The HepQuant SHUNT Test for Monitoring Liver Disease and Treatment Effects by Measuring Liver Function and Physiology
Phase: PHASE2
Conditions: NASH - Nonalcoholic Steatohepatitis
Sponsor: HepQuant, LLC

HepQuant, LLC 
1 
The HepQuant SHUNT Test for Monitoring Liver Disease and 
Treatment Effects by Measuring Liver Function and Physiology 
Protocol Version: 
Protocol Date: 
Protocol ID: 
NCT03294941 
Original Version 5.0 
Amended Version 6.0 
Original Date September 5, 2017 
Amendment 1 Date May 15, 2018 
HQ-US-SHUNT-1701 
Co-enrollment Clinical Trials: 
GS-US-384-1943 (STELLAR 3) 
GS-US-384-1944 (STELLAR 4) 
GS-US-454-4378 (NASH F3/F4) 
Author: 
Gregory T. Everson, MD  
NCT Number:

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Clinical Investigator and Clinical Testing Site 
Study Title:  The HepQuant SHUNT Test for Monitoring Liver Disease and Treatment Effects by Measuring 
Liver Function and Physiology 
Principal Investigator:  
Dr R Bhamidimarri, Dr N Alkhouri, Dr A Sanyal, Dr R Reddy, Dr M Fuchs, Dr K Bhamba, Dr P Mantry, Dr J Trotter   
Dr S Harrison, Dr M Waters       
Sponsor:  
HepQuant LLC 
Device: 
HepQuant SHUNT Liver Diagnostic Kit 
Funding and  
Investigational device  
Provided by:  
HepQuant LLC 
Indication:
Monitoring Liver Disease and Treatment Effects 
Parallel Drug Trials: 
GS-US-384-1943 / Gilead Sciences; 
IND 129570 
GS-US-384-1944 / Gilead Sciences; 
IND 129570 
GS-US-454-4378 / Gilead Sciences; 
INDs Pending 
Compounds used in HepQuant SHUNT Test Kit: 
[24-13C]cholic acid (13C-cholate or 13C-CA) 
[2,2,4,4-2H(D)]cholic acid (4D-cholate or 4D-CA) 
Drug used in the Clinical Drug Trials: 
Selonsertib; GS-0976; GS-9674 
IDEs (HepQuant): 
G170034-S001 for GS-US-384-1943 (NASH F3, STELLAR 3) 
G170034-S002 for GS-US-384-1944 (NASH F4, STELLAR 4) 
Pending for GS-US-454-4378 (NASH F3 / F4) 
Protocol ID: 
HQ-US-SHUNT-1701 
Protocol Version: 
Original Version 5.0 
Amended Version 6.0 
Date of protocol: 
Original date September 5, 2017 
Amendment 1 date May , 2018 
Participating institutions: 
Schiff Center for Liver Diseases at the University of Miami 
Texas Liver Institute 

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Virginia Commonwealth University 
University of Pennsylvania 
Hunter Holmes McGuire VA Medical Center 
University of Washington 
Methodist Dallas  
Baylor University 
eStudy Site 
Pinnacle Clinical Research 
 
 
 
IDE Compliance: This study will be conducted under US Food & Drug Administration IDE regulations (21 CFR Part 
812). 
GCP Compliance:  This study will be conducted in compliance with Good Clinical Practice, and applicable 
regulatory requirements. 
Confidentiality Statement:  The information in this document contains trade secrets and commercial information 
that are privileged or confidential and may not be disclosed unless such disclosure is required by applicable law or 
regulations.  In any event, persons to whom the information is disclosed must be informed that the information is 
privileged or confidential and may not be further disclosed by them. These restrictions on disclosure will apply 
equally to all future information supplied to you that is indicated as privileged or confidential. 
 
 

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Table of Contents  
 
Title Page 
 
 
 
 
 
 
 
 
 
 
1 
 
Clinical Investigator and Clinical Testing Site  
 
 
 
 
 
2 
 
Table of Contents 
 
 
 
 
 
 
 
 
 
4 
 
Synopsis 
 
 
 
 
 
 
 
 
 
 
7 
 
HepQuant Contact Information 
 
 
 
 
 
 
 
11 
 
Glossary of Abbreviations 
 
 
 
 
 
 
 
 
12 
 
1.0 
Introduction 
 
 
 
 
 
 
 
 
 
14 
 
 
1.1 
Parallel Study Design  
 
 
 
 
 
 
14 
 
 
1.1.1 
Use of the HepQuant SHUNT test in Clinical Trials 
 
 
14 
 
 
1.1.2 
The HepQuant Study as a Parallel Study 
 
 
 
14 
 
 
1.1.3 
Coordination and Sponsorship 
 
 
 
 
15 
 
 
1.1.4 
Co-Enrollment  
 
 
 
 
 
 
15 
 
1.2 
The HepQuant SHUNT Test 
 
 
 
 
 
 
16 
 
1.3 
The Gilead Clinical Trials 
 
 
 
 
 
 
16 
 
2.0 
Primary and Secondary Objectives 
 
 
 
 
 
 
17 
 
 
2.1 
Primary Objective 
 
 
 
 
 
 
 
17 
 
2.2 
Secondary Objective  
 
 
 
 
 
 
18 
 
3.0 
Background and Significance  
 
 
 
 
 
 
19 
 
 
3.1 
The HepQuant SHUNT Test 
 
 
 
 
 
 
19 
 
 
3.1.1 
Overall Experience with the HepQuant SHUNT Test  
 
19 
 
 
3.1.2 
Test Administration 
 
 
 
 
 
 
19 
 
 
3.1.3 
Test Analysis and Outputs 
 
 
 
 
 
19 
 
 
3.1.4 
The HepQuant SHUNT Liver Diagnostic Kit 
 
 
 
20 
 
 
3.1.5 
Unmet Need 
 
 
 
 
 
 
 
21 
 
 
3.1.6 
Measuring Hepatocyte Function 
 
 
 
 
21 
 
 
3.1.7 
Measuring the Portal Circulation 
 
 
 
 
22 
 
 
3.1.8 
Measuring Liver Disease Severity by the HepQuant SHUNT Test 
23 
 
 
3.1.9 
Measuring Treatment Effect by the HepQuant SHUNT Test 
 
23 
 
3.2 
Selonsertib 
 
 
 
 
 
 
 
 
24 
 
 
3.2.1 
ASK-1 Inhibitor 
 
 
 
 
 
 
24 
 
 
3.2.2 
GS-0976 (ACC inhibitor) 
 
 
 
 
 
26 
 
 
3.2.3 
GS-9674 (FXR inhibitor) 
 
 
 
 
 
27 
 
 
 
3.2.4 
The Combination of Selonsertib with either GS-0976 or GS-9674 
28 
 
3.3 
Non-Alcoholic Steatohepatitis (NASH) 
 
 
 
 
28 
 
 
3.3.1 
Burden of NAFLD and NASH  
 
 
 
 
28 
 
 
3.3.2 
Clinical Presentation of NAFLD and NASH 
 
 
 
29 
 
 
3.3.3 
Diagnosis of NAFLD and NASH 
 
 
 
 
29 
 
 
3.3.4 
Measuring Disease Severity in NAFLD and NASH 
 
 
29 

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3.3.5 
Treatment of NASH 
 
 
 
 
 
 
30 
 
 
3.3.6 
Pitfalls in Measuring Treatment Effects in NASH 
 
 
30 
 
 
3.3.7 
The HepQuant SHUNT Test in NASH  
 
 
 
31 
 
4.0 
Preliminary Results 
 
 
 
 
 
 
 
 
32 
 
4.1 
Measuring Disease Severity by the HepQuant SHUNT Test 
 
 
32 
 
 
4.1.1 
Prior Studies  
 
 
 
 
 
 
32 
 
4.2 
Measuring Treatment Effects by the HepQuant SHUNT Test  
 
32 
 
 
4.2.1 
Prior Studies  
 
 
 
 
 
 
32 
 
 
4.2.2 
SOLAR 1 study 
 
 
 
 
 
 
33 
 
 
4.2.3 
HALT-C and PSC studies 
 
 
 
 
 
33 
 
5.0 
Research Plan  
 
 
 
 
 
 
 
 
34 
 
5.1 
Subjects 
 
 
 
 
 
 
 
 
34 
 
5.2 
Inclusion/Exclusion Criteria 
 
 
 
 
 
 
35 
 
5.3 
Study Design  
 
 
 
 
 
 
 
35 
 
 
5.3.1 
Subjects and Dosing of Selonsertib 
 
 
 
 
35 
 
 
5.3.2 
Timing of HepQuant SHUNT testing 
 
 
 
 
35 
 
 
5.3.3 
Treatment and procedures 
 
 
 
 
 
37 
 
 
5.3.4 
Administration of the HepQuant SHUNT Test  
 
 
38 
 
 
5.3.5 
Procedures by Study Visit 
 
 
 
 
 
38 
 
6.0 
Descriptions, Risks, Justifications 
 
 
 
 
 
 
42 
 
 
6.1 
Drug Safety Definitions 
 
 
 
 
 
 
42 
 
6.2 
Test Administration Risks 
 
    
 
 
 
             45 
 
6.3 
Cholates 
 
 
 
 
 
 
 
 
45 
 
 
6.3.1 
Stable Isotope labels (13C and 4D) 
 
 
 
 
45 
 
 
6.3.2 
Manufacturing of Labeled Cholates 
 
 
 
 
46 
 
 
6.3.3 
Formulations  
 
 
 
 
 
 
46 
 
 
6.3.4 
Tolerability and Safety  
 
 
 
 
 
47 
 
 
6.3.5 
Unknown / Unexpected Risks  
 
 
 
 
48 
 
 
6.3.6 
Reporting 
 
 
 
 
 
 
 
48 
 
6.4 
Albumin 
 
 
 
 
 
 
 
 
49 
 
 
6.4.1 
Use of Albumin in the HepQuant SHUNT Test  
 
 
49 
 
 
6.4.2 
Reported Reactions to Human serum Albumin 
 
 
49 
 
 
      6.4.2.1     Hypersensitivity  
 
 
 
 
 
49 
 
 
      6.4.2.2     Risk of Transmissible Agents      
 
 
 
50 
 
6.5 
Intravenous Catheter  
 
 
 
 
 
 
50 
6.6 
Other  
 
 
 
 
 
 
 
 
51 
6.6.1 
Data Security  
 
 
 
 
 
 
51 
 
 
6.6.2 
Data Security Protocol 
 
 
 
 
 
51 
 
 
6.6.3 
Management of Abnormal Test Results 
 
 
 
54 
 
 
6.6.4 
HepQuant Study Versus Gilead Clinical Trial  
 
 
54 
 
6.7 
Pregnancy Protocol 
 
 
 
 
 
 
 
55 
 
7.0 
Potential Scientific Problems  
 
 
 
 
 
 
55 
 
7.1 
Potential Interfering Substances in Lab Assay 
 
 
 
55 
 
 
7.1.1 
Assay Performance                     
 
 
 
 
55 
 
7.2 
Potential Complicating Medical Issues 
 
 
 
 
56 
 
 
7.2.1 
Concomitant Medications 
 
 
 
 
 
56 
 
 
7.2.2 
Associated Conditions 
 
 
 
 
 
56 

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7.3 
Potential Interaction between Cholate and Selonsertib 
 
 
56 
 
8.0 
Data Analysis and Statistics  
 
 
 
 
 
 
56 
 
8.1 
Sample Size Calculations 
 
 
 
 
 
 
56 
 
8.2 
Primary Objective 
 
 
 
 
 
 
 
57 
 
 
8.2.1 
Outline of Approach 
 
 
 
 
 
 
57 
 
 
8.2.2 
Comparators from Gilead Trial 
 
 
 
 
58 
 
 
8.2.3 
Statistical Considerations 
 
 
 
 
 
59 
 
8.3 
Secondary Objectives  
 
 
 
 
 
 
60 
 
 
8.3.1 
Outline of Approach 
 
 
 
 
 
 
60 
 
 
8.3.2 
Correlating baseline tests of liver disease severity 
 
 
60 
 
 
8.3.3 
Baseline DSI as predictor of clinical outcome  
 
 
60 
 
9.0 
Summary 
 
 
 
 
 
 
 
 
 
61 
 
10.0 
References 
 
 
 
 
 
 
 
 
 
63 
 
11.0 
Appendices 
 
 
 
 
 
 
 
 
 
69 
 
Appendix A:  HepQuant IDEs (letters from FDA) 
 
 
 
 
70 
 
Appendix B:  IFU for HepQuant Liver Diagnostic Kit  
 
 
 
81 
 
Appendix C:  PRO Questionnaire 
 
 
 
 
 
 
90 
 
Appendix D:  Flow Charts of Study Procedures 
 
 
 
             92 
 
Appendix E:  Investigator Agreement  
 
 
 
 
             97 
 
Appendix F:  IB for HepQuant Liver Diagnostic Kit 
 
 
 
             99  
 
 
 
 
 
 
 
 
 
 

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Protocol Synopsis 
 
Study Title:    
The HepQuant SHUNT Test for Monitoring Liver Disease and Treatment 
Effects by Measuring Liver Function and Physiology 
 
Sponsor: 
HepQuant LLC 
 
Device: 
HepQuant SHUNT Liver Diagnostic Kit 
 
Funding BY: 
HepQuant LLC  
Device Provided by: 
HepQuant LLC 
 
Protocol: 
ID: 
 
HQ-US-SHUNT-1701 
 
 
Version: 
Original Version 5.0 
 
 
 
 
Amended Version 6.0 
 
 
Date: 
 
Original date September 5, 2017 
 
 
 
 
Amendment date May 15, 2018 
 
Compounds used in HepQuant SHUNT Test Kit: 
[24-13C]cholic acid (13C-cholate or 13C-CA) 
 
 
 
 
 
 
 
[2,2,4,4-2H(d4)]cholic acid (d4-cholate or d4-CA) 
 
Indication for Use: 
 
Monitoring effects of Liver Disease and Treatment on Liver Function 
 
Gilead Parallel Clinical Trials: GS-US-384-1943 (STELLAR 3) / Gilead Sciences:  
IND 129570 
 
 
 
 
GS-US-384-1944 (STELLAR 4) / Gilead Sciences;  
IND 129570 
 
 
 
 
GS-US-454-4378 (NASH F3/F4) / Gilead Sciences; 
INDs pending 
 
Drug used in the Parallel Drug Trials:  
 
Selonsertib, GS-0976, and GS-9674 
 
IDE Numbers (for use of the HepQuant SHUNT Test Kit in subjects co-enrolled in the Parallel Drug Trials): 
 
 
    
 
G170034-S001 for GS-US-384-1943 (STELLAR 3) 
 
 
 
 
G170034-S002 for GS-US-384-1944 (STELLAR 4) 
 
 
 
 
IDE pending for GS-US-454-4378 (NASH F3 / F4) 
 
Study Centers/PIs: 
This HepQuant SHUNT study is run in parallel to three Gilead clinical trials. Subjects are 
simultaneously participating in this HepQuant SHUNT study while participating in a Gilead-sponsored clinical trials 
for treatment of NASH. Because the HepQuant study will focus on the subjects with more advanced liver disease, 
the clinical sites participating in the HepQuant study must also be participating in one of the NASH F3 (STELLAR 
3), NASH F4 (STELLAR 4), or GS-US-454-4378 (NASH F3 and F4) studies. The maximum number of clinical sites 
is 15 and the maximum number of study subjects is 100. 
 

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Primary Objective: 
To determine the utility of the Disease Severity Index (DSI) from the HepQuant SHUNT 
test for monitoring liver disease and treatment effects, DSI and the change in DSI (∆DSI) will be measured across 
all treatment arms. 
 
 
The clinical significance of ∆DSI will be evaluated by analyzing the relationships of ∆DSI to changes in 
standard laboratory tests, clinical models, histologic stage of disease, and risk for clinical outcome.   
 
We will also evaluate whether the HepQuant SHUNT test and DSI identify a treatment effect in the 
combined NASH F3 + NASH F4 cohorts, after unblinding of treatment assignments and completion of the primary 
analysis for Gilead’s clinical trials.  Treatment effect, as measured by DSI and ∆DSI, will be defined from the 
differences in DSI between treatment and placebo arms; and, compared to the treatment effect defined by changes 
in histology, standard laboratory tests, clinical models and by clinical outcomes. 
 
Secondary Objectives:    To determine the ability of baseline DSI, performed prior to treatment, to assess the 
severity of liver disease by:  1. correlation with other baseline tests of liver disease severity; and, 2. prediction of 
risk for future clinical outcome. 
 
Study Design: 
Open Label HepQuant SHUNT Testing in All Subjects 
 
 
 
STELLAR 3:   
Baseline and Weeks 24, 48, and 240 
 
 
 
 
STELLAR 4:   
Baseline and Weeks 24, 48, and 240 
 
GS-US-454-4378 (NASH F3 / F4):  Baseline and Weeks 24 and 48 
 
Subjects, N: 
Up to a total of 100 for all trials 
Clinical Sites, N: 
Up to 15 
 
Target Populations: 
NASH F3: 
NASH Clinical Research Network (CRN) Fibrosis Stage 3 
 
NASH F4: 
NASH CRN Fibrosis Stage 4 (compensated cirrhosis) 
 
Compounds used in HepQuant SHUNT Test Kit: 
[24-13C]cholic acid, 20 mg, intravenous injection 
 
 
 
 
 
 
 
[2,2,4,4-2H(D)]cholic acid, 40 mg, oral solution 
 
Drug Treatment: 
STELLAR 3: 
 
Selonsertib vs Placebo 
 
STELLAR 4: 
 
Selonsertib vs Placebo 
 
GS-US-454-4378 (NASH F3/F4): Selonsertib, GS-0976, GS-9674 alone or in combination 
versus placebo. 
 
Duration of Drug Treatment:  
STELLAR 3: 
 
 
 
240 weeks 
 
 
 
STELLAR 4: 
 
 
 
240 weeks 
 
 
 
GS-US-454-4378 (NASH F3/F4):  
48 weeks 
 
Duration of Study and Drug Trial: 
STELLAR 3: 
 
 
 
252 weeks 
 
 
 
STELLAR 4: 
 
 
 
252 weeks 
 
 
 
GS-US-454-4378 (NASH F3/F4): 
52 weeks 
 

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Diagnostic and Eligibility Criteria: 
Enrollment in the corresponding Gilead Trial, listed above 
 
 
 
Intravenous access for catheter placement 
 
 
 
Able to take orally administered solution 
 
 
 
No known allergy to any component of the HepQuant SHUNT test kit 
 
 
 
Procedures and Frequency of the HepQuant SHUNT Testing: 
 
 
 
 
 
 
 
STELLAR 3: 
 
 
 
Baseline and Weeks 24, 48, and 240 
 
 
 
STELLAR 4: 
 
 
 
Baseline and Weeks 24, 48, and 240 
 
 
 
GS-US-454-4378 (NASH F3/F4): 
Baseline and Weeks 24 and 48 
 
Dose and Mode of Administration of Drug used in Drug Trial: 
 
 
 
 
 
 
 
STELLAR 3 and 4: 
Selonsertib 6 or 18 mg orally daily 
 
 
 
GS-US-454-4378: 
Selonsertib 18 mg, GS-0976 20 mg, or GS-9674 30 mg for both  
 
 
 
 
 
 
monotherapy and combinations; Orally; Daily dosing 
 
Reference Therapy in Drug Trial, Dose, Mode of Administration: 
Placebo tablets; Orally; Daily 
 
 
 
 
Criteria for Evaluation (HepQuant test parameters are the same for all studies): 
 
 
 
 
Baseline DSI: DSI measured prior to randomization 
 
 
 
∆DSIi:  
Change in DSI from baseline to time point, i 
 
 
 
 
 
Treatment Effect:  ∆DSIi Treatment versus ∆DSIi Placebo 
 
Primary Clinical Outcomes to link to baseline DSI and ∆DSIi: 
 
 
 
 
 
STELLAR 3: 
Wk48: 1 Stage improvement in fibrosis without worsening NASH 
 
 
 
 
Wk48: Freedom from progression to cirrhosis 
 
 
 
 
Wk48: Freedom from decompensation, transplant, or death 
 
 
 
 
Wk240: 1 Stage improvement in fibrosis without worsening NASH 
 
 
 
 
Wk240: Freedom from progression to cirrhosis 
 
 
 
 
Wk240: Freedom from decompensation, transplant, or death 
 
 
 
STELLAR 4: 
Wk48: 1 Stage improvement in fibrosis without worsening NASH 
 
 
 
 
Wk48: Freedom from decompensation, transplant, or death 
 
 
 
 
Wk240: 1 Stage improvement in fibrosis without worsening of NASH 
 
 
 
 
Wk240: Freedom from decompensation, transplant, or death 
 
 
 
GS-US-454-4378:   Endpoints as above for F3 and G]F4 cases but only for 48 Weeks 
 

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Statistical Methods: 
 
We will determine the utility of the Disease Severity Index (DSI) from the HepQuant SHUNT test to monitor liver 
disease and treatment effects by measuring liver function and physiology.  We propose a three-step analytical 
process to achieve this goal.  First, without knowledge of treatment arm, we will track DSI and the change in DSI, 
∆DSI, from baseline to on-treatment and follow-up timepoints. Second, we will apply separate survival models to 
evaluate ∆DSI as a continuous variable and a 2 point or more change in DSI as dichotomous variable in the 
prediction of changes in other tests or models of liver disease severity and clinical outcomes.  In HCV, an increase 
in DSI of 2 or more has been associated with disease progression; and, a decrease in DSI of 2 or more has been 
associated with improvement after viral clearance.  Third, once the treatment arms have been unblinded we will 
evaluate if the use of the HepQuant SHUNT test and DSI identify a treatment effect in the combined cohort of 
NASH F3 plus NASH F4; and we will compare DSI to the primary measures of efficacy in each of the clinical trials.  
In terms of DSI, a treatment effect will be defined from the difference between the ∆DSI of the treatment arm versus 
the ∆DSI of the placebo arm.  The significance of this difference will be evaluated using a two-sided t-test.  
Furthermore, we will link treatment effect defined by DSI to effects of treatment on other tests and clinical outcomes 
(bleeding from varices or portal hypertensive gastropathy, ascites, encephalopathy, SBP, progression to cirrhosis, 
and patient mortality or liver transplantation).  We will use both generalized estimating equations (GEE) and profile 
analysis – profile of DSI between treatment and placebo groups at each time point – for additional primary endpoint 
analyses.   
 
We will also determine the ability of baseline DSI, performed prior to treatment, to assess the severity of liver 
disease by:  1. correlation with other baseline tests of liver disease severity; and, 2. prediction of risk for future 
clinical outcome. 
 
Correlation of Baseline DSI with other baseline tests: 
 
 
 
 
 
 
 
STELLAR 3: 
 
Liver histology; standard labs; MELD & CP scores 
 
 
 
STELLAR 4: 
 
Liver histology, standard labs, MELD & CP scores 
 
 
 
GS-US-454-4378:  
As above for F3 and F4 disease 
 
In an analysis of the combined NASH F3 and F4 cohorts, we will correlate the baseline DSI with histology, lab tests, 
and clinical models by linear regression and Spearman’s correlation coefficients. Regression analysis will be used 
to obtain prediction and 95% CI of baseline B-K fibrosis score, standard lab tests, and clinical models based on 
DSI.  In a secondary analysis of the combined NASH trials, we will use a discriminant analysis to compare the 
ability of baseline DSI to distinguish NASH F3 from NASH F4 to the ability of other tests and models to discriminate 
F3 from F4. 
 
Baseline DSI as Predictor of Risk for future clinical outcome: 
We will perform survival analysis for freedom from clinical outcome using Kaplan-Meier plots.  Cox models will be 
used to estimate hazard ratios corresponding to 1 unit of DSI, controlling for other known risk factors of mortality 
(age, gender, smoking, etc).  We will also apply separate survival models to evaluate DSI as a continuous variable 
and as above or below mean DSI as dichotomous variable in the prediction of clinical outcomes.  In a secondary 
exploratory analysis of the combined NASH trials, we will use a discriminant analysis to compare the ability of 
baseline DSI to the ability of other tests and models to predict clinical outcomes. 
 
 
 

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HepQuant Contact Information 

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Glossary 
β-hCG β-human chorionic gonadotropin 
AE adverse event 
AH alcoholic hepatitis 
ALT alanine aminotransferase (also SGPT) 
ANC absolute neutrophil count 
APTT activated partial thromboplastin time 
AST aspartate aminotransferase (also SGOT) 
BID twice a day 
BLQ below the limit of quantitation 
BMI body mass index 
BW body weight 
CI Confidence Interval 
CLD chronic liver disease 
CP Child-Pugh (for clinical/laboratory classification of cirrhosis) 
CrCL creatinine clearance 
CT Computed Tomography 
CPK Creatine phosphokinase 
Cr serum creatinine 
CRF case report form(s) 
CYP Cytochrome P450 
dL Deciliter 
DNA deoxyribonucleic acid 
DSI Disease Severity Index (from HepQuant SHUNT test) 
DSPH Drug Safety and Public Health 
ECG Electrocardiogram 
eCRF Electronic case report form(s) 
EOT End of Treatment 
EU European Union 
FDA (United States) Food and Drug Administration 
GCP Good Clinical Practice (Guidelines) 
GGT gamma glutamyl transferase 
GI Gastrointestinal 
GT Genotype (viral) 
Hgb Hemoglobin 
HgbA1c Hemoglobin A1c 
HBsAg hemoglobin surface antigen 
HBV Hepatitis B virus 
HCC Hepatocellular carcinoma 
HCV Hepatitis C virus 
HFR Hepatic Filtration Rate 
IB Investigator Brochure 
ICH International Conference on Harmonisation 
IDE Investigational device exemption 
IEC independent ethics committee 
IMP Investigational Medicinal Product 
IND Investigational New Drug (Application) 
INR International Normalized Ratio of prothrombin time 
IRB institutional review board 

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IUD intrauterine device 
IU international units 
IV Intravenous 
IWRS interactive web response system 
Kg Kilogram 
L Liter 
LDL low-density lipoprotein 
LLN lower limit of the normal range 
LLOQ Lower limit of quantification 
LLT Lower-Level Term 
MCV mean corpuscular volume or mean cell volume 
MedDRA Medical Dictionary for Regulatory Activities 
mg Milligram 
mM Millimeter 
MH Mantel-Haenszel 
mL Milliliter 
min Minute 
mmHg millimeters mercury 
NAFLD Non-Alcoholic Fatty Liver Disease 
NASH Non-Alcoholic Steato-Hepatitis 
OTC Over the counter 
P-gp P-glycoprotein 
PG Pharmacogenomic 
PH portal hypertension 
PI Protease inhibitor 
PO by mouth 
QD once daily (use only in tablets) 
PK Pharmacokinetic 
PSC Primary sclerosing cholangitis 
PT prothrombin time 
RBC red blood cell count 
RBV Ribavirin 
RNA ribonucleic acid 
RVR rapid virologic response 
SAE serious adverse event 
SD Standard deviation 
SEL selonsertib 
SMV Olysio/Simeprevir  
SOC Standard of Care 
SOF Sovaldi/Sofosbuvir 
SOP Standard operating procedure 
SUSAR Suspected Unexpected Serious Adverse Reaction 
SVR Sustained Virologic Response 
SVR 4 Sustained viral response 4 weeks after discontinuation of study treatment 
SVR 12 Sustained viral response 12 weeks after discontinuation of study treatment 
SVR 24 Sustained viral response 24 weeks after discontinuation of study treatment 
TVR Telaprevir 
US United States 
WBC white blood cell count 
 
 

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1.0 Introduction 
 
1.1 
Parallel Study Design 
 
1.1.1 Use of the HepQuant SHUNT Test in Clinical Trials 
 
The HepQuant SHUNT test, which is provided as a HepQuant SHUNT Liver Diagnostic 
Kit, is a minimally-invasive test of liver function and physiology which has been 
designated by the FDA as an investigational drug/device combination product.  Because 
the HepQuant SHUNT test is an investigational product, its use in clinical trials requires 
an investigational device exemption (IDE) issued from FDA.  HepQuant has already 
secured IDEs for performing the HepQuant SHUNT test in subjects enrolled in Gilead’s 
clinical trials.  HepQuant’s IDE numbers for the Gilead trials are: 
 
 
 G170034-S001 for the GS-US-384-1943 trial of non-alcoholic 
steatohepatitis (NASH) fibrosis stage 3 (STELLAR-3),  
 
 G170034-S002 for the GS-US-384-1944 trial of NASH and compensated 
cirrhosis (STELLAR-4), and 
 
 IDE pending for the GS-US-454-4378 trial of NASH fibrosis stage 3 and 
NASH fibrosis stage 4. 
 
 
1.1.2 The HepQuant study as a Parallel Study 
 
In this HepQuant study the HepQuant SHUNT test will be performed in subjects enrolled 
in the three Gilead trials noted above.  The HepQuant study protocol, informed consent, 
and documents govern the administration, monitoring, and analyses of the HepQuant 
SHUNT test.   
 
The HepQuant study is to run parallel to the Gilead clinical trials.  The time points for the 
HepQuant SHUNT tests coincide with pre-specified time points within the Gilead clinical 
trials.  Subjects enrolled in GILEAD’s STELLAR and GS-US-454-4378 (NASH F3/F4) 
trials may participate concurrently in this HepQuant sponsored investigational device 
study at participating US sites only once approved by the applicable IRB/IEC. 

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1.1.3 Coordination and Sponsorship 
 
The main eligibility criteria for enrollment into the HepQuant study is enrollment into one 
of the three Gilead clinical trials listed above in Section 1.1.1 and criteria noted in Section 
5.2.  A participant in the HepQuant study will consent to one of the Gilead clinical trials 
and the HepQuant study at the same visit or when the subject qualifies for one of the 
above listed Gilead studies.  Enrollment into one of the Gilead clinical trials is required for 
enrollment into the HepQuant study. 
 
The HepQuant study covers all procedures, oversight, and monitoring related to the 
HepQuant SHUNT test.  HepQuant LLC is the sole Sponsor of the HepQuant study.  
Gilead has contracted with HepQuant for the results of all the HepQuant tests performed 
in the subjects enrolled in the Gilead trials. 
 
All other aspects of Gilead’s clinical trials are covered under Gilead’s main protocols and 
contracts with clinical sites.  Gilead Sciences is the sponsor of the three clinical trials 
noted above.  HepQuant test results will be sent to Gilead and Gilead will conduct all 
correlation analyses with other parameters being collected in the Gilead protocols.  
HepQuant will request the results of these analyses from Gilead.   
 
1.1.4 Co-enrollment 
 
Subjects enrolled in this HepQuant study of the diagnostic utility of the HepQuant SHUNT 
test are co-enrolled in a Gilead clinical trial of treatment with selonsertib.  Justification for 
co-enrollment includes: 
 Complementary, not competing, interests and endpoints for the two studies 
 Collaboration agreement between HepQuant and Gilead 
 Independent informed consents 
 Gilead will conduct correlation analysis of study results 
 
Co-enrollment into the two studies does not violate any of the ethical principles of respect 
for persons, beneficence, and justice that guide research involving human subjects. 
 
 
 
 

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1.2 
Rationale for Conducting this Study 
 
Why conduct this research study?  There is an unmet medical need for a non-invasive or 
minimally-invasive test that accurately measures liver function and physiology.  The HepQuant 
SHUNT Test is minimally-invasive and measures hepatocyte function and inflow to the liver from 
the simultaneous clearances (hepatic filtration rates, HFRs) of cholate from systemic and portal 
circulations.  The Test quantifies portal-systemic shunting (SHUNT) and generates a liver 
disease severity index (DSI) [1].  DSI is a score from 0 (no disease) to 50 (terminal illness) that 
is a composite of both HFRs and correlates with stage of fibrosis, presence of varices, 
especially large varices, and risk for future clinical outcomes [2-8].  DSI is the primary output 
variable from the HepQuant SHUNT test.  The HepQuant SHUNT test potentially satisfies the 
unmet medical need for a minimally-invasive test of global liver function and physiology. 
 
HepQuant believes that DSI from baseline and serial HepQuant SHUNT tests will be useful in 
defining baseline disease severity and that the change in DSI, ∆DSI, will detect clinically 
meaningful changes in liver disease and responses to treatment. 
 
1.3 
The Gilead Clinical Trials 
 
As stated above, HepQuant believes that baseline DSI and change in DSI, ∆DSI, are useful for 
monitoring liver disease and treatment effects.  HepQuant proposes collaborative parallel 
studies with pharmaceutical drug trials to validate DSI and ∆DSI for monitoring. 
 
The Gilead trials are double-blind, randomized, placebo-controlled clinical trials – the HepQuant 
study will be run in parallel and coordinated with three Gilead trials: 
 
1. 
GS-US-384-1943 (STELLAR-3); subjects with non-alcoholic steatohepatitis (NASH) 
and bridging (F3) fibrosis 
2. 
GS-US-384-1944 (STELLAR-4); subjects with NASH and compensated cirrhosis (F4) 
3. 
GS-US-454-4378; subjects with NASH and bridging (F3) fibrosis or compensated 
cirrhosis (F4) 
 
The Gilead IND for selonsertib is 129570. The INDs for GS-0976 and GS-9674 are pending.  
This is provided to HepQuant for cross-referencing.  HepQuant’s letters of FDA’s issuance of 
the IDEs for each of the trials are provided in Appendix A. 
 

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2.0 Primary and Secondary Objectives 
 
As described in Section 1.2, the Disease Severity Index (DSI) from the HepQuant SHUNT test is 
a score from 0 to 50 based upon the dual simultaneous clearance of cholates (CAs) from 
systemic (13C-CA) and portal circulations (4D-CA).  DSI is the primary output from the 
HepQuant SHUNT test; and, both DSI and change in DSI (∆DSI) may be useful in monitoring 
liver disease and treatment effects. 
 
2.1 
Primary Objective 
 
The primary objective is to determine the utility of the Disease Severity Index (DSI) 
derived from the HepQuant SHUNT test, which measures liver function and physiology, 
for monitoring liver disease and treatment effects. 
 
 Utility will be defined by measuring DSI and the change in DSI (∆DSI) across all 
treatment arms.  DSI and ∆DSI will be evaluated in real time. 
 
 The clinical significance of changes in DSI will be evaluated by analyzing the 
relationship of ∆DSI to changes in histologic stage of disease, standard laboratory 
tests, clinical models, or risk for clinical outcome.  Some of the proposed 
comparative analyses will only be performed after unblinding of treatment arms 
and completion of analyses for the main Gilead trials and may be conducted by 
Gilead. 
 
 Treatment effects will only be determined after unblinding the treatment arms and 
completion of the analyses for the main Gilead trials.  In terms of DSI, treatment 
effects will be determined from the differences in DSI and ∆DSI between treatment 
and placebo arms. The analyses will be conducted by Gilead. 
 
Clinically-meaningful outcomes.  As noted above, the subjects will be followed for 
clinical outcomes in the Gilead trials.  In the case of STELLAR 3 and 4, the follow-up for 
clinical outcomes (bleeding from varices or portal hypertensive gastropathy, ascites, 
encephalopathy, liver-related death) will be up to 240 Wks – the ∆DSI associated with 
development of clinical outcome at both Wk48 and Wk240 will be analyzed. In the case 
of GS-US-454-4378, the follow-up for clinical outcomes will be for 48 Weeks. 
 

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Unblinding Treatment Arms.  Each of the Gilead NASH trials is placebo-controlled.  
Once the allocation code for the Gilead clinical trial is unblinded, HepQuant will request 
the treatment allocation from Gilead in order to analyze the HepQuant SHUNT results in 
the context of treatment versus placebo.  The ∆DSI of the treatment arm will be 
compared to the ∆DSI of the placebo arm.  We anticipate that the ∆DSI in the placebo 
arm will either not change reflecting stability, or be positive, reflecting the natural 
progression of liver disease.  If treatment is effective the ∆DSI of the treatment arm 
should be negative – implying a lowering of disease severity and improvement in liver 
function and physiology.  A treatment effect based on DSI will be determined from 
comparison of ∆DSI of the treatment and control arms. 
 
2.2 
Secondary Objective 
 
The secondary objective is to determine the ability of baseline DSI, performed prior to 
treatment, to assess the severity of liver disease by: 
 
 correlation with other baseline tests of liver disease severity; and, 
 
 prediction of risk for future clinical outcome. 
 
Tests of liver disease severity to compare to baseline DSI.  Standard tests for 
assessing the severity of liver disease include liver histology (stage of fibrosis), standard 
blood tests (elevated bilirubin and INR, and reduced albumin and platelet count), and 
clinical models (MELD and CTP scores).  Subjects enrolled in the Gilead trials will 
undergo a battery of these tests, at baseline. Through requests to Gilead for specific data 
analyses, HepQuant’s baseline DSI will be compared to the baseline results of these 
other measurements, such as fibrosis score on liver biopsy, clinical laboratory tests and 
models, and other exploratory tests of stage of fibrosis (elastography [FibroScan, MRE] 
and biomarkers). 
 
Clinically-meaningful outcomes. The subjects will be followed for clinical outcomes.  In 
the case of F3/F4 NASH, the rates of development of clinical outcomes (progression to 
cirrhosis, bleeding from varices or portal hypertensive gastropathy, ascites, 
encephalopathy, liver-related death) will be relatively slow so the follow-up is up to 240 
weeks – the baseline DSI associated with development of clinical outcome at both Wk48 
and Wk240 will be analyzed.  
 

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3.0 Background and Significance 
 
3.1 
The HepQuant SHUNT Test 
 
3.1.1 Overall Experience with the HepQuant SHUNT Test 
 
The HepQuant SHUNT test is blood-based, minimally-invasive, well tolerated by 
subjects, simple to administer, and test administration does not require expensive 
equipment or technology.  Over 1400 tests have been performed in over 650 persons 
without adverse event related to the HepQuant SHUNT test. 
 
This experience encompasses healthy controls, and subjects with chronic hepatitis C, 
non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH), primary 
sclerosing cholangitis (PSC), hepatocellular carcinoma (HCC), polycystic liver disease, 
and spans the spectrum from minimal to advanced fibrosis and compensated to 
decompensated disease [1-21]. 
 
3.1.2 Test Administration 
 
The HepQuant SHUNT test is performed after an overnight fast or after at least 5 hours 
of fasting during the daytime.  The HepQuant SHUNT test requires venous access via a 
standard indwelling intravenous catheter, preferably placed in an antecubital vein.  An 
oral solution of 4D-CA and an injectable solution of 13C-CA mixed with 25% human 
serum albumin are administered simultaneously.  Blood samples are obtained at baseline 
and at 5±1, 20±2, 45±5, 60±5, and 90±5 minutes after dosing.  The serum is separated 
and sent to the HepQuant laboratory for quantification of concentrations of endogenous 
cholate, 4D-CA and 13C-CA.  Clearances are calculated from AUCs and dose and DSI 
and SHUNT are derived from the clearances. 
 
3.1.3 Test Analysis and Outputs 
 
The HepQuant SHUNT test outputs are: 
 
 Systemic HFR. The intravenous clearance (Cliv, mL min-1) is defined as the 
dose/AUC for 13C-CA. The Systemic Hepatic Filtration Rate (Systemic 

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HFR) is defined as the Cliv per kg of body weight and is expressed as mL 
min-1 kg-1. 
 
 Portal HFR.  The apparent oral clearance (Cloral, mL min-1) is defined as the 
dose/AUC for 4D-cholate. The Portal Hepatic Filtration Rate (Portal HFR) is 
defined as the Cloral per kg of body weight and is also expressed as mL  
min-1 kg-1. 
 
 SHUNT.  SHUNT, the portal-systemic shunt fraction, is calculated as the 
ratio Systemic HFR/Portal HFR x 100%. 
 
 DSI.  The calculation for Disease Severity Index is a proprietary formula 
derived from Systemic HFR and Portal HFR.  DSI 0 implies no hepatic 
impairment and is based on the means (+3 SDs) for Systemic and Portal 
HFRs of normal weight healthy volunteers.  DSI 50 represents severe 
hepatic impairment as measured in terminally ill CP (Child-Pugh) C cases 
with clinically advanced liver disease. Subjects with intermediate severity of 
liver disease have intermediate DSI scores. 
 
 STAT.  STAT is defined as the 60 minute d4-CA concentration from the 
SHUNT test, normalized to an ideal body weight of 75 kg.  STAT was found 
in previous studies to correlate closely with DSI (r2 = 0.88). Utilizing this 
single blood sample will also be evaluated as a simplified testing approach.  
 
For the purpose of the proposed analyses, DSI is considered the primary output variable 
from the HepQuant SHUNT test.  However, in exploratory analyses we will determine the 
performance of the other test parameters – Systemic HFR, Portal HFR, SHUNT, and 
STAT. 
 
3.1.4 The HepQuant SHUNT Liver Diagnostic Kit 
 
The test compounds, 13C-CA and d4-CA, and 25% human serum albumin are included 
in a HepQuant SHUNT Liver Diagnostic test kit with appropriate Instructions for Use.  In 
brief, the contents of the kit include: 
 
 Sealed vial of sterile solution of d4-CA (40 mg in 10 mL sodium 
bicarbonate) for oral use 

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 Sealed vial of sterile solution of 13C-CA (22 mg in 5.5 mL sodium 
bicarbonate) for intravenous use 
 Sealed vial of 20 mL of 25% human serum albumin for intravenous use – 5 
mL is added to 5 mL of the 13C-CA solution prior to intravenous injection 
 6 blood collection tubes (blood is allowed to clot and serum separated by 
centrifugation) 
 6 transfer tubes (for transport of serum to HepQuant designated lab) 
 Labels 
 Instructions for Use 
 Mailer 
 
3.1.5 Unmet Need 
 
The HepQuant SHUNT test fulfills an unmet medical need by providing a minimally-
invasive test of global liver function and physiology.  With progression, chronic liver 
disease (CLD) impairs hepatocyte function and the portal circulation - abnormalities 
which manifest as portal hypertension and portal-systemic shunting.  HepQuant SHUNT 
measures the changes occurring in both the liver’s function and the portal circulation. 
 
In recognition of the limitations of standard lab tests, liver biopsy, and HVPG, the trans-
NIH Action Plan for Liver Disease Research and the RFA for New Technologies for Liver 
Disease have clearly stated the need for “non-invasive means of assessing the liver” that 
would “obviate the need for liver biopsy in diagnosis, staging and grading of liver 
diseases” and that would “accurately reflect the stage of liver disease and can detect mild 
to moderate degrees of fibrosis before the onset of cirrhosis”. The minimally-invasive 
HepQuant SHUNT test fulfills most, if not all, of these criteria. 
 
3.1.6 Measuring Hepatocyte Function 
 
For decades, investigators have used tests of hepatic metabolism to quantify hepatic 
function – most are insensitive, lack sufficient reproducibility, and require complex 
detection systems or analytical methods.  However, none can measure the portal 
circulation or portal-systemic shunting – the latter being a key determinant of clinical 
complications of liver disease (varices, ascites and encephalopathy).  The 13C-
methacetin breath test is currently undergoing evaluation in trials; but it suffers from the 
same issues as other metabolic tests because it requires purchase of expensive bedside 

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equipment, and is influenced by gender, age, and use of many prescribed and OTC 
drugs or medications [22-25]. 
 
In head-to-head comparisons, the HepQuant SHUNT test performed favorably in 
comparison to a battery of metabolic tests in prediction of clinical outcomes.  The 
metabolic substrates used in the metabolic tests as comparators were caffeine, 
antipyrine, lidocaine-MEGX, galactose, 13C-methionine, and 14C-erythromycin [7,8]. 
 
Key differences in the HepQuant SHUNT test, compared to metabolic tests, may explain 
its superior performance: 
 HepQuant SHUNT is a dual clearance method tagging both systemic and 
portal inflow to the liver, i.e., it is a global clearance test. 
 HepQuant SHUNT uses cholate, a compound with multiple hepatic 
transporters and relatively high first-pass hepatic extraction. 
 Because multiple transporters are involved, a single nucleotide 
polymorphism in one transporter is not likely to influence overall cholate 
uptake. 
 Cholate uptake is not dependent upon hepatocyte metabolism -  drugs, 
medications, or other factors affecting cytochrome P450 metabolism. 
 By comparing portal to systemic clearance of cholate, HepQuant SHUNT 
quantifies portal-systemic shunting, a key variable associated with and 
reflecting the meaningful clinical outcomes of varices, ascites, and 
encephalopathy. 
 
3.1.7 Measuring the Portal Circulation 
 
Hepatic inflammation and fibrosis within the sinusoid and space of Disse increase the 
resistance to sinusoidal perfusion.  The increase in hepatic resistance to portal inflow 
raises portal pressure - as CLD progresses, fibrosis accumulates, further raising hepatic 
resistance and leading to portal hypertension.  The splanchnic bed adapts to the rise in 
portal pressure by vasodilatation and formation of portal-systemic collaterals leading to 
portal-systemic shunting [26]. 
 
Underlying portal hypertension (PH) is a risk factor for poor outcome in chronic liver 
disease (CLD).  PH can be measured directly by percutaneous puncture of the liver to 
access the portal vein or by threading a catheter from the jugular vein into the portal vein 
to measure the hepatic venous pressure gradient (HVPG).  Increased portal pressure or 

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HVPG correlates with risk of cirrhosis, varices, ascites, and decompensation.  However, 
these tests are invasive, time consuming, risky, cumbersome, expensive, require 
specialized equipment and expertise, and are not embraced by subjects [27-29]. 
 
HepQuant SHUNT is a global function test capable of monitoring hepatocellular function, 
total hepatic perfusion, portal inflow to the liver, and portal-systemic shunting.  Similar to 
HVPG, HepQuant SHUNT assesses the portal circulation.  In contrast to HVPG, 
HepQuant SHUNT accomplishes this minimally-invasively with high patient tolerability 
and lower cost. These characteristics suggest that HepQuant SHUNT could be a 
minimally-invasive alternative to HVPG in monitoring the effects of liver disease on the 
portal circulation.  In addition, these characteristics suggest that the HepQuant SHUNT 
test could be useful for detecting changes in response to treatments or interventions. 
 
3.1.8 Measuring Disease Severity by the HepQuant SHUNT Test 
 
In our prior studies of chronic hepatitis C, DSI correlated with ISHAK and METAVIR 
stages of fibrosis and predicted likelihood of cirrhosis, varices, and risk for clinical 
outcome.  DSI has performed similarly in subjects with chronic hepatitis C, non-alcoholic 
fatty liver disease (NAFLD), and primary sclerosing cholangitis [2-21]. 
 
3.1.9 Measuring Treatment Effect by the HepQuant SHUNT Test 
 
Serial changes in DSI have been assessed in subjects with chronic hepatitis C, during 
and after antiviral treatment, and in subjects with PSC.  Two studies of HepQuant 
SHUNT for measuring impact of sustained viral response (SVR) are highlighted, 
GILEAD-sponsored SOLAR-1 and NIH-sponsored HALT-C.  In SOLAR-1, subjects were 
treated with ledipasvir/sofosbuvir/ribavirin, and in HALT-C, subjects were treated with 
peginterferon/ribavirin.  HepQuant SHUNT was performed at baseline and at weeks 4, 
24, 36, and 48 in SOLAR-1; and, at baseline and after approximately 2 years in HALT-C.  
In the PSC study, subjects underwent HepQuant SHUNT tests at baseline and after 1 
year. 
 
In SOLAR-1, DSI improved significantly by week 4.  The improvement was greatest in 
transplant recipients with necro-inflammation and non-cirrhotic stages of fibrosis (∆DSI -
4.4±3.7, p<0.0001) and in transplant recipients with cirrhosis (∆DSI -1.7±2.5, p<0.03).  
DSI did not change in non-transplant subjects with end-stage decompensated disease 

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(∆DSI 0.2±2.8, p=NS).  Standard laboratory tests, MELD score, and CTP score failed to 
detect the improvement in liver function at week 4 [20]. 
 
The results of SOLAR-1 suggest that the decrease in DSI by week 4 was likely due to 
improvement in the hepatic microcirculation.  First, ALT declined in parallel with the 
clearance of HCV RNA indicating reduction in necro-inflammation.  Second, platelet 
count increased in LT Fibrosis and LT Cirrhosis, most probably from a reduction in 
splenic sequestration as hepatic resistance declined and portal hypertension improved.  
Third, the increase in platelet count correlated with the decrease in DSI.  Fourth, although 
improvement in hepatocyte uptake of cholate might also account for the increases in 
HFRs and decline in DSI, there was no improvement in blood tests of hepatocyte 
function, such as bilirubin, albumin and INR.  These findings are consistent with the 
interpretation that early hepatic improvement measured by drop in DSI reflects 
improvement in the hepatic microcirculation. 
 
Because in CLD, necro-inflammation is a hallmark of the injury, baseline DSI will define 
the severity of the hepatic impairment at start of treatment.  With successful treatment by 
selonsertib, necro-inflammation should be reversed and be manifested as improved 
hepatic perfusion and hepatocyte uptake – events that should be detected by drop in DSI 
from the baseline test.  If selonsertib has a very rapid onset of action, we could possibly 
detect improvement at the earliest time point after initiating treatment. 
 
The serial studies in HALT-C and PSC may not be as relevant to detection of early 
treatment effect since the follow-up DSI in these studies was either 1 year (PSC) or 2 
years (HALT-C) after baseline testing.  Nonetheless, the results in HALT-C demonstrated 
significant improvement in DSI after SVR and worsening of DSI in both untreated 
subjects and treated subjects who remained infected [4].  The study of DSI in PSC 
subjects identified 3 progressor groups of PSC subjects, slow, moderate, or rapid, based 
on DSI relative to age and the change in DSI over one year.  The serial changes in DSI 
over one year confirmed slower progression in the slow progressor group compared to a 
more rapid rate of progression in the moderate to rapid progressor groups [18]. 
 
3.2 
Investigational Drugs 
 
3.2.1 Selonsertib (ASK1 Inhibitor) 
 

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Selonsertib (GS-4997) is an orally bioavailable inhibitor of the apoptosis signal-regulating 
kinase 1 (ASK1). Upon oral administration, the ASK1 inhibitor selonsertib targets and 
binds to the catalytic kinase domain of ASK1 in an ATP-competitive manner, thereby 
preventing its phosphorylation and activation. This prevents the phosphorylation of 
downstream kinases, such as c-Jun N-terminal kinases (JNKs) and p38 mitogen-
activated protein kinase (p38 MAPK). By preventing the activation of ASK1-dependent 
signal transduction pathways, selonsertib prevents the production of inflammatory 
cytokines, down-regulates the expression of genes involved in fibrosis, suppresses 
excessive apoptosis and inhibits cellular proliferation. 
 
Selonsertib undergoes hepatic metabolism and biliary excretion.  In a mass balance 
study of radiolabeled selonsertib, the mean overall recovery of radioactivity was 95%, 
with recovery primarily in feces (~58%) versus urine (~37%). Radioactivity was eliminated 
as a combination of metabolites and unchanged parent drug. Selonsertib metabolism 
involved oxidation, hydrolysis, N-dealkylation, methylation, and glucuronidation with N-
dealkylation as a major pathway. 
 
Selonsertib is a very weak P-gp inhibitor and may increase exposures of sensitive P-gp 
substrates such as digoxin. Selonsertib is not a sensitive substrate of hepatic uptake 
transporters OATP1B1/1B3 and may be co-administered with OATP1B1/1B3 inhibitors. 
Because rifampin significantly decreased selonsertib plasma exposure, strong CYP3A4 
inducers in combination with selonsertib in long-term clinical studies are excluded.  There 
is no anticipated interaction of selonsertib with the cholates used in the HepQuant 
SHUNT test. 
 
Selonsertib was evaluated in an open-label Phase 2 study, alone or in combination with 
the monoclonal antibody simtuzumab (SIM) for 24 weeks, in patients with nonalcoholic 
steatohepatitis (NASH) and moderate to severe liver fibrosis (fibrosis stages F2 or F3). 
The data demonstrated regression in fibrosis that was, in parallel, associated with 
reductions in other measures of liver injury in patients treated with selonsertib.  
Patients receiving selonsertib demonstrated improvements in several measures of liver 
disease severity, including fibrosis stage, progression to cirrhosis, liver stiffness 
(measured by magnetic resonance elastography, MRE) and liver fat content (measured 
by magnetic resonance imaging (MRI)-proton density fat fraction, PDFF). Data for these 
efficacy endpoints are summarized in the table below. As no differences were observed 
between combination and monotherapy, results are presented for selonsertib (18 mg and 
6 mg) with/without SIM and for SIM alone. Additionally, patients with fibrosis improvement 

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demonstrated reductions in hepatic collagen content, liver biochemistry (e.g., serum ALT) 
and the apoptosis marker, cytokeratin-18, supporting the biological activity of selonsertib. 
 
Endpoint (Week 24)  
  Selonsertib
18 mg ± SIM
Selonsertib 
6 mg ± SIM 
  
SIM 
 
 
 
  
 
Fibrosis Improvement ≥1 Stage from Baseline*  
43% (n=13/30)
30% (n=8/27) 
20% (n=2/10) 
Progression to Cirrhosis  
3% (n=1/30) 
7% (n=2/27) 
20% (n=2/10) 
≥15% Reduction in Liver Stiffness by MRE  
20% (n=5/25) 
32% (n=7/22) 
0% (n=0/7) 
≥30% Reduction in Liver Fat by MRI-PDFF  
26% (n=8/31) 
13% (n=3/24) 
10% (n=1/10) 
   
*Fibrosis staged according to the NASH Clinical Research Network (CRN) classification.  
 
There were no dose-related increases in treatment-emergent adverse events or serious 
adverse events. Headache, nausea and sinusitis were the most common adverse events 
in patients receiving selonsertib.  
 
3.2.2 GS-0976 (ACC inhibitor) 
 
GS-0976 is an oral, investigational inhibitor of Acetyl-CoA carboxylase (ACC), one of 
several biologically relevant pathways associated with disease progression in NASH. 
ACC catalyzes the first step in hepatic de novo lipogenesis, the synthesis of fatty acids 
that contribute to hepatic steatosis and, subsequently, inflammation and liver fibrosis. 
Recent clinical trials have demonstrated that GS-0976 (20 mg taken orally once daily) 
when administered for 12 weeks was associated with statistically significant reductions in 
hepatic steatosis and a noninvasive marker of fibrosis (TIMP-1) compared to placebo. 
The study included 126 patients who were randomized to receive GS-0976 20 mg 
(n=49), GS-0976 5 mg (n=51), or placebo (n=26) once daily for 12 weeks. All patients in 
the study were diagnosed with NASH and liver fibrosis stages F1 through F3 based on 
biopsy, or by magnetic resonance elastography (MRE) and MRI proton density fat 
fraction (MRI-PDFF). 
 
Patients receiving GS-0976 20 mg demonstrated significant decreases in liver fat content 
(measured by MRI-PDFF) compared to placebo after 12 weeks of treatment. Patients 
treated with GS-0976 20 mg also experienced a significant decrease in TIMP-1, a serum 
marker associated with liver fibrosis. Differences between GS-0976 5 mg and placebo 
were not statistically significant. Data for these efficacy endpoints are summarized in the 
table below. 
 

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Relative (%) Changes in Imaging, ALT and Serum Fibrosis Markers at Week 12* 
Endpoint (Week 12)  
 GS‐0976
20 mg  
(n=49)  
 GS‐0976
5 mg  
(n=51)  
 
Placebo
(n=26) 
 P‐values  
20 mg vs. 
Placebo     5 mg vs. 
Placebo 
MRI‐PDFF  
‐28.9 
‐13.0 
‐8.4 
0.002 
 
0.142 
≥30% reduction in MRI‐PDFF, % (n/N) 
48% 
(22/46)
23% 
(11/47)
15% 
(4/26)
0.004 
 
0.433 
MRE‐stiffness  
‐5.5 
‐9.6 
‐12.5 
0.100 
 
0.743 
Liver stiffness by FibroScan  
‐11.1 
‐8.4 
‐3.1 
0.212 
 
0.364 
ALT  
‐20.5 
‐9.8 
‐6.7 
0.176 
 
0.765 
TIMP‐1  
‐7.9 
‐2.9 
‐1.5 
0.022 
 
0.301 
PIII‐NP  
‐13.9 
‐7.0 
‐0.5 
0.107 
 
0.605 
* Unless indicated, all data are median relative (%) changes from baseline. 
 
In other measures, including liver stiffness by FibroScan, liver stiffness by MRE, serum 
ALT and PIII-NP, a serum marker of fibrogenesis, no statistically significant differences 
were observed between the treatment and placebo arms of the study. 
 
At week 12, a median relative change in triglycerides (TG) from baseline of +11 percent, 
+13 percent and -4 percent was observed in patients receiving GS-0976 20 mg, GS-0976 
5 mg and placebo, respectively. Asymptomatic Grade 3 or 4 TG elevations (>500 mg/dL) 
were observed in 16 patients receiving GS-0976 20 mg (n=7) or 5 mg (n=9); the primary 
factor associated with such elevations was a baseline TG level >250 mg/dL (p<0.001). 
The majority of patients with such elevations either responded to fibrate or fish oil therapy 
(n=4) or resolved without additional treatment or cessation of GS-0976 (n=7). GS-0976 
was well-tolerated. Nausea, abdominal pain and diarrhea were the most common 
adverse events. 
 
3.2.3 GS-9674 (FXR inhibitor) 
 
GS-9674 is a selective, non-steroidal agonist of the Farnesoid X receptor (FXR), a 
nuclear hormone receptor that is highly expressed in the gastrointestinal tract and liver. 

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FXR is the primary regulator of bile acid synthesis and plays important roles in glucose 
and lipid metabolism. Pre-clinical studies in animal models demonstrated that GS-9674 
reduced hepatic steatosis and fibrosis, as well as serum levels of cholesterol, ALT and 
AST compared with untreated animals, and also had a dose-dependent anti-fibrotic 
effect, associated with lowering of portal pressure. 
 
 
3.2.4 The Combination of Selonsertib with either GS-0976 or GS-9674 
 
This combination has been evaluated in patients with advanced fibrosis due to 
nonalcoholic steatohepatitis (NASH) - the apoptosis signal-regulating kinase 1 (ASK1) 
inhibitor selonsertib with either the Acetyl-CoA carboxylase (ACC) inhibitor GS-0976 or 
the selective, non-steroidal Farnesoid X receptor (FXR) agonist GS-9674. 
 
In this proof-of-concept study, 70 patients were treated with either selonsertib 18 mg plus 
GS-0976 20 mg (n=20), selonsertib 18 mg plus GS-9674 30 mg (n=20), or each 
monotherapy (n=10 per group) once daily for 12 weeks. All patients in the study were 
diagnosed with NASH and liver fibrosis stages F2 to F3 based on biopsy, or by magnetic 
resonance elastography (MRE) and MRI proton density fat fraction (MRI-PDFF). The 
greatest changes observed after 12 weeks of treatment in the study were decreases in 
liver fat content (measured by MRI-PDFF), which occurred in regimens containing GS-
0976. Improvements in liver biochemistry and/or markers of fibrosis were also observed 
across both combination arms of the study compared to baseline. In patients treated with 
selonsertib plus GS-0976, kinetic labeling revealed the largest reduction in the fractional 
synthesis rate of lumican, a marker of fibrogenesis. Similar rates of adverse events were 
observed between patients treated with single-agent and combination therapies. No 
patient discontinued treatment prematurely. 
 
 
3.3 
Non-alcoholic Steatohepatitis (NASH) 
 
3.3.1 Burden of Nonalcoholic Fatty Liver Disease (NAFLD) and Steatohepatitis 
(NASH) 
 
NAFLD is the term encompassing all forms of fatty liver disease from benign steatosis to 
the necroinflammation and progressive fibrosis associated with NASH.  The prevalence 
of NAFLD in North America ranges from 27 to 34% and the prevalence of NASH in the 

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United States ranges from 3 to 5% [52-54].  Similar prevalence has been described in 
studies from Europe and Asia. 
 
Cirrhosis caused by NASH is an increasing indication for liver transplantation and is a 
common risk factor for hepatocellular carcinoma [55,56].  Patients with NASH are also at 
increased risk for cardiovascular disease, type 2 diabetes, and chronic kidney disease 
[55]. 
 
3.3.2 Clinical Presentation of NAFLD and NASH 
 
Most individuals with NAFLD are typically asymptomatic and have normal liver blood 
tests.  However, some individuals will have nonspecific symptoms or minimal elevations 
of liver enzymes; and, a minority will present with obvious clinical or laboratory features of 
advanced disease.  Older age, hypertension, concomitant type 2 diabetes, obesity, 
dyslipidemia, and AST:ALT >1 are risk factors for NASH with fibrosis [52-55]. 
 
3.3.3 Diagnosis of NAFLD and NASH 
 
NAFLD and NASH are suspected after exclusion of other causes of liver disease.  The 
diagnosis of NAFLD is typically based on clinical, laboratory, and imaging criteria but the 
definitive diagnosis of NASH requires histological evaluation of a liver biopsy [45,57]. 
 
Given the poor patient acceptance and potential morbidity of liver biopsy a safer 
minimally-invasive alternative for NASH diagnosis is desirable.  Clinical models, such as 
the NASH Clinical Scoring System or the NASH Predictive Index, incorporate 
demographic features and laboratory tests to generate a risk score for NASH [58,59]. 
 
Other minimally-invasive approaches include biomarkers, fibrosis biomarkers, liver 
stiffness measurements, and quantitative tests of liver function [60-65].  The HepQuant 
SHUNT test to be used in this study simultaneously measures clearance from portal and 
systemic circulations to detect changes in the portal circulation. 
 
3.3.4 Measuring Disease Severity in NAFLD and NASH 
 
Disease severity in NAFLD and NASH is based upon clinical, laboratory, and histologic 
criteria.  Clinical and laboratory criteria are useful in defining disease severity only in the 

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late stages of disease.  Histologic criteria define the spectrum from minimal change, 
through stages of advancing fibrosis and inflammation to cirrhosis [58,59]. 
 
In multi-variable analyses of clinical, laboratory, and histologic features, hepatic fibrosis 
(NASH CRN fibrosis stage >2) is the only factor that consistently predicts risk for liver-
related mortality in NASH [57-65].  MELD (model for end-stage liver disease) and CTP 
(Child-Turcotte-Pugh) clinical models can define which cirrhotic NASH patients are at 
highest risk for death [65]. 
 
A non- or minimally-invasive method for measuring disease severity in NAFLD and NASH 
is desirable.  The HepQuant SHUNT test to be used in this study provides a disease 
severity index (DSI) which correlates with other measures of disease severity.  DSI can 
define baseline hepatic impairment and serial DSI measurements can track changes in 
response to treatments. 
 
3.3.5 Treatment of NASH 
 
There is currently no FDA-approved treatment for NASH.  In the PIVENS trial of 
nondiabetic patients with NASH, Vitamin E, 800 IU/d x 96 weeks, reduced hepatic 
steatosis and inflammation but not hepatic fibrosis [59].  Clearly, new treatment options 
are needed for patients with NASH. 
 
3.3.6 Pitfalls in Measuring Treatment Effect in NASH 
 
A key pitfall in determining a positive (or negative) effect of any treatment of NASH is the 
lack of reliable minimally-invasive diagnostic tools.  Generally, the efficacy of any 
intervention in NASH has been defined by histologic improvement on serial liver biopsies.  
However, poor tolerability and sampling error limit the accuracy and usefulness of liver 
biopsy as a monitoring tool. 
 
Early identification of patients with a substantial improvement in hepatic function is of 
interest in the management of NASH.  It is possible, even in relatively small trials, to link 
the results from an exploratory diagnostic tool, such as HepQuant SHUNT, to the 
histologic outcomes. 
 
Standard blood tests, such as liver enzymes, bilirubin, albumin, and clotting times, can 
vary widely in concentration or activity due to a number of factors including non-hepatic 

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diseases, hemolysis, inflammation, diet, medications, gender, and age. These concerns  
make their use for monitoring treatment effects problematic. 
 
Although liver biopsy is useful for establishing the diagnosis of NASH and can yield 
prognostic information it is invasive and risky [45].  Biopsy also suffers from sampling 
error, subjective scoring, and a potential for significant clinical morbidity. 
 
Non-invasive elastography has virtually replaced liver biopsy in assessing disease 
severity in chronic hepatitis C but has not been validated in NASH.  Elastography 
measures liver stiffness which is related to severity of liver fibrosis.  However, the fat and 
inflammation which are the hallmark of NASH will likely alter the relationship of 
elastography to fibrosis and prognosis. 
 
3.3.7 The HepQuant SHUNT Test in NASH 
 
The HepQuant SHUNT test fulfills an unmet medical need because it is a minimally-
invasive test of global liver function and physiology.  With progression, liver diseases 
impair hepatocyte function and the portal circulation and these abnormalities become 
manifest as portal hypertension and portal-systemic shunting.  The clinical consequences 
are coagulopathy, jaundice, varices, ascites and encephalopathy. HepQuant SHUNT 
quantifies the changes in liver function and the portal circulation from early on through 
later stages of disease. 
 
Hepatic inflammation and hepatic fibrosis, which are hallmarks of NASH, impair 
hepatocyte function and hepatic perfusion.  Most patients with NASH have underlying 
portal fibrosis which contributes to hepatic functional impairment via changes in the portal 
circulation.  The progressive fibrosis and dysfunction ultimately leads to cirrhosis, portal 
hypertension, and portal-systemic shunting.  Hepatocellular impairment, portal circulatory 
changes, portal hypertension, and portal-systemic shunting account for the major 
manifestations of NASH cirrhosis – jaundice, coagulopathy, fluid retention, varices and 
encephalopathy. 
 
Underlying portal hypertension (PH) is a risk factor for poor outcome in NASH.  PH can 
be measured by threading a catheter from the jugular vein into the portal vein to measure 
the hepatic venous pressure gradient (HVPG).  Increased HVPG correlates with risk of 
cirrhosis, varices, ascites, and decompensation [27-29]. 
 

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In NASH patients with PH, PH may reverse with resolution of the NASH.  So why not use 
HVPG?  First, HVPG is only effective as a monitoring tool in the patient with PH – many 
patients with NASH will not have PH and, accordingly, they will have normal HVPG which 
won’t change with treatment.  Second, HVPG is invasive, expensive, and requires 
specialized monitoring and specially trained professionals.  As a result, HVPG can only 
be used selectively in specialized centers. 
 
4.0 Preliminary Results 
 
4.1 
Measuring Disease Severity by the HepQuant SHUNT Test 
 
4.1.1 Prior Studies 
 
Over 1400 HepQuant SHUNT tests have been performed in over 650 persons.  This 
experience encompasses healthy controls, and subjects with chronic hepatitis C, non-
alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH), primary 
sclerosing cholangitis (PSC), hepatocellular carcinoma (HCC), polycystic liver disease, 
and spans the spectrum from minimal to advanced fibrosis and compensated to 
decompensated disease [1-21].  In our prior studies of chronic hepatitis C, DSI correlated 
with ISHAK and METAVIR stages of fibrosis and predicted the likelihood of cirrhosis, 
varices, and risk for clinical outcome.  DSI performed similarly in subjects with non-
alcoholic fatty liver disease (NAFLD) and primary sclerosing cholangitis.  Our data 
indicates that DSI scoring is effective in a wide range of liver diseases. 
 
4.2 
Measuring Treatment Effect by the HepQuant SHUNT Test 
 
4.2.1 Prior Studies 
 
Serial changes in DSI have been assessed in subjects with chronic hepatitis C, during 
and after antiviral treatment, and in subjects with PSC.  There were two studies of 
HepQuant SHUNT measuring impact of sustained viral response (SVR), SOLAR-1 and 
HALT-C.  In SOLAR-1, subjects were treated with ledipasvir/sofosbuvir/ribavirin, and in 
HALT-C, subjects were treated with peginterferon/ribavirin.  HepQuant SHUNT was 
performed at baseline and at weeks 4, 24, 36, and 48 in SOLAR-1; and, at baseline and 
after approximately 2 years in HALT-C.  In the PSC study, subjects underwent HepQuant 
SHUNT tests at baseline and after 1 year. 

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4.2.2 SOLAR-1 Clinical Trial 
 
In SOLAR-1, DSI improved significantly by week 4.  The improvement was greatest in 
transplant recipients with necro-inflammation and non-cirrhotic stages of fibrosis (∆DSI -
4.4±3.7, p<0.0001) and in transplant recipients with cirrhosis (∆DSI -1.7±2.5, p<0.03).  
DSI did not change in non-transplant subjects with end-stage decompensated disease 
(∆DSI 0.2±2.8, p=NS).  Standard laboratory tests, MELD score, and CTP score failed to 
detect the improvement in liver function at week 4 [20]. 
 
The results in SOLAR-1 suggested that the decrease in DSI by week 4 was likely due to 
improvement in the hepatic microcirculation.  First, ALT declined in parallel with the 
clearance of HCV RNA indicating reduction in necro-inflammation.  Second, platelet 
count increased in LT Fibrosis and LT Cirrhosis, most probably from a reduction in 
splenic sequestration as hepatic resistance declined and portal hypertension improved.  
Third, the increase in platelet count correlated with the decrease in DSI.  Fourth, although 
improvement in hepatocyte uptake of cholate might also account for the increases in 
HFRs and decline in DSI, there was no improvement in blood tests of hepatocyte 
function, such as bilirubin, albumin and INR.  These findings are consistent with the 
interpretation that early hepatic improvement measured by drop in DSI reflects 
improvement in the hepatic microcirculation. 
 
In CLD, necro-inflammation is a hallmark of the injury – baseline DSI will define the 
severity of the hepatic impairment at start of treatment.  With successful treatment, necro-
inflammation should reverse and be manifest as improved hepatic perfusion and 
hepatocyte uptake – events that should be detected by drop in DSI from the baseline 
test.  If treatment has a very rapid onset of action, DSI could detect fibrosis reversal at 
the earliest time point after initiating treatment. 
 
4.2.3 HALT-C and PSC Studies 
 
The serial studies in HALT-C and PSC may not be as relevant to detection of early 
treatment effect since the follow-up DSI in these studies was either 1 year (PSC) or 2 
years (HALT-C) after baseline testing.  Nonetheless, the results in HALT-C demonstrated 
significant improvement in DSI after SVR and worsening of DSI in both untreated 
subjects and treated subjects who remained infected [4].  The study of PSC subjects 
identified 3 groups of PSC subjects:  slow, moderate, and rapid progressors.  The data in 
follow-up, one year later, confirmed slow progression in the slower progressor group 

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compared to the faster rate of progression in the moderate to rapid progressor groups 
[18]. 
 
5.0 Research Plan 
 
5.1 
Subjects 
 
The subjects for the HepQuant study will be recruited from the patient cohorts for the 
three Gilead clinical trials with no more than N = 100:   
 
 
 
 
 STELLAR 3:  
 
 
Adult subjects with NASH 
 
 
 
 
 
NASH CRN Stage F3 
 
 
 
 
 
 STELLAR 4:   
 
 
Adult subjects with NASH 
 
 
 
 
 
NASH CRN Stage F4 
 
 
 
 
 
Compensated Cirrhosis 
 
 GS-US-454-4378 (NASH F3/F4) Adult subjects with NASH 
 
 
 
 
 
 
NASH CRN Stage F3 or F4 (compensated) 
 
 
 
 
  
5.2 
Inclusion and Exclusion Criteria 
 
To be enrolled in the HepQuant Study the subject must be co-enrolled in one of 3 Gilead 
trials noted in Section 5.1 and also: 
 
 lack all exclusion criteria for the Gilead trial, 
 not have had any previous dosing with Selonsertib, GS-0976 or GS-9674 
 have adequate intravenous access for catheter placement and 6 blood draws, 
 be able to drink the oral solution of 4D-cholate, and 
 not have known hypersensitivity to albumin preparations, any ingredient in the 
formulation, or component of the HepQuant SHUNT Test Kit. 
 
Subjects or persons with serious intercurrent medical or surgical illness, such as acute 
myocardial infarction, acute cerebral hemorrhage, sepsis, or other immediate life-
threatening illness will be excluded.  Subjects with extensive resection of large segments 

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of small intestine (short gut) or severe gastroparesis might not be able to absorb the oral 
dose of 4D-CA and are excluded. 
 
Patients with cirrhotic stage of disease may not have had any clinical decompensating 
events (e.g., variceal hemorrhage, ascites, encephalopathy, spontaneous bacterial 
peritonitis). 
 
5.3 
Study Design 
 
5.3.1 Dosing of Drugs 
 
Dosing of drugs in the clinical trials is dictated by the Gilead clinical trial and is not further 
modified by the HepQuant Study.  The dosing schedules are: 
 
 
 STELLAR 3:   
Selonsertib 6 mg/d for 240 Wks, or 
 
 
 
 
Selonsertib 18 mg/d for 240 Wks, or 
 
 
 
 
Placebo 
 
 STELLAR 4:  
Selonsertib 6 mg/d for 240 Wks, or 
 
 
 
 
Selonsertib 18 mg/d for 240 Wks, or 
 
 
 
 
Placebo 
 
 GS-US-454-4378: Selonsertib 18 mg/d, or 
 
 
 
 
GS-0976 20 mg/d, or 
 
 
 
 
GS-9674 30 mg/d, or 
 
 
 
 
A combination thereof, or 
 
 
 
 
Placebo 
 
The duration of treatment in the GS-US-454-4378 study is 48 weeks. 
 
5.3.2 Timing of HepQuant SHUNT testing 
 
The basic design for HepQuant SHUNT testing is similar across the clinical trials: 
 
 In all trials, the HepQuant SHUNT Test is initially performed at baseline, prior to 
any treatment, to assess liver function and physiology at entry to the trial, time t0. 

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 The HepQuant SHUNT Test is then performed serially to monitor the change in 
function and physiology, relative to baseline, during the placebo/treatment and 
post-treatment follow-up periods of the study. In all cases, the study visits for the 
HepQuant tests will coincide and align with the protocol-defined visit windows for 
the GILEAD clinical trials. 
 
In the STELLAR 3 and STELLAR 4 trials, the HepQuant SHUNT test will be 
administered at baseline and Weeks 24, 48, and 240.  The total length of time subject 
will be in the SHUNT study is approximately 252 weeks. The study design for the 
STELLAR studies is shown in the figure below. 
 
 
 
In the GS-US-454-4378 (NASH F3/4) trial, the HepQuant SHUNT test will be 
administered at baseline and Weeks 24 and 48.  The total length of time subject will be in 
SHUNT study is 52 weeks. The study design for the GS-US-454-4378 (NASH F3/4) trial 
is shown in the following figure. 
 
 

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The specific comparative data collected in the HepQuant study are given in Appendix C.  
The results of the HepQuant SHUNT test will be transferred to Gilead who will analyze 
the test results in comparison to other procedures and clinical outcomes recorded in the 
Gilead trials.  HepQuant will query Gilead for the results of these analyses. 
 
5.3.3 Treatment and Procedures 
 
The HepQuant Study encompasses the administration of the HepQuant SHUNT test, use 
of the HepQuant SHUNT Liver Diagnostic Kit, and monitoring of the test and test 
outcome. The procedures, components, and outputs of the HepQuant SHUNT test have 
been previously summarized in Section 3.1 of this document. 
 
All other aspects of the subject’s care, treatments, management, and additional 
procedures are governed by the Gilead main clinical trial protocols.  HepQuant will 
upload its test results to Gilead via secured portals and subsequently make requests of 
Gilead for specific analyses as defined via a collaborative research agreement between 
HepQuant and Gilead.  
 
 

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5.3.4 Administration of the HepQuant SHUNT test 
 
Detailed instructions are provided in the Instructions for Use (Appendix B).  The 
HepQuant SHUNT test is performed after an overnight fast or after at least 5 hours of 
fasting during the daytime.  The morning dose of drug(s) is held until after completion of 
the HepQuant SHUNT test.  The HepQuant SHUNT test requires venous access via a 
standard indwelling intravenous catheter, preferably placed in an antecubital vein.  An 
oral solution of 4D-CA and an injectable solution of 13C-CA mixed with 25% human 
serum albumin are administered simultaneously over approximately 1 minute. If a 
problem is encountered with administering compounds simultaneously, the test is to be 
re-scheduled.  If a rescheduled visit falls outside of the Gilead protocol-defined windows, 
sites should contact their study monitor for further instruction. 
 
Blood samples are obtained at baseline and at 5±1, 20±2, 45±5, 60±5, and 90±5 minutes 
after cholate administration.  The serum is separated and sent to the HepQuant 
laboratory for quantification of concentrations of endogenous cholate, 4D-CA and 13C-
CA.  Clearances are calculated from AUCs and dose and DSI and SHUNT are derived 
from the clearances. 
 
5.3.5 Procedures by Study Visit (Also refer to Schedule of Events) 
 
Week -8 to Day -1 (Screening) 
 
 Informed Consent 
 Review Inclusion/exclusion criteria  
 Review continued eligibility 
 Demographics 
 Medical History 
o Hepatorenal Syndrome (Type I or Type II, treatment [if applicable])  
o Infection Related to Spontaneous Bacterial Peritonitis or SHUNT test or IV 
catheter (type, organism, treatment) 
o Hepatic Encephalopathy (treatment [if applicable], treatment type, whether 
controlled with treatment) 
o Ascites (treatment [if applicable], treatment type, whether controlled with 
treatment) 
o Variceal Hemorrhage (treatment [if applicable], treatment type) 
 Physical Exam  

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Baseline/Day 1 
 
 Informed Consent (if not performed Week -8 to Day -1/Screening) 
 Review inclusion / exclusion criteria (if not performed Week -8 to Day -
1/Screening) 
 Review continued eligibility  
 Demographics (if not performed Week -8 to Day -1/Screening) 
 Medical history (if not performed Week -8 to Day -1/Screening) 
o Hepatorenal Syndrome (Type I or Type II, treatment [if applicable])  
o Infection Related to Spontaneous Bacterial Peritonitis or SHUNT test or IV 
catheter (type, organism, treatment) 
o Hepatic Encephalopathy (treatment [if applicable], treatment type, whether 
controlled with treatment) 
o Ascites (treatment [if applicable], treatment type, whether controlled with 
treatment) 
o Variceal Hemorrhage (treatment [if applicable], treatment type) 
o Varices and Endoscopy (whether performed, date, presence of varices, size 
of largest varix) 
 Any EGD results that were collected as part of the subject’s standard 
of care. Window is ± 18 months of the SHUNT test date. These 
reports will be de-identified of any HIPAA information and sent to 
HepQuant with the samples. 
 Physical exam (if not performed Week -8 to Day -1/Screening) 
 Vital signs (respiratory rate, temperature, blood pressure, heart rate, weight, and 
height) 
 Prothrombin time, INR (see table below) 
 Hematology (see table below)  
 Chemistry (see table below) 
 HepQuant SHUNT Test 
 Patient survey  
 Adverse events 
 
Week 24 
 
 Review continued eligibility 
 Medical History 

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o Hepatorenal Syndrome (Type I or Type II, treatment [if applicable])  
o Infection Related to Spontaneous Bacterial Peritonitis or SHUNT test or IV 
catheter (type, organism, treatment) 
o Hepatic Encephalopathy (treatment [if applicable], treatment type, whether 
controlled with treatment) 
o Ascites (treatment [if applicable], treatment type, whether controlled with 
treatment) 
o Variceal Hemorrhage (treatment [if applicable], treatment type) 
o Varices and Endoscopy (whether performed, date, presence of varices, size 
of largest varix) 
 Any EGD results that were collected as part of the subject’s standard 
of care. Window is ± 18 months of the SHUNT test date. These 
reports will be de-identified of any HIPAA information and sent to 
HepQuant with the samples. 
 Vital signs (respiratory rate, temperature, blood pressure, heart rate, weight, and 
height) and laboratory tests 
o Prothrombin time, INR (see table below)  
o Hematology (see table below)  
o Chemistry (see table below) 
 HepQuant SHUNT Test 
 Patient survey  
 Adverse events 
 
Week 48  
 
 Review continued eligibility  
 Medical history  
o Hepatorenal Syndrome (Type I or Type II, treatment [if applicable])  
o Infection Related to Spontaneous Bacterial Peritonitis or SHUNT test or IV 
catheter (type, organism, treatment) 
o Hepatic Encephalopathy (treatment [if applicable], treatment type, whether 
controlled with treatment) 
o Ascites (treatment [if applicable], treatment type, whether controlled with 
treatment) 
o Variceal Hemorrhage (treatment [if applicable], treatment type) 
o Varices and Endoscopy (whether performed, date, presence of varices, size 
of largest varix) 

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 Any EGD results that were collected as part of the subject’s standard 
of care. Window is ± 18 months of the SHUNT test date. These 
reports will be de-identified of any HIPAA information and sent to 
HepQuant with the samples. 
 Physical exam  
 Vital signs (respiratory rate, temperature, blood pressure, heart rate, weight, and 
height) 
 Prothrombin time, INR (see table below) 
 Hematology (see table below)  
 Chemistry (see table below) 
 HepQuant SHUNT Test 
 Patient survey  
 Adverse events 
 
Week 240 (Not applicable to GS-454-4378) 
 
 Review continued eligibility  
 Demographics  
 Medical history  
 Hepatorenal Syndrome (Type I or Type II, treatment [if applicable])  
 Infection Related to Spontaneous Bacterial Peritonitis or SHUNT test or IV 
catheter (type, organism, treatment) 
 Hepatic Encephalopathy (treatment [if applicable], treatment type, whether 
controlled with treatment) 
 Ascites (treatment [if applicable], treatment type, whether controlled with 
treatment) 
 Variceal Hemorrhage (treatment [if applicable], treatment type) 
 Varices and Endoscopy (whether performed, date, presence of varices, size of 
largest varix) 
o Any EGD results that were collected as part of the subject’s standard of 
care. Window is ± 18 months of the SHUNT test date, for the final study 
visit. These reports will be de-identified of any HIPAA information and sent 
to HepQuant with the samples. 
 Physical exam  
 Vital signs (respiratory rate, temperature, blood pressure, heart rate, weight, and 
height) 

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 Prothrombin time, INR (see table below) 
 Hematology (see table below)  
 Chemistry (see table below) 
 HepQuant SHUNT Test 
 Patient survey  
 Adverse events 
 
Local Laboratory Tests* 
Hematology 
Chemistry 
Coagulation Profile 
WBC 
Sodium 
Prothrombin time 
RBC 
Potassium 
INR 
Hemoglobin 
Calcium 
 
Platelets 
Urea Nitrogen 
 
Neutrophils 
Creatinine 
 
Lymphocytes 
Albumin 
 
Monocytes 
Bilirubin 
 
Eosinophils 
Alanine Aminotransferase 
 
Basphils 
Aspartate Aminotransferase
 
Neutrophils (%) 
Alkaline Phosphatase 
 
Lymphocytes (%) 
Glucose 
 
 
Chloride  
 
*To be collected prior to cholate/SHUNT test administration at each study visit 
 
 
6.0 Descriptions, Risks, Justifications 
 
6.1. Drug Safety Definitions 
 
Abuse: Persistent or sporadic intentional excessive use of a medicinal product by a 
patient or clinical trial subject. 
 
Adverse Event (“AE”): Any untoward medical occurrence in a patient or clinical 
investigation subject administered a medicinal product and which does not necessarily 
have to have a causal relationship with this treatment. An Adverse Event (AE) can 
therefore be any unfavourable and/or unintended sign (including an abnormal laboratory 
finding, for example), symptom, or disease temporally associated with the use of a 

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medicinal product, whether or not considered related to the medicinal product.  AEs may 
also include pre- or post-treatment complications that occur as a result of protocol 
mandated procedures, lack of efficacy, overdose or drug Abuse/Misuse reports. Pre-
existing events that increase in severity or change in nature during or as a consequence 
of participation in the clinical study shall also be considered AEs. 
 
Adverse Reaction (“AR”): An untoward medical occurrence (unintended or noxious 
responses) considered causally related to an investigational or authorized medicinal 
product at any dose administered. Adverse Reactions may arise from Medication Errors, 
uses outside what is foreseen in the protocol or prescribing information (off-label use), 
Misuse and Abuse of the product, Overdose or Occupational Exposure where applicable. 
 
Development Safety Update Report (“DSUR”): A report providing an annual review and 
evaluation of pertinent safety information collected during the reporting period to 
summarise the current understanding and management of identified and potential risks, 
describe new safety issues that could impact clinical trial subjects and provide an update 
on the status of the development program. 
 
Medication Error: Any unintentional error in the prescribing, dispensing or administration 
of a medicinal product while the medication is in the control of a healthcare professional, 
patient or consumer. 
 
Misuse: Use of a medicinal product that is intentional and inappropriate and not in 
accordance with its authorized product information.  
 
Occupational Exposure: Exposure to a medicinal product as a result of one’s 
professional or non-professional occupation. 
 
Off-label Use: Where a medicinal product is intentionally prescribed by a Health Care 
Professional for a medical purpose not in accordance with the authorized product 
information with respect to indication, route, dose or patient population (e.g. the elderly).  
For avoidance of doubt, Off-Label Use will not apply in clinical trials. 
 
Overdose: Administration of a quantity of a medicinal product given per administration or 
cumulatively which is above the maximum recommended dose as per the protocol or in 
the product labelling. The Parties agree that in the course of conducting a clinical study, 

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the terms of the clinical study protocol (as fully approved by all applicable bodies) 
overrides the local product labelling. 
 
Pregnancy Reports: Reports of pregnancy following maternal or paternal exposure to 
the product.  
 
Product Complaints: Complaints arising from potential deviations in the manufacture, 
packaging or distribution of the medicinal product. 
 
Serious Adverse Event (“SAE”) / Serious Adverse Reaction (“SAR”):  An event or 
any untoward medical occurrence that at any dose either: 
 
a) 
Results in death; or 
b) 
Is life-threatening 
 
The term “life-threatening” in the definition of “serious” refers to an event in which the 
patient was at risk of death at the time of the event; it does not refer to an event which 
hypothetically might have caused death if it were more severe; or: 
 
c) 
Requires in-patient hospitalisation or prolongation of existing hospitalisation; or 
d) 
Results in persistent or significant disability/incapacity; or 
e) 
Results in a congenital anomaly/birth defect; or  
f) 
Results in a medically important event or reaction. 
 
Explaining a medically important event: AEs requiring medical and scientific judgment 
to determine if expedited reporting is appropriate.  Such events may not be immediately 
life-threatening or result in death or hospitalisation but may jeopardise the patient or may 
require intervention to prevent one of the other outcomes constituting SAEs.  Medical and 
scientific judgement should be exercised in deciding whether an event is a medically 
important event.  Examples of medically important events include intensive treatment in 
an emergency room or at home for allergic bronchospasm; blood dyscrasias or 
convulsions that do not result in hospitalisation; or development of drug dependency or 
drug Abuse.  For the avoidance of doubt, infections resulting from contaminated 
medicinal product shall be considered a medically important event and subject to 
expedited reporting requirements. 
 

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Special Situation Reports (“SSR”): One of a) Pregnancy, b) Abuse, c) Medication 
Error, d) Misuse, e) Off-Label Use, f) Overdose, g) Lack of Effect, h) AEs in infants 
following exposure from breastfeeding, i) AEs associated with Product Complaints or 
arising from Occupational Exposure.  For the avoidance of doubt this applies to all 
reports including reports in a pediatric or elderly population.   
 
Unexpected:  An AE or AR where the nature and/or severity of the reaction is not 
consistent with the term or description used in the investigator brochure or product 
labeling. 
 
NOTE: This is not an exhaustive list and any safety information will be reported to Gilead 
DSPH and appropriate regulatory bodies. 
  
6.2 
Test Administration Risks 
 
BLOOD DRAWS  
Drawing blood from a vein may cause local pain, bruising, occasional lightheadedness, 
fainting, and very rarely, infection at the site of the blood draw. 
 
FASTING 
Fasting could cause dizziness, headache, stomach discomfort, or fainting. 
 
INTRAVENOUS CATHETER 
In some cases, having an intravenous catheter inserted into your vein can cause infection 
or inflammation where it goes under the skin. In rare cases, it can cause a blood clot in 
the vein. 
 
 
6.3 
Cholates 
 
6.3.1 Stable Isotope Labels (13C and 4D) 
 
The labels used in the HepQuant test are cold and stable – they are NOT RADIOACTIVE 
and do not expose the individual to any radioactivity.  No special monitoring is required.  
The HepQuant tests measure the clearance of 2 cholates labeled with molecular probes 
(carbon-13 (13C), and 4 deuteriums (4D)). Cholates are naturally occurring endogenous 
compounds found in the human body and 13C and D are also naturally occurring non-

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radioactive cold stable isotopes.  Cholates labeled with stable isotopes in the amounts 
administered in the HepQuant tests have no known harmful effects.  Because clearance 
of cholate from the blood is a liver specific flow-dependent function, measuring cholate 
clearance is a minimally-invasive method for assessing the severity of liver disease in 
intact humans. 
 
Cholates are naturally-occurring in the human body.  The cholates in the HepQuant 
SHUNT test are modified by the addition of stable, cold (NON-RADIOACTIVE) isotopes.  
Neither naturally occurring nor the modified cholates used in the HepQuant SHUNT test 
have any known deleterious or adverse effects when given intravenously or orally in the 
doses used in HQ tests. The serum cholate concentrations that are achieved by either 
the intravenous or oral doses are similar to the serum concentrations of bile acids that 
occur after the ingestion of a fatty meal. 
 
6.3.2 Manufacturing of Labeled Cholates 
 
The cholate testing compounds will be purchased. The test compound 2,2,4,4-d4-cholate 
(d4-CA) has been previously studied under FDA Investigational New Drug (IND) 
application 65,123. The test compound 24-13C-cholate (13C-CA) has been previously 
studied under FDA IND 65,121. No adverse events occurred in over 1400 test 
administrations.  These INDs are legacies to the current IDEs that govern use of the 
HepQuant SHUNT test in the Gilead trials. To date, the cholates used in this study have 
not been associated with any adverse events. However, they are still considered 
experimental and there may be unknown risks. 
 
6.3.3 Formulations 
 
Cholates are purchased in powder form and both powders are dissolved in sodium 
bicarbonate solution (USP grade) to achieve concentrations of 4 mg/mL and passed 
through 0.22 micron filters. The absolute concentrations of 4D-cholate in the 4D-cholate 
solution and the 13C-cholate in the 13C-cholate solution will be defined by LCMS 
methods – concentrations typically range from 3.5 to 4.2 mg/mL. 
 
d4-CA aliquots of 10 mL (~40 mg) will be transferred to sterile glass vials and stored at 
ambient temperature. For 13C-CA aliquots of 5.5 mL (~22 mg) will be transferred to 
sterile glass vials and stored at ambient temperature.  Sterility and absence of pyrogens 
will be confirmed prior to use. 

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A central pharmacy will formulate the test compounds, perform the sterile filtering, and 
prepare the sterile aliquots.  Once concentration and sterility are confirmed the cholate 
solutions are transferred to a kit maker for assemblage of the HepQuant SHUNT Liver 
Diagnostic Kit. 
 
NOTE:  The vial containing the oral dose of 4D-CA contains the exact amount of 10 mL 
of 4D-CA (~40 mg).  But, in contrast, the vial containing the intravenous dose of 13C-CA 
contains approximately 5.5 mL of 13C-CA (~22 mg) – an excess of 0.5 mL (2 mg) to 
allow for aspiration of exactly 5 mL into a syringe.  Accurate testing requires 
administration of the entire 10 mL of 4D-CA (~40 mg) oral solution; and, exactly 5 mL of 
13C-CA (~20 mg) intravenously. 
 
6.3.4 Tolerability and Safety 
 
Over 1400 HepQuant SHUNT tests have been performed in over 650 subjects without 
any untoward effects.  In the GILEAD SOLAR-1 study of ledipasvir/sofosbuvir/ribavirin 
treatment of hepatitis C in decompensated cirrhosis or in liver transplant recipients, 
HepQuant SHUNT tests were performed 5 times over 48 weeks in each of 31 study 
participants. All 31 subjects completed a survey of their experience with the HepQuant 
SHUNT test after a mean (±SD) of 4.5±0.6 tests. The patient reported outcome survey is 
given in Appendix C.  Pain was rated 0.5±0.8, where 0 was no pain and 10 was severe 
pain.  Discomfort was rated 0.3±0.5, where 0 was no discomfort and 10 was severe 
discomfort.  Inconvenience was rated 0.4±0.9, where 0 was no interference with daily 
activity and 10 was complete inability to conduct usual daily activity. 
 
The recording of time commitment indicated <3 hours in 23 cases and >3 h in 8 cases.  
The overall experience with the test was rated 9.5±1.1, where 0 was very negative and 
10 was very positive.  Willingness to undergo additional testing was rated 9.1±1.8, where 
0 was definitely not willing and 10 was very willing. 
 
Ten subjects (32%) experienced 14 SAEs during the treatment course.  The SAEs were 
2 bouts of spontaneous bacterial peritonitis in the same patient, two admissions for 
anasarca in the same patient, and one each of dysphagia, myocardial infarction, biliary 
stricture, incarcerated umbilical hernia, nausea, hepatic encephalopathy, chest pain, 
diarrhea, anemia, and fever.  None of the SAEs were attributed to HepQuant tests and 
only one, encephalopathy, was attributed to study drug (ribavirin). 
 

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6.3.5 Unknown / Unexpected Risks 
The two cholates used in the HepQuant test for this study are labeled with stable (non-
radioactive) forms of carbon and hydrogen that are found in nature and can be measured 
in blood. These forms of cholate have been registered with the FDA since 2002, and their 
use in humans has been monitored since that time. To date, the cholates used in this 
study have not been associated with any complaints or side effects. However, they are 
still considered experimental and there may be unknown risks. 
The subject will be told of any new information that might cause him/her to change 
his/her mind about continuing to take part in this study. 
6.3.6 Reporting 
HepQuant is not collecting any information regarding adverse events, serious adverse 
events, and Special Situation Reports (SSR) with respect to the GILEAD products used 
in the GILEAD clinical trials.  HepQuant is only monitoring the SHUNT test and procedure 
for AEs, SAEs, and Special Situations that are solely related to the HepQuant SHUNT 
test.  HepQuant will inform study sites and GILEAD of AEs, SAEs, and SSRs that are 
documented to occur and are related to the HepQuant SHUNT test. 
HepQuant will be responsible for the management of safety data from the Study and any 
associated regulatory reporting obligations for individual or periodic safety reports to the 
appropriate authorities and clinical investigators and applicable IRB/EC, in compliance 
with all applicable laws and the requirements of the IRB/EC. 
The contact information for reporting of safety issues or adverse reactions is: 
Andrea Herman 
Manager, Clinical Accounts & Quality 

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U.S.A. 
 
The contact information for reporting of safety issues, product concerns or feedback is: 
 
Ring Central 
800-793-8534 
complaints@hepquant.com 
 
6.4 Albumin 
 
6.4.1 Use of Albumin in the HepQuant SHUNT Test 
 
For the intravenous 13C-CA dose, exactly 5 mL of the 13C-CA intravenous solution are 
mixed with 5 ml of the albumin solution (25% w/v human serum albumin, Albutein ®-25 
from Grifols Therapeutic, Inc, BLA 102478 – NOTE that the vial of albumin contains 20 
mL; only 5 mL is added to the 13C-CA in the syringe, 15 mL is discarded). The 13C-
CA/Albumin mixture is administered intravenously through the indwelling intravenous 
catheter over 1 minute by the nurse administering the test. The 4D-cholate/flavored 
solution is administered orally simultaneously. 
 
6.4.2 Reported Reactions to Human Serum Albumin 
 
6.4.2.1 
Hypersensitivity 
 
Albumin is given with the 13C cholate that is given intravenously.  Albumin is a 
protein which is a normal part of your blood. In rare cases, hypersensitivity 
reactions to albumin have occurred. Some things that happen during any 
hypersensitivity reaction are:  
 
 rash 
 having a hard time breathing 
 wheezing when you breathe 
 sudden drop in blood pressure 
 swelling around the mouth, throat, or eyes 
 fast pulse 
 sweating 

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 severe reactions are very rare (<0.01%; less than one chance in 
10,000) but a severe reaction (called anaphylaxis) can lead to 
profoundly low blood pressure and even death. 
 
Rare Hypersensitivity Reactions have been reported to human albumin 
preparations and include anaphylaxis or anaphylactoid reactions, fever, chills, 
rash, urticaria, pruritus, angioneurotic edema, and erythema or flushing.  
 
6.4.2.2 
Risk of Transmissible Agents 
 
Because human albumin is prepared from pooled human plasma, there is a 
potential to pass human viruses (e.g., hepatitis viruses, HIV) and may carry a risk 
of transmitting Creutzfeldt-Jakob disease (CJD) or its variant CJD (vCJD).  
Through donor plasma screening and special procedures, like pasteurization to 
remove or inactivate any possible causes, reduce, but do not entirely eliminate the 
risk of transmission of disease causing agents.  Nonetheless, the risk of 
transmission of viral disease with plasma-derived human albumin is considered 
extremely low.  No cases of transmission of HBV, HCV, or HIV have been 
documented following use of commercially available human albumin.  There are 
no documented cases of CJD or vCJD transmitted through plasma-derived 
preparations (including plasma-derived human albumin); the theoretical risk for 
disease transmission of CJD with commercially available human albumin is 
considered extremely low. 
 
However, no purification method has been shown to be totally effective in 
removing the risk of viral infectivity from plasma-derived preparations and because 
new blood-borne viruses or other disease agents may emerge which may not be 
removed or inactivated by current manufacturing processes, the risks of human 
albumin are not entirely known. 
 
6.5 Intravenous Catheter 
 
Placing the indwelling intravenous catheter will cause minor pain and discomfort. With 
any blood draw there is a small risk of hematoma and a very small risk of a blood clot (1 
in 100) or infection (1 in 1000). Over 1500 HepQuant SHUNT tests or the prototypical 
dual cholate research tests have been performed on over 600 individuals and most had 
the test multiple times. There has never been an adverse event - the risk from the test is 

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very small.  A questionnaire will be completed by the subject at each of the testing 
periods to log their experience and tolerability of HQ tests (Appendix C). 
 
6.6 Other 
 
6.6.1 Data Security 
 
Data Security:  HepQuant study data will be collected and entered by the site staff into 
OmniComm TrialMaster Electronic Data Capture system. Additionally, HepQuant test and 
analytic data will be entered by HepQuant into EXCEL, ACCESS or REDCAP databases 
on HepQuant’s HIPAA-compliant and encrypted servers that have been configured for 
21CFR11 compliance. 
 
HepQuant will transfer test results of the HQ shunt test to Gilead.  The corresponding 
studies will be linked by de-identified study IDs.   
The technology used for backing up the HepQuant file servers is Veeam. Backups run 
every 12 hours and are stored on a dedicated storage appliance. Onsite backups are 
retained for 90 days before expiring and being over written. All backups are stored with 
AES 256 encryption. Every night a backup copy is sent offsite to a secure datacenter in 
Arizona. Backups are retained in Arizona with the following schedules: 
 
 180 Daily backups 
 26 Full Weekly backups 
 24 Full Monthly Backups (First Sunday of the month) 
 16 Quarterly backups (First Sunday of the quarter) 
 5 Annual backups 
 
Backup data replicated offsite is encrypted in transit and has hardware level encryption at 
rest. 
 
6.6.2 Data Security Procedures 
 
Storing and Securing Collected Data 
 
 Chromatograms and associated peak areas are printed out during each LCMS 
analytical run and are considered the official raw data. 

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 Electronic copies (Agilent ChemStation software files) of the raw data from each 
run are stored on the local permanent disk storage (C drive) on the controlling 
computer attached to the LCMS system. 
 
The ChemStation sequence used to direct the instrument is named for the day of the run 
(year as XX, month as XX, day as XX) and the analyst’s initials. For example, the 
sequence of May 22, 2017, performed by SMH is named “170522 SMH” and the raw data 
generated from that sequence is automatically stored by ChemStation in a folder of that 
same name within the folder “C:\Chem32\1\data\”. The sequence may be initiated to test 
system suitability or other parameters and only the first vial (system suitability standard) 
is injected and then the sequence is stopped if required (For example, the system 
suitability standard fails or the macro is not set up correctly). Each time the sequence is 
initiated a new subfolder is created. The subfolder that is created when the full sequence 
is completely executed is considered the run raw data set and at the completion of the 
run, it is renamed “XXXXXX” the date of the run. For example, the run Raw Data set 
would be “170522”. 
 
 During each run, a macro within Agilent Chemstation simultaneously generates in 
the folder “C:\Chem32\CORE\” a file called “rcvdata” containing a subset of the 
raw data (samples injection file #, injection time, vial #, sample name, injection #, 
peak retention time, peak area). After each run this raw data subset file is saved 
with a new name “rcvdata XXXXXX” with the same date as the raw data folder. 
 
 For example, this file would be “rcvdata 170522”. The “rcvdata XXXXXX” file is 
transferred to the analyst’s encrypted laptop and used in the analysis to generate 
standard curves, QC results, and patient testing results. 
 
 These data and folders, as well as all other laboratory data collection and analysis 
software on all the laboratory computers are only accessible when logged in by an 
authorized analyst. Each analyst is required to log in with a unique user name and 
unique password. 
 
Backing Up Data 
 
 After each run the raw data folder, “XXXXXX” is backed up by transferring it by the 
secure DSL directly from the C drive to the secure HepQuant server at RFC (Z 
drive). The raw data folders are also periodically backed up by transferring them to 

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read only DVDs that cannot be overwritten.  These DVDs are then securely 
archived. 
 
 After each run, the raw data subset file “rcvdata XXXXXX” is backed up by 
transferring it by the secure DSL directly from the C drive to the secure HepQuant 
server at RFC (Z drive). 
 
 The secure server at RFC (Z drive) is itself backed up every night by CyberTrails 
to their secure server in Arizona. 
 
Recovery of Data Backups 
 
 When raw data backed up on the secure HepQuant server at RFC (Z drive) needs 
to be recovered, either for new analysis, supporting documentation, laboratory 
administration or integrity verification and validation, the raw data will be recovered 
from the secure HepQuant server at RFC (Z drive) via the secure DSL. 
 
 Prior to transfer, the raw data folder, “XXXXXX”, on the Z drive will be saved with 
the new name “XXXXXX recover” and this folder will be transferred via the secure 
DSL to the laboratory LCMS computer folder which contains the original data. 
 
 The reprocess function of Agilent Chemstation will then be activated on the file 
“XXXXXX recover” so that the macro generates a new raw data subset file and 
this file then will be saved with the name “rcvdata XXXXXX recover”. 
 
Validation of Data Backups 
 
 At intervals, dependent on project requirements, data backed up to the remote 
HepQuant server at RFC (Z drive) will be retrieved solely for the purpose of testing 
their completeness and integrity for validation of the data backup process. 
 
 The SOP 001101 Form: “Laboratory Data Backup Validation Form” must also be 
completed by appropriate members of the validation team. 
 
 On the form, paste screenshots of the file properties of the original raw data file 
folder and a recovered version of the raw data file folder and validate that the 

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recovered file folder is the same size as the original and contains the same 
number of files and subfolders. 
 
 Open the “rcvdata XXXXXX recover” file and label every row of data as “recover” 
to distinguish it from the original data. Copy and paste the peak areas from the 
original “rcvdata XXXXXX” onto a new column and perform a test for equality [ =IF 
(recover area = original area, TRUE, FALSE)] and check that all values return a 
result of TRUE to validate that peak areas of the file “rcv data XXXXXX recovery 
test” match original peak areas. 
 
 Transfer of the “rcvdata XXXXXX recover” file will be to an analyst’s encrypted 
laptop and used it to generate standard curves and QC results. Validate that 
standard curves generated from the “rcvdata XXXXXX recover” match the original 
data standard curves in linearity (R2), slope, and back-calculated values. Validate 
that QC results values generated from the “rcvdata XXXXXX recover” match the 
original data QC results values. 
 
6.6.3 Management of Abnormal Test Results 
 
HepQuant will review all HepQuant SHUNT test results but will not distribute results to 
investigators, coordinators, GILEAD study personnel, primary care physicians, care 
providers, or subjects and no treatment or management decisions will be made on the 
basis of the HepQuant SHUNT results. 
 
6.6.4 HepQuant Study Versus Gilead Clinical Trial 
 
The HepQuant Study encompasses only the administration of the HepQuant SHUNT 
test, use of the HepQuant SHUNT Liver Diagnostic Kit, and monitoring of the test and 
test outcome.  We do not anticipate any additional risks or issues related to the 
HepQuant SHUNT test other than those described above.  AEs, SAEs, and SUSARs 
related to the HepQuant SHUNT test will be evaluated by HepQuant and reported by 
HepQuant to the appropriate regulatory authorities, including local IRBs and FDA and 
Gilead drug safety. 
 
 
 

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6.7 Pregnancy Protocol 
 
Because cholates are naturally occurring with a pool size in humans of 1 to 5 g, the 20 
and 40 mg doses of labeled cholates used in the HQ tests are unlikely to be harmful to a 
fetus. However, the effects of these compounds on the fetus are not definitively known. 
 
Subjects will be co-enrolled in a placebo-controlled Gilead clinical trial of selonsertib.  
Subjects found to be pregnant during the Gilead clinical trial must discontinue study drug 
immediately.  In this case, the subject must also stop participation in the HepQuant study. 
 
It is recommended that all participants use a reliable form of birth control throughout the 
study.  Double contraception using hormonal plus barrier methods is required. Hormonal 
birth control pills, intrauterine device (IUD), DepoProvera, Norplant, barrier methods 
(condom or diaphragm) plus a spermicidal agent, surgical sterilization, and complete 
abstinence are examples of reliable methods of birth control.  Study participants will 
comply with the pregnancy precautions, and use one of the approved contraceptive 
methods, if applicable, as specified in the clinical protocols. 
 
7.0 Potential Scientific Problems 
 
7.1 
Potential Interfering Substances in Laboratory Assay 
 
7.1.1 Assay Performance 
 
Use of concomitant medications could theoretically present potential interference in 
serum cholate measurement. The liquid chromatography-mass spectrometry (LCMS) 
technique used to measure cholate levels was validated according to FDA guidelines [66] 
for selectivity, accuracy, precision, recovery, stability, and freedom from interferences by 
serum components or medications. Freedom from interference was tested on blanks and 
at the LLOQ for each analyte with a number of metabolites and medications including: 
Bilirubin, Cholesterol, Carbamazepine, Oxazepam, Diazepam, Nordiazepam, Lorazepam, 
Temazepam, Flunitrazepam, Nitrazepam, Clonazepam, Alprazolam, Ephedrine, Codeine, 
Diphenhydramine, Nortriptyline, Propoxyphene, d-Amphetamine, d-Methamphetamine, 
Phenylpropanolamine, Phenmetrazine, Caffeine, Phencyclidine, Imipramine, 
Spironolactone, Furosemide, and dl-Propranolol.  There was no interference at the 

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concentrations tested which were above those usually observed in patient serum 
samples. 
 
7.2 
Potential Complicating Medical Issues 
 
7.2.1 Concomitant Medications 
 
There are no known interactions of drugs or medications with the cholates used in the 
HepQuant SHUNT test.  β-blockers or ACE inhibitors could affect the blood flow to the 
liver, so subjects who are currently taking either a beta blocker or an ACE inhibitor will be 
asked to delay taking their normal dose the morning of their testing.  They can take the 
morning dose of these medications immediately after the 90 minute sample for the 
HepQuant SHUNT test is obtained. Delaying these medications could cause a temporary 
elevation in blood pressure but the risk would be minimal, similar to that of subjects who 
delay doses of medications in everyday life. 
 
7.2.2 Associated Conditions 
 
Subjects with serious intercurrent medical or surgical illness, such as acute myocardial 
infarction, acute cerebral hemorrhage, sepsis, or other immediate life-threatening illness 
will be excluded.  Subjects with extensive resection of large segments of small intestine 
(short gut) or severe gastroparesis might not be able to absorb the oral dose of 4D-CA 
and are excluded. 
 
7.3 
Potential Interaction between Cholate and Study Drugs 
 
Cholate, especially in the low doses used in the HepQuant SHUNT test, does not 
interfere with hepatic metabolism.  Given these considerations there would be no 
suspected interaction between study drugs and cholate. 
 
8.0 Data Analysis and Statistics 
 
8.1 
Sample Size Calculation 
 
An important offshoot of our prior serial studies of the HepQuant SHUNT test was 
defining an average standard deviation for ∆DSI (average SD of ∆DSI = 3.5) that could 

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be used to determine sample sizes.  The following table demonstrates the sample sizes 
required to detect a range of changes in DSI at 80% power or higher – for the case of the 
NASH trials.  A sample size of 60 total subjects (N in following table), well below the size 
of our projected number of evaluable NASH patients could provide sufficient power to 
detect a difference in the ∆DSI of 2 to 3. 
 
 
Actual 
 
 
 
 
 
 
 
Power 
N1 
N2 
N 
∆DSI 
σ 
Alpha 
 
0.80010 
112 
112 
224 
1.00 
3.00 
0.050 
 
0.80084 
199 
199 
398 
1.00 
4.00 
0.050 
 
0.80590 
29 
29 
58 
2.00 
3.00 
0.050 
 
0.80590 
51 
51 
102 
2.00 
4.00 
0.050 
 
0.82409 
14 
14 
28 
3.00 
3.00 
0.050 
 
0.80486 
23 
23 
46 
3.00 
4.00 
0.050 
 
0.81316 
8 
8 
16 
4.00 
3.00 
0.050 
 
0.82409 
14 
14 
28 
4.00 
4.00 
0.050 
 
0.85055 
6 
6 
12 
5.00 
3.00 
0.050 
 
0.81383 
9 
9 
18 
5.00 
4.00 
0.050 
 
0.80153 
4 
4 
8 
6.00 
3.00 
0.050 
 
0.84086 
7 
7 
14 
6.00 
4.00 
0.050 
 
NOTE:  Once the treatment for the clinical trial is unblinded, if DSI is unchanged or 
worsens by 1 in the placebo arm, then one could detect as little as a 1 or 2 point 
reduction in DSI by selonsertib in the treatment arm.  
 
8.2 
Primary Objective 
 
8.2.1 Outline of Approach 
 
The primary objective is to determine the utility of the Disease Severity Index (DSI), 
derived from the HepQuant SHUNT test, for monitoring liver disease and treatment 
effects. 
 
 Utility will be defined by measuring DSI and the change in DSI (∆DSI) across all 
treatment arms. 
 
 The clinical significance of a change in DSI will be evaluated by analyzing the 
relationships between changes in DSI and changes in histologic stage of disease, 
clinical models, and risk for clinical outcome. 

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Treatment effect will be determined after the completion of the clinical trial and after 
completion of Gilead’s analysis of the results of the main clinical trial.  Treatment effect 
will be defined from the difference in ∆DSI between treatment and placebo arms.  
Treatment effect defined by ∆DSI will also be compared to the treatment effect defined by 
changes in other tests and by clinical outcomes. 
 
8.2.2 Comparators from the Gilead Clinical Trials 
 
The primary clinical or histologic outcomes for the Gilead Clinical Trials are: 
 
 
 STELLAR 3: 
 
Wk48: Decrease fibrosis stage ≥1; no worsening NASH 
 
 
 
 
Wk48: Freedom from progression to cirrhosis 
 
 
 
 
Wk48: Freedom from decompensation, transplant, death 
 
 
 
 
Wk240: Decrease fibrosis stage ≥1; no worsening NASH 
 
 
 
 
Wk240: Freedom from progression to cirrhosis  
 
 
 
 
 
Wk240: Freedom from decompensation, transplant, death 
 
 STELLAR 4: 
 
Wk48: Decrease fibrosis stage ≥1; no worsening NASH 
 
 
 
 
Wk48: Freedom from decompensation, transplant, death 
 
 
 
 
Wk240: Decrease fibrosis stage ≥1; no worsening NASH 
 
 
 
 
Wk240: Freedom from decompensation, transplant, death 
 
 GS-US-454-4378 (F3): Wk48: Decrease fibrosis stage ≥1; no worsening NASH 
 
 
 
 
Wk48: Freedom from progression to cirrhosis 
 
 
 
 
Wk48: Freedom from decompensation, transplant, death 
 
 GS-US-454-4378 (F4): Wk48: Decrease fibrosis stage ≥1; no worsening NASH 
 
 
 
 
Wk48: Freedom from decompensation, transplant, death 
 
The definition of worsening of NASH is a ≥1 point increase in hepatocellular ballooning or 
lobular inflammation using histologic criteria of the NASH CRN. 
 
 
 
 

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8.2.3 Statistical Considerations 
 
Our Primary Objective is to determine the utility of the Disease Severity Index (DSI), 
derived from the HepQuant SHUNT test, to monitor liver disease and treatment effects.  
We propose a three-step analytical process to achieve this goal: 
 
o First, without knowledge of treatment arm and for the group of subjects as a 
whole, we will track DSI and the change in DSI, ∆DSI, from baseline to on-
treatment and follow-up timepoints. 
 
o Second, we will apply separate survival models to evaluate ∆DSI as a 
continuous variable and a 2 point or more drop in DSI as dichotomous variable 
in the prediction of changes in other tests or models of liver disease severity 
and clinical outcomes. 
 
o Third, after unblinding the study and completion of Gilead’s analysis of the 
main clinical trial, we will make request of Gilead for allocation code to 
determine if there is a treatment effect, by comparing the difference between 
the ∆DSI of the treatment arm versus the ∆DSI of the placebo arm.  The 
significance of this difference will be evaluated using two-sided t-tests and 
survival models. 
 
Due to short enrollment period, only 5 STELLAR 3 and 4 STELLAR 4 subjects were 
enrolled into HQ-SHUNT-1701.  STELLAR 3 and STELLAR 4 had two treatment arms (6 
and 18 mg/d doses) and one placebo arm.  Of the 9 subjects enrolled in HQ-SHUNT-
1701, we would project that 6 would have been treated with selonsertib and 3 would have 
be taking placebo.  Enrollment criteria, diagnosis, and F3/F4 stage of NASH is identical 
between the STELLAR studies and Gilead 454-4378. For the final analysis of treatment 
effects, once the code on all treatment arms from all studies is unblinded the projected 6 
cases enrolled in HQ-SHUNT-1701 on selonsertib treatment from STELLAR studies will 
be added to the cases from Arm C of 454-4378 (selonsertib monotherapy) and the 
projected 3 cases enrolled in HQ-SHUNT-1701 on placebo from STELLAR studies will be 
added to the cases from Arm F (placebo) of the 454-4378 study. 
 
Furthermore, we will link treatment effect defined by the change in DSI to effects of 
treatment on other tests and clinical outcomes (bleeding from varices or portal 
hypertensive gastropathy, ascites, encephalopathy, SBP, progression to cirrhosis, and 

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patient mortality or liver transplantation).  In addition, we will use both generalized 
estimating equations (GEE) and profile analysis – profile of DSI between treatment and 
placebo groups at each time point – for additional primary endpoint analyses.  The two 
NASH trials will be combined in the analysis of treatment effect. 
 
8.3 
Secondary Objective 
 
8.3.1 Outline of Approach 
 
The secondary objective will be to determine the ability of baseline DSI, performed prior 
to treatment, to assess the severity of liver disease by: 
 
 correlation with other baseline tests of liver disease severity; and, 
 
 prediction of risk for future clinical outcome. 
 
8.3.2 Correlating baseline tests of liver disease severity 
 
Our goal is to correlate the baseline DSI as a measure of liver disease severity with other 
measurements of liver disease severity. 
 
The other measurements include: 
 
 STELLAR 3: 
 
Liver histology, std labs, MELD & CP scores 
 STELLAR 4: 
 
Liver histology, std labs, MELD & CP scores 
 GS-US-454-4378: 
Liver histology, std labs, MELD & CP scores 
 
Regression analysis will be used to obtain prediction and 95% CI of baseline NASH CRN 
fibrosis score, standard laboratory tests, and clinical models based on DSI.  In a 
secondary analysis of the combined NASH trials, Gilead may use a discriminant analysis 
to compare the ability of baseline DSI to distinguish NASH F3 from NASH F4 to the ability 
of other tests and models to discriminate F3 from F4. 
 
8.3.3 Baseline DSI as a predictor of Clinical Outcome 
 

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Our goal is to correlate the baseline DSI with risk for clinical outcomes.  The primary 
clinical outcomes in the Gilead clinical trials include: 
 STELLAR 3: 
Wk48: Decrease fibrosis stage ≥1; no worsening NASH 
 
 
 
Wk48: Freedom from progression to cirrhosis 
 
 
 
Wk48: Freedom from decompensation, transplant, death 
 
 
 
Wk240: Decrease fibrosis stage ≥1; no worsening NASH 
 
 
 
Wk240: Freedom from progression to cirrhosis 
 
 
 
Wk240: Freedom from decompensation, transplant, death 
 
 STELLAR 4: 
Wk48: Decrease fibrosis stage ≥1; no worsening NASH 
 
 
 
Wk48: Freedom from decompensation, transplant, death 
 
 
 
Wk240: Decrease fibrosis stage ≥1; no worsening NASH 
 
 
 
Wk240: Freedom from decompensation, transplant, death 
 GS-US-454-4378 (F3): Wk48: Decrease fibrosis stage ≥1; no worsening NASH 
 
 
 
 
Wk48: Freedom from progression to cirrhosis 
 
 
 
 
Wk48: Freedom from decompensation, transplant, death 
 
 GS-US-454-4378 (F4): Wk48: Decrease fibrosis stage ≥1; no worsening NASH 
 
 
 
 
Wk48: Freedom from decompensation, transplant, death 
 
We will perform survival analysis for freedom from clinical outcome by Kaplan-Meier 
plots.  Cox models will be used to estimate hazard ratios corresponding to 1 unit of DSI, 
controlling for other known risk factors of mortality (age, gender, smoking, etc).  We will 
also apply separate survival models to evaluate DSI as a continuous variable and as 
above or below mean DSI as dichotomous variable in the prediction of clinical outcomes.  
In a secondary analysis of the combined NASH trials, we will use a discriminant analysis 
to compare the ability of baseline DSI to the ability of other tests and models to predict 
clinical outcomes. 
 
9.0 Summary 
 
This HepQuant study, conducted in parallel with three Gilead clinical trials, will determine 
the utility of DSI scores derived from the HepQuant SHUNT test for monitoring liver 
disease and treatment effects by measuring liver function and physiology.  The results of 
DSI may be linked by Gilead to clinically meaningful changes in histology, laboratory 
tests, clinical models, and patient clinical outcomes.  No more than 100 subjects will be 

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studied. The proposed HepQuant study and analytical plan will specifically measure the 
ability of DSI and ∆DSI to: 
1. Define baseline disease severity, and 
2. Detect clinically-meaningful changes in DSI by relating them to: 
a. changes in histology, 
b. changes in laboratory tests, 
c. changes in clinical models, and 
d. risk for clinical outcome. 
 
The performance of DSI relative to other measurements made in these clinical trials 
could potentially establish DSI as an important tool for monitoring liver disease and 
treatment effects. 
 
The HepQuant study will address key issues in NASH – is HepQuant SHUNT suitable for 
defining severity of the liver disease and monitoring the response of the liver to NASH 
treatment? 
 
o The comparison of ∆DSI to change in NASH CRN fibrosis stage could provide 
evidence that supports use of ∆DSI as a potential surrogate for assessing 
changes in fibrosis stage – i.e., using ∆DSI as a minimally-invasive alternative 
to biopsy for monitoring treatment effects. 
 
o Examining the baseline heterogeneity in functional impairment in NASH might 
identify high and low risk subgroups with different treatment response or 
clinical outcomes (such as progression to cirrhosis, or risk for clinical 
outcomes). 
 
o Comparing the ∆DSI of the treatment arms to the ∆DSI of the placebo arm will 
demonstrate the effect of treatment.  Not only baseline DSI but also ∆DSI will 
be correlated to the other tests, clinical models, and clinical outcomes.  
Changes in individual parameters of the HepQuant SHUNT test may identify 
the specific hepatic effects of the treatment. 
 
Overall, the results from this study may support the use of HepQuant SHUNT as a tool or 
even endpoint for assessing efficacy of drug therapy of fibrotic stages of NASH in future 
trials. 
 

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