Conditions: Acute Liver Failure
Keywords: acute liver failure methacetin breath test severe acute liver injury hepatic encephalopathy ALFSG Registry acetaminophen toxicity
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 76
Healthy volunteers: no

Brief summary:
The ALFSG-MBT protocol is for a multicenter, open label, non-randomized study to determine the value of Breath Identification® (BreathID®) N-(4-Methoxy-13C-phenyl)acetamide (13C-Methacetin) Breath Test System in predicting the outcome of patients diagnosed with severe acute liver injury that is not related to acetaminophen overdose or acute liver failure who meet inclusion/exclusion criteria.

Up to 200 evaluable patients will be enrolled. An evaluable patient is one who has completed one or more breath tests for at least 30 minutes after administration of the 13C-Methacetin solution (test substrate).

The Breath Test will be performed up to five times during the study period on all enrolled patients. The first Breath Test will be performed upon admission into the study (Day 1) and repeated on Days 2, 3, 5 and 7 provided no contra-indications are present. Each test continuously measures changes in the metabolism of the 13C-Methacetin in order to assess the improvement or deterioration in liver metabolic function about improvement or deterioration in liver metabolic function. If an enrolled non-APAP ALI or ALF patient receives a liver transplant, is discharged /transferred from the hospital or dies prior to Day 7, additional Breath Tests will not be performed.

Patients will be contacted for the Day 21 follow up (21 days after enrollment into the trial) to determine spontaneous survival, transplantation and occurrence of serious adverse events since the patient's last study treatment.

Detailed description:
The importance of identifying the patient with with ALI or ALF who is likely to die without a liver transplant cannot be overstated and has remained a primary focus of clinical investigation for 25 years. A recent analysis also conducted by the Acute Liver Failure Study Group (ALFSG) found that poor outcomes in the ALI patients are less frequent than is observed in the ALF population. However, in cases where ALI was not related to an acetaminophen (APAP) overdose, progression to poor outcomes was similar. Traditional scoring systems and prognostic models, such as King's College Criteria (KCC), Sequential Organ Failure Assessment (SOFA) and Acute Physiology and Chronic Health Evaluation II (APACHE II), currently used to monitor patients with ALF lack individual sensitivity and specificity and do not provide direct information about the liver's metabolic function, which is a key variable in assessing liver status and potential disease progression versus recovery in ALF patients. Despite recent advances used by the ALFSG Prognostic Index (ALFSG-PI), ALI Prognostic Index (ALI-PI) and Model for End-Stage Liver Disease (MELD), better predictive modalities are still needed.

The 13C-Methacetin breath test is a rapid, reproducible, point-of-care test of liver metabolic function. After oral or naso-enteric/orogastric tube administration, the 13C labeled Methacetin is O-demethylated by cytochrome P450 1A2 (liver enzyme name) in the liver and further biotransformed into carbon dioxide labeled with carbon 13 (13CO2), which is expired in breath. The BreathID® Molecular Correlation Spectroscopy (MCS) device captures and quantifies expired 13CO2 and standardizes recovery against expired normal carbon dioxide (12CO2) through a nasal cannula (in conscious patients) or an adaptor connected to the ventilator line (for intubated patients). The results obtained from the device are expressed as delta over baseline (DOB), which expresses the change in 13CO2/12CO2 ratio in comparison to the baseline measurement. It can be transformed into the percentage of 13C dose recovered over time (PDR) after the ingestion of Methacetin, and the cumulative PDR (CPDR), the rate at which 13C substrate is metabolized, derived from the breath 13C/12C ratio.

This is a multicenter, open label, non-randomized study of the MBT to assess functional trends of liver metabolism in patients diagnosed with severe acute liver injury not related to acetaminophen overdose (non-APAP ALI) or acute liver failure (ALF). Up to 200 evaluable patients with non-APAP ALI or ALF present at the time of enrollment into the ALFSG Registry will be consecutively enrolled. An evaluable patient is one who has completed one or more Breath Tests measured for a minimum of 30 (and ideally 60) minutes after administration of the 13C-Methacetin solution. Study sites will include up to 11 of the clinical sites located in the United States that are involved in the ALFSG.

The Breath Test will be performed up to five times during the study period on all enrolled subjects. The first Breath Test will be performed as close to the time of study enrollment as possible upon admission into the study (Day 1). The Breath Test will be repeated on Days 2, 3, 5 and 7 as close as possible to the same time of day as the first Breath Test. If a subject who is enrolled into the ALFSG-MBT Trial with non-APAP ALI converts to ALF, breath test collection will continue until a maximum of five Breath Tests have been performed. If an enrolled non-APAP ALI or ALF subject receives a liver transplant, is discharged/transferred from the hospital or dies prior to Day 7, no additional Breath Tests will be performed. Enrolled patients will be contacted for the Day 21 follow up (21 days after the subject's enrollment into the trial) to determine spontaneous survival, transplantation and occurrence of serious adverse events since the subject's last study treatment.

Arms:
arm 1: All patients enrolled into the ALFSG Registry with the duration of illness \<26 weeks with (1) severe acute liver injury; International Normalized Ratio (INR) ≥2.0) and not related to acetaminophen overdose, with no evidence of hepatic encephalopathy (HE); and (2) acute liver failure; INR ≥1.5 with presence of any degree of HE will perform the Breath Test.

Interventions: intervention 1: 13C-Methacetin
Intervention descriptions: intervention 1: The test substrate in this study, ¹³C-methacetin solution for single-use oral administration (75 mg in 150 ml purified water), is administered orally or via feeding tube, rapidly absorbed, exclusively metabolized by hepatic mixed function oxidase via O-demethylation, mainly by cytochrome P450 enzyme, subtype 1A2, into acetaminophen and formaldehyde. The formaldehyde is then transformed through two successive oxidative steps to ¹³carbon dioxide, the quantity of which is measured in exhaled breath as a ratio of 13C to 12C.

The nasal or intubated breath sampling investigational device (ID) circuit continuously transports the breath sample from the patient to the BreathID® MCS device before and following administration of the 13C-methylacetanilide test substrate.

Protocol document:
Prot_SAP_001.pdf: 
Acute Liver Failure Study Group 13C-Methacetin Breath Test for the Prediction 
of Outcome in Acute Liver Injury or Acute Liver Failure (ALFSG-MBT) 
NCT02786836 
Protocol Date: 15-March-2018 
 
 


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Acute Liver Failure Study Group 
13C-Methacetin Breath Test for the Prediction of Outcome  
in Acute Liver Injury or Acute Liver Failure 
(ALFSG-MBT) 
 
 
Protocol Number: 
 
ALFSG-MBT-003 
IDE Number: 
 
 
G150226 
Study Chair: 
Robert J. Fontana, M.D. 
 
University of Michigan 
 
Ann Arbor, MI 
Study Intervention Provided by: 
Exalenz Bioscience Ltd. 
Sponsor of IDE: 
William M. Lee, M.D. 
UT Southwestern Medical Center at Dallas 
 
for the Acute Liver Failure Study Group 
Protocol Version: 
3.0 
Protocol Date: 
15-March-2018 
 
 
 


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Investigator Statement of Agreement 
I have read the 13C-Methacetin Breath Test for the Prediction of Outcome in Acute Liver 
Injury or Acute Liver Failure (ALFSG-MBT) clinical protocol version 3.0 dated 15-March-2018 
and agree to implement and conduct the protocol as written in this document. 
I agree to comply with the Declaration of Helsinki/Tokyo/Venice on Experimentation in 
Humans as required by the United States Food and Drug Administration regulations; the Code of 
Federal Regulations Title 21 parts 50, 56, 312, 800, as applicable; the Code of Federal Regulations 
Title 45 part 46; International Council on Harmonization Good Clinical Practice Guidelines; and 
all other applicable laws, regulations and guidelines. 
I understand this document contains confidential information of Exalenz Bioscience Ltd., 
the ALFSG-MBT Executive Committee and the Statistical and Data Coordinating Center at the 
Medical University of South Carolina that cannot be disclosed to anyone other than members of 
my staff conducting this trial and members of my Institutional Review Board or Ethical 
Committee. 
I agree to ensure that this information will not be used for any purpose other than the 
evaluation or conduct of this clinical trial without the prior written permission of the ALFSG-MBT 
Executive Committee. 
 
 
___________________________________  
_____________________________ 
Signature of Site Principal Investigator  
 
Date 
 
 
 
___________________________________ 
Printed name of Site Principal Investigator 
 
 
 
___________________________________ 
Institution 
 
 
 


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TABLE OF CONTENTS 
1 
ABBREVIATIONS ............................................................................................................... 5 
2 
PROTOCOL SYNOPSIS ........................................................................................................ 7 
3 
INTRODUCTION ............................................................................................................... 12 
3.1 
Background and Rationale ............................................................................................. 12 
3.2 
Summary of BreathID® and MBT Clinical Data ........................................................... 13 
3.3 
BreathID® 13C-Methacetin Breath Test (MBT) System Description ............................. 15 
3.3.1 
BreathID® MCS Unit .............................................................................................. 15 
3.3.2 
Cannula ................................................................................................................... 16 
3.3.3 
13C-Methacetin Solution ......................................................................................... 17 
4 
OBJECTIVES AND ENDPOINTS .......................................................................................... 18 
4.1 
Primary Objective .......................................................................................................... 18 
4.2 
Endpoints ........................................................................................................................ 18 
4.2.1 
Primary Endpoint .................................................................................................... 18 
4.2.2 
Secondary Endpoints .............................................................................................. 18 
4.2.3 
Safety Endpoints ..................................................................................................... 19 
5 
INVESTIGATIONAL PLAN ................................................................................................. 20 
5.1 
Study Design .................................................................................................................. 20 
5.2 
Study Population ............................................................................................................ 20 
5.3 
Study Sites ...................................................................................................................... 20 
5.4 
Investigational Product ................................................................................................... 21 
5.4.1 
Preparation of 13C-Methacetin Solution .................................................................. 21 
5.4.2 
Investigational Product Handling ........................................................................... 21 
5.4.3 
13C-Methacetin Accountability ............................................................................... 21 
5.4.4 
Investigational Device Accountability .................................................................... 22 
5.5 
Risk Based Monitoring................................................................................................... 22 
6 
ELIGIBILITY CRITERIA .................................................................................................... 22 
6.1 
Inclusion Criteria ............................................................................................................ 22 
6.2 
Exclusion Criteria ........................................................................................................... 22 
6.3 
Prohibited During Study Period ..................................................................................... 24 
7 
SUBJECT ENROLLMENT ................................................................................................... 24 
7.1 
Eligibility Assessment .................................................................................................... 24 
7.2 
Presentation of Informed Consent .................................................................................. 24 


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8 
STUDY PROCEDURES ....................................................................................................... 26 
8.1 
Preamble ......................................................................................................................... 26 
8.2 
Administration of the Breath Test .................................................................................. 26 
8.2.1 
Requirements for Administration of Test Substrate ............................................... 27 
8.2.2 
Performance of the Breath Collection..................................................................... 28 
8.2.3 
Early Termination of the Breath Test ..................................................................... 28 
8.2.4 
Interruptions and Confounders of the Breath Test .................................................. 29 
8.3 
Performance of the APAP Adduct Assays and APAP Concentration Assays ............... 29 
8.4 
Clinical and Laboratory Assessments ............................................................................ 29 
8.5 
Schedule of Assessments ............................................................................................... 30 
9 
DISCONTINUATION OF PARTICIPATION ........................................................................ 31 
9.1 
Withdrawal of Consent................................................................................................... 31 
9.2 
Withdrawal from Study Procedures ............................................................................... 31 
9.3 
 Subject Lost to Follow-Up ............................................................................................ 32 
10 
SAFETY AND ADVERSE EVENTS .................................................................................... 32 
10.1 
Safety Sub-study ............................................................................................................ 32 
10.2 
Adverse Events ............................................................................................................... 32 
10.3 
Data Safety Monitoring Board (DSMB) ........................................................................ 33 
11 
DATA MANAGEMENT .................................................................................................... 33 
11.1 
Data Handling ................................................................................................................ 33 
12 
STATISTICAL CONSIDERATIONS ................................................................................... 34 
12.1 
Sample Size .................................................................................................................... 34 
12.2 
Statistical Analyses ........................................................................................................ 34 
12.2.1 Futility Analysis ......................................................................................................... 34 
12.2.2 Final Analyses ............................................................................................................ 35 
13 
OBLIGATIONS OF THE INVESTIGATOR .......................................................................... 36 
13.1 
Compliance with Good Clinical Practice (GCP)............................................................ 36 
13.2 
Access to Source Data and Documents .......................................................................... 36 
13.3 
Retention of Documentation .......................................................................................... 36 
13.4 
Confidentiality of Material Provided by Exalenz Bioscience Ltd. ................................ 37 
13.5 
Publication ...................................................................................................................... 37 
14 
LIST OF REFERENCES .................................................................................................... 38 
 
 


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1 
ABBREVIATIONS 
AASLD 
American Association for the Study of Liver Disease  
AE 
adverse event 
ALF  
acute liver failure 
ALF-MBT  
Acute Liver Failure 13C-Methacetin Breath Test 
ALFSG 
Acute Liver Failure Study Group 
ALFSG-PI  
ALFSG Prognostic Index 
ALI 
acute liver injury 
ALT 
alanine transaminase 
APACHE II 
Acute Physiology and Chronic Health Evaluation II 
APAP 
acetaminophen (acetyl-para-aminophenol) 
ARDS 
Acute Respiratory Distress Syndrome 
AST 
aspartate aminotransferase 
AUROC 
area under the ROC curve 
CPDR  
cumulative percentage of 13C dose recovered over time 
CRF 
case report form 
CTCAE 
Common Terminology Criteria for Adverse Events 
CVP 
central venous pressure 
DCR 
Data Clarification Request 
DCU 
Data Coordination Unit 
DIC  
disseminated intravascular coagulation 
DHHS 
Department of Health and Human Services 
DOB  
delta over baseline 
DSMB 
Data Safety Monitoring Board 
EC 
Ethics Committee 
eCRF 
electronic case report form 
ET 
endotracheal  
FDA 
U.S. Food and Drug Administration 
FEV1 
forced expiratory volume in one second 
FiO2 
fraction inspiratory oxygen 
GCP 
Good Clinical Practice 
GI 
gastro-intestinal 
HE  
hepatic encephalopathy 
ICH/GCP 
International Conference on Harmonization Good Clinical Practice Guidelines  


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ICU 
Intensive Care Unit 
IDE 
Investigational Device Exemption 
INR  
International Normalized Ratio 
IRB 
Institutional Review Board 
KCC  
King’s College Criteria 
LAR 
legally authorized representative 
LT 
liver transplant 
MAP 
mean arterial pressure 
MBT  
13C-Methacetin Breath Test  
MCS 
molecular correlation spectrometry 
MedDRA  
Medical Dictionary for Regulatory Activities 
MELD  
Model for End-Stage Liver Disease 
MOP 
Manual of Procedures 
MSM 
medical safety monitor 
NAC 
n-acetyl cysteine 
NIDDK 
National Institute of Diabetes and Digestive and Kidney Diseases 
NIH 
National Institutes of Health 
PaO2 
partial arterial oxygen 
PDR 
percentage of dose recovered over time 
PDRPeak 
the maximum value of PDRs 
PEEP 
positive end expiratory pressure 
PI 
principal investigator 
RBC 
red blood cell 
ROC 
receiver operating characteristic 
RRT 
renal replacement therapy 
SAE 
serious adverse event 
SAR 
suspected adverse reaction 
ScvO2 
central venous oxygen saturation 
SDCC/MUSC 
Statistical Data Coordinating Center at the Medical University of South 
Carolina 
SOFA  
Sequential Organ Failure Assessment 
SS  
spontaneous survivors 
SSL 
secure socket layer 
WBC 
white blood cells 
 
 


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2 
PROTOCOL SYNOPSIS 
Protocol Number/ 
Title 
ALFSG-MBT-003:  13C-Methacetin Breath Test for the Prediction of 
Outcome in Acute Liver Injury or Acute Liver Failure (ALFSG-MBT) 
IDE # 
G150226 
Version and Date 
Version 3.0; 15-March-2018 
Study Chair 
Robert J. Fontana, MD, University of Michigan, Ann Arbor, MI 
Co-Principal 
Investigators 
Valerie Durkalski, PhD, Medical University of South Carolina,  
Charleston, SC 
R. Todd Stravitz, MD, VCU Medical Center, Richmond, VA 
Sponsor of IDE & 
Administrative PI 
William M. Lee, MD, UT Southwestern Medical Center, Dallas, Texas 
Investigative Sites 
Approximately 11 investigative sites in the United States. 
Objective &  
Outcomes 
Primary Objective: To assess the relationship between Day 1 PDRPeak 
values and Day 21 spontaneous survival in adult patients with ALI that is 
not related to acetaminophen overdose (non-APAP ALI) or ALF. This 
outcome requires only one measurement per subject and examines whether 
the distributions (means) of enrollment PDRPeak values vary between those 
that spontaneously survive at Day 21 and those that either die or get a 
transplant by Day 21.  
Secondary Outcomes:  
1. Using a continuous variable, we will determine the optimal cut point in 
the PDRPeak that best distinguishes between outcomes. 
2. There are several exploratory aims that will be conducted for future 
hypothesis generation. These analyses include: assessing the change in 
MBT measurements (CPDR20, PDRPeak, PDR20 and CPDR30) over a 
maximum of 7 days from enrollment; assessing the relationship 
between single time points of MBT measurements (PDRPeak, PDR20 
and CPDR20) and clinical outcome (both early and late outcomes, Day 
7 and Day 21 SS); and, evaluating the use of the PDRPeak as a prognosis 
for non-APAP ALI and ALF outcomes (Day 7 and Day 21 SS) either 
alone or in conjunction with other clinical parameters including 
etiology, laboratory measurements and other available prognostic 
indices. 
Investigational  
Product 
BreathID® 13C-Methacetin Breath Test System  
Supplied to the Investigator by Exalenz Bioscience Ltd. 


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Study Population 
Approximately 200 male and female patients, aged 18 to 80 years (have not 
reached their 81st birthday) with severe acute liver injury that is not related 
to acetaminophen overdose or acute liver failure present at the time of 
enrollment into the ALFSG Registry will participate. 
Study Design 
This is a multicenter, open label, non-randomized study to determine the 
value of BreathID® 13C-Methacetin Breath Test System (MBT) in 
predicting the outcome of patients diagnosed with severe acute liver injury 
that is not related to acetaminophen overdose and acute liver failure who 
meet inclusion/exclusion criteria. 
Informed consent will be obtained from the patient and/or patient’s legally 
authorized representative (LAR) or family member as defined in 
21CFR50.3(m). All patients will receive medical care for severe acute liver 
injury or acute liver failure according to the institution’s standards of care.  
Up to 200 evaluable patients will be consecutively enrolled.  The MBT (also 
referred to as “Breath Test”) will be performed up to five times during the 
study period on all enrolled patients.  An evaluable patient is one who has 
completed one or more Breath Tests measured for a minimum of 30 (and 
ideally 60) minutes after administration of the 13C-Methacetin solution (also 
referred to as “test substrate”). The first Breath Test will be performed upon 
admission into the study (Day 1) and repeated on Days 2, 3, 5 and 7, as 
close to the same time of day as possible, provided no contra-indications 
are present.  
If a patient who is enrolled into the ALFSG-MBT Trial with ALI converts 
to ALF, breath test collection will continue until the maximum of five 
Breath Tests have been performed.  Once the ALI patient has been enrolled 
into the ALFSG Registry with ALF, subsequent Breath Tests should be 
performed as close to the same time of day as the previous Breath Tests 
provided no contra-indications are present.  During the initial consent 
process, the enrolled ALI patient should indicate consent to continue 
participation into the ALFSG-MBT study should the disease progress to 
ALF.   
If an enrolled ALI or ALF patient receives a liver transplant, is discharged 
/transferred from the hospital or dies prior to Day 7, additional Breath Tests 
will not be performed. 
Subjects will be contacted to determine Day 21 spontaneous survival, 
transplantation and occurrence of serious adverse events since the last 
Breath Test.  
Only trained study personnel will perform the breath test procedure and 
administer the test substrate. Site principal investigators (PIs) and clinical 
staff will be blinded to the results of the MBT so that there cannot be any 
reliance on the MBT for clinical decision-making.   


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Inclusion Criteria 
1. Adult men or women (18-80 years of age)  
2. Severe acute liver injury (ALI), defined as the development of 
coagulopathy (International normalized ratio [INR] ≥2.0) and not 
related to acetaminophen overdose, with no evidence of hepatic 
encephalopathy (HE); these patients may provide consent themselves. 
3. Acute liver failure, defined as the development of coagulopathy (INR 
≥1.5) with HE  
4. Duration of illness <26 weeks 
5. Enrolled into the ALFSG Registry  
6. Written informed consent from the patient or patient’s legally 
authorized representative or family member 
as defined in 
21CFR50.3(m) 
Exclusion Criteria 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1. Evidence of pre-existing chronic liver disease  
2. Pre-existing New York Heart Association stage III/IV heart failure   
3. Evidence of pre-existing chronic renal failure requiring hemodialysis 
4. Chronic hemodialysis prior to hospital admission 
5. Evidence of cirrhosis (unless clinically acute Wilson disease or 
autoimmune non-APAP or ALF)  
6. Severe obstructive lung disease (FEV1 <50% of predicted on previous 
spirometry) 
7. Severe shock, defined as MAP <70 mmHg despite >15 µg/kg/min 
dopamine, >0.1 µg/kg/min epinephrine, or >0.1 norepinephrine 
µg/kg/min 
8. Extensive small bowel resection (>50 cm)  
9. Any evidence of upper GI bleeding at enrollment requiring intervention 
(endoscopy or RBC transfusion specifically for upper GI bleeding) 
10. Liver transplantation prior to enrollment (Note: Listing for LT does not 
preclude participation in the trial.) 
11. Pregnancy or breastfeeding women (Note: Pregnancy related non-
APAP ALI or ALF may be considered for entry following the delivery 
of the baby and assuming the mother does not wish to breastfeed or 
collect breast milk during the study period.)  
12. Allergic to acetaminophen (such as Tylenol® or any other 
acetaminophen-containing medications)  
13. Participation in other clinical studies evaluating other experimental 
treatments or procedures. (Note: Participation in observatory studies is 
not an exclusion.) 
14. Patients in whom enteral drugs or fluids are contra-indicated or the 
patient either does not have an appropriately placed naso-


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Exclusion Criteria 
Continued 
enteric/orogastric tube in situ or cannot tolerate taking the drug 
preparation orally (200 ml)  
15. Budd-Chiari Syndrome  
16. Non-APAP ALI or ALF caused by malignancy  
17. Moderate and severe ARDS, as defined by Berlin Criteria (see ALFSG-
MBT Manual of Procedures (MOP)) 
18. Subjects who have received amiodarone in the 30 days prior to study 
enrollment 
19. Consumption of any food or beverage that contains caffeine in the 24 
hours prior to enrollment 
20. Consumption of any of the following drugs that may interfere with the 
metabolism of 13C-Methacetin in the 48 hours prior to study enrollment 
including:  allopurinol, carbamazepine, cimetidine, ciprofloxacin, 
daidzein, disulfiram, Echinacea, enoxacin, fluvoxamine, methoxsalen, 
mexilitene, montelukast, norfloxacin, phenylpropanolamine, phenytoin, 
propafenone, 
rifampin, 
terbinafine, 
ticlodipine, 
thiabendazole, 
verapamil, zileuton or oral contraceptives  
21. Consumption of alcohol in the 24 hours prior to enrollment 
22. Smoking cigarettes in the 8 hours prior to enrollment 
Study Procedures 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
The MBT requires administration of an oral dose of 13C-Methacetin 
solution, followed by retrieval of expired 13CO2 over a period of one hour 
following the test substrate administration, representing first pass kinetics 
and the ability of the liver to withdraw and metabolize 13C-Methacetin, 
yielding the product, 13CO2. 
Subjects will have up to five Breath Tests performed once enrolled into the 
trial and all requirements for the administration of the test substrate have 
been met.  Subjects will be fasting for a minimum of 6 hours from solid 
food or a minimum of 4 hours from naso-enteric/orogastric tube feeding 
and tested, in most instances, in the morning hours. The test substrate may 
be administered in 200 ml of water by mouth or by naso-enteric/orogastric 
tube.  
Breath is collected automatically for a total of 75 minutes (up to 15 minutes 
for baseline measurement and 60 minutes following enteral administration 
of 13C-Methacetin solution) using the BreathID® MCS device at the 
subject’s bedside.  Exhaled breath is collected via a nasal retrieval cannula 
or via connection of the BreathID® MCS device to the endotracheal tube in 
intubated patients.   The BreathID® MCS device will record data over the 
75 minute interval and then the cannula or ET tube connection is removed.  
During the study hour, all other care may be administered to the patient, 
including renal replacement therapy, other food intake as necessary; 
however, it is preferred that the test substrate and Breath Test are not 


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Study Procedures 
Continued 
administered during dialysis treatments and subjects remain fasting for the 
duration of the breath test collection. 
Consumption of the following drugs that may interfere with (compete with 
or induce) the metabolism of 13C-Methacetin will be recorded on the case 
report forms and include the following: 
a. HMG-CoA reductase (Statin) drugs in the 30 days prior to study 
enrollment;  
b. Acyclovir and famotidine taken in the 48 hours prior to study enrollment; 
and 
c. Any of the following taken during the study period: allopurinol, 
carbamazepine, 
cimetidine, 
ciprofloxacin, 
daidzein, 
disulfiram, 
Echinacea, 
enoxacin, 
fluvoxamine, 
methoxsalen, 
mexilitene, 
montelukast, 
norfloxacin, 
phenylpropanolamine, 
phenytoin, 
propafenone, 
rifampin, 
terbinafine, 
ticlodipine, 
thiabendazole, 
verapamil, zileuton or oral contraceptives. 
Safety Evaluations 
This is not a therapy trial; however, the safety of administration of 13C-
Methacetin solution in this setting will be evaluated. Safety assessments 
will be performed by assessing for acetaminophen levels and levels of 
acetaminophen-Cys adducts, by collecting daily serum samples for adduct 
and APAP testing in the laboratory of Dr. Laura James, University of 
Arkansas for Medical Sciences, Little Rock, AR, on the first 20 evaluable 
ALF patients. 
Safety Analysis 
The detection of serum APAP-Cys adducts in those ALF patients not 
thought secondary to APAP toxicity will be considered significant if a value 
> 1.0 nmol/L is determined.  Likewise, the detection of any level serum 
APAP in these patients will also be considered significant.  An increase of 
significance in the adduct level in APAP cases or in serial serum APAP 
levels will also require adjudication as a possible indicator of an additional 
adverse effect of 13C-Methacetin administration.  In addition, standard 
laboratory 
parameters 
including 
serum 
electrolytes 
and 
Liver 
biochemistries will be measured in all subjects through a maximum of 7 
days (See Schedule of Assessments, Section 8.5). 


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3 
INTRODUCTION 
3.1 
Background and Rationale 
The importance of identifying the patient with ALI or ALF who is likely to die without a liver 
transplant cannot be overstated and has remained a primary focus of clinical investigation for 25 
years. A recent analysis also conducted by the Acute Liver Failure Study Group (ALFSG) found 
that poor outcomes in the ALI patients are less frequent than is observed in the ALF population.  
However, in cases where ALI was not related to an acetaminophen (APAP) overdose, progression 
to poor outcomes was similar:  41% of patients with non-APAP ALI progressed to ALF, transplant 
and/or death compared to 7% of patients with APAP ALI (1).  Unfortunately, analyses have shown 
that the King’s College Criteria (KCC) are inaccurate predictors of prognosis. Clinically based 
criteria such as the Sequential Organ Failure Assessment (SOFA) and Acute Physiology and 
Chronic Health Evaluation II (APACHE II) scores offer improved but still inadequate accuracy 
(2-4).  These traditional scoring systems and prognostic models currently used to monitor patients 
with ALF lack individual sensitivity and specificity and do not provide direct information about 
the liver’s metabolic function, which is a key variable in assessing liver status and potential disease 
progression versus recovery in ALF patients.  The Acute Liver Failure Study Group (ALFSG) 
completed an analysis of predictors of outcome in the largest cohort ever studied, 1,974 patients, 
half of whom were randomly assigned to participate in a model development cohort, and half in a 
validation cohort (5). The ALFSG Prognostic Index (ALFSG-PI) identified coma grade, ALF 
etiology, vasopressor use, and the log transformations of admission bilirubin and INR as 
independent clinical predictors of spontaneous survival in patients with ALF, with a c-statistic of 
0.84 by AUROC curve analysis. The ALFSG-PI was also significantly better than Model for End-
Stage Liver Disease (MELD) score (0.70) or KCC (0.65). The group also developed a prognosis 
index for ALI patients that identified etiology, admission values of bilirubin, INR, APAP level and 
duration of jaundice prior to enrollment as variables associated with prediction of progression to 
ALF, transplant or death within 21days (1).  The ALI prognosis index had a c-statistic of 0.84.  
Despite these advances, better predictive modalities are still needed.  
To more precisely assess the functional reserve of the liver and its capacity to regenerate, we 
propose to use an actual measure of liver metabolic function, the 13C-Methacetin Breath Test 
(MBT; Breath ID®, Exalenz Bioscience Ltd., Modi’in, Israel). MBT is a rapid, reproducible, point-


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of-care test of liver metabolic function. After oral or naso-enteric/orogastric tube administration, 
the 13C labeled Methacetin is O-demethylated by cytochrome P4501A2 in the liver and further 
biotransformed into 13CO2, which is expired in breath. The BreathID® MCS device captures and 
quantifies expired 13CO2 and standardizes recovery against expired 12CO2 through a nasal cannula 
(in conscious patients) or an adaptor connected to the ventilator line (for intubated patients). The 
results obtained from the device are expressed as delta over baseline (DOB), which expresses the 
change in 13CO2/12CO2 ratio in comparison to the baseline measurement.  It can be transformed 
into the percentage of 13C dose recovered over time (PDR) after the ingestion of Methacetin, and 
the cumulative PDR (CPRD), the rate at which 13C-Methacetin solution is metabolized, derived 
from the breath 13C/12C ratio.  
3.2 
Summary of BreathID® and MBT Clinical Data 
The BreathID® MBT has been used to assess the functional capacity of the liver in several clinical 
situations. In a study of 165 patients with cirrhosis which included two ALFSG sites, MBT 
predicted liver-related death and the appearance/exacerbation of ascites better than the Model for 
End-Stage Liver Disease (MELD) score (6, copy attached).  
MBT has also been used in a pilot study of patients with ALF performed at Kings College Hospital 
and in Israel (7). Sixty-two patients with severe ALI/ALF of diverse etiologies were followed with 
the MBT during the acute phase of their illness. During their hospital course, 62% of patients 
spontaneously survived, 21% underwent liver transplantation, and 18% died with a mean follow-
up of 7 days after baseline testing. In patients who spontaneously recovered, admission PDR and 
CPDR at 20 minutes after Methacetin ingestion were higher than those who died or underwent 
liver transplantation, with p-value of approximately 0.08 for the difference for both indices. 
Moreover, in the 38 patients in whom post-admission MBTs were available, PDR20 and CPDR20 
were both much higher in spontaneous survivors (SS) than those who died or underwent transplant 
(p=0.0002 and 0.001, respectively), as were the changes in both parameters between Day 1 and 
Day 2-4. (The choice of the later day was not standardized. Values were used in the following 
order of preference depending upon their availability: Day 3, Day 4, or Day 2, if neither Day 3 nor 
4 were available). Subgroup analyses clearly showed that patients who died or underwent 
transplant had similar PDR 20 and CPDR 20 on Day 1, but both were significantly lower than SS. 
These data strongly support the hypothesis that the MBT may assess the severity of ALF at 


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admission and the likelihood of hepatic recovery within 7 days of admission more accurately than 
laboratory tests regardless of the etiology of liver injury.   
Table 1. Methacetin breath test (MBT) results in 62 patients with ALI/ALF on admission 
(Day 1) and in 38 patients at one time point after admission (Day 3, or Day 2 or 4 if Day 3 is 
missing). 
MBT 
Parameter Day 
N 
Total 
Population 
Spontaneous 
Survivors 
Death/  
Transplant 
P* 
Mean±SD/ 
Median[range] 
Mean±SD/ 
Median[range] 
Mean±SD/ 
Median[range] 
PDR 20 
1 
62 
2.60[-1.71 - 33.02] 
3.31[-1.71 - 
13.52] 
1.13[-1.22 - 
12.80] 
0.088 
PDR 20 
2-4 
38 
5.05[-1.41 - 28.68] 
7.19[0.58 - 
28.68] 
1.63[-1.41 - 
6.11] 
0.0002 
PDR 20 
Delta 
1-4 
38 
3.06±8.29 
5.28±8.96 
-1.74±3.46 
0.013 
 
 
 
 
 
 
 
CPDR 20 
1 
62 
0.52[-0.32 - 5.72] 
0.58[-0.29 - 
5.72] 
0.19[-0.32 - 
3.23] 
0.079 
CPDR 20 
2-4 
38 
1.13[-0.41 - 20.83] 
1.54[0.12 - 
20.83] 
0.46[-0.41 - 
1.31] 
0.001 
CPRD 20 
Delta 
1-4 
38 
0.73[-2.10 - 20.32] 
0.85[-2.10 - 
20.32] 
0.0[-1.98 - 1.04] 0.0009 
* ANOVA for normally distributed data; Wilcoxon for non-normally-distributed data. PDR 20, percent dose 
recovery of 13CO2 against 12CO2 in expired breath at 20 minutes after administration of 13C-Methacetin. PDR 
20 Delta refers to the change between Day 1 and post-admission. CPDR 20, cumulative percent dose recovery 
in expired breath over 20 minutes after administration of 13C-Methacetin. CPDR 20 Delta refers to the change 
between Day 1 and post admission.  Adapted from (7). 
The currently marketed BreathID® Hp System was used in previous clinical trials related to liver 
applications under IDEs.  This system has been modified to allow for longer test duration, resulting 
in the BreathID® MCS System. The Acute Liver Failure Study Group 13C-Methacetin Breath Test 
(ALFSG-MBT) Trial using the BreathID® MCS device will allow several tests of each patient at 
point-of-care, in real-time, without requiring active patient participation (breath is collected 
passively).  Each patient’s baseline measurement is compared to the Breath Test conducted that 
day.  The objective is to determine if these results reflect improvement or deterioration of liver 
metabolic function during the course of the disease, which can assist the physician in the 
management of patients with non-APAP ALI or ALF.   


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Since current treatments used to stabilize and care for patients with ALI and ALF may obscure the 
main parameters used to delineate the prognosis (i.e., INR, bilirubin, grade of hepatic 
encephalopathy and renal/metabolic status), the MBT may show superiority over the current 
prognosis models. For example, INR interpretation can be confounded by blood products 
administered to a patient or may be impacted upon by a variety of medical interventions, such as 
disseminated intravascular coagulation (DIC); in contrast, the MBT is not affected by blood 
product administration or development of DIC.  Additionally, MBT monitoring could be 
potentially performed every 24 hours over the patient’s course in the Intensive Care Unit (ICU), 
providing dynamic data that may show earlier changes than routine blood tests, since it measures 
directly the immediate metabolism of the test substrate, as opposed to assessing the presence of 
blood components that have accumulated over a greater period of time, such as total bilirubin 
levels. 
3.3 
BreathID® 13C-Methacetin Breath Test (MBT) System Description  
The ¹³C-Methacetin Breath Test (MBT) is a non-invasive test for assessing liver metabolic function 
in order to aid in management of patients with non-APAP ALI or ALF. The BreathID® MCS 
System consists of the BreathID® MCS device and a test kit containing a breath collection cannula 
and a non-radioactive isotope 13C-Methacetin solution. It measures and computes the ratio between 
13CO2 and 12CO2 in the patient’s exhaled breath. The MBT System has inherent design features 
and accessories for this indication including a small footprint and portability for deployment in an 
ICU environment.  
The components of the MBT system include the (1) BreathID® MCS unit; (2) Cannula; and the 
(3) 13C-Methacetin solution.  A dedicated flash memory stick for automatic raw data download 
will be provided to all participating investigative sites. All components of the MBT system will be 
provided by Exalenz Bioscience Ltd., and a representative from Exalenz will provide the initial 
on-site training to the study team at each investigative site. 
 
3.3.1 BreathID® MCS Unit 
Exalenz Bioscience Ltd. has developed a molecular correlation spectrometer, based on specific 
optical-radiation emission and absorption by 13CO2 and 12CO2 gases. This device continuously 
senses exhaled breath and analyzes CO2 in real-time through a breath collection cannula connected 
to the patient. Based on Molecular Correlation Spectrometry (MCS), the BreathID® MCS device 


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continuously measures 13CO2 and 12CO2 concentrations from the patient’s breath and establishes 
the 13CO2/12CO2 ratio. The cannula continuously transports the breath sample from the patient to 
the BreathID® MCS device. Depending upon the status of the patient, the cannula may be nasal 
(for conscious patients) or an adaptor may be used for connection to the respiratory line (for 
intubated patients). Results are displayed in real time on the device screen and are printed after the 
completion of the test.  The BreathID® MCS device calculates the delta over baseline (DOB), 
which can be translated into the percent dose recovery (PDR) and the cumulative percent dose 
recovery (CPDR) for each time point derived from the ratio difference compared to baseline 
(normalized/adjusted to the patient’s body weight and height as well as test substrate dose) 
throughout the course of the Breath Test. The maximum value of the PDR is called the PDRPeak. 
The 13C-Methacetin metabolism begins almost immediately since liquid passes through the 
stomach to the duodenum, where it is absorbed, without delay. The default breath collection test 
time is one hour after ingestion of 13C-Methacetin solution.  
The light sources are 13CO2 and 12CO2 discharging lamps. By using 13CO2 and 12CO2 discharging 
lamps as light sources, light absorption will be solely due to the existence of 13CO2 and 12CO2 in 
the gas mixtures. Furthermore, by using 13CO2 and 12CO2 discharging lamps as light sources, 
background radiation will be substantially reduced, leading to highly sensitive absorption curves. 
These highly sensitive absorption curves enable detection of very small variations in 13CO2 and 
12CO2 concentrations and the 13CO2/12CO2 ratio. The BreathID® MCS System can detect variations 
of less than 1/1000 in the 13CO2/12CO2 ratio measurement. 
The actual breath collection is automatically done by the device and is not operator dependent. If 
the patient is not connected properly (e.g. the breath does not reach the device), the BreathID® 
MCS device will prompt the operator to adjust the cannula. 
 
3.3.2 Cannula 
There are two test kit options included in the kit; one containing a breath collection cannula with 
nasal prongs to be used for breath collection in non-intubated/conscious patients, and the other 
containing a cannula that has an airway adapter to facilitate connection to the respiratory line in 
intubated patients.  The cannula transports the breath sample from the patient to the BreathID® 
MCS device. The cannula test kit is a single-use kit and is comprised of a plastic sampling line 


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with an in-line hydrophobic filter (used to reduce the amount of moisture present from the patient’s 
breath) and a Luer-lock connector for connection to the BreathID® MCS device. 
Most subjects arriving at the ICU with ALF are unconscious and intubated. As patient status 
improves and the patient is removed from the respirator, the nasal cannula can be used to capture 
breath while they are spontaneously breathing.  Both types of cannulae are necessary so as to 
enable regular monitoring and trending of results as patient's status changes during their stay in 
the ICU. No significant difference in measurements is expected between those captured by the 
intubated or the nasal cannulae, since the internal capnograph-like instrument of the device 
automatically sorts out the breath collected and allows through the system only the end tidal 
portion of the breath exhaled. The device is designed to accommodate an extended range of 
respiratory rates.   
 
3.3.3 
13C-Methacetin Solution 
Exalenz Bioscience Ltd. will supply a ready to use, non-radioactive isotope ¹³C-Methacetin 
solution for single-use oral administration (75 mg in 150 ml purified water), dispensed in 
thermoplastic polyester (PTE) bottles sealed with a plastic child resistant stopper. The ¹³C-
Methacetin solution can be administered through a feeding tube (used in standard ALF patient 
management), as well as via typical oral administration for ALI and ALF patients able to ingest 
the test substrate orally. 13C-Methacetin meets all of the qualifications for a liver function test 
substrate: it is a nontoxic small molecule; is administered orally; is rapidly absorbed; and is 
exclusively metabolized by the liver. Furthermore, 13C can be easily synthesized into a key location 
within this molecule. No related serious adverse events have been reported when using this 
substance, including in vulnerable populations, and the compound remains stable over time. 
The 13C-Methacetin solution is a well-known diagnostic reagent that has been described in the 
literature and used for over 30 years by researchers around the world (8-10). ¹³C-Methacetin is 
rapidly absorbed and metabolized by the hepatic mixed function oxidase, via O-demethylation. 
This process is carried out by hepatic cytochrome P450 enzymes that produce two products, 
acetaminophen and formaldehyde, which is transformed through two successive oxidative steps to 
13CO2.   
Toxicology testing results in animals and other clinical information support the safe use of 
Methacetin in humans. Based on the acute toxicity studies in mice and rats where relatively high 


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LD50 values of 1190mg/kg were administered, the Methacetin dose administered in human breath 
tests in adults of 75 mg, or approximately 1mg/kg, has a safety ratio in excess of 1000-fold (11). 
There have been no reports of any complications with the use of this substance in over 2500 
patients tested worldwide (see IDE filing). The main metabolite of Methacetin is acetaminophen 
which has wide routine clinical use at much higher doses than orally administered dose of 13C-
Methacetin of 75mg used in this study.  Testing of sera after exposure to 75mg 13C-Methacetin in 
23 ALF patients at King’s College disclosed no increase in serum acetaminophen-cysteine adducts 
that would suggest further hepatocyte injury due to the 13C-Methacetin (12). 
Many studies using 13C-Methacetin for liver function assessment have been published. 
Representative references have been cited that support the conclusion that 13C-Methacetin solution 
is a “safe” test substrate and that it has been used on high-risk population groups such as the 
elderly, neonates or pregnant women, and patients with cirrhosis (13-16). This provides further 
assurance that 13C-Methacetin is appropriate to use in breath tests intended for liver function 
assessment. 
4 
OBJECTIVES AND ENDPOINTS 
4.1 
Primary Objective 
The primary aim of the study is to assess the relationship between Day 1 PDRPeak values and Day 
21 spontaneous survival in patients with ALF or non-APAP ALI. This outcome requires only one 
measurement per subject and examines whether the distributions (means) of enrollment PDRPeak 
values vary between those that spontaneously survive at Day 21 and those that either die or get a 
transplant by Day 21. 
4.2 
Endpoints 
 
4.2.1 Primary Endpoint 
 The primary end-point of the study is PDRPeak at Day 1 that will be treated as a 
continuous variable.     
 
4.2.2 Secondary Endpoints 
1. Using the Day 1 PDRPeak as a continuous variable, we will determine a cut point value that best 
distinguishes between those that do and do not spontaneously survive at Day 21.   


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2. There are several exploratory aims that will be conducted for future hypothesis generation. 
These analyses include: assessing the change in MBT measurements (CPDR20, PDRPeak, 
PDR20 and CPDR30) over a maximum of 7 days from enrollment; assessing the relationship 
between single time points of MBT measurements (PDRPeak, PDR20 and CPDR20) and clinical 
outcome (both early and late outcomes, Day 7 and Day 21 SS); and, evaluating the use of the 
PDRPeak as a prognosis for Day 7 and Day 21 SS either alone or in conjunction with other 
clinical parameters including etiology, laboratory measurements and other available prognostic 
indices. 
 
4.2.3 Safety Endpoints 
The administration of Methacetin (75mg/dose) results in the generation of acetaminophen 
(75mg/dose), and therefore presents a modest safety concern in patients with ALF, particularly in 
those with APAP overdose. Use of NAC in patients admitted with APAP overdose or non-APAP 
ALF should obviate minor adverse effect potential of this small dose.  In a retrospective study, 
sera from ALF patients who were given the 13C-Methacetin MBT at King’s College were assayed 
for presence of acetaminophen adducts.  Participants of this study received MBT daily up to 7 
days.  No de novo appearance of adducts was observed for non-APAP subjects, nor was there an 
increase in adduct levels in acetaminophen toxicity subjects.  
To confirm the safety profile of the MBT, a prospective safety sub-study is planned for this 
protocol as outlined in Section 10.1. This safety sub-study will include determining APAP and 
APAP-adduct concentrations in sera from study participants with ALF immediately prior to each 
administration of the 13C-Methacetin solution. A positive safety endpoint is defined as: 
1. the absence of decline in APAP or APAP-adduct concentration between two consecutive 
determinations for patients with ALF due to APAP overdose;  
2. presence of a significant (toxic quantity, ≥ 1 µmol/L) APAP-adducts in patients with non-
APAP ALF; 
3. adjudicated cases where APAP (non-adduct, parent compound) is detected in the serum of a 
patient with non-APAP ALF. It should be noted that in some instances, APAP appears to be 
detected in patients with non-APAP ALF if the total bilirubin is ≥10 mg/dl; these are 
considered false-positive due to the cross-reactivity between APAP reactants and bilirubin in 
some colorimetric APAP assays (Polson, et al.). Such cases of detectable APAP in patients 
with non-APAP ALF will be adjudicated on an individual basis by the study’s appointed 


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independent Medical Safety Monitor to determine the likelihood of Methacetin toxicity.  
Results of adduct testing will initially be reviewed internally and forwarded in real time to the 
MSM for review. These data will then be reported in summary fashion to the DSMB.  
5 
INVESTIGATIONAL PLAN 
5.1 
Study Design 
This is a multicenter, open label, non-randomized study of the MBT to assess functional trends of 
liver metabolism in patients diagnosed with ALI not related to acetaminophen overdose and 
patients diagnosed with ALF.  The Breath Test will be performed up to five times on all enrolled 
subjects. The first Breath Test will be performed upon admission into the study (Day 1) and 
repeated on Days 2, 3, 5 and 7, as close to the same time of day as possible provided no contra-
indications are present.  If an enrolled ALI or ALF subject receives a liver transplant, is 
discharged/transferred from the hospital or dies prior to Day 7, no additional Breath Tests will be 
performed. The study ends after the subject has been contacted to determine Day 21 spontaneous 
survival, transplantation and occurrence of serious adverse events since the last Breath Test. 
If a subject who is enrolled into the ALFSG-MBT Trial with ALI converts to ALF, breath test 
collection will continue until a maximum of five Breath Tests have been performed. Once the ALI 
subject has been enrolled into the ALFSG Registry with ALF, subsequent Breath Tests should be 
performed as close to the same time of day as the previous Breath Tests provided no contra-
indications are present. 
Site principal investigators (PIs) and clinical staff will be blinded to the results of the MBT so that 
there cannot be any reliance on the MBT for clinical decision-making. 
5.2 
Study Population 
Up to 200 evaluable male and female patients, aged 18 to 80 years (have not reached their 81st 
birthday) with severe acute liver injury not related to acetaminophen overdose or acute liver failure 
present at the time of enrollment into the ALFSG Registry will be consecutively enrolled.  An 
evaluable patient is one who has completed one or more Breath Tests measured for a minimum of 
30 (and ideally 60) minutes after administration of the 13C-Methacetin solution.  
5.3 
Study Sites 


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Study sites will include up to 11 of the clinical sites located in the United States that are involved 
in the ALFSG. 
5.4 
Investigational Product 
 
5.4.1 Preparation of 13C-Methacetin Solution  
Exalenz will provide 75 mg 13C-Methacetin in a 0.05% solution in 150mL purified water supplied 
in amber thermoplastic polyester (PET) bottles. No preparation is needed. 
 
5.4.2 Investigational Product Handling 
The Investigator and/or Research Pharmacist (if relevant) will be provided with Investigational 
Product Handling Guidelines that will provide details regarding the packaging and labeling 
requirements, receipt of investigational product, dispensing and accountability procedures, 
preparation instructions, storage and stability of Investigational product, Disposition of 
Investigational product, with the following forms required: Proof of receipt form, Temperature 
logs, Accountability logs, Investigational product and material transfer/disposition form and 
pharmacy staff identification log (if applicable).  
Local forms may be authorized for use after being approved by the Sponsor or his assigned 
representative. 
 
5.4.3 
13C-Methacetin Accountability 
The Investigator and/or Lead Study Pharmacist are responsible for ensuring that all test substrate 
supplies received at the site are inventoried and accounted for throughout the study. The dispensing 
of test substrate to the subject must be documented in the respective accountability form. The study 
Investigational Product must be handled in strict accordance with the protocol and the container 
label and will be stored in a limited access area or in a locked cabinet under appropriate 
environmental conditions. Unused study materials will be verified by the Sponsor’s site monitor 
when on-site monitoring is conducted. The destruction of unused study materials (both, expired or 
unexpired) will be documented on the return/disposition form. The Sponsor will authorize to 
destroy excess supplies on site according to local policy. In this case, before proceeding, the site 
must seek authorization from the Sponsor using the return/destruction form and this must also be 
documented on the Study Supply Return Form. 


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The 13C-Methacetin solution should be dispensed under the supervision of the investigator, a 
qualified member of the investigational staff, or by hospital clinical pharmacist. 
5.4.4 Investigational Device Accountability 
The investigational devices will bear an identification number and their accountability will be filed 
in the Investigator’s Study File. Study supplies logged in will be kept by the investigator or the 
delegated persons in a secure place. All supplies (device, test substrate and cannulae) will be used 
for this study only. After completion of the study, the device, drug and all unused accessories must 
be returned to Exalenz Bioscience Ltd. as per their request or alternatively, destroyed according to 
local regulations after receiving explicit authorization by Sponsor to do so and provide the Sponsor 
with written confirmation. 
5.5 
Risk Based Monitoring 
To ensure compliance with GCP, local regulations and scientific integrity, monitoring of this trial 
will be managed and oversight retained by the Sponsor or designee.  The scope of monitoring 
includes on-site visits, remote and central review as described in the ALFSG-MBT Monitoring 
Plan. 
6 
ELIGIBILITY CRITERIA 
6.1 
Inclusion Criteria 
1. Adult men or women (18-80 years of age)  
2. Severe acute liver injury, (ALI) defined as the development of coagulopathy (International 
normalized ratio [INR] ≥2.0) and not related to acetaminophen overdose, with no evidence of 
hepatic encephalopathy (HE); ALI patients may provide consent themselves. 
3. Acute liver failure, defined as the development of coagulopathy (INR ≥1.5) with presence of 
any degree of hepatic encephalopathy  
4. Duration of illness <26 weeks 
5. Enrolled into the ALFSG Registry.  
6. Written informed consent from the patient or patient’s legally authorized representative or 
family member as defined in 21CFR50.3(m) 
6.2 
Exclusion Criteria  
1. Evidence of pre-existing chronic liver disease  


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2. Pre-existing New York Heart Association stage III/IV heart failure   
3. Evidence of pre-existing chronic renal failure 
4. Chronic hemodialysis prior to hospital admission 
5. Evidence of cirrhosis (unless clinically acute Wilson disease or autoimmune non-APAP ALI 
or ALF)  
6. Severe obstructive lung disease (FEV1 <50% of predicted on previous spirometry) 
7. Severe shock, defined as MAP <70 mmHg despite >15 µg/kg/min dopamine, >0.1 µg/kg/min 
epinephrine, or >0.1 norepinephrine µg/kg/min 
8. Extensive small bowel resection (>50 cm)  
9. Any evidence of upper GI bleeding at enrollment requiring intervention (endoscopy or RBC 
transfusion specifically for upper GI bleeding) 
10. Liver transplantation prior to enrollment. (Note: Listing for LT does not preclude participation 
in the trial.) 
11. Pregnancy or breastfeeding women (Note: Pregnancy related non-APAP ALI or ALF may be 
considered for entry following the delivery of the baby and assuming the mother does not wish 
to breastfeed or collect breast milk during the study period.)  
12. Allergic to acetaminophen (such as Tylenol® or any other acetaminophen-containing 
medications)  
13. Participation in other clinical studies evaluating other experimental treatments or procedures. 
(Note: Participation in observatory studies is not an exclusion.) 
14. Patients in whom enteral drugs or fluids are contra-indicated or the patient either does not have 
an appropriately placed naso-enteric/orogastric tube in situ or cannot tolerate taking the drug 
preparation orally (200 ml)  
15. Budd-Chiari Syndrome  
16. Non-APAP ALI or ALF caused by malignancy  
17. Moderate and severe ARDS, as defined by Berlin Criteria ALFSG-MBT MOP 
18. Subjects who have received amiodarone in the 30 days prior to study enrollment 
19. Consumption of any food or beverage that contains caffeine in the 24 hours prior to enrollment 
20. Consumption of any of the following drugs that may interfere with the metabolism of 13C-
Methacetin in the 48 hours prior to study enrollment including:  allopurinol, carbamazepine, 
cimetidine, ciprofloxacin, daidzein, disulfiram, Echinacea, enoxacin, fluvoxamine, 


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methoxsalen, mexilitene, montelukast, norfloxacin, phenylpropanolamine, phenytoin, 
propafenone, rifampin, terbinafine, ticlodipine, thiabendazole, verapamil, zileuton or oral 
contraceptives  
21. Consumption of alcohol in the 24 hours prior to enrollment 
22. Smoking cigarettes in the 8 hours prior to enrollment.  
6.3 
Prohibited During Study Period 
There are no specific contraindications in this protocol.  However, consumption of drugs that may 
interfere with (competes or induces) the metabolism of 13C-Methacetin will be recorded on the 
case report forms and include the following: 
a. HMG-CoA reductase (Statin) drugs in the 30 days prior to study enrollment;  
b. Acyclovir and famotidine taken in the 48 hours prior to study enrollment; and 
c. Any of the following taken during the study period: allopurinol, carbamazepine, cimetidine, 
ciprofloxacin, daidzein, disulfiram, Echinacea, enoxacin, fluvoxamine, methoxsalen, 
mexilitene, montelukast, norfloxacin, phenylpropanolamine, phenytoin, propafenone, 
rifampin, terbinafine, ticlodipine, thiabendazole, verapamil, zileuton or oral contraceptives. 
7 
SUBJECT ENROLLMENT 
7.1 
Eligibility Assessment 
All patients with ALI not related to acetaminophen overdose and patients with ALF enrolled into 
the ALFSG Registry should be screened for eligibility for entry into this trial. As in all trials, the 
goal is to achieve a high level of compliance with protocol requirements by assuring during the 
eligibility assessment that the potential subject or legally authorized representative (LAR) is fully 
informed and agrees to the protocol requirements. Careful assessment of the patient’s or LAR’s 
understanding of the trial is required prior to enrollment. 
7.2 
Presentation of Informed Consent 
Patients who meet criteria for ALI are, by definition, not encephalopathic and will be able to 
consent for enrollment without additional consents being obtained from the LAR or family 
member, although both consents may be obtained.  During the initial consent process, ALI patients 
should indicate consent to continue participation into the ALFSG-MBT study should the disease 
progress to ALF.   


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In some cases, patients with ALF may have already returned to their baseline mental status and 
will be able to provide consent for themselves.  In all other cases, the LAR of patients with ALF 
will sign a consent/assent form prior to study participation. Due to the critical state of many 
patients with ALF and the fact that patients are not always escorted by a relative or other 
representative, if local IRB will allow, a documented telephone consent with a patient’s LAR may 
suffice to provide consent. 
Informed consent will be obtained by either the site Principal Investigator or by individuals 
approved by the site Principal Investigator and whose names have been submitted to the ALFSG-
MBT regulatory database.  The consent should be the current IRB-approved version corresponding 
to the version of the protocol approved when the screening was initiated. All elements listed in the 
International Council on Harmonization Good Clinical Practice guidelines (ICH/GCP), to the 
extent that it is compatible with the US FDA and DHHS regulations, must be included in the 
informed consent.   
In accordance with US FDA regulations (21 CFR 50) and guidelines (Federal Register, May 9, 
1997, Vol. 62, Number 90 - ICH Good Clinical Practice Consolidated Guideline) it is the 
investigator’s responsibility to ensure that informed consent is obtained from the patient or 
patient’s LAR before participating in an investigational study, after an adequate explanation of the 
purpose, methods, risks, potential benefits and subject responsibilities of the study. Procedures that 
are to be performed as part of the practice of medicine and which would be done whether or not 
study entry was contemplated, such as for diagnosis or treatment of a disease or medical condition, 
may be performed and the results subsequently used for determining study eligibility without first 
obtaining consent.  
On the other hand, informed consent must be obtained prior to initiation of any screening 
procedures that are performed solely for the purpose of determining eligibility for research.  For 
the ALFSG-MBT study, there is no activity required in the screening process that is not typically 
included within the reasonable scope of standard care evaluation for patients with acute liver injury 
and acute liver failure; patients would be approached for consent only after the clinical screening 
process had established eligibility. 
Informed consent will be obtained from the patient or patient’s LAR after the details of the protocol 
have been reviewed. The individual responsible for obtaining consent will assure, prior to signing 


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of the informed consent, that the patient has had all questions regarding study procedures and the 
protocol answered. During the initial consent process, ALI patients should indicate consent to 
continue participation into the ALFSG-MBT study should the disease progress to ALF.   
Each subject/LAR must be given a copy of the informed consent. The original signed consent must 
be retained in the institution’s records and is subject to review by the Sponsor, SDCC/MUSC, the 
FDA or representative from another agency that performs the same function, and the IRB 
responsible for the conduct of the institution.  
8 
STUDY PROCEDURES 
8.1 
Preamble 
The study will be conducted in compliance with this protocol, with GCP standards, and applicable 
regulatory requirements. Only trained personnel on the delegation of authority log (doctors, nurses, 
study coordinators) will perform the breath test procedure.  The 13C-Methacetin solution should 
be administered by a medical practitioner registered on the delegation log or a research coordinator 
with specific training in test substrate administration.  The site will contact the subject to determine 
Day 21 spontaneous survival, transplantation and occurrence of serious adverse events since the 
subject’s last study procedure. 
All investigators will be blinded to the MBT score results until the end of the study. The MBT 
results will therefore not affect the current patient management. In order to maintain the blind, the 
site Study Coordinator will perform the MBT and transfer the data to the DCU directly, without 
review by the site-PI.  
8.2 
Administration of the Breath Test 
The Breath Test will be performed up to five times during the study period on all enrolled subjects. 
The first Breath Test will be performed as close to the time of study enrollment as possible upon 
admission into the study (Day 1).  The Breath Test will be repeated on Days 2, 3, 5 and 7 as close 
as possible to the same time of day as the first Breath Test.  If a subject who is enrolled into the 
ALFSG-MBT Trial with ALI converts to ALF, breath test collection will continue until a 
maximum of five Breath Tests have been performed. Once the ALI subject has been enrolled into 
the ALFSG Registry with ALF, subsequent Breath Tests should be performed as close to the same 
time of day as the first Breath Test.  If an enrolled ALI or ALF subject receives a liver transplant, 


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is discharged/transferred from the hospital or dies prior to Day 7, no additional Breath Tests will 
be performed. 
The following characteristics will be collected on each day of the MBT before administration of 
test substrate:  
 The mechanism of ventilation (spontaneous or mechanical); 
 Route of administration of test substrate (by mouth or by naso-enteric or orogastric tube); 
 Location of the distal end of the naso-enteric tube (intragastric or intra-duodenal) or 
orogastric tube; 
 Concurrent use of vasopressors, acyclovir, famotidine and statins; 
 Last meal or tube feeding; 
 Subject’s weight; 
 Concurrently administered medications, including NAC; and 
 Use of renal replacement therapy during the MBT.   
8.2.1 Requirements for Administration of Test Substrate 
1. Fasting 
Subjects will perform the breath test once enrolled into the trial and when the following criteria 
have been met: 
 Protocol-specified labs are completed prior to the administration of the test substrate. 
 Oral administration of test substrate:  The subject should be fasting from solid food for a 
minimum 6 hours and from oral medications for a minimum of 1 hour. 
 Naso-enteric and orogastric tube administration of test substrate. In subjects who are 
receiving tube feeding, feeding should be discontinued for a minimum of 4 hours. Gastric 
contents should be aspirated prior to the instillation of 13C-Methacetin solution via the 
naso-enteric or orogastric tube. 
Feeding may be resumed after the MBT has been completed (i.e., at 60 minutes after 
administration of the test substrate). 
2. Subject has not ingested acetaminophen-related medications (e.g. Tylenol) within the past 24 
hours (subjects with acetaminophen intoxication may be included 24 hours after ingestion). 
3. Subject has not received general anesthesia in the last 24 hours. This does not include 
appropriate utilization of sedative and analgesic drugs for subjects within a critical care 


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environment, such as propofol sedation or sedation related to liver biopsy. In those instances, 
all enterally and parenterally administered medications will be recorded in the study CRF. 
4. Evidence of ileus or presenting with contra-indication to administration of oral drugs. 
5. The MBT should not be performed in cases where there is a suspicion that aspiration may 
occur. 
6. Subjects who require RRT (intermittent and continuous) after enrollment into the study will 
not be excluded; however, it is preferred that the test substrate and Breath Test are not 
administered during dialysis treatments. 
Note: Subjects with gastroparesis may be included as the delayed emptying has negligible impact 
on fluids (17). The presence of evidence of delayed gastric emptying and/or ileus should 
be noted on the CRF for further analysis to show that the impact is negligible for MBT 
measures. Naso-enteric and orogastric tubes should be clamped for 10 minutes after 
administration of the 13C-Methacetin solution. 
8.2.2 Performance of the Breath Collection 
The breath test collection will be performed after the test substrate is administered to the subject; 
no waiting period is required once the test substrate has been ingested.  The entire contents of the 
test substrate need to be ingested by the subject; if the subject is not able to ingest the entire amount, 
the Breath Test should not be performed.  
Breath is collected automatically via a nasal cannula, or if the subject is intubated, by a ventilator 
hose adaptor at the subject’s bedside for a total of 75 minutes (up to 15 minutes for baseline 
measurement and 60 minutes following ingestion of 13C-Methacetin solution). All test-specific 
equipment will be supplied by Exalenz.   
8.2.3 Early Termination of the Breath Test 
Key personnel will be trained how to terminate the breath test early if a subject is unable to 
complete the full 75 minutes of breath collection. In the following situations, the breath test 
collection will be terminated: 
 The subject vomits. 
 The subject has to be disconnected from the nasal cannula/ collection device for more than 1 
minute due to an urgent procedure. 
 The subject is inadvertently disconnected from the BreathID® MCS device. 


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 The BreathID® MCS device malfunctions. 
 
8.2.4 Interruptions and Confounders of the Breath Test 
Breath Tests should be timed to anticipate interruptions and possible confounders. Recognizing 
that patients with ALI and ALF are critically ill, guidelines if deviations occur can be found in the 
ALFSG-MBT Manual of Procedures.  
Should an interruption in breath collection due to removal of the cannula or its disconnection occur 
during the administration of the MBT and the device cannot compute the delta over baseline 
(DOB) results, the next MBT will not be repeated until 24-hours after the interruption time of the 
previous Breath Test.  If the interruption occurred at the beginning of the Breath Test (during 
baseline breath collection) and before ingestion of the 13C-Methacetin solution, the test can be 
repeated immediately.  If the interruption occurs on Days 3 or 5, then the study team should 
perform the MBT on the next study day as outlined in the Schedule of Assessments.  The MBT is 
considered ‘performed’ and the subject “evaluable” if it has been measuring the subject’s breath 
for a minimum of 30 (and preferably 60) minutes after test substrate has been administered. 
8.3 
Performance of the APAP Adduct Assays and APAP Concentration Assays 
Serum for acetaminophen (APAP) and APAP-adduct levels will be collected with routine morning 
labs on each day from Day 2-7, as well as Day 1 as a baseline value, from the first 20 ALF subjects 
enrolled. Serum samples will be reserved for the sole purpose of determining whether APAP or 
APAP-adducts accumulate during repeated administrations of the 13C-Methacetin solution in 
patients with ALF. The collected samples will be shipped to and analyzed by Dr. Laura James at 
the University of Arkansas.  
8.4 
Clinical and Laboratory Assessments 
Subjects will routinely undergo laboratory tests as part of the ALFSG Registry.  Specific to this 
study, sites will be required to collect lactate, ammonia, phosphate, AST/ALT, creatinine, bilirubin 
and INR on a daily basis.  
 
 


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8.5 
Schedule of Assessments 
 
Screening/ 
Subject 
Enrollment 
Day 1 Day 2 Day 3 Day 5 Day 7 Day 21 
Demographics 
X 
 
 
 
 
 
 
Medical History 
X 
 
 
 
 
 
 
Confirm Eligibility 
Criteria^ 
X 
 
 
 
 
 
 
Prior Medications 
X 
 
 
 
 
 
 
Concomitant Medications  
 
X 
X 
X 
X 
X 
 
Grade of HE 
X 
X 
X 
X 
X 
X 
 
Labs 
(lactate, bicarbonate, INR, 
total bilirubin, phosphate, 
Creatinine, WBC, Platelet 
count, ammonia-arterial or 
venous (if clinically 
indicated) 
 
X 
X 
X 
X 
X 
 
RRT 
(record time/tx prior to 
MBT) 
 
X 
X 
X 
X 
X 
 
MBT 
 
X 
X 
X 
X 
X 
 
Serum for APAP and 
APAP-adduct levels* 
 
X 
X 
X 
X 
X 
 
AE Reporting 
 
X 
X 
X 
X 
X 
(SAEs 
only) 
End of Study 
 
 
 
 
 
 
X 
Collection of data for 
MELD, KCC, and ALFSG-
PI 
X 
X 
X 
X 
X 
X 
 
* Only in the first 20 consecutive ALF subjects enrolled 
^ In women of child-bearing potential; Urine pregnancy test or blood human chorionic 
gonadotropin (hCG) is acceptable. 
 
 


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9 
DISCONTINUATION OF PARTICIPATION 
9.1 
Withdrawal of Consent 
The subject or the subject’s LAR has the right to voluntarily withdraw consent from the study at 
any time for any reason without prejudice to his/her future medical care by the physician or at the 
institution. The Investigator must withdraw any subject from the study if requested. 
For the occasional subject who withdraws consent, the date, time and reason for consent 
withdrawal should be documented. Subject data will be included in the analysis up to the date of 
the consent withdrawal. No further study evaluations should be performed and no additional data 
or biological samples should be collected once consent is withdrawn by the subject or the subject’s 
LAR. 
Below is the procedure to be followed at the time a subject/LAR withdraws consent from the trial: 
(1) 
Check for the development of adverse events. 
(2) 
Complete the End-of-Study form and include an explanation of why the subject is 
withdrawing consent. 
(3) 
Subject or subject’s LAR will be required to document in writing his or her desire 
to withdraw consent. 
9.2 
Withdrawal from Study Procedures 
As participation in the ALFSG-MBT Trial is voluntary, the subject or subject’s LAR may request 
to discontinue study procedures at any time. In addition, the treating physician may stop the study 
procedures if there is a safety concern; reasons that subjects may have procedures discontinued 
include, but are not limited to the following: 
 The subject has recovered or has been discharged from the hospital prior to the Day 7 MBT. 
The subject will be considered to have recovered if hepatic encephalopathy has resolved, 
INR < 1.6 and/or AST < 150 U/L and/or Bilirubin < 50% of its peak value. Alternately, if 
the subject has been discharged from hospital the subject will be considered to have 
recovered.  
 Liver transplantation during the study procedure period (excluding the Day 21 visit) 
 The subject’s naso-enteric/orogastric tube is removed and oral administration of the drug 
preparation is not tolerated. 
 Plasma transfusion 


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Subjects who had an early discontinuation of study procedure period (excluding the Day 21 visit) 
will continue to be followed carefully through the 21-day study period unless consent is withdrawn 
and all follow-up data will be included in the analysis. 
9.3 
 Subject Lost to Follow-Up  
In the event that all possible attempts to locate the subject for the Day 21 follow up interview have 
failed, all efforts made by the site study personnel to contact the subject will be documented and 
included in the subject’s study binder. 
10 
SAFETY AND ADVERSE EVENTS 
10.1 
Safety Sub-study 
The safety of the MBT has been demonstrated in several clinical applications. However, the 
repeated administration of the test substrate, which represents the equivalent of 75 mg of APAP 
per administration, will be closely monitored to ensure that this small dose of APAP will not result 
in additional liver injury (particularly in the presence of NAC). The results of the Retrospective 
Safety Study were examined before enrollment in the prognosis study began, as outlined in Section 
4.2.3 of this protocol.  A second, prospective safety study will be performed and sera collected 
from the first 20 ALF subjects enrolled into the ALF-MBT Trial as described herein, in whom we 
propose to administer MBT on Day 2, 3, 5, and 7 unless the subject is discharged/transferred, 
transplanted or dies. Serum for acetaminophen (APAP) and APAP-adduct levels will be collected 
with routine AM labs on each day from Day 2-7, as well as Day 1 as a baseline value. Serum 
samples will be reserved for the sole purpose of determining APAP or APAP-adducts levels in 
patients with ALF during repeated administrations of the 13C-Methacetin solution and will be 
performed by Dr. James at the University of Arkansas for Medical Sciences, Little Rock, AR.  
10.2 
Adverse Events 
Adverse event reporting and procedures are described in detail in the MOP. 
In the ALFSG-MBT Trial, any medical conditions not present prior to the initial administration of 
the test substrate but that emerge after the initial test substrate is administered are considered AEs.  
Adverse Events encountered from Day 1 through Day 7 of the study period will be recorded, and 
Serious Adverse Events from Day 1 through the Day 21 of the study period will be recorded.  


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Designated study personnel at all investigative sites are responsible for entering any and all non-
serious and serious adverse events, regardless of severity or relationship to the 13C-Methacetin 
solution and device, and including updates to adverse event information (e.g., date of resolution, 
action taken) as needed, into the WebDCUTM database within timelines defined in the MOP and in 
the subject’s source documents. Upon data submission of the SAE eCRF, the system will trigger 
an automatic e-mail notification to the independent Medical Safety Monitor (MSM) stating that an 
SAE has occurred. The MSM will access the information via the password protected web based 
system for review and report safety concerns to the DSMB. 
Adverse events will be defined and severity graded according to the Common Terminology 
Criteria for Adverse Events (CTCAE) found in the MOP. AEs will be submitted online through 
WebDCU™ and coded centrally into standard terminology using AEs will be using the Medical 
Dictionary for Regulatory Activities (MedDRA). Guidelines for report content and structure are 
provided in the MOP. 
10.3 
Data Safety Monitoring Board (DSMB) 
The Data Safety Monitoring Board (DSMB) used for this study will be that designated by NIDDK 
to oversee the ALFSG.  Semi-annual data quality and safety reports will be provided to the DSMB. 
This timeline can be changed by the DSMB if requested. The analysis of all adverse events will 
include incidence tables and summary of AEs and SAEs by severity and relationship to device or 
drug.   
11 
DATA MANAGEMENT 
11.1 
Data Handling 
The raw data from the individual tests on the BreathID® MCS device will be analyzed using 
Exalenz’s trending software. This program provided by Exalenz will combine all tests for any 
given subject bearing the same identification number and will plot the trend of the breath test 
results with the percent change displayed. This analysis will be carried out only after the subject 
has completed his/her participation in the study and in order not to bias the treating physician, the 
results will not be shared until the study is completed. 
For all other study data, sites will enter case report form data into the password protected study 
database housed in WebDCU™. The WebDCU™ is a validated web-based clinical trials 


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management system that provides the infrastructure for real-time data capture and data sharing and 
includes designated web servers and supporting database servers. This user-friendly web-based 
database system will be used for subject enrollment, data entry, data validation, project progress 
monitoring, user customizable report generation, and secure data transfer. The web-based data 
capturing system allows for study data to be directly entered into the study specific database by 
the site via a secure internet connection. Secure Socket Layer (SSL) is used for data encryption.  
The web system combines all study tools into one system which includes study database, online 
training and help desk, subject calendar, electronic data clarification request (DCR) process, data 
entry, case report form (CRF) and participate tracking system, audit trail, and report generation 
mechanisms. This is the system used for the currently ongoing ALFSG Registry. Clinical and 
laboratory data will be collected by the site in order to derive MELD, ALFSG-PI, ALI-PI and KCC 
scores on a daily basis. These clinical data and laboratories are mandatory as they will be needed 
for calculation of the clinical outcome predictor indices. 
12 
STATISTICAL CONSIDERATIONS 
12.1 
Sample Size 
The relationship between the Day 1 PDRPeak and clinical outcome (21-day spontaneous survival) 
will be assessed using logistic regression with a two-sided alpha of 0.10. Based on previous 
enrollment rates from the ALFSG Registry, we estimate an accrual rate of approximately 100 ALF 
or non-APAP ALI patients per year into the ALFSG-MBT Trial.  The power of the logistic model 
predicting ‘good’ outcome (21 Day SS) is based on the proportion of ‘good’ outcome at the mean 
MBT value.  Assuming this proportion is 0.50, we determined that approximately 200 enrolled 
non-APAP ALI and ALF patients are needed in order to ensure sufficient power (80%) to detect 
odds ratios of 1.5 or higher. 
12.2 
Statistical Analyses 
12.2.1 Futility Analysis 
A futility analysis will be conducted after 100 consecutive enrollments with 21-day outcome 
obtained.  This analysis is based on the error spending function method with O'Brien and Fleming 
(OBF) type stopping guidelines.  The goal of this analysis is to consider stopping the study early 
if there is no signal of a difference in the PDRPeak distributions between those that SS at 21days 


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and those that do not SS.  If the p-value of a two-sided t-test for the difference in means at an alpha 
level of 0.10 is 0.807 or greater, then the study may be considered stopped for futility. 
12.2.2 Final Analyses 
Preliminary analyses will be conducted to describe the collected variables. Univariate descriptive 
statistics and frequency distributions will be calculated, as appropriate for all collected variables 
along with two-sided 95% confidence intervals.  The primary endpoint, Day 1 PDRPeak will be 
compared between those that spontaneously survive (SS) by Day 21 and those that either die or 
receive a transplant by Day 21 with a logistic regression model at a two-sided alpha level of 0.10.  
All enrolled patients with a value at Day 1 will be included in the analysis.  The primary analysis 
will not stratify by non-APAP ALI/ALF but exploratory analyses will be conducted that examine 
potential differences as described below.  It is not anticipated that any enrolled patient will be 
missing the outcome of SS.   
There are several exploratory aims that will be conducted for future hypothesis generation. In 
addition to the comparison of mean PDRPeak values between the two cohorts, a cutpoint for the 
PDRPeak value will be examined using a receiver operating curve. The identified cutpoint will be 
compared to other known prognostic indices including Kings College criteria (KCC), Model of 
End-Stage Liver Disease (MELD), and the ALFSG-PI (prognostic index).  As a secondary analysis 
and where serial measurements are available, the trends in the MBT results (PDRPeak, PDR20, 
CPDR20) will be related to outcome (Day 21 SS) using repeated measures analysis.  A 
multivariable regression will be performed to assess if PDRPeak, in combination with traditional 
prognostic measures provides an accurate prediction of Day 21 outcome. These pre-specified 
exploratory analyses will be conducted using a significance level of 0.05. 
With the inclusion of non-APAP ALI patients, we will conduct exploratory subgroup analyses and 
examine the association with MBT results for each subgroup (non-APAP ALI and ALF).  Based 
on current registry enrolments, we anticipate the study population to consist of 17-20% non-APAP 
ALI enrollments.  A total of 35-40 non-APAP ALIs will not provide definitive evidence but will 
provide further hypothesis generating information.  For the non-APAP ALI subgroup, we will also 
examine the PDR peak when poor outcome is defined as progression to ALF, transplant or death 
within 21days.  Point estimates and confidence intervals will be used for interpretation of these 
analyses more than pvalues due to the exploratory nature of the analyses. We anticipate roughly 


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10% will convert to ALF.  All statistical analyses will be conducted using SAS V9.2 or higher 
(SAS Institute, Inc., Cary, NC).  The specified analyses will be repeated as an exploratory aim 
with the Day 7 outcome of SS.   
13 
OBLIGATIONS OF THE INVESTIGATOR 
13.1 
Compliance with Good Clinical Practice (GCP) 
The investigator is responsible for ensuring that the clinical study is performed in accordance with 
the protocol, current ICH guidelines on GCP, and applicable regulatory requirements. Compliance 
with GCP provides public assurance that the rights, safety, and well-being of study subjects are 
protected, consistent with the principles that originated in the Declaration of Helsinki and that the 
clinical study data are credible. 
13.2 
Access to Source Data and Documents 
The PI and designees agree to maintain accurate CRFs and source documentation as part of the 
case histories.  Source documents are the originals of any documents used by the PI or Sub-
Investigator or hospital/institution that allow verification of the existence of the patient and 
substantiate the integrity of the data collected during the trial. The study CRFs will be available in 
printable format on the study website. The CRFs for each patient must be completed only by 
persons designated by the PI and who have data entry permissions for the study database.  All data 
entered into the CRF must also be available in the source documents. Once a subject completes 
the study, the PI must review the completed study book and sign the End of Study Form that 
acknowledges this review. 
The Investigator will permit study-related monitoring, audits, EC review, and regulatory 
inspections (where appropriate) by providing direct access to source data and other documents (i.e. 
patients’ case sheets, blood test reports, histology reports etc.). 
13.3 
Retention of Documentation 
In June 2005, Federal law extended the statute of limitations to six (6) years to bring forward an 
allegation of research misconduct. In response to this extension, research records must be retained 
for a sufficient period to investigate an allegation of research misconduct--a minimum period of 
six (6) years. 


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Additionally, existing Federal regulations [56 CFR 56.115(b)] require that IRB records be retained 
for at least 3 years after completion of the research. All records must be accessible for inspection 
and copying by authorized representatives of DHHS and FDA at reasonable times and in a 
reasonable manner. At the end of three years, the IRB records may be boxed, labeled and sent to 
central storage for an additional 3-10 years. A log of stored records is maintained in the IRB office 
for retrieval if files are needed for audit or other purposes. 
An agreement must be in place between the clinical site Principal Investigator and the Principal 
Investigator/Sponsor regarding records that may be destroyed. Records will be maintained in a de-
identified manner in a locked location to ensure confidentiality. 
13.4 
Confidentiality of Material Provided by Exalenz Bioscience Ltd. 
Any written materials provided by Exalenz Bioscience Ltd., as well as documentation, data, and 
all other information generated as part of this study will be held in strict confidence by the 
Investigator and participating clinical site staff.  Conduct of this study will comply with all 
provisions of the Health Insurance Portability and Accountability Act of 1996. 
13.5 
Publication 
Publication of the results of this trial will be governed by the policies and procedures developed 
by the ALFSG Executive Committee. All manuscripts pertaining to this study will be forwarded 
to Exalenz Bioscience Ltd. and the NIDDK staff for review before submission for publication. 
The Publication Policy will be fully compliant with the voluntary NIH Public Access Policy 
mandated by the Consolidated Appropriations Act of 2008 (Division G, Title II, Section 218 of 
PL 110-161). The Executive Committee will follow NIH policies on data-sharing (as described 
at 
the 
site: 
http://grants2.nih.gov/grants/policy/data_sharing/data_ 
sharing_guidance.htm and any updates thereto). 
 
 


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14 
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12. Lee WM, James L, Wendon J, Stravitz RT, Pop OT, Audimoolam VK. Does Methacetin 
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