mg/kg or placebo 30 minutes after decannulation from CPB 
immediately followed by a 9 mg/kg/hr continuous infusion of L-citrulline or placebo for 48 hours. 
The study drug or placebo infusion will be discontinued once invasive arterial blood pressure 
monitoring is discontinued or at 48 hours, whichever comes first. Subjects will be followed until 
discharge from the hospital, and a final assessment via telephone will be conducted at Day 28. 
1.2 
Objectives 
The primary objective of the study is to determine if L-citrulline delivery given perioperatively 
reduces clinical sequelae of acute CPB-induced lung injury in pediatric subjects undergoing 
repair of congenital heart defects, as evidenced by the reduction of post-operative need for 
mechanical ventilation and inotrope therapy. 
The primary efficacy parameter is a composite variable consisting of the longer of either (1) 
length of time on mechanical ventilation or (2) length of time of inotrope use. The definition of 
mechanical ventilation shall include invasive mechanical ventilation or non-invasive mechanical 
ventilation including bilevel (biphasic) positive airway pressure (BPAP) or continuous positive 
airway pressure (CPAP). The definition of inotrope use includes medications which are 
considered within the derivation of the total inotrope score (dopamine, dobutamine, milrinone, 
epinephrine, phenylephrine, norepinephrine).  
The following hypothesis will be tested: 
H0: Composite endpoint within the L-citrulline group is equal to that of the placebo group 
H1: Composite endpoint within the L-citrulline group is unequal to that of the placebo group 
 
 


Statistical Analysis Plan  
Final Analysis 
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October 01, 2018 
SAP (version 2.0) 
Secondary objectives are to evaluate the effect of L-citrulline compared with placebo regarding 
 safety and tolerability, 
 hemodynamic improvement post-operatively, 
 the usage and length of time on vasodilators, 
 length of hospitalization, 
 the duration of chest tube placement, 
 the plasma concentrations of citrulline. 
1.3 
Sample Size Calculation 
Sample size was calculated with nQuery 7.0. Assuming a standard deviation of 50 and mean 
values of 44 for placebo and 14 for the L-citrulline group, the effect size is estimated being 0.600, 
which yields a sample size of N=92 per group when applying a two-sided Wilcoxon-rank-sum test 
with a 0.01 significance level and a power of 90%. The assumptions concerning standard 
deviation and mean values are based on results of the preceding study CIT-002-01. 
To allow for subjects being enrolled but not assigned to the full analysis set (FAS), an overall 
N=95 subjects per group will be enrolled. A sample size re-estimation will be done within the 
framework of an interim analysis (see section 1.4). 
1.4 
Interim Analysis 
One interim analysis is planned to be carried out when the first 95 consecutively randomized FAS 
patients (i.e. 50% of the planned patients) will have undertaken their Follow-Up Visit (Day 28 or 
Hospital Discharge), data required for the analysis of the composite endpoint have been entered 
into the eCRF, and those data have been cleaned appropriately (See external sample size 
reestimation document). During this interim analysis, only a sample size re-estimation will be 
carried out. No other statistical analyses will be conducted. The sample size re-estimation will be 
done by an independent, unblinded statistician following the same procedures and criteria as for 
the initial sample size estimate. Only the re-estimated variance values will be used for the sample 
re-calculation, while the assumed mean values will not be re-estimated but kept. Only the results 
of the sample size re-estimation will be provided to Asklepion Pharmaceuticals. 
In the event that the sample size re-estimation leads to a reduction of the sample size due to 
lower re-estimated variance compared to the assumed variance values the sample size as 
currently planned will be kept but not reduced to ensure robust estimations for safety and efficacy 
variables. If the trial is to be continued with the re-estimated sample size, an appropriate protocol 
amendment will be submitted to the Food and Drug Administration (FDA) as soon as feasible. 
Final analysis will be based on the population of all randomized patients without taking the 
sample size re-estimation results into consideration. Due to the fact that only the interim variance 
and mean values will be assessed, no alpha adjustment of statistical analyses is required. 
 
 


Statistical Analysis Plan  
Final Analysis 
7/24 
CIT-003-01 
 
BS-FRM-001_01-2.0 
October 01, 2018 
SAP (version 2.0) 
1.5 
Visit Terminology 
The notation displayed in table 1 will be used for table, listing, and graph (TLG) presentation of 
visits. 
Table 1: Visit terminology 
Notation in protocol 
Notation used for 
TLG presentation 
Notation for 
treatment phases 
Screening visit  (Day -14 to Day 0) 
Screening 
Pre-Treatment 
Baseline visit (Day of Surgery, Pre-operation) 
Day 0 – Pre-Op 
 
Baseline1 
Day 0, Surgery (Intra-operative) 
Day 0 – Intra-Op 
Treatment 
Day 0, Post-surgery, 0 hours 
Day 0 – 0 hours 
Day 0, Post-surgery, 6 hours 
Day 0 – 6 hours 
Day 0, Post-surgery, 12 hours 
Day 0 – 12 hours 
Day 1, Post-surgery, 24 hours  
Day 1 – 24 hours 
Day 2, Post-surgery, 48 hours 
Day 2 – 48 hours 
Follow-Up Visit (Day 28/ Hospital Discharge) 
Day 28/ Discharge 
Follow-up 
Follow-Up Phone Assessment2 
Day 28 - Phone call 
1Baseline will be defined as the most recent non-missing value prior to surgery. 
2 Patients discharged from hospital prior to Day 28 will be contacted by phone on Day 28 to 
assess their survival status. 


Statistical Analysis Plan  
Final Analysis 
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October 01, 2018 
SAP (version 2.0) 
2 
Efficacy and Safety Variables 
2.1 
Primary Efficacy Variable 
The primary efficacy parameter for this study is a composite variable consisting of the longer of 
either (1) length of time on mechanical ventilation or (2) length of time of inotrope use. 
The definition of mechanical ventilation shall include invasive mechanical ventilation or non-
invasive mechanical ventilation including BPAP or CPAP. The definition of inotrope use includes 
medications which are considered within the derivation of the total inotrope score (dopamine, 
dobutamine, milrinone, epinephrine, phenylephrine, norepinephrine). 
2.2 
Secondary Efficacy Variables 
The following secondary efficacy parameters will be analyzed: 
 Length of time on mechanical ventilation 
 Length of time on positive pressure ventilation 
 Length of time of inotrope use 
 Inotrope score  
 Hemodynamic improvement  
 Length of time of intubation 
 Length of pediatric intensive care unit (PICU) stay 
 Length of time on vasodilators 
 Length of hospitalization 
 Thoracotomy output - duration and volume of chest tube drainage 
 L-citrulline plasma levels 
2.3 
Secondary Safety Variables 
 Occurrence of adverse and serious adverse events 
 Incidence of refractory hypotension 
 Change from Baseline in laboratory values 
2.4 
Exploratory Variables 
 PF ratio 
 28-day mortality 
2.5 
Other Relevant Variables 
 Arterial blood gas (ABG) 
 
 
 
 


Statistical Analysis Plan  
Final Analysis 
9/24 
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BS-FRM-001_01-2.0 
October 01, 2018 
SAP (version 2.0) 
3 
Statistical Analysis Sets 
3.1 
Safety Analysis Set  
All randomized subjects who received surgery including undergoing CPB and study medication 
(independent of whether it is L-citrulline or placebo) will be valid for the safety analysis set (SAF). 
Within the SAF a subject will be considered for the treatment actually received and not for the 
treatment assigned by randomization, if different.  
3.2 
Full Analysis Set  
The full analysis set (FAS) includes all subjects who underwent surgery including undergoing 
CPB. Within the FAS a subject will be considered for the treatment assigned by randomization 
and not for the treatment actually received, if different, i.e. following the intent-to-treat (ITT) 
principle.  
3.3 
Modified Full Analysis Set 
The modified full analysis set (mFAS) includes all subjects who underwent surgery including 
undergoing CPB and received study medication (independent of whether it is L-citrulline or 
placebo). Within the mFAS, a subject will be considered for the treatment assigned by 
randomization and not for the treatment actually received, if different, i.e. following the ITT 
principle.  
3.4 
Per-Protocol Set  
The per-protocol set (PP) includes all subjects included in the SAF who had no significant 
protocol deviations and completed through Day 28 or until discharge from the hospital (end of 
study follow-up). Protocol deviations will be identified and classified for each subject during a 
blind data review meeting. For the PP all subjects will be assigned to the treatment actually 
received.  
3.5 
Assignment of Analysis Sets to Analysis 
The FAS serves as the primary efficacy analysis set. All efficacy analyses are based on the FAS. 
The mFAS and the PP serve as sensitivity populations for the efficacy analyses.  
The SAF will be used for the evaluation of the safety assessments. 


Statistical Analysis Plan  
Final Analysis 
10/24 
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BS-FRM-001_01-2.0 
October 01, 2018 
SAP (version 2.0) 
4 
Statistical Evaluation 
Continuous variables will be summarized by mean, standard deviation, median, lower and upper 
quartiles, minimum and maximum. Frequencies and percentages will be used to summarize 
categorical variables. 
Analyses will be performed by visit and treatment group, if not stated otherwise. Baseline will be 
defined as the most recent non-missing value prior to surgery.  
For time to event analyses censoring information will be used for statistical analyses. Handling of 
missing values is described in detail in the following sections. If no method is described, no 
imputation of missing values will be applied. 
Any parameter describing a “duration” or “length” is calculated as stop date/time - start date/time 
+ 1. 
4.1 
Dispositions of Subjects and Analysis Sets 
Disposition of subjects and analysis sets 
The disposition of subjects and analysis sets, subjects per visit, subjects per site, subject 
eligibility (informed consent signed, subject failed inclusion and/or exclusion criteria), and subject 
status (discontinuation and reasons for early study drug and study discontinuation, hospital 
discharge prior to Day 28) will be tabulated. 
No inferential assessments will be performed on disposition data. 
4.2 
Demographics and Other Covariates 
Demographic data 
Demographic data (age, gender, race, ethnicity, body height, body weight, body mass index [BMI; 
calculated: (kg/m2)]) will be tabulated by treatment group. Age, body weight and BMI will be 
additionally stratified by visit.  
Menarche and pregnancy test 
The proportion of female patients who reached the age of menarche will be presented. For 
females with childbearing potential conduction of pregnancy test will be tabulated. For females 
who conducted a pregnancy test the test results will be tabulated. 
Medical history 
The proportion of subjects with medical history conditions or events will be tabulated. 
Concomitant medication 
Concomitant medications should be recorded starting from the date of informed consent until end 
of follow-up. Inotropes and vasoactive medications are captured separately and will be analyzed 
separately. Medications will be coded by the World Health Organization drug dictionary (WHO-
DD).  
Concomitant medication will be tabulated by Anatomic Group (Anatomical therapeutic chemical 
classification [ATC] level 1), ATC level 4, and WHO-DD preferred term.  
Echocardiography 
The results of the transthoracic echocardiogram specifying the congenital cardiac anomaly 
(AVSD / ASD / VSD) will be tabulated. 
 
 


Statistical Analysis Plan  
Final Analysis 
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October 01, 2018 
SAP (version 2.0) 
Physical examination 
Physical examination assessments (Normal / Abnormal, not clinically significant / Abnormal, 
clinically significant / Not done) will be tabulated by body system. 
No inferential assessments will be performed on demographics and other covariates.  
4.3 
Study Drug Administration and Intra-operative Details 
Study drug administration 
Details regarding study drug administration (infusion access, interruption, dosage, and duration) 
will be tabulated. 
Intra-operative details 
Details regarding surgery, transfusion, procedural fluids, and cardiopulmonary bypass will be 
listed. 
No inferential assessments will be performed on study drug administration data.  
4.4 
Efficacy Analysis 
4.4.1 Analysis of Primary Efficacy Variable 
The primary efficacy endpoint is a composite variable consisting of the longer of either (1) length 
of time on mechanical ventilation or (2) length of time of inotrope use.  
The definition of mechanical ventilation shall include invasive mechanical ventilation or non-
invasive mechanical ventilation including BPAP or CPAP. The definition of inotrope use includes 
medications which are considered within the derivation of the total inotrope score (dopamine, 
dobutamine, milrinone, epinephrine, phenylephrine, norepinephrine). 
The length of time on mechanical ventilation will be measured until the subject is discharged from 
the hospital or at Day 28. The length will be derived as the time in minutes from separation from 
CPB (variable “End time” of parameter “CPB” from section “Post incision” on CRF “Surgery”) until 
discontinuation of all mechanical ventilation including non-invasive support (variables “Stop 
Time/Date of last use of non-invasive ventilation” and “Stop Time/Date of last use of invasive 
ventilation” of parameter “CPB” from section “Post incision” on CRF “Mechanical ventilation”). If 
invasive or non-invasive mechanical ventilation is stopped but has to be restarted, time will 
continue to accrue by subtracting the total duration of all ventilation breaks (variables “Stop 
date/time” and “Restart date/time” from CRF “Ventilation breaks”).  
 Length of time on mechanical ventilation (min) =  
(Time mechanical ventilation discontinuation - End time (CPB) + 1) -  
sum of all ventilation breaks with any ventilation break = Restart time - Stop time + 1 
If a subject did not receive any mechanical ventilation the length is set to 0. For subjects who died 
before discharge from the hospital or before Day 28, respectively, the observed length of time on 
mechanical ventilation will be used. As sensitivity analyses subjects who died will (1) be excluded 
from analysis and (2) be assigned a length of time on mechanical ventilation of 28 days.  
Length of time of inotrope use will be measured until the subject is discharged from the hospital 
or until Day 28, whichever occurs first. The length will be derived as the time in minutes from 
separation from CPB (variable “End time” of parameter “CPB” from section “Post incision” on 
CRF “Surgery”) until last use (last “end date/time” of any medication as documented in section 
“Inotropes” on CRF “Inotropes and Vasoactive Medications”).  
 Length of time of inotrope use (min) =  
Last “end date/time” of any inotrope - End time (CPB) + 1 


Statistical Analysis Plan  
Final Analysis 
12/24 
CIT-003-01 
 
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October 01, 2018 
SAP (version 2.0) 
Any interruptions will discontinue to accrue, any duration of re-use will continue to accrue. If a 
subject did not use any inotropes the length is set to 0. For subjects who died before discharge 
from the hospital or before Day 28, respectively, the observed length on inotropes will be used. 
As sensitivity analyses subjects who died will (1) be excluded from analysis and (2) be assigned 
a length of time on inotropes of 28 days.Hypothesis to be tested: 
H0: Composite endpoint within the L-citrulline group is equal to that of the placebo group 
H1: Composite endpoint within the L-citrulline group is unequal to that of the placebo group 
H0 will be tested using a Wilcoxon-rank-sum test. The level of significance is 1% (two-sided). 
As additional sensitivity analyses, the composite endpoint will be derived with (3) length of time of 
inotrope use starting on first “start date/time” of inotrope use and (4) length of time of inotrope use 
starting on first “start date/time” of inotrope use and length of time on mechanical ventilation 
starting on “start date/time” of mechanical ventilation. For sensitivity analyses (3) and (4) the 
observed length on mechanical ventilation/ inotropes will be used for subjects who died. 
The FAS will be the primary analysis set for the analysis on the composite endpoint. Analyses 
using the mFAS and the PP serve as sensitivity analyses. 
Two-sided 95% confidence intervals using Hodges-Lehman method will be additionally 
calculated.  
Survival analyses using Kaplan-Meier estimates and plots, log-rank test (significance level of 5%) 
and Cox-regression modeling including factor treatment (no further factors to be included) will 
serve as sensitivity analyses. For subjects who died before discharge from the hospital or before 
Day 28, respectively, the observed length of time on mechanical ventilation/inotropes will be 
used. The sensitivity analyses (1) and (2) as described for the primary analysis (subjects who 
died will (1) be excluded from analysis and (2) be assigned a length of time on inotropes of 28 
days) will not be applied for survival analyses. The sensitivity analyses (3) and (4) as described 
for the primary analysis (composite endpoint will be derived with (3) length of time of inotrope use 
starting on first “start date/time” of inotrope use and (4) length of time of inotrope starting on first 
“start date/time” of inotrope use and length of time on mechanical ventilation starting on “start 
date/time” of mechanical ventilation) will be applied for survival analyses. For survival analyses 
different sets of analyses will be done: 
(1) The composite variable for subjects without any mechanical ventilation and without any use of 
inotropes is set to 0, observations will not be censored 
(2) The composite variable for subjects without any mechanical ventilation and without any use of 
inotropes is set to 0, observations will be censored 
(3) Subjects without any mechanical ventilation and without any use of inotropes will be deleted 
from analysis 
For sensitivity analyses (3) and (4) the first set of analyses (i.e., (1) The composite variable for 
subjects without any mechanical ventilation and without any use of inotropes is set to 0, 
observations will not be censored) will be used. 
Subjects who receive mechanical ventilation or are still using inotropes on the last day of 
observation (which can be Day 28, Day of early discontinuation, or Day of death) will be censored 
for survival analyses. 
4.4.2 Analysis of Secondary Efficacy Variables 
No formal statistical hypotheses are specified for the secondary endpoint variables. Nonetheless 
statistical testing methods will be applied to compare treatment groups. A significance level of 5% 
(two-sided) will be used for such analyses. Secondary efficacy endpoints will be analyzed using 
FAS, mFAS and PP. All secondary analyses are considered exploratory. 


Statistical Analysis Plan  
Final Analysis 
13/24 
CIT-003-01 
 
BS-FRM-001_01-2.0 
October 01, 2018 
SAP (version 2.0) 
 
Length of time on mechanical ventilation 
The same definitions and analyses as described for the primary endpoint will be applied. As 
sensitivity analysis, length of time on mechanical ventilation will be calculated starting on “start 
date/time” of mechanical ventilation. 
Length of time on positive pressure (non-invasive) ventilation 
Positive pressure ventilation describes non-invasive mechanical ventilation. The same definitions 
(only using non-invasive ventilation stop date/time and non-invasive ventilation breaks: BPAP, 
CPAP, HFNC, and other non-invasive) and analyses as described for the primary endpoint will be 
applied. 
Length of time of inotrope use 
The same definitions and analyses as described for the primary endpoint will be applied. As 
sensitivity analysis, length of time of inotrope use will be calculated starting on first “start 
date/time” of inotrope use. 
 
 
 


Statistical Analysis Plan  
Final Analysis 
14/24 
CIT-003-01 
 
BS-FRM-001_01-2.0 
October 01, 2018 
SAP (version 2.0) 
Inotrope score 
The total inotrope score at each hour post-operatively from the time of separation from bypass 
until the completion of study drug will be calculated as: 
Total Inotrope Score = Dopamine + Dobutamine + 10*Milrinone + 100*Epinephrine 
+100*Phenylephrine + 100*Norepinephrine. 
The score will be calculated for each hour post separation from CPB (see section 4.4.1) until 48 
hours post separation. If within the time interval of hours x after the separation from CPB any 
inotrope was given, the total score will be calculated and used for analysis. 
Example for a patient’s inotrope documentation:  
 12:00: Separation from CPB 
 Dopamine: 100 mcg/kg/min from 12:00 to 13:00,  
 Dobutamine 100 mcg/kg/min from 13:00 to 13:30, 14:01 to 14:59, and 16:00 to 17:00.  
The times of interest at 1, 2, 3,  … hrs will be derived as time of separation from CPB + 59, 119, 
179, … minutes. I.e., the times of interest and the total inotrope score would then be calculated 
as follows:  
 1 hrs (12:00-12:59) = 100  ( Dopamine (100)  
documented 12:00-13:00) 
 2 hrs (13:00-13:59)  = 200  ( Dopamine (100)  
documented 12:00-13:00 
 
   Dobutamine (100)  documented 13:00-13:30 ) 
 3 hrs (14:00-14:59)  = 100  ( Dobutamine (100)  documented 14:01-14:59 ) 
 4 hrs (15:00-15:59)  =  
0 ( no inotropes  
documented 15:00-15:59 ) 
 5 hrs (16:00-16:59)  = 100 ( Dobutamine  
documented 16:00-17:00 ) 
 6 hrs (17:00-17:59)  = 100 ( Dobutamine  
documented 16:00-17:00 ) 
 7 hrs (18:00-18:59)  =  
0 ( no inotropes  
documented 17:01-18:00 ) 
 … 
The total inotrope score will be imputed to zero after the bypass pump has been removed 
(separation from CPB). It will also be imputed as zero for patients not using inotropes at all.  
The total inotrope score will be summarized at each post-operative hour and analyzed using a 
Wilcoxon-Rank sum test and an analysis of variance (ANOVA) with a fixed effect for treatment 
group. Additionally, a repeated measures analysis of variance will be used to compare total 
inotrope score between placebo and L-citrulline over time.  
Hemodynamic improvement 
Hemodynamic evaluations include heart rate, systemic arterial blood pressure, oxygen saturation, 
and central venous pressure. The absolute changes from Baseline at hours 1, 2, 4, 12, 24, and 
48 will be compared between groups using an ANOVA with a fixed effect for treatment group and 
Baseline level. 
The last non-missing hemodynamic assessment before start of surgery will be used as Baseline 
level; i.e. “Date and time of assessment” from section “Hemodynamics” on CRF “Hemodynamics” 
before “Date of surgery” and “Start time (incision time)” from section “Surgery” on CRF “Surgery”. 
Documented assessments as closest to the target hours 1, 2, 4, 12, 24, and 48 using the 
following analysis time windows will be used for analysis: 
  
1 hrs = 
 1 min  to  
89 min   
 ( 
>0 hrs  to  <1.5 hrs) 
  
2 hrs =  
90 min  to  179 min   
 ( ≥1.5 hrs  to  <3.0 hrs) 
 
  
4 hrs =  180 min  to  479 min 
 ( ≥3.0 hrs  to  <8.0 hrs) 
  12 hrs =  480 min  to  1079 min 
 ( ≥8.0 hrs  to  <18.0 hrs) 
  24 hrs =  1080 min to  2159 min 
 
 ( ≥18.0 hrs  to  <36.0 hrs) 
  48 hrs =  2160 min to  3599 min 
 ( ≥36.0 hrs  to  <60.0 hrs) 


Statistical Analysis Plan  
Final Analysis 
15/24 
CIT-003-01 
 
BS-FRM-001_01-2.0 
October 01, 2018 
SAP (version 2.0) 
An ANOVA per hour adding site and site-by-treatment group interaction will be conducted as a 
sensitivity analysis. If the p-value for the site-by-treatment group interaction is lower than 0.1 the 
analysis will also be provided by site. Absolute values and absolute change from Baseline values 
will be summarized for all observed time points. 
Length of time on intubation (invasive ventilation) 
The same definitions (only using invasive ventilation stop date/time and invasive ventilation 
breaks: FVS, PVS and other invasive) and analyses as described for the primary endpoint will be 
applied. 
Length of PICU stay 
The length of PICU stay will be calculated as the total number of days postoperative until 
discharge from PICU. The length will be derived as the number of days from day after surgery 
(variable “Date of surgery” on CRF “Surgery” + 1) until discharge from PICU (variable “Date 
transferred to floor” on CRF “Post-operative observation”). If subject has to be readmitted to 
PICU, time will continue to accrue (variables “Date of readmission” and “Date transferred to floor” 
(second assessment) from CRF “Post-operative observation”).  
 Length of PICU stay (days) =  
(Date transferred to floor - (Date of surgery + 1) + 1) +  
(Date transferred to floor (2nd assessment) - Date of readmission + 1)  
For subjects who died before discharge from PICU or before Day 28, respectively, the observed 
length of PICU stay will be used. As sensitivity analyses subjects who died will (1) be excluded 
from analysis and (2) be assigned a length of PICU stay of 28 days. 
For the length of PICU stay the same analyses as described for the primary endpoint will be 
applied. 
Length of time on vasodilators 
Length of time on vasodilators will be measured from first use following separation from bypass, 
until the subject is discharged from the hospital or at Day 28. The length will be derived as the 
time in minutes from separation from CPB (variable “End time” of parameter “CPB” from section 
“Post incision” on CRF “Surgery”) until last use (last “end date/time” of any medication as 
documented in sections “Vasodilators” or “Other vasodilators”  on CRF “Inotropes and Vasoactive 
Medications”).  
 Length of time of vasodilators (min) =  
Last “end date/time” of any vasodilators - End time (CPB) + 1 
Any interruptions will discontinue to accrue, any duration of re-use of vasodilators will continue to 
accrue. If a subject did not use any vasodilators the length is set to 0. As a sensitivity analysis, 
subjects with no use of vasodilators will be excluded. For subjects who died before discharge 
from the hospital or before Day 28, respectively, the observed length of time on vasodilators will 
be used. As sensitivity analyses subjects who died will (1) be excluded from analysis and (2) be 
assigned a length of time on vasodilators of 28 days. 
For the length of time on vasodilators the same analyses as described for the primary endpoint 
will be applied. 
Length of hospitalization 
The length of hospitalization will be calculated as the total number of days postoperative until 
discharge from the hospital. The length will be derived as the number of days from day after 
surgery (variable “Date of surgery” on CRF “Surgery” + 1) until discharge from the hospital 
(variable “Hospital Discharge date” on CRF “End of study”).  
 Length of hospitalization (days) =  
Hospital Discharge date - (Date of surgery + 1) + 1  


Statistical Analysis Plan  
Final Analysis 
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BS-FRM-001_01-2.0 
October 01, 2018 
SAP (version 2.0) 
For subjects who died before discharge from the hospital or before Day 28, respectively, the 
observed length of hospitalization will be used. As sensitivity analyses subjects who died will (1) 
be excluded from analysis and (2) be assigned a length of hospitalization of 28 days. 
For the length of hospitalization, the same analyses as described for the primary endpoint will be 
applied. 
Thoracotomy output - duration and volume of chest tube drainage 
The total postoperative duration (in minutes) that the chest tube will be used will be calculated as 
the time from the end of the surgery (variables “Date of surgery” and “End time” from section/CRF 
“Surgery”) to the time the chest tube will be removed (variable “Date/time of removal” from 
section/CRF “Thoracotomy Output”).  
If an additional chest tube is required or reinserted (until discharge from the hospital or at Day 28) 
the duration that the additional chest tube was used (from time of insertion to time of removal) will 
be added to the time the original chest tube was used for the total postoperative duration.  
 Length of chest tube drainage (min) =  
(Time removal - End time (surgery) + 1) +  
(Time removal (second assessment) - Time reinsertion + 1) 
If a subject did not use any chest tube the duration is set to 0. As a sensitivity analysis, subjects 
with no use of chest tube will be excluded. For subjects who died before discharge from the 
hospital or before Day 28, respectively, the observed duration of chest tube drainage will be 
used. 
For the total postoperative duration, the same analyses as described for the primary endpoint will 
be applied.  
Again, as sensitivity analyses subjects who died will (1) be excluded from analysis and (2) be 
assigned a length of time of chest tube placement of 28 days.  
The total amount of chest tube drainage (mL), adding up each duration of chest tube as 
described above, will be summarized using descriptive measures and compared between groups 
using an ANOVA with a fixed effect for treatment group. 
L-citrulline plasma levels 
L-citrulline plasma levels will be obtained on each patient at set times. Individual concentration-
time curves will be plotted to confirm that they are ≥ the threshold level of 100 μmol/L. In addition, 
summary statistics using descriptive measures will be tabulated. 
4.5 
Safety Analysis 
Safety endpoints will be analyzed using the SAF. 
Adverse events 
Pre-treatment adverse events (PTAE) and treatment adverse events (TAE) will be analyzed 
separately. TAE are defined as all adverse events (AE) with time of onset or worsening on or 
after the time of first drug administration. If unclear due to incomplete start time / date of AE or 
drug administration, AEs will be assumed as TAEs. 
Adverse events (AE) will be summarized by system organ class (SOC) and preferred term (PT) 
according to Medical Dictionary for Regulatory Activities (MedDRA) version 19.0. The number of 
events, as well as the number and rate of affected subjects will be reported for each treatment 
group. AEs will be summarized also by severity, and by relationship to study medication. 
Separate analyses will be done for serious AEs and AEs leading to death. 


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SAP (version 2.0) 
Preferred terms of AEs with a proportion of ≥ 5% in either L-citrulline or placebo group will be 
additionally tabulated in descending order of frequency together with the relative risk (RR), risk 
difference (RD), and Odds ratio (OR). 
SAEs will additionally be tabulated by time of onset after time from the end of the surgery 
(variables “Date of surgery” and “End time” from section/CRF “Surgery”). The following 
timeframes will be used: 
 ≤ 0 hours:  
Occurrence from time of first drug administration until and inclusive  
 
time of end of the surgery 
 >0 to ≤12 hours:  Occurrence after time of end of the surgery until and inclusive +  
 
12 hours 
 >12 to ≤24 hours:  Occurrence after time of end of the surgery + 12 hours until and  
 
inclusive + 24 hours 
 >24 to ≤36 hours:  Occurrence after time of end of the surgery + 24 hours until and  
 
inclusive + 36 hours 
 >36 to 48 hours:  Occurrence after time of end of the surgery + 36 hours until and  
 
inclusive + 48 hours 
 >48 hours:  
Occurrence after time of end of the surgery + 48 hours until  
If unclear due to incomplete start time / date of AE or drug administration, AEs will be assigned to 
category >0 to ≤12 hours. 
No statistical testing procedures will be applied to the analysis of AEs.  
Incidence of refractory hypotension 
Refractory hypotension is defined as dropping of mean arterial (blood) pressure below specific 
age-related criteria (see below) for more than 30 minutes. I.e., 2 or more successive mean 
arterial blood pressure (MAP) measurements within a time frame of at least 30 minutes have to 
be below the criteria as listed below. 
The number of subjects with any refractory hypotension from end of surgery until 48 hours will be 
compared between groups using Fishers’ exact test. Additionally, a logistic regression analysis 
will be applied including factors treatment and site. 
Hypotension assessment (Refractory hypotension is defined as a 20% drop of MAP below 
specific age-related criteria (Haque and Zaritsky (2007) for greater than 30 minutes):  
 Infants: MAP of 40 
 Age 1 year: MAP of 40 
 Age 2 years: MAP of 44 
 Age 3 years: MAP of 47 
 Age 4 years: MAP of 52 
 Age 6 years: MAP of 53 
 Age 7 years: MAP of 52 
 Age 8 years: MAP of 54 
 Age 9 years: MAP of 55 
 Age 10 years: MAP of 56 
 Age 11 years: MAP of 57 
 Age 12 years: MAP of 58 
 Age 13 years: MAP of 59 
 Age 14 years: MAP of 61 
 Age 15 years: MAP of 62 
 Age 16 years: MAP of 63 
 Age 17-18 years: MAP of 64 
 
 
 


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Laboratory values 
Laboratory values will be analyzed descriptively. For quantitative parameters, absolute values as 
well as absolute and percentage changes from Baseline will be tabulated using summary 
statistics for all observed time points. For qualitative variables, frequency tables will be generated 
for all observed time points. 
A conversion to the given standard units will be performed if necessary. Concentrations below the 
limit of quantification will be set to zero. 
All laboratory values will be classified as normal or abnormal according to the laboratories normal 
ranges. Abnormal values will be indicated as clinically significant or not clinically significant by the 
investigator. Shift tables (normal / abnormal, not clinically significant / abnormal, clinically 
significant) will be created illustrating changes with respect to the laboratories normal ranges and 
clinical significance between Baseline and post Baseline visits Day 1, Day 2, and Day 28/ 
Discharge. 
The following laboratory parameters were determined and will be described for the corresponding 
visits: 
Hematology  
Type of all parameters is quantitative. 
 Hemoglobin 
 Hematocrit 
 Red blood cell (RBC) count 
 White blood cell (WBC) count (total and differential) 
 Platelet count 
Serum Electrolytes, BUN, and creatinine  
Type of all parameters is quantitative. 
 Serum electrolytes (Na, K, Ca, Mg and Cl) 
 BUN 
 Creatinine 
Liver function tests  
Type of all parameters is quantitative. 
 Bilirubin (total) 
 AP 
 AST 
 ALT 
 LDH 
Coagulation  
Type of all parameters is quantitative. 
 ACT 
Urinalysis  
Specific gravity and pH are quantitative, the remaining parameters are qualitative.  
 Visual: Color, Clarity/Turbidity 
 Chemical: pH, specific gravity, glucose, ketones, nitrites 
 Microscopic: RBCs, WBCs, epithelial cells, casts, bacteria 
No statistical testing procedures will be applied to the analysis of laboratory values. 


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SAP (version 2.0) 
4.6 
Analysis of Exploratory Variables 
PF ratio 
PF ratio is defined as PaO2 [mmHg] divided by FiO2 in [%]. PaO2 and FiO2 are measured at 
varying times. Since FiO2 is measured more often, PF ratio will only be calculated for the times 
when a corresponding PaO2 measurement is available (defined as FiO2 value measured at time 
of PaO2 value + 0.5h).  As denominator, the FiO2 value measured with minimum time difference 
will be used. If there is no FiO2 value measured at time of PaO2 ± 0.5h, no PF ratio will be 
calculated.  
The PF ratio will be listed. In addition, the mean PF ratio per patient and visit will be calculated 
and displayed using basic statistics.  
Example: 
ID 
Date/time of FiO2 
FiO2 
value 
 
Date/time of PaO2 
PaO2 
value 
PF ratio 
ID of matched 
FiO2 
1 
2018-01-08T11:57 
100 
 
2018-01-08T11:15  
73 
no matching value 
 
2 
2018-01-08T13:43 
80 
 
2018-01-08T12:32  
236 
no matching value 
 
3 
2018-01-08T14:00 
80 
 
2018-01-08T14:49  
267  
267/80  
4 
4 
2018-01-08T14:43 
80  
 
2018-01-08T16:50  
147  
147/75  
6 
5 
2018-01-08T15:00 
70 
 
2018-01-08T17:59  
213  
213/75  
7 
6 
2018-01-08T17:11 
75  
 
2018-01-08T19:04  
273  
273/75  
8 
7 
2018-01-08T18:00 
75  
 
2018-01-08T20:15  
326  
326/75  
9 
8 
2018-01-08T19:00 
75  
 
2018-01-08T22:27  
336 
no matching value 
 
9 
2018-01-08T20:18 
75  
 
 
 
 
 
10 
2018-01-08T23:33 
75 
 
 
 
 
 
 
28-day mortality 
The percentage of patients who have died at Day 28 will be presented by treatment group. 
4.7 
Analysis of Other Relevant Variables 
Arterial blood gases (ABG) 
The number of patients with any low or high values for AGB parameters PaO2, PaCO2, HCO3, 
pH per 6 hour time window and overall will be evaluated. 
6 hour time windows from time of post-op admission to the PICU will be defined as: 
 Time of AGB evaluation = Time collected AGB - Time of admission to PICU 
  >0 to  ≤6  hrs ( 
>0min  to  ≤360min ) 
  >6  to ≤12  hrs ( >360min  to  ≤720min ) 
  >12  to ≤18  hrs ( >720min  to  ≤1080min ) 
 … 
  >42  to ≤48  hrs ( >2520min  to  ≤2880min ) 
  
   >48  hrs (   
   
>2880min ) 
Normal ranges for the AGB parameters are defined as: 
 PaO2:  <70 mmHg = low; 70-100 mmHg = normal; >100 mmHg = high 
 PaCO2: <35 mmHg = low; 35-45 mmHg = normal; >45 mmHg = high 
 HCO3: <22 mmol/L = low; 22-26 mmol/L = normal; >26 mmol/L = high 
 pH: <7.35 = low; 7.35-7.45 = normal; >7.45 = high 
Patients will be categorized per AGB parameter and per time window (and overall) as follows: 


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 Low: At least one value at one visit was low 
 Normal: All values at all visit were normal 
 High: At least one value at one visit was high 
 Low/High: At least one value at one visit was low, and at least one value at one visit was 
high 
The number of patients for those categories will be tabulated per AGB parameter and per time 
window (and overall). 
4.8 
Missing Values 
Efficacy endpoints describing length of time 
If a subject did not receive any mechanical ventilation / inotropes / positive pressure ventilation / 
intubation / vasodilators / chest tube, the corresponding length of time is set to 0. For subjects 
who died before discharge from the hospital or before Day 28, respectively, the observed length 
of time will be used for analysis of all time variables. 
For time to event analyses censoring information will be used for statistical analyses.  
No other imputation of missing values will be used. 
Efficacy endpoint inotrope score 
The total inotrope score will be set to zero after the bypass pump has been removed. It will also 
be set to zero for patients not using inotropes at all.  
Further secondary efficacy endpoints 
No imputation of missing values will be applied for the secondary endpoints hemodynamic 
improvement, volume of chest tube drainage, and L-citrulline plasma levels. 
Safety endpoints 
For laboratory data, concentrations below the limit of quantification will be set to zero. Missing 
safety data or safety assessments will not be estimated nor imputed. All safety summaries and 
analyses will be based on observed data.  
No other imputation of missing values will be used. 
4.9 
Data Base Closure and Data Review 
The statistical analysis of the clinical observation period will be performed after cleaning and 
database lock of all data and final unblinding.  
A blind data review meeting (BDRM) will be conducted based on all data to check for protocol 
deviations and to allocate the subjects to the analysis sets FAS, mFAS, SAF, and PP. These 
evaluations and assessments will be done together and in agreement with the Sponsor, however 
FGK will provide the Sponsor with the appropriate subject listings. BDRM will be done via 
WebEx. 
Data unblinding on the basis of the randomization listing and the analysis will be done after 
BDRM has been conducted and BDRM minutes have been signed by both the Sponsor and FGK.  
 
 


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SAP (version 2.0) 
4.10 
Miscellaneous 
For qualitative variables the frequencies (absolute and relative) are calculated. If no further 
remark is given in the description of the tables following format will be used for all tables with 
qualitative variables: 
 
 
Y-variable(s) (e.g., treatment group) 
 
Category 1 
Category 2 
Total 
X-variable(s) 
N 
% 
N 
% 
N 
% 
category 1 
xx 
xx.x 
xx 
xx.x 
xx 
xx.x 
category 2 
xx 
xx.x 
xx 
xx.x 
xx 
xx.x 
Missing 
xx 
xx.x 
xx 
xx.x 
xx 
xx.x 
Total 
xx 
100.0 
xx 
100.0 
xx 
100.0 
 
For this standard format the description of the tables in Appendix A determines only the X- and Y-
variables. If another format of table is described in the details to the tables, the real design will be 
determined by the technical possibilities within SAS and may not look identical to the provided 
example. However, all information as displayed will be included. 
Quantitative parameters will be described by declaring the mean value, standard deviation, 
minimum, first quartile, median, third quartile, and maximum. In the description of the tables this 
will be denoted by „basic statistics“. 
The listings are always sorted by treatment group, center, and subject. If a different sorting order 
should be used for some listings this will be remarked separately. The variables for the special 
listings are explicitly given in the description of listings. All listings will be presented for the full 
analysis set, if not stated differently. Indicator variables for further analysis sets, e.g., safety 
analysis set, will be added to listing “Disposition of subjects and analysis sets”.  
The following title will be used for all generated tables, listings, and graphs: 
CIT-003-01: L-CITRULLINE 
Page # of # 
<Table/Listing/Graph NNN: Description of contents>  
<Subtitle for description of contents - if applicable> 
<Analysis set> 
 
The numbering NNN of the tables/listings/graphs will be stated in the detailed description 
(Appendix A). 
Following footnote will be used for all generated tables, listings, and graphs: 
Date: <Actual date> 
Program: <Name of program> 
 
The statistical evaluation will be performed using SAS version 9.3 or higher. 
5 
Changes from Protocol 
Length of time of inotrope therapy 
According to protocol length of time of inotrope use will be measured until the subject is 
discharged from the hospital or until Day 28, whichever occurs first. The length will be derived as 
the time of first use until last use (in hours).  
Actually the exact start and end date/times of inotropes will be documented in the CRF, therefore 
the length of time of inotrope use will be derived in minutes.  
Additionally, to be consistent to the time of mechanical ventilation, the time of separation from 
CPB will be used as starting point instead of time of first use. 


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Also, it is stated “For any missing scores between two non-missing scores they will be imputed 
using the most recent non-missing score (the last observation carried forward principle).” Due to 
the inotrope documentation as described above the LOCF principle is not to be applied. 
 
 


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6 
Literature 
n/a 
 
7 
Appendices 
 Appendix A: List of generated summary tables, listings, and graphs