Clinical Study of the BreathID® LF System to Train the Algorithm for the ¹³C-Methacetin Breath Test (MBT) in Assessment of Portal Hypertension in Patients With Compensated Liver Cirrhosis
Phase: NA
Conditions: Patients With Compensated Liver Cirrhosis
Sponsor: Meridian Bioscience, Inc.

 
 
 
Clinical Study of the BreathID® MCS System to train the 
algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No. CSPH-EX-0414 
 
 
NCT#: NCT02143778 
 
 
 

Exalenz Bioscience Ltd. 
Last update on: Nov 21, 2016 
Study Title: Clinical Study of the BreathID® MCS System to 
train the algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
Version: 1.5 
Document No: CSD00076 
Page 2 of 72 
 
Exalenz Bioscience Ltd.     
 
CONFIDENTIAL AND PROPRIETARY 
 
Clinical Study of the BreathID® MCS System to train the 
algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No. CSPH-EX-0414 
 
Document No: CSD00076 
Total pages: 72 
Version Level: 1.5 
Last update on: Nov 21, 2016 
 
 
Name 
Position 
Author / 
Owner 
Avraham Hershkowitz 
Clinical Trial Manager 
Supervisor / 
Revised by 
Gil Guggenheim 
Clinical Application Development 
Specialist and Database Manager 
Approved by 
Melina Arazy 
Director of Clinical Affairs 
Electronic signatures and approvals, effective information and Lifecycle Status can be 
obtained by accessing the system generated approval page associated with this document.  
The system generated approval page can be retrieved from the PDM system. 
This publication, in whole or in part, may not be reproduced, disclosed, translated into any 
language or computer language or distributed without the prior written consent of: 
Exalenz Bioscience Ltd. 4 Ha’Maayan St. Modiin, Israel 7177872 
Versions Control: 
Version 
Date 
Responsible Person 
Description of Change 
01 
02-JUN-14 
Avraham Hershkowitz 
Initial Release 
1.1 
10-SEP-14 
Avraham Hershkowitz 
Correction of 3 typos 
1.2 
27-OCT-14 
Avraham Hershkowitz 
Addition of US Investigator 
1.3 
11-NOV-14 
Avraham Hershkowitz 
Amendments 
based 
on 
FDA  
feedback 
1.4 
13-NOV-14 
Gil Guggenheim 
Additional amendments based on 
FDA feedback from 12-NOV-2014 
1.5 
21-NOV-16 
Avraham Hershkowitz 
Amendment of eligibility criteria: 
statins included, HCV treatment 

Exalenz Bioscience Ltd. 
Last update on: Nov 21, 2016 
Study Title: Clinical Study of the BreathID® MCS System to 
train the algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
Version: 1.5 
Document No: CSD00076 
Page 3 of 72 
 
Exalenz Bioscience Ltd.     
 
CONFIDENTIAL AND PROPRIETARY 
excluded 
1 TABLE OF CONTENTS 
 
1 Table of Contents ................................................................................................................. 2 
2 Abbreviations ....................................................................................................................... 6 
3 Protocol Synopsis ................................................................................................................. 9 
4 Visit Schedule Overview .................................................................................................... 15 
5 Background ......................................................................................................................... 16 
5.1 
Portal Hypertension and Hepatic Decompensation ...................................................................... 16 
5.2 
Hepatic Venous Pressure Gradient (HVPG) assessing Portal Hypertension ................................ 17 
5.3 
HVPG ≥10 mmHg defines Clinically Significant Portal Hypertension........................................ 20 
5.4 
Unmet need for a noninvasive tool for assessment of CSPH ....................................................... 21 
5.5 
13C-Breath Tests ............................................................................................................................ 22 
5.5.1 
13C-Methacetin Breath Tests - MBT .............................................................................................. 22 
5.6 
Rationale to use the MBT for assessment of portal hypertension ................................................. 23 
6 Intended Use / Indication for Use ..................................................................................... 28 
7 Proposed Clinical Algorithm using the MBT .................................................................. 28 
8 MBT Short Description ..................................................................................................... 29 
8.1 
MBT Overview ............................................................................................................................. 29 
8.2 
13C-labelled Substrate: 13C-Methacetin ......................................................................................... 29 
8.2.1 
13C-Methacetin Safety .................................................................................................................... 30 
8.3 
MBT Components......................................................................................................................... 31 
9 Study Design ....................................................................................................................... 32 

Exalenz Bioscience Ltd. 
Last update on: Nov 21, 2016 
Study Title: Clinical Study of the BreathID® MCS System to 
train the algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
Version: 1.5 
Document No: CSD00076 
Page 4 of 72 
 
Exalenz Bioscience Ltd.     
 
CONFIDENTIAL AND PROPRIETARY 
10 Study Objectives ................................................................................................................. 32 
10.1 Efficacy Objective ........................................................................................................................ 32 
10.2 Safety Objective ............................................................................................................................ 32 
10.3 Secondary Objectives ................................................................................................................... 32 
10.4 Efficacy Endpoints ........................................................................................................................ 33 
10.4.1 
Primary efficacy endpoint .............................................................................................................. 33 
10.4.2 
Secondary efficacy endpoint .......................................................................................................... 33 
10.5 Safety Endpoints ........................................................................................................................... 33 
11 Subject Selection ................................................................................................................ 33 
11.1 Inclusion Criteria .......................................................................................................................... 34 
11.2 Exclusion Criteria: ........................................................................................................................ 34 
11.3 On the day of the MBT: ................................................................................................................ 35 
11.4 Consenting .................................................................................................................................... 36 
11.5 Safety Termination of Subjects or Study ...................................................................................... 36 
11.6 Early Withdrawal from the Study ................................................................................................. 36 
11.7 Expected Duration of Recruitment ............................................................................................... 36 
12 Statistical Considerations .................................................................................................. 37 
12.1 Study Design and Objectives ........................................................................................................ 37 
12.2 Primary endpoints ......................................................................................................................... 37 
12.3 Secondary efficacy endpoint ......................................................................................................... 37 
12.4 Safety Endpoints ........................................................................................................................... 37 
12.5 Sample Size estimation ................................................................................................................. 37 
12.6 Analysis sets ................................................................................................................................. 38 
12.7 Statistical Analysis ........................................................................................................................ 38 

Exalenz Bioscience Ltd. 
Last update on: Nov 21, 2016 
Study Title: Clinical Study of the BreathID® MCS System to 
train the algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
Version: 1.5 
Document No: CSD00076 
Page 5 of 72 
 
Exalenz Bioscience Ltd.     
 
CONFIDENTIAL AND PROPRIETARY 
12.7.1 
General Considerations .................................................................................................................. 38 
12.7.2 
Demographic and Other Baseline Characteristics .......................................................................... 38 
12.7.3 
Disposition of Subjects .................................................................................................................. 38 
12.7.4 
Safety Analysis .............................................................................................................................. 39 
12.7.5 
Performance Analysis .................................................................................................................... 39 
12.7.6 
Interim Analysis ............................................................................................................................. 39 
12.7.7 
Pooling ........................................................................................................................................... 39 
12.7.8 
Handling of Missing Data .............................................................................................................. 39 
13 Study Procedures ............................................................................................................... 40 
13.1 General .......................................................................................................................................... 40 
13.2 Masking procedures ...................................................................................................................... 42 
13.3 Breath Test Procedure ................................................................................................................... 42 
13.4 Principles of the Hepatic Venous Pressure Gradient (HVPG) ...................................................... 42 
13.4.1 
Laboratory Tests ............................................................................................................................ 43 
13.5 Investigational Product Handling ................................................................................................. 44 
13.6 Investigational Product Accountability ......................................................................................... 44 
14 Ethics & Regulatory Considerations ................................................................................ 45 
14.1 Ethics & Regulatory Approvals .................................................................................................... 45 
15 Investigator Responsibilities ............................................................................................. 45 
16 Safety Considerations ........................................................................................................ 46 
16.1 Adverse Event Definitions ............................................................................................................ 46 
16.2 Serious Adverse Event (SAE), Unanticipated Adverse Device Effect (UADE) and Suspected 
Unexpected Serious Adverse Reactions (SUSAR) ....................................................................... 46 
16.3 Relationship to Study Medical Product ........................................................................................ 48 
16.4 Adverse Event Severity ................................................................................................................ 48 

Exalenz Bioscience Ltd. 
Last update on: Nov 21, 2016 
Study Title: Clinical Study of the BreathID® MCS System to 
train the algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
Version: 1.5 
Document No: CSD00076 
Page 6 of 72 
 
Exalenz Bioscience Ltd.     
 
CONFIDENTIAL AND PROPRIETARY 
16.5 Adverse Event Reporting .............................................................................................................. 48 
16.6 Study-Specific Exceptions ............................................................................................................ 49 
17 Access to Source Data and Documents............................................................................. 50 
17.1 Availability of Source Data and Documents ................................................................................ 50 
17.2 Patient Confidentiality .................................................................................................................. 50 
18 Monitoring and Quality Assurance .................................................................................. 50 
18.1 Data Collection and Monitoring ................................................................................................... 50 
19 Publication Policy and Finance ......................................................................................... 50 
20 Financial Aspects ................................................................................................................ 51 
21 Study termination .............................................................................................................. 51 
22 References ........................................................................................................................... 52 
Appendix I - Protocol Approval Signature Page ................................................................. 59 
Appendix II – Detailed HVPG procedure ............................................................................ 60 
Appendix III – Detailed MBT procedure ............................................................................. 70 
 
 
 

Exalenz Bioscience Ltd. 
Last update on: Nov 21, 2016 
Study Title: Clinical Study of the BreathID® MCS System to 
train the algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
Version: 1.5 
Document No: CSD00076 
Page 7 of 72 
 
Exalenz Bioscience Ltd.     
 
CONFIDENTIAL AND PROPRIETARY 
2 ABBREVIATIONS 
 
AASLD 
- American Association for the Study of Liver Diseases 
AE 
- Adverse Event 
ALF 
- Acute Liver Failure 
ALT 
- Alanine aminotransferase 
APAP 
- n-acetyl-p-aminophenol (acetaminophen) 
aPTT 
- activated Partial Thromboplastin Time 
AST 
- Aspartate aminotransferase 
AUC 
- Area Under the Curve 
BUN 
- Blood Urea Nitrogen 
CFR 
- Code of Federal Regulation 
CO2 
- Carbon dioxide 
CPDR 
- Cumulative Percentage Dose Recovery (expressed as %) 
CRA 
- Clinical Research Associate 
CRF 
- Case Report Form 
CRO 
- Clinical Research Organization 
CSPH 
- Clinically Significant Portal Hypertension 
CT 
- Computed Tomography 
CTCAE 
- Common Terminology Criteria for Adverse Events 
CVP 
- Central Venous Pressure 
DOB 
- Delta over baseline 
EASL 
- European Association for the Study of the Liver 
EC 
- Ethics Committee 
eCRF 
- Electronic Case Report Form 
EDC 
- Electronic Data Capture 
FDA 
- Food and Drug Administration 

Exalenz Bioscience Ltd. 
Last update on: Nov 21, 2016 
Study Title: Clinical Study of the BreathID® MCS System to 
train the algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
Version: 1.5 
Document No: CSD00076 
Page 8 of 72 
 
Exalenz Bioscience Ltd.     
 
CONFIDENTIAL AND PROPRIETARY 
FHVP 
- Free Hepatic Venous Pressure 
GCP 
- Good Clinical Practice 
GI 
- Gastrointestinal 
GTTP 
- Gamma-Glutamyl Transpeptidase 
HCC 
- Hepatocellular Carcinoma 
HCO3 
- Bicarbonate 
HE 
- Hepatic Encephalopathy 
HVPG 
- Hepatic Venous Pressure Gradient 
ICH 
-  International Conference of Harmonization 
IDE 
- Investigational Device Exemption 
INR 
- International Normalized Ratio (for prothrombin time) 
IRB 
- Institutional Review Board 
KOL 
- Key Opinion Leaders 
K+ 
- Potassium 
LF 
- Liver Function 
MBT 
- 
13C-Methacetin Breath Test 
MCS 
- Molecular Correlation Spectrometry  
MELD 
- Model for End-stage Liver Disease 
Na+ 
- Sodium 
NIH 
- National Institute of Health 
NPV 
- Negative Predictive Value 
NSBB 
- Non-Selective Beta Blocker 
OUS 
- Out of the United States 
O2 
- Oxygen 
PAP 
- Pulmonary Artery Pressure 
PEEP 
- Positive End Expiratory Pressure 
PHT 
- Portal Hypertension 

Exalenz Bioscience Ltd. 
Last update on: Nov 21, 2016 
Study Title: Clinical Study of the BreathID® MCS System to 
train the algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
Version: 1.5 
Document No: CSD00076 
Page 9 of 72 
 
Exalenz Bioscience Ltd.     
 
CONFIDENTIAL AND PROPRIETARY 
PPV 
- Positive Predictive Value 
PDR 
- Percent Dose Recovered (expressed as %  per hour) 
ROC 
- Receiver Operating Characteristic 
SAE 
- Serious Adverse Event 
SUSAR 
- Suspected Unexpected Serious Adverse Reactions 
TIPS 
- Transjugular Intrahepatic Portosystemic Shunt 
US 
- United States (of America) 
USA 
- United States of America 
WBC 
- White Blood Cells 
WHVP  
- Wedged Hepatic Venous Pressure 
 
 
 

Exalenz Bioscience Ltd. 
Last update on: Nov 21, 2016 
Study Title: Clinical Study of the BreathID® MCS System to 
train the algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
Version: 1.5 
Document No: CSD00076 
Page 10 of 72 
 
Exalenz Bioscience Ltd.     
 
CONFIDENTIAL AND PROPRIETARY 
3 PROTOCOL SYNOPSIS 
 
Protocol Title: 
Clinical Study of the BreathID® MCS System to train the algorithm 
for the ¹³C-Methacetin Breath Test (MBT) in assessment of Portal 
Hypertension in Patients with Compensated Liver Cirrhosis 
Protocol Reference Number: 
CSPH-EX-0414  
Version and Date: 
v 1.5, 21 Nov, 2016 
 
Phase of Development:  
Phase II (training) 
Sponsor: 
Exalenz Bioscience Ltd. 
4 Hama'ayan Street 
Modi'in, Israel 71700 
Tel: +972-8-9737500 
Fax: +972-8-9737501 
 
Principle Investigators: 
a. Dr. Juan Carlos García Pagán, Barcelona, Spain 
b. Dr. Agustín Albillos, Madrid, Spain 
c. Dr. Christophe Bureau, Toulouse, France 
d. Dr. Didier Lebrec, Dr. Pierre Ratou, Paris (Clichy), France 
e. Dr. Guadalupe Garcia-Tsao, New Haven , CT , USA 
f. Dr. Velimir Luketic, Richmond, VA, USA 
g. Dr. Annalisa Berzigotti, Bern, Switzerland 
h. Dr. Manal Abdelmalek, Durham, NC, USA 
 
Institutions: 
a. Hospital Clinic, , Barcelona, Spain. 
 
b. Hospital Santiago Ramón y Cajal, Madrid, Spain 
 
c. Hopital Purpan, Toulouse, France 
 
d. Hopital Beaujon, Paris (Clichy), France 
 
e. Yale University School of Medicine, New Haven, CT / VA 
Connecticut Health Care   System, USA 
 
f. Virginia Commonwealth University Medical Hospital, VA, USA 
 
g.  Inselspital University Hospital, Bern, Switzerland 
 
h. Duke University, Durham, NC, USA 

Exalenz Bioscience Ltd. 
Last update on: Nov 21, 2016 
Study Title: Clinical Study of the BreathID® MCS System to 
train the algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
Version: 1.5 
Document No: CSD00076 
Page 11 of 72 
 
Exalenz Bioscience Ltd.     
 
CONFIDENTIAL AND PROPRIETARY 
 
Investigated disease: 
Clinically Significant Portal Hypertension (CSPH) in patients with 
compensated liver cirrhosis 
 
Combination Product  
BreathID® MCS system together with ¹³C-labeled Methacetin  
 
Comparator:   
 
HVPG - Hepatic Vein Portal Pressure Gradient 
 
Primary Objective: 
The primary objective of the study is to develop an algorithm and its 
cut-off to detect CSPH, defined as HVPG ≥10mmHg, based on the 
MBT. 
Primary Efficacy  
Endpoint: 
Binary diagnosis of CSPH, defined as HVPG ≥10 mmHg, correlated 
with an MBT result to be developed.  
Secondary: 
1. Binary diagnosis of portal hypertension with HVPG ≥ 12mmHg 
2. Binary diagnosis of portal hypertension with HVPG ≥ 20mmHg 
3. Correlation of MBT with HVPG readings 
4. Assess the effect of beta blockers or other confounding factors on 
the Correlation of MBT with HVPG readings 
5. Examine the effect of a single small (<3cm) HCC (hepatocellular 
carcinoma) on the correlation of MBT with HVPG tracings 
Safety Endpoints: 
Safety will be assessed by all adverse and serious adverse events 
occurring in all subjects enrolled throughout the duration of the study.  
Study Design:  
A multicenter, non-randomized, blinded, cross-sectional comparative 
exploratory study comparing various MBT measures to HVPG. 
This study will be used to train an algorithm using MBT measures and 
to select a cut-off to determine presence or absence of CSPH as defined 
by HVPG ≥ 10mmHg. 

Exalenz Bioscience Ltd. 
Last update on: Nov 21, 2016 
Study Title: Clinical Study of the BreathID® MCS System to 
train the algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
Version: 1.5 
Document No: CSD00076 
Page 12 of 72 
 
Exalenz Bioscience Ltd.     
 
CONFIDENTIAL AND PROPRIETARY 
Study Populationa:  
At least 200 consecutive compensated cirrhotic patients, defined as 
cirrhotic 
patients 
without 
complicationsb 
and 
meeting 
all 
inclusion/exclusion criteria will be enrolled on a walk-in basis. If beta 
blockers or HCC or other confounding factors affect the correlation 
between MBT and HVPG the sample size may be enlarged following 
interim analysis. 
Inclusion Criteria:  
 
1. 
Adult men or women (>18 years of age) 
2. 
Patient (or legal guardian) can sign an Informed Consent Form 
3. 
Known chronic liver disease with cirrhosis confirmed by either: 
a. liver biopsy or 
b. clinical (palpable left lobe, splenomegaly) and laboratory 
(platelets <150,000/mm3 or albumin< 3.8g/dL, or INR >1.3) 
evidence of cirrhosis and/or 
c. imaging studies by abdominal sonography, computer assisted 
axial tomography, or magnetic resonance imaging, showing a 
nodular liver and/or enlarged spleen and/or portosystemic 
collaterals with portal vein patency and/or minimal ascites, 
and/or colloid shift on a colloid-isotope liver-spleen scan or 
measurements of liver stiffness suggestive of cirrhosis 
 
4. 
Europe: Indicated to undergo HVPG testing 
US: Consented for HVPG  
5. 
Can tolerate an overnight (8-hour) fast  
6. 
For patients treated with statins: They have to be on a stable dose for 
at least 4 weeks prior to any study related tasks (MBT or HVPG 
measurement) 
7. 
For patients treated with beta blockers: They have to be on a stable 
dose for at least 6 weeks prior to any study related tasks (MBT or 
HVPG measurement) 
                                                     
a Most patients to be enrolled in the proposed study will be from centers in Europe. Based on expert opinion in the 
field of hepatology, the population to be enrolled from Spain and France is representative of patients with chronic 
liver disease in the United States. The management of patients in the United States is based upon and derived from 
the experience and guidelines developed in Barcelona (Spain). Patients in these countries have similar etiologies and 
prognosis and are being used interchangeably in multicenter clinical trials using HVPG testing 
 
b See eligibility criteria. 

Exalenz Bioscience Ltd. 
Last update on: Nov 21, 2016 
Study Title: Clinical Study of the BreathID® MCS System to 
train the algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
Version: 1.5 
Document No: CSD00076 
Page 13 of 72 
 
Exalenz Bioscience Ltd.     
 
CONFIDENTIAL AND PROPRIETARY 
8. 
For patients who stopped their treatment with beta blockers: Their 
last dose should be at least 6 weeks prior to any study related tasks 
(MBT or HVPG measurement) 
Note: In case patient has HCC, breath test has to be performed on 
the day of HVPG measurement. 
 
Exclusion Criteria:  
1. 
Decompensated cirrhosis as clinically defined by the occurrence of 
any of the following: ascites, hepatic encephalopathy, variceal 
bleeding (active upper GI bleeding) or hepatorenal syndrome 
2. 
Renal failure (creatinine > 2.5 mg/dl)  
3. 
Known acute renal tubular disease 
4. 
Known hypotension (Systolic Pressure <100mmHg)  
5. 
Hypocoagulablity defined as PT >6 and INR >2.3. 
6. 
Congestive heart failure (assessed clinically as NIHA >2)  
7. 
Known pulmonary hypertension (right ventricular systolic pressure > 
45 mm Hg)  
8. 
Uncontrolled diabetes mellitus (HBA1C >9.5gr%)  
9. 
Concurrent prednisone or immunosuppressive treatment, if therapy 
and/or response to treatment are not stable for at least 3 months. 
10. 
Documented hepatocellular carcinoma lesion larger than 3cm and/or 
multifocal lesions and/or evidence of vascular invasions 
11. 
Gastric bypass surgery or extensive small bowel resection 
12. 
Total parenteral nutrition 
13. 
Any organ transplant recipient 
14. 
Pregnant or breast feeding 
15. 
Allergy to acetaminophen and/or other related medications. 
16. 
Documented drug-related concurrent hepatotoxicity or drug-related 
silent steatosis or drug-related fibrosis (e.g. amiodarone, methotrexate 
and tamoxifen) 
17. 
Uncontrolled malabsorption or diarrhea  
18. 
Documented non-cirrhotic PHT, partial/complete portal venous 
occlusion, hepatic venous occlusion, previous PHT surgery, or 
placement of a transjugular intrahepatic portosystemic shunt (TIPS) 
19. 
Primary or secondary biliary cirrhosis, primary or secondary 
sclerosing cholangitis, hepatic sarcoidosis, or other cholestatic 
disorders  
20. 
Subjects unable to perform the MBT within 7 days of HVPG 
procedure.  

Exalenz Bioscience Ltd. 
Last update on: Nov 21, 2016 
Study Title: Clinical Study of the BreathID® MCS System to 
train the algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
Version: 1.5 
Document No: CSD00076 
Page 14 of 72 
 
Exalenz Bioscience Ltd.     
 
CONFIDENTIAL AND PROPRIETARY 
21. 
Subject should not have taken any of the following for at least 48 
hours prior to the breath test: Acyclovir , allopurinol, carbamazepine, 
cimetidine, ciprofloxacin, daidzein, (herbal) disulfiram, echinacea, 
enoxacin, 
famotidine, 
fluvoxamine, 
methoxsalen, 
mexiletine, 
montelukast, 
norfloxacin, 
phenylpropanolamine, 
phenytoin, 
propafenone, 
rifampin, 
terbinafine, 
ticlopidine, 
thiabendazole, 
verapamil, zileuton or any medication that might interfere with 
Methacetin metabolism or might affect CYP 1A2 
22. 
Subject should not have taken amiodarone within the last 30 days 
prior to the breath test or HVPG procedure. Splenomegaly 
secondary to inflammation/infection or hematologic malignancy. 
23. 
Last dose of HCV treatment received within the past 3 years 
On the day of the MBT: 
1. 
8 hours fasting prior to MBT including all PO morning medications 
2. 
No Smoking on the day of the MBT 
3. 
No caffeine within 24 hours prior to MBT 
4. 
No paracetamol (acetaminophen) based medications within 24 hours 
prior to MBT 
5. 
No general anesthesia or sedation within 24 hours prior to MBT 
6. 
No alcohol within 24 hours prior to MBT  
7. 
Patient should be symptom free of any prior intervention (e.g. biopsy) 
Listing of Study Procedures:  
1. IC and eligibility  
2. Baseline and Physical Examination 
3. HVPG procedurec 
4. MBT - ¹³C-Methacetin Breath Test (within 7 days of HVPG) 
Note1: The MBT may be performed prior to HVPG as long as it is 
done within 7 days. 
Note2: If patient is using a stable dose of beta blockers both the 
MBT and the HVPG procedures should be performed while 
continuing the stable dose even on the morning of each test. 
Note3: If patient has HCC, breath test has to be performed on the 
day of HVPG measurement. 
 
                                                     
c The measurement will be performed in a uniform way including following a standardized procedure to measure 
HVPG pressures (see Appendix II), either before or after the MBT, with certain limitations detailed in the protocol. 

Exalenz Bioscience Ltd. 
Last update on: Nov 21, 2016 
Study Title: Clinical Study of the BreathID® MCS System to 
train the algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
Version: 1.5 
Document No: CSD00076 
Page 15 of 72 
 
Exalenz Bioscience Ltd.     
 
CONFIDENTIAL AND PROPRIETARY 
Rules of discontinuations (Subject and/or Study): 
1. 
Withdrawal of patients from the study if they have a CTCAE  
(Common Terminology Criteria for Adverse Events) Grade 3 or 
higher regardless of whether the event is attributed to the drug unless 
it is caused by an accident that could not reasonably be attributable to 
the drug (CTCAE: Common Terminology Criteria for Adverse Events 
Version 4.0). 
2. 
Discontinuance of study, if ≥ 2 patients on Methacetin develop the 
same CTCAE Grade 3 event or 1 patient develops a CTCAE Grade 4 
or higher event due to the breath test. 
 
Data Analysis / Statistics: 
A ROC analysis will be used to determine the optimal parameter and cut-
offs for required performance measures sensitivity and specificity. 
 
 
 
All efficacy analysis will include the point estimates of sensitivity, 
specificity, PPV and NPV with respective exact 95% confidence 
intervals. The respective Positive and Negative Predictive Values (PPV 
and NPV) will be presented as a function of possible CSPH prevalence 
and will include the prevalence of the enrolled population. 
 
 

Exalenz Bioscience Ltd. 
Last update on: Nov 21, 2016 
Study Title: Clinical Study of the BreathID® MCS System to 
train the algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
Version: 1.5 
Document No: CSD00076 
Page 16 of 72 
 
Exalenz Bioscience Ltd.     
 
CONFIDENTIAL AND PROPRIETARY 
4 VISIT SCHEDULE OVERVIEW 
Screening (Baseline) 
 
 
 
 
   
 
 
 
Day 1-7 
 
 
 
 
 
 
 
 
 
 
• 
Informed Consent 
• 
Inclusion/exclusion criteria 
• 
Safety urine sample (pregnancy)* 
• 
Medical/disease history 
• 
Concomitant medications 
• 
Eligibility screening for HVPG 
• 
Physical examination and vital signs 
• 
HVPG procedure  
• 
13C-methacetin breath test 
• 
Concomitant medications  
• 
Adverse event reporting 

Exalenz Bioscience Ltd. 
Last update on: Nov 21, 2016 
Study Title: Clinical Study of the BreathID® MCS System to 
train the algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
Version: 1.5 
Document No: CSD00076 
Page 17 of 72 
 
Exalenz Bioscience Ltd.     
 
CONFIDENTIAL AND PROPRIETARY 
5 BACKGROUND 
Exalenz Biosciences Ltd. has developed diagnostic breath test products consisting of 
combinations of a medical device and various 
13C-labeled diagnostic substrates for 
gastrointestinal and liver applications.  
The proposed device is a molecular correlation based spectrometer, using a patented technology 
based on specific light source emissions and absorption of 13CO2 and 12CO2 gases. This 
technology is already implemented in a similar device already approved for marketing in Europe 
and cleared in the USA. The 13C-labeled substrates (in this case 13C-Methatecin) are metabolized 
by the target organ under investigation, producing 13CO2 which in turn leads to changes in 
13CO2/12CO2 ratio in patient's exhaled breath over time. These ratio changes are displayed in real 
time on the device's screen and printed at end of test.  
The rate and pattern of the 13CO2/12CO2 ratio curve reflect substrate metabolism, i.e. target 
organ’s metabolic capacity. 
The aim of the Company in the field of liver diseases is to provide a non-invasive, point-of-care, 
breath test to assess disease severity using 13C-Methacetin.  
5.1 Portal Hypertension and Hepatic Decompensation 
Cirrhosis results from continuous liver injury and repair, and is defined pathologically as hepatic 
regeneration constrained into a disorganized nodular architecture by bridging bands of fibrosis.(1, 
2) Early cirrhosis showing no functional abnormality is said to be “compensated”.(3) However, as 
fibrosis worsens and further distorts liver architecture, the naturally highly compliant liver 
sinusoids exhibit increasing resistance to blood flow. These sinusoidal changes are marked by 
loss of the fenestrae (pores), sub-endothelial collagen deposition and by contractile 
myofibroblasts derived from activated stellate cells(4) as well as by nodules.(1, 5) Progressive 
impairment of liver performance follows.  
Increased intrahepatic vascular resistance raises sinusoidal and subsequently portal venous 
pressure. When the pressure gradient between the portal and systemic venous systems rises,  
patients are said to be afflicted by Portal Hypertension (PHT). The rise in pressure causes the 
opening and enlargement of existing porto-systemic connections and the creation of new porto-
systemic collateral veins (neovascularization). The shunting vasculature is known as varices 
when they line mucosal surfaces of the esophagus, stomach and elsewhere in the gastrointestinal 
tract.(6) An additional shunting effect is present within the liver by the formation of intrahepatic 
shunts which in-fact are bypassing the functional liver. PHT may result in the transudation of 
extracellular fluid from the liver into the peritoneal cavity (ascites), and may also be responsible 

Exalenz Bioscience Ltd. 
Last update on: Nov 21, 2016 
Study Title: Clinical Study of the BreathID® MCS System to 
train the algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
Version: 1.5 
Document No: CSD00076 
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for both the opening and enlargement of existing porto-systemic connections and the creation of 
new porto-systemic collateral veins (neovascularization) that are known as varices when they 
line mucosal surfaces of the esophagus, stomach and elsewhere in the gastrointestinal tract.(6) 
When ascites is present or varices bleed, the cirrhotic patient is said to be “decompensated”, 
which correlates with increased patient morbidity and mortality.(3) Progressive PHT may lead to 
splenomegaly and subsequently systemic vasodilatation as well as changes in systemic 
hemodynamics affecting the heart (hyperdynamic circulation) and blood flow to vital organs, 
notably the kidneys, lungs and brain.(7, 8) Whereas PHT and its complications may be recognized 
clinically and/or with non-invasive imaging, actual portal pressure can only be measured 
invasively. 
5.2 Hepatic Venous Pressure Gradient (HVPG) assessing Portal 
Hypertension 
Sinusoidal pressure can be estimated by catheterizing the hepatic veins (most commonly right 
hepatic vein via transjugular access). The pressure is then measured in the hepatic venous 
radicles once when the vein is open and a second time when it is occluded.(9, 10) The difference 
between the free (unoccluded) and wedged pressure is the hepatic venous pressure gradient 
(HVPG), which gives a reliable estimate of sinusoidal portal pressure. (The hepatic venous 
pressure method does not detect presinusoidal portal hypertension, e.g. due to portal vein 
obstruction).  
HVPG > 5 mm Hg defines PHT, whereas HVPG ≥ 10mm Hg is recognized as “clinically 
significant portal hypertension (CSPH)”, as it is the pressure threshold that predicts many of the 
complications of cirrhosis.(6, 11) As shown in a recent review based on the recommendations of 
the AASLD (American Association for the Study of Liver Disease) and EASL (European 
Association for the Study of the Liver), supported by over 80 references,(12) increments in HVPG 
are significantly associated with liver decompensation and patient death. Increments in HVPG 
occuring over time are positive predictors of the development of varices, variceal hemorrhage, 
non-variceal complications and death.  
For a better understanding of HVPG and its utility, a citation from the current American 
Association for the Study of Liver Disease (AASLD) Guidelines(13) is quoted:  
“The method for assessing portal pressure is the wedged hepatic venous pressure 
(WHVP) measurement, which is obtained by placing a catheter in the hepatic vein 
and wedging it into a small branch or, better still, by inflating a balloon and 
occluding a larger branch of the hepatic vein. The WHVP has been shown to 
correlate very closely with portal pressure both in alcoholic and non-alcoholic 

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Study Title: Clinical Study of the BreathID® MCS System to 
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assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
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cirrhosis.(14) The WHVP is always corrected for increases in intra-abdominal 
pressure (e.g., ascites) by subtracting the free hepatic vein pressure (FHVP) or the 
intra-abdominal inferior vena cava pressure, which act as internal zeroes. The 
resultant pressure is the hepatic venous pressure gradient (HVPG), which is best 
accomplished with the use of a balloon catheter, usually taking triplicate readings 
and, when measured with a proper technique, is very reproducible and reliable.(10) 
Since it is a measure of sinusoidal pressure, the HVPG will be elevated in 
intrahepatic causes of portal hypertension, such as cirrhosis, but will be normal in 
pre-hepatic causes of portal hypertension, such as portal vein thrombosis. The 
normal HVPG is 3-5 mmHg. The HVPG and changes in HVPG that occur over time 
have predictive value for the development of esophagogastric varices,(15, 16) the risk 
of variceal hemorrhage,(7, 17, 18) the development of non-variceal complications of 
portal hypertension,(7, 19, 20) and death.(3, 18, 20, 21) Single measurements are useful in 
the prognosis of both compensated and decompensated cirrhosis, while repeat 
measurements are useful to monitor response to pharmacological therapy and 
progression of liver disease. Limitations to the generalized use of HVPG 
measurement are the lack of local expertise and poor adherence to guidelines that 
will ensure reliable and reproducible measurements,(10) as well as its invasive 
nature.”  
The reproducibility of HVPG in skilled hands was recently assessed in a study in which repeated 
HVPG measurements were performed between 20 and 720 minutes from baseline demonstrating 
a mean change in HVPG of only 0.4%.(22) In addition, another recent study showed excellent 
inter-observer agreement (r = 0.98) in tracing interpretation.(12) A study performed in post-
transplantation recurrent hepatitis C showed HVPG to be more accurate in predicting the 
occurrence of a decompensation event than liver biopsy.(23) Based on these results, HVPG is 
regarded as the “standard of care” for assessment of the severity of liver disease.(12)  
Currently, HVPG measurement is considered the standard of care for measuring portal 
hypertension.(13, 24-29)  
Many peer-reviewed publications support the use of HVPG as a valuable tool for evaluating liver 
disease severity and prognosis.(30)  HVPG measurements have been discussed in approximately 
600 publications, showing high correlation with the degree of liver disease severity, development 
of complications and patient survival. HVPG has been called “the best and only surrogate 
biomarker in Hepatology”.(26) Recent comprehensive reviews even refer colloquially to the 
HVPG measurement as the “gold standard” for the determination of CSPH.(25, 31) In a recent 
review(25) leading experts in the field stated: 

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Study Title: Clinical Study of the BreathID® MCS System to 
train the algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
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Bosch J, Abraldes JG, Berzigotti A, Garcia-Pagan JC(30): “Measurement of hepatic 
venous pressure gradient (HVPG) and upper GI endoscopy are considered the gold 
standards for portal hypertension assessment in patients with cirrhosis. However, 
both types of investigation are invasive and HVPG measurement is routinely 
available and/or performed with adequate standards only in expert centers. 
Castera L, Pinzani M, Bosch J:(25) There is thus a need for non-invasive methods able 
to predict, with acceptable diagnostic accuracy, the progression of portal 
hypertension ….” 
An editorial in Hepatology by other leading hepatologists stated: 
Burroughs AK, Thalheimer U:(27, 32) “At present, HVPG measurement is the best 
surrogate of portal pressure and also the best single prognostic marker available for 
patients with cirrhosis.” 
Another review by Triantos CK, Nikolopoulou V, Burroughs AK (32) concluded that: “The 
prognostic and therapeutic value of HVPG is established beyond portal hypertensive 
bleeding for which there are some clinical surrogates. [e.g. the development of gastro-
oesophageal varices and bleeding, ascites, hepatorenal syndrome and hepatic 
encephalopathy] HVPG measurement should now be part of everyday clinical practice.” 
Furthermore, it is clinically accepted that reduction of portal pressure is the ultimate goal in the 
treatment of patients with cirrhosis.(13) HVPG has been shown to be highly correlative with the 
response to portal pressure-lowering drugs and their beneficial effects on liver disease 
complications. These studies demonstrate the ability of HVPG to predict the clinical course of 
these patients, as well as to enable follow-up of treatment in patients with chronic liver disease. 
Hepatology experts concluded(33) HVPG can serve as a valid tool for follow up of disease 
progression in patients with chronic liver disease.  
There are over 40 clinical trials listed in clinicaltrial.gov, including several drug development 
studies in the US approved by FDA (NCT: 00737594, 01644656, 01714609) as well as multiple 
Phase 4 studies (01769040, 00563602, 00596414 00570973, 00570622, 00188045) in which 
HVPG is the primary outcome for evaluating portal hypertension. 
A recent review(30), by Jaime Bosch, M.D., summarizes the clinical value of single and repeated 
HVPG measurements, including stepwise HVPG cut-off values and changes in time, with repeat 
measurements (7, 15, 17, 19, 34-42) and further supports the cut-off: ≥10mmHg of CSPH as a predictor 

Exalenz Bioscience Ltd. 
Last update on: Nov 21, 2016 
Study Title: Clinical Study of the BreathID® MCS System to 
train the algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
Version: 1.5 
Document No: CSD00076 
Page 21 of 72 
 
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"for the risk of developing varices, ascites, clinical decompensation, and hepatocellular 
carcinoma (HCC), and the risk of decompensation after liver resection for HCC". 
The following table (Table 1) summarizes some of the clinical indications and the change in 
management as a result of HVPG measurements:  
 
Table 1 - Selected literature based utilities of HVPG measurements 
HVPG - MBT 
CLINICAL UTILITY 
 
PATIENT 
POPULATION 
INDICATION 
CHANGE IN 
MANAGEMENT 
REFERENCES 
Patients with 
cirrhosis 
Patients at risk for varices and 
bleeding (based on 
HVPG ≥10 mmHg) to determine 
necessity for upper endoscopy 
and frequency of endoscopy. 
1. May avoid the need for 
Endoscopy in low risk 
patients 
2. Define frequency of 
Surveillance for varices  
and their management 
(16, 
43) 
(44, 
45) 
(46-48) 
Patients with 
chronic liver disease 
and varices being 
treated with beta 
blockers 
Response to Beta Blocker  
1.  Dose adjustment for 
responders  
2. Stop therapy and possibly 
refer to alternative therapy 
(e.g. banding) for non-
responders 
(18, 
20, 
43) 
Patients with HCC 
Determine the risk of resection 
(HVPG<10) 
Performance of surgery 
(49, 50) 
Patients at risk for 
HCC (Cirrhosis or 
HBV) 
Assess the risk for developing 
HCC 
Enhance HCC surveillance 
with 
more 
expensive 
imaging studies 
(11) 
Patients with 
cirrhosis treated in 
the community 
Classify patients into low and 
high risk groups (HVPG ≥10) 
Refer higher risk group to 
transplant center although 
MELD is not elevated 
(44) 
Patients with 
cirrhosis and acute 
bleeding 
Determine if urgent TIPS 
should be performed  
Performance 
of 
TIPS 
procedure 
(40, 41)  
 
5.3 HVPG ≥10 mmHg defines Clinically Significant Portal Hypertension 
As mentioned above, HVPG can distinguish sub-stages within compensated and decompensated 
cirrhosis. HVPG > 5 mm Hg defines portal hypertension (PHT), whereas HVPG ≥ 10 mmHg is 

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Last update on: Nov 21, 2016 
Study Title: Clinical Study of the BreathID® MCS System to 
train the algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
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widely recognized as “clinically significant portal hypertension (CSPH)”,(48) as it is the pressure 
threshold that predicts many of the complications of cirrhosis.(6, 11, 33) As shown in a recent 
review,(22) including over 80 references, HVPG of 10 mmHg and higher is significantly 
associated with liver decompensation, hepatocellular carcinoma (HCC) and patient death. 
 
5.4 Unmet need for a noninvasive tool for assessment of CSPH 
Although HVPG is highly clinically valuable,(26) and the method used to measure HVPG is 
accurate, reproducible and reasonably safe,(15) limitations to the generalized use of HVPG are a 
lack of local expertise and poor adherence to guidelines to ensure reliable and reproducible 
measurements.(10, 13) Moreover, HVPG is an invasive intravascular procedure involving imaging, 
contrast media injection and catheterization. Hence HVPG is limited to large medical facilities 
requiring resources beyond the hepatology clinic and point of care physician and holds 
significant added risk to the patients. Berzigotti et al. in their recent review (31) determined that 
HVPG is the best method to assess CSPH, but they also cite the limitations of the procedure, 
especially in the context of therapy monitoring:  
“HVPG measurements are not available in all centers, the technique is invasive and 
some patients are unwilling to be submitted to it. This is even more relevant when the 
repetition of the procedure is needed to monitor treatment response. These issues have 
raised interest to non-invasively determine when CSPH is present, so allowing 
defining a patient at risk of developing portal hypertension-related complications.” (31) 
Hence, HVPG is an impractical test for everyday clinical use. Serial HVPG measurements are 
likely to be useful but not practicable in clinical practice. Furthermore, key opinion leaders 
(KOL) have commented that many more HVPG measurements would be done if it was not for 
the practical drawbacks and limitations since it is felt to be a good predictor of complications of 
cirrhosis.(12, 22) It is clearly desirable and necessary to have an equally reliable surrogate test that 
is non-invasive. 
Moreover, although HVPG is clinically highly valuable,(26) even in experienced hands, 
complications can occur including large hematomas of the neck or groin being the most common 
(as reported in a leading Spanish center that regularly measures HVPG with a complication rate 
of 2.3%). 

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Study Title: Clinical Study of the BreathID® MCS System to 
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assessment of Portal Hypertension in Patients with 
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Protocol No.: CSPH-EX-0414 
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5.5 
13C-Breath Tests 
Breath testing with 13C-labeled substrates provides a safe, non-invasive means for evaluating 
hepatic impairment as it pertains to liver metabolic function. 13C is a stable, non-radioactive 
isotope, which can be incorporated into a specific location within a test substrate so it can be 
metabolized into 13CO2 by the liver. Ideally, the 13C-compound would be administered orally, 
rapidly absorbed and exclusively metabolized by the liver. 13C-Methacetin has been identified as 
such a substrate. 
5.5.1 13C-Methacetin Breath Tests - MBT 
Methacetin had been available and described in literature for over 100 years. Within the last 30 
years its use for Breath testing has been described as well. No risks have been reported in the 
literature using much higher doses of Methacetin than currently proposed to be used in this 
protocol. No adverse events have been reported in performing 13C-Methacetin Breath Test 
(MBT) using the proposed device or substrate to date including in more vulnerable populations. 
Additional safety assessments will be made during this clinical study. 
Methacetin metabolism involves an initial O-demethylation process carried out exclusively by 
hepatic cytochrome CYP450 Iso-enzyme 1A2 producing two products: O-demethylated 
Methacetin, similar to acetaminophen, and formaldehyde.(51-56) Formaldehyde is then 
transformed into 13CO2, through additional fast enzymatic steps. The rate limiting factor in this 
process is the O-demethylation performed by the liver cytochrome CYP450.(57) More details 
regarding the Methacetin metabolism are presented in section ‎8.2. 
The ability of MBT to assess liver's function had been demonstrated in several applications. The 
13C-Methacetin metabolism performed exclusively by the liver and resulting in 13CO2 exhaled as 
described in section ‎8.2. In a study using MBT for the prediction of postoperative outcome after 
hepatectomy,(58) results demonstrated 64 patients showing close correlation between MBT results 
and liver volume (as measured by CT volumetry) during regeneration (r=0.94, p<0.0001). MBT 
was the only predictor of outcome, namely liver failure and/or mortality for these patients prior 
to surgery. The concept of MBT as an overall liver function test, has also been demonstrated in 
rats,(59) where MBT correlated with liver mass following partial hepatic resection; as the liver 
mass increased with regeneration, so did the MBT values. Furthermore, MBT was assessed for 
the hepatic specificity of Methacetin metabolism in 5 patients undergoing liver transplantation, 
by injecting 13C-Methacetin intravenously at the anhepatic stage (just after removal of the liver) 
and before reperfusion of the graft.(58) Liver function was assessed with an MBT, and a clear 
increase in 13CO2 was observed after reperfusion, as can be seen in Figure 1. This clearly 
demonstrated that the metabolism of Methacetin was solely liver-dependent. 

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Study Title: Clinical Study of the BreathID® MCS System to 
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assessment of Portal Hypertension in Patients with 
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Figure 1: 13C-Methacetin Breath Test during Orthotopic Liver Transplant 
 
It has been further demonstrated that MBT can be used to assess liver reserve and function 
before and after transplantation(60) and for pre-operative assessment of liver reserve to determine 
the probability of success.(61) The same group has shown utility of the MBT in predicting 
prognosis in the ALF population.(62) 
Furthermore, several large clinical trials have shown the correlation of MBT with severity of 
disease in cirrhotic patients compared to other tests such as CTP, MELD and ICG.(63-67) 
Several factors may affect 13C measurements in cirrhotic patients. These include: alteration in 
blood flow, both intra-hepatic and shunting(30) of blood due to portal hypertension thereby 
decreasing the rate of metabolism; the presence of fibrotic barriers in peri-sinusoidal spaces 
causing a possible delay the release of CO2 into the blood; damage to hepatocytes which further 
decreases the metabolism of Methacetin. Accordingly, the Methacetin breath test can potentially 
be a tool for overall assessment of liver disease, portal hypertension and their consequences. 
5.6 Rationale to use the MBT for assessment of portal hypertension  
Tests of true hepatic function that rely on the metabolism of administered exogenous compounds 
have not yet been widely adopted in clinical practice because they have been cumbersome to 
perform and acquisition of results is slow. The recent development of the BreathID® analyzer 
offers a unique opportunity to demonstrate a metabolism-based test showing agreement with 
HVPG.  
Low values of Methacetin metabolism may be suggestive of patients who are likely to have 
increased portal pressure. In advanced liver disorders, the liver has reduced 13C-Methacetin 
metabolic capacity therefore the MBT values are low. Several MBT output variables (Percentage 

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Last update on: Nov 21, 2016 
Study Title: Clinical Study of the BreathID® MCS System to 
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assessment of Portal Hypertension in Patients with 
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Dose Recovery rate and Cumulative Percentage Dose Recovered - PDR/CPDR values at 
different time points) could be used to assess CSPH, as they were shown to be proportionally 
lowered in the presence of CSPH in two independent studies and in a meta-analysis. 
Exalenz completed a study performed in eight large US centers and three OUS centers entitled 
“Pivotal study to evaluate the efficacy and safety of the BreathID® system for detection of 
cirrhosis using the ¹³C-Methacetin Breath Test MBT” (G080107) during which a subset of 21 
patients with MBT data had undergone HVPG at the time of their liver biopsy. When comparing 
MBT variables and HVPG, significant correlations were noted.. For example, a statistically 
significant inverse relationship (r=-0.75) was found between the PDR values at 45 minutes 
(PDR45) and HVPG values  as depicted in Figure 2. It can be concluded that the correlation 
does not seem to be affected by the use of non-selective beta-blockers or the presence of varices. 
It was also observed that ascites was observed in those patients with CSPH. 
 
 
Figure 2: Correlation between HVPG and MBT measure (PDR45) 
 

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Study Title: Clinical Study of the BreathID® MCS System to 
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assessment of Portal Hypertension in Patients with 
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In addition, using the Receiver Operating Characteristic (ROC) Area Under the Curve (AUC) to 
assess the ability of an MBT variable to identify CSPH, demonstrated the Cumulative Percent 
Dose Recovered at 15 minutes (CPDR15) MBT parameter distinguished HVPG ≥10mmHg 
(CSPH) from lower HVPG values (non-CSPH). The AUC of the MBT parameter (CPDR15) was 
0.89 (95% CI [0.72-1.00], p < 0.0001)(68) (see Figure 3). The patient population tested included 
5 non-CSPH subjects and 16 subjects with CSPH. 
 
 
Figure 3: ROC curve for the detection of CSPH as measured by MBT Measure CPDR15 
Based on these findings, an additional study was conducted in India to compare MBT to HVPG 
in 31 subjects clinically identified as cirrhotic. All subjects underwent both tests within 2 days of 
each other. The results showed a statistically significant correlation of approximately r= -0.6 for 
most MBT variables with CSPH. The ROC AUC remained at 0.90 for CPDR15 (Cumulative 
PDR at 15 minutes) (see Figure 4). There were, however only two subjects with HVPG 
measures lower than 10mmHg. 

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Study Title: Clinical Study of the BreathID® MCS System to 
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assessment of Portal Hypertension in Patients with 
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Figure 4: ROC curve for the detection of CSPH as measured by MBT Measure CPDR15 in 
Indian data 
 
The Company conducted a meta-analysis of all accumulated data (in the US and in India) 
including several patients from an additional study including cirrhotic patients conducted under 
IDE G080227, who had HVPG   done as part of their clinical care. A total of 58 subjects were 
included in this analysis to assess the correlation between MBT and HVPG as well as the ability 
of the MBT to identify CSPH (51 with CSPH and 7 without CSPH). The analysis demonstrated a 
statistically significant negative correlation between HVPG and MBT (r=-0.57) as seen in 
Figure 5.  
 

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Study Title: Clinical Study of the BreathID® MCS System to 
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assessment of Portal Hypertension in Patients with 
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Figure 5: Correlation between HVPG and MBT measure CPDR15 on 58 patients 
The AUC in this meta-analysis remained at 0.88 for CPDR15. The ability of CPDR15 to assess 
CSPH can be seen in a boxplot in Figure 6. 
 
 
Figure 6: Boxplot showing the ability of CPDR15 to assess CSPH in 58 patients 

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Study Title: Clinical Study of the BreathID® MCS System to 
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assessment of Portal Hypertension in Patients with 
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The aim of the proposed protocol is to collect prospective clinical data and complete the 
statistical assessments to identify the most appropriate, sensitive and specific measurement 
and/or algorithm to use to identify patients at high risk for de-compensation as defined by HVPG 
≥ 10mmHg as the basis for the device labeling requested. 
The company’s current focus is to discriminate between the presence and absence of CSPH, 
defined as HVPG ≥ 10mmHg in the cirrhotic patient as a dichotomous test. As noted above, this 
will provide a simple non-invasive repeatable method of determining the onset/presence of 
CSPH, thereby contributing to the efficient identification and management of patients with 
advanced liver disease. 
It had been proposed that additional endpoints will be evaluated as well, including: the 
dichotomous discrimination of HVPG lower or greater than 20 mmHg and the assessment of 
response to treatment (beta blockers) of patients with proven CSPH acutely, during the test and / 
or at steady state of the prescribed treatment.  
 
6 INTENDED USE / INDICATION FOR USE 
The BreathID® System 13C-Methacetin Breath Test (MBT) is to be used for the identification of 
clinically significant portal hypertension (CSPH), as defined by an Hepatic Venous Pressure 
Gradient (HVPG) ≥ 10 mmHg in patients with liver cirrhosis independent of disease etiology, 
without a history of clinical decompensation (variceal bleeding, uncontrolled ascites, hepatic 
encephalopathy and/or hepatorenal syndrome). 
 
7 PROPOSED CLINICAL ALGORITHM USING THE MBT 
The company's goal is to develop a point-of-care device for the identification of clinically 
significant portal hypertension (CSPH).  
The company proposes to train a selected MBT-based variable that can reliably identify 
clinically significant portal hypertension (defined as HVPG ≥ 10mmHg) in patients with 
compensated liver cirrhosis. This will enable the use of a dichotomous measure from MBT to 
assess CSPH by means of rule-in or rule-out or both. 
 

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assessment of Portal Hypertension in Patients with 
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8 MBT SHORT DESCRIPTION 
8.1  
MBT Overview 
The MBT is a non-invasive test for assessing liver metabolic capacity to metabolize 13C-
Methacetin in order to assess presence of CSPH. The BreathID® MCS System consisting of the 
BreathID® MCS device and a test kit containing a breath collection cannula and a non-
radioactive isotope 13C-Methacetin solution, which measures and computes the ratio between 
13CO2 and 12CO2 in the patient’s exhaled breath. 
For more details please refer to the study Investigator Brochure.  
The device is based on an FDA cleared device (510k#: K130524) for assessment of H. pylori 
infection in the stomach utilizing 13C-Urea as a substrate for the bacterial urease. Performance 
and safety of the device utilizing the 13C-Methacetin substrate for assessment of liver function, 
have been studied in thousands of patients worldwide, including in a large US pivotal study – 
under IDE G080107 – of over 400 patients with chronic liver disease from 11 participating sites 
(including 141 patients with biopsy proven cirrhosis),(69) which validated the safety of the MBT. 
8.2 
13C-labelled Substrate: 13C-Methacetin 
The 13C-labeled Methacetin has been selected as the test substrate. 13C-Methacetin is a white, 
crystalline powder bearing the chemical name [N-(4-Methoxy phenyl) acetamide]. 
13C-Methacetin is rapidly absorbed and metabolized exclusively by hepatic microsomal function 
oxidase via O-demethylation, mainly by cytochrome P450 1A2, into acetaminophen (also known 
as paracetamol, TylenolTM, and n-acetyl-p-aminophenol or APAP) and formaldehyde.(51-56) 
Formaldehyde is then transformed, through two successive oxidative steps, into ¹³CO2. The rate 
limiting factor in this process is the O-demethylation performed by the liver cytochrome 
CYP450.(57) The general reaction is described in the following scheme: 

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Figure 7: 
Oxidative Cleavage of Methacetin to Acetaminophen and Formaldehyde 
The resultant ¹³CO2 can be measured in the exhaled breath. The amount of metabolized 
Methacetin indicates the capability of the liver to accomplish one of its main physiological tasks 
and has been shown to correlate with liver fibrosis and cirrhosis.(66, 70-72) 
13C-Methacetin meets all of the qualifications for a liver function test substrate: It is a non-toxic 
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 
adverse events have been reported when using this substance, including in vulnerable 
populations, and the compound remains stable over time.  
8.2.1 13C-Methacetin Safety 
The 13C-Methacetin substrate is a well-known diagnostic reagent that has been described in the 
literature and used for over 100 years in general and for over 30 years specifically for breath 
testing by leading researchers around the world (US, Europe and Japan). As previously 
mentioned, there have been no reports of any complications with the use of this substance. The 

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Last update on: Nov 21, 2016 
Study Title: Clinical Study of the BreathID® MCS System to 
train the algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
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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.  
In current literature regarding pre-clinical and clinical studies performed with Methacetin, results 
from a total of 2987 individuals (2021 chronic liver disease patients including critically ill 
patients and 966 controls) were reported in 73 publications, including neonates,(73) infants,(74) 
healthy adults, adults with various forms of liver disease (including toxic injury), pregnant 
women(75) and the elderly - with no reports of adverse events. MBT has also been used in patients 
with Acute Liver Failure. Some of these data were developed utilizing the Exalenz device and 
substrate, while the majority were performed with other similar systems. 
Regarding internal unpublished data using our device and Methacetin, 1466 chronic liver patients 
(of these 278 cirrhotic - some before transplant) and 74 healthy controls/subjects without liver 
disease, have also been tested. No adverse events related to the substrate were experienced by the 
subjects 
Toxicology testing results in animals and other information support the safe use of Methacetin in 
humans. Based on the acute toxicity studies in mice and rats where relatively high LD50 values 
of 1190mg/kg were administrated,(76) 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. 
Many studies using 13C-Methacetin for liver function assessment have been published. 
Representative references have been cited that support the conclusion that 13C-Methacetin is a 
“safe” molecule and that it has been used on high-risk population groups such as the elderly, 
neonates or pregnant women.(60, 65, 77, 78) This provides further assurance that the 13C-Methacetin 
substrate is appropriate to use in breath tests intended for liver function assessment. 
8.3 MBT Components  
The following are the components of the MBT System: The BreathID® MCS device, the 13C-
Methacetin substrate and the sampling kit containing breath collection cannulae. 
The BreathID® MCS device will be provided by Exalenz Bioscience Ltd. The cannulae, 
accessories and the test substrate will be supplied in proper packaging and will bear all required 
labeling based on applicable regulatory requirements. 
The solution administered orally to the patient is a 0.05% solution of ¹³C-Methacetin in purified 
water, supplied in amber thermoplastic polyester (PET) bottles with a child resistant plastic 
stopper (75mg of ¹³C-Methacetin in 150mL purified water). All bottles are for single use only 
and must be used solely for per-protocol investigational product administration. Each bottle 

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assessment of Portal Hypertension in Patients with 
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contains a single test dose. The solution is manufactured at Taro Pharmaceutical Industries, Ltd., 
14 Hakitor St., Haifa Bay 26110, Israel and will be provided under the responsibilities of 
Exalenz Bioscience Ltd, 4 Ha` Ma'ayan St., Modiin, 71700, Israel. 
All study test components must be stored in an environment according to the Investigator 
Brochure and product labeling guidance.  
 
9 STUDY DESIGN  
This study is a multicenter, non-randomized, blinded, cross-sectional comparative exploratory 
study comparing various MBT measures to HVPG. 
This study will be used to train an algorithm using MBT measures and to select a cut-off to 
determine presence or absence of CSPH as defined by HVPG ≥ 10mmHg. 
 
10 STUDY OBJECTIVES  
10.1 Efficacy Objective 
The primary objective of the study is to develop an algorithm and its cut-off to detect CSPH, 
defined as HVPG ≥10mmHg, based on the MBT. 
 
10.2 Safety Objective 
All adverse events, serious adverse events (SAE) and suspected unexpected serious adverse 
reactions (SUSAR) will be reported according to local regulations. The actual reporting is 
discussed in section ‎14. No breath-test related adverse events are expected. For more information 
please refer to the Investigator Brochure. 
10.3 Secondary Objectives 
1. To assess the ability of the MBT model to diagnose portal hypertension with HVPG ≥ 
12mmHg 
2. To assess the ability of the MBT model to diagnose portal hypertension with HVPG ≥ 
20mmHg 
3. To assess the correlation of MBT with HVPG readings 

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Study Title: Clinical Study of the BreathID® MCS System to 
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assessment of Portal Hypertension in Patients with 
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4. To Assess the effect of beta blockers or other confounding factors on the correlation of 
MBT with HVPG readings 
5. Examine the effect of a single small (<3cm) HCC (hepatocellular carcinoma) on the 
correlation of MBT with HVPG tracings 
 
10.4 Efficacy Endpoints 
10.4.1 Primary efficacy endpoint 
Binary diagnosis of CSPH, defined by HVPG ≥10 mmHg, correlated with an MBT result to be 
developed.  
The relationship will be assessed on both sides of the definitive HVPG value to obtain a 
clinically significant MBT model/algorithm and its cut-off to be validated in a separate study.  
10.4.2 Secondary efficacy endpoint 
1. Binary diagnosis of portal hypertension with HVPG ≥ 12mmHg 
2. Binary diagnosis of portal hypertension with HVPG ≥ 20mmHg 
3. Correlation of MBT with HVPG readings 
4. Assess the effect of beta blockers or other confounding factors on the correlation of MBT 
with HVPG readings 
5. Examine the effect of a single small (<3cm) HCC (hepatocellular carcinoma) on the 
correlation of MBT with HVPG tracings 
 
10.5 Safety Endpoints 
Adverse Event reporting. 
 
11 SUBJECT SELECTION 
The study population will be essentially enrolled on a consecutive basis of able and willing 
subjects meeting all inclusion/exclusion criteria with clinically, imaging or biopsy proven liver 
cirrhosis without decompensation events in the past. 
Most patients to be enrolled in the proposed study will be from centers in Europe with the 
addition of site(s) in the US. Based on expert opinion in the field of hepatology, the population to 
be enrolled from Spain and France is representative of patients with chronic liver disease in the 

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Study Title: Clinical Study of the BreathID® MCS System to 
train the algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
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Protocol No.: CSPH-EX-0414 
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United States. It is presumed patients in both countries have similar etiologies and prognosis and 
are being used interchangeably in multicenter clinical trials using HVPG testing.(7, 8, 18) 
 
11.1  Inclusion Criteria  
1. 
Adult men or women (>18 years of age) 
2. 
Patient (or legal guardian) can sign an Informed Consent Form. 
3. 
Known chronic liver disease with cirrhosis confirmed by either: 
a. liver biopsy or 
b. clinical (palpable left lobe, splenomegaly) and laboratory (platelets 
<150,000/mm3 or albumin< 3.8g/dL, or INR >1.3) evidence of cirrhosis 
and/or 
c. imaging studies by abdominal sonography, computer assisted axial 
tomography, or magnetic resonance imaging, showing a nodular liver 
and/or enlarged spleen and/or portosystemic collaterals with portal vein 
patency and/or minimal ascites, and/or colloid shift on a colloid-isotope 
liver-spleen scan or measurements of liver stiffness suggestive of cirrhosis 
4. 
Europe: Indicated to undergo HVPG testing 
US: Consented for HVPG  
5. 
Can tolerate an overnight (8-hour) fast  
6. 
For patients treated with statins: They have to be on a stable dose for at least 4 
weeks prior to any study related tasks (MBT or HVPG measurement) 
7. 
For patients treated with beta blockers: They have to be on a stable dose for at least 
6 weeks prior to any study related tasks (MBT or HVPG measurement) 
8. 
For patients who stopped their treatment with beta blockers: Their last dose should 
be at least 6 weeks prior to any study related tasks (MBT or HVPG measurement) 
Note: In case patient has HCC, breath test has to be performed on the day of HVPG measurement. 
11.2 Exclusion Criteria:  
1. 
Decompensated cirrhosis as clinically defined by the occurrence of any of the 
following: ascites, hepatic encephalopathy, variceal bleeding (active upper GI 
bleeding ) or hepatorenal syndrome 
2. 
Renal failure (creatinine > 2.5 mg/dl)  
3. 
Known acute renal tubular disease 
4. 
Known hypotension (Systolic Pressure < 100mmHg)  
5. 
Hypocoagulablity defined as PT >6 and INR >2.3 
6. 
Congestive heart failure (assessed clinically as NIHA >2)  

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assessment of Portal Hypertension in Patients with 
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7. 
Known pulmonary hypertension (right ventricular systolic pressure > 45 mm Hg,)  
8. 
Uncontrolled diabetes mellitus (HBA1C >9.5gr%)  
9. 
Concurrent prednisone or immunosuppressive treatment, if therapy and/or 
response to treatment are not stable for at least 3 months 
10. 
Documented hepatocellular carcinoma lesion larger than 3cm and/or multifocal 
lesions and/or evidence of vascular invasions 
11. 
Gastric bypass surgery or extensive small bowel resection 
12. 
Total parenteral nutrition 
13. 
Any organ transplant recipient 
14. 
Pregnant or breast feeding 
15. 
Allergy to acetaminophen and/or other related medications. 
16. 
Documented drug-related concurrent hepatotoxicity or drug-related silent steatosis 
or drug-related fibrosis (e.g. amiodarone, methotrexate and tamoxifen). 
17. 
Uncontrolled malabsorption or diarrhea  
18. 
Documented non-cirrhotic PHT, partial / complete portal venous occlusion, hepatic 
venous occlusion, previous PHT surgery, or placement of a transjugular 
intrahepatic portosystemic shunt (TIPS) 
19. 
Primary or secondary biliary cirrhosis, primary or secondary sclerosing 
cholangitis, hepatic sarcoidosis, or other cholestatic disorders  
20. 
Subjects unable to perform the MBT within 7 days of HVPG procedure  
21. 
Subject should not have taken any of the following for at least 48 hours prior to the 
breath test: Acyclovir , allopurinol, carbamazepine, cimetidine, ciprofloxacin, 
daidzein, (herbal) disulfiram, echinacea, enoxacin, famotidine, fluvoxamine, 
methoxsalen, 
mexiletine, 
montelukast, 
norfloxacin, 
phenylpropanolamine, 
phenytoin, propafenone, rifampin, terbinafine, ticlopidine, thiabendazole, 
verapamil, zileuton or any medication that might interfere with Methacetin 
metabolism or might affect CYP 1A2 
22. 
Subject should not have taken amiodarone within the last 30 days prior to the breath 
test or HVPG procedure 
23. 
Splenomegaly secondary to inflammation/infection or hematologic malignancy. 
24. 
Last dose of HCV treatment received within the last 3 years. 
 
11.3 On the day of the MBT: 
1. 
8 hours fasting prior to MBT including all PO morning medications except for beta 
blockers 
2. 
No Smoking on the day of the MBT 

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Study Title: Clinical Study of the BreathID® MCS System to 
train the algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
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3. 
No caffeine within 24 hours prior to MBT 
4. 
No paracetamol (acetaminophen) based medications within 24 hours prior to MBT 
5. 
No general anesthesia or sedation within 24 hours prior to MBT 
6. 
No alcohol within 24 hours prior to MBT  
7. 
Patient should be symptom free of any prior intervention (e.g. biopsy) 
11.4 Consenting 
Patients’ and/or their representative will sign a consent form prior to study participation which 
must be signed and witnessed. 
The consent will include willingness to allow acquisition and collation of blood, clinical and 
imaging data taken on entry to the study and incorporate all other data from time of admission 
until the patient's termination of the study. 
 
11.5 Safety Termination of Subjects or Study 
1. 
Withdrawal of patients from the study if they have a CTCAE Grade 3 or higher 
regardless of whether the event is attributed to the drug unless it is caused by an 
accident that could not reasonably be attributable to the drug (CTCAE: Common 
Terminology Criteria for Adverse Events Version 4.0). 
2. 
Discontinuance of study, if ≥ 2 patients on Methacetin develop the same CTCAE 
Grade 3 event or 1 patient develops a CTCAE Grade 4 or higher event due to the 
breath test. 
11.6 Early Withdrawal from the Study 
Patients, their relatives, their representatives or the patients’ physician may withdraw the patient 
from the study at any time if they feel the study is not in the patients’ best interest, or they may 
be withdrawn by the investigator or sponsor for safety, behavioral, or administrative reasons. All 
withdrawals must be fully documented. 
If the patient withdraws from the study and also withdraws consent for the disclosure of future 
information, no further evaluations should be performed and no additional data should be 
collected. Previously acquired data will be analyzed. This will not have any effect on the 
treatment that the patient receives. 
11.7 Expected Duration of Recruitment 
The study is planned for a duration of approximately 12-18 months  

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Study Title: Clinical Study of the BreathID® MCS System to 
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assessment of Portal Hypertension in Patients with 
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12 STATISTICAL CONSIDERATIONS 
12.1 Study Design and Objectives 
This is a multicenter, non-randomized, blinded, cross-sectional comparative exploratory study 
comparing various MBT measures to HVPG. 
This study will be used to train an algorithm using MBT measures and to select a cut-off to 
determine presence or absence of CSPH as defined by HVPG ≥ 10mmHg. 
The modeled algorithm will be validated in a separate study  
12.2 Primary endpoints 
Binary diagnosis of CSPH, defined as HVPG ≥10 mmHg, correlated with an MBT result to be 
developed.  
The relationship will be assessed on both sides of the definitive HVPG value to obtain a 
clinically significant MBT model/algorithm and its cut-off to be validated in a separate study.  
12.3 Secondary efficacy endpoint 
1. Binary diagnosis of portal hypertension with HVPG ≥ 12mmHg 
2. Binary diagnosis of portal hypertension with HVPG ≥ 20mmHg 
3. Correlation of MBT with HVPG readings 
4. Assess the effect of beta blockers or other confounding factors on the correlation of MBT 
with HVPG readings 
5. Examine the effect of a single small (<3cm) HCC (hepatocellular carcinoma) on the 
correlation of MBT with HVPG tracings 
 
12.4 Safety Endpoints 
All Adverse Events. 
12.5 Sample Size estimation 
As presented in section ‎5.6 above the MBT parameter CPDR15 was found to have an AUC of 
about 0.9. Two hundred (200) subjects of which at least 50 will be positive for CSPH and at least 
50 will be negative for CSPH will enable the estimation of a more conservative AUC of 0.85 
with a half width of the 95% confidence interval of 0.1, if the ratio of positive to negative 
subjects is as high as 3:1 and as low as 1:8 (1 pos. per 7 neg.). 

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Last update on: Nov 21, 2016 
Study Title: Clinical Study of the BreathID® MCS System to 
train the algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
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During the study the correlation between MBT and HVPG will be assessed periodically in order 
to see if the intake of beta blockers, HCC or any other confounding factors have an effect on the 
breath test result. If the population using beta blockers, HCC or if some other confounding factor 
shows a significantly different correlation than the rest of the population, the study population 
may be increased up to 400 patients. 
12.6 Analysis sets 
All subjects enrolled to the study.  
12.7 Statistical Analysis  
12.7.1 General Considerations 
Statistical analyses will be performed using SAS® v9.3 or higher (SAS Institute, Cary NC, 
USA). 
If any statistical tests are performed, they will be two-sided. The required significance level of 
findings will be equal to or lower than 5%. Where confidence limits are appropriate, the 
confidence level will be 95%. 
Baseline values are defined as the last valid value prior to study treatment start. 
All statistical analyses of safety and performance measures will be descriptive in nature. 
Continuous variables will be summarized by a mean, standard deviation, minimum, median and 
maximum, and categorical variables by a count and percentage. Confidence intervals will be 
provided where relevant.  
If multiple measurements are taken in a single patient, statistics described below will be 
appropriately modified to accommodate the within patient correlation.  
12.7.2 Demographic and Other Baseline Characteristics 
Demographic, medical and clinical history variables will be tabulated. Continuous variables will 
be summarized by a mean, standard deviation, minimum, median and maximum, and categorical 
variables by a count and percentage. 
12.7.3 Disposition of Subjects 
The numbers of patients who were enrolled will be provided, as well as the reasons for all 
enrollment discontinuations, grouped by major reason (e.g., lost to follow-up, adverse event, 
poor compliance). A list of discontinued patients, protocol deviations, and patients excluded 
from the efficacy analysis will be provided as well 

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assessment of Portal Hypertension in Patients with 
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Protocol No.: CSPH-EX-0414 
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12.7.4 Safety Analysis 
The events related to study drug and/or device AE’s rate will be presented along with a 95% 
exact binomial two sided 95% confidence interval. The analysis of all adverse events will 
include incidence tables and will include analyses by severity, relationship to device or drug and 
baseline variables. 
12.7.5 Performance Analysis 
A model/algorithm will be developed for the differentiation between subjects with and without 
CSPH, defined as HVPG ≥10mmHg. It will be developed using either logistic regression or 
neural network or any other known methodology. Using a cut-point on the score a binary 
diagnosis of CSPH (Y/N) will be obtained. The minimal criterion for sufficient efficacy is an 
Area under the Receiver Operating Curve (AUC) ≥0.85 in this patient population. A potential 
clinical utility was designated for the algorithm/model output; where this algorithm could aid the 
clinician in ruling-out CSPH. This will be translated into finding a model prediction cut-point 
that provides a high sensitivity (true positive rate) and at least moderate specificity (true negative 
rate), which in turn achieves a high negative predictive value (NPV). High NPV is considered to 
be near 90% (i.e. 88%) thus sensitivity should be at least 90% and specificity should be at least 
50%. Cross validation methods will be used to test algorithm stability. 
12.7.6 Interim Analysis 
This study is an open label study for training purposes. The data may be assessed prior to 
achieving the full sample size. This will be done specifically in order to assess the effect of beta 
blockers, HCC, or any other confounding factor on the correlation between MBT and HVPG. If 
beta blocker, HCC or if some other confounding factor shows significantly different correlation 
than the rest of the population, the study population may be increased up to 400 patients. 
12.7.7 Pooling 
Subgroup analysis of the primary performance endpoint by center via a regression model will be 
used to evaluate the poolability of the results. If the variable by center analysis is found 
significant, the reason for this will be further explored and rationalized. This evaluation may 
include demographic features, symptoms at presentation, clinical and treatment history, and 
center comparability in the features found to be associated with the primary efficacy variable 
12.7.8 Handling of Missing Data 
The study variables cannot be evaluated for patients for whom MBT device results or HVPG 
results are not available and therefore these subjects will be left out of the efficacy analysis. 

Exalenz Bioscience Ltd. 
Last update on: Nov 21, 2016 
Study Title: Clinical Study of the BreathID® MCS System to 
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assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
Version: 1.5 
Document No: CSD00076 
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Demographic clinical characteristics and safety data of patients with missing data will be 
compared to patients with complete data. 
12.7.8.1 Procedure 
After all the relevant data will be entered into the database, a soft lock to the database will be 
performed. The study statistician will perform the assessment of the primary and safety 
endpoints. 
12.7.8.2 Decision Rules 
Stop the study in case of severe safety concern. 
 
13 STUDY PROCEDURES  
13.1  General 
1. The study will be conducted in compliance with this protocol, with GCP 
standards, and the applicable regulatory requirements. 
2. This study will be cross-sectional, where patients will be enrolled, if 
possible, in a consecutive manner so as to prevent any bias. All patients 
with cirrhosis and without clinical signs of complications should be 
screened and reason for non-suitability documented. 
3. Patients’ relative or other representative will sign a consent/assent form 
prior to study participation.  
4. A CRF will be filled out for each patient and all data will be collated 
incorporating all demographics, detailed clinical history, physiological 
variables, radiological data, biochemical data and organ support provided 
(cardiovascular, respiratory, renal, hepatic, neurological) and all baseline 
characteristics. 
5. HVPG procedure will be performed in a standardized fashion in all study 
centers (see section ‎13.4 and Appendix II).  
6. In addition, the tracings will be read at a centralized location by an 
independent expert reader. This independent analysis will be done by an 
expert reader blinded to the breath test results. 
7. The MBT will be performed within 7 days (prior or post) of the HVPG 
procedure using a single dose of 75mg 13C-Methacetin. Note that the same 

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Last update on: Nov 21, 2016 
Study Title: Clinical Study of the BreathID® MCS System to 
train the algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
Version: 1.5 
Document No: CSD00076 
Page 42 of 72 
 
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policy should be applied with regards to the use of beta blockers as for the 
HVPG procedure for those patients that are treated with beta blockers. In 
case patient has a HCC, breath test has to be performed on the day of 
HVPG measurement. 
8. If patient is using a stable dose of beta blockers, both the MBT and the 
HVPG procedures should be performed while continuing the stable dose 
even on the morning of each test. 
9. All investigators will be blinded to the MBT score results until the end of 
the study recruitment. The MBT results will therefore not affect the current 
patient management. 
10. Safety follow up will be done during the entire period of the study, and 
any adverse events will be recorded according to GCP ICH guidelines. 
Section ‎16.6 relates to the reportable and non-reportable SAEs.  
11. Results of the individual blood tests and other clinical assessments, 
standard prognostic criteria and clinical condition of the patient will be 
collected in the CRF.  
12. The raw data will be saved on the BreathID® MCS device and will be 
downloaded to a flash memory stick and uploaded to a computer for 
further analyses.  
13. All concurrent medication will be recorded. Certain drugs will be pre-
recorded in the CRF. 
14. Patients’ survival will be ascertained 30 days following their last breath 
test. 
15. The breath test procedure is described in detail in the main protocol and it 
requires minimal operator and patient participation. BreathID® MCS 
automatic breath collection eliminates virtually all breath collection 
related errors, as described in section ‎13.3 and Appendix III.  
 
 

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Study Title: Clinical Study of the BreathID® MCS System to 
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assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
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13.2   Masking procedures 
In order to ensure unbiased interpretation of assay results, the following steps for masking will 
be taken: 
 The authorized medical staff performing the HVPG procedure will be blinded to 
the breath test results. 
 The principal investigator or co-investigator(s) will not be blinded to the results of 
HVPG and other diagnostic tests, since he may be the one performing the 
procedure and needs to evaluate the eligibility of the subject for treatment and/or 
possible monitoring as part of standard of care management. 
 The blinding procedure is applicable only for the output of the BreathID® MCS 
device. The breath is collected and analyzed by the BreathID® MCS device and the 
results are printed out automatically at the end of the test. Hard copies of the device 
printouts will be stored with the CRFs as the source data.  
 All HVPG tracing print outs will be stored as a hard copy or downloaded to a 
computer at the end of participation of each subject in the study, so that an 
independent analysis by a third party may be done. 
 
13.3 Breath Test Procedure 
Please refer to Appendix III and the IB for the MBT procedure 
13.4 Principles of the Hepatic Venous Pressure Gradient (HVPG)  
In order to standardize the measurements of the HVPG in the study the main procedure steps, 
derived from the common standards of care practice(10), are described below in Appendix II. 
Under conscious sedation and with non-invasive vital sign monitoring (ECG, arterial blood 
pressure and pulse oximetry) the right jugular vein (or the femoral or antecubital vein) is 
catheterized, a venous introducer is placed and a balloon-tipped catheter is advanced under 
fluoroscopic control into the inferior vena cava and a hepatic vein, to measure pressures. This is a 
moderately invasive technique that is only being performed as part of the clinical evaluation of the 
patient (and not solely for the purpose of the clinical trial) in common with other routine 
procedures that are carried out in patients with chronic liver injury. 

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Free hepatic venous pressure is measured by maintaining the tip of the catheter “free” in hepatic 
vein, 2-4 cm from its opening into the vena cava. Wedge hepatic venous pressure is measured by 
occluding the hepatic vein by inflating a balloon at the tip of the catheter. The wedged hepatic 
venous pressure is measured until the value remains stable over 30-40 seconds. All measurements 
are performed in triplicate, permanent tracings are captured on a computer or chart recorder. The 
difference between the free and wedged hepatic venous pressures is the HVPG. Adequate 
occlusion of the hepatic vein is confirmed after pressure measurement, by slowly injecting 5cc of 
contrast into the vein that should show a typical “wedged” pattern without reflux of the contrast or 
its washout through communications with other hepatic veins. 
Major complications have been limited to local injury at the puncture site, including leakage, 
hematoma and rarely an arteriovenous fistula or Horner syndrome. Ultrasonographic guidance 
reduces the risk of procedural complications. Passage of the catheter through the right atrium might 
cause supraventricular dysrhythmias that are self-limited in more than 90% of cases. Although 
coagulation disorders are common in patients with cirrhosis, only severe thrombocytopenia 
(platelets <20,000/mm3) calls for platelet transfusion. The procedure entails very little discomfort, is 
carried out under moderate conscious sedation that does not influence HVPG measurement. The 
procedure’s acceptability is comparable to that of upper gastrointestinal endoscopy.  
Complications of the HVPG measurement will be reported as anticipated Adverse Events. The test 
procedure in this protocol is the MBT. The HVPG should be performed for clinical cause according 
to local standards of care; the HVPG will not be performed for the purpose of enrolling a patient 
under this protocol. For the purpose of the study, in US centers the HVPGs is considered an 
investigational procedure since HVPG is not used as common practice in the US. 
13.4.1 Laboratory Tests 
The patients will routinely undergo laboratory tests in preparation for their HVPG procedure. All 
liver related blood tests will be recorded along with other available laboratory information (as 
described below). If more than one blood collection is performed on a given day, the blood test 
results at the time closest to the time of the breath test will be recorded. 
 
Recent (most recent, but within 6 months): blood test results will be recorded if available  
Blood tests used as part of the pre-procedure safety evaluation for the HVPG procedure should 
be within 2 months (or sooner, if indicated by the patient’s co-morbid conditions). 
1. aPTT, fibrinogen  
2. Hepatic panel (AST, ALT, alkaline phosphatase, GGTP, total bilirubin, direct 
bilirubin, serum albumin and total protein) 

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3. Renal and electrolyte panel (BUN, creatinine, electrolytes (Na+ and K+), total 
calcium, and PO4 (phosphate corrected), magnesium  
4. Blood count (WBC, Hemoglobin, Platelets) 
5. Ammonia – arterial (preferred) or venous (if venous must be 
sampled from central vein not femoral or peripheral vein) 
 
Additionally, assessments of the following variables will be made (not necessarily on days of 
breath test, but most recent within the past 6 months):  
1. Ultrasound, CT (liver outline, vasculature, evidence of fat etc.), imaging of major hepatic 
vessels, pancreas, guts, and liver volume results if calculated; a single assessment of 
these variables when available.  
2. Clinical data including: previous medical history, presentation, etiological factors, special 
diets and likely diagnosis on entry and update as available. 
3. Habits and history of alcohol, substance and smoking will be recorded. 
4. Liver Biopsy – if undertaken 
5. Etiology, including etiological screening tests and diagnosis 
 
13.5 Investigational Product Handling  
The Investigator and 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. 
13.6 Investigational Product Accountability 
The Investigator and Study Pharmacist (if relevant) are responsible for ensuring that all study 
supplies received at the site are inventoried and accounted for throughout the study. The 
dispensing of study 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 

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assessment of Portal Hypertension in Patients with 
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appropriate environmental conditions. Unused study materials must be available for verification 
by the sponsor’s site monitor during on-site monitoring. 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. 
Study substrate should be dispensed under the supervision of the investigator, a qualified 
member of the investigational staff, or by hospital clinical pharmacist. 
 
14 ETHICS & REGULATORY CONSIDERATIONS 
14.1 Ethics & Regulatory Approvals 
The study will be conducted in both the United States of America and Europe (Spain and 
France). As such, regulatory requirements that are relevant for all countries will be discussed in 
this section. 
The study will be conducted in compliance with the principles of the Declaration of Helsinki 
(1996), the principles of GCP and in accordance with all applicable regulatory requirements. 
This protocol and related documents will be submitted for review to all relevant ethics 
committees delegated to approve the study at their respective sites. Any amendment to the 
protocol may be proposed by the principal investigator. The amendment must be submitted to the 
Sponsor and, when approved to the relevant IRB or EC. When applicable, the implementation of 
the amendment will take place only once approved by the appropriate ethics committee. 
Annual progress and safety reports and a final report at conclusion of the study will be submitted 
by Exalenz Bioscience (or on behalf of the Sponsor) to the applicable regulatory bodies within 
the timelines defined in the Regulations. 
 
15 INVESTIGATOR RESPONSIBILITIES 
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. GCP 
is an international ethical and scientific quality standard for designing, conducting, recording, 
and reporting studies that involve the participation of human subjects. Compliance with this 
standard provides public assurance that the rights, safety, and well-being of study subjects are 

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Protocol No.: CSPH-EX-0414 
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Document No: CSD00076 
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protected, consistent with the principles that originated in the Declaration of Helsinki, and that 
the clinical study data are credible. 
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, 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 Exalenz to do so and 
provide Exalenz with a confirmation. 
The Principal Investigator will act as custodian for the study data. Patient data will be 
anonymized and all the anonymized data will be stored on a password protected computer. All 
data will be compliant with CFR and GCP.  
 
16 SAFETY CONSIDERATIONS 
16.1 
Adverse Event Definitions  
An adverse event (AE) is any undesirable or unintentional event that occurs in a patient or 
clinical investigation subject, whether or not considered related to the investigational product; 
this includes clinically significant changes in laboratory values. As this study is not therapeutic 
in nature, there will be no therapeutic failures to report.  
Regardless of severity or relationship to the study investigational product, all diagnoses, 
symptom(s), sign(s) or finding(s) with a start date after the first study test has begun (Breath 
test/EGD) and until the last study procedure is being completed will be documented, assessed by 
the investigator and recorded in the subject's CRF.  
A surgical procedure that was planned prior to the first study test by any physician treating the 
subject should not be recorded as an AE. 
16.2 
 Serious Adverse Event (SAE), Unanticipated Adverse Device Effect 
(UADE) and Suspected Unexpected Serious Adverse Reactions (SUSAR) 
A serious adverse event (SAE) is an event that is either: 
a) Fatal 
b) Life-threatening 

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Life threatening is defined as an event in which the patient is at risk of death at the time of 
the event. It does not refer to an event that hypothetically might have caused death if it was 
more severe. 
c) Results in persistent or significant disability/incapacity  
Disability is defined as a substantial disruption of a person’s ability to conduct normal life 
functions.  
d) Requires or prolongs inpatient hospitalization 
e) A congenital anomaly or birth defect. 
f) Another serious or important medical event as judged by the investigator. 
An Unanticipated Adverse Device Effect (UADE) or a Suspected Unexpected Serious Adverse 
Reactions (SUSAR) is an SAE, related to the investigational product, for which the nature or 
severity is not consistent with the expected outcomes of the treatment/testing being offered. 
An unexpected adverse event is one that has not been previously observed, or one that is of a 
specificity or severity not consistent with the current investigator brochure. 
As with every procedure risks and discomforts may occur with relations to the Breath ID test. 
The potential risks/complications and discomforts which may be associated with all study 
procedures are listed below.  
 Allergic and/or other reactions to BreathID reagent 
 Aspiration (inhaling) of food or fluids into the lungs 
 Discomfort  
When an adverse event occurs, the following information and assessments should be 
documented in the subject’s file and reported in the adverse event section of the CRF: 
 The signs, symptoms, or diagnosis of the event if available 
 The date and time of onset of the event using the 24 hour clock where midnight is 00:00 
and noon is 12:00 
 The adverse event severity using the criteria outlined below  
 The relationship of the event to the study medical device or drug as outlined below 
 Describe any action taken regarding study medical device or drug disposition  
 List any required therapy, medication, treatment, or diagnostic procedure. 

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16.3 
Relationship to Study Medical Product 
An investigator, who is a qualified physician, should assess the relationship to the study medical 
device and the substrate, based on all information available at the time of assessment.   
The following definitions should be used: 
 Not Related:  
1. The event is clearly related to other factors such as the patient's clinical state, therapeutic 
interventions or concomitant drugs.  
2. There is a highly likely alternative explanation. 
 Related:  
1. The event AE is reasonably associated with the use of the investigational product (device 
or substrate). 
16.4 
Adverse Event Severity 
 Mild Adverse Events 
A mild adverse event is one that the symptoms are barely noticeable to the patient. It does not 
influence performance, require drug treatment or prevent the patient from carrying on with 
normal life activities. 
 Moderate Adverse Events 
A moderate adverse event is one that the symptoms make the patient uncomfortable and causes 
some impairment to normal life activities. Treatment for symptom(s) may be required. 
 Severe Adverse Events 
A severe adverse event is one that the symptoms cause severe discomforts to the patient and 
severely limits the patient’s normal daily activities. Treatment for symptom(s) is given.  
Note: Serious and severe are not synonymous. A serious adverse event must fulfill the 
requirements listed in the definition above.  
16.5 
Adverse Event Reporting 
The investigator should report all adverse events in the case report form. The investigator is 
responsible for the appropriate medical management of all adverse events.  

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The investigator must report any serious adverse event to the sponsor immediately, within 24 
hours of awareness, via email. Full details of the event, severity, outcome (if available) and an 
assessment of the relationship to the study investigational product must be provided in the report.  
All SAEs must be followed up until resolution or stabilization by submission of updated reports. 
Follow-up SAE reports will be reported according to the same timelines as initial reports, as 
soon as new significant information becomes available.  
All SAEs that fulfill UADE or SUSAR definitions will be reported to the relevant authorities by 
expedited means. 
Notification of the IECs/IRBs and regulatory authorities 
Notification to the IECs/IRBs about all relevant events (e.g. SAEs, UADEs, SUSARs) will be 
performed by the sponsor and/or by the investigator according to all applicable regulations. 
The processing and reporting of all relevant events to the regulatory authorities will be done by 
the sponsor according to all applicable regulations. 
If unexpected safety issues are identified, specific amendments will be implemented. 
16.6 Study-Specific Exceptions 
Disease complications as a result of disease progression is expected and it is not necessary to 
report them as SUSARs, but they should be reported in the follow-up section of the case report 
form (CRF). 
 
The following situations and non-fatal toxicities that fulfill the definition of an SAE are excluded 
from expedited notification on an SAE form, but should be reported in the follow-up section of 
the CRF form: 
 
 Elevation of bilirubin 
 Elevation liver enzymes 
 Nausea, vomiting , diarrhea  
 Bleeding following line insertion  
 
Although these events are anticipated to occur as part of the disease process, they will be 
collected and periodically analyzed to assess whether they are occurring at a higher rate than 
would be expected in this population. If certain events occur at a higher rate than expected for a 
given stage or severity of the disease, then the adverse event analysis will be reported under 

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Study Title: Clinical Study of the BreathID® MCS System to 
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assessment of Portal Hypertension in Patients with 
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Protocol No.: CSPH-EX-0414 
Version: 1.5 
Document No: CSD00076 
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expedited reporting requirements. (FDA Guidance: Safety Reporting Requirements for INDs and 
BA/BE Studies. http://www.fda.gov/downloads/Drugs/.../Guidances/UCM227351.pdf) 
17 ACCESS TO SOURCE DATA AND DOCUMENTS 
17.1 Availability of Source Data and Documents 
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.). 
17.2 Patient Confidentiality 
The patient’s name and personal data will remain confidential and will not be published in any 
way. All data will be coded and stored in locked offices or on password protected computers. 
 
18 MONITORING AND QUALITY ASSURANCE 
Monitoring of this study will be to ensure compliance with GCP, local regulations and scientific 
integrity and will be managed and oversight retained by Exalenz Bioscience (the Sponsor) or its 
assigned representative.  
18.1 Data Collection and Monitoring 
An eCRF will be completed for each patient.  The EDC (electronic data capture) system will be 
21 CRF Part 11 compliant and the site staff and all involved parties will be trained on the system 
prior to patient enrolment.   
IRB and EC members and regulatory authorities will be permitted to review study documents at 
the site according to local and federal regulations. 
 
19 PUBLICATION POLICY AND FINANCE 
It is intended that the results of the study will be reported and disseminated at international 
conferences and in peer-reviewed scientific journals. The policy regarding publications appears 
in the non-disclosure agreement signed by each study participant prior to signing of the contract. 
 

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20 FINANCIAL ASPECTS  
The device and compound will be provided by the Sponsor. Funding for regulatory approvals 
and administration will also be provided by the Sponsor. Funding will also be provided for a 
study support staff at local sites. 
 
21 STUDY TERMINATION 
The study may be terminated after appropriate consultation between the study sponsor and the 
chief investigator and co investigators. Conditions warranting termination include, but are not 
limited to: 
 Failure of the investigator to enroll patients at an acceptable rate 
 Insufficient adherence to protocol requirements 
 Failure to adhere to GCP 
 Decision by the study sponsor to suspend or discontinue development of the device 
or kit 
 
 

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assessment of Portal Hypertension in Patients with 
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22 
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Exalenz Bioscience Ltd. 
Last update on: Nov 21, 2016 
Study Title: Clinical Study of the BreathID® MCS System to 
train the algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
Version: 1.5 
Document No: CSD00076 
Page 54 of 72 
 
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Study Title: Clinical Study of the BreathID® MCS System to 
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assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
Version: 1.5 
Document No: CSD00076 
Page 55 of 72 
 
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Hepatol 2005;43:167-176. 

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Consensus Workshop on definitions, methodology and therapeutic strategies in portal 
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13C-methacetin breath test in evaluating hepatic injury in rats. J Gastroenterol Hepatol 
2008;23:1762-1768. 

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M. 13C-methacetin breath test for monitoring hepatic function in cirrhotic patients 
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Predicting the prognosis in acute liver failure: results from a retrospective pilot study 
using the LiMAx test. Ann Hepatol 2013;12:556-562. 
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critically ill]. Dtsch Med Wochenschr 2012;137:2212-2216. 
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transplantation. Alimentary Pharmacology & Therapeutics 2004;19:243-243. 
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A, et al. Measurement of hepatic functional mass by means of 13C-methacetin and 13C-
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Metabolism, Measured Using Exalenz BreathID, Correlates Well with the Hepatic Venous 
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Shiffman ML LG, Reuben A, Gordon SC, McCullough AJ, Reddy R, Poordad F, Younossi 
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cirrhosis with a non-invasive 13C Methacetin breath test: A prospective phase 3 
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Braden B, Faust D, Sarrazin U, Zeuzem S, Dietrich CF, Caspary WF, Sarrazin C. 13C-
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Braun M, Ben-Ari, Z. The unique breath ID test system for the diagnosis and prediction 
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assessment of fibrosis in chronic hepatitis C. In: EASL, DDW. Vienna, Los Angeles; 2006. 
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[13C]methacetin breath test for evaluation of liver damage. Dig Dis Sci 1987;32:344-348. 
 
 
 

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APPENDIX I - PROTOCOL APPROVAL SIGNATURE PAGE 
 
 
Protocol No: CSPH-EX-0414 
 
Protocol Title: Clinical Study of the BreathID® MCS System to train the algorithm for the 
¹³C-Methacetin Breath Test (MBT) in assessment of Portal Hypertension in 
Patients with Compensated Liver Cirrhosis 
 
Version:  
1.5 
 
Date of Protocol: 21-NOV-2016 
 
Site Name: 
_____________ 
 
Principal Investigator: __________________ 
 
 
 
 
      Print name 
 
I have read this protocol and agree to conduct the study as outlined herein and as per GCP and 
local regulations. 
 
Principal/ Chief Investigator____________________ Date_____________________ 
 
 

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APPENDIX II – DETAILED HVPG PROCEDURE  
 
 
HVPG procedure: Instruction for a correct measurement  
 
 
Jaime Bosch, MD, PhD, FRCP 
 
Professor of Medicine, Hospital Clínic-IDIBAPS, University of Barcelona  
 
 
 
 
 
Abbreviations: 
 
HVPG : hepatic venous pressure gradient 
WHVP: wedged hepatic vein pressure 
FHVP: free hepatic vein pressure 
IVCP : inferior vena cava pressure 
RAP: right atrial pressure 
 
 
 

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HVPG: What is it? 
 
HVPG stand for hepatic vein pressure gradient, which is calculated as the difference between the 
“wedged” and the “free” hepatic vein pressure (Figure 1). 
 
Figure 1. Typical measurement of the hepatic venous pressure gradient (HVPG) with a balloon 
catheter. With the balloon deflated, the “free” pressure (FHVP) is recorded. After occluding the 
hepatic vein by inflating the balloon (arrows), the “wedged” pressure (WHVP) is recorded. Between 
measurements the “zero” pressure is verified by opening the transducer to air. HVPG is calculated as 
the difference between WHVP (32 mmHg) and FHVP (8 mmHg); in this case it was very high, of 
24 mmHg. Note that the two measurements shown in the figure are identical, and that in this patient 
it took about 45 seconds for WHVP to reach a stable plateau after inflating the balloon.  
 
The wedged hepatic vein pressure is obtained by occluding the lumen of the hepatic vein by inflating a 
balloon at the tip of the catheter (or by advancing an end-hole catheter as far as it could go into the 
hepatic vein, a technique that is no longer used as it is less accurate and has more variability that the 
balloon catheter technique).  Once the hepatic vein has been occluded, the static column of blood makes 
that the pressure sensed by the catheter corresponds to that existing in the hepatic sinusoids. Because of 
that, the wedged pressure reflects the hepatic sinusoidal pressure. In the normal liver, the hepatic 
sinusoids are a very uniformly communicated vascular bed. By contrast, in advanced liver disease, 
especially in cirrhosis, fibrosis and nodule formation makes that the intersinusoidal communications are 
lost, which makes that when the hepatic vein is occluded, the static column of blood extends beyond the 
sinusoids and thus reflects portal pressure (Figure 2).  It has long been known that in different forms of 
cirrhosis measurement of WHVP equals to the direct measurement of portal pressure, which is much 
more invasive as requires the transhepatic or surgical catheterization of the portal vein. 
0                    30                   60                   90                  120                 150                 180                 210                 240 
 
 
 
mmHg 
 
 
 
 
 
 
 
0 

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Figure 2. Measurement of wedged hepatic venous pressure (WHVP) in the normal 
and cirrhotic liver. (A) In the normal liver, after occlusion of blood flow in a hepatic 
vein with a balloon catheter, the pressure of the static column of blood (WHVP) 
equilibrates through the interconnected sinusoids. Therefore WHVP is a measurement 
of the hepatic sinusoidal pressure, which is slightly lower than portal pressure (-1 
mmHg). (B) In the cirrhotic liver, the pressure of the static column of blood created by 
balloon inflation cannot be decompressed at the sinusoidal level due to the sinusoidal 
narrowing and disruption of the normal intersinusoidal architecture by fibrosis and 
nodule formation. Because of this, WHVP equilibrates with portal pressure. 
 
The “free” hepatic vein pressure is obtained by deflating the balloon of the hepatic vein catheter, and is 
very close to the pressure in the inferior vena cava. If the difference between the free and the IVC 
pressure is ≥ 2 mmHg it usually reflects that the catheter is advanced too much into an irregular hepatic 
vein. Because of this the free pressure should always be obtained also in a “retired” position, close to the 
IVC (Figure 3, below) 

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Figure 3. Free pressures should be measured in a “retired” position, 
close to the IVC. 
In hemodynamic terms, absolute pressures have much less meaning than pressure gradients, the 
difference of pressure between two points in a vascular system. The pressure gradient (P) is 
mathematically related to blood flow (Q) and to vascular resistance (r) by Ohms law in the equation:  P 
= R x Q. 
Expressing pressures as pressure gradients beyond being physiologically the correct way has the practical 
advantage of being independent of external influences. For instance the presence of ascites or obesity 
may increase the intra-abdominal pressure; this will equally increase the portal pressure and the IVC 
pressure (or its equivalents, the wedged and free hepatic vein pressure) but will not affect the pressure 
gradient. The same happens when the electronic pressure transducer is placed to low or to high with 
respect to the recumbent patient; this will make all pressures to be increased or decreased, but will not 
alter the pressure gradient.  An important final point regarding pressure gradients is that this should be 
obtained between the WHVP and FHVP (or IVC), not with the right atrial pressure! Using the right atrial 
pressure introduces a systematic error that overestimates the true pressure gradient and it has been shown 
that doing this detract the prognostic value of the measurement. In short, the right atrial pressure should 
not be used to assess portal pressure or hepatic hemodynamics.   
 
 

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The procedure  
 
Hepatic vein catheterization is a moderately invasive procedure that should be done by well-trained 
personnel.  It is an ambulant procedure, although it is done with a day-hospital admission in most centers 
(see video “HVPG measurement” in Garcia-Tsao and Bosch, NEJM 2010, also available in You Tube). 
The patients usually accept to have repeat HVPG measurements over the time, as the procedure is usually 
very well tolerated.  The factor more closely related to a less good tolerance is the duration over 45 
minutes (which most frequently reflect lack of expertise or need for associated procedures such as a 
transjugular liver biopsy).  The median duration of the procedure is 25 minutes.  
The procedure should be done preferably in the morning, after fasting overnight. This is so since there is 
a circadian variation of the HVPG that increases during the day and decreases over the night. On this 
rhythm there is a superimposed postprandial increase of HVPG that lasts 1-2 hours. If a patient is to 
undergo repeat measurements these should always be done at approximately the same hour.  
The favorite access points are either the right internal jugular vein or the right femoral vein. The 
procedure is done under slight conscious sedation.  Deep sedation should be avoided as it results in 
exaggerated deep breathing that cause artifacts precluding accurate pressure measurements. Direct 
ultrasonography guidance should be used, as it is saves time and reduces the risk of the more common 
complications (hematomas, local bleeding, or vascular lesions such as arteriovenous fistulae). 
After the access vein has been punctured, a guide wire is advanced into the vein allowing the placement 
of a venous introducer sheath allowing advancing the balloon catheter under fluoroscopic guidance into a 
hepatic vein. This usually takes less than 5 minutes. 
Once the catheter is in the hepatic vein (preferably, the main right hepatic vein) (Figure 2) an adequate 
occlusion of the hepatic vein should be checked after inflating the balloon by injecting by hand a small 
amount of iodinated contrast media. This should show the occluded hepatic vein, without reflux of 
contrast through the occluded vein or through communications with another hepatic vein. If present, 
these communications prevent an adequate estimation of the portal pressure. This is extremely 
uncommon in cirrhosis but very frequent in idiopathic portal hypertension or nodular regenerative 
hyperplasia (Figure 3). 
 
 
 
 

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Figure 3. The presence of communicating vessels between different hepatic veins (arrows) 
prevents an effective occlusion of the vein and precludes a correct measurement of WHVP. This 
should be checked in every patient. Such communications are very frequent in patients with 
idiopathic portal hypertension, but very rare in alcoholic, viral or metabolic cirrhosis. 
 
After documenting the correct occlusion of the hepatic vein, the balloon is released and the catheter is 
thoroughly rinsed with saline or 5% dextrose and connected to a multichannel pressure recorder for the 
measurement of the wedged and free hepatic venous pressures (see next section). 
 
Pressure measurements 
Correct measurement of intravascular pressures requires fulfilling a series of steps and to have adequate 
equipment. This is available almost in all hospitals, but not all monitors in IR rooms are adequate for 
accurate measurements of HVPG.  
1. Pressure recorder: The adequate pressure recorder should have the following capabilities: 
a) Adjusting the pressure scale for a relatively low range 1-40 or 1-50 mmHg. The pressure 
scale should be printed together with the pressure tracings, and preferably a milimetric grid 
should also be disclosed. 
b) Adjusting the recording speed to low velocity (1-6 mm/second). 
c) Capacity of obtaining permanent pressure tracings, either using an on-line printing device, or 
by generating a printed report from the digitally stored data after completing the procedure. 
 
If the monitor you have is not able of doing this, your center does not qualify for the study.  
In addition, a non-invasive monitor for EKG, BP and O2 saturation should be used. 

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2. Pressure transducer: Nowadays most centers use disposable pre-calibrated transducers that are very 
reliable.  
If you are using another type of transducer or you have doubts on its correct functioning, the transducer 
should be calibrated against a know pressure. This is done by connecting the transducer to a column of 
saline of 544 mm height (where it should read 40 mmHg); 272 mm height (where is should read 20 
mmHg), and then to 136 mm (where it should read 10 mmHg). 
The pressure transducer should be placed in a fixed position that should be set at the mid-axillary line of 
the recumbent patient. This is the conventional level for the right atrium. Neither the transducer nor the 
patient should move during the procedure. 
 
3. Balloon catheter: These should have a small (8 – 11 mm inflated balloon diameter), preferably not 
entirely distal (Figure 4). 
 
4. Obtaining the wedged (occluded) hepatic vein pressure (WHVP): After having verified that the 
catheter is at an adequate position, the hepatic vein is occluded by inflating the balloon while 
continuously recording the pressure. The hepatic vein pressure will rapidly increase from the free 
pressure until it finally stabilizes at the final WHVP. This requires usually some 45-60 seconds but 
sometimes it requires longer periods (Figures 1 and 5). The pressure measurement should be 
continuously recorded for at least 60 seconds (longer if the pressure is still increasing). Because it needs 
such a long time it is mandatory to run the recorder at very low speed, if not one cannot notice if the 
Figure 4. Balloon catheters suitable for HVPG measurements. The balloon is of 8-11 
mm in diameter and is not totally distal, so a fully inflated balloon will not occlude the 
central lumen that will be centered in the axis of the vein. 

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pressure is still increasing or has achieved a plateau. For adequate pressure measurements the patient 
should be relaxed, asked not to move or talk, and to breathe smoothly. 
 
 
Figure 5. Typical HVPG tracing 
 
Most recorders will allow printing either (or both) the “phasic” oscillating pressure or the “mean” 
electronically averaged pressure (Figure 6). If possible, both should be recorded. “Mean” pressures are 
easier to read, but are slow reacting and therefore less adequate to recognize artifacts. 
The WHVP should be recorded by triplicate. This allows averaging the values to get a more robust 
measurement. 

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Figure 6. Oscillating and “mean” pressures recorded simultaneously 
5. Obtaining the free hepatic vein pressure (FHVP) and the inferior vena cava (IVC) pressure. After 
completing the measurement of WHVP the catheter should be rinsed, the zero level checked by opening 
the transducer to atmospheric pressure, and then the FHVP is recorded while the patient is quiet and 
breathing smoothly. The recording should be run for 20 - 30 seconds.  
After obtaining the FHVP the catheter should be slightly withdrawn to obtain another FHVP at a 
“retired” position, with the catheter tip 2-3 cm from the IVC, and from here withdrawn to the IVC 
where pressures are measured again (at the level of the hepatic veins), and finally into the right atrium, 
to measure RAP. 
Again, remember that all pressures should be measured by triplicate.  
 
Frequent mistakes 
The most frequent mistakes in the measurement of HVPG are due to the fact that the measurement is 
done at a IR department forgetting to discuss with the radiologist that the main aim of the procedure is to 
obtain reliable, accurate measurements, with a precision of 0.5 mmHg.  The more frequent mistakes are 
listed and illustrated below: 
a) Inadequate scale: many monitors are pre-calibrated for measuring arterial blood pressure. 
This scale (usually 0 – 150/200 mmHg) is not adequate for measuring HVPG as the 
deflections would be too small to allow enough accuracy. The preferred scale should be one 
in which 1 mmHg equals one division of the grid. Figure n shows an example of a correct 
and of an incorrect measurement. 

Exalenz Bioscience Ltd. 
Last update on: Nov 21, 2016 
Study Title: Clinical Study of the BreathID® MCS System to 
train the algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
Version: 1.5 
Document No: CSD00076 
Page 70 of 72 
 
Exalenz Bioscience Ltd.     
 
CONFIDENTIAL AND PROPRIETARY 
 
b) Inadequate speed: it is common to run the recorder at a speed of 25-50 mm/sec. This is 
adequate for EKG but not for HVPG, as a full study would require strips of about 15 foot 
length, or show only a few seconds of recording.  
 
c) Inadequate length: a variant of the previous mistake is to run the recording at correct speed 
but for a too short period, not allowing the pressure to stabilize. This results in 
underestimation of the true HVPG.   
 
d) Artifacts due to deep respiration are very common if the patient is excessively sedated and 
almost invariable when using IV fentanyl. Typically this results in a wide variation of all 
venous pressures during the respiratory cycle, precluding accurate measurements of HVPG (-
-, Figure). The patient moving, talking or coughing during the measurements may also cause 
artifacts. Whenever an artifact occurs, this should be annotated in the tracing and the 
pressure measurement discarded and repeated. 
 
 
 
Sending the tracings of a HVPG measurement to the central reader. 
The original tracings are important source documents that should be kept in the patient’s charts.  Please, 
do not send original materials by mail or courier. It is safer, quicker and cheaper to send by e-mail a 
scanned copy of the tracings as a PDF file. 
Each tracing should be clearly identified with the name/code number of the local Principal Investigator 
(PI); patient’s code number and initials, and date.  
The tracings should include the calibration used, and 3 sets of measurements of the WHVP (balloon 
occluded), FHVP, “retired” FHVP, IVC and RAP. Remember to set the recorder at a low speed (1-6 
mm/sec), an adequate scale (0-40 or 0-50 mmHg), and to run the measurements at least for 20 seconds 
for FHVP, IVC and RAP and at least for 60 seconds for WHVP. 
 
0 
1
2
3
Figure 6. Marked respiratory oscillation due to excessive sedation 

Exalenz Bioscience Ltd. 
Last update on: Nov 21, 2016 
Study Title: Clinical Study of the BreathID® MCS System to 
train the algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
Version: 1.5 
Document No: CSD00076 
Page 71 of 72 
 
Exalenz Bioscience Ltd.     
 
CONFIDENTIAL AND PROPRIETARY 
APPENDIX III – DETAILED MBT PROCEDURE 
 
Preparation of the study subject 
Once enrolled, the patients will perform the breath test if they meet the criteria of the patient 
population. In preparation for each breath test the patient will be asked to comply with the 
following instructions: 
1. Fast 8 hours prior to the breath test. 
2. Refrain from use of caffeine, propranolol, within the last 24 hours. Refrain from use of 
acetaminophen-related medications (e.g. Tylenol) 24 hours prior to the breath test.  
3. Refrain from use of alcohol 24 hours prior to the breath test.  
Preparation of 13C-Methacetin 
Exalenz Bioscience Ltd. will provide 75 mg 13C-Methacetin doses in a 0.05% solution of ¹³C-
Methacetin in purified water, supplied, in amber thermoplastic polyester (PET) bottles with a 
child resistant plastic stopper (75mg of ¹³C-Methacetin in 150mL purified water). No preparation 
is needed other than pouring the contents of the solution into a cup for ingestion if administered 
orally. 
Performance of the breath test 
Only trained personnel (doctors, nurses) will perform the breath test procedure. The actual breath 
collection is automatically done by the device and is not operator dependent. If the patient was 
not connected properly with the cannula (e.g. the breath does not reach the device), the 
BreathID® MCS device will prompt the operator to adjust the cannula. 
1. Turn on device from switch in rear and allow up to 1 hour for warm-up to complete. To 
perform the test, ensure that the BreathID® MCS console screen shows that the device is 
in the ‘Ready’ state.  
2. The cannula will be attached to the BreathID® MCS device and to the patient. The 
BreathID® MCS device will be activated and will collect the patient’s baseline exhaled
 
breath for approximately 15-20 minutes. 
3. The solution 13C-Methacetin is poured into a disposable cup and administrated to the 
patient. The solution should be administered by a medical practitioner registered on the 
delegation log or a research nurse if specific training for administration has been given. 
Immediately after ingestion, the operator will press the "continue" button and activate the 

Exalenz Bioscience Ltd. 
Last update on: Nov 21, 2016 
Study Title: Clinical Study of the BreathID® MCS System to 
train the algorithm for the ¹³C-Methacetin Breath Test (MBT) in 
assessment of Portal Hypertension in Patients with 
Compensated Liver Cirrhosis 
Protocol No.: CSPH-EX-0414 
Version: 1.5 
Document No: CSD00076 
Page 72 of 72 
 
Exalenz Bioscience Ltd.     
 
CONFIDENTIAL AND PROPRIETARY 
actual measurement. CO2 production with 13C may be visible within a few minutes in 
cases with relatively functional livers. 
Note: In rare cases the administration of fluids may cause vomiting. If this happens the 
test should be aborted and repeated the next day. The expected adverse event should be 
reported in the appropriate CRF section. 
4. The patient should remain in a seated position breathing in a normal manner for up to 
75 minutes, whilst data are collected.  
5. The BreathID® MCS device continuously measures and analyzes the patient’s exhaled 
breath in real time. As the 13C-Methacetin is metabolized, the value of the 13CO2/12CO2 
ratio in the exhaled breath will change and will be calculated in real time by the 
BreathID® MCS device. The device will prompt the operator to enter patient details such 
as weight and height, age and gender. The BreathID® MCS device will calculate the MBT 
variables.  
6. If at any time the device does not detect patient’s breath, or if there is any other deviation 
from the desired test requirements, the device will produce an appropriate warning signal. 
7. At the completion of the procedure, the cannula is removed and the patient is 
disconnected from the BreathID® MCS device. 
The patient will be under the supervision of the physician or any other qualified medical staff 
during the entire test.  
The operators will be trained how to terminate the breath test early. In the following situations, 
the MBT test will be terminated and a test termination form will be completed: 
1. The patient vomits.  
2. The patient is inadvertently disconnected. 
3. The BreathID® MCS device malfunctions (In this case, the operator will complete a 
technical complaint form in addition to the test termination form and contact Exalenz 
immediately for further instructions)  
In all these cases, the next MBT cannot be repeated the same day. An entry will be made in the 
drug/kit accountability log and the bottles will be kept for inspection by the study monitor.