Short-Term Outcome of N-Carbamylglutamate in the Treatment of Acute Hyperammonemia
Phase: PHASE2
Conditions: Propionic Acidemia, Type I and/or Type II, Methylmalonic Acidemia, Carbamoyl-Phosphate Synthase I Deficiency Disease, Ornithine Carbamoyltransferase Deficiency
Sponsor: Mendel Tuchman

 
 
 
 
 
 
 
 
 
Clinical Protocol Title:  
Short-Term Outcome of N-Carbamylglutamate in the Treatment of Acute 
Hyperammonemia.  
 
 
Study Chair:  
 
Nicholas Ah Mew, MD 
Director, Inherited Metabolic Disorders  
Children’s National Research Institute  
Associate Professor of Pediatrics  
The George Washington University 
111 Michigan Ave, NW  
Washington, DC 20010 
nahmew@childrensnational.org 
202-476-5863 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Phase of Clinical Investigation: Phase II 
 
IND Number:   68,185 
 
Investigational Drug(s):  N-carbamyl-L-glutamate, N-carbamylglutamate, NCG, Carglumic acid, 
“Carbaglu” (Orphan Europe) 
 
Sponsor:  National Institutes of Health (NIH), Recordati Rare Diseases 
This protocol is for research purposes only and should not be copied, redistributed, or used for any 
other purpose. The procedures in this protocol are intended only for use by study investigators in 
carefully controlled settings. The Study Chair of this study should be consulted before using this 
protocol. 

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Short-Term Outcome of N-Carbamylglutamate in the Treatment of Acute Hyperammonemia 
 
 
Investigator Agreement 
 
Protocol Version 1.0 ……………………………………………………………..... Version Date: May 4, 2012 
Protocol Version 1.1 …………………………………………………...… Version Date: September 27, 2012 
Protocol Version 1.2 …………………….……………………………………….. Version Date: July 30, 2013 
Protocol Version 2.0 …………………………………………….………… Version Date: September 9, 2014 
Protocol Version 2.1 ……………………………………………………............. Version Date: April 14, 2015 
Protocol Version 3.0 ………………………………………………………. Version Date: September 9, 2016 
Protocol Version 3.1 ……………………………………………………….. Version Date: November 8, 2016 
Protocol Version 4.0 …………………………………………………………..... Version Date: March 6, 2017 
Protocol Version 4.1 ……………………………………………………..………. Version Date: May 24, 2017  
Protocol Version 4.2 ………………………………………………………...…. Version Date: August 4, 2017 
Protocol Version 5.0 ………………………………………………………… Version Date: October 17, 2018 
Protocol Version 6.0 ………………………………………………..…………. Version Date: March 20, 2019 
Protocol Version 7.0 …………………………………………………………... Version Date: March 31, 2020  
Protocol Version 7.1 …………………………………………………………….… Version Date: May 1, 2020 
 
The signature below represents my approval of: 
Protocol Version 7.1 ……………………………………………………………... Version Date: May 1, 2020 
 
 
Study Chair: Nicholas Ah Mew, MD 
Director, Inherited Metabolic Disorders 
Children's National Research Institute 
Associate Professor of Pediatrics 
The George Washington University 
 
 
 
 
 
Signature: 
_____________________________________________            _________________ 
 
 
 
 
 
 
 
 
 
 
 
 
Date 
 
 
Site PI:  
_____________________________________________          
 
 
Site:     
_____________________________________________          
 
 
Signature: 
_____________________________________________            _________________ 
 
 
 
 
 
 
 
 
 
 
 
 
Date 
 
 
 
 
 

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IND Number:   68,185 
 
Sponsor:  National Institutes of Health (NIH), Recordati Rare Diseases 
Table of Contents 
LIST OF PARTICIPATING INSTITUTIONS ........................................................................................... 6 
LIST OF CENTRAL ENTITIES ............................................................................................................... 8 
LIST OF DATA AND SAFETY MONITORING BOARD (DSMB) MEMBERS ........................................ 10 
LIST OF ABBREVIATIONS.................................................................................................................. 11 
STUDY SYNOPSIS AND SCHEMA ..................................................................................................... 12 
1 INTRODUCTION .............................................................................................................................. 14 
 Background .............................................................................................................................. 14 
1.1
1.1.1 
The Urea Cycle ............................................................................................................. 14 
1.1.2 
Carbamyl Phosphate Synthetase I Deficiency (CPSD) ................................................. 14 
1.1.3 
Ornithine Transcarbamylase Deficiency (OTCD) .......................................................... 14 
1.1.4 
Propionic Acidemia (PA) ............................................................................................... 15 
1.1.5 
Methylmalonic Acidemia (MMA) .................................................................................... 15 
1.1.6 
Mechanism of Hyperammonemia in PA and MMA ........................................................ 15 
1.1.7 
Current Therapies for Hyperammonemia ...................................................................... 15 
 Study Rationale ........................................................................................................................ 16 
1.2
 Study Treatment: N-carbamyl-L-glutamate (NCG) .................................................................... 17 
1.3
1.3.1 
Preclinical Profile .......................................................................................................... 17 
1.3.2 
Clinical Profile ............................................................................................................... 18 
1.3.3 
Carbaglu Formulation and Dosage ............................................................................... 19 
1.3.4 
Safety and Side Effects of Carbaglu ............................................................................. 20 
2 OBJECTIVES ................................................................................................................................... 21 
2.3.1.1 .............................................................................................................................................. 21 
 Assess Efficacy of NCG Treatment .......................................................................................... 21 
2.1
 Safety ....................................................................................................................................... 21 
2.2
3 STUDY DESIGN ............................................................................................................................... 22 
3.3.1.1 .............................................................................................................................................. 22 
 Overview of Study Design ........................................................................................................ 22 
3.1
4 STUDY POPULATION ...................................................................................................................... 24 
4.3.1.1 .............................................................................................................................................. 24 
 Inclusion Criteria ...................................................................................................................... 24 
4.1
 Exclusion Criteria ..................................................................................................................... 24 
4.2
5 STUDY PROCEDURES ................................................................................................................... 25 
5.3.1.1 .............................................................................................................................................. 25 
 Screening of Potential Subjects ................................................................................................ 25 
5.1
5.1.1 
Confirmation of Diagnosis of PA, MMA, CPSD or OTCD .............................................. 25 
 Informed Consent Procedures .................................................................................................. 25 
5.2
 Retention Procedures ............................................................................................................... 26 
5.3
 Duration of Study and Visit Schedule ....................................................................................... 27 
5.4
5.4.1 
Hyperammonemia Episodes ......................................................................................... 27 
 Study Assessments .................................................................................................................. 30 
5.5
5.5.1 
Laboratory Tests ........................................................................................................... 30 
5.5.2 
Electrocardiogram Test (EKG) ...................................................................................... 31 
5.5.3 
Transfusion Information ................................................................................................ 31 
5.5.4 
Clinical Assessments .................................................................................................... 31 
5.5.5 
Efficacy Assessments ................................................................................................... 32 
5.5.6 
Safety Assessments ..................................................................................................... 32 

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 Treatment Compliance ............................................................................................................. 33 
5.6
 Study Termination/Stopping Rules ........................................................................................... 33 
5.7
 Subject Discontinuation ............................................................................................................ 33 
5.8
 Blinding .................................................................................................................................... 33 
5.9
5.9.1 
Type of Blinding ............................................................................................................ 33 
5.9.2 
Maintenance of Blinding ............................................................................................... 33 
5.9.3 
Breaking the Blinding .................................................................................................... 34 
6 STUDY TREATMENTS .................................................................................................................... 35 
6.1 Study Intervention .................................................................................................................... 35 
6.2 Control (Placebo) Intervention .................................................................................................. 35 
6.3 Dispensing of Study Medications.............................................................................................. 35 
6.4 Provisions for Access to Investigational Treatment after Study ................................................ 35 
6.5 Drug Packaging/Handling/Storage/Accountability ..................................................................... 35 
6.6 Concomitant Therapy ............................................................................................................... 36 
6.6.1 
Therapies Prohibited Before/During the Trial ................................................................ 36 
6.6.2 
Therapies Allowed During the Trial ............................................................................... 36 
7 SAFETY MONITORING .................................................................................................................... 37 
7.1 Adverse Events (AEs) .............................................................................................................. 37 
7.1.1 
Definitions ..................................................................................................................... 37 
7.1.2 
Collection and Reporting of Adverse Events ................................................................. 39 
7.2 Data and Safety Monitoring Board (DSMB) .............................................................................. 41 
7.3 Medical Monitor ........................................................................................................................ 41 
8 STATISTICAL ANALYSIS ................................................................................................................. 42 
8.1 Study Aims ............................................................................................................................... 42 
8.1.1 
Primary Aim ................................................................................................................. 42 
8.1.2 
Secondary Aim ............................................................................................................ 42 
8.2 Factors for Stratification ........................................................................................................... 42 
8.3 Statistical Considerations ......................................................................................................... 42 
8.3.1 
Interim Analysis ............................................................................................................ 43 
8.4 Rationale for Sample Size and Statistical Power ...................................................................... 44 
9 REGULATORY COMPLIANCE AND SAFETY MONITORING .......................................................... 45 
9.1 Regulatory Compliance ............................................................................................................ 45 
9.2 Statement of Compliance ......................................................................................................... 45 
9.3 Informed Consent ..................................................................................................................... 45 
9.4 Records Retention and Requirements ...................................................................................... 46 
9.5 Financial Disclosure ................................................................................................................. 46 
9.6 Inclusion of Minorities and Children .......................................................................................... 46 
9.7 Confidentiality........................................................................................................................... 46 
9.7.1 
Protection Against Risk ................................................................................................. 46 
9.7.2 
Health Insurance Portability and Accountability Act (HIPAA) ........................................ 47 
9.8 Regulatory Files ....................................................................................................................... 47 
9.9 Source Data Verification Monitoring ......................................................................................... 48 
9.10 Modification of the Protocol ...................................................................................................... 48 
9.11 Protocol Deviations and Violations ........................................................................................... 48 
10 DATA MANAGEMENT AND PROCEDURES ................................................................................. 50 
10.1 Data Collection ......................................................................................................................... 50 
10.2 Data Processing ....................................................................................................................... 50 
10.3 Design and Development of REDCap for Study Data Management ......................................... 50 
10.3.1 REDCap Time Event Matrix .......................................................................................... 50 
10.3.2 REDCap Protocol Management .................................................................................... 51 
10.3.3 REDCap Facilitated Screening Features ...................................................................... 51 

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10.3.4 REDCap Randomization ............................................................................................... 51 
10.4 Recruitment and Retention Monitoring ..................................................................................... 51 
10.5 Site Data and Study Responsibilities ........................................................................................ 52 
10.6 CNMC Coordinating Center Responsibilities ............................................................................ 52 
10.7 Data Security............................................................................................................................ 53 
10.8 Data Acquisition and Entry ....................................................................................................... 53 
10.9 Data Editing ............................................................................................................................. 54 
10.10 Data Lock and Freeze ........................................................................................................... 54 
10.11 Data Entry Training ............................................................................................................... 55 
10.12 Data Quality Assurance ......................................................................................................... 55 
11 PUBLICATIONS AND OTHER RIGHTS ......................................................................................... 56 
12 REFERENCES ............................................................................................................................... 57 
 
 
 

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LIST OF PARTICIPATING INSTITUTIONS   
 
Boston Children’s Hospital  
Gerard T. Berry, MD 
Institution: 
Boston Children’s Hospital  
Address: 
Department of Pediatric Endocrinology 
300 Longwood Ave., # FEGAN10 
Boston, MA 02115 
Phone: 
857-218-4636 
Fax: 
617-730-4874 
 
University Hospitals of Cleveland Medical Center  
Laura Konczal, MD 
Institution: 
University Hospitals of Cleveland Medical Center 
Address: 
Center for Human Genetics 
11100 Euclid Avenue 
Lakeside 1500 
Cleveland, OH 44106-6055 
Phone: 
216-844-8567 
Fax: 
216-844-7497 
 
The Children’s Hospital of Colorado 
Shawn McCandless, MD 
Institution: 
The Children’s Hospital of Colorado 
Address: 
13123 E. 16th Ave, B300 
Aurora, CO 80045 
Phone: 
303-724-2310  
Fax: 
720-777-7322   
 
University of California Los Angeles  
Derek Wong, MD 
Institution: 
University of California Los Angeles 
Address: 
Division of Genetics 
10833 Le Conte Avenue, CHS 32-231 
Los Angeles, CA 90095 
Phone: 
310-206-6581 
Fax: 
310-206-8616 
 
The Children's Hospital of Philadelphia (CHOP)  
Can Ficicioglu, MD, PhD 
Institution: 
The Children's Hospital of Philadelphia 
Address: 
Division of Human Genetics and Metabolism 
3501 Civic Center Blvd, #9054 
Philadelphia, PA 19104 
Phone: 
215-590-3376 
Fax: 
267-425-0121 
 
 
 

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Lucile Packard Children's Hospital at Stanford University  
Gregory Enns, MD 
Institution: 
Lucile Packard Children’s Hospital at Stanford University 
Address: 
Pediatric Genetics 
730 Welch Rd, 2nd Floor 
Palo Alto, CA 94304 
Phone: 
650-721-5804 
Fax: 
650-498-4555 
 
Children’s National Hospital  
Nicholas Ah Mew, MD 
Institution: 
Children’s National Hospital 
Address: 
Division of Genetics and Metabolism 
111 Michigan Avenue NW, 
Washington, DC 20010 
Phone: 
202-476-5863 
Fax: 
202-476-5650 
 
Children’s Hospital Pittsburgh 
Gerard Vockley, MD, PhD 
Institution: 
Children’s Hospital Pittsburgh 
Address: 
4401 Penn Avenue 
Suite 1200 
Pittsburgh, PA 15224 
Phone: 
412-692-7746 
Fax: 
412-692-7816 
 
Mount Sinai Hospital 
George Diaz, MD 
Institution: 
Mount Sinai Hospital 
Address: 
1428 Madison Avenue 
1st Floor, Room AB1-12 
New York, NY 10029 
Phone: 
212-241-6947 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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LIST OF CENTRAL ENTITIES 
 
Study Principal Investigator 
Nicholas Ah Mew, MD 
Institution: 
Children’s National Hospital 
Address: 
Division of Genetics and Metabolism 
111 Michigan Avenue NW 
Washington, DC 20010 
Phone: 
202-476-5863 
Fax: 
202-476-5650 
 
 
 
 
Project Manager 
Katie Rice, MPH, CCRP  
Institution: 
Children’s National Hospital 
Address: 
Children’s National Research Institute 
111 Michigan Avenue NW 
Washington, DC 20010 
Phone: 
202-476-6191 
Fax: 
202-476-5650 
 

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Study Biostatistician  
Robert J. McCarter, ScD 
Institution: 
Children’s National Hospital 
Address: 
Center for Translational Science 
111 Michigan Avenue NW 
Washington, DC 20010 
Phone: 
202-476-3140 
Fax: 
202- 476-3425 
Pager: 
202-259-4567 
 
 
Central Pharmacy   
Henry Choi, PharmD  
Institution: 
Children’s National Hospital 
Address: 
Pharmacy Department 
111 Michigan Avenue NW 
Washington, DC 20010 
Phone: 
202-476-2088 
Fax: 
202-476-6984 
Pager: 
202-259-4132 
 
 
Medical Monitor  
Barbara Burton, MD 
Institution: 
Lurie Children’s Hospital of Chicago 
Address: 
225 E Chicago Avenue 
Box MC 59 
Chicago, IL 60611 
Phone: 
312-227-6120  
Cell: 
312-919-2056 
 
 

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LIST OF DATA AND SAFETY MONITORING BOARD (DSMB) MEMBERS 
 
DSMB Chair Joan Conry, MD 
Institution: 
Children’s National Hospital 
Address: 
Center for Translational Science 
111 Michigan Avenue NW 
Washington, DC 20010 
Phone: 
202-476-2120 
 
John Van Den Anker, MD, PhD 
Institution: 
Children’s National Hospital 
Address: 
Center for Translational Science 
111 Michigan Avenue NW 
Washington, DC 20010 
Phone: 
202-476-4875 
 
An Massaro, MD 
Institution: 
Children’s National Hospital 
Address: 
Center for Neurosciences Research 
111 Michigan Avenue NW 
Washington, DC 20010 
Phone: 
202-476-5225 
 
Tomas Silber, MD 
Institution: 
Children’s National Hospital 
Address: 
Department of Adolescent and Young Adult Medicine 
111 Michigan Avenue NW 
Washington, DC 20010 
Phone: 
202-476-3066 
 
Samuel Simmens, PhD, MA 
Institution: 
The George Washington University 
Address: 
Department of Epidemiology and Biostatistics 
School of Public Health and Health Services 
The George Washington University 
2100-W Pennsylvania Ave, NW 
8th Floor 
Washington, DC 20037 
Phone: 
202-994-7887 

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LIST OF ABBREVIATIONS 
AE – Adverse Event 
ALT – Alanine Aminotransferase 
AST – Aspartate Aminotransferase 
BCH – Boston Children’s Hospital 
CBC – Complete Blood Count 
CFR – Code of Federal Regulations 
CHOP – Children’s Hospital of Philadelphia 
CPS1 – Carbamyl Phosphate Synthetase 1 
CPSD – Carbamyl Phosphate Synthetase deficiency 
CRA – Clinical Research Associates 
CRP – Central Research Pharmacy 
CTCAE – Common Terminology Criteria for Adverse Events 
DSMB – Data Safety Monitoring Board 
EKG – Electrocardiogram  
eCRF – electronic Case Report Form 
FDA – Food and Drug Administration 
FSS – Functional Status Scale 
GCMS – Gas Chromatography-Mass Spectrometry 
G-tube – Gastrostomy tube 
HA - Hyperammonemia 
HHS – Department of Health and Human Services 
HHS – Hyperinsulinism Hyperammonemia Syndrome 
HIPAA – Health Insurance Portability and Accountability Act 
ICH – International Conference on Harmonization 
INR – International Normalized Ratio 
IRB – Institutional Review Board 
MMA – Methylmalonic Acidemia 
NAG – N-acetylglutamate 
NAGS – N-acetylglutamate synthase 
NCG – N-carbamyl-L-glutamate, N-carbamylglutamate 
NG-tube – Nasogastric tube 
NIH – National Institutes of Health 
PA – Propionic Acidemia 
PHI – Protected Health Information 
PI – Primary Investigator 
PID – Participant Identifier 
PLBO – Placebo 
PTT – Partial Thromboplastin Time 
REDCap – Research Electronic Data Capture 
SAE – Serious Adverse Event 
SOP – Standard Operating Procedures 
STD – Standard Therapy 
TCHC – The Children’s Hospital of Colorado 
StGr – Study Group 
UCD – Urea Cycle Disorder 
UCLA – University of California Los Angeles 
UHCMC – University Hospitals of Cleveland Medical Center 

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STUDY SYNOPSIS AND SCHEMA 
 
Protocol Number: 
2894 
Protocol Title: 
N-carbamylglutamate in the Treatment of Hyperammonemia: Accelerating the 
Resolution of Hyperammonemia and Clinical Recovery and Safety 
Study Chair: 
Nicholas Ah Mew, MD 
Statistician: 
Robert McCarter, ScD 
Consortium: 
N-carbamylglutamate Consortium (NCGC) 
Participating Sites: 
Boston Children’s Hospital (BCH), University Hospitals of Cleveland Medical 
Center (UHCMC), The Children’s Hospital of Colorado (CHCO), the University of 
California Los Angeles (UCLA), Children’s Hospital of Philadelphia (CHOP), Lucile 
Packard Children’s Hospital at Stanford (SU), Children’s National Hospital 
(Children’s National), Children’s Hospital Pittsburgh (UPMC), and Mount Sinai 
Hospital (MSSM). 
Activation Date: 
 July 2nd, 2012 
Sample Size: 
36 patients experiencing approximately 114 hyperammonemia episodes across 
diagnoses (with approval to enroll up to 80 patients to allow for dropouts, losses to 
follow-up, and lower than expected incidence of hyperammonemic episodes) 
Target Enrollment 
Period: 
5 years 
Study Design: 
Phase II 
Primary Study 
Objective: 
The overall objective of this project is to determine whether treatment of acute 
hyperammonemia with N-carbamyl-L-glutamate (N-carbamylglutamate, NCG, 
Carglumic acid, Carbaglu) in propionic acidemia (PA), methylmalonic acidemia 
(MMA), late-onset CPS1 deficiency (CPSD) and late-onset Ornithine 
transcarbamylase deficiency (OTCD) accelerates the resolution of 
hyperammonemia.  
Study Population 
and Main Eligibility/ 
Exclusion Criteria: 
Inclusion Criteria 
o Aged older than 1 week with an established diagnosis of CPSD or OTCD 
(as follows):  
 
Diagnosed with late-onset CPSD confirmed by detection of pathogenic 
mutation(s), and/or decreased (<20% of control) CPS enzyme activity 
in liver OR 
 
Diagnosed with late-onset OTCD by detection of pathogenic OTC 
mutation, OR decreased (<20% of control) OTC enzyme activity in 
liver OR elevated urinary orotate (greater than 20 µM/mM creatinine) 
following allopurinol loading with absence of argininosuccinic acid 
 
 
AND: Subject or subject’s first-degree relative had initial plasma 
ammonia level ≥100mcmol/L >1 week of age 
 
OR 
 
o An established diagnosis of PA or MMA (as follows): 
 
Diagnosed with PA by semi-quantitative urine organic acid analysis, 
defined as presence of elevated methylcitric acid and normal 

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methylmalonic acid levels and no evidence of biotin-related disorders 
in the organic acid analysis OR 
 
Diagnosed with MMA by semi-quantitative urine organic acid analysis, 
defined as elevation of methylmalonic acid and no evidence of vitamin 
B12 dependent disorder on plasma amino acid analysis (B12 
dependency is defined by documented B12 responsiveness) 
 
 
AND: Subject or subject’s first-degree relative had plasma ammonia 
level at any time ≥100 mcmol/L 
 
o Able to receive medications orally, by nasogastric (NG)-tube or by Gastric 
(G)-tube 
o No concomitant illness which would preclude safe participation as judged by 
the investigator 
o If post-menarcheal must have a negative pregnancy test prior to 
administration of study drug at each episode 
o Signed informed consent by the subject or the subject's legally acceptable 
representative 
 
Exclusion criteria 
o Administration of NCG within 7 days of participation in the study 
o Use of any other investigational drug, biologic, or therapy 
o Planned participation in any other clinical trial 
o Diagnosis of any medical condition causing hyperammonemia which is not 
PA, MMA, CPSD, OTCD 
o Any clinical or laboratory abnormality or medical condition that, at the 
discretion of the investigator, may put the subject at an additional risk by 
participating in this study 
o Had a liver transplant  
o Is pregnant 
o Is not expected to be compliant with the study 
 
Patients enrolled in the now-closed long-term outcomes study being conducted in 
parallel are eligible to enroll in this study provided that all other inclusion and 
exclusion criteria are satisfied. 
Primary Outcome 
Measures: 
Composite of the shortest of the time to reach an ammonia level <50 mcmol/L or time 
to discharge from the hospital after the first dose of NCG. Safety of NCG treatment 
as measured by serious adverse events, electrocardiogram test and laboratory blood 
tests 
Statistical 
Considerations 
(sample size and 
analysis plan): 
Based on 2-tailed testing assuming a type 1 error of 5% and using comparisons of 
treatment effects across episodes, a sample size of 36 patients, experiencing an 
average of <4 episodes each (total 114 episodes) would provide 80% power to 
detect hazard ratio at least as large as 1.7 favoring a higher average probability of 
the composite outcome in the NCG group. A sample size of 80 will allow for drop 
outs, losses to follow-up, and lower than expected incidence of hyperammonemic 
episodes. 
Sponsors (federal, 
state, foundation and 
industry support): 
National Institutes of Health (NIH), Recordati Rare Diseases 

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1 INTRODUCTION 
 
 
Background 
1.1
1.1.1 The Urea Cycle 
The urea cycle consists of several biochemical steps required for the conversion of ammonia 
into urea (Figure). The first and rate-limiting step of the urea cycle is catalyzed by the 
mitochondrial enzyme, carbamyl phosphate synthetase (CPS1), which converts ammonia, 
bicarbonate, and ATP into carbamyl phosphate. Subsequently, the enzyme ornithine 
transcarbamylase (OTC) conjugates ornithine and carbamyl phosphate to form citrulline, which 
then continues through the rest of the urea cycle. CPS1 has as an essential allosteric enzyme, 
N-acetylglutamate, produced from the enzyme N-acetylglutamate synthase. 
 
 
 
1.1.2 Carbamyl Phosphate Synthetase I Deficiency (CPSD) 
Carbamyl phosphate synthetase I is the first and rate-limiting step of the urea cycle and 
converts ammonia, bicarbonate, and adenosine triphosphate (ATP) into carbamyl phosphate. 
Mutations which completely abolish CPS activity, eliminate flux through this step, and lead to 
hyperammonemia. However, mutations resulting in reduced but not abolished CPS enzyme 
function may result in an attenuated form of hyperammonemia, characterized by a later 
presentation, plasma ammonia better amenable to dietary and pharmacotherapy or fewer 
admissions for hyperammonemia. Nevertheless, episodes of hyperammonemia may still be life-
threatening, and if untreated, may result in coma or death. Clinical features of hyperammonemia 
may also include encephalopathy, brain edema, tachypnea, lethargy, vomiting, hypotonia, and 
hypothermia. 
 
1.1.3 Ornithine Transcarbamylase Deficiency (OTCD) 
Ornithine transcarbamylase (OTC) conjugates carbamyl phosphate and ornithine to form 
citrulline. Deficiency of the OTC enzyme results in hyperammonemia and may result in 
encephalopathy, brain edema, tachypnea, lethargy, vomiting, hypotonia, hypothermia, and if 
untreated, coma and/or death. The OTC gene is located on the X-chromosome, thus males 
have only one copy of the OTC gene, whereas females have two copies, but due to lyonization 
have only one active copy per cell. The X-chromosome in each cell that is inactivated is 

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determined randomly and prenatally, at an early embryological stage. A partial deficiency of 
OTC may be observed in hemizygous males only if a mutation in the OTC gene is hypomorphic. 
However, in affected females, a partial deficiency is typically only observed with mutations that 
entirely abolish OTC activity. This is because due to lyonization, affected females have two 
populations of hepatocytes – those that express the mutant OTC allele, and those that express 
the normal allele. 
 
1.1.4 Propionic Acidemia (PA) 
Propionic acidemia (PA) is caused by a complete or partial deficiency of the enzyme, propionyl-
CoA carboxylase, or a defect in the utilization or salvage of its cofactor, biotin. In the neonatal 
period, this condition can present with lethargy, poor feeding, vomiting, ketoacidosis, and 
hyperammonemia, and may result in death if not treated appropriately. Ammonia can reach 
extremely high levels, approaching those seen in patients with urea cycle disorders (ref). 
 
1.1.5 Methylmalonic Acidemia (MMA) 
Methylmalonic acidemia/aciduria (MMA) is caused by a complete or partial deficiency of the 
enzyme methylmalonyl-CoA mutase, a defect in the transport or synthesis of its cofactor, 
adenosyl-cobalamin, or deficiency of the enzyme methylmalonyl-CoA epimerase. In the 
neonatal period, the disease can present with lethargy, vomiting, hypotonia, hypothermia, 
respiratory distress, severe ketoacidosis, and hyperammonemia, and can result in death if not 
appropriately treated. Hyperammonemia also occurs in methylmalonic acidemia, in particular in 
the neonatal period, and like in PA, can approach levels seen in patients with urea cycle 
disorders. 
 
1.1.6 Mechanism of Hyperammonemia in PA and MMA 
Hyperammonemia in PA and MMA are thought to result from the inhibition of the N-
acetylglutamate (NAG) synthase reaction. This may be either the result of direct competitive 
inhibition of the NAG synthase reaction by the organic acid, from decreased availability of 
acetyl-Coenzyme A or from Coenzyme A depletion (ref). Any of these mechanism lead to 
decreased mitochondrial concentration of NAG. Because NAG is the essential allosteric 
cofactor to carbamylphosphate synthase (CPS1) reaction (the first step of the urea cycle), a 
decrease in NAG concentration leads to decreased flux through the CPS1 reaction and results 
in hyperammonemia. 
 
1.1.7 Current Therapies for Hyperammonemia  
Very few drugs to treat hyperammonemia have been developed and brought to market. Dietary 
protein restriction and administration of alternate pathway acylating agents are the current 
primary treatments, but they frequently fail to avert brain damage. Current alternate pathway 
therapies remove excess nitrogen stoichiometrically, eliminating a fixed number of nitrogen 
atoms (1 or 2) for every molecule of drug administered, reactions, which require the adequate 
liver function to be effective.  
  
 
Orthotopic liver transplantation cures hyperammonemia, but organ availability has been limited 
and the procedure is highly invasive and requires life-long immunosuppression.  
 
Since hyperammonemia is critical in the pathophysiology of screenable disorders such as urea 
cycle defects and possibly organic acidemias, which are currently, or will be in the near future 
detectable by expanded newborn screening, developing new innovative treatments for these 
disorders is essential if early diagnosis is to change the natural course of these conditions. 

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 Study Rationale   
1.2
As described above, current drug therapy for inherited hyperammonemia relies mainly on the 
activation of alternate pathways for nitrogen elimination. In contrast, N-carbamylglutamate (NCG), a 
stable analog of N-acetylglutamate, catalytically stimulates the urea cycle, thus converting many more 
molecules of ammonia to urea with a relatively small dose of the drug. This represents a paradigm 
shift in the medical treatment of hyperammonemia.  
 
A drug that could repair or stimulate a dysfunctional urea cycle would have several advantages over 
current therapy. First, it would intervene through the normal pathway of ureagenesis, which is 
catalytic rather than stoichiometric. Second, it may not need to replace current treatment but would be 
complementary to it. Third, the ability to stimulate residual ureagenesis could benefit many conditions 
that cause hyperammonemia, not just a single disorder. Enhancement of residual ureagenesis 
capacity in patients with hyperammonemia would markedly alter the natural disease course resulting 
in a manageable phenotype and better clinical outcome. 
 
Our recent studies show that ureagenesis is augmented by NCG in healthy individuals and in more 
than half of all individuals with inborn errors that were tested. 
 
The eligibility for the studies in this study specifically includes severe PA and MMA and “milder” 
CPSD and OTCD, which may appear paradoxical. However, the rationale for these selection criteria 
is based on the different mechanism(s) of action in these disorders. Significant hyperammonemia in 
PA or MMA is usually seen only in the most severely affected patients who present as newborns. 
Patients with hypomorphic alleles have milder disease and are unlikely to have serious 
hyperammonemia.72 In severely affected patients with PA and MMA, NCG works to alleviate 
deficiency of NAG created by the inhibition of NAGS by the mitochondrial accumulation of propionyl-
CoA, hence, the more profound the NAG deficiency, the more robust the NCG effect. In CPSD and 
OTCD however, the putative effect of NCG relies on stimulating residual CPS1 or OTC activity. 
Indeed, when these enzymes are totally absent, no amount of NCG would help, hence, the need to 
enroll patients with partial rather than complete enzyme deficiencies. 
 
If the proposed studies show that the use of NCG is effective in enhancing or completely restoring 
ureagenesis in patients with PA, MMA, CPSD and/or OTCD, they can be treated long-term with this 
therapeutic agent either during acute episodes or chronically. Carbaglu is FDA-approved (03-2010). 
Thus, responsive participants could receive this medication off-label if their physician obtains a single 
patient IND. There can be an important direct benefit for these participants. If the underlying 
hypothesis is correct, this study could improve the medical condition of some or all of the patients, 
also preventing life-threatening events in those who respond.  
 
The study will provide the first reliable information of whether NCG could potentially reduce the 
hyperammonemia associated with the 4 disorders to be studied. Such knowledge could provide a 
new treatment for some patients with hyperammonemia. In addition, the information gained could 
also be relevant to better understanding the regulation of the urea cycle, which may impact also the 
treatment of hyperammonemia due to generalized liver disease or other causes. 
 
This emerging concept posits that NCG will be effective as an adjunct treatment for hyperammonemia 
in several conditions. A few case reports provide anecdotal evidence for the effectiveness of NCG in 
reversing hyperammonemia in propionic acidemia (PA) and methylmalonic acidemia (MMA) 17-22. 
Although these observations may be factually correct, they do not represent credible scientific 
evidence and are unlikely to be accepted by regulatory agencies. The studies proposed herein, are 

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placebo-controlled, randomized, and blinded addressing this question using multiple true and 
surrogate endpoints, increasing scientific reliability.   
 
 Study Treatment: N-carbamyl-L-glutamate (NCG) 
1.3
N-carbamylglutamate, Carglumic acid, and Carbaglu are synonyms for the study treatment, N-
carbamyl-L-glutamate (NCG). 
 
N-carbamyl-L-glutamate (NCG) has recently been found to have virtually curative of a urea cycle 
defect, N-acetylglutamate synthase (NAGS) deficiency (FDA IND# 68,185). In this disorder, treatment 
with NCG alone normalizes ureagenesis, blood ammonia, and glutamine levels, allows normal protein 
tolerance, and restores health. Knowledge from the study of NCG in inborn errors could be applied to 
acquired hyperammonemia, specifically in patients with propionic acidemia (PA) and methylmalonic 
acidemia (MMA), to improve neurodevelopmental outcomes and reduce nitrogen load.  
 
1.3.1 Preclinical Profile 
Carbaglu® is synthesized by Synth-Innove and was confirmed to activate CPS I in vitro. 
Labeled NCG injected into mice can be detected in the liver. 
Toxicology studies in rats, at doses of up to 1000 mg/kg did not show any neurobehavioral, 
neurovegetative, neurotoxic or psychotropic effects nor any effects on body temperature. 
Respiratory function, including respiratory rate, peak respiratory and inspiratory flows, 
inspiration and expiration time, tidal volume and airway resistance also showed no statistically 
significant change.  
 
In isolated canine Purkinje fibers, there was no statistically significant effect on action potential 
parameters, and no dearly depolarization was observed at any concentration. Additionally NCG 
at concentrations up to 0.1 mM had no effect on action potential duration. 
 
Toxicology studies on the cardiovascular system in dogs showed no change in blood pressure, 
heart rate and cardiac conduction times including QT interval and QTc after administration of up 
to 1000 mg/kg of Carbaglu.  
 
Carbaglu was administered enterally once up to 2806 mg/kg or intravenously up to 238.6mg/kg, 
in Sprague-Dawley rats, and no mortality was observed. 
 
No clastogenic activity was found in a study with human lymphocytes, and the micronucleus test 
in rats was negative. The impurity hydantoin-5-propionic acid demonstrated clastogenic activity 
at concentrations > 2500 µg/ml. The quality profile of Carbaglu is less than 0.1% hydantoin-5-
propionic acid. NCG does not seem to affect male or female fertility.  
 
1.3.1.1 
Pediatric Animal Studies 
In newborn rats, NCG given at 2000 mg/kg/day was lethal to all pups between 2-14 days. At 
1000 mg/kg/d, orange colored feces, a slight reduction of body weight gain, and a 
decreased thymus weight were observed. At doses of <500 mg/kg/d, NCG was well 
tolerated, and no adverse effects were observed. 
 
NCG was also administered to 4-week old Sprague-Dawley rats daily for 6 months at doses 
of 500 or 1000 mg/kg/d. At 1000 mg/kg/d, ptyalism (excess salivation) was the only adverse 
effect observed. 
 

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1.3.2 Clinical Profile  
 
1.3.2.1 Carbaglu in Treatment of NAG Synthase Deficiency 
Carbaglu corrects the biochemical defect and normalizes all biochemical parameters, 
including ammonia, in patients with a primary deficiency of NAG, as observed in NAG 
Synthase (NAGS) deficiency. Retrospective data from 13 patients with NAGS who were 
treated for acute hyperammonemia were collected by Orphan Europe. Mean plasma 
ammonia for these patients at initial presentation, as well as each day following Carbaglu 
administration, is shown in Figure 1, below: 
 
Figure 1. Mean ammonia vs. time in n=13 subjects with NAGS deficiency receiving 
Carbaglu 
 
 
1.3.2.2 Carbaglu in Secondary Deficiencies of NAG  
Several publications have pointed out the potential of NCG as a therapeutic adjunct to the 
treatment of hyperammonemia in secondary deficiencies of NAG (i.e., not resulting from 
NAGS deficiency). All these reports were uncontrolled case reports or case series10-13,15,20. 
 
Our first project was initially funded for 34 months from 8/5/2008 through 5/31/2011. The 
original specific aims were: 1) To determine whether 3-day treatment with NCG improves or 
restores ureagenesis in patients with NAGS deficiency, Carbamyl phosphate synthase 
(CPSD), PA, MMA and hyperinsulinism and hyperammonemia syndrome (HHS), and 2) To 
evaluate the safety of short-term (3-day) treatment with NCG. The accomplishments thus far 
indicate that 3-day treatment with NCG: 
 
Normalizes ureagenesis rate, plasma ammonia, glutamine and urea in NAGS 
deficiency10,21  
 
Enhances ureagenesis and reduces plasma ammonia and glutamine levels in PA9 
 
Improves ureagenesis and decreases plasma ammonia and glutamine in some 
patients with CPSD 
 
Improves ureagenesis in some patients with HHS 

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 
Is safe at the prescribed dose of 100/mg/k/d or 2.2 g/m2/d 
In addition, we found that a single dose of NCG rapidly augments ureagenesis rate in 
healthy adults8. 
1.3.2.3 Carbaglu in PA and MMA 
As described above, limited case reports have suggested the use of NCG in PA and MMA9-
14.  
 
We applied the same study design as described above to investigate the effect of NCG in 
severe PA and MMA. These patients usually present as newborns and have marked and 
recurrent hyperammonemia. As of the time of this reporting, we completed studies of 7 
patients with PA and 1 patient with MMA. We specifically wanted to determine whether NCG 
reduces plasma levels of ammonia and glutamine and increases ureagenesis rate, even 
though plasma glutamine levels are typically not elevated in PA.   
 
Results from longitudinal mixed effects linear regression (Figure 2) show that peak 
[13C]urea increased following 3 days of NCG treatment (from 2.2 μM to 3.8 μM p<0.0005). 
There were corresponding decreases in mean plasma ammonia (59 to 43 μM p<0.0005) 
and glutamine (552 to 331 μM p<0.0005). This study demonstrated conclusively that NCG 
enhances ureagenesis and reduces plasma ammonia and glutamine concentrations in 
patients with PA. We obtained similar results in the patient with MMA, thus, this protocol 
represents a systematic approach to validating these findings a controlled clinical trial. 
 
Figure 2. [13C]-urea before (pre-NCG) and after (post-NCG) a 3-d NCG treatment in 7 
patients with PA9 
 
 
1.3.3 Carbaglu Formulation and Dosage 
Chemical Composition:  N-carbamoyl-L-glutamic acid (NCG) 
 

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Study drug will be prepared for administration according to the package insert. Briefly, study 
drug tablets will be dispersed in water and ingested or administered immediately via nasogastric 
or gastrostomy tube. 
 
The daily dose will be 150 mg/kg/ day or 3.3 g/m2/day for patients >15 kg. The doses are to be 
divided into 2 equal doses and administered orally or enterally by nasogastric or gastrostomy 
tube. Standard care will prevail when choosing the mode of drug administration. 
 
1.3.4 Safety and Side Effects of Carbaglu 
The safety of Carbaglu for the treatment of hyperammonemia due to NAGS deficiency was 
evaluated in a retrospective review of 23 patients (14 males and 9 females) (Carbaglu 
[prescribing information]. Orphan Europe; 2010). The most common adverse reactions 
(occurring in >13% of patients), regardless of causality, were infections, vomiting, abdominal 
pain, pyrexia, tonsilitis, anemia, ear infection, diarrhea, nasopharyngitis, and headache.   
 
 

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2 OBJECTIVES 
 
To determine whether NCG treatment of acute hyperammonemia in severe, neonatal-onset PA and 
MMA, partial CPSD, and OTCD is efficacious and whether it is safe.  
 
 Assess Efficacy of NCG Treatment   
2.1
The objective of this study is to assess whether NCG is efficacious in treating hyperammonemia and 
improving the following outcomes: 
 
The primary efficacy outcome of this study is the resolution of the ammonia levels or earlier 
discharge following the hyperammonemia episode(s). 
 
The secondary efficacy outcomes of this study include the Functional Status Scale, 
measured daily during each hyperammonemic episode, and the length of hospitalization. 
 
This goal will be realized by randomizing each hyperammonemic episode from every subject to  NCG 
plus standard treatment (NCG-STD) versus placebo plus standard treatment (PLBO-STD) and 
subsequently gauging response with the primary outcome of plasma ammonia levels, in addition to 
the Functional Status Scale and the length of hospitalization. 
 
 Safety 
2.2
The primary safety outcome of the study will be the rate of Serious Adverse Events (SAEs) during 
study episodes defined in this study as death or substantial prolongation of hospitalization, as 
patients are hospitalized as part of having a qualifying hyperammonemia episode. Safety tests 
consisting of complete blood count (CBC), liver and kidney function tests, coagulation profile 
(PTT/INR) will be performed before treatment, on the third day of treatment,  just prior to episode 
treatment completion of NCG and thirty days (up to six months) after the last study dose. Patients in a 
hyperammonemia crisis frequently experience transient anemia once appropriately hydrated during 
an emergency room visit. In order to monitor the transient anemia, in addition to collecting CBC’s, if a 
patient receives a blood transfusion during hospitalization this information will also be collected. An 
EKG test will be performed before treatment and between days 3-5 of treatment or before discharge if 
earlier.  

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3 STUDY DESIGN 
 
 Overview of Study Design  
3.1
This will be a double-blinded, placebo controlled randomized clinical trial to evaluate the efficacy of 
NCG in the treatment of two organic acidemias (severe PA and MMA), and two urea-cycle disorders 
(late-onset CPSD and OTCD). Figure 3 summarizes the design of the proposed trial.  
 
Figure 3. Study Overview Diagram 
 
 
Protocol Overview  
 
Eligibility – Briefly, subjects (a) aged >1 week with verified diagnosis of CPSD or OTCD or 
(b) subjects of any age with verified diagnosis of PA or MMA and ammonia level 
≥70mcmol/L. All subjects (or a first-degree relative) must have had a recorded plasma 
ammonia ≥100 mcmol/L and must have presented initially at >1 week of age for CPSD or 
OTCD. Subjects must not be pregnant. A complete listing of eligibility criteria is included in 
Section 4 (Study Population). 
 
Enrollment – Informed consent from the participant or one parent or legal guardian.  
 
Randomization –  At each hyperammonemic episode, participants will be randomized to 
either placebo plus standard therapy (PLBO-STD) or NCG plus standard therapy (NCG-
STD) at  a dose of 150 mg/kg/ day or 3.3  g/m2/day for patients >15 kg, divided into 2 equal 
doses and administered enterally by nasogastric or gastrostomy tube or by oral syringe.  
 
Study Treatment – The NCG/PLBO dose will be administered for metabolic 
decompensation for 7 days or until discharge, whichever is shorter. 
 
Concomitant Standard Treatment – Participants will continue receiving standard treatment 
which may include intravenous glucose (and insulin if required), L-carnitine dosing and/or 
dialysis. Ammonia scavengers may only be administered in patients with urea cycle 
disorders. 

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 
Laboratory Measurements – Plasma ammonia testing should be performed as clinically 
indicated, including checking the level at admission and at least every 6-12 hours for the first 
48 hours and every day thereafter if the previous level was ≥50 mcmol/L, for a total of 7 
days or until discharge, whichever is shorter. Plasma amino acids should also be checked 
as clinically indicated at admission and at least every 2 days thereafter, if ammonia levels 
are measured. Safety laboratory assessments including complete blood cell count, liver and 
kidney function tests, amylase and lipase testing, as well as a coagulation profile (if clinically 
indicated) will be done at the time of initial admission, at 3-5 days, at 6-7 days or at 
discharge, whichever is shorter. 
 
Electrocardiogram (EKG) Test – At each episode, participants will have an EKG at 
admission and repeated on day 3-5 of treatment (48 hours following the initial drug 
administration, before discharge if earlier) to check for cardiac toxicity.   
 
Functional Scale Score (FSS) – Determined for each episode at admission and every day 
of hospitalization for a total of 7 days or until discharge, whichever is shorter. 
 
Recurrent Episodes – Each subsequent admission for metabolic decompensation will 
follow the same above procedure but has to occur at least 7 days after the last discharge to 
guarantee study drug wash out before the next administration should patient have received 
study drug.  
 
 

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4 STUDY POPULATION 
 
Patients enrolled in the long-term outcomes study being conducted in parallel are eligible to enroll in 
this study when the long-term outcomes study closes, provided that all other inclusion and exclusion 
criteria are satisfied. 
 
 Inclusion Criteria 
4.1
o Aged older than 1 week with an established diagnosis of CPSD or OTCD (as follows):  
 
Diagnosed with late-onset CPSD confirmed by detection of pathogenic mutation(s), 
and/or decreased (<20% of control) CPS enzyme activity in liver OR 
 
Diagnosed with late-onset OTCD by detection of pathogenic OTC mutation, OR 
decreased (<20% of control) OTC enzyme activity in liver OR elevated urinary 
orotate (greater than 20 µM/mM) following allopurinol loading with absence of 
argininosuccinic acid 
 
 
AND: Subject or subject’s first-degree relative had plasma ammonia level 
≥100mcmol/L >1 week of age 
 
OR 
 
o An established diagnosis of PA or MMA (as follows): 
 
Diagnosed with PA by semi-quantitative urine organic acid analysis, defined as 
presence of elevated methylcitric acid and normal methylmalonic acid levels and 
no evidence of biotin-related disorders in the organic acid analysis OR 
 
Diagnosed with MMA by semi-quantitative urine organic acid analysis, defined as 
elevation of methylmalonic acid and no evidence of vitamin B12 dependent 
disorder on plasma amino acid analysis  (B12 dependency is defined by 
documented B12 responsiveness) 
 
 
AND: Subject or subject’s first-degree relative had plasma ammonia level at 
any time ≥100mcmol/L 
 
o Able to receive medications orally, by nasogastric (NG)-tube or by gastric (G)-tube 
o No concomitant illness which would preclude safe participation as judged by the 
investigator 
o If post-menarcheal must have a negative pregnancy test prior to administration of study 
drug at each episode 
o Signed informed consent by the subject or the subject's legally acceptable 
representative 
 
 Exclusion Criteria 
4.2
o Administration of NCG within 7 days of participation in the study 
o Use of any other investigational drug, biologic, or therapy. 
o Planned participation in any other clinical trial 
o Diagnosis of any medical condition causing hyperammonemia that is not PA or MMA. 
CPSD or OTCD. Other urea cycle disorders will be excluded from this study. 
o Any clinical or laboratory abnormality or medical condition that, at the discretion of the 
investigator, may put the subject at an additional risk by participating in this study 
o Has had a liver transplant  
o Is not expected to be compliant with this study in terms of returning to site for 
subsequent episodes of hyperammonemia crises 
Is pregnant 

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5 STUDY PROCEDURES 
 
 Screening of Potential Subjects 
5.1
Prior to enrollment, patients must be deemed by the site PI, or his assigned surrogate, to meet 
eligibility criteria.  
 
5.1.1 Confirmation of diagnosis of PA, MMA, CPSD or OTCD 
5.1.1.1 
Diagnosis of PA or MMA 
Results from the subject’s urine organic acid analysis, performed via semi-quantitative 
methods by gas chromatography–mass spectrometry (GCMS), should be reviewed. Patients 
who do not meet these laboratory diagnostic criteria will not be enrolled. 
5.1.1.1.1 Laboratory Diagnostic Criteria for PA 
1) Elevation of methylcitric acid and propionylglycine 
2) No elevation in methylmalonic acid 
3) No evidence of biotin-related disorders 
5.1.1.1.2 Laboratory Diagnostic Criteria for MMA 
1) Elevation of methylmalonic acid  
2) No elevation of homocysteine, total homocysteine, or homocysteine-cysteine 
disulfide by plasma amino acid analysis  
3) No response to 1 mg IM hydroxocobalamin injections 
5.1.1.1.3 Laboratory Diagnostic Criteria for CPSD 
 
Detection of pathogenic CPS1 mutation(s) OR 
 
Decreased (<20% of control) CPS enzyme activity in liver 
5.1.1.1.4 Laboratory Diagnostic Criteria for OTCD 
 
Detection of pathogenic OTC mutation OR 
 
Decreased (<20% of control) OTC enzyme activity in liver OR 
 
Elevated urinary orotate (greater than 20 µM/mM) following allopurinol loading, 
with absence of argininosuccinic acid on plasma amino acid analysis 
 
5.1.1.2 
Documentation of Hyperammonemia 
1) The subject (or subject’s first-degree relative) should have a previously 
documented ammonia level at any time of ≥100 mcmol/L 
2) If diagnosed with CPSD or OTCD (as per above), initial hyperammonemia 
should have occurred >1 week of age 
 
In some cases, a subject may have been diagnosed prenatally, or before the manifestation 
of symptoms, because of previously-identified affected family member. As a result, the 
subject may have been treated prospectively and averted any early hyperammonemic 
events. In such cases, the previously-identified first-degree relative must meet the above 
criteria instead. 
 
 Informed Consent Procedures 
5.2
Participants will be recruited from all participating sites and their metabolic services. Ideally, patients 
will be recruited to this study during periods of clinical and metabolic stability, rather than at the time 
of presentation of acute illness or hyperammonemia.  Whenever possible, the participants (or 

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guardians) will be given a copy of the consent form for review, several days before the formal 
informed consent process takes place.  
 
For participants who can provide informed consent: 
The licensed physician investigator will meet with the potential study participant to describe the study. 
Patients will be brought to a private, quiet room where the benefits and risk of study will be explained 
thoroughly, and any questions will be answered. The investigator will then give the patient time to 
read the consent form and consider participation. The investigator will return to answer questions, 
ensure that the patient understands the information provided, and obtain informed consent. 
  
For participants unable to provide informed consent who arrive with parents/guardians:  
The site PI will meet with the parent(s) or guardian(s) to describe the study. Parents will be brought to 
a private, quiet room where the benefits and risks of the study will be explained thoroughly, and any 
questions will be answered. The investigator will then give the parent(s) time to read the consent form 
and consider participation.  The investigator will return to answer questions, ensure that the 
parent/guardian understands the information provided, and obtain informed consent.  
 
For participants unable to provide informed consent arriving without parents/guardians:   
Some of these patients may be transferred directly from another institution without parents or 
guardians. Patients’ families will be first contacted by phone by clinical personnel to discuss the 
possibility of participating in a clinical trial. Should they be interested, informed consent will be 
obtained by the site PI by telephone before the study can begin. The investigator will speak with the 
parent/guardian by phone, and provide them with a copy of the consent form (e.g., by fax, e-mail). 
When the parent/guardian receives the consent form, it will be immediately signed and sent/faxed 
back to the investigator. No research-related activities will occur until a signed hard copy of the 
consent form has been received by the investigator.     
 
For participants who are being recruited during a period of clinical and metabolic stability: 
Whenever possible, patients will be recruited to participate during periods of clinical and metabolic 
stability. The site PI  will meet with these patients (and their families, if applicable) following their 
routine clinical visits to discuss study participation, to answer questions, to ensure that all information 
provided is understood, and to obtain informed consent. Patients who are not scheduled for a routine 
clinical visit within the next 3 months will be contacted by phone by clinical personnel to discuss the 
possibility of participating in a clinical trial. Should they be interested, informed consent will be 
obtained by the site PI by telephone. The investigator will speak with the patient or parent/guardian by 
phone, and provide them with a copy of the consent form (e.g., by fax, e-mail, or post). When the 
patient or parent/guardian receives the consent form, it will be signed and returned to the investigator. 
No research-related activities will occur until a signed hard copy (original or facsimile) of the consent 
form has been received by the investigator.     
  
Informed consent documents will be available in English. If patients or their parents/guardians are 
only fluent in another language, and the site has a standard operating procedure for informed consent 
in that language (such as an on-call interpreter and if available abbreviated consent form in 
respective language) and the site IRB has approved the process for this study in advance, non-
English speaking patients, and parents/guardians will also be included. 
 
 Retention Procedures 
5.3
Every effort will be made to identify patients who have enrolled in this study at their next presentation 
to the emergency department or another medical facility. 
 
At the time of enrollment, patient families will be provided with a card, indicating that they 
are participating in an investigational drug trial, to be presented to hospital staff at 

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subsequent episodes of illness. They will also be reminded to inform the on-call genetics or 
metabolism attending physician of their participation in this trial. 
 
The last note from the treating genetics and/or metabolism physician will indicate in the plan, 
or in an addendum, that the patient is participating in a clinical investigational drug trial 
 
The patient charts will be flagged, if possible, indicating that the patient is participating in a 
clinical investigational drug trial 
 Duration of Study and Visit Schedule  
5.4
5.4.1 Hyperammonemia Episodes 
At any time after the initial enrollment, participants may present to the hospital with PA-, MMA-, 
CPSD- or OTCD- associated symptoms (see Sections 1.1.2-1.1.5). Indicated clinically, the 
plasma ammonia level should be verified. 
 
Subjects are not eligible to receive study drug if they have received NCG or study drug 
any time in the preceding 7 days. 
 
If plasma ammonia is ≥70 mcmol/L at presentation (confirmed on site) or during a 
hospitalization for an acute decompensation, participants should receive the study drug. 
The time from an episode- qualifying ammonia level to randomization should not be 
longer than a maximum of 4 hours.  
 
5.4.1.1 Random Group Assignment 
At each hyperammonemia episode, subjects will be randomized to a treatment arm. 
Randomization will be performed online using a stored, pre-generated, encrypted 
randomization schedule created by the CNMC Division of Biostatistics and Study 
Methodology. To complete the process, the Site Pharmacy will simultaneously receive an 
automated notification of the assignment to enable them to prepare the correct prescription 
for the participant. The time from randomization to study treatment administration should be 
as brief as possible and no longer than a maximum of 6 hours. 
 
5.4.1.2 Concurrent with Randomization 
Pre-Treatment 
1. Indicated clinically, a blood sample will be drawn for plasma ammonia and amino 
acid analysis. All patients are expected to have clinically indicated vascular access. If 
vascular access is inadequate, blood will be drawn by venipuncture. All ammonia 
levels measured up to 24 hours prior to first treatment administration will be 
recorded. Ammonia levels measured >24 hours prior to first treatment administration 
will not be recorded in this study. 
2. Laboratory safety assessments: complete blood count (CBC), liver function tests 
(LFT), renal function test (RFT), amylase and lipase. If indicated clinically, a 
coagulation profile (PTT and INR) should also be done. A pregnancy test is required 
(if applicable). 
3. Electrocardiogram (EKG) test 
4. Functional Status Scale (FSS) assessment 
 
 
 
 
 
Table 1. Hospitalization Study Procedures 

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Day 1 
Day 2 
Day 3 
Day 4 
Day 5 
Day 6 
Day 7 
Day 30 
(≤6 Months) 
Time (hours) 
0 
Post 
Dialysis 
6 
12 
18 
24 
36 
48 
60 
72 
84 
96 
108 
120 
132 
144 
156 
 
Confirm 
Eligibility 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Pregnancy 
Testb 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Randomization 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Plasma 
Ammonia 
Levelc  
X 
(X) 
X 
X 
Xd 
X 
X 
X 
 
Xf 
 
Xf 
 
Xf 
 
Xf 
 
 
Plasma Amino 
Acidsc 
X 
(X) 
 
 
 
X 
 
 
 
Xg 
 
 
 
Xg 
 
 
 
 
Safety Labsc 
X 
 
 
 
 
 
 
Xh 
X 
 
EKG Testi 
X 
 
 
 
 
 
 
Xi 
 
 
 
Functional 
Status Scalec 
X 
 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
Concomitant 
Medications 
Log 
 
 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
Adverse 
Events Log 
 
 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
Study Drug 
(NCG or 
PLBO) 
X 
 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Treatment 
Administration 
Log 
X 
 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Blood 
Transfusion 
Log 
X 
 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
NCG=N-carbamylglutamate; PLBO=Placebo 
a 
 Suggested timeline to be followed for 7 days, or until discharge if discharged prior to 7 days of drug administration 
b  For women of childbearing potential only. A serum pregnancy test will be performed in the event of a positive or equivocal urine pregnancy 
test result, or if the patient is anuric 
c  Allowable windows for day 3 (days 3-5) and day 7 (days 6-7), unless otherwise indicated 
d  Plasma ammonia should be drawn as frequently as clinically indicated, for example, every 6 hours during the first 24-hour window if the 
patient has a central or peripheral venous catheter. 
e  Only to be drawn if dialysis is performed. Blood draws should be performed immediately upon termination of hemodialysis or hemofiltration, 
even if the regular time-point labs have been drawn. 
f  Plasma ammonia should be drawn as frequently as clinically indicated, for example, after 48 hours, follow-up ammonia should be obtained 
if the prior measurement was ≥50 mcmol/L  
g  Amino acids should be drawn as frequently as clinically indicated, for example, obtained only if ammonia also obtained (see f) 
h  Safety Lab blood draw to be obtained on day 6-7, or if discharged <7d, within 24h prior to discharge 
i 
EKG should be done before treatment begins and repeated once between Days 3-5 or before discharge 
 
 
5.4.1.3 Post-Randomization 
Day 1 

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1. As per their assignment, the participant will be started on NCG (150 mg/kg/day 
for patients ≤15 kg or 3.3 g/m2/day for patients >15 kg in two divided doses) OR 
PLBO (three-quarters tablet/kg/d for patients ≤15 kg or 16.5 tablets/m2/day for 
patients >15 kg, in two divided doses) to be administered through a nasogastric 
tube, or if not available, by oral syringe, as described in Section 6.1. The 
participant will also continue to receive standard therapy. 
2. Following the first drug administration, the subsequent drug administration should 
be given between 6 and 12 hours after the first dose, at a standard drug 
administration time for the site. All subsequent doses should be given every 12 
hours. 
3. Indicated clinically, plasma ammonia testing will be performed every 6 hours 
following study drug administration if the patient has a central or peripheral 
catheter. In all other cases, plasma ammonia testing will be performed every 12 
hours, as indicated clinically. 
4. Clinical monitoring FSS 
5. Treatment administration log, concomitant medications log, blood transfusion log, 
and adverse events log will be completed daily 
 
 
Day 2 
1. NCG/PLBO Rx. & Standard Rx every 12 hours. 
2. Plasma ammonia every 12 hours (as indicated clinically) 
3. Amino acids (as indicated clinically) 
4. Clinical monitoring FSS 
5. Treatment administration log, concomitant medications log, blood transfusion log, 
and adverse events log will be completed daily 
 
Day 3 (if not yet discharged) 
1. NCG/PLBO Rx. & Standard Rx every 12 hours. 
2. Plasma ammonia (as indicated clinically)  
3. Safety labs should be performed between days 3 - 5 
4. EKG test should be performed between days 3-5 
5. Clinical monitoring FSS 
6. Treatment administration log, concomitant medications log, blood transfusion log, 
and adverse events log will be completed daily 
 
Day 4 (if not yet discharged) 
1. NCG/PLBO Rx. & Standard Rx every 12 hours. 
2. Plasma ammonia  (as indicated clinically) 
3. Amino acids (as indicated clinically) 
4. Clinical monitoring FSS 
5. Treatment administration log, concomitant medications log, blood transfusion log, 
and adverse events log will be completed daily 
 
Day 5 (if not yet discharged) 
1. NCG/PLBO Rx. & Standard Rx every 12 hours. 
2. Plasma ammonia (as indicated clinically) 
3. Clinical monitoring FSS 
4. Treatment administration log, concomitant medications log, blood transfusion log, 
and adverse events log will be completed daily 
 
Day 6 (if not yet discharged) 

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1. NCG/PLBO Rx. & Standard Rx every 12 hours. 
2. Plasma ammonia (as indicated clinically) 
3. Safety labs should be performed between days 6 – 7 
4. EKG test should be performed between days 6-7 (if not done already between 
days 3-5) 
5. Amino acids (as indicated clinically) 
6. Clinical monitoring FSS 
7. Treatment administration log, concomitant medications log, blood transfusion log, 
and adverse events log will be completed daily 
 
Day 7 (if not yet discharged) 
1. NCG/PLBO Rx. & Standard Rx. 
2. Plasma ammonia (as indicated clinically)  
3. Safety labs if not done on Day 6 
4. EKG if not already done 
5. Clinical monitoring FSS 
6. Treatment administration log, concomitant medications log, blood transfusion log, 
and adverse events log will be completed daily 
 
 
Day 8 (if not yet discharged) 
1. The final dose of NCG/PLBO Rx. & Standard Rx. 
2. Plasma ammonia (as indicated clinically) 
3. Treatment administration log, concomitant medications log, blood transfusions 
log and adverse events log will be completed a final time. 
 
5.4.1.4 Discharge Procedures 
If still on NCG/PLBO, the subjects will be discontinued from the study drug at the time of 
discharge from the hospital. 
 
We will contact subjects or their families to collect any adverse events that occurred in the 
72 hours following episode treatment completion. 
 
 
 Study Assessments 
5.5
5.5.1 Laboratory Tests 
 
Plasma ammonia (as indicated clinically) 
 
Plasma amino acids (as indicated clinically) 
 
Complete blood cell count 
o White blood cells and differential 
o Platelets 
o Hemoglobin 
o Hematocrit 
 
Liver function tests 
o ALT (Alanine Aminotransferase) 
o AST (Aspartate Aminotransferase) 
o Alkaline phosphatase 
o Direct bilirubin 
o Total bilirubin 
o Albumin 
o Total protein 

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 
Kidney function tests 
o Blood urea nitrogen 
o Creatinine 
 
Amylase and lipase 
o Amylase 
o Lipase 
 
Coagulation profile (as indicated clinically) 
o PTT (Partial Thromboplastin Time) 
o INR (International Normalized Ratio) 
 
Pregnancy test (if applicable) 
 
5.5.2 Electrocardiogram test (EKG) 
QT interval and QTc interval will be collected from each EKG, along with any abnormalities 
noted in the interpretation 
 
5.5.3 Transfusion Information 
Type of transfusion (e.g., packed red blood cells, fresh frozen plasma) and number of units will 
be collected 
 
5.5.4 Clinical Assessments 
 
The secondary objective of this study is to evaluate the impact of NCG treatment on the 
functional status scale. During each admission, the functional status scale will be evaluated 
prior to the start of study drug, as well as every day thereafter, until discharge or until episode 
treatment completion of the study drug. 
 
5.5.4.1 Pharmacokinetic Assessments (CNMC & CHOP) 
 
*** THIS PROTOCOL SECTION ONLY PERTAINS TO CHILDREN’S NATIONAL 
HOSPITALAND CHILDREN’S HOSPITAL OF PHILADELPHIA (CHOP) *** 
 
Left over plasma or serum from any sample drawn for clinical laboratory tests will be saved 
for pharmacokinetic (PK) analysis to be paired with study drug dosage and time of 
administration. The plasma and/or serum samples will be stored frozen at -20˚C and batch-
shipped on dry-ice to Atlanbio Laboratory (Rue Graham Bell, ZI de Brais – BP 40309, 44605 
Saint-Nazaire, Cedex, France) for determination of N-carbamylglutamate levels at the end of 
each patient episode. Since study participants experience a high number of blood draws for 
clinical tests, no additional blood will be drawn specifically for PK analysis. There will be no 
schedule of assessments for PK samples. Instead, study staff at CNMC and CHOP sites will 
be encouraged to collect the left over plasma or serum samples during each 
hyperammonemia episode. The exact time and date of the blood draw is recorded (and will 
correspond to the medical record) for each sample. These samples will be stripped of 
hospital identifiers and will be labeled with the study number and episode number before 
being shipped to the reference laboratory. 
 
 

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5.5.5 Efficacy Assessments 
For the primary outcome, we will evaluate the plasma ammonia trajectory during episodes in 
which patients receive NCG vs. PLBO. Secondary outcomes will consist of FSS, and length of 
hospitalizations. 
 
5.5.5.1 Functional Status Scale 
The Functional Status Scale (FSS) will be used to assess changes in the subject’s global 
functioning in the hospital setting. The measure will be obtained at baseline (i.e., prior to 
administration of study drug or placebo at each episode) and then daily during 
hospitalization up to discharge or for 7 days whichever is shorter. The scale includes 6 
domains; mental status, sensory functioning, communication, motor functioning, feeding, 
and respiratory status. Each domain is rated on a 5-point scale from 1 (normal) to 5 (severe 
dysfunction), thus the range of overall scores is 6 to 30 (Table 4). The measure was 
designed to be quantitative, rapid, reliable, minimally dependent on subjective assessments, 
applicable to as broad an age range as possible, and pertinent to hospitalized patients in as 
many inpatient environments as possible22. All individuals collecting data will be trained in 
the measure and can include medical professionals such as a nurse, genetic counselor, or 
physician investigator at the site.   
 
 
Table 2. The Functional Status Scale (from Pollack et al., 2009) 
 
 
5.5.6 Safety Assessments 
Safety assessments will consist of serious adverse events, adverse events, EKG tests, and 
safety laboratory measurements in the NCG vs. the PLBO group. 
 

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 Treatment Compliance  
5.6
Treatment logs will capture the timing and dosage of each occurrence of study drug administration. 
All treatments will be administered in the hospital so non-compliance with the medication is not a 
concern. 
 
 
 Study Termination/Stopping Rules  
5.7
The study PI and Coordinating Center will seek input from the DSMB regarding the need to stop the 
study if either of the following events occurs: (a) Life-threatening adverse event that is clearly 
associated with drug hypersensitivity and/or (b) Unexplained serious illness or death of one of the 
participants. 
 
The NIH and local IRBs (at their local site) or the site PI has the authority to stop or suspend this trial 
at any time. The Medical Monitor, DSMB, or FDA may recommend suspending or stopping the trial 
after a thorough review, due to evidence related to safety or efficacy based on planned interim 
analyses (see Section 7 on Safety Monitoring and Section 8.3.1 on Interim Analysis).  
 
 
 Subject Discontinuation 
5.8
A subject will be discontinued from the study if s/he:  
 
Is determined not to have PA, MMA, CPSD, or OTCD (as defined in Section 5.1.1.1) 
 
Voluntarily withdraws or is voluntarily withdrawn from the study by his/her parents 
 
The study drug will also be discontinued if a subject: 
 
Experiences a serious adverse event, deemed by the Medical Monitor and site PI to be 
related to or likely related to study drug administration. 
 
Has received a hepatic transplantation 
 
Relocates to a location, not within the catchment area of one of the other participating 
institutions 
 
If a participant drops from the study before its completion, a narrative description of the reason(s) for 
discontinuation will be recorded on the Study Closeout Form (see Manual of Procedures).  
 
 
 
Blinding 
5.9
This is a double-blind, placebo-controlled trial performed in children’s hospitals across the country. 
Handling, storage, dispensing, and record keeping of all study medication will follow a Pharmacy 
Manual, which will be developed by the Central Research Pharmacy at Children’s National 
Hospital. The Pharmacy Manual will define the responsibilities and procedures of the Central 
Research Pharmacy and of the Site Pharmacies both of which will be unblinded.  
 
5.9.1 Type of Blinding 
The participants, their families, caregivers, and all research personnel at the sites, as well as the 
study chair and co-chair, will be blinded to the identity of the drug (NCG or PLBO). The 
appearance of active and placebo tablets is nearly identical. 
 
5.9.2 Maintenance of Blinding 
 
NCG and PBLO will be sent to each site pharmacy by the Central Pharmacy (CNMC).  

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 
Assigned NCG/PLBO doses will be prepared individually for each subject by the Site 
Pharmacy.  
 
Identically labeled NCG/PLBO doses prepared by the Site Pharmacy will be dispensed 
by blinded hospital staff based on a prescription from the blinded study physician.  
 
An additional supply of NCG/PLBO will be provided as needed based on each site’s 
enrollment rate to provide the doses of study medication for all newly enrolled subjects 
as well as a small supply to meet emergency needs at each site.  
 
The individual Site Pharmacy will prepare, blind and dispense unit doses of study 
medication for each enrolled subject from the drug supply while the subject is 
hospitalized. 
 
All unopened medications will be stored at controlled refrigerated temperature (2-8° C; 
36-46° F), and opened medications will be stored at controlled room temperature (15-30° 
C; 59-86° F) in a secure area in the site’s research pharmacy to which access will be 
limited to the research pharmacist and designated assistants. To maintain blinding, only 
the site pharmacist and designated assistants will have access to the secured area.  
 
5.9.3 Breaking the Blinding 
Treatment assignment should not be unblinded unless the Medical Monitor assesses that 
knowledge of drug assignment is required in order to assess how to treat a safety concern 
within a patient. The site investigator should contact the Coordinating Center immediately if 
he/she believes that knowledge of the treatment assignment will affect their decision regarding 
treatment of a safety concern. 
 
 

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6 STUDY TREATMENTS 
 
6.1 Study Intervention 
Carbaglu® 200 mg Dispersible Tablets (N-carbamylglutamate), scored in three places, allowing each 
tablet to be broken into four 50 mg pieces. 
 
The target dose is 150 mg/kg/d for patients ≤15kg or 3.3 g/m2/day for patients >15kg, divided into two 
equal doses 12 hours apart. Each dose will be rounded to the nearest 50mg using the dosing 
nomograms provided. The maximum variance from the calculated target daily dose and the actual 
daily dose will be 23.5% or less. Study drug will be prepared for administration according to the 
package insert.  The suspension has a slightly acidic taste and a white, cloudy appearance. 
Participants will either receive PLBO or the study intervention, NCG (Carbaglu®) 150 mg/kg/day for 
patients ≤15kg or 3.3 g/m2/day for patients >15 kg.   
 
 
6.2 
Control (Placebo) Intervention  
The placebo and NCG tablets, which are produced by the same manufacturer and include the same 
inactive components look and taste similar. When the two tablets are suspended in solution, there is 
a slight difference in a hue that is only distinguishable when the two solutions are observed side by 
side. 
 
 
6.3 Dispensing of Study Medications  
 
Handling, storage, dispensing, and record keeping of all study medication will follow a 
Pharmacy Manual, which will be developed by the Central Research Pharmacy (CRP) at 
the Children’s National Hospital. The CRP Pharmacy Manual will define the 
responsibilities and procedures of the CRP and of the site pharmacies both of which will 
be unblinded.  
 
The CRP will receive study medication (NCG) and matched placebo (PLBO) from 
Orphan Europe. 
 
Once the CRP and the Coordinating Center at Children’s National Hospital determine a 
site pharmacy is appropriately certified and ready to receive the drug, the CRP will ship 
an initial quantity of NCG and PLBO to the individual site research pharmacy.  
 
All doses of NCG/PLBO (as assigned) will be prepared, blinded, and dispensed in unit 
doses individually for each subject by each site pharmacy while the subject is 
hospitalized. 
 
 
6.4 Provisions for Access to Investigational Treatment after Study  
The results of the study will be shared with the patient, his/her family, and his/her treating physician. If 
the study reveals that NCG is effective, the treating physician will have the prerogative for treating the 
patient off-label with NCG, which is already approved for use in NAGS deficiency, until it is approved 
by the FDA for additional indications.  
 
6.5 Drug Packaging/Handling/Storage/Accountability 
 
Oral NCG and matching PLBO will be supplied by Orphan Europe and distributed to 
each site by the CRP 
 
The designated research pharmacist, investigator, or coordinator at each participating 
site will be required to acknowledge receipt of study drug shipments/doses and notify the 
CRP of any problems with shipments or with the on-site supply of study medication.   

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 
S/he will sign receipts for medication supplies, which will be kept in a secure area in the 
site’s research pharmacy to which access will be limited to the research pharmacist and 
their designated assistants. To maintain blinding, only the site pharmacist and 
designated assistants will have access to the secured area. 
 
Unopened bottles of medications will be stored at controlled refrigerated temperatures 
(2-8° C; 36-46° F).  
 
Opened bottles of medications will be stored at controlled room temperature (15-30° C; 
59-86° F). The date of first opening a bottle will be recorded on the tablet container.  
 
Drug accountability logs will be completed by the site pharmacists and the study 
coordinators.    
 
The sites will update their monthly supplies logs and expiration dates and the information 
will be monitored by the central pharmacy. 
 
Unopened bottles of medications will not be used after the expiration date stated on the 
tablet container. Opened containers of medications will not be used after 30 days of first 
opening. 
 
All unused study medication will be returned by the site to the central pharmacy and be 
documented in the study database. After reconciliation, the drug will be destroyed or 
retained and used in animal research.   
 
At the conclusion of the study or as required for review, supplies of received, dispensed 
and returned medication will be reconciled to account for all distributed supplies of 
PLBO/NCG.  
 
 
6.6 Concomitant Therapy  
 
6.6.1 Therapies Prohibited Before/During the Trial  
All investigational drugs, biologics, and therapies are prohibited following enrollment in this trial. 
 
This includes the use of alternative pathway medications, including sodium benzoate, and any 
medication with phenylacetate as an active metabolite (e.g., Buphenyl, Ammonul, Ravicti) in 
propionic acidemia, and methylmalonic acidemia; these medications have not been approved 
for this indication and there is no evidence for their effectiveness in these conditions.  
 
No other changes in the special diet or medications are called for in this protocol. 
 
6.6.2 Therapies Allowed During the Trial  
Standard therapy, including but not limited to intravenous fluids, dextrose, intralipids, biotin, 
hydroxocobalamin, 
levocarnitine, 
metronidazole, 
and 
metabolic 
specialty 
formulas. 
Hemodialysis or hemofiltration may be administered as determined by the treating physician. 
 
In CPSD and OTCD, FDA approved alternative pathway medications (e.g., Na-benzoate, Na-
phenylacetate, Buphenyl, Ammonul, Ravicti) are allowed and are indicated in these conditions.  

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7 SAFETY MONITORING 
 
The study protocol will be submitted to the FDA under IND # 68,185 and will be reviewed by the study 
DSMB (Section 7.2) and the Medical Monitor (Section 7.3). Participant enrollment may only begin after 
final IRB approval.  
 
7.1 Adverse Events (AEs) 
 
 
7.1.1 Definitions 
 
7.1.1.1 Adverse Event and Suspected Adverse Reaction 
As defined in title 21 of the Code of Federal Regulations Part 312, an adverse event (AE) is 
“any untoward medical occurrence associated with the use of a drug, whether or not 
considered drug related”. All adverse events will be classified using version 4.0 of Common 
Terminology Criteria for Adverse Events (CTCAE), developed and maintained by CTEP at 
the National Cancer Institute (http://evs.nci.nih.gov/ftp1/CTCAE/About.html). 
 
A suspected adverse reaction is any adverse event for which there is a reasonable 
possibility that the drug (NCG) caused the adverse event. "Reasonable possibility" means 
that there is evidence, such as a temporal relationship, to suggest a causal relationship 
between administration of NCG and the adverse events. It is less certain about causality 
than adverse reaction. 
 
7.1.1.2 Serious Adverse Events 
Title 21 of the CFR also provides a definition for serious adverse events (SAEs), described 
as those events that result in death; or are life-threatening; or require prolonged inpatient 
hospitalization or prolongation of existing hospitalization, or create persistent or significant 
disability/incapacity, or a congenital anomaly/birth defects. However, application of this 
definition is difficult in subjects who, because of their underlying congenital disorder, present 
with a life-threatening illness, requiring lengthy inpatient hospitalization.  
 
Administration of NCG has not been described to result in serious adverse events (see side 
effects section 1.3.4), and using the standard definition of SAEs is likely to result in capture 
of events secondary to the underlying acute metabolic disease, and which would not be 
helpful in the collection of data pertaining to NCG or its administration. Therefore, for this 
study, we modify the standard definition and define serious adverse events as those 
events that: 
 
Result in death 
 
Are life-threatening, that is, places the patient at immediate risk of death from the 
event as it occurred. Because acutely ill patients with PA or MMA are likely to 
experience life-threatening events as part of their underlying condition and meeting 
eligibility criteria, this category will be restricted to events that arise as new events 
subsequent to administration of NCG, not having started prior to administration of 
NCG. Life-threatening events resulting from processes, which based on the opinion 
of the investigator likely began prior to administration of NCG but had not been 
identified because the diagnostic investigation had not yet been performed, will not 
be considered serious adverse events. 

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 
Require prolongation of existing hospitalization. This category will be restricted to 
only complications that clearly extend the hospitalization that was anticipated at 
randomization to an episode. 
 
Serious Adverse Events in between episodes 
 
Result in death 
 
Are life–threatening 
 
Require hospitalization (except for elective hospitalizations for e.g. elective 
procedures and hospitalizations for a hyperammonemic event which are episodes 
and not SAEs) 
 
Note: if a hospitalization occurred without an episode-qualifying ammonia level, but 
later in the course of the hospitalization, the ammonia level rises to a qualifying 
episode, the participant will be randomized to treatment and the hospitalization will 
not be considered an SAE. 
 
Cause prolongation of existing hospitalization 
 
Cause persistent or significant disability/incapacity 
 
Cause a congenital anomaly/birth defects   
 
 
7.1.1.3 Expected and Unexpected Adverse Events 
An unexpected adverse event or reaction is defined as any adverse experience, the 
specificity or severity of which is not consistent with the risks described in the protocol (see 
Section 1.3.4 on Safety and Side Effects of Carbaglu) or in the Carbaglu investigator’s 
brochure. Expected adverse events are those that are identified in the research protocol as 
having been previously associated with or having the potential to arise as a consequence of 
participation in the study or are part of the clinical spectrum of the underlying disorder. 
Common acute and chronic complications of propionic and methylmalonic acidemia include: 
 
Anorexia 
 
Vomiting 
 
Lethargy 
 
Stupor 
 
Coma 
 
Seizures 
 
Ketosis 
 
Acidosis 
 
Hyperventilation 
 
Hyperglycemia 
 
Hypoglycemia 
 
Hypothermia 
 
Neutropenia; bone marrow suppression  
 
Electrolytes abnormalities  
 
Amino acid abnormalities 
 
Pancreatitis (PA) 
 
Renal dysfunction (MMA) 
 
Cardiomyopathy (PA) 
 
 
7.1.1.4 Relationship to Intervention Assessment 

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Relationship of NCG to the adverse event or suspected adverse reaction is defined as 
follows: 
 
Unrelated:   
Adverse event is clearly due to extraneous causes (e.g., underlying disease) 
 
 
Unlikely related (must have 2 of the below):  
Does not have temporal relationship to NCG administration 
Could readily have occurred due to subject's clinical state 
Could have been due to environment or other interventions 
Does not follow known pattern of response to NCG 
Does not reappear or worsen with reintroduction of NCG 
 
 
Possibly related (must have 2 of below):  
NCG administration and the occurrence of the AE are reasonably related in time  
Could not readily have occurred due to subject's clinical state 
Could not readily be due to environment or other interventions 
Follows a known pattern of response to NCG  
 
 
Probably related (must have 3 of the below):  
NCG administration and the occurrence of the AE are reasonably related in time  
Could not readily have occurred due to subject's clinical state 
Could not readily be due to environment or other interventions 
Follows a known pattern of response to NCG 
 
 
Definitely related (must have all 4 of the below):  
NCG administration and the occurrence of the AE are reasonably related in time  
Could not readily have occurred due to subject's clinical state 
Could not readily be due to environment or other interventions 
Follows a known pattern of response to NCG 
 
7.1.2 Collection and Reporting of Adverse Events 
The site study coordinator is responsible for collecting and recording all clinical and laboratory 
data. Adverse events will be collected on subjects on each day of admission, as well as by 
phone or e-mail communication with parents when the study coordinator contacts subjects or 
their families to collect any adverse events that occurred in the 72 hours following episode 
treatment completion following each admission. The reporting period for new AEs is the period 
from the start of study drug administration until the patient completes their participation in the 
study (i.e., until the end of the study period or until discontinuation if early withdrawal occurs). 
Follow-up reports on AEs should continue up to 30 days after the administration of study 
treatment if the AE did not resolve. 
 
For each AE, the site investigator will assess severity, and whether the event meets the 
definition of a serious adverse event (see Section 7.1.1) and will report whether study treatment 
was interrupted or stopped, as well as the outcome of the AE. If the event is an SAE, both the 
site PI and the Medical Monitor will assess the relationship to study treatment. As described in 
5.7.3, treatment assignment should not be unblinded unless the Medical Monitor assesses that 
knowledge of drug assignment is required in order to assess how to treat a safety concern 
within a patient. 
 
If a non-serious adverse event is unresolved at the time of discharge from the study, the site PI 
will make a clinical assessment as to whether continued follow-up of the AE is warranted. The 

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site PI may request input from the Medical Monitor. Resolution is defined as the return to 
baseline status or stabilization of the condition with the expectation that it will remain chronic.  
 
The CNMC-CC will prepare aggregate reports of all adverse events (serious/not serious and 
expected, unexpected) for the DSMB as required and for the FDA on an annual basis. Local site 
investigators are also required to fulfill all reporting requirements to their IRB and to the CNMC-
CC. 
 
7.1.2.1 Reporting of Serious Adverse Events  
Serious adverse events (SAEs) must be reported by each site PI to the CNMC-CC within  
24 hours of learning of the event. The CNMC-CC has a  joint phone & email tree for 
reporting SAEs to the CNMC-CC at any time. Those involved in the phone & email tree are 
aware that they may expect to receive a phone call or email at any time for an SAE. The site 
reporting the SAE must attempt to call or email first the CNMC-CC Principal Investigator 
(PI), followed by the Project Manager (PM), and then the Data Manager (DM) until someone 
has been reached in real time. It will be the responsibility of the CNMC-CC PI, PM, and DM 
to have a backup point of contact for instances of sick leave or vacation. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
The Medical Monitor (or their delegate) will then be contacted by the CNMC-CC and be 
informed of the details of the SAE. The CNMC-CC will immediately report unexpected SAEs 
to the CNMC IRB according to local guidelines. 
 
Sites are required to submit follow-up reports to the initial report as promptly as is feasible. 
The CNMC-CC will submit a report of the SAE to the FDA within 15 calendar days of the 
event if the event meets the reporting requirement of a serious and unexpected suspected 
CNMC-CC Principal Investigator 
Nicholas Ah Mew, MD 
nahmew@cnmc.org 
202-577-9971 
202-476-5863 
CNMC-CC Project Manager 
Katie Rice, MPH, CCRP 
krice3@cnmc.org 
202-621-0062 
CNMC-CC Data Manager 
Sneha Saraf, MS 
ssaraf@cnmc.org 
315-751-7760 
202-476-2353 

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adverse reaction (SUSAR). In those cases, the CNMC-CC will also report the event to the 
DSMB. In all other cases, the CNMC-CC will submit the report as part of periodic reporting 
to the DSMB and FDA. 
 
The Medical Monitor will review causality (unrelated, not likely related, possibly related, 
probably related, definitely related) of the serious adverse event. The Medical Monitor may 
request further information if necessary and possibly request changes to the protocol or 
consent form as a consequence of the adverse event.  
 
Additionally, site investigators will be responsible for reporting SAEs to their IRB within the 
time mandated by the site IRB. The CNMC-CC will inform all sites of any safety updates or 
changes, and the site investigators will be responsible for notifying their local IRB of these 
modifications. The CNMC-CC will ensure that all SAEs and any safety updates or changes 
are reported to the site’s IRB by collecting a copy of all IRB-related correspondence and 
reviewing regulatory submissions during monitoring visits.  
 
7.2 Data and Safety Monitoring Board (DSMB) 
A DSMB will be assembled by the investigators. Typically, the Board members represent the 
following specialties, all of whom will have no formal involvement with the participants or the 
investigators: a medical doctor with experience in inherited metabolic disorders, a neonatologist, a 
biostatistician, and the clinical trial coordinator. This Board is responsible for safety and accuracy 
monitoring of the data entered by the investigators. The DSMB will regularly evaluate trial 
performance, monitor interim data for safety and effectiveness of study regimens, review any protocol 
modifications, and advise the investigators regarding early termination or continuation of a study 
based on the interim monitoring or scientific findings. The DSMB will receive and respond to reports 
of any serious adverse events (SAEs) and will be immediately notified of fatal or life-threatening 
events. Based on the review of safety, efficacy, and performance data, the DSMB will make 
recommendations regarding the conduct of the study. It is expected that the DSMB will meet at least 
biannually in Washington, D.C., with conference calls as necessary.  
 
7.3 Medical Monitor 
A Medical Monitor has been identified by the investigators. The Medical Monitor is responsible for 
being available by pager to monitor study safety issues and must identify an appropriate delegate to 
provide coverage whenever he is not available. The Medical Monitor (or designee) will be contacted 
whenever a critical situation with a patient arises and a decision about unblinding the treatment 
assignment must be made (i.e., in the event that unblinding the treatment assignment would affect 
subsequent management of the patient). The Medical Monitor may be unblinded whenever he deems 
it necessary. The Medical Monitor will also conduct an independent blinded review of all Serious 
Adverse Events (SAEs) and will contribute to the preparation of all SAE reports. The Medical Monitor 
will communicate any identified safety concerns to the study PI and Coordinating Center. The Medical 
Monitor may consult with the DSMB. 

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8 STATISTICAL ANALYSIS 
 
8.1 Study Aims 
 
8.1.1 
Primary Aim 
The primary aim is to evaluate the impact of NCG treatment during hyperammonemic events on 
the resolution of ammonia levels, among patients diagnosed with severe PA or MMA and partial 
CPSD or OTCD.   
 
8.1.2 
Secondary Aim 
Secondary analyses will also evaluate the safety of NCG. Safety is a secondary aim because 
there is already evidence that NCG, which is approved by the FDA for use in the treatment of 
NAGS, poses no serious risks to patient safety. Therefore, the focus of this research is less on 
safety than on its effectiveness in treating PA and MMA, which share the common risks 
associated with hyperammonemia with NAGS. The analysis of safety will focus on three 
outcomes; these include the level of selected laboratory markers related especially to organ 
health, the frequency of reported symptoms, and the frequency of reported adverse events by 
seriousness. The analysis of laboratory markers will compare the time specific and time 
averaged mean levels of each marker by study group. In addition, we will compare the 
frequency of symptoms and adverse events, relying mainly on tabulations by a study group of 
reports by organ system paying most attention to the magnitude of the difference between 
groups and consistency of the evidence than on formal hypothesis testing.  
 
 
8.2 Factors for Stratification 
Because clinical management practices and referral patterns may vary from site to site, stratification 
during randomization by the site will ensure that episodes will be balanced across study groups within 
each site.   
 
 
8.3 Statistical Considerations 
Experience with models evaluating the overall change in ammonia level during episodes following 
randomization (not divided by treatment group) has demonstrated that the shape of the ammonia 
curve is more variable and more complex than envisioned during study design. This circumstance 
has made it difficult to see how to generate a stable model to properly reflect and compare the slope 
of ammonia decline by treatment group as the primary outcome. 
Challenges included the following: 
1. The need to introduce curvilinearity in order to model change appropriately; and  
2. The need to also rely on piecewise regression models to adequately model bends or 
points of inflection in the curve. 
 
The latter has proven especially difficult to manage because methods vary in their recommendation 
regarding the number (1 vs 2) and position (time point of each bend) to define piecewise regression 
segments. This variability would lead to the need to create a number of alternative models that would 
affect the interpretation of results with no clear way to prioritize the choice of the final model.  
 
This situation prompted us to choose an alternative composite outcome and modeling strategy based 
on time to reach an ammonia level of 50 mcmol/L or time to discharge whichever occurs first. This 
approach avoids the complexity of modeling the correct shape of the ammonia decline curve but 
provides a rigorous, well-defined, and clinically-relevant outcome for comparison of treatment effects. 

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The choice of 50 mcmol/L as the target for ammonia decline is based on the extensive experience of 
metabolic physicians that a patient reaching this level of ammonia is no longer at risk of acute 
hyperammonemic injury and would be a candidate for discharge in the absence of other 
complications, which are not hypothesized to be affected by treatment with NCG.   
 
We propose to use Cox Proportional Hazard modeling to implement the primary analysis to compare 
the average differences in time to reach the composite outcome and allow us to control for 
differences in severity (ammonia level and or FSS) at episode baseline. This method will also allow 
us to take account of and adjust statistical testing for the correlation between results of participants 
who contributed more than one event to the analysis. The comparison of slopes of the ammonia 
decline curves by treatment group will shift to secondary analysis, which will allow us to focus on 
consistency of results rather than choosing the precise model for significance testing for the purpose 
of rejecting the null hypothesis of no treatment effect.  
 
Study analyses will be based on an intention-to-treat paradigm. In analyzing the primary outcome, we 
will evaluate the difference over time in the probability of reaching an ammonia level of 50 mcmol/L or 
hospital discharge. For this purpose, we will use a z-test to assess the evidence that the hazard 
(event) ratio (HR) differs from 1 (no effect) to test for a difference in the probability of the composite 
outcome over time by the group. We will account for the correlation between repeat episodes per 
participant by treating participant episodes with the same ID as a cluster, which will adjust standard 
error estimates for the intraclass correlation between episodes in the same cluster. The primary 
analysis will be implemented using the sts cox procedure in Stata 14 (StataCorp. Stata Statistical 
Software: Release 14, College Station, TX: StataCorp LP, 2015).  
 
Secondary efficacy outcomes are based on groupwise comparisons of the trajectory (slope) of 
ammonia decline, FSS, and time-to-hospital-discharge during hyperammonemia episodes. For 
analysis of the ammonia decline trajectory and FSS analysis, we will implement longitudinal linear 
hierarchical models. The models will enable us to estimate and compare the slope(s) based on 
ammonia change trajectory over time, as well as the time, averaged FSS across all episodes treated 
with NCG vs. PLBO. For time to discharge comparisons, we will rely on the same Cox Regression 
Analysis described for the primary outcome except the outcome event will be limited to time to 
discharge. As in the primary analysis, we will adjust model error for the clustering of observations by 
patient and control for severity based on ammonia level or FSS at admission to each episode. To 
assess NCG safety we will compare the levels/frequency of laboratory test results, reported 
symptoms, and adverse events. Secondary analyses will control for indicators of episode severity, 
e.g. peak ammonia. 
  
8.3.1 Interim Analysis 
In addition to the proposed final analysis, we will conduct an interim analysis using the same 
methodology described above when at least 50% (70 episodes) or when at least 60 episodes 
have occurred after the study midpoint based on the projected 4 hyperammonemic events per 
participant have been completed  (participant discharged). Allowing for one interim look for both 
efficacy and futility when half the information becomes available, using an O’Brien-Fleming 
stopping rule and a Lan-DeMets approach to the use function of the p-value. If conducted 
exactly at the 50% outcome, the boundary for rejecting the null hypothesis and concluding that 
Carbaglu is either efficacious or worse that placebo is a z-score of +/-2.9626, corresponding to 
a p-value <0.0031 at the interim look. The boundary for deciding futility, i.e., that Carbaglu is not 
expected to be significantly different than placebo is +/-0.3557, corresponding to a p-value of 
>0.72 at the interim look. With these boundaries, the decision rules at the conclusion of the 
study will be +/-1.9686 or more extreme to conclude that Carbaglu is either efficacious or worse 

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than placebo, corresponding to a p-value of <0.049. These values were calculated using East 
5.3 (Cytel, Inc.). 
 
A report of the interim analyses will be provided to the DSMB, which will review and discuss the 
results considering all evidence available, including magnitude of difference between groups 
and p-value, as well as results on secondary outcomes and safety to consider whether the 
results warrant stopping the trial either from persuasive evidence of benefit or harm.  
 
8.4 Rationale for Sample Size and Statistical Power 
All sample size and power estimates are based on 2-tailed testing, which assumes a type 1 error of 
5% and achieving 80% power to detect an HR difference by treatment group and that there is no 
censoring as each episode will contribute information to one of the composite outcomes. We used the 
Cox Regression procedure in PASS (Hintze, J. PASS 11. NCSS, LLC. Kaysville, Utah, USA. 
www.ncss.com, 2011) to evaluate the composite time to event outcome of the earlier of the time to 
reach an ammonia level of 50 mcmol/L or time to discharge within hyperammonemic episodes (the 
unit of randomization and analysis). We evaluated HR estimates above 1, reflecting that the NCG 
group achieved the higher probability of achieving the composite event. Based on these 
considerations, a total sample size of N=146 episodes can detect an HR of 1.6, one of N=114 can 
detect an HR of 1.7, one of N=93 can detect an HR of 1.8 and one of N=78 can detect an HR of 1.9. 
These sample size estimates account for an inflated R-square as large as 0.05 between the time to 
event outcome and covariates reflecting baseline severity at the start of an episode. We used the 
inflated R-square to account for the modest effect of clustering of episodes within a person. 
Therefore, we consider the originally-targeted sample size of 144 episodes to be more than sufficient 
to meet the aims of the study. Based on our re-analysis of the time-to-event outcome, an HR of 1.7 is 
realistic and a sample size of 114 episodes is sufficient to detect that effect. Therefore, the target 
sample size will be 114 episodes. 
 

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9 REGULATORY COMPLIANCE AND SAFETY MONITORING 
 
9.1 Regulatory Compliance 
This clinical trial will be conducted in accordance with the protocol, the ICH Harmonised Tripartite 
Guideline “Note for Guidance on Good Clinical Practice,” and the applicable local regulatory 
requirements. 
 
9.2 Statement of Compliance 
The CNMC IRB and all participating site IRBs comply with Good Clinical Practices as defined by the 
U.S. Food and Drug Administration (FDA) regulations and the International Conference on 
Harmonization (ICH) guidelines.  
 
Investigator Assurances 
 
For quality assurance reasons, the Coordinating Center reserves the right to perform site-
monitoring visits.  
 
Monitoring shall ensure that the study is planned, conducted, evaluated, and reported 
according to this protocol and the applicable SOPs of the Coordinating Center, the ethical 
principles that have their origin in the Declaration of Helsinki (1996), the requirements of 
the ICH Harmonised Tripartite Guidelines.  
 
Monitoring shall also ensure that the documentation of the study is available, complete, 
organized, and valid. 
 
The investigator agrees to give the study monitor access to all relevant documents, 
including source documents, for review. The same applies in the case of an inspection of 
federal authorities or the relevant IRB. 
 
9.3 Informed Consent 
 
It is the responsibility of the investigator to ensure that no subject is enrolled in any study-
related examination or activity before written informed consent has been obtained.  
 
Written informed consent will be obtained from subjects or their legal representative(s) in 
compliance with 21 CFR Part 50 and the ethical principles that have their origin in the 
Declaration of Helsinki. 
 
For underage subjects, written informed consent will be obtained from the subject’s legal 
representative(s) (e.g., his/her parent(s) or legal guardian(s)) in compliance with 21 CFR 
Part 50 and the ethical principles that have their origin in the Declaration of Helsinki.  
 
If the subject is younger than the age of consent but old enough to provide assent, it is the 
investigator’s responsibility to ensure that the site’s assent procedures are followed and 
that there is documentation of the fact that the assent procedures were followed. The 
subject and the subject’s legal representative(s) will be informed that they are completely 
free to refuse to enter the study or to withdraw from it at any time and that this will not 
affect their overall care at the institution. 
 
If the subject’s mental age or psychological state is such that they cannot assent or if there 
is potential medical benefit to the subject by participating in the study an assent waiver 
must be signed by a parent or legal representative. 
 
Subjects, whose legal representative(s) refuse to give written informed consent or 
withdraw their informed consent, later on, must not be included in the trial or must be 
excluded from further participation, respectively.  
 
Before personally dating and signing the informed consent form, the subject’s legal 
representative(s) will be informed in detail by the investigator about all pertinent aspects of 
the trial according to 21 CFR Part 50 and ICH GCP.  

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 
The subject’s legal representative(s) should be given sufficient time to request further 
details about the trial before signing the informed consent form, in accordance with the 
ICH GCP Guidelines (1997). Under certain circumstances, when a patient cannot provide 
informed consent and is not accompanied by parents/guardians,  the investigator will 
speak with the parent/guardian by phone, and provide them with a copy of the consent 
form (e.g., by fax, e-mail). When the parent/guardian receives the consent form, it will be 
immediately signed and sent/faxed back to the investigator. 
 
The receipt of the informed consent from the legal representative(s) must be documented 
on the appropriate page of the subject’s CRF and in the source documents.  
 
One copy of the consent form signed and dated by the subject’s legal representative(s) 
and by the physician who informed the legal representative(s) will be kept at the study site; 
a second copy will be provided to the subject’s legal representative(s). 
 
9.4 Records Retention and Requirements 
 
The investigators and the sponsor(s) must archive all essential records and documents 
including but not limited to CRF, informed consent, and identification codes for subjects 
and other original records.  
 
These documents of the study will be retained at least 3 years after study close and data 
lock. 
 
The site investigators will be informed by the Coordinating Center (CNMC-CC) when the 
documents need not be retained any longer. 
 
9.5 
Financial Disclosure 
Financial disclosure information will be collected per Part 54 of Title 21 of the CFR and ICH E6. In 
addition, the site investigators must provide to the study PI a commitment to promptly update this 
information if any relevant changes occur during the course of the investigation and for 1 year 
following completion of the study. 
 
9.6 Inclusion of Minorities and Children 
The disorders to be investigated affect both genders and all different ethnic groups and we, therefore, 
expect to have proportional representation among the participants of the trials. These are rare 
disorders, and we will attempt to recruit all presenting potentially eligible patients. Participants of this 
study will include neonates, infants, children, adolescents, and adults. There is no upper age limit. 
 
9.7 Confidentiality 
By conducting this study, the investigator pledges that he/she will keep all information pertaining to 
the study strictly confidential, including data generated from this study, except as exempted for 
regulatory purposes. Blood samples and neuropsychological testing will be collected and diagnostic 
records will be reviewed. Data recorded will include demographics, clinical summary, diagnostic tests 
and study results. All research data will be accessible only to the research team and as required by law.  
9.7.1 Protection Against Risk 
 
The NCG drug and placebo will be handled by the respective site investigational drug 
pharmacy and will be dispensed by that pharmacy.  
 
The participants and their families will be provided psychological and emotional support 
to deal with the results of the developmental testing.  
 
Venipuncture:  The vein in which the needle has been inserted to draw blood may 
become sore and red. A temporary bruise may develop, and rarely fainting may occur.  
 
Confidentiality is protected to the full extent required by law and applicable local, State 
and Federal regulations and guidance including HIPAA.  

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 
All medical records, including case report forms, will be kept in locked files accessible 
only to the health professionals involved in the clinical research or responsible for the 
participant’s care, governmental agencies (e.g., FDA, NIH), and local convening 
authorities (e.g., IRB) for the purpose of audit regarding scientific validity and/or aspects 
pertaining to the ethical conduct of human clinical investigation. Identifier data are not 
released without the parent/participant’s knowledge and consent. Electronic databases 
are user ID/password protected.  
 
The results of the study will be shared with the participant of the study as soon as they 
become available.  
 
9.7.2 Health Insurance Portability and Accountability Act (HIPAA) 
The Standards for Privacy of Individually Identifiable Health Information (“Privacy Rule”) 
establishes a set of national standards for the protection of certain health information. The U.S. 
Department of Health and Human Services (HHS) issued the Privacy Rule to implement the 
requirement of the Health Insurance Portability and Accountability Act of 1996 (HIPAA). 
 
The Privacy Rule standards address the use and disclosure of individuals’ health information—
called protected health information (PHI) by organizations subject to the Privacy Rule — called 
“covered entities”, as well as standards for individuals' privacy rights to understand and control 
how their health information is used.      
 
Under HIPAA, Protected health information encompasses 18 identifiers, which must be treated 
with special care, including information on a patient's medical chart or a patient's test results, 
contact information, as well as an individual's billing information for medical services rendered. 
PHI also includes identifiable health information about subjects of clinical research gathered by 
a researcher who is a covered health care provider. This study does not collect any PHI except 
dates, and other are considered a limited data set. 
 
For this portion of the study, which involves a minimal data set of PHI for recruitment purposes, 
only the minimum necessary information required to accomplish the research objectives will be 
extracted from the electronic medical record and recorded in the research database. Allowing 
the study team to use the electronic medical record to identify and capture information about all 
potentially eligible subjects is necessary to meet the research objectives. 
 
9.8 Regulatory Files 
Prior to beginning the study, the study Principal Investigator and Site Investigators will be asked to 
comply with ICH E6 8.2 and Title 21 of the CFR by providing the following essential documents, 
including but not limited to: 
 
An original investigator-signed Investigator Agreement page of the protocol. 
 
An IRB-approved informed consent, samples of site advertisements for recruitment for 
this study, and any other written information regarding this study that is to be provided to 
the subject or legal guardians. 
 
IRB approval of the protocol and amendments. 
 
Form FDA 1572, fully executed, and all updates on a new fully executed form FDA 1572 
 
Curriculum vitae (CV) for the site Principal Investigator and each Sub-Investigator listed 
on Form FDA 1572. Current licensure must be noted on the CV. They will be signed and 
dated by the site Principal Investigators and Sub-Investigators at study start up, 
indicating that they are accurate and current. 
 
Financial disclosure information to allow the sponsor to submit complete and accurate 
certification or disclosure statements required under Part 54 of Title 21 of the CFR and 

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ICH E6. In addition, the Investigators must provide to the study PI a commitment to 
promptly update this information if any relevant changes occur during the course of the 
investigation and for 1 year following the completion of the study. 
 
Laboratory certifications and normal ranges for any test or assay being performed. 
 
9.9 Source Data Verification Monitoring 
 
The study PI will authorize the Coordinating Center (CNMC-CC) to monitor the study 
according to the relevant standard operating procedures (SOPs) and GCP guidelines as 
frequently as deemed necessary by the CNMC-CC to verify that data entries into the 
CRF are correct and that the study is conducted in accordance with this protocol. The 
first monitoring visit will take place approximately 12 months after the Site Initiation Visit. 
 
During each monitoring visit, the monitor will check the entries made in the CRFs. The 
monitor will compare these entries with the source data, e.g., the subject’s medical 
records or laboratory results (source data verification). The case number, informed 
consent, demographic data, inclusion/exclusion criteria, concomitant diseases, 
concomitant treatment, and all AEs will be verified.  
 
The monitor will check all data for plausibility and completeness in collaboration with the 
site investigator. At the same time, a data check for medical plausibility and conformity 
with ICH GCP will be performed. 
 
9.10 Modification of the Protocol 
Protocol modifications that affect the safety of the subjects or that alter the scope of the investigation, 
the scientific quality of the study, the experimental design, dosages, assessment variable(s), the 
number of subjects treated, or the subject selection criteria may be proposed by the study Principal 
Investigator and the Medical Monitor. Any change to the protocol can be made only in the form of a 
written amendment to this study protocol. Such amendments must be approved by the DSMB and the 
institutional IRB of the study PI and the CNMC-CC first. Once approved, these amendments will be 
circulated to the sites for their IRB approval prior to implementation at each site.  The IRB at each site 
will determine if the changes are such to warrant that the informed consent form must be revised and 
re-signed by the legal representatives of all subjects enrolled in the trial, or only applied to new 
subjects in the study. 
 
9.11 Protocol Deviations and Violations 
Protocol deviations or violations are any non-adherences to the procedures outlined in this document. 
After a subject has been enrolled, it is the investigator’s responsibility to make any reasonable effort 
to avoid and, if necessary, correct protocol deviations or violations 
 
Protocol Deviation 
A protocol deviation is any change, divergence, or departure from the IRB-approved protocol, 
which does not affect the subject's rights, safety or well-being, or the completeness, accuracy, 
and reliability of primary study data. 
 
Protocol Violation 
A protocol violation is any change, divergence, or departure from the IRB-approved protocol that 
may affect the subject's rights, safety, or well-being and/or the completeness, accuracy, and 
reliability of primary study data. 
 
If any of the following five criteria are met, a protocol violation has occurred (examples 
provided): 
 

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1. The deviation has harmed or posed a significant or substantive risk of harm to the 
research subject. 
• A research subject received the wrong treatment or incorrect dose or expired drug 
• A research subject met withdrawal criteria during the study but was not withdrawn 
• A research subject received an excluded concomitant medication 
2. The deviation compromises the scientific integrity of the data collected for the 
study. 
• A research subject was enrolled but does not meet the protocol's eligibility criteria 
• Changing the protocol without prior IRB approval 
3. The deviation is a breach of human subject protection regulations, policies, or 
procedures on the part of the investigator(s). 
• Failure to obtain informed consent prior to initiation of study-related procedures 
• Falsifying research or medical records 
• Performing tests or procedures beyond the individual's professional scope or 
privilege status (credentialing) 
4. The deviation involves a serious or continuing noncompliance with federal, state, 
local, or institutional human subject protection regulations, policies, or 
procedures. 
• Working under an expired professional license or certification 
• Failure to follow federal and/or local regulations, and intramural research or 
Coordinating Center policies 
• Repeated minor deviations 
5. The deviation is inconsistent with the NIH Human Research Protection Program’s 
research, medical, and ethical principles. 
• A breach of confidentiality. 
• Inadequate or improper informed consent procedure 
 
All protocol violations will be reported by the site to the CNMC-CC immediately upon discovery. The 
CNMC-CC will report to the study PI any violations reported by sites or discovered through 
monitoring. All protocol violations will be listed and the admissibility of the subjects’ data will be 
reviewed and assessed by the CNMC-CC. In case any emergency or AE occurs that requires a 
protocol deviation in the particular case, the investigator should contact the Medical Monitor and 
DSMB as soon as possible to enable a decision on whether the subject’s participation in the study 
may be continued. 

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10 DATA MANAGEMENT AND PROCEDURES 
 
All study data will be collected via systems created at the CNMC Coordinating Center (CNMC-CC) and 
will comply with all applicable guidelines regarding patient confidentiality and data integrity.   
 
10.1 Data Collection 
Data can be captured either on paper source documentation and then entered into the Research 
Electronic Data CAPture (REDCap) system or entered directly (https://redcap.vanderbilt.edu). The 
web-based transmission of data is encrypted and enclosed within a firewall. 
Copies of all source records (original consent documents, contact notes, laboratory evaluation, and 
other diagnostic reports) must be kept in the participants’ medical record or in a separate research 
chart as allowable by institutional guidelines. At a minimum, the participants’ medical record and 
study chart must contain a copy of the informed consent. The participant’s medical record must 
contain the source documents. 
 
10.2 Data Processing  
Data will be entered into REDCap by site study staff and using data validity and cleaning applications 
within REDCap, all data will be checked against data specifications. Data exceptions (i.e., invalid 
values, outliers, etc.) or missing data are flagged and described to give the site coordinator an 
opportunity to review and make corrections directly in the database. All changes to the database will 
be logged in the REDCap data audit trail. Nevertheless, an eCRF containing data exceptions can be 
submitted and is logged. Data exceptions, detected at multiple check levels, are queried and 
followed. The Coordinating Center will also perform regular data queries.   
 
10.3 Design and Development of REDCap for Study Data Management 
We will collect and manage study data using the Research Electronic Data CAPture System 
(REDCap) which has been used to successfully support more than  700  single and multi-centered 
clinical research studies at CNMC and The George Washington University as part of the collaboration 
between these two institutions for the Clinical and Translational Research Institute at Children's 
National (CTSI-CN). REDCap contains tools that are designed to significantly enhance study 
management and improve the completeness, quality, security, and integrity of research data. The 
system provides support for all facets of an investigation including protocol oversight, screening, and 
enrollment of volunteers, a collection of protocol-driven data, as well as reporting and analysis of 
interim and final results. REDCap also assists with monitoring patient safety and data quality. At 
enrollment, REDCap supports the random allocation of participants to study groups. In addition to the 
study data itself, the database houses information about each protocol including information 
describing each item of data to be collected, targeted enrollment quotas, eligibility criteria, as well as 
the detailed schedule of visits with information to be collected at each visit (event-time matrix). This 
enables the system to help with study management and to help improve study quality. The system 
provides for Web-based data collection from professionally designed forms that can be accessed for 
online entry directly into the database. Using the Data Access Groups, REDCap also confines each 
site to view and edit only their own participant dataset. At entry, data checks are performed and data 
exceptions are logged and flagged for correction. Corrections are implemented based on data edits 
applied to online forms by approved personnel. All changes are logged and subjected to the same 
validity and consistency checking of the original data and are applied directly to stored data. 
10.3.1 REDCap Time Event Matrix 
A key feature of REDCap are the event grids, which define and help to manage the study 
protocol, it is used as the fundamental view for both scheduling and management of protocol 

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events, including access to eCRFs. It provides per protocol, a complete and up-to-date picture 
of each participant’s status regarding expected and completed events by the target date.  
 
10.3.2 REDCap Protocol Management 
In terms of protocol management, the event grid also lists and tracks each of the data capture 
events that must take place to implement the research protocol and it provides monitoring to 
help assure that each is completed accurately (see Section 10.6) and that the requisite data 
reside in the database within the designated timeframe.  During the encounter, it serves as a list 
to ensure that events that need to occur are completed according to protocol. It also provides a 
“just in time” link to the eCRF used to record, view, and or edit the data.  Symbols are used to 
show the status of each event at each time point in the event grid. Initially, the symbol for each 
form will be red if the form has not been completed, yellow if the form has been completed but 
data have not been verified, and green when the form has been completed.   Visit window data 
will be used to identify any data collected outside of the allowable timeframes; these will be 
noted as protocol violations.    
 
10.3.3 REDCap Facilitated Screening Features 
REDCap will track study screenees from the point of contact through enrollment. During the 
screening, REDCap will assign each screenee a unique numeric study Screening ID, which is 
distinct from any personal identifiers.  The screening identifier will be selected from a range 
dedicated to each site. Screenees are classified as “pending eligibility” while they are being 
evaluated per eligibility criteria. Subsequently, they are labeled as “eligible” or “ineligible”, noting 
the reasons for ineligibility. Those meeting eligibility criteria are classified as “pending 
enrollment” while they are being processed and later as “enrolled” or “refused” noting the 
reasons for their refusal once they have made their decisions. At enrollment, each participant is 
randomly assigned to a study group and receives a Participant ID (PID), which is separate from 
the Screening ID. In addition to changes in the subject's recruitment status, the time of such 
changes is also recorded. 
 
10.3.4 REDCap Randomization  
At each episode, online checks ensure all episode eligibility criteria are met, and if so, access is 
provided to the REDCap randomization button. When pressed,  the button implements an online 
program that accesses a pre-generated, encrypted, randomization schedule created by the 
CNMC Division of Biostatistics and Study Methodology to make the appropriate treatment 
assignment for that subject. This procedure will provide a balanced allocation of assignments to 
each treatment arm over the episodes. Once an assignment is made, to maintain the blind the 
person enrolling the subject will be notified simply that the site pharmacy is preparing the 
assigned medication. These procedures will ensure tamper-proof, blinded randomization as well 
as overall balance in assignment to treatment groups. The Site Pharmacy will simultaneously 
receive online or fax notification of the assignment and will be requested to prepare the 
appropriate dosing kit for pick-up or delivery to the floor (depending on site logistics). 
 
10.4 Recruitment and Retention Monitoring  
REDCap reports will monitor recruitment overall and by the site. It will warn of recruitment problems 
by plotting actual vs. expected screens, recruits, and enrollees. Should problems arise, REDCap can 
help diagnose the source of such problems, and identify possible remedies. It is also possible to 
compare the demographics of all those eligible to all enrollees. These monitoring reports will be 
forwarded on a monthly basis to study leadership and will be discussed at the weekly calls. Retention 
rates at each site will be evaluated on a monthly basis using REDCap reporting modules. Should a 

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site show an unexpectedly high loss to follow-up, causes of the problem will be reviewed, and 
possible changes to retention strategies will be evaluated.  
 
10.5 Site Data and Study Responsibilities 
Each site will: 
 
Submit protocol and any protocol amendments or modifications to site IRBs for approval 
 
Maintain regular communications with the Coordinating Center regarding IRB protocol 
approvals 
 
AEs or other study related issues 
 
Attend regular conference calls to discuss milestones and administrative issues 
 
Maintain study charts 
 
Recruit, consent and enroll study participants 
 
Conduct all study visits in accordance with the protocol and the Manual of Operating 
Procedures 
 
Receive, log and account for all study drug from the central pharmacy, and will dispense 
drug to the study participants and will return unused or expired drug to the central pharmacy 
 
Enter and verify all study data 
 
10.6 CNMC Coordinating Center Responsibilities 
In order to ensure that data are of the highest quality, special attention will be paid to project 
monitoring. Site performance monitoring will be done by the Project Manager. Monitoring of 
milestones and data quality will be done both in-house at the CNMC-CC and during on-site 
monitoring. 
 
The internal component will consist of the CNMC-CC data manager performing a clinical review of the 
eCRFs. This process will supplement the range checks performed at the time of data entry by the 
sites and real-time review of adverse events and safety data. The internal component will also include 
tracking of regulatory documents in an ongoing fashion. If the internal processes indicate a problem 
at a site, the need for additional training or oversight will be assessed. A plan will be developed to 
address the needs/issues identified, including addressing the needs via remote methods 
(teleconferences or video conferences) in order to minimize the associated costs. If problems persist, 
the CNMC-CC will be updated.  
 
The external component will involve the Project Manager visiting each site at least annually if the site 
has had an episode within that year. The first visit will be for site initiation. The next visit will occur 
approximately 12 months later. At each visit s/he will monitor the site’s compliance with operational 
protocols, evaluate the site’s data tracking and maintenance procedures, and review all safety and 
efficacy data points as well as to review all other data in the database against source patient records, 
in compliance with GCP. This visit provides an opportunity to resolve any outstanding queries and 
deal with potential challenges or problems related to protocol adherence, and participant recruitment 
and retention. After each site visit, a monitoring report will be prepared and copies sent to the Study 
File, the CNMC-CC, and the Executive Committee. A follow-up will be initiated to ensure that any 
concerns raised at a monitoring visit are resolved. 
 
Additional reporting and site monitoring will be conducted remotely on a regular basis, including the 
following:   
 
Site reports that include simple tabulations of the number of admissions, screened patients, 
eligible patients, ineligible patients, and refusals, by month and cumulatively, as well as the 
observed and expected number of weeks of follow-up on study 
 
Subject retention and attrition reports 

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 
Adverse event reports 
 
Pharmacy and study drug accounting activity reports 
 
Summaries of the paper-based data captured (e.g., number of forms completed, number of 
forms expected, and the number of missing pages or fields) 
 
Summaries of the electronic data captured (e.g., amount of data captured, amount of data 
expected, and the number of missing fields) 
 
DSMB reports 
 
The Coordinating Center will also: 
 
Confirm current contact information for all site personnel and study teams 
 
Confirm that site delegation logs are up-to-date 
 
Review IRB approvals and continuing reviews 
 
Obtain documentation of study drug shipment and receipt and sample shipment and receipt 
 
Assign database IDs 
 
Oversee training for data entry, data management, and pharmacy randomization 
 
10.7 Data Security 
There are several layers of security that protect the (data) stored in REDCap. The first layer consists 
of network security, through firewalls, and is designed to limit access to the wide-area network (WAN) 
to specific computers and authorized users and to deny access to all others. The firewall blocks 
malware and prevents flooding. The second layer consists of database authentication. This limits 
access to the database to those individuals passing layer one who has an assigned database 
account and can enter the correct, suitably complex, the user-defined password for that account. The 
third layer consists of controlling a user’s capabilities, what a user can see and do, based on the 
user’s role in each investigation. Each user needing access to the data for a study must be included 
on the list of key staff for that specific study in the protocol and must be assigned a study role. If a 
user is not in the key staff list of the study, s/he will not be granted access to that study’s data even if 
s/he has a valid password associated with another study. Key staff will only be allowed to perform 
those tasks that are dictated by the person’s defined role in the study. For instance, a person who 
enters data will only be able to add records to specific data files; s/he cannot peruse or make 
changes to existing records in the database. Only those persons who have clinical responsibilities for 
the patient would be able to see the patient identifying information.   
 
There are also two other components to security and they are monitoring and system administration. 
The network is closely monitored and all activity is logged. Both the real-time monitor and the logs are 
reviewed by eye and by the computer to identify suspicious activity so that it can be dealt with 
appropriately. Furthermore, all access to research data is audited and all activities are logged so that 
if a problem occurs it will be possible to identify who was responsible and how it happened. The 
system and database software is kept up-to-date with the latest security protections released for the 
operating system and for the database. These measures help us to keep ahead of the threats to 
security or to close loopholes before they can be more widely exploited. Finally, there is excellent 
physical security; access to the systems is keypad controlled, there is a backup generator to provide 
power to keep systems operating in the event of power failure, all computer rooms housing database 
and web servers are environmentally controlled and protected against flood and fire.  
 
10.8 Data Acquisition and Entry 
 
Data collection for this study will be accomplished with online electronic case report forms.   
 
On-line forms will be developed that contain the requisite data fields.   
 
For each subject enrolled all study-related episodes of the enrollment, diagnostic, 
application, observation, termination and follow-up phases will be recorded in the CRF.  

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This CRF must be completed and signed by the investigator for every subject on whose behalf written 
informed consent was given. This also applies to subjects who fail to complete the study. If a subject 
is being withdrawn from the study by his/her parent(s)/legal guardian(s), the reason must be stated in 
the CRF, if the parent(s)/legal guardian(s) are willing to provide such reason. If a subject is withdrawn 
from the study because of a limiting AE, reasonable efforts should be made to clearly document the 
outcome.  
 
All subjects will be identified by their PID.  
 
Each investigator will be responsible for ensuring that the identification of his/her subjects is 
possible at any time.  
 
The investigator will be provided with CRF Completion Guidelines from the Coordinating 
Center.  
 
These guidelines will provide instructions on how to complete, correct, and archive final 
copies of the CRFs. 
 
Prior to the beginning of the study, the investigator must establish a site delegation log, a list 
of the individuals with the trial-related duties and of the persons authorized to make entries 
and data changes in the CRFs. These persons will be listed, including details on their 
function in the study, their full names, initials, and signatures, and the date of their 
assignment to (and, if applicable, their demission from) their respective duties on this study. 
 
10.9 Data Editing 
A number of data checks and procedures may be applied during data collection that either prevent 
errors or detect them with the requirement for later review and adjudication. No matter what the data 
source all data must pass through all predefined data checks and all potential errors must be 
reviewed and corrected or accepted with a comment using the Data Quality tool in REDCap. Any 
discrepancies are reported in a data exception feedback report for correction and are monitored to 
ensure that they are accepted or corrected. Predefined procedures and data checks include: 
 
Limiting the availability of electronic data entry forms to a defined list of enrollees indexed by 
assigned PID; 
 
Implementing validation and consistency checks before data are sent to the database to 
allow correction but logging of all uncorrected exceptions for required later review;  
 
Checking for and recording on each form the rate of missing data that can be used to set 
criteria for subsequent forms processing, e.g. to reject forms with high missing data rates 
from entering the database;  
 
Flagging out of range values; and 
 
Creating a context sensitive comment window that allows subsequently retrievable text to be 
linked to any form or data item, e.g., to explain an unusual data response.   
 
Web-based, forms allow data to be viewed and edited. Quality assurance checks are applied during 
data entry and editing.  In addition to maintaining a time-stamped audit trail of all logins and 
transactions, the system has the capability to be proactive, for example in requiring all unflagged data 
corrections be accompanied by a justification as well as to be adjudicated, reviewed, accepted or 
rejected, by a designee, e.g. a data manager at the Coordinating Center. Together these options and 
procedures promote a high degree of completeness, and accuracy of study data.  
 
10.10 Data Lock and Freeze 
Study data may be preserved by applying electronic security procedures such as ‘locking’ and 
‘freezing’. Locking data (via either partial or final lock) implies a ‘soft lock’ by which approved users 
can make updates to the database. Freezing data disallows users to update the data and it only 
allows browsing access. The terms partial lock, final lock, and database freeze are explained below. 

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Partial Lock: 
 
May be used to lock subsets of data (by site, by the participant, by demographic strata). 
 
Is flexible and may be used to: 
o Lock an entire set of participant records at a site; 
o Lock a single participant record; or 
o Lock a single form. 
 
Only allows a designated user with update privileges to make updates to the locked 
data. 
 
May be applied if data are not readily available to the sites during study completion (e.g., 
biomarker results produced at the end of the study). 
 
Final Lock: 
 
Locks all data entered for a study. 
 
Only allows a designated user with update privileges to make updates to the locked 
data. 
 
May require entered data to exist in the locked state until the final manuscript is 
submitted to the Coordinating Center.  
 
Database Freeze: 
 
Once the CNMC-CC has confirmed the data are satisfactory, the database will be 
frozen.  
 
10.11 Data Entry Training  
Data entry training will be conducted online using the REDCap database. Study coordinators 
will be trained and certified in the on-line use of the REDCap database. Training sessions will be 
scheduled and organized by the Coordinating Center. 
 
10.12 Data Quality Assurance 
Data quality is assessed at the data entry point using intelligent review and controls during on-line 
data entry or edits via study forms. Quality assurance (QA) reports assess data quality post-data 
entry. As we note, data quality begins with the design of the data collection forms and procedures and 
incorporates reasonable checks to minimize transcription and omission errors.   Equally important 
quality assurance measures are the internal checks for reasonableness and consistency that will be 
implemented via simple tabulations and cross-tabulations that should reveal any remaining data 
quality issues. 
 

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11 PUBLICATIONS AND OTHER RIGHTS 
 
Since this project is conducted in UCDC sites, we will subscribe to the data and research resources 
sharing plan that have been developed and approved by the Rare Disease Clinical Research Network.  
According to this plan, datasets will be made available to the scientific community after publication(s) of 
planned analyses (as set forth in the protocol) of the clinical trial results or no later than 3 years after 
the final visit of the last participant to a clinical trial site, whichever comes first. Data obtained in this trial 
will be presented at national and international meetings and will be published in the medical literature 
with no delays. The investigators are not under any agreement with the provider of the drug to limit or 
delay the publication of data from this trial. Results from this study will also be posted on 
www.clinicaltrials.gov. 

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