Clinical trial NCT03656068
Conditions: Non-alcoholic Steatohepatitis Fatty Liver Fibrosis, Liver Compensated Cirrhosis
Sponsor: Pinnacle Clinical Research, PLLC
Start date: 2018-12-04; Completion date: 2020-11-25
Enrollment: 21 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
To evaluate the safety and tolerability of Nitazoxanide (NTZ) 500mg Twice Daily (BID) after 24 weeks of treatment in patients with NASH induced Stage 2 or Stage 3 fibrosis

Detailed description:
Based on the anti-fibrotic properties demonstrated in the animal models of fibrosis, this proof of concept clinical study aims at evaluating NTZ in patients with non-alcoholic steatohepatitis (NASH) and fibrosis stage 2 and 3. Although NTZ has been evaluated in liver disease populations up to 60 weeks, this is the first study evaluating NTZ treatment in a population with NASH induced stage 2 and 3 fibrosis. The aim of this study is to evaluate the safety and tolerability of NTZ 500 mg BID after 24 weeks of treatment in this population.

This proof of concept study will also evaluate the anti-fibrotic effect of NTZ as a secondary objective.

The methods of evaluation of fibrosis will include an innovative method of metabolic labeling.This approach is based on the concept that liver status can be determined by measuring the ratio of newly synthesized/pre-existing proteins.The turn-over rate of newly synthesized collagen and proteins represents the hepatic fibrogenic disease activity. Patients will be given "heavy water" to drink. Heavy water contains D20, deuterium being a stable isotope of hydrogen. Mass spectrometry is used to identify individual proteins and to quantify the ratio of labeled protein to total protein. The results are expressed as fractional synthesis rate of these proteins (FSR). This method has been previously published (Decaris et al, 2017).

Other non-invasive methods will be used to evaluate the liver stiffness changes after NTZ treatment: Magnetic Resonance Elastography (MRE) and FibroScan®.

Arms: arm 1: Open label. All patients will receive study drug
Interventions: intervention 1: Nitazoxanide 500mg BID
Intervention descriptions: intervention 1: Patients will receive 500mg of Nitazoxanide BID daily for 24 weeks

Protocol documents:
SAP_006.pdf: 
Statistical Analysis Plan 
 
A Monocentric, Open-Label, Proof of Concept Study to Evaluate the 
Safety and Efficacy of NTZ at 500mg Twice Daily on Collagen Turnover 
in Plasma in NASH Patients with Fibrosis Stage 2 or 3. 
 
 
NCT Number: 03656068 
Unique Protocol ID: NTZ-218-1 
Version Date : 30 April 2019 


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STATISTICAL ANALYSIS PLAN 
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Pinnacle Clinical Research 
CONFIDENTIAL 
Statistical Analysis Plan 
Protocol No: NTZ-218-1 
 
Version 1.0 / 30-APR-2019 
Page 3 of 15 
 
VERSION HISTORY 
Version 
Version Date 
Description 
1.0 
30-APR-2019 
Original signed version 
 
 
 
 
 


Pinnacle Clinical Research 
CONFIDENTIAL 
Statistical Analysis Plan 
Protocol No: NTZ-218-1 
 
Version 1.0 / 30-APR-2019 
Page 4 of 15 
 
TABLE OF CONTENTS 
1 
Introduction ......................................................................................................................... 7 
2 
Study Overview ................................................................................................................... 7 
2.1 
Study Objectives and Endpoints .................................................................................. 7 
2.2 
Study Design ............................................................................................................... 8 
2.2.1 
Overview ............................................................................................................... 8 
2.2.2 
Randomization and Blinding ................................................................................. 8 
2.2.3 
Study Drug ............................................................................................................ 8 
2.2.4 
Sample Size Determination ................................................................................... 8 
3 
Statistical Methodology ....................................................................................................... 9 
3.1 
General Considerations ............................................................................................... 9 
3.1.1 
Analysis Day ......................................................................................................... 9 
3.1.2 
Analysis Visits ....................................................................................................... 9 
3.1.3 
Definition of Baseline ............................................................................................ 9 
3.1.4 
Summary Statistics ............................................................................................... 9 
3.1.5 
Hypothesis Testing ............................................................................................... 9 
3.1.6 
Handling of Dropouts and Missing Data ................................................................ 9 
3.2 
Analysis Populations .................................................................................................... 9 
3.2.1 
Full Analysis Set ................................................................................................... 9 
3.2.2 
Safety Analysis Set ............................................................................................... 9 
3.3 
Subject Data and Study Conduct ................................................................................10 
3.3.1 
Subject Disposition and Analysis Populations ......................................................10 
3.3.2 
Protocol Deviations ..............................................................................................10 
3.3.3 
Demographic and Baseline Characteristics ..........................................................10 
3.3.4 
Medical History ....................................................................................................10 
3.3.5 
Concomitant Medications .....................................................................................10 
3.3.6 
Study Drug Exposure and Compliance ................................................................11 
3.4 
Efficacy Assessments .................................................................................................11 
3.4.1 
Fractional Synthesis Rate ....................................................................................11 
3.4.2 
Non-invasive Markers of Fibrosis .........................................................................12 
3.4.3 
Fibrosis Scores ....................................................................................................12 
3.4.4 
Exploratory Analysis ............................................................................................12 
3.5 
Safety Assessments ...................................................................................................13 
3.5.1 
Adverse Events ....................................................................................................13 
3.5.2 
Clinical Laboratory Tests .....................................................................................13 
3.5.3 
Vital Signs ............................................................................................................14 
3.5.4 
Electrocardiograms ..............................................................................................14 
3.5.5 
Physical Examinations .........................................................................................14 
3.5.6 
Other Safety Assessments ...................................................................................14 
4 
Data Safety Monitoring Board ............................................................................................14 


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Statistical Analysis Plan 
Protocol No: NTZ-218-1 
 
Version 1.0 / 30-APR-2019 
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5 
Analysis Timing ..................................................................................................................14 
5.1 
Interim Analysis ..........................................................................................................14 
5.2 
Pre-Final Analysis .......................................................................................................14 
5.3 
Final Analysis ..............................................................................................................14 
6 
Changes from Protocol-Specified Statistical Analyses .......................................................15 
7 
Programming Specifications ..............................................................................................15 
 
 
 


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Statistical Analysis Plan 
Protocol No: NTZ-218-1 
 
Version 1.0 / 30-APR-2019 
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LIST OF ABBREVIATIONS 
Abbreviation 
Definition 
ADaM 
Analysis Data Model 
AE 
Adverse event 
ALT 
Alanine Aminotransferase 
AST 
Aspartate aminotransferase 
ATC 
Anatomical therapeutic chemical 
BID 
Twice daily 
BMI 
Body mass index 
CI 
Confidence interval 
CK 
Creatine Kinase 
CRF 
Case report form 
CSR 
Clinical Study Report 
DSMB 
Data Safety Monitoring Board 
ECG 
Electrocardiogram 
EOS 
End of study 
FSR 
Fractional Synthesis Rate 
MedDRA 
Medical Dictionary for Regulatory Activities 
MRE 
Magnetic Resonance Elastrography 
MRI 
Magnetic resonance imaging 
NAFLD 
Non alcoholic fatty liver disease 
NASH 
Non alcoholic steatohepatitis 
Q1 
First quartile 
Q3 
Third quartile 
SAE 
Serious adverse event 
SAP 
Statistical Analysis Plan 
SDTM 
Study Data Tabulation Model 
TEAE 
Treatment-emergent adverse event 
TFL 
Table, figure, and listing 
ULN 
Upper limit of normal 
WHO 
World Health Organization 
 
 
 


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Statistical Analysis Plan 
Protocol No: NTZ-218-1 
 
Version 1.0 / 30-APR-2019 
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1 INTRODUCTION 
The purpose of this Statistical Analysis Plan (SAP) is to provide a description of the statistical 
methods to be implemented for the analysis of data from the study with protocol number 
NTZ-218-1.  The SAP will be finalized prior to database lock.  Any deviations from the SAP after 
database lock will be documented in the final Clinical Study Report (CSR). 
2 STUDY OVERVIEW 
2.1 Study Objectives and Endpoints 
Primary 
Objective 
Endpoint 
To evaluate the safety and tolerability of 
NTZ 500 mg twice daily (BID) after 24 weeks 
of treatment in patients with non alcoholic 
steatohepatitis (NASH)-induced stage 2 or 
stage 3 fibrosis. 
Adverse events (AEs) including treatment-
emergent AEs (TEAEs) and serious AEs 
(SAEs), laboratory evaluations, vital signs, 
electrocardiograms (ECGs), and physical 
examinations.   
Secondary 
Objective 
Endpoint 
To determine the effect of oral administration 
of NTZ 500 mg BID on de novo collagen 
synthesis through Fractional Synthesis Rate 
(FSR) of circulating plasma proteins in 
patients with NASH induced stage 2 or stage 
3 fibrosis. 
Percent change in FSR from baseline to end 
of treatment (24 weeks of treatment). 
To assess changes in liver stiffness as 
measured by FibroScan® and by Magnetic 
Resonance Elastography (MRE). 
Observed change in liver stiffness by 
FibroScan from baseline to 24 weeks of 
treatment 
Observed change in liver stiffness by MRE 
from baseline to 12 weeks and 24 weeks of 
treatment. 
To assess the change from baseline in non-
invasive markers of fibrosis: ELFTM test 
score, PIIINP, Hyaluronic acid, CK18, 
TIMP-1, YKL-40, Alpha 2 macroglobulin, 
miRNA, ProC3, ProC6, FGF19, and FGF21. 
Observed change in non-invasive biomarkers 
of fibrosis from baseline to 12 weeks and 24 
weeks of treatment. 
To assess changes in additional non-
invasive tests: fibrosis scores: non alcoholic 
fatty liver disease (NAFLD) fibrosis score 
and FIB-4. 
Observed change in fibrosis scores from 
baseline to 12 weeks and 24 weeks of 
treatment. 


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Protocol No: NTZ-218-1 
 
Version 1.0 / 30-APR-2019 
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2.2 Study Design 
2.2.1 Overview 
This is a Phase 2 proof of concept study to evaluate the safety and efficacy of open-label 
NTZ 500 mg BID in a NASH population with NASH induced Stage 2 or 3 fibrosis. 
The study will include a screening visit within 6 weeks of randomization (i.e. Day 1) followed by 
a 24-week treatment period consisting of daily, BID, oral administration of NTZ and an end of 
treatment follow up period of 4 weeks.   
Patients will come to the research center and complete the following visits: 
 
Screening Visit within 6 weeks of Randomization (i.e. Day 1) 
 
Day -14 Deuterated Water Run-In 
o Day-14, Day-11, Day-7 and Day 1: sampling for labeling of Deuterated water 
during run-in, 
 
Repeat baseline labs for alanine aminotransferase (ALT), aspartate aminotransferase 
(AST), total bilirubin, alkaline phosphatase, creatine kinase (CK) collected at least 4 
weeks from initial screening. 
 
Day 1 randomization: start of treatment  
 
Every 4 weeks after randomization until 20 weeks post randomization 
 
22 weeks post enrollment and Deuterated Water Administration Period 
o 22 weeks plus 3 days 
o Sampling for labeling of Deuterated Water on Day 155, Day 158, Day 162, and 
Day 169 
 
24 weeks post randomization: End of Treatment 
 
Week Post Treatment Follow Up 
2.2.2 Randomization and Blinding 
There is only one treatment group; there is no randomization. Any use of “randomization” refers 
to enrollment Day 1 (i.e. subject passed screening and will enter the treatment period). This is 
an open-label study; there is no blinding. 
2.2.3 Study Drug 
Patients will receive 500 mg NTZ (ALINIA®) twice daily for 24 weeks. NTZ will be supplied as 
500 mg of ALINIA (nitazoxanide) tablets, for oral use.  The tables are round, yellow, film-coated 
and debossed with ALINIA on one side and 500 on the other side. Each tablet contains 500 mg 
of nitazoxanide. 
2.2.4 Sample Size Determination 
The sample size of this Phase 2 proof of concept study is based on clinical assessment. Twenty 
male and female subjects with histologically confirmed NASH and fibrosis stage 2 or 3 will 


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Protocol No: NTZ-218-1 
 
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provide sufficient information to assess the safety and tolerability of NTZ 500mg BID over 24 
weeks. 
3 STATISTICAL METHODOLOGY 
3.1 General Considerations 
3.1.1 Analysis Day 
Analysis day will be calculated from the date of first dose of study drug.  The day of the first 
dose of study drug will be Day 1, and the day immediately before Day 1 will be Day -1.  There 
will be no Day 0.  
3.1.2 Analysis Visits 
Scheduled visits will be assigned to analysis visits as recorded on the case report form (CRF). 
Where applicable, early termination visits will be summarized with Visit 10 Week 24 (i.e. Visit 10 
Week 24/Early Termination).  
3.1.3 Definition of Baseline  
Baseline is defined as the last measurement prior to the first dose of study drug (generally 
should be the Day 1 pre-dose assessment). 
3.1.4 Summary Statistics 
Categorical data will generally be summarized with counts and percentages of subjects.  The 
denominator used for the percentage calculation will be clearly defined in the table.  Continuous 
data will generally be summarized with descriptive statistics including n (number of non-missing 
values), mean, standard deviation, first quartile (Q1), median, third quartile (Q3), minimum, and 
maximum. 
3.1.5 Hypothesis Testing 
This is a proof of concept study to assess the safety and tolerability of NTZ 500 mg BID over 24 
weeks. There will be no formal hypothesis testing. 
3.1.6 Handling of Dropouts and Missing Data 
All data will be analyzed as observed. Dropouts and missing data will not be imputed.    
3.2 Analysis Populations 
3.2.1 Full Analysis Set 
The Full Analysis Set consists of all subjects that meet the eligibility criteria and enroll into the 
study (Visit 1 Day 1). 
The Full Analysis Set will be the primary population for all efficacy analyses. Only subjects with 
results for the specified efficacy parameter will be included in each analysis.  
3.2.2 Safety Analysis Set 
The Safety Analysis Set consists of all enrolled subjects who receive at least one dose of study 
drug. 


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Protocol No: NTZ-218-1 
 
Version 1.0 / 30-APR-2019 
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The Safety Analysis Set will be the primary population for all safety analyses. 
3.3 Subject Data and Study Conduct 
3.3.1 Subject Disposition and Analysis Populations 
Counts and percentages of subjects who were screened (signed informed consent), 
discontinued early during screening (screen failures), and enrolled (Full Analysis Set) will be 
summarized in total based on all screened subjects.  Reasons for early discontinuation will also 
be summarized. 
Counts and percentages of subjects in the Full Analysis Set, Safety Analysis Set, discontinued 
early from the study, and completed the study will be summarized based on the Full Analysis 
Set.  Reasons for early discontinuation will also be summarized. 
3.3.2 Protocol Deviations 
Protocol deviations will be logged in the Medpace ClinTrak system. All protocol deviations will 
be listed. 
3.3.3 Demographic and Baseline Characteristics 
The following demographic and baseline characteristics will be summarized based on the Full 
Analysis Set: 
 
Age (years) at informed consent and age categories (<65 years, ≥65 years),  
 
Sex,  
 
Race,  
 
Ethnicity,  
 
Height (cm) at Screening,  
 
Baseline weight (kg), and 
 
Baseline body mass index (BMI) (kg/m2). 
3.3.4 Medical History 
All medical history will be listed. 
3.3.5 Concomitant Medications 
Prior and concomitant medications will be coded to anatomical therapeutic chemical (ATC) 
class and preferred term using the World Health Organization (WHO) Drug Dictionary version 
Global B3 September 2018.  For summary purposes, medications will be considered prior 
medications if they stopped prior to the first dose of study drug and concomitant medications if 
they were taken at any time after the first dose of study drug (i.e. started prior to the first dose of 
study drug and were ongoing at the time of the first dose of study drug, or started after the first 
dose of study drug). Medications cannot be counted as both prior and concomitant. 
If a medication has incomplete start or stop dates, dates will not be imputed to determine 
whether a medication should be considered prior or concomitant. If the date is missing but it is 
known that the medication was taken within the same month as the first dose of study drug, it 
will be considered concomitant. If the date and month are missing but it is known that the 
medication was taken within the same year as the first dose of study drug, it will be considered 
concomitant. 


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Counts and percentages of subjects taking prior and concomitant medications by ATC class and 
preferred term will be summarized based on the Safety Analysis Set.   
3.3.6 Study Drug Exposure and Compliance 
Days of exposure to study drug will be calculated as date of last dose of study drug as recorded 
on the end of study form – date of first dose of study drug + 1.  Note that the exposure 
calculation is intended to describe the length of time a subject was exposed to study drug and 
therefore does not take study drug interruptions into account.  Days of exposure to study drug 
will be summarized based on the Safety Analysis Set with descriptive statistics and with counts 
and percentages of subjects with exposure in the following categories: 
 
<4 weeks (<29 days) 
 
4 to <8 weeks (29 – 56 days) 
 
8 to <12 weeks (57 – 84 days) 
 
12 to <16 weeks (85 – 112 days) 
 
16 to <20 weeks (113 – 140 days) 
 
20 to <24 weeks (141 – 168 days) 
 
≥24 weeks (≥169 days) 
Percent compliance to the study drug regimen will be calculated as 100 x number of actual 
capsules taken / number of expected capsules taken.  The number of actual capsules taken will 
be calculated as number of capsules dispensed – number of capsules returned.  If study drug is 
not returned, the number of capsules returned will be considered 0 for the compliance 
calculation, i.e. it will be assumed that the subject took all capsules.  The number of capsules 
expected will be calculated as 2 x days of exposure.  Percent compliance to the study drug 
regimen will be summarized based on the Safety Analysis Set with descriptive statistics and 
with counts and percentages of subjects with compliance in the following categories: 
 
<80% 
 
80-120% 
 
>120% 
3.4 Efficacy Assessments 
3.4.1 Fractional Synthesis Rate 
FSR of circulating plasma proteins will be measured at baseline and after 24 weeks of 
treatment.  The observed change and percent change in FSR from baseline to Visit 10 Week 24 
between the first and second deuterated water loading periods will be assessed to determine 
the effects of NTZ 500 mg BID on de novo collagen synthesis in the Full Analysis Set.   
Wilcoxon signed-rank test will be used for statistical inference. Summary statistics of change in 
FSR (observed change and percent change) from baseline in FSR will be reported and 
evaluated at a significance level of 0.05. The p-value from Wilcoxon signed rank test will be 
reported. Only subjects with FSR at both baseline and Visit 10 Week 24 will be included in 
analysis.Liver Stiffness 


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FibroScan 
Summary statistics for liver stiffness as measured by FibroScan will be provided at baseline and 
Visit 10 Week 24/Early Termination, including the change from baseline, based on the Full 
Analysis Set. 
Student’s T-test will be used for statistical inference. Summary statistics of change in liver 
stiffness as measured by FibroScan from baseline in FSR will be reported and evaluated at a 
significance level of 0.05. The 95% confidence interval (CI) and p-value from Student’s T-test 
will be reported. Only subjects with FibroScan at both baseline and Visit 10 Week 24/Early 
Termination will be included in analysis.  
MRE 
Summary statistics for liver stiffness as measured by MRE will be provided at baseline, Visit 4 
Week 12, and Visit 10 Week 24/Early Termination, including the changes from baseline, based 
on the Full Analysis Set. Student’s T-test will be used for statistical inference in the same 
manner as for FibroScan. 
3.4.2 Non-invasive Markers of Fibrosis 
Biomarkers of fibrosis include ELF test score, PIIINP, Hyaluronic acid, CK18, TIMP-1, YKL-40, 
Alpha 2 macroglobulin, miRNA, ProC3, ProC6, FGF19, and FGF21. 
Summary statistics for biomarkers will be provided at baseline, Visit 4 Week 12, Visit 10 Week 
24/Early Termination, and at end of study (EOS) Week 28, including the changes from baseline, 
based on the Full Analysis Set. Student’s T-test will be used for statistical inference in the same 
manner as for FibroScan. 
3.4.3 Fibrosis Scores 
NAFLD Fibrosis Score 
Summary statistics for NAFLD fibrosis score will be provided at baseline, Visit 4 Week 12, and 
Visit 10 Week 24/Early Termination, including changes from baseline, based on the Full 
Analysis Set. Student’s T-test will be used for statistical inference in the same manner as for 
FibroScan. 
FIB-4 
Summary statistics for FIB-4 will be provided at baseline, Visit 4 Week 12, and Visit 10 Week 
24/Early Termination, including changes from baseline, based on the Full Analysis Set. 
Student’s T-test will be used for statistical inference in the same manner as for FibroScan. 
3.4.4 Exploratory Analysis 
Additional serum and plasma samples will be maintained for future analysis.  Exploratory 
measures for magnetic resonance imaging (MRI)/MRE sequence development will be 
maintained for future analysis. 
Exploratory scatter plots will be provided for FSR vs. ProC3, FSR vs. liver stiffness as measured 
by MRE, and FSR vs. ELF test score, including the Pearson Correlation Coefficients and p-
values. Additional exploratory analyses may also be performed to evaluate the association of 
individual exploratory biomarkers or combination of biomarkers with clinical measurements and 
other risk factors.   


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3.5 Safety Assessments 
3.5.1 Adverse Events 
All AEs regardless of seriousness or relationship to study drug, including those occurring during 
the Screening Period, are to be recorded on the corresponding page(s) of the CRF and in the 
patient’s medical record from the informed consent form signature until study end for each 
patient. All AEs will be coded to system organ class and preferred term using Medical Dictionary 
for Regulatory Activities (MedDRA) version 21.0.   
TEAEs are defined as AEs that worsen in severity or relationship to study drug or commence on 
or after the time of start of first study drug administration. This may include AEs that start during 
follow-up after the last dose of study drug. 
Study drug related AEs are defined as AEs that are possibly related or related. 
An overview of AEs based on the Safety Analysis Set will be provided including counts and 
percentages of subjects with the following: 
 
Any TEAEs (overall and by maximum severity) 
 
Any study drug related TEAEs (overall and by maximum severity) 
 
Any SAEs 
 
Any study drug related SAEs 
 
Any deaths due to AEs 
 
Any AEs leading to withdrawal from study or study drug 
 
Any study drug related AEs leading to withdrawal from study or study drug 
Counts and percentages of subjects will also be presented by system organ class and preferred 
term for TEAEs and study drug related TEAEs. In addition, summaries will be provided by 
system organ class, preferred term, and maximum severity. 
Listings will be presented specifically for SAEs and TEAEs leading to withdrawal from study or 
study drug. 
3.5.2 Clinical Laboratory Tests 
Blood and urine samples for chemistry, hematology, coagulation, urinalysis, and other safety 
markers will be processed by a central laboratory. Values and observed changes from baseline 
will be summarized at each scheduled visit (Visit 2 Week 4, Visit 3 Week 8, Visit 4 Week 12, 
Visit 5 Week 16, Visit 6 Week 20, Visit 10 Week 24/Early Termination, and EOS Week 28) by 
laboratory test based on the Safety Analysis Set. 
Shift tables from baseline to maximum post-baseline value (including consideration of 
unscheduled visits) will be presented for ALT and AST (≤1x upper limit of normal [ULN], 
>1xULN to ≤3xULN, >3xULN to ≤5xULN, >5xULN), CK (≤1xULN, >1xULN to ≤2.5xULN, 
>2.5xULN to ≤5xULN, >5xULN), and total bilirubin (≤1xULN, >1xULN to ≤1.5xULN, >1.5xULN to 
≤2xULN, >2xULN). 


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3.5.3 Vital Signs 
Values and observed changes from baseline will be summarized at baseline and each 
scheduled visit (Visit 4 Week 12, Visit 10 Week 24/Early Termination, and EOS Week 28) for 
vital signs (systolic blood pressure, diastolic blood pressure, and heart rate), weight, and BMI 
based on the Safety Analysis Set. 
3.5.4 Electrocardiograms 
Values at baseline and Visit 10 Week 24/Early Termination, including the observed change from 
baseline, for 12-lead ECG parameters (heart rate, PR interval, QRS duration, QT interval, QTc 
interval, QTcF interval, and RR interval) will be summarized based on the Safety Analysis Set. 
The overall interpretations (normal, abnormal not clinically significant, or abnormal clinically 
significant) will be summarized by visit. 
3.5.5 Physical Examinations 
Physical examination findings (normal, abnormal not clinically significant, or abnormal clinically 
significant) at baseline, Visit 4 Week 12, Visit 10 Week 24/Early Termination, and EOS Week 28 
will be summarized by body system based on the Safety Analysis Set.  
3.5.6 Other Safety Assessments 
Other safety assessments such as pregnancy tests will be listed. 
4 DATA SAFETY MONITORING BOARD 
An independent Data Safety Monitoring Board (DSMB) composed of 3 liver experts will review 
the safety of patients enrolled in the trial. The DSMB will perform a safety data review every 3 
months, the first review occurring 3 months after first patient randomized. A DSMB charter will 
define the role, responsibilities, rules, and tasks of the DSMB. 
5 ANALYSIS TIMING 
5.1 Interim Analysis 
No interim analysis is planned.   
5.2 Pre-Final Analysis 
After the database is locked, the pre-final analysis will be generated.  Pre-final tables, figures, 
and listings (TFLs) will be provided approximately 3 weeks after database lock.   
5.3 Final Analysis 
After all comments on the pre-final analysis have been resolved and the study analysis 
database is declared final, the final analysis will be generated.  Final TFLs will be provided 
approximately 1 week after the study database is declared final.  If there were no changes to the 
pre-final analysis or the study database, the pre-final TFLs may be considered final.  In addition 
to TFLs, Study Data Tabulation Model (SDTM) data and Analysis Data Model (ADaM) data 
along with associated files will be provided.   


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6 CHANGES FROM PROTOCOL-SPECIFIED STATISTICAL ANALYSES 
There are no changes from the protocol-specified statistical analysis. 
7 PROGRAMMING SPECIFICATIONS 
Analyses will be performed using SAS® version 9.4.  All available data will be presented in 
subject data listings which will be sorted by subject and visit date as applicable.  Detailed 
Programming Specifications will be provided in a separate document. 
 

Prot_003.pdf: 
Protocol 
 
A Monocentric, Open-Label, Proof of Concept Study to Evaluate the 
Safety and Efficacy of NTZ at 500mg Twice Daily on Collagen Turnover 
in Plasma in NASH Patients with Fibrosis Stage 2 or 3. 
 
 
NCT Number: 03656068 
Unique Protocol ID: NTZ-218-1 
Version Date : 24 August 2019 


 
 
Protocol No: NTZ-218-1 
Version: V 24August 2019 
 
 
 
 
 
Page 1 
 
 
 
 
 
 
 
 
 
A Monocentric, Open-Label, Proof of Concept Study to Evaluate the Safety 
and Efficacy of NTZ at 500mg Twice Daily on Collagen Turnover in Plasma in 
NASH Patients with Fibrosis Stage 2 or 3. 
  
 
 
 
Protocol Number: NTZ-218-1 
Version Date: 24August 2019 
 


 
 
Protocol No: NTZ-218-1 
Version: V 24August 2019 
 
 
 
 
 
Page 2 
List of Abbreviations 
  
ACR 
 
Albumin to Creatinine Ratio 
AE             
Adverse Event  
ALT            
Alanine Aminotransferase/GPT   
AST            
Aspartate aminotransferase  
BMI            
Body Mass Index   
CDAA  
Choline-deficient L-amino Acid Defined Diet 
CFR 
 
Code of Federal Regulations 
CSR 
 
Clinical Study Report 
CPK            
Creatine phosphokinase  
CRF            
Case Report/Record Form  
CRN            
Clinical Research Network  
CTCAE  
Common Terminology Criteria for Adverse Events 
D2O 
 
Deuterated Water 
DILI 
 
Drug Induce Liver Injury 
DSMB  
Data Safety Monitoring Board 
GGT 
 
Gamma-glutamyltransferase 
eCRF           
Electronic case report form  
eGFR            
estimated Glomerular Filtration Rate   
EC            
Ethics Committee   
ECG            
Electrocardiogram  
ET             
Early Termination  
EOS 
 
End Of Study 
EU             
European Union  
FDA            
Food and Drug Administration  
FSR 
 
Fractional Synthesis Rate 
GCP            
Good Clinical Practice  
Hb             
Hemoglobin  
HBV           
Hepatitis B Virus  
HCG 
 
Human Chorionic Gonadotropin 
HCV           
Hepatitis C Virus  
HDPE  
High-density polyethylene 
HDL            
High Density Lipoprotein  
HIV            
Human Immunodeficiency Virus  
HOMA           Homeostasis Model Assessment  
HSC 
 
Hepatic Stellate Cell 
hs-CRP            High Sensitivity C-reactive protein  
ICH           
International Conference on Harmonization  
IEC 
 
Independent Ethics Committee 
IMP            
Investigational medical product 


 
 
Protocol No: NTZ-218-1 
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INR 
 
International Normalized Ratio  
IRB            
Institutional Review Board  
LDL            
Low density lipoprotein 
MedDRA          Medical Dictionary for Regulatory Activity  
MRE 
 
Magnetic Resonance Elastography 
MRI            
Magnetic Resonance Imaging  
NAFLD            Non Alcoholic Fatty Liver Disease  
NAS            
NAFLD Activity Score  
NASH            
Non Alcoholic Steatohepatitis  
NTZ 
 
Nitazoxanide 
PI             
Principal Investigator  
PK             
Pharmacokinetic 
PT/INR            Prothrombin Time/International Normalized Ratio  
 
RNA            
Ribonucleic Acid  
SAE            
Serious Adverse Event 
SAP 
 
Statistical Analysis Plan  
TQT 
 
Thorough-QT study 
  
 
 
 


 
 
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Study Synopsis 
Protocol Number: 
NTZ-218-1 
Study Title: 
A Monocentric, Open-Label, Proof of Concept Study to Evaluate the Safety 
and Efficacy of NTZ at 500mg Twice Daily on Collagen Turnover in Plasma in 
NASH Patients with Fibrosis Stage 2 or 3. 
Phase: 
Proof of Concept 
Study Drug: 
Nitazoxanide 
Indication: 
NASH induced fibrosis 
Principal Investigator & Sponsor 
Stephen Harrison, MD 
Pinnacle Clinical Research 
5109 Medical Drive, Suite 200 
San Antonio, TX 78229 
Study Duration: 
34 weeks 
Objectives: 
The primary objective of the study is: 
• 
To evaluate the safety and tolerability of NTZ 500mg bid after 24 
weeks of treatment in patients with NASH-induced stage 2 or 
stage 3 fibrosis  
 
 
The secondary objectives of the study are: 
• 
To determine the effect of oral administration of NTZ 500mg bid 
on de novo collagen synthesis through Fractional Synthesis Rate 
(FSR) of circulating plasma proteins in patients with NASH-
induced stage 2 or stage 3 fibrosis. 
• 
To assess changes in liver stiffness as measured by FibroScan® 
and by Magnetic Resonance Elastography (MRE). 
• 
To assess the change from baseline in non-invasive markers of 
fibrosis: ELFTM test score, PIIINP, Hyaluronic acid, CK18, TIMP-1, 
YKL-40, Alpha 2 macroglobulin,, ,miRNA, ProC3, ProC6, FGF19, 
FGF21. 
• 
To assess changes in additional non-invasive tests: fibrosis scores: 
NAFLD fibrosis score, FIB-4 
 
Study Design: 
This is a Phase 2 proof of concept study to evaluate the safety and efficacy 
of open-label NTZ 500mg bid in a NASH population with NASH induced 
Stage 2 or 3 fibrosis. 
 
The study will include a screening visit within 6 weeks of randomization 
(Day 1) followed by a 24-week treatment period consisting of daily, BID, 
oral administration of NTZ and an end of treatment follow up period   of 4 
weeks.   
Patients will come to the research center and complete the following visits: 
• 
Screening Visit within 6 weeks of Randomization 
• 
Day -14 Deuterated Water Run-In 
o 
Day-14, Day-11, Day-7 and Day 1: sampling for labeling 
of Deuterated water during run-in, 
• 
Repeat baseline labs for ALT, AST, total bilirubin, ALP, CPK 
collected at least 4 weeks from initial screening. 


 
 
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• 
Day 1 randomization: start of treatment 
• 
Every 4 weeks after randomization until 20 weeks post 
randomization 
• 22 weeks post randomization and Deuterated Water       
Administration Period 
o 
22 weeks plus 3 days 
o 
sampling for labeling of Deuterated water on Day 155, 
Day 158, Day 162 and Day 169  
• 
24 weeks post randomization: End of Treatment 
• 
4 Week Post Treatment Follow Up 
Number of Sites: 
1 site  
Number of Subjects: 
20 evaluable subjects 
Drug Dosage / Frequency: 
500 mg NTZ BID 
Route of Administration: 
Oral Administration 
Inclusion Criteria: 
Patients must meet all of the following inclusion criteria to be eligible for 
enrollment in the trial:  
1. Males or females aged from 18 to 75 years inclusive at the Screening 
Visit.  
2. Must provide signed written informed consent and agree to comply 
with the study protocol.  
3. Females participating in this study must be of non-childbearing 
potential or using highly efficient contraception for the full duration of 
the study.  Childbearing potential refers to those female subjects who 
have not had a hysterectomy, bilateral oophorectomy, or medically-
documented ovarian failure. Female subjects are considered 
postmenopausal and not of child-bearing potential if they are >55 years 
of age, or >50 years of age with >12 months of amenorrhea or have 
serum follicle stimulating hormone (FSH) values >40 mIU/ml with >12 
months of amenorrhea.  Highly efficient contraception is defined as the 
use of one of the following methods of birth control in addition to a 
male partner using a condom (unless surgically sterile) from Screening 
to 90 days after the last dose of study medication: 
(1) hormone-containing contraceptive 
(2) intrauterine device 
(3) cervical cap or diaphragm with spermicidal agent 
4. Histological confirmation of steatohepatitis on a diagnostic liver biopsy 
(biopsy obtained within 6 months prior to Screening or during the 
Screening Period) with at least 1 in each component of the NAS 
(steatosis scored 0-3, ballooning degeneration scored 0-2, and lobular 
inflammation scored 0-3).   
5.    Fibrosis stage of 2 or 3, according to the NASH CRN fibrosis staging 
system on a diagnostic liver biopsy (biopsy obtained within 6 months 
prior to Screening or during the Screening Period).   
6. Two assessments of ALT, AST, Total bilirubin, ALP, CPK will be collected 
during screening at least 4 weeks apart. To be eligible the second value 
cannot be ≥2x the first value. 
Exclusion Criteria: 
Patients who meet any of the following criteria will be excluded from 
entering the study:  


 
 
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1. History of efficient bariatric surgery within 5 years prior to Screening, 
or planned bariatric surgery in the course of the study.  
2. Patients with HbA1c >10.0%. If abnormal at the first Screening Visit, the 
HbA1c measurement can be repeated. A repeated abnormal HbA1c 
(HbA1c >10.0%) leads to exclusion.  
3. Patients with a history of clinically significant acute cardiac event, or 
cerebrovascular event within 6 months prior to Screening such as: 
stroke, transient ischemic attack, or coronary heart disease (angina 
pectoris, myocardial infarction, revascularization procedures).  
4. Weight loss of more than 10% within 6 months prior to Randomization.  
5. Patient with any history or presence of cirrhosis. 
6. Current or recent history (within a <1 year prior to screening ) of 
significant alcohol consumption. For men, significant consumption is 
typically defined as higher than 30 g pure alcohol per day. For women, 
it is typically defined as higher than 20 g pure alcohol per day.  
7. Current or history of other substance abuse within 1 year prior to 
screening. 
8. Pregnant or lactating females or females planning to become pregnant 
during the study period.  
9. Other well documented causes of chronic liver disease according to 
standard diagnostic procedures including, but not restricted to:  
a. Positive hepatitis B surface antigen (HBsAg)  
b. Positive HCV RNA, (tested for in case of known cured HCV 
infection, or positive HCV Ab at Screening)  
c. 
Suspicion of drug-induced liver disease  
d. Alcoholic liver disease  
e. Autoimmune hepatitis  
f. 
Wilson’s disease   
g. 
Primary biliary cirrhosis 
h. Primary sclerosing cholangitis  
i. 
Genetic homozygous hemochromatosis  
j. 
Known or suspected HCC  
k. 
History or planned liver transplant, or current MELD 
score >15.  
10. Patients who cannot be contacted in case of emergency.  
11. Known hypersensitivity to the investigational product or any of its 
formulation excipients.  
12. Patients who are taking warfarin or other highly plasma protein-bound 
drugs with narrow therapeutic indices. 
13. Patients who are currently participating in, plan to participate in, or 
have participated in an investigational drug trial or medical device trial 
containing active substance within 30 days or five half-lives, whichever 
is longer, prior to Screening. Patients who have screened for, and failed 
to qualify, for another clinical trial are eligible for screening for the NTZ-
218-1, even if the Screening Visit for the NTZ-218-1 study is less than 30 
days from the failed study screening visit, provided that the subject 
received no investigational product within 30 days of 5 half-lives of the 
investigational product prior to screening for NTZ-218-1. 


 
 
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14. Evidence of any other unstable or, untreated clinically significant 
immunological, endocrine, hematological, gastrointestinal, 
neurological, neoplastic, or psychiatric disease.  
15. Mental instability or incompetence, such that the validity of informed 
consent or ability to be compliant with the study is uncertain.  
16.  History of noncompliance with medical regimens, or patients who are 
considered to be unreliable 
17. Positive anti-human immunodeficiency virus (HIV) antibody. 
18. AST and/or ALT >5 x upper limit of normal (ULN).  
19. Total bilirubin >1.3 mg/dL due to altered hepatic function.  
20. Direct bilirubin > ULN 
Note: Gilbert Disease patients are allowed into the study.  
21. INR >1.2 in the absence of anticoagulant therapy.  
22. Platelet count <150,000/mm3 in the   context of portal hypertension.  
23. Significant renal disease, including nephritic syndrome, chronic kidney 
disease (defined as patients with markers of kidney damage or eGFR of 
less than 60 ml/min/1.73 m2). 
 
Evaluation Criteria 
Primary Endpoint: 
To assess the safety and tolerability of NTZ after 24 weeks of treatment  
 
Secondary Endpoints: 
To assess the % change in FSR from baseline to end of treatment (24 weeks 
of treatment) 
To assess the change in liver stiffness by MRE from baseline to 12 weeks 
and 24 weeks of treatment 
To assess the change in liver stiffness by fibroscan from baseline to 24 
weeks of treatment  
To assess the change from baseline to 12 weeks and 24 weeks of treatment 
of fibrosis serum biomarkers and scores. 
 
 


 
 
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TABLE 1: Schedule of events 
 
Screening period 
 
 
Treatment period 
Visit 
SV 
SV2 
SV3 
SV4 
V1 
V2 
V3 
V4 
V5 
V6 
V7 
V8 
V9 
V10 
EOS 
ET 
Day 
-42 
-14 
-11 
-7 
1 
29 
57 
85 
113 
141 
155 
158 
162 
169 
+28 
days 
 
Window 
 
+3 
+1 
+1 
+3 
+3 
+3 
+3 
+3 
+3 
+3 
+1 
+1 
+1 
+3 
 
Week 
-6 
-2 
 
 
0 
4 
8 
12 
16 
20 
22 
 
 
24 
28 
 
Informed 
Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical 
History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inclusion 
Exclusion 
Criteria 
X 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
Height 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Weight, BMI 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
Physical Exam 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
12-Lead ECG 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
X 
Vital Signs 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
Con Meds 
X 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
AEs 
X 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Liver Biopsy1 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
MRE 
 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
 
X 
Fibroscan 
 
 
 
 
X 
 
 
 
 
 
 
 
 
X 
 
X 
Dispense 
deuterated 
water 
 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
FSR 
 
X 
X 
X 
X 
 
 
 
 
 
X 
X 
X 
X 
 
 
Study Drug 
Dispense 
 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
Study Drug 
Compliance 
 
 
 
 
 
X 
X 
X 
X 
X 
 
 
 
X 
 
 
X 
 
 
                                                          
1 Required only if there is no history of a liver biopsy within the past 6 months.  


 
 
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TABLE 2: Biological assessments 
 
Screening period 
 
 
Treatment period 
Visit 
SV 
SV2 
SV3 
SV4 
V1 
V2 
V3 
V4 
V5 
V6 
V7 
V8 
V9 
V10 
EOS 
ET 
Day 
-42 
-14 
-11 
-7 
1 
29 
57 
85 
113 
141 
155 
158 
162 
169 
+28 
days 
 
Week 
-6 
-2 
 
 
0 
4 
8 
12 
16 
20 
22 
 
 
24 
28 
 
Labs - 
Haematology 
haemoglobin, 
haematocrit, RBC, 
WBC, differential 
count, platelet 
count, 
reticulocytes 
count, MCV 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Coagulation (PT 
(INR)) 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Labs- Urinary 
Pregnancy tests2 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
 
X 
Labs – Serology 
HIV, HBs and HCV 
serology 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
                                                          
2 If Urine pregnancy test is positive, a serum β-hCG is required.  
 
 
 


 
 
Protocol No: NTZ-218-1 
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Screening period 
 
 
Treatment period 
Visit 
SV 
SV2 
SV3 
SV4 
V1 
V2 
V3 
V4 
V5 
V6 
V7 
V8 
V9 
V10 
EOS 
ET 
Day 
-42 
-14 
-11 
-7 
1 
29 
57 
85 
113 
141 
155 
158 
162 
169 
+28 
days 
 
Labs – 
Biochemistry 
HbA1c, fasting 
plasma glucose, 
insulin, HOMA-IR, 
fructosamine, 
alkaline 
phosphatase, ALT, 
AST, GGT, CPK, 
total and 
conjugated 
bilirubin, ferritin, 
creatinine, eGFR, 
uric acid, BUN, 
total cholesterol, 
HDL-C, LDL-C, 
triglycerides, 
albumin, lipase, 
amylase, 
electrolytes 
(sodium, 
potassium, 
chloride, calcium), 
MELD 
X 
 
 
X3 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Inflammatory 
markers 
hsCRP, fibrinogen, 
haptoglobin, 
resistin,  TNF-, 
TGF-, IL-6, PAI-1 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
Biomarkers of 
fibrosis 
ELF Score, PIIINP, 
Hyaluronic acid, 
CK18, TIMP-1, YKL-
40, Alpha 2 
macroglobulin,, 
,miRNA, ProC3, 
ProC6, FGF19, 
FGF21 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
NAFLD Fibrosis 
Score (NFS) &  
FIB-4 
 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
 
X 


 
 
Protocol No: NTZ-218-1 
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Screening period 
 
 
Treatment period 
Visit 
SV 
SV2 
SV3 
SV4 
V1 
V2 
V3 
V4 
V5 
V6 
V7 
V8 
V9 
V10 
EOS 
ET 
Day 
-42 
-14 
-11 
-7 
1 
29 
57 
85 
113 
141 
155 
158 
162 
169 
+28 
days 
 
Labs – Urinalysis 
dipstick  
Specific gravity, 
pH, protein, 
glucose, ketones, 
bilirubin, 
urobilinogen, 
blood, nitrite, 
leukocytes 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
 
X 
Safety markers 
Serum Cystatin C, 
troponin T, NT 
proBNP 
Urinary albumin, 
urinary creatinine, 
urinary ACR 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Labs -- FSR 
 
X 
X 
X 
X 
 
 
 
 
 
X 
X 
X 
X 
 
 
Sampling for 
additional 
parameters 
X 
 
 
 
X 
 
 
X 
 
X 
 
 
 
X 
X 
X 
 
3 ALT, AST, Total bilirubin, Alkaline phosphatase and CPK must be retested at least 4 weeks after initial 
screening labs. . If these values at SV4 are ≥2x Screening values SV, then the patient cannot be 
randomized. A repeat test may be performed if a screening value is considered an error. 
 
 


 
 
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Table of Contents 
1. 
Introduction .......................................................................................................................... 17 
1.1. 
Background ................................................................................................................... 17 
1.2. 
General Information for Nitazoxanide ......................................................................... 18 
1.2.1. 
Nitazoxanide (NTZ) ............................................................................................... 18 
1.2.2. 
Clinical Safety & Toxicology .................................................................................. 18 
1.2.3. 
Non clinical pharmacology ................................................................................... 20 
1.3. 
Rationale for This Study................................................................................................ 23 
2. 
Objectives .............................................................................................................................. 23 
2.1. 
Primary Objective ......................................................................................................... 23 
2.2. 
Secondary Objectives: .................................................................................................. 23 
3. 
Study Design.......................................................................................................................... 24 
3.1. 
Study Design.................................................................................................................. 24 
3.2. 
Treatment Plan and Regimen ....................................................................................... 25 
4. 
Subject Population ................................................................................................................ 25 
4.1. 
Number of Subjects & Subject Selection ..................................................................... 25 
4.2. 
Inclusion Criteria ........................................................................................................... 25 
4.3. 
Exclusion Criteria .......................................................................................................... 26 
5. 
Investigational Medicinal Products ...................................................................................... 28 
5.1. 
Description .................................................................................................................... 28 
5.2. 
Packaging ...................................................................................................................... 29 
5.3. 
Labeling ......................................................................................................................... 29 
5.4. 
Storage and Handling ................................................................................................... 29 
5.5. 
Dosage and Administration .......................................................................................... 29 
5.6. 
Dose Adjustment .......................................................................................................... 30 
5.7. 
Prior and Concomitant Medications ............................................................................ 30 
5.7.1. 
Non-permitted medication ................................................................................... 30 
5.7.2. 
Permitted Medications ......................................................................................... 30 
5.8. 
Study Drug Accountability ............................................................................................ 31 
6. 
Study Procedures .................................................................................................................. 31 


 
 
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6.1. 
Duration of Study Participation ................................................................................... 31 
6.2. 
Screening Assessment .................................................................................................. 31 
Screening Visit ....................................................................................................................... 31 
6.3. 
Day -14 Deuterated Water Run In ................................................................................ 32 
6.4. 
Treatment Period Assessments .................................................................................... 33 
6.4.1. 
Day 1 Visit.............................................................................................................. 33 
6.4.2. 
Week 4 and Week 8 Visit + 3 days ........................................................................ 33 
6.4.3. 
Week 12 Visit + 3 days .......................................................................................... 34 
6.4.4. 
Week 16 and Week 20 visits ±3 days ................................................................... 34 
6.4.5. 
Week 22 Visit + 3 days (Day 155) ......................................................................... 35 
6.4.6. 
Week 24 Visit +1 ................................................................................................... 35 
6.5. 
Follow Up Period ........................................................................................................... 36 
6.5.1. 
4-Week Post Treatment Follow up (EOS – End of study visit) ±3 days ............... 36 
6.5.2. 
Early Termination (ET) .......................................................................................... 36 
6.5.3. 
Unscheduled Visits ................................................................................................ 37 
6.6. 
Assessment for Premature Discontinuation from Study ............................................. 37 
6.6.1. 
Pre-specified Patient Discontinuation rules ........................................................ 38 
6.6.2. 
Pre-specified Trial Discontinuation rules ............................................................. 38 
6.6.3. 
Dose Adjustment .................................................................................................. 38 
6.6.4. 
Specific Liver Function Monitoring after Start of Treatment .............................. 39 
6.6.5. 
Data Safety Monitoring Board ............................................................................. 40 
6.7. 
Description of Assessments .......................................................................................... 41 
6.7.1. 
Histological Assessments ...................................................................................... 41 
6.7.2. 
Assessment of newly synthesized proteins ......................................................... 41 
6.7.3. 
Biological Assessments (Table 2) .......................................................................... 41 
Table 2 Biological assessments ...................................................................................................... 43 
6.7.4. 
Electrocardiogram ................................................................................................. 44 
6.7.5. 
MRE ....................................................................................................................... 44 
6.7.6. 
Fibroscan ............................................................................................................... 45 
6.7.7. 
Medical History ..................................................................................................... 45 
6.7.8. 
Physical Examination ............................................................................................ 45 


 
 
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6.7.9. 
Vital Signs .............................................................................................................. 45 
6.7.10. 
Pregnancy Testing ................................................................................................. 46 
7. 
Adverse Events and Toxicity Management .......................................................................... 46 
7.1. 
Definitions of Adverse Events, Serious Adverse Events and Unexpected Events ...... 46 
7.1.1. 
Adverse Events ...................................................................................................... 46 
7.1.2. 
Clinical Laboratory Abnormalities and Other Abnormal Assessments as Adverse 
Events or Serious Adverse Events ........................................................................................ 47 
7.1.3. 
Serious Adverse Events ......................................................................................... 47 
7.1.4. 
Unexpected adverse event ................................................................................... 48 
7.2. 
Assessment of Adverse Events & Serious Adverse Events .......................................... 48 
7.2.1. 
Intensity assessment ............................................................................................ 48 
7.2.2. 
Relation to the study treatment .......................................................................... 49 
7.2.3. 
Action taken and outcome ................................................................................... 50 
7.3. 
Reporting ....................................................................................................................... 51 
7.3.1. 
Reporting an adverse event ................................................................................. 51 
7.3.2. 
Initial reports......................................................................................................... 51 
7.3.3. 
Follow-up reports ................................................................................................. 52 
7.3.4. 
Follow-up .............................................................................................................. 52 
7.4. 
Post Study Reporting Requirements ............................................................................ 53 
7.5. 
Special Situation Reports .............................................................................................. 53 
7.5.1. 
Pregnancy .............................................................................................................. 53 
7.5.2. 
Medication error ................................................................................................... 53 
7.5.3. 
Misuse ................................................................................................................... 54 
7.5.4. 
Overdose ............................................................................................................... 54 
7.5.5. 
Abuse ..................................................................................................................... 54 
8. 
Statistical Considerations ..................................................................................................... 54 
8.1. 
Analysis Endpoints ........................................................................................................ 54 
8.1.1. 
Primary Endpoint .................................................................................................. 54 
8.1.2. 
Secondary Endpoints ............................................................................................ 54 
8.2. 
Analysis Sets .................................................................................................................. 55 
8.3. 
Data Handling Conventions .......................................................................................... 55 
8.4. 
Demographic Data and Baseline Characteristics ......................................................... 55 


 
 
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8.5. 
Primary Safety Analysis ................................................................................................ 55 
8.5.1. 
Adverse Events ...................................................................................................... 56 
8.5.2. 
Laboratory Evaluations ......................................................................................... 56 
8.5.3. 
Other Safety Evaluations ...................................................................................... 56 
8.6. 
Secondary Efficacy Analyses ......................................................................................... 56 
8.6.1. 
FSR ......................................................................................................................... 56 
8.6.2. 
Liver stiffness ........................................................................................................ 57 
8.6.3. 
Exploratory Analysis ............................................................................................. 57 
8.6.4. 
Interim Analysis .................................................................................................... 57 
8.7. 
Sample Size ................................................................................................................... 57 
9. 
Responsibilities ..................................................................................................................... 57 
9.1. 
Investigator Responsibilities ........................................................................................ 58 
9.2. 
Good Clinical Practice ................................................................................................... 58 
9.3. 
Institutional Review Board Review & Approval .......................................................... 59 
9.4. 
Informed Consent ......................................................................................................... 59 
9.5. 
Confidentiality .............................................................................................................. 59 
9.6. 
Study Files and Retention of Records .......................................................................... 60 
9.7. 
Electronic Case Report Forms ....................................................................................... 61 
9.8. 
Investigational Medicinal Product Accountability and Return ................................... 62 
9.9. 
Inspections .................................................................................................................... 62 
9.10. Protocol Compliance ..................................................................................................... 62 
9.11. Protocol Modifications ................................................................................................. 62 
9.12. Study Reports ................................................................................................................ 63 
9.13. Study Termination ........................................................................................................ 63 
10. 
References ......................................................................................................................... 64 
10.1. Literature ....................................................................................................................... 64 
10.2. WHO Fact Sheet 312 ..................................................................................................... 67 
10.3. Studies on ClinicalTrials.gov ......................................................................................... 67 
 
 
 


 
 
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1. Introduction 
1.1. 
Background 
Non-alcoholic fatty liver disease (NAFLD) has become one of the most prominent forms 
of chronic liver disease worldwide, reflecting the epidemic of global obesity. Those with 
the progressive variant of NAFLD, non-alcoholic steatohepatitis (NASH), are at significantly 
increased risk of multisystem morbidity and mortality. However, there are currently no 
approved pharmacologic therapies for NASH (Konerman et al, 2018). The World Health 
Organization estimates the prevalence of obesity has more than doubled since 1980 with 
>600 million people (13%) having a body mass index (BMI)) ≥30 (Konerman et al, 2018). 
In this setting, the global prevalence of diabetes among adults has also increased from 
4.7% in 1980 to 8.5% as of 2014 (WHO Fact Sheet 312) . Prevalence rates of NAFLD among 
those with metabolic disease are notably higher.  Approximately a third of patients with 
hypertension, half of patients with dyslipidaemia, up to two-thirds of patients with type II 
diabetes, and  >90% of patients undergoing bariatric surgery had evidence of NAFLD (Leite 
et al, 2009; Fruci et al, 2013; Assy et al, 2000; Wu  et al, 2016; Sasaki et al, 2014 Subichin 
et al; 2015 ; Donati et al ,2004). 
The underlying pathophysiologic mechanisms that contribute to the development and 
progression of NAFLD and NASH are complex and include modulation of metabolic 
pathways, inflammatory cascades, and/or mechanisms impacting fibrosis.  During disease 
progression fibrosis may worsen, while features of steatohepatitis resolve or ‘‘burnout”.    
As a result, NASH and fibrosis need to be evaluated independently to ensure a beneficial 
impact on one parameter does not simultaneously result in a negative impact on another 
endpoint of interest, particularly given that individual treatments tend to focus on one 
primary mechanism of action (i.e. metabolic or NASH disease modifier vs. antifibrotic) ( 
Konerman et al, 2018). 
Persistent and progressive fibrosis is a characteristic of many chronic liver diseases in 
which excessive accumulation of collagen-rich extracellular matrix is a key feature. 


 
 
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Following liver injury, quiescent Hepatic Stellate Cells (HSC) undergo a process of 
activation that is characterized by a differentiation into (α-SMA)-positive contractile 
myofibroblasts which secrete high amounts of collagen and play a central role in the 
fibrogenic process (Friedman et al, 2008, Carpino et al, 2005). Nitazoxanide may be a 
potent antifibrotic agent that can potentially be used for the treatment of different fibrotic 
pathologies, including liver fibrosis. (Belanger et al;2017) 
1.2. 
General Information for Nitazoxanide 
1.2.1. Nitazoxanide (NTZ)  
Nitazoxanide (NTZ) is a broad-spectrum antiparasitic and broad-spectrum antiviral drug 
used for the treatment of various helminthic, protozoal, and viral infections.  It was 
approved in the United States by the Food and Drug Administration (Alinia®, Romark 
Laboratories) as an Orphan Drug for treatment of diarrhea caused by C. parvum and 
Giardia intestinalis in adults and children at least 12 months of age [Rossignol, 2014]. 
The approved Alinia dose for adults is 500 mg twice daily (BID) for three days.  
1.2.2. Clinical Safety & Toxicology 
Nitazoxanide is rapidly metabolized in the liver to an active metabolite tizoxanide (TZ) in 
vivo. TZ undergoes conjugation to form tizoxanide glucuronide. Nitazoxanide is not 
detectable in the serum following oral administration.  Peak concentrations in blood 
plasma of TZ and TZ glucuronide are seen between 1-4 hours.  NTZ is excreted in both 
Urine (~33%) and feces (~67%).   
The safety of ALINIA was evaluated in 2177 HIV-uninfected subjects 12 months of age 
and older who received ALINIA Tablets or ALINIA for Oral Suspension at the 
recommended dose for at least three days. In pooled controlled clinical trials involving 
536 HIV-uninfected subjects treated with ALINIA Tablets or ALINIA for Oral Suspension, 
the most common adverse reactions were abdominal pain, headache, chromaturia and 
nausea (≥2%).[Alinia USPI] 


 
 
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Safety data were analyzed separately for 280 HIV-uninfected subjects ≥12 years of age 
receiving Alinia at the recommended dose for at least 3 days in 5 placebo-controlled 
clinical trials and for 256 HIV-uninfected subjects 1 through 11 years of age in 7 
controlled clinical trials. There were no differences between the adverse reactions 
reported for Alinia-treated subjects based upon age [Alinia USPI]. 
Twenty-one clinical studies of NTZ were conducted in patients with chronic hepatitis C 
(14 studies: Rossignol et al, 2008; Rossignol et al, 2009; Bacon et al, 2010, Rossignol et 
al, 2010; Shiffman et al, 2010 ; Laufer et al, 2011; Kabil et al, 2011 ; Amorosa et al, 2013; 
Shebab et al, 2014; Macias et al, 2015; Kohla et al, 2016; Basu et al, 2013; 
NCT01185028; NCT01770483), hepatic encephalopathy in chronic liver failure (1 study: 
Basu et al, 2008), acquired immune deficiency syndrome (AIDS)-related 
cryptosporidiosis (3 studies: Rossignol et al, 1998; Rossignol et al, 2006; Zulu et al, 
2005), or Helicobacter pylori (2 studies: Abd-Elsalam et al, 2016; Shehata et al., 2017), 
and Clostridium difficile infections (1 study: Musher et al, 2009) in the literature. The 
dose of NTZ administered in 19 studies was 500 mg BID; in one study 1000 mg NTZ BID 
was administered, whereas in one compassionate-use study the NTZ dose administered 
ranged from 500 to 1500 mg BID. Most studies administered NTZ treatment for 48 to 60 
weeks, whereas the duration of treatment in the compassionate-use study ranged from 
1 day to 4 years.  
The studies identified from the literature review demonstrate the safety of NTZ in the 
treatment of chronic hepatitis C, hepatic encephalopathy in chronic liver failure, AIDS-
related cryptosporidiosis, persistent diarrhea in patients with AIDS, and Helicobacter 
pylori and Clostridium difficile infections.  
The adverse event (AE) profile of NTZ was predominantly GI in nature and included 
nausea, vomiting, diarrhea, abdominal pain, dyspepsia, constipation, flatulence, and 
dysphagia. These GI AEs were generally mild in intensity and transient. Other AEs 
related to NTZ treatment were fatigue, rash, headache, myalgia, and arthralgia. When 


 
 
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reported, no significant changes were observed in the electrocardiograms (ECGs), vital 
signs, and clinical laboratory parameters after administration with NTZ [Stockis et al, 
2002a; Stockis et al, 2002b]. A formal International Council on Harmonization (ICH) E14 
compliant thorough QT (TQT) study [Taubel et al, 2014] completed the cardiac safety 
evaluation and showed that neither a therapeutic single dose of 675 mg nor a 2700 mg 
(supra-therapeutic) dose of NTZ prolonged the QT interval in healthy male and female 
volunteers.   
The safety profile of NTZ reported in all these studies therefore provides supporting 
evidence for the potential use of NTZ for the treatment of liver fibrosis in patients with 
NASH.  
The following adverse reactions have been identified during post approval use of ALINIA 
as described in the Package Insert:  
• Gastrointestinal disorders: diarrhea, gastroesophageal reflux disease 
• Nervous System disorders: dizziness 
• Respiratory, thoracic and mediastinal disorders: dyspnea 
• Skin and subcutaneous tissue disorders: rash, urticaria 
1.2.3. Non clinical pharmacology 
Nonclinical pharmacology studies were conducted to determine whether NTZ and its 
main metabolites (TZ and TZ glucuronide [TZ glu]) exhibit anti-fibrotic properties in vitro, 
and in vivo. 
NTZ and its main metabolite, TZ, significantly (p<0.001 from 0.3 µM) inhibited, in a dose-
dependent manner, the expression of αSMA protein in TGF-β-induced human HSCs, but 
TZ glu had no anti-fibrotic properties. NTZ and TZ demonstrated good potency as the 
IC50 was in the micromolar range.  


 
 
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The CDAA diet-induced rodent model develops a fibrosing NASH within a relatively short 
period [Takeuchi-Yorimoto et al, 2013], which makes it a suitable model for studying the 
treatment of both NASH pathology and hepatic fibrosis. 
In studies to determine the effects of NTZ on liver fibrosis in C57BL/6J mice induced by a 
CDAA diet supplemented with 1% cholesterol, microscopic examination revealed that 
NTZ at doses of 11.4 mg/kg/day and 33.9 mg/kg/day tended to reduce, in a dose-
dependent manner, CDAA + 1% cholesterol diet-induced NASH. Although NTZ did not 
have a beneficial effect on hepatic steatosis and lobular inflammation grade, a tendency 
to reduce hepatocyte ballooning was observed, which resulted in a slight reduction in 
NASH prevalence.  
At 12 weeks, NTZ significantly prevented, in a dose-dependent manner, hepatic collagen 
storage induced by the CDAA + 1% cholesterol diet. However, upon histological 
examination, this beneficial effect was not reflected by a dose-dependent improvement 
of the fibrosis grade. A significant decrease of the fibrosis area was observed with NTZ at 
doses of 11.4 mg/kg/day (p<0.05) and 78.1 mg/kg/day (p<0.001) and correlated with 
the significant decrease of the hepatic collagen content.  
The CDAA + 1% cholesterol diet notably induces a significant augmentation in circulating 
markers of liver injury such as AST, ALT, and PIIINP, which is directly associated with 
fibrosis [Tanwar et al, 2013]. Moreover, the hepatic content is dramatically modified as 
illustrated by the significant increase of collagen. Mice treated with NTZ at 
78.1 mg/kg/day exhibited lower plasma levels of ALT (-17%, p>0.05), AST (-24%, p<0.05), 
and PIIINP (-25%, p<0.01). These data suggest a potential preventive action of NTZ on 
liver damage induced by the CDAA + 1% cholesterol diet in mice.  
The hepatic expression of fibrotic markers (collagens, MMP2), but also inflammatory 
markers (TNFα) were enhanced in the animals fed the CDAA + 1% cholesterol diet as 
compared with animals fed a CSAA control diet. Chronic administration of NTZ at 
78.1 mg/kg/day to mice fed a CDAA + 1% cholesterol diet led to the inhibition of the 


 
 
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Col1α1 (-17%, p>0.05), Col1α2 (-25%, p<0.05), MMP2 (-32%, p<0.05), and TNFα  
(-28%, p<0.01) gene expression.  
In the studies with CCl4-treated mice, NTZ significantly prevented hepatic fibrosis. This 
was shown at the dose of 95.3 mg/kg/day on the hepatic collagen content decrease in 
the first study [SSR_NTZ_PHA1_004], whereas no significant improvement was observed 
upon histological examination. Conversely, in the second study [SSR_NTZ_PHA1_005], 
no significant improvement of CCl4-induced hepatic collagen content was observed, 
whereas the histological evaluation of hepatic fibrosis revealed a significant effect of 
NTZ at 32 and 104 mg/kg/day. 
There was no significant beneficial effect of NTZ treatment on plasma markers of 
hepatotoxicity in CCl4-treated mice.  
These studies demonstrate the anti-fibrotic effect of NTZ, which suggests that NTZ may 
have potential as an anti-fibrotic drug. These studies also suggest that NTZ may show 
some beneficial therapeutic properties for NASH, such as a tendency to reduce 
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1.3. 
Rationale for This Study 
Based on the anti-fibrotic properties demonstrated in the animal models of fibrosis, this proof 
of concept clinical study aims at  evaluating NTZ in patients with non-alcoholic steatohepatitis 
(NASH) and  fibrosis stage 2 and 3.Although NTZ has been evaluated in liver disease 
populations (viral hepatitis C) up to 60 weeks, this is the first study evaluating NTZ treatment in 
a population with NASH induced stage 2 and 3 fibrosis.  The aim of this study is to evaluate the 
safety and tolerability of NTZ 500 mg BID after 24 weeks of treatment in this population. This 
proof of concept study will also evaluate the anti-fibrotic effect of NTZ as a secondary 
objective. 
The methods of evaluation of fibrosis will include an innovative method of metabolic labeling. 
This approach is based on the concept that liver status can be determined by measuring the 
ratio of newly synthesized/pre-existing proteins. The turn-over rate of newly synthesized 
collagen and proteins represents the hepatic fibrogenic disease activity.  Patients will be given 
“heavy water” to drink. Heavy water contains D20, deuterium being a stable isotope of 
hydrogen. Mass spectrometry is used to identify individual proteins and to quantify the ratio of 
labeled protein to total protein. The results are expressed as fractional synthesis rate of these 
proteins (FSR). This method has been previously published (Decaris et al, 2017). 
Other non-invasive methods will be used to evaluate the liver stiffness changes after NTZ 
treatment: Magnetic Resonance Elastography (MRE) and FibroScan®.  
2. 
Objectives 
2.1. Primary Objective 
 
The primary objective of the study is: 
• To evaluate the safety and tolerability of NTZ 500mg bid after 24 weeks of treatment in 
patients with NASH induced Stage 2 or Stage 3 fibrosis. 
2.2. 
Secondary Objectives: 
The secondary objectives of the study are: 


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• To determine the effect of daily oral administration of NTZ 500mg bid on de novo 
collagen synthesis through Fractional Synthesis Rate (FSR) of circulating plasma 
proteins in patients with NASH-induced Stage 2 or Stage 3 fibrosis. 
• To evaluate changes in liver stiffness as measured by FibroScan® and by Magnetic 
Resonance Elastography (MRE). 
• To assess the change from baseline in non-invasive markers of fibrosis: ELFTM test 
score, PIIINP, Hyaluronic acid, CK18, TIMP-1, YKL-40, Alpha 2 macroglobulin,, ,miRNA, 
ProC3, ProC6, FGF19, FGF21. 
• To assess changes in additional non-invasive tests: fibrosis scores: NAFLD fibrosis 
score, FIB-4 
3. 
Study Design 
3.1. 
Study Design 
This is a phase 2 proof of concept study to evaluate the efficacy and safety of open-label NTZ 
500mg bid in a NASH population with NASH induced Stage 2 or 3 fibrosis. 
The study will include a screening visit within 6 weeks of randomization (Day 1) followed by a 
24-week treatment period consisting of daily, BID, oral administration of NTZ and an end of 
treatment follow up period of 4 weeks.   
Patients will come to the research center and complete the following visits: 
• 
Screening Visit within 6 weeks of Randomization 
• 
Day -14 Deuterated Water Run-In 
o Day-14, Day-11, Day-7 and Day 1: sampling for labelling of Deuterated water 
during run-in 
• 
Repeat baseline labs for ALT, AST, total bilirubin, ALP, CPK collected at least 4 
weeks from initial screening.  
• 
Day 1 Randomization: start of treatment 
• 
Every 4 weeks after randomization until 20 weeks post randomization 


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• 
 22 weeks post randomization and Deuterated Water Administration Period:  
o 22 weeks plus 3 days 
o sampling for labelling of Deuterated water on Day 155, 158, 162, and Day 
169/24 weeks post randomization: End of Treatment 
• 
4 Week Post Treatment Follow Up 
3.2. 
Treatment Plan and Regimen 
NTZ will be administered in an open label fashion to participating subjects as 500mg bid, for 24 
weeks. 
4. 
Subject Population 
4.1. 
Number of Subjects & Subject Selection 
Twenty male and female subjects with histologically confirmed NASH and fibrosis stage 2 or 3 
will be enrolled in the study at a single site in the United States. 
4.2. 
Inclusion Criteria 
Patients must meet all of the following inclusion criteria to be eligible for enrollment in the 
trial:  
1. 
Males or females aged from 18 to 75 years inclusive the Screening Visit.  
2. 
Must provide signed written informed consent and agree to comply with the 
study protocol.  
3. Females participating in this study must be of non-childbearing potential or using 
highly efficient contraception for the full duration of the study.   Childbearing 
potential refers to those female subjects who have not had a hysterectomy, 
bilateral oophorectomy, or medically-documented ovarian failure. Female subjects 
are considered postmenopausal and not of child-bearing potential if they are >55 
years of age, or >50 years of age with >12 months of amenorrhea, or have serum 
follicle stimulating hormone (FSH) values >40 mIU/ml with >12 months of 
amenorrhea.  Highly efficient contraception is defined as the use of one of the 


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following methods of birth control in addition to a male partner using a condom 
(unless surgically sterile) from Screening to 90 days after the last dose of study 
medication: 
• hormone-containing contraceptive 
• intrauterine device 
• cervical cap or diaphragm with spermicidal agent 
4. 
 Histological confirmation of steatohepatitis on a diagnostic liver biopsy (biopsy 
obtained within 6 months prior to Screening or during the Screening Period) 
with at least 1 in each component of the NAS (steatosis scored 0-3, ballooning 
degeneration scored 0-2, and lobular inflammation scored 0-3).   
5. 
Fibrosis stage of 2 or 3, according to the NASH CRN fibrosis staging system on a 
diagnostic liver biopsy (biopsy obtained within 6 months prior to Screening or 
during the Screening Period).   
6. 
Two assessments of ALT, AST, Total bilirubin, ALP, CPK will be collected during 
screening at least 4 weeks apart. To be eligible the second value cannot be ≥2x 
the first value. 
4.3. 
Exclusion Criteria 
Patients who meet any of the following criteria will be excluded from entering the study:  
1. 
History of efficient bariatric surgery within 5 years prior to Screening, or 
planned bariatric surgery in the course of the study.  
2. 
Patients with HbA1c >10.0%. If abnormal at the first Screening Visit, the HbA1c 
measurement can be repeated. A repeated abnormal HbA1c (HbA1c >10.0%) 
leads to exclusion.  
3. 
Patients with a history of clinically significant acute cardiac event, or 
cerebrovascular event, within 6 months prior to Screening such as: stroke, 
transient ischemic attack, or coronary heart disease (angina pectoris, 
myocardial infarction, revascularization procedures).  
4. 
Weight loss of more than 10% within 6 months prior to Randomization.  
5. 
Patient with any history or presence of cirrhosis. 


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6. 
Current or recent history (within a year of screening) of significant alcohol 
consumption. For men, significant consumption is typically defined as higher 
than 30 g pure alcohol per day. For women, it is typically defined as higher than 
20 g pure alcohol per day.  
7. 
Current or history of other substance abuse within 1 year prior to screening. 
8. 
Pregnant or lactating females or females planning to become pregnant during 
the study period.  
9. 
Other well documented causes of chronic liver disease according to standard 
diagnostic procedures including, but not restricted to:  
a) Positive hepatitis B surface antigen (HBsAg)  
b) Positive HCV RNA, (tested for in case of known cured HCV infection, or positive 
HCV Ab at Screening)  
c) Suspicion of drug-induced liver disease  
d) Alcoholic liver disease  
e) Autoimmune hepatitis  
f) Wilson’s disease   
g) Primary biliary cirrhosis 
h) Primary sclerosing cholangitis  
i) Genetic homozygous hemochromatosis  
j) Known or suspected HCC  
k) History or planned liver transplant, or current MELD score >15.  
10. 
Patients who cannot be contacted in case of emergency.  
11. 
Known hypersensitivity to the investigation product or any of its formulation 
excipients.  
12. 
Patients who are taking warfarin or other highly plasma protein-bound drugs 
with narrow therapeutic indices. 
13. 
Patients who are currently participating in, plan to participate in, or have 
participated in an investigational drug trial or medical device trial containing 
active substance within 30 days or five half-lives, whichever is longer, prior to 


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Screening. Patients who have screened for, and failed to qualify, for another 
clinical trial are eligible for screening for the NTZ-218-1, even if the Screening 
Visit for the NTZ-218-1 study is less than 30 days from the failed study screening 
visit, provided that the subject received no investigational product within 30 
days of 5 half-lives of the investigational product prior to screening for NTZ-218-
1. 
14. 
Evidence of any other unstable or, untreated clinically significant 
immunological, endocrine, hematological, gastrointestinal, neurological, 
neoplastic, or psychiatric disease.  
15. 
Mental instability or incompetence, such that the validity of informed consent 
or ability to be compliant with the study is uncertain.  
16. 
 History of noncompliance with medical regimens, or patients who are 
considered to be unreliable. 
17. 
Positive anti-human immunodeficiency virus (HIV) antibody. 
18. 
AST and/or ALT >5 x upper limit of normal (ULN).  
19. 
Total bilirubin >1.3 mg/dL due to altered hepatic function. 
20. 
Direct bilirubin > ULN 
 Note: Gilbert Disease patients are allowed into the study.  
21. 
INR >1.2 in the absence of anticoagulant therapy.  
22. 
Platelet count <150,000/mm3 in the context of portal hypertension.  
23. 
Significant renal disease, including nephritic syndrome, chronic kidney disease 
(defined as patients with markers of kidney damage or eGFR of less than 60 
ml/min/1.73 m2). 
5. 
Investigational Medicinal Products 
Patients will receive 500 mg NTZ (ALINIA®) twice daily for 24 weeks. 
5.1. Description  


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NTZ will be supplied as 500 mg of ALINIA® (nitazoxanide) tablets, for oral use.  The tables are 
round, yellow, film-coated and debossed with ALINIA on one side and 500 on the other side. 
Each tablet contains 500 mg of nitazoxanide. 
For additional information see the Package Insert for ALINIA®. 
5.2. Packaging  
ALINIA® tablets are packaged in HDPE bottles of 12 tablets. Period boxes will be delivered 
every 4 weeks, each box containing 6 bottles. 
5.3. 
Labeling 
All labels for study drugs meet all applicable requirements of the US Food and Drug 
Administration (FDA) and the EU annex 13 of Good Manufacturing Practices: Manufacture of 
Investigational Medicinal Products (February 2010) and /or other local regulations, as 
applicable.  
Distribution of study drug will be performed according to the Good Distribution Practices.  
Product boxes for each period will be labeled with the protocol number, Sponsor’s name and 
address, description of contents, storage conditions, expiry date, dosage instructions, and any 
other applicable items required by national and regional guidelines/regulations. The label will 
contain the statements “Caution – new drug, limited by Federal Law to investigational use” or 
other similar/appropriate statements as well as the following instructions “Please return 
empty packaging and unused products to your doctor at your next visit.”  
5.4. 
Storage and Handling 
The tablets are to be stored at 20-25oC (68-77oF). Storage conditions are specified on the label. 
5.5. 
Dosage and Administration 
Patients will be instructed to take one tablet of 500mg NTZ mg orally, twice a day with water 
during a meal, approximately 12 hours apart.    
The study staff will administer the first dose of the medication at the research center.  Patients 
will self-administer the evening dose on Day 1 and twice a day thereafter, when not attending 


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a visit at the research center.  On scheduled follow up visit days while the patient is being 
treated with study drug, the patient will be directed to bring back all used and unused study 
medication containers. Compliance will be checked by the Investigator or designee during 
those visits and registered in the electronic Case Report Form (eCRF).  
If treatment is interrupted, whatever the cause, duration and reason of the interruption will be 
documented. 
5.6. 
Dose Adjustment 
Enrolled subjects who experience treatment emergent adverse events may have their dose 
adjusted, at the discretion of the Principal Investigator, in accordance with section 6.6.3. 
5.7. 
Prior and Concomitant Medications 
In a general manner, patients should be discouraged from starting any new medication 
without consulting the Investigator unless the new medication is required for emergency 
purpose.  In the same way, any qualitative or quantitative change in concomitant therapy 
should be avoided, when possible. In the event that it becomes necessary during the study, 
this should be recorded by the Investigator or designee in the eCRF (including concomitant 
medications taken within 6 months prior to Screening). This includes drugs used on a chronic 
as well as on an “as needed” basis. 
5.7.1.  Non-permitted medication 
The following medications are not allowed within the timeframe given:  
• Warfarin or other highly plasma protein-bound drugs with narrow therapeutic indices 
are not permitted from Day 1 until end of treatment 
5.7.2.  Permitted Medications 
Any medications other than those listed above are permitted. However, the dosage of a 
current medication for a chronic disease should remain unchanged as far as possible in order 
to reduce the risk of unknown drug-drug interactions.  
In the event that additional concomitant therapy becomes necessary during the study, this will 
be recorded by the Investigator in the eCRF. This includes drugs used on a chronic as well as on 


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an “as-needed” basis. Patients should be discouraged from starting any new medication 
without consulting the Investigator unless the new medication is required for emergency 
purpose. 
5.8. Study Drug Accountability 
The Investigator will acknowledge receipt for each study treatment on the day of receipt. A 
drug accountability record will be maintained by the person responsible for dispensing the trial 
medication to the patient.  
All partially used or unused treatments will be inventoried during and at the conclusion of the 
study.  
The Drug Distribution Center will organize the retrieval of all treatments (used or unused) and 
will proceed to their destruction only after written authorization and review of accountability 
and distribution records.  
6. 
Study Procedures 
The procedures performed at each visit are summarized in the Schedule of Events (Table 1).  
A patient may be seen at any time for reasons of safety.  
During each visit, safety evaluations will be done, and the patient will be queried in the form of 
an open question regarding new or continuing events.  
6.1. 
Duration of Study Participation 
The estimated duration of the study will be approximately 34 weeks inclusive of the screening 
period. 
6.2. 
Screening Assessment 
Screening Visit 
The following screening procedures will be performed for all potential patients at the 
Screening Visit and prior to Day 1:  
• 
Informed consent witnessed by the Investigator or designated person.  


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• 
Medical history & demographics.  
• 
Assessment of inclusion/exclusion criteria 
• 
Complete physical examination. 
• 
Record vital signs  
• 
Record height, weight, BMI  
• 
Check concomitant/prior medication (within 6 months prior to Screening)  
• 
12-lead ECG 
• 
Liver biopsy.  A historical liver biopsy with confirmed NASH and fibrosis is 
acceptable if performed within 6 months prior to the Screening Visit.  
• 
Adverse Events assessment  
• 
Blood samples will be collected for 
o 
Biochemistry 
o 
Hematology 
o 
Serology (HIV, HBs, HCV). In the event of a positive HCV Ab, HCV RNA 
can be tested for confirmation. In case of known cured HCV infection, HCV RNA 
can be tested directly at Screening.  
• 
Repeat baseline labs for ALT, AST, total bilirubin, ALP, CPK collected at least 4 
weeks from initial screening. 
• 
Urine samples will be collected for Urinalysis  
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
If needed, a retesting of any laboratory results may be performed during the screening 
window to determine the eligibility for the study.  
6.3. 
Day -14 Deuterated Water Run In 
Potential Subjects who complete all of the Screening Visit assessments and who 
continue to be eligible for the study, will return to the research center to complete the 
Deuterated Water Run In.  The visit is to be done on Day -14 + 3 days, provided that the 
subject completes 7 consecutive days of deuterated water administration.  The 
following procedures will be performed at the Day -14 Visit: 


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• Dispense deuterated water (D2O). Instruct patient to drink 50ml of deuterated 
water 3 times per day starting on Day-14 for 7 consecutive days (until day-8).  
• Blood sample for baseline labeling by D2O (kinetic biomarkers) (patients should 
be instructed to fast the evening prior to the visit to ensure a 10-hour fast) 
•  Adverse Events assessment  
• Record concomitant medication taken 
Patients will return at Day-11, Day-7 (+ 1 day) and Day 1 for blood sampling (labeled 
D2O) 
6.4. 
Treatment Period Assessments 
6.4.1. Day 1 Visit 
The following procedures will be performed at the Day 1 Visit prior to dose administration:  
• 
Review inclusion/exclusion criteria 
• 
Complete physical examination 
• 
Weight, BMI 
• 
Record vital signs  
• 
Record concomitant medication taken  
• 
Adverse Events assessment  
• 
MRE (may be done within + 5 days of the visit)  
• 
Fibroscan 
• 
Blood samples will be collected  
• 
Urine samples will be collected for Urinalysis  
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
• 
Dispense the study drug as directed 
• 
Review study drug administration instructions with subject  
• 
Administer 500 mg of NTZ with water & food 
6.4.2. Week 4 and Week 8 Visit + 3 days 


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The following procedures will be performed at these Visits:  
• 
Record concomitant medication taken  
• 
Adverse Events assessment  
• 
Blood samples will be collected  
• 
Urine samples will be collected for Urinalysis  
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
• 
Dispense the study drug as directed 
• 
Review study drug compliance and drug administration instructions with subject  
• 
Reconcile study drug administration using pill counts  
• 
Administer 500 mg of NTZ with water & food 
6.4.3. Week 12 Visit + 3 days 
The following procedures will be performed at the Visit:  
• 
Complete physical examination 
• 
Weight, BMI 
• 
Record vital signs  
• 
Record concomitant medication taken  
• 
Adverse Events assessment  
• 
MRE (may be done within + 5 days of the visit) 
• 
Blood samples will be collected  
• 
Urine samples will be collected for Urinalysis  
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
• 
Dispense the study drug as directed 
• 
Review study drug compliance and drug administration instructions with subject  
• 
Reconcile study drug administration using pill counts  
• 
Administer 500 mg of NTZ with water and food 
6.4.4. Week 16 and Week 20 visits ±3 days 
The following procedures will be performed at these Visits:  


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• 
Record concomitant medication taken  
• 
Adverse Events assessment  
• 
Blood samples will be collected  
• 
Urine samples will be collected for Urinalysis  
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
• 
Dispense the study drug as directed 
• 
Review study drug compliance and drug administration instructions with subject  
• 
Reconcile study drug administration using pill counts  
• 
Administer 500 mg of NTZ with water & food 
6.4.5. Week 22 Visit + 3 days (Day 155) 
The following procedures will be performed at the Visit:  
• 
Record concomitant medication taken  
• 
Adverse Events assessment  
• 
Blood samples will be collected for D2O labeling 
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]) 
• Dispense Deuterated water. Instruct patient to drink 50ml of deuterated water 
3 times per day starting on Day 155 for 7 consecutive days (until day 162).  
• Blood sample for kinetic biomarkers analysis (patients should be instructed to 
fast the evening prior to the visit to ensure a 10-hour fast) 
Patients will return for blood sampling for kinetic biomarker analysis at 22weeks/+ 3 days),  
Day 158, Day 162 (+ 1 day), and at day 169 (Week 24). 
6.4.6. Week 24 Visit +1 
The following procedures will be performed at the visit: 
• 
Complete physical examination 
• 
12-lead ECG 
• 
Record vital signs  
• 
Record weight, BMI  


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• 
Record concomitant medication taken  
• 
MRE (may be done within + 5 days of the visit) 
• 
Fibroscan  
• 
Adverse Events assessment  
• 
Blood samples will be collected  
• 
Urine samples will be collected for Urinalysis  
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
• 
Review study drug compliance and drug administration instructions with subject  
• 
Reconcile study drug administration using pill counts  
6.5. 
Follow Up Period 
6.5.1. 4-Week Post Treatment Follow up (EOS – End of study visit) ±3 days 
The following procedures will be performed at the visit: 
• Complete physical examination 
• Weight, BMI 
• Record vital signs  
• Record concomitant medication taken  
• Adverse Events assessment  
• Blood samples will be collected Urine samples will be collected for Urinalysis  
• Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
6.5.2. Early Termination (ET) 
Patients who have discontinued from the study after the first dose of study medication and for 
any reason will have the following procedures completed: 
• 
Complete physical examination 
• 
Record vital signs 
• 
Weight, BMI  
• 
ECG 
• 
Record concomitant medication taken  
• 
MRE (may be done within + 5 days of the visit) 


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• 
Fibroscan 
• 
Adverse Events assessment  
• 
Blood samples will be collected  
• 
Urine samples will be collected for Urinalysis  
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]) 
• 
Review study drug compliance with subject  
• 
Reconcile study drug administration using pill counts  
6.5.3. 
Unscheduled Visits 
An unscheduled visit is defined as any visit to the study unit outside of the protocol-evaluation 
time points where the patient is seen by study unit personnel, e.g., when follow-up 
assessments are required for safety reasons or when repeat measurements are required out of 
the screening period (either to confirm a measurement or in case of errors, measuring device 
failure, etc.).  
Unscheduled visits will be needed for patients who may require further follow-up due to 
safety. 
6.6. 
Assessment for Premature Discontinuation from Study 
Study medication may be discontinued in the following instances: 
• Intercurrent illness that would, in the judgment of the investigator, affect 
assessments of clinical status to a significant degree. Following resolution of 
intercurrent illness, the subject may resume study dosing at the discretion of the 
investigator. 
• Subject noncompliance  
• Investigator discretion  
• Study medication must be discontinued in the following instances:  
o 
Unacceptable toxicity,  or toxicity that, in the judgment of the 
investigator, compromises the ability to continue study-specific 
procedures or is considered to not be in the subject’s best interest  
o 
Subject request to discontinue for any reason  


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o 
Pregnancy  
o 
Discontinuation of the study at the request of a regulatory agency or an 
institutional review board or independent ethics committee (IRB/IEC) 
Pre-specified stopping criteria are defined at the patient level and at the study level. These are 
described in the following sections 6.6.1., 6.6.2., and 6.6.3. In all cases, when the stopping 
criteria are met, the Data Safety Monitoring Board will perform causality assessment, and 
decide whether the study drug/study trial may be continued (See section 6.6.4.). 
 
6.6.1. 
Pre-specified Patient Discontinuation rules 
If a patient experiences a Grade IV Common Terminology Criteria for Adverse Event (CTCAE) or 
meets liver injury criteria as listed in section 6.6.3., the study drug should be interrupted. After 
causality assessment by the DSMB, if the AE or lab abnormality is considered unrelated to 
study drug, and the event has resolved or returned to baseline, then the study drug may be re-
started. If the event reoccurs after restart of study drug, then the study drug will be 
permanently discontinued. 
6.6.2. 
Pre-specified Trial Discontinuation rules 
If one patient experiences a Grade V CTCAE, or 2 patients experience the same Grade IV 
CTCAE, or 3 patients experience the same Grade III CTCAE, or 4 patients experience the same 
Grade II CTCAE, the trial will be paused. The DSMB will assess causality of these adverse 
events; if the events are deemed unrelated to study drug, the trial will continue.  
6.6.3. 
Dose Adjustment 
Enrolled subjects who experience treatment emergent adverse events should be monitored 
closely and may have the NTZ dose adjusted at the Principal Investigator’s discretion. The 
following procedure will be followed for dose adjustments: 
1. Patient will be asked to hold study drug for 3 days. 
2. After the 3 day drug hold, patients will be restarted on a reduced dose of 500 mg NTZ, 
once a day for 7 days. 
3. Patients will be evaluated for treatment emergent adverse events and consideration 
for resuming the dosage of 500 mg NTZ twice a day after 7 day of reduced dose.  PI 


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may determine that  the patient should remain at the 500 mg NTZ once a day following 
7 days of reduced dose. In such scenarios, the patient will be monitored and 
consideration for resuming the dosage of 500 mg NTZ twice daily will be evaluated at 
each subsequent visit.    
 
6.6.4. 
Specific Liver Function Monitoring after Start of Treatment 
Liver function monitoring guidelines are detailed below. The cases will be reviewed by the 
DSMB, who will assess potential Drug Induced Liver Injury (DILI), and apply discontinuation 
rules as described in 6.6.1., 6.6.2., and in this section. 
Study drug interruption 
Study drug must be interrupted in the following cases:  
• ALT >8x ULN  (if ALT is normal at baseline) 
• ALT > 5x Baseline (if ALT is elevated at baseline) OR ALT>500 U/L 
• ALT >5x ULN (if ALT is normal at baseline) AND Total Bilirubin>2xULN 
• ALT >3x Baseline (if ALT is elevated at baseline) OR ALT>300 U/L AND total 
bilirubin>2xULN  
• ALT >5x ULN (if ALT is normal at baseline) AND liver-associated symptoms 
• ALT >3x Baseline (if ALT is elevated at baseline) OR ALT>300 U/L AND liver-associated 
symptoms 
In all these cases, close monitoring should be initiated, with repeat liver biochemistries within 
24-72 hours. Continue monitoring twice/week until liver biochemistries resolve, stabilize, or 
return within baseline values. If abnormalities stabilize and patient is asymptomatic, then 
monitoring should be performed once a week. History of symptoms, disease, concomitant 
medication, or other conditions such as alcohol consumption or alcohol hepatitis should be 
obtained for competing etiologies. The cases will be reviewed by the DSMB, who will 
determine if the patient may restart study drug (if causality assessment is deemed to be not 
related to study drug). 
 


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Management of liver biochemistries elevations 
In the following cases of liver enzyme increase, liver biochemistries including ALT, AST, Total 
bilirubin, ALP, should be repeated within 2-5 days. Follow up of symptoms should be carefully 
monitored. Frequency of monitoring should continue once/week until liver abnormalities 
resolve, stabilize, or return to values below the thresholds mentioned below: 
• ALT >5x ULN  (if ALT is normal at baseline) 
• ALT >3x Baseline (if ALT is elevated at baseline) OR ALT >300 U/L 
NOTE: Laboratory assessments should be done centrally. If the patient lives remotely, he/she 
can have laboratory tests performed locally, and the results communicated to the investigator 
promptly. 
 
6.6.5. 
Data Safety Monitoring Board 
An independent Data Safety Monitoring Board (DSMB) composed of 3 liver experts will review 
the safety of patients enrolled in the trial. The DSMB will perform a safety data review every 3 
months, the first review occurring 3 months after first patient randomized. A DSMB charter 
will define the role, responsibilities, rules, and tasks of the DSMB. 
The DSMB will ensure that the patient and trial discontinuation rules are respected and will 
determine the causality assessment of the events described in sections 6.6.1, 6.6.2., and 6.6.3., 
in order to make the recommendation for trial /patient discontinuation. If the events are 
deemed unrelated to the study drug by the DSMB, then the patient or the trial will continue. 
 
 
 


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6.7. 
Description of Assessments 
6.7.1. Histological Assessments 
All possible attempts should be made to acquire a liver biopsy specimen of at least 2.0 cm in 
length, from a 16 gauge needle to ensure accurate staging of fibrosis and other histological 
lesions.  A historical biopsy within 6 months of the Screening visit may be accepted as the 
Screening biopsy. The liver biopsy sample must be deemed adequate for evaluation by the 
pathologist for inclusion. Liver biopsies will be assessed by a local reader.  This assessment will 
include an assessment of the adequacy of the specimen as well as the fibrosis stage and a 
determination that the biopsy is consistent with NASH. If either the historical or screening liver 
biopsy is deemed unacceptable, it may be repeated. 
6.7.2. Assessment of newly synthesized proteins 
Kinetic biomarkers will be analyzed to evaluate the pharmacodynamic effect of NTZ on 
fibrogenesis. Blood samples analyses will be performed after deuterated water intake before 
and after 24 weeks of treatment. The assessment will involve the analysis of the change 
(absolute and relative) from baseline between the post-dose and pre-dose deuterated water 
loading periods. 
6.7.3. Biological Assessments (Table 2) 
Chemistry:  
HbA1c, fasting plasma glucose, insulin, HOMA-IR, fructosamine, alkaline phosphatase, ALT, 
AST, GGT, CPK, total and conjugated bilirubin, ferritin, creatinine, eGFR, uric acid, BUN, total 
cholesterol, HDL-C, LDL-C, triglycerides, albumin, lipase, amylase, electrolytes (sodium, 
potassium, chloride, calcium), MELD 
Hematology:  
Hematocrit (Hct), hemoglobin (Hb), platelet count, red blood cell count (RBC), white blood cell 
count (WBC) with differential (absolute and percentage) including lymphocytes, monocytes, 


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neutrophils, eosinophils, and basophils and, reticulocyte count and mean corpuscular volume 
(MCV).  
Inflammatory markers:  
hsCRP, fibrinogen, haptoglobin, resistin, TNF-α, TGF-β, IL-6, PAI-1 
Coagulation Panel:  
Prothrombin time (PT), and international normalized ratio (INR)  
Pregnancy Tests:  
Urine β-hCG (if positive, requires immediate confirmation with Serum β-hCG)  
Urinary Tests:  
Urinary dipstick analysis, urinary albumin, urinary creatinine, urinary ACR 
Additional Tests:  
Serology (HIV-1, HBsAg, HCVAb & HCV RNA (as needed)), serum cystatin C, troponin T, NT 
proBNP 
Fibrosis Biomarker tests:  
ELFTM test score, PIIINP, Hyaluronic acid, CK18, TIMP-1, YKL-40, Alpha 2 macroglobulin, miRNA, 
FGF19, FGF21, ProC3 and ProC6.  
A laboratory manual will be provided 
The manual will outline the collection and processing requirements for the laboratory. Blood 
sampling will be performed by trained personnel. Blood samples will be processed and shipped 
as outlined in the laboratory manual. Refer to the laboratory manual for exact amounts of 
blood required for each test.  
The option to retest during the study is left to the Investigator’s judgment.  
Additional serum and plasma samples will be maintained for future analysis 
 
 


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Table 2 Biological assessments 
 
Screening period 
 
 
Treatment period 
Visit 
SV 
 
S
V2 
SV3 
S
V
4 
V
1 
 
V
2 
V
3 
V4 
 
V5 
V6 
V7 
V8 
V9 
V10 
 
EOS 
 
ET 
Day 
-42 
-
14 
-11 
-7 
1 
29 
57 
85 
113 
141 
155 
158 
162 
169 
+28 
days 
 
Week 
-6 
-2 
 
 
0 
4 
8 
12 
16 
20 
22 
 
 
24 
28 
 
Labs - 
Haematology 
haemoglobin, 
haematocrit, RBC, 
WBC, differential 
count, platelet 
count, reticulocytes 
count, MCV) 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
 
 
X 
Coagulatio (PT, 
INR) 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Labs- Urinary 
Pregnancy tests 
X 
 
 
 
 
X 
X 
X 
X 
X 
 
 
 
X 
X 
 
X 
Labs – Serology 
HIV, HBs and HCV 
serology 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Labs – 
Biochemistry 
HbA1c, fasting 
plasma glucose, 
insulin, HOMA-IR, 
fructosamine, 
alkaline 
phosphatase, ALT, 
AST, GGT, CPK, 
total and conjugated 
bilirubin, ferritin, 
creatinine, eGFR, 
uric acid, BUN, 
total cholesterol, 
HDL-C, LDL-C, 
triglycerides, 
albumin, lipase, 
amylase, 
electrolytes 
(sodium, potassium, 
chloride, calcium), 
MELD 
X 
 
 
X1 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Inflammatory 
markers 
hsCRP, fibrinogen, 
haptoglobin, 
resistin,  TNF-, 
TGF-, IL-6, PAI-1 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 


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Screening period 
 
 
Treatment period 
Visit 
SV 
 
S
V2 
SV3 
S
V
4 
V
1 
 
V
2 
V
3 
V4 
 
V5 
V6 
V7 
V8 
V9 
V10 
 
EOS 
 
ET 
Day 
-42 
-
14 
-11 
-7 
1 
29 
57 
85 
113 
141 
155 
158 
162 
169 
+28 
days 
 
Biomarkers of 
fibrosis 
ELF Score, PIIINP, 
Hyaluronic acid, 
CK18, TIMP-1, 
YKL-40, Alpha 2 
macroglobulin,, 
,miRNA, ProC3, 
ProC6, FGF19, 
FGF21 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
NAFLD Fibrosis 
Score (NAS) & 
FIB-4 
 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
 
X 
Labs – Urinalysis 
dipstick  
Specific gravity, pH, 
protein, glucose, 
ketones, bilirubin, 
urobilinogen, blood, 
nitrite, leukocytes 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
 
 
 
X 
Safety markers 
Serum Cystatin C, 
troponin T, NT 
proBNP 
Urinary albumin, 
urinary creatinine, 
urinary ACR 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Labs -- FSR 
 
X 
X 
X 
X 
 
 
 
 
 
X 
X 
X 
X 
 
 
Sampling for 
additional 
parameters 
X 
 
 
 
X 
 
 
X 
 
X 
 
 
 
X 
X 
X 
 
6.7.4. Electrocardiogram 
Standard 12-lead electrocardiogram (ECG) assessments will be performed. The Investigator 
will review the ECGs for any clinically significant abnormalities to ensure subject safety. 
Abnormal ECG findings that are considered clinically significant by the Investigator and meet 
the definition of an AE should be reported and recorded in the AE eCRF page.  
6.7.5. MRE 


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Liver stiffness will be assessed by MRE. It is recommended that each subject’s radiological 
assessment is performed using the same procedure for each study visit.  Exploratory measures 
for MRI/MRE sequence development will be maintained for future analysis.  
6.7.6. Fibroscan 
Liver stiffness will be assessed by FibroScan®. It is required that each subject’s FibroScan® 
assessments be done with the same type of probe at each study visit.  
6.7.7. Medical History 
Medical history including details regarding illnesses and allergies, date(s) of onset, and 
whether condition(s) is currently ongoing, and medication history, including nicotine and 
alcohol use, will be collected on all subjects during screening. 
6.7.8. Physical Examination 
A complete physical examination should include source documentation of general appearance, 
and the following body systems: head, neck, and thyroid; eyes, ears, nose, throat, mouth, and 
tongue; chest (excluding breasts); respiratory; cardiovascular; lymph nodes; abdomen; skin, 
hair, nails; musculoskeletal and neurological.  
The patient’s weight will be measured under the same conditions at each visit where the 
procedure is required. Where possible, the scale for weight must be the same for a given 
patient throughout the visits. 
6.7.9. Vital Signs 
Blood pressure (mmHg) and pulse rate (beats per minute) will be measured at each visit using 
automated blood pressure equipment. 
Important points for clinical blood pressure measurement  
• 
The patient should be seated comfortably with the back supported and the 
upper arm bare without constrictive clothing. The legs should not be crossed. 
• 
The arm should be supported at heart level, and the bladder of the cuff should 
encircle at least 80% of the arm circumference.  


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• 
Neither the patient nor the observer should talk during the measurement.  
Systolic BP and diastolic BP will be measured after 5 minutes rest in the seating.  Where 
possible, the blood pressure equipment should be the same for a given patient throughout the 
visits. 
6.7.10. Pregnancy Testing 
Urinary pregnancy tests will be done on WOCBP.  
7. Adverse Events and Toxicity Management 
7.1. Definitions of Adverse Events, Serious Adverse Events and Unexpected Events 
7.1.1. Adverse Events 
An adverse event (AE) is any untoward medical occurrence in a clinical study subject 
administered a medicinal product, which does not necessarily have a causal relationship with 
the treatment. An AE can therefore be any unfavorable and/or unintended sign, symptom, or 
disease temporally associated with the use of a 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 specified procedures, overdose, drug abuse/misuse reports, 
or occupational exposure. Preexisting events that increase in severity or change in nature 
during or as a consequence of participation in the clinical study will also be considered AEs.  
An AE does not include the following:  
• 
Medical or surgical procedures such as surgery, endoscopy, tooth extraction, 
and transfusion. The condition that led to the procedure may be an adverse 
event and must be reported  
• 
Pre-existing diseases, conditions, or laboratory abnormalities present or 
detected before the Screening visit that do not worsen  
• 
Situations where an untoward medical occurrence has not occurred (eg., 
hospitalization for elective surgery, social and/or convenience admissions)  
• 
Overdose without clinical sequelae.   


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• 
Any medical condition or clinically significant laboratory abnormality with an 
onset date before the consent form is signed and not related to a protocol-
associated procedure is not an AE. It is considered to be pre-existing and should 
be documented on the medical history eCRF. 
7.1.2.  Clinical Laboratory Abnormalities and Other Abnormal Assessments as Adverse 
Events or Serious Adverse Events 
Laboratory abnormalities without clinical significance are not recorded as AEs or SAEs. 
However, laboratory abnormalities (eg, clinical chemistry, hematology, and urinalysis) that 
require medical or surgical intervention or lead to study drug interruption, modification, or 
discontinuation must be recorded as an AE, as well as an SAE, if applicable. In addition, 
laboratory or other abnormal assessments (eg, electrocardiogram, x-rays, vital signs) that are 
associated with signs and/or symptoms must be recorded as an AE or SAE if they meet the 
definition of an AE or SAE as described above.  If the laboratory abnormality is part of a 
syndrome, record the syndrome or diagnosis (eg, anemia), not the laboratory result (ie, 
decreased hemoglobin). 
7.1.3. Serious Adverse Events 
A serious adverse event (SAE) is defined as an event that, at any dose, results in the following:  
• 
Death  
• 
Life-threatening (Note: The term “life-threatening” in the definition of 
“serious” refers to an event in which the subject was at risk of death at the 
time of the event; it does not refer to an event that hypothetically might 
have caused death if it were more severe.) 
• 
In-patient hospitalization or prolongation of existing hospitalization 
• 
Persistent or significant disability/incapacity  
• 
A congenital anomaly/birth defect  
• 
A medically important event or reaction: such events may not be 
immediately life-threatening or result in death or hospitalization but may 
jeopardize the subject or may require intervention to prevent one of the 


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other outcomes constituting SAEs. Medical and scientific judgment must be 
exercised to determine whether such an event is a reportable under 
expedited reporting rules. 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 
hospitalization; and development of drug dependency or drug abuse. For the 
avoidance of doubt, infections resulting from contaminated medicinal 
product will be considered a medically important event and subject to 
expedited reporting requirements. 
7.1.4. Unexpected adverse event  
Expectedness is assessed by the Sponsor-Investigator. An unexpected AE is defined as an event 
that has a nature of severity or specificity that is not consistent with the applicable Package 
Insert or that is symptomatically and pathophysiologically related to a known toxicity but 
differs because of a greater severity or specificity.  
“Unexpected” refers to an event that has not been previously observed and reported rather 
than an event that has not been anticipated based on the properties of the drug. 
7.2. 
Assessment of Adverse Events & Serious Adverse Events 
The Investigator will establish whether or not any AE have occurred at each visit from the date 
of consent. The patient will be questioned in a general manner to determine specific 
symptoms without offering the patient any suggestion. The investigator or qualified sub-
investigator is responsible for assessing AEs and SAEs for causality and severity, and for final 
review and confirmation of accuracy of event information and assessments. 
7.2.1.  Intensity assessment  
The intensity of the AE will be graded as follows:  
• 
Mild: Awareness of signs or symptoms, but easily tolerated and are of 
minor irritant type causing no loss of time from normal activities. 


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Symptoms do not require therapy or a medical evaluation; signs and 
symptoms are transient.  
• 
Moderate: Events introduce a low level of inconvenience or concern to 
the participant and may interfere with daily activities, but are usually 
improved by simple therapeutic measures; moderate experiences may 
cause some interference with functioning. 
• 
Severe: Events interrupt the participant’s normal daily activities and 
generally require systemic drug therapy or other treatment; they are 
usually incapacitating. 
7.2.2.  Relation to the study treatment  
The Investigator will make a clinical and scientific judgment regarding whether or not the AE 
was related to study treatment. The Investigator will evaluate any changes in laboratory 
values, make a determination as to whether or not the change is clinically important, and 
whether or not the changes were related to study drug. However, even if the Investigator feels 
there is no relationship to the study drug, the AE or clinically significant laboratory abnormality 
must be recorded in the eCRF.  
The Investigator will record the relation to the study treatment according to the following 
causality terms:  
• 
Related: the AE follows a reasonable temporal sequence from the time 
of drug administration and it cannot be explained by the patient's 
clinical state or the study procedures/conditions. The AE abates upon 
discontinuation of the study drug and reappears when the study drug is 
introduced.  
• 
Possibly related: the AE follows a reasonable temporal sequence from 
the time of drug administration, but could have been produced by the 
patient's clinical state or the study procedures/conditions.  


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• 
Unlikely related: the temporal association between the AE and the study 
drug is such that the study drug is not likely to have any reasonable 
association with the AE. The relationship is not likely because of other 
plausible explanations.  
• 
Not related: the AE must definitely be caused by the patient's clinical 
state or the study procedure/conditions. A reasonable explanation must 
be given, e.g., no investigational product taken, preplanned elective 
medical intervention, or incompatible temporal relationship.  
• 
Not assessable: the report suggesting an adverse reaction cannot be 
judged because information is insufficient or contradictory and data 
cannot be supplemented or verified. 
7.2.3. 
 Action taken and outcome  
The Investigator will record the action taken with drug and outcome of the event for each AE 
according to the following:  
Action taken with investigational drug  
• 
Drug permanently withdrawn – in case a patient is permanently 
withdrawn from the study drug  
• 
Drug temporarily withdrawn – in case the study drug is temporarily 
withdrawn  
• 
Dose not changed – in case no action is taken regarding the study drug  
• 
Unknown  
• 
Not applicable – an AE started before initiation of treatment with study 
drug, the treatment had been completed prior to reaction/event, or the 
patient has died. 
Outcome  
• 
Recovered/resolved  


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• 
Recovering/resolving  
• 
Not recovered/not resolved  
• 
Recovered/resolved with sequelae  
• 
Fatal  
• 
Unknown.  
Note: In case of irreversible congenital anomalies the choice not recovered/not resolved 
should be used. “Fatal” should be used when death is possibly related to the reaction/event. 
7.3. Reporting 
7.3.1.  Reporting an adverse event  
All AEs regardless of seriousness or relationship to study drug, including those occurring during 
the Screening Period, are to be recorded on the corresponding page(s) of the eCRF and in the 
patient’s medical record from the ICF signature until study end for each patient. Whenever 
possible, symptoms should be grouped as a single syndrome or diagnosis. The Investigator 
should specify the date of onset, maximal intensity, action taken with respect to study drug, 
corrective therapy given, outcome, and his/her opinion as to whether there is a reasonable 
possibility that the AE was caused by the study drug.  
Adverse event reporting begins from signature of the patient ICF at the first Screening Visit and 
ends at study end for each patient. 
7.3.2. Initial reports 
All SAEs occurring from the time of informed consent until 30 days following the 
last administration of study drug must be reported to Medpace Clinical Safety 
within 24 hours of the knowledge of the occurrence (this refers to any adverse 
event that meets any of the aforementioned serious criteria). All SAEs that the 
Investigator considers related to study drug occurring after the 30-day follow-up 
period must be reported to the Sponsor. 
To report the SAE, complete the SAE form electronically in the electronic data 
capture (EDC) system for the study. When the form is completed, Medpace Safety 


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personnel will be notified electronically and will retrieve the form. If the event 
meets serious criteria and it is not possible to access the EDC system, send an 
email to Medpace Safety at medpace safetynotification@medpace.com or call the 
Medpace SAE hotline (phone number listed below), and fax the completed paper 
SAE form to Medpace (fax number listed below) within 24 hours of awareness. 
When the EDC system becomes available, the SAE information must be entered 
within 24 hours of the system becoming available.  
7.3.3. Follow-up reports 
The Investigator must continue to follow the patient until the SAE has subsided or 
until the condition becomes chronic in nature, stabilizes (in the case of persistent 
impairment), or the patient dies. 
Within 24 hours of receipt of follow-up information, the Investigator must update 
the SAE form electronically in the EDC system for the study and submit any 
supporting documentation (eg, patient discharge summary or autopsy reports) to 
Medpace Clinical Safety via fax or e-mail. If it is not possible to access the EDC 
system, refer to the procedures outlined above for initial reporting of SAEs. 
All SAEs independent of the circumstances or suspected cause must be reported on a SAE 
Form/captured in EDC. The SAE Form should include a clearly written narrative describing 
signs, symptoms, and treatment of the event, diagnostic procedures, as well as any relevant 
laboratory data and any sequelae, in order to allow a complete medical assessment of the case 
and independent determination of the possible causality.  
It is critical that the information provided on the initial or follow-up SAE Form matches the 
information recorded in the source documents and the eCRF for the same event.  
Any unexpected safety issue that changes the risk benefit analysis and is likely to have an 
impact on the patients who have participated in the trial will be reported as soon as possible 
to the Competent Authority(ies) concerned together with proposed actions. 
7.3.4. Follow-up  


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The Investigator should take all appropriate measures to ensure the safety of the patients, 
notably he/she should follow up the outcome of any AE until the return to normal or until 
stabilization of the patient’s condition.  
The patient must be followed up until clinical recovery is complete and laboratory results have 
returned to normal, or until progression has been stabilized. This information should be 
documented in the patient’s medical records.  
7.4. Post Study Reporting Requirements 
Any SAEs and deaths that occur within 30 days of the last dose of the study drug, regardless of 
causality, should be reported.  
Any SAE that is brought to the attention of the Investigator at any time after the reporting 
period and which is considered by him/her to be caused by the study drug within a reasonable 
possibility, should be reported.  
7.5. Special Situation Reports 
Special situations reports include pregnancy reports, reports of medication error, abuse, 
misuse or overdose, and reports associated with product complaints.  
7.5.1. Pregnancy  
In case of pregnancy, the Investigator will notify regulatory authorities and IRB within 24 hours 
of his/her knowledge of the pregnancy.  
Female patients must be instructed to discontinue the study drug immediately and inform the 
Investigator as soon as possible once they are aware of being pregnant or suspect that they 
are pregnant during the study or within 30 days of the last dose of the study drug.  
Female patients will be requested, as part of the general ICF, to provide informed consent to 
allow reasonable attempts to be made to obtain information on any possible medicinal 
product exposure to an embryo or fetus and to follow up on the outcome of the pregnancy.  
The Investigator will contact the patient at the expected time of delivery for follow-up.  
7.5.2. Medication error  


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Medication error is defined as an unintentional error in the prescribing, dispensing, or 
administration of a medicinal product while in the control of the healthcare professional, 
patient, or consumer. All medication errors will be documented in the eCRF and, in case of any 
potential risk to patient safety, would be reported as appropriate.  
7.5.3. Misuse  
This refers to situations where the medicinal product is intentionally and inappropriately used 
not in accordance with the authorized product information and will be reported in the eCRF. 
All misuse will be documented in the eCRF and, in case of any potential risk to patient safety, 
would be reported as appropriate. 
7.5.4. 
 Overdose  
This refers to the administration of a quantity of a medicinal product given per administration 
or cumulatively, which is above the maximum recommended dose according to the authorized 
product information. Clinical judgment should always be applied. Doses >4000 mg in one day 
are considered overdose. 
7.5.5. 
 Abuse  
This corresponds to the persistent or sporadic, intentional excessive use of a medicinal 
product, which is accompanied by harmful physical or psychological effects. 
8. 
Statistical Considerations 
This section is an overview of the principal elements of the statistical analysis for this study. 
Further details will be contained in a separate statistical analysis plan (SAP). 
8.1. 
Analysis Endpoints 
8.1.1. Primary Endpoint 
The primary endpoint is to assess the safety and tolerability of NTZ after 24 weeks of treatment 
8.1.2. Secondary Endpoints 
The secondary endpoints are: 


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• To assess the % change in FSR from baseline to end of treatment (24 weeks of 
treatment) 
• To assess the change in liver stiffness by MRE from baseline to 12 weeks and 24 weeks 
of treatment 
• To assess the change in liver stiffness by fibroscan from baseline to 24 weeks of 
treatment  
• To assess the change from baseline to 12 weeks and 24 weeks of treatment of fibrosis 
serum biomarkers and scores  
8.2. 
Analysis Sets 
The Full Analysis Set will consist of all subjects that meet the eligibility criteria and enroll into 
the study (Visit 1 Day 1). 
The Safety Set will consist of all enrolled subjects who receive at least one dose of study drug.  
8.3. 
Data Handling Conventions 
Continuous variables will be summarized with descriptive statistics (i.e., sample size, mean, 
standard deviation, minimum, maximum, median, and quintiles). Categorical variables will be 
summarized with descriptive statistics (i.e., sample size, frequency counts, and percentage). 
Statistical testing on change from baseline values will be performed using analysis of variance 
techniques with a term for baseline in the model. Statistical significance will be considered to 
have been reached at an alpha level of 0.05. 
Baseline value is measured on Day 1 of the treatment period before the administration of NTZ. 
8.4. 
Demographic Data and Baseline Characteristics 
Demographic and baseline characteristics will be descriptively summarized. Quantitative 
and/or categorical summaries will be presented for demographics and other baseline 
characteristics. Demographic and baseline characteristics will be summarized using the Full 
Analysis Set. 
8.5. 
Primary Safety Analysis 
Safety and tolerability will be assessed by the following parameters:  


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AEs including TEAEs and SAEs, laboratory evaluations, vital signs, ECGs, and physical 
examinations.  
8.5.1. Adverse Events 
Treatment-emergent adverse events (TEAEs) are defined as AEs that worsen or commence on 
or after the time of start of first study drug administration. AEs, including SAEs, will be coded 
using MedDRA. The number and percentage of subjects experiencing TEAE, including TEAEs 
leading to study drug discontinuation and SAEs, will be summarized for each system organ 
class (SOC) and Preferred Term. In addition, AEs will be tabulated according to severity, 
causality, and relation to the study drug.  
Individual data listings for AEs will be presented for each subject.  
8.5.2. Laboratory Evaluations 
Absolute and change from baseline values for laboratory parameters will be summarized by 
visit. In addition, shift from baseline tables will be presented for selected laboratory 
abnormalities.  
8.5.3. Other Safety Evaluations 
Observed values and change from baseline will be summarized descriptively for all vital sign 
parameters and ECG parameters by visit. Individual listings will be presented for each subject. 
Individual data listings for physical examination results will be presented for each subject. 
Safety data will be summarized using descriptive statistics. Further details will be provided in 
the SAP. 
 All safety analyses will be conducted using the Safety Population. 
8.6. 
Secondary Efficacy Analyses 
8.6.1. FSR 
Fractional Synthesis Rate of circulating plasma proteins will be measured at Baseline, and after 
24 weeks of treatment.  


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The change in FSR (absolute and relative) from baseline to end of treatment between the first 
and second deuterated water loading periods will be assessed to determine the effects of NTZ 
500mg BID on de novo collagen synthesis in the Full Analysis Set.  
Wilcoxon signed-rank test will be used for statistical inference. Estimated mean, standard error 
and 95% confidence interval of change in FSR (absolute and relative) change from baseline in 
FSR will be reported and evaluated at a significance level of 0.05. Exploratory analyses may 
also be performed to evaluate the association of individual exploratory biomarkers or 
combination of biomarkers with clinical measurements and other risk factors. 
Additional details will be provided in the SAP. 
8.6.2. Liver stiffness 
The change in liver stiffness from baseline at 24 weeks as measured by FibroScan, and at 12 
weeks and 24 weeks by MRE, the change in non-invasive markers of fibrosis from baseline to 
24 weeks, and the change in fibrosis scores from baseline to 12 weeks, to 24 weeks for the Full 
Analysis Set will be assessed. Summary statistics for the Full Analysis Set will be provided at all 
visits and the change from baseline will be provided. 
8.6.3. Exploratory Analysis 
Additional serum and plasma samples will be maintained for future analysis.  Exploratory 
measures for MRI/MRE sequence development will be maintained for future analysis.  
8.6.4. Interim Analysis 
 An interim analysis is not planned. 
8.7. 
Sample Size 
The sample size of this Phase 2 proof of concept study is based on clinical assessment. Twenty 
male and female subjects with histologically confirmed NASH and fibrosis stage 2 or 3) will 
provide sufficient information to assess the safety and tolerability of NTZ 500mg BID over 24 
weeks. 
9. 
Responsibilities 


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9.1. 
Investigator Responsibilities 
By signing this protocol, the Investigator agrees to conduct the study in an efficient and 
diligent manner and in conformance with this protocol; generally accepted standards of GCP; 
and all applicable local laws, rules and regulations relating to the conduct of the clinical study.  
The Investigator also agrees to allow monitoring, audits, IRB review and regulatory agency 
inspection of trial-related documents and procedures and provide for direct access to all study 
related source data and documents.  
The Investigator shall prepare and maintain complete and accurate study documentation in 
compliance with GCP standards and applicable local laws, rules and regulations; and, for each 
subject participating in the study, provide all data, and upon completion or termination of the 
clinical study submit any other reports to the Funder as required by this protocol or as 
otherwise required pursuant to any agreement with the Funder.  
Study documentation will be promptly and fully disclosed to regulatory authorities by the 
Investigator upon request and also shall be made available at the Investigator’s site upon 
request for inspection, copying, review and audit at reasonable times by representatives any 
regulatory agencies.   
Persons debarred from conducting or working on clinical studies by any court or regulatory 
agency will not be allowed to conduct or work on this study.   
9.2. 
Good Clinical Practice 
The investigator will ensure that this study is conducted in accordance with the principles of 
the Declaration of Helsinki (as amended in Edinburgh, Tokyo, Venice, Hong Kong, and South 
Africa), International Conference on Harmonisation (ICH) guidelines.  These standards are 
consistent with the European Union Clinical Trials Directive 2001/20/EC and Good Clinical 
Practice Directive 2005/28/EC. The investigator will ensure adherence to the basic principles of 
Good Clinical Practice, as outlined in 21CFR312, subpart D, “Responsibilities of Sponsors and 
Investigators,” 21CFR, part50, 1998, and 21CFR, part56, 1998. The investigator and all 
applicable sub investigators will comply with 21CFR, Part54, 1998, providing documentation of 


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their financial interest or arrangements with any study funder, or proprietary interests in the 
investigational drug under study. This documentation must be provided prior to the 
investigator’s (and any sub investigator’s) participation in the study. The investigator and sub 
investigator agree to notify study funder of any change in reportable interests during the study 
and for 1 year following completion of the study. Study completion is defined as the date 
when the last subject completes the protocol-defined activities 
9.3. 
Institutional Review Board Review & Approval 
The investigator will submit this protocol, informed consent form, and any accompanying 
material to be provided to the subject (such as advertisements, subject information sheets, or 
descriptions of the study used to obtain informed consent) to an IRB/IEC.  The investigator will 
not begin any study subject activities until approval from the IRB/IEC has been documented 
and provided as a letter to the investigator. Before implementation, the investigator will 
submit to and receive documented approval from the IRB/IEC any modifications made to the 
protocol or any accompanying material to be provided to the subject after initial approval, 
with the exception of those necessary to reduce immediate risk to study subjects. 
9.4. 
Informed Consent 
The investigator is responsible for obtaining written informed consent from each individual 
participating in this study after adequate explanation of the aims, methods, objectives, and 
potential hazards of the study and before undertaking any study-related procedures. The 
investigator must use the most current IRB/IEC approved consent form for documenting 
written informed consent. Each informed consent will be appropriately signed and dated by 
the subject and the person conducting the consent discussion, and also by an impartial witness 
if required by local requirements. 
9.5. 
Confidentiality 
The investigator will assure that subjects’ anonymity will be strictly maintained and that their 
identities are protected from unauthorized parties. Only subject initials, date of birth, another 
unique identifier (as allowed by local law) and an identification code will be recorded on any 
form or biological sample submitted to the study funder, IRB/IEC or laboratory. Laboratory 


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specimens will be labeled in such a way as to protect subject identity while allowing the results 
to be recorded to the proper subject. Refer to specific laboratory instructions for further 
details. NOTE: The investigator will keep a screening log showing codes, names, and addresses 
for all subjects screened and for all subjects enrolled in the trial. Subject data will be processed 
in accordance with all applicable regulations. The investigator agrees that all information 
received from the study funder, including but not limited to the Package Insert, the IMP, and 
any other study information, remain the sole and exclusive property of the study funder during 
the conduct of the study and thereafter. This information is not to be disclosed to any third 
party (except employees or agents directly involved in the conduct of the study or as required 
by law) without prior written consent from the study funder.  The investigator further agrees 
to take all reasonable precautions to prevent the disclosure by any employee or agent of the 
study site to any third party or otherwise into the public domain. 
9.6. 
Study Files and Retention of Records 
The investigator will maintain adequate and accurate records to enable the conduct of the 
study to be fully documented and the study data to be subsequently verified. These 
documents should be classified into at least the following two categories: (1) investigator’s 
study master file, and (2) subject clinical source documents. The investigator’s study file will 
contain the protocol/amendments, CRF and query forms, IRB/IEC and governmental approval 
with correspondence, informed consent, drug records, staff curriculum vitae and authorization 
forms, and other appropriate documents and correspondence. The required source data 
should include sequential notes containing at least the following information for each subject:  
• 
Subject identification (name, date of birth, gender);  
• 
Documentation that subject meets eligibility criteria, i.e., history, physical 
examination, and confirmation of diagnosis (to support inclusion and exclusion 
criteria); 
• 
Documentation of the reason(s) a consented subject is not enrolled  
• 
Participation in study (including study number);  


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• 
Study discussed and date of informed consent;  
• 
Dates of all visits;  
• 
Documentation that protocol specific procedures were performed;  
• 
Results of efficacy parameters, as required by the protocol;  
• 
Start and end date (including dose regimen) of IMP, including dates of 
dispensing and return;  
• 
Record of all adverse events and other safety parameters (start and end date, 
and including causality and severity);  
• 
Concomitant medication (including start and end date, dose if relevant; dose 
changes); 
• 
Date of study completion and reason for early discontinuation, if it occurs.  
All clinical study documents must be retained by the investigator until at least 2years or 
according to local laws, whichever is longer, after the last approval of a marketing application 
in an ICH region (i.e., United States, Europe, or Japan) and until there are no pending or 
planned marketing applications in an ICH region; or, if no application is filed or if the 
application is not approved for such indication, until 2years after the investigation is 
discontinued and regulatory authorities have been notified. Investigators may be required to 
retain documents longer if specified by regulatory requirements, by local regulations, or by an 
agreement with study funder. The investigator must notify study funder before destroying any 
clinical study records. Should the investigator wish to assign the study records to another party 
or move them to another location, study funder must be notified in advance. If the 
investigator cannot provide for this archiving requirement at the study site for any or all of the 
documents, special arrangements must be made between the investigator and study funder to 
store these records securely away from the site so that they can be returned sealed to the 
investigator in case of an inspection. When source documents are required for the continued 
care of the subject, appropriate copies should be made for storage away from the site. 
9.7. 
Electronic Case Report Forms 


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For each subject consented, an eCRF will be completed by an authorized study staff member 
whose training for this function is documented according to study procedures. The eCRF will 
be completed in a timely manner. Original entries as well as any changes to data fields will be 
stored in the audit trail of the system. At the conclusion of the trial, a read-only archive copy of 
the data entered will be generated. This archive will be stored in accordance with the records 
retention requirements. 
9.8. 
Investigational Medicinal Product Accountability and Return 
The investigator or designee is responsible for ensuring adequate accountability of all used and 
unused study drug.  This includes acknowledgment of receipt of each shipment of study 
product (quantity and condition), subject dispensing records, and returned or destroyed study 
product. Dispensing records will document quantities received from study funder and 
quantities dispensed to subjects, including lot number, date dispensed, subject identifier 
number, subject initials, and the initials of the person dispensing the medication. Drug may be 
returned or destroyed on an ongoing basis during the study, if appropriate.  At the end of the 
study, following final drug inventory reconciliation, the study site will arrange for the return of 
unused investigational medicinal product supplies. All drug supplies and associated 
documentation will be periodically reviewed and verified by the study funder over the course 
of the study. 
9.9. 
Inspections 
The investigator understands that source documents for this trial should be made available to 
appropriately qualified personnel from study funder and its representatives, to IRBs/IECs, or to 
regulatory authority or health authority inspectors. 
9.10. Protocol Compliance 
The investigator is responsible for ensuring the study is conducted in accordance with the 
procedures and evaluations described in this protocol. 
9.11. Protocol Modifications 


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Protocol modifications, except those intended to reduce immediate risk to study subjects, may 
be made by the investigator in collaboration with the study funder. All protocol modifications 
must be submitted to the IRB/IEC in accordance with local requirements. Approval must be 
obtained before changes can be implemented. 
9.12. Study Reports 
A clinical study report (CSR) will be prepared and provided to the regulatory agency (ies). The 
Investigator will ensure that the report meets the standards set out in the ICH Guideline for 
Structure and Content of Clinical Study Reports (ICHE3). Note that an abbreviated report may 
be prepared in certain cases. After conclusion of the study, Investigators in this study may 
communicate, orally present, or publish in scientific journals or other scholarly media only 
after the following conditions have been met:  
• 
The results of the study in their entirety have been publicly disclosed by or with 
the consent of the study funder in an abstract, manuscript, or presentation form. 
9.13. Study Termination 
The Investigator reserves the right to terminate the study at any time. Should this be 
necessary, the investigator will arrange discontinuation procedures and notify the appropriate 
regulatory authorities, IRBs, IECs, and the Funder. In terminating the study, the investigator 
will assure that adequate consideration is given to the protection of the subjects’ interests. 


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10. References 
 
10.1. 
Literature  
Abd-Elsalam, S., Kobtan, A., El-Kalla, F., Elkhalawany, W., Nawasany, S.E., Saif, S.A., Yousef, M., 
Ali, L.A., Soliman, S., Mansour, L., et al. (2016). A 2-week Nitazoxanide-based quadruple 
treatment as a rescue therapy for Helicobacter pylori eradication: A single center experience. 
Medicine (Baltimore) 95, e3879. 
 
Alinia (nitazoxanide): US Prescribing Information. Tampa, FL. Romark, L.C., 2016 
 
Amorosa, V.K., Luetkemeyer, A., Kang, M., Johnson, V.A., Umbleja, T., Haas, D.W., Yesmin, S., 
Bardin, M.C., Chung, R.T., Alston-Smith, B., et al. (2013). Addition of nitazoxanide to PEG-IFN 
and ribavirin to improve HCV treatment response in HIV-1 and HCV genotype 1 coinfected 
persons naive to HCV therapy: results of the ACTG A5269 trial. HIV Clin Trials 14, 274-283. 
 
Assy, N., Kaita, K., Mymin, D., Levy, C., Rosser, B., and Minuk, G. (2000). Fatty infiltration of 
liver in hyperlipidemic patients. Dig Dis Sci 45, 1929-1934. 
 
Bacon, B.R., Shiffman, M.L., Lim, J.K., Berman, A., Rustgi, V.K., and Keeffe, E.B. (2010). A Phase 
II, Randomized, Double-Blind, Placebo-Controlled Study of Nitazoxanide Plus Peginterferon 
and Ribavirin in Naive Patients With Chronic Hepatitis C Genotype 1 Infection: Final Report. 
Gastroenterology 139, e18. 
 
Basu, P.P., Rayapudi, K., Shah, N.J., Krishnaswamy, N., and Brown, R.S. (2013). Effects of triple-
drug therapy with nitazoxanide, high-dose ribavirin and peginterferon-alpha-2a in patients 
with chronic hepatitis C. Hepatol Res 43, 589-595. 
 
Basu PP, Rayapudi K, Esteves J, Brown R. A pilot study utilizing nitazoxanide for hepatic 
encephalopathy in chronic liver failure. Abstract S151 (392) for the 59th Annual Meeting of the 
American Association for the Study of Liver Diseases. 31 Oct-4 Nov. 2008. p1742 
 
Belanger, C., Foucart, C., Negro, E., Dubernet, M., Hum, D.W., Staels, B., and Walczak, R. 
(2017). Drug repurposing screen identifies novel small molecule compounds with potent 
antifibrotic properties. Journal of Hepatology 66, S605. 
 
Carpino, G., Morini, S., Ginanni Corradini, S., Franchitto, A., Merli, M., Siciliano, M., Gentili, F., 
Onetti Muda, A., Berloco, P., Rossi, M., et al. (2005). Alpha-SMA expression in hepatic stellate 
cells and quantitative analysis of hepatic fibrosis in cirrhosis and in recurrent chronic hepatitis 
after liver transplantation. Dig Liver Dis 37, 349-356. 
 
Decaris, M.L., Li, K.W., Emson, C.L., Gatmaitan, M., Liu, S., Wang, Y., Nyangau, E., Colangelo, 
M., Angel, T.E., Beysen, C., et al. (2017). Identifying nonalcoholic fatty liver disease patients 


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with active fibrosis by measuring extracellular matrix remodeling rates in tissue and blood. 
Hepatology 65, 78-88. 
 
Donati, G., Stagni, B., Piscaglia, F., Venturoli, N., Morselli-Labate, A.M., Rasciti, L., and Bolondi, 
L. (2004). Increased prevalence of fatty liver in arterial hypertensive patients with normal liver 
enzymes: role of insulin resistance. Gut 53, 1020-1023. 
 
Friedman, S.L. (2008). Hepatic stellate cells: protean, multifunctional, and enigmatic cells of 
the liver. Physiol Rev 88, 125-172. 
 
Fruci, B., Giuliano, S., Mazza, A., Malaguarnera, R., and Belfiore, A. (2013). Nonalcoholic Fatty 
liver: a possible new target for type 2 diabetes prevention and treatment. Int J Mol Sci 14, 
22933-22966. 
 
Kabil, S.M., Soliman, M.S., Youssef, S.M., and Mounier, B.I. (2011). A controlled study of 
nitazoxanide (NTZ) 3 years after treatment of hepatitis C genotype 4. J Egypt Soc Parasitol 41, 
251-261. 
 
Kohla, M.A., El-Said, H., El-Fert, A., Ehsan, N., Ezzat, S., and Taha, H. (2016). Impact of 
nitazoxanide on sustained virologic response in Egyptian patients with chronic hepatitis C 
genotype 4: a double-blind placebo-controlled trial. Eur J Gastroenterol Hepatol 28, 42-47. 
 
Konerman, M.A., Jones, J.C., and Harrison, S.A. (2018). Pharmacotherapy for NASH: Current 
and emerging. J Hepatol 68, 362-375. 
 
Laufer, N., Abusamra, L., Bolcic, F., Gun, A., Rolon, M.J., Perez, H., Krolewiecki, A., Salomon, H., 
Quarleri, J., and Cahn, P. (2011). No reduction of HCV viral load in HIV patients co-infected with 
HCV genotype 1 during a 30 days course of nitazoxanide monotherapy. Antiviral Res 92, 497-
499. 
 
Leite, N.C., Salles, G.F., Araujo, A.L., Villela-Nogueira, C.A., and Cardoso, C.R. (2009). Prevalence 
and associated factors of non-alcoholic fatty liver disease in patients with type-2 diabetes 
mellitus. Liver Int 29, 113-119. 
 
Macias, J., Lopez-Cortes, L.F., Tellez, F., Recio, E., Ojeda-Burgos, G., Rios, M.J., Rivero-Juarez, 
A., Delgado, M., Rivas, J., and Pineda, J.A. (2015). Low Efficacy of Pegylated Interferon plus 
Ribavirin plus Nitazoxanide for HCV Genotype 4 and HIV Coinfection. PLoS One 10, e0143492. 
 
Musher, D.M., Logan, N., Bressler, A.M., Johnson, D.P., and Rossignol, J.F. (2009). Nitazoxanide 
versus vancomycin in Clostridium difficile infection: a randomized, double-blind study. Clin 
Infect Dis 48, e41-46. 
 


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Rossignol, J.F. (2006). Nitazoxanide in the treatment of acquired immune deficiency syndrome-
related cryptosporidiosis: results of the United States compassionate use program in 365 
patients. Aliment Pharmacol Ther 24, 887-894. 
 
Rossignol, J.F. (2014). Nitazoxanide: a first-in-class broad-spectrum antiviral agent. Antiviral 
Res 110, 94-103. 
 
Rossignol, J.F., Elfert, A., El-Gohary, Y., and Keeffe, E.B. (2009). Improved virologic response in 
chronic hepatitis C genotype 4 treated with nitazoxanide, peginterferon, and ribavirin. 
Gastroenterology 136, 856-862. 
 
Rossignol, J.F., Elfert, A., and Keeffe, E.B. (2010). Treatment of chronic hepatitis C using a 4-
week lead-in with nitazoxanide before peginterferon plus nitazoxanide. J Clin Gastroenterol 
44, 504-509. 
 
Rossignol, J.F., Hidalgo, H., Feregrino, M., Higuera, F., Gomez, W.H., Romero, J.L., Padierna, J., 
Geyne, A., and Ayers, M.S. (1998). A double-'blind' placebo-controlled study of nitazoxanide in 
the treatment of cryptosporidial diarrhoea in AIDS patients in Mexico. Trans R Soc Trop Med 
Hyg 92, 663-666. 
 
Rossignol, J.F., Kabil, S.M., El-Gohary, Y., Elfert, A., and Keeffe, E.B. (2008). Clinical trial: 
randomized, double-blind, placebo-controlled study of nitazoxanide monotherapy for the 
treatment of patients with chronic hepatitis C genotype 4. Aliment Pharmacol Ther 28, 574-
580. 
 
Sasaki, A., Nitta, H., Otsuka, K., Umemura, A., Baba, S., Obuchi, T., and Wakabayashi, G. (2014). 
Bariatric surgery and non-alcoholic Fatty liver disease: current and potential future treatments. 
Front Endocrinol (Lausanne) 5, 164. 
 
Shehab, H.M., Elbaz, T.M., and Deraz, D.M. (2014). Nitazoxanide plus pegylated interferon and 
ribavirin in the treatment of genotype 4 chronic hepatitis C, a randomized controlled trial. Liver 
Int 34, 259-265. 
 
Shehata, M.A., Talaat, R., Soliman, S., Elmesseri, H., and Abd-Elsalam, S. (2017). Randomized 
controlled study of a novel triple nitazoxanide (NTZ)-containing therapeutic regimen versus the 
traditional regimen for eradication of Helicobacter pylori infection. Helicobacter 22. 
 
Shiffman, M.L., Ahmed, A., Jacobson, I.M., Pruitt, R.E., and Keeffe, E.B. (2010). 1188  Phase 2 
randomized, double-blind, placebo-controlled study of nitazoxanide with peginterferon alfa-2a 
and ribavirin in nonresponders (NR) with chronic hepatitis C genotype 1: final report. Journal 
of Hepatology 52, S461. 
 


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Stockis, A., Allemon, A.M., De Bruyn, S., and Gengler, C. (2002a). Nitazoxanide 
pharmacokinetics and tolerability in man using single ascending oral doses. Int J Clin 
Pharmacol Ther 40, 213-220. 
 
Stockis, A., De Bruyn, S., Gengler, C., and Rosillon, D. (2002b). Nitazoxanide pharmacokinetics 
and tolerability in man during 7 days dosing with 0.5 g and 1 g b.i.d. Int J Clin Pharmacol Ther 
40, 221-227. 
 
Subichin, M., Clanton, J., Makuszewski, M., Bohon, A., Zografakis, J.G., and Dan, A. (2015). Liver 
disease in the morbidly obese: a review of 1000 consecutive patients undergoing weight loss 
surgery. Surg Obes Relat Dis 11, 137-141. 
 
Takeuchi-Yorimoto, A., Noto, T., Yamada, A., Miyamae, Y., Oishi, Y., and Matsumoto, M. 
(2013). Persistent fibrosis in the liver of choline-deficient and iron-supplemented L-amino acid-
defined diet-induced nonalcoholic steatohepatitis rat due to continuing oxidative stress after 
choline supplementation. Toxicol Appl Pharmacol 268, 264-277. 
 
Tanwar, S., Trembling, P.M., Guha, I.N., Parkes, J., Kaye, P., Burt, A.D., Ryder, S.D., Aithal, G.P., 
Day, C.P., and Rosenberg, W.M. (2013). Validation of terminal peptide of procollagen III for the 
detection and assessment of nonalcoholic steatohepatitis in patients with nonalcoholic fatty 
liver disease. Hepatology 57, 103-111. 
 
Taubel, J., Lorch, U., Rossignol, J.F., Ferber, G., and Camm, A.J. (2014). Analyzing the 
relationship of QT interval and exposure to nitazoxanide, a prospective candidate for influenza 
antiviral therapy--A formal TQT study. J Clin Pharmacol 54, 987-994. 
 
Wu, K.T., Kuo, P.L., Su, S.B., Chen, Y.Y., Yeh, M.L., Huang, C.I., Yang, J.F., Lin, C.I., Hsieh, M.H., 
Hsieh, M.Y., et al. (2016). Nonalcoholic fatty liver disease severity is associated with the ratios 
of total cholesterol and triglycerides to high-density lipoprotein cholesterol. J Clin Lipidol 10, 
420-425 e421. 
 
Zulu, I., Kelly, P., Njobvu, L., Sianongo, S., Kaonga, K., McDonald, V., Farthing, M., and Pollok, R. 
(2005). Nitazoxanide for persistent diarrhoea in Zambian acquired immune deficiency 
syndrome patients: a randomized-controlled trial. Aliment Pharmacol Ther 21, 757-763. 
 
10.2. 
WHO Fact Sheet 312 
World Health Organization. Media Centre- Diabetes. 
http://www.who.int/mediacentre/factsheets/fs312/en/  
 
10.3. 
Studies on ClinicalTrials.gov 


Protocol No: NTZ-218-1 
Version: 24August 2019 
 
 
68 
NCT01185028. A safety and tolerability study of nitazoxanide in HIV-HCV treatment failures. 
Last updated: March 21, 2018  
https://clinicaltrials.gov/ct2/show/results/NCT01185028?sect=X430156#othr 
NCT01770483. The role of nitazoxanide, interferon alfa and ribavirin in treatment of hepatitis C 
infected Type 2 diabetic patients (HEP-C-FM). Last updated: September 20, 2013 
https://clinicaltrials.gov/ct2/show/results/NCT01770483 
 
 



Prot_005.pdf: 
Protocol 
 
A Monocentric, Open-Label, Proof of Concept Study to Evaluate the 
Safety and Efficacy of NTZ at 500mg Twice Daily on Collagen Turnover 
in Plasma in NASH Patients with Fibrosis Stage 2 or 3. 
 
 
NCT Number: 03656068 
Unique Protocol ID: NTZ-218-1 
Version Date : 15 April 2020 


 
 
Protocol No: NTZ-218-1 
Version: V 15 APRIL 2020 
 
 
 
 
 
Page 1 
 
 
 
 
 
 
 
 
 
A Monocentric, Open-Label, Proof of Concept Study to Evaluate the Safety 
and Efficacy of NTZ at 500mg Twice Daily on Collagen Turnover in Plasma in 
NASH Patients with Fibrosis Stage 2 or 3. 
  
 
 
 
Protocol Number: NTZ-218-1 
Version Date: 15 APRIL 2020 
 


 
 
Protocol No: NTZ-218-1 
Version: V 15 APRIL 2020 
 
 
 
 
 
Page 2 
List of Abbreviations 
  
ACR 
 
Albumin to Creatinine Ratio 
AE             
Adverse Event  
ALT            
Alanine Aminotransferase/GPT   
AST            
Aspartate aminotransferase  
BMI            
Body Mass Index   
CDAA  
Choline-deficient L-amino Acid Defined Diet 
CFR 
 
Code of Federal Regulations 
COVID-19 
Corona Virus Disease 2019 
CSR 
 
Clinical Study Report 
CPK            
Creatine phosphokinase  
CRF            
Case Report/Record Form  
CRN            
Clinical Research Network  
CTCAE  
Common Terminology Criteria for Adverse Events 
D2O 
 
Deuterated Water 
DILI 
 
Drug Induce Liver Injury 
DSMB  
Data Safety Monitoring Board 
GGT 
 
Gamma-glutamyltransferase 
eCRF           
Electronic case report form  
eGFR            
estimated Glomerular Filtration Rate   
EC            
Ethics Committee   
ECG            
Electrocardiogram  
ET             
Early Termination  
EOS 
 
End Of Study 
EU             
European Union  
FDA            
Food and Drug Administration  
FSR 
 
Fractional Synthesis Rate 
GCP            
Good Clinical Practice  
Hb             
Hemoglobin  
HBV           
Hepatitis B Virus  
HCG 
 
Human Chorionic Gonadotropin 
HCV           
Hepatitis C Virus  
HDPE  
High-density polyethylene 
HDL            
High Density Lipoprotein  
HIV            
Human Immunodeficiency Virus  
HOMA           Homeostasis Model Assessment  
HSC 
 
Hepatic Stellate Cell 
hs-CRP            High Sensitivity C-reactive protein  
ICH           
International Conference on Harmonization  
IEC 
 
Independent Ethics Committee 


 
 
Protocol No: NTZ-218-1 
Version: V 15 APRIL 2020 
 
 
 
 
 
Page 3 
IMP            
Investigational medical product 
INR 
 
International Normalized Ratio  
IRB            
Institutional Review Board  
LDL            
Low density lipoprotein 
MedDRA          Medical Dictionary for Regulatory Activity  
MRE 
 
Magnetic Resonance Elastography 
MRI            
Magnetic Resonance Imaging  
NAFLD            Non Alcoholic Fatty Liver Disease  
NAS            
NAFLD Activity Score  
NASH            
Non Alcoholic Steatohepatitis  
NTZ 
 
Nitazoxanide 
PI             
Principal Investigator  
PK             
Pharmacokinetic 
PT/INR            Prothrombin Time/International Normalized Ratio  
 
RNA            
Ribonucleic Acid  
SAE            
Serious Adverse Event 
SAP 
 
Statistical Analysis Plan  
TQT 
 
Thorough-QT study 
  
 
 
 


 
 
Protocol No: NTZ-218-1 
Version: V 15 APRIL 2020 
 
 
 
 
 
Page 4 
Study Synopsis 
Protocol Number: 
NTZ-218-1 
Study Title: 
A Monocentric, Open-Label, Proof of Concept Study to Evaluate the Safety 
and Efficacy of NTZ at 500mg Twice Daily on Collagen Turnover in Plasma in 
NASH Patients with Fibrosis Stage 2 or 3. 
Phase: 
Proof of Concept 
Study Drug: 
Nitazoxanide 
Indication: 
NASH induced fibrosis 
Principal Investigator & Sponsor 
Stephen Harrison, MD 
Pinnacle Clinical Research 
5109 Medical Drive, Suite 200 
San Antonio, TX 78229 
Study Duration: 
34 weeks 
Objectives: 
The primary objective of the study is: 
• 
To evaluate the safety and tolerability of NTZ 500mg bid after 24 
weeks of treatment in patients with NASH-induced stage 2 or 
stage 3 fibrosis  
 
 
The secondary objectives of the study are: 
• 
To determine the effect of oral administration of NTZ 500mg bid 
on de novo collagen synthesis through Fractional Synthesis Rate 
(FSR) of circulating plasma proteins in patients with NASH-
induced stage 2 or stage 3 fibrosis. 
• 
To assess changes in liver stiffness as measured by FibroScan® 
and by Magnetic Resonance Elastography (MRE). 
• 
To assess the change from baseline in non-invasive markers of 
fibrosis: ELFTM test score, PIIINP, Hyaluronic acid, CK18, TIMP-1, 
YKL-40, Alpha 2 macroglobulin,, ,miRNA, ProC3, ProC6, FGF19, 
FGF21. 
• 
To assess changes in additional non-invasive tests: fibrosis scores: 
NAFLD fibrosis score, FIB-4 
 
Study Design: 
This is a Phase 2 proof of concept study to evaluate the safety and efficacy 
of open-label NTZ 500mg bid in a NASH population with NASH induced 
Stage 2 or 3 fibrosis. 
 
The study will include a screening visit within 6 weeks of randomization 
(Day 1) followed by a 24-week treatment period consisting of daily, BID, 
oral administration of NTZ and an end of treatment follow up period   of 4 
weeks.   
Patients will come to the research center and complete the following visits: 
• 
Screening Visit within 6 weeks of Randomization 
• 
Day -14 Deuterated Water Run-In 
o 
Day-14, Day-11, Day-7 and Day 1: sampling for labeling 
of Deuterated water during run-in, 
• 
Repeat baseline labs for ALT, AST, total bilirubin, ALP, CPK 
collected at least 4 weeks from initial screening. 


 
 
Protocol No: NTZ-218-1 
Version: V 15 APRIL 2020 
 
 
 
 
 
Page 5 
• 
Day 1 randomization: start of treatment 
• 
Every 4 weeks after randomization until 20 weeks post 
randomization 
• 22 weeks post randomization and Deuterated Water       
Administration Period 
o 
22 weeks plus 3 days 
o 
sampling for labeling of Deuterated water on Day 155, 
Day 158, Day 162 and Day 169  
• 
24 weeks post randomization: End of Treatment 
• 
4 Week Post Treatment Follow Up 
Number of Sites: 
1 site  
Number of Subjects: 
20 evaluable subjects 
Drug Dosage / Frequency: 
500 mg NTZ BID 
Route of Administration: 
Oral Administration 
Inclusion Criteria: 
Patients must meet all of the following inclusion criteria to be eligible for 
enrollment in the trial:  
1. Males or females aged from 18 to 75 years inclusive at the Screening 
Visit.  
2. Must provide signed written informed consent and agree to comply 
with the study protocol.  
3. Females participating in this study must be of non-childbearing 
potential or using highly efficient contraception for the full duration of 
the study.  Childbearing potential refers to those female subjects who 
have not had a hysterectomy, bilateral oophorectomy, or medically-
documented ovarian failure. Female subjects are considered 
postmenopausal and not of child-bearing potential if they are >55 years 
of age, or >50 years of age with >12 months of amenorrhea or have 
serum follicle stimulating hormone (FSH) values >40 mIU/ml with >12 
months of amenorrhea.  Highly efficient contraception is defined as the 
use of one of the following methods of birth control in addition to a 
male partner using a condom (unless surgically sterile) from Screening 
to 90 days after the last dose of study medication: 
(1) hormone-containing contraceptive 
(2) intrauterine device 
(3) cervical cap or diaphragm with spermicidal agent 
4. Histological confirmation of steatohepatitis on a diagnostic liver biopsy 
(biopsy obtained within 6 months prior to Screening or during the 
Screening Period) with at least 1 in each component of the NAS 
(steatosis scored 0-3, ballooning degeneration scored 0-2, and lobular 
inflammation scored 0-3).   
5.    Fibrosis stage of 2 or 3, according to the NASH CRN fibrosis staging 
system on a diagnostic liver biopsy (biopsy obtained within 6 months 
prior to Screening or during the Screening Period).   
6. Two assessments of ALT, AST, Total bilirubin, ALP, CPK will be collected 
during screening at least 4 weeks apart. To be eligible the second value 
cannot be ≥2x the first value. 
Exclusion Criteria: 
Patients who meet any of the following criteria will be excluded from 
entering the study:  


 
 
Protocol No: NTZ-218-1 
Version: V 15 APRIL 2020 
 
 
 
 
 
Page 6 
1. History of efficient bariatric surgery within 5 years prior to Screening, 
or planned bariatric surgery in the course of the study.  
2. Patients with HbA1c >10.0%. If abnormal at the first Screening Visit, the 
HbA1c measurement can be repeated. A repeated abnormal HbA1c 
(HbA1c >10.0%) leads to exclusion.  
3. Patients with a history of clinically significant acute cardiac event, or 
cerebrovascular event within 6 months prior to Screening such as: 
stroke, transient ischemic attack, or coronary heart disease (angina 
pectoris, myocardial infarction, revascularization procedures).  
4. Weight loss of more than 10% within 6 months prior to Randomization.  
5. Patient with any history or presence of cirrhosis. 
6. Current or recent history (within a <1 year prior to screening ) of 
significant alcohol consumption. For men, significant consumption is 
typically defined as higher than 30 g pure alcohol per day. For women, 
it is typically defined as higher than 20 g pure alcohol per day.  
7. Current or history of other substance abuse within 1 year prior to 
screening. 
8. Pregnant or lactating females or females planning to become pregnant 
during the study period.  
9. Other well documented causes of chronic liver disease according to 
standard diagnostic procedures including, but not restricted to:  
a. Positive hepatitis B surface antigen (HBsAg)  
b. Positive HCV RNA, (tested for in case of known cured HCV 
infection, or positive HCV Ab at Screening)  
c. 
Suspicion of drug-induced liver disease  
d. Alcoholic liver disease  
e. Autoimmune hepatitis  
f. 
Wilson’s disease   
g. 
Primary biliary cirrhosis 
h. Primary sclerosing cholangitis  
i. 
Genetic homozygous hemochromatosis  
j. 
Known or suspected HCC  
k. 
History or planned liver transplant, or current MELD 
score >15.  
10. Patients who cannot be contacted in case of emergency.  
11. Known hypersensitivity to the investigational product or any of its 
formulation excipients.  
12. Patients who are taking warfarin or other highly plasma protein-bound 
drugs with narrow therapeutic indices. 
13. Patients who are currently participating in, plan to participate in, or 
have participated in an investigational drug trial or medical device trial 
containing active substance within 30 days or five half-lives, whichever 
is longer, prior to Screening. Patients who have screened for, and failed 
to qualify, for another clinical trial are eligible for screening for the NTZ-
218-1, even if the Screening Visit for the NTZ-218-1 study is less than 30 
days from the failed study screening visit, provided that the subject 
received no investigational product within 30 days of 5 half-lives of the 
investigational product prior to screening for NTZ-218-1. 


 
 
Protocol No: NTZ-218-1 
Version: V 15 APRIL 2020 
 
 
 
 
 
Page 7 
14. Evidence of any other unstable or, untreated clinically significant 
immunological, endocrine, hematological, gastrointestinal, 
neurological, neoplastic, or psychiatric disease.  
15. Mental instability or incompetence, such that the validity of informed 
consent or ability to be compliant with the study is uncertain.  
16.  History of noncompliance with medical regimens, or patients who are 
considered to be unreliable 
17. Positive anti-human immunodeficiency virus (HIV) antibody. 
18. AST and/or ALT >5 x upper limit of normal (ULN).  
19. Total bilirubin >1.3 mg/dL due to altered hepatic function.  
20. Direct bilirubin > ULN 
Note: Gilbert Disease patients are allowed into the study.  
21. INR >1.2 in the absence of anticoagulant therapy.  
22. Platelet count <150,000/mm3 in the   context of portal hypertension.  
23. Significant renal disease, including nephritic syndrome, chronic kidney 
disease (defined as patients with markers of kidney damage or eGFR of 
less than 60 ml/min/1.73 m2). 
 
Evaluation Criteria 
Primary Endpoint: 
To assess the safety and tolerability of NTZ after 24 weeks of treatment  
 
Secondary Endpoints: 
To assess the % change in FSR from baseline to end of treatment (24 weeks 
of treatment) 
To assess the change in liver stiffness by MRE from baseline to 12 weeks 
and 24 weeks of treatment 
To assess the change in liver stiffness by fibroscan from baseline to 24 
weeks of treatment  
To assess the change from baseline to 12 weeks and 24 weeks of treatment 
of fibrosis serum biomarkers and scores. 
 
 


 
 
Protocol No: NTZ-218-1 
Version: V 15 APRIL 2020 
 
 
 
 
 
Page 8 
TABLE 1: Schedule of events 
 
Screening period 
 
 
Treatment period 
Visit 
SV 
SV2 
SV3 
SV4 
V1 
V2 
V3 
V4 
V5 
V6 
V7 
V8 
V9 
V10 
EOS 
ET 
Day 
-42 
-14 
-11 
-7 
1 
29 
57 
85 
113 
141 
155 
158 
162 
169 
+28 
days 
 
Window 
 
+3 
+1 
+1 
+3 
+3 
+3 
+3 
+3 
+3 
+3 
+1 
+1 
+1 
+3 
 
Week 
-6 
-2 
 
 
0 
4 
8 
12 
16 
20 
22 
 
 
24 
28 
 
Informed 
Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical 
History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inclusion 
Exclusion 
Criteria 
X 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
Height 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Weight, BMI 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
Physical Exam 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
12-Lead ECG 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
X 
Vital Signs 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
Con Meds 
X 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
AEs 
X 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Liver Biopsy1 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
MRE 
 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
 
X 
Fibroscan 
 
 
 
 
X 
 
 
 
 
 
 
 
 
X 
 
X 
Dispense 
deuterated 
water 
 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
FSR 
 
X 
X 
X 
X 
 
 
 
 
 
X 
X 
X 
X 
 
 
Study Drug 
Dispense 
 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
Study Drug 
Compliance 
 
 
 
 
 
X 
X 
X 
X 
X 
 
 
 
X 
 
 
X 
 
 
 
1 Required only if there is no history of a liver biopsy within the past 6 months.  


 
 
Protocol No: NTZ-218-1 
Version: V 15 APRIL 2020 
 
 
 
 
 
Page 9 
TABLE 2: Biological assessments 
 
Screening period 
 
 
Treatment period 
Visit 
SV 
SV2 
SV3 
SV4 
V1 
V2 
V3 
V4 
V5 
V6 
V7 
V8 
V9 
V10 
EOS 
ET 
Day 
-42 
-14 
-11 
-7 
1 
29 
57 
85 
113 
141 
155 
158 
162 
169 
+28 
days 
 
Week 
-6 
-2 
 
 
0 
4 
8 
12 
16 
20 
22 
 
 
24 
28 
 
Labs - 
Haematology 
haemoglobin, 
haematocrit, RBC, 
WBC, differential 
count, platelet 
count, 
reticulocytes 
count, MCV 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Coagulation (PT 
(INR)) 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Labs- Urinary 
Pregnancy tests2 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
 
X 
Labs – Serology 
HIV, HBs and HCV 
serology 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
2 If Urine pregnancy test is positive, a serum β-hCG is required.  
 
 
 


 
 
Protocol No: NTZ-218-1 
Version: V 15 APRIL 2020 
 
 
 
 
 
Page 10 
 
Screening period 
 
 
Treatment period 
Visit 
SV 
SV2 
SV3 
SV4 
V1 
V2 
V3 
V4 
V5 
V6 
V7 
V8 
V9 
V10 
EOS 
ET 
Day 
-42 
-14 
-11 
-7 
1 
29 
57 
85 
113 
141 
155 
158 
162 
169 
+28 
days 
 
Labs – 
Biochemistry 
HbA1c, fasting 
plasma glucose, 
insulin, HOMA-IR, 
fructosamine, 
alkaline 
phosphatase, ALT, 
AST, GGT, CPK, 
total and 
conjugated 
bilirubin, ferritin, 
creatinine, eGFR, 
uric acid, BUN, 
total cholesterol, 
HDL-C, LDL-C, 
triglycerides, 
albumin, lipase, 
amylase, 
electrolytes 
(sodium, 
potassium, 
chloride, calcium), 
MELD 
X 
 
 
X3 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Inflammatory 
markers 
hsCRP, fibrinogen, 
haptoglobin, 
resistin,  TNF-α, 
TGF-β, IL-6, PAI-1 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
Biomarkers of 
fibrosis 
ELF Score, PIIINP, 
Hyaluronic acid, 
CK18, TIMP-1, YKL-
40, Alpha 2 
macroglobulin,, 
,miRNA, ProC3, 
ProC6, FGF19, 
FGF21 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
NAFLD Fibrosis 
Score (NFS) &  
FIB-4 
 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
 
X 


 
 
Protocol No: NTZ-218-1 
Version: V 15 APRIL 2020 
 
 
 
 
 
Page 11 
 
Screening period 
 
 
Treatment period 
Visit 
SV 
SV2 
SV3 
SV4 
V1 
V2 
V3 
V4 
V5 
V6 
V7 
V8 
V9 
V10 
EOS 
ET 
Day 
-42 
-14 
-11 
-7 
1 
29 
57 
85 
113 
141 
155 
158 
162 
169 
+28 
days 
 
Labs – Urinalysis 
dipstick  
Specific gravity, 
pH, protein, 
glucose, ketones, 
bilirubin, 
urobilinogen, 
blood, nitrite, 
leukocytes 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
 
X 
Safety markers 
Serum Cystatin C, 
troponin T, NT 
proBNP 
Urinary albumin, 
urinary creatinine, 
urinary ACR 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Labs -- FSR 
 
X 
X 
X 
X 
 
 
 
 
 
X 
X 
X 
X 
 
 
Sampling for 
additional 
parameters 
X 
 
 
 
X 
 
 
X 
 
X 
 
 
 
X 
X 
X 
 
3 ALT, AST, Total bilirubin, Alkaline phosphatase and CPK must be retested at least 4 weeks after initial 
screening labs. . If these values at SV4 are ≥2x Screening values SV, then the patient cannot be 
randomized. A repeat test may be performed if a screening value is considered an error. 
 
 


 
 
Protocol No: NTZ-218-1 
Version: V 15 APRIL 2020 
 
 
 
 
 
Page 12 
 
Table of Contents 
1. 
Introduction .......................................................................................................................... 16 
1.1. 
Background ................................................................................................................... 16 
1.2. 
General Information for Nitazoxanide ......................................................................... 17 
1.2.1. 
Nitazoxanide (NTZ) ............................................................................................... 17 
1.2.2. 
Clinical Safety & Toxicology .................................................................................. 17 
1.2.3. 
Non clinical pharmacology ................................................................................... 19 
1.3. 
Rationale for This Study................................................................................................ 22 
2. 
Objectives .............................................................................................................................. 22 
2.1. 
Primary Objective ......................................................................................................... 22 
2.2. 
Secondary Objectives: .................................................................................................. 22 
3. 
Study Design.......................................................................................................................... 23 
3.1. 
Study Design.................................................................................................................. 23 
3.2. 
Treatment Plan and Regimen ....................................................................................... 24 
4. 
Subject Population ................................................................................................................ 24 
4.1. 
Number of Subjects & Subject Selection ..................................................................... 24 
4.2. 
Inclusion Criteria ........................................................................................................... 24 
4.3. 
Exclusion Criteria .......................................................................................................... 25 
5. 
Investigational Medicinal Products ...................................................................................... 27 
5.1. 
Description .................................................................................................................... 27 
5.2. 
Packaging ...................................................................................................................... 28 
5.3. 
Labeling ......................................................................................................................... 28 
5.4. 
Storage and Handling ................................................................................................... 28 
5.5. 
Dosage and Administration .......................................................................................... 28 
5.6. 
Dose Adjustment .......................................................................................................... 29 
5.7. 
Prior and Concomitant Medications ............................................................................ 29 
5.7.1. 
Non-permitted medication ................................................................................... 29 
5.7.2. 
Permitted Medications ......................................................................................... 29 
5.8. 
Study Drug Accountability ............................................................................................ 30 
6. 
Study Procedures .................................................................................................................. 30 


 
 
Protocol No: NTZ-218-1 
Version: V 15 APRIL 2020 
 
 
 
 
 
Page 13 
6.1. 
Duration of Study Participation ................................................................................... 30 
6.2. 
Screening Assessment .................................................................................................. 30 
Screening Visit ....................................................................................................................... 30 
6.3. 
Day -14 Deuterated Water Run In ................................................................................ 31 
6.4. 
Treatment Period Assessments .................................................................................... 32 
6.4.1. 
Day 1 Visit.............................................................................................................. 32 
6.4.2. 
Week 4 and Week 8 Visit + 3 days ........................................................................ 32 
6.4.3. 
Week 12 Visit + 3 days .......................................................................................... 33 
6.4.4. 
Week 16 and Week 20 visits ±3 days ................................................................... 33 
6.4.5. 
Week 22 Visit + 3 days (Day 155) ......................................................................... 34 
6.4.6. 
Week 24 Visit +1 ................................................................................................... 34 
6.5. 
Follow Up Period ........................................................................................................... 35 
6.5.1. 
4-Week Post Treatment Follow up (EOS – End of study visit) ±3 days ............... 35 
6.5.2. 
Early Termination (ET) .......................................................................................... 35 
6.5.3. 
Unscheduled Visits ................................................................................................ 36 
6.6. 
Missed or Partially Completed Visits due to COVID-19 ............................................... 36 
6.7. 
Assessment for Premature Discontinuation from Study ............................................. 37 
6.6.1. 
Pre-specified Patient Discontinuation rules ........................................................ 37 
6.6.2. 
Pre-specified Trial Discontinuation rules ............................................................. 38 
6.6.3. 
Dose Adjustment .................................................................................................. 38 
6.6.4. 
Specific Liver Function Monitoring after Start of Treatment .............................. 39 
6.6.5. 
Data Safety Monitoring Board ............................................................................. 40 
6.8. 
Description of Assessments .......................................................................................... 41 
6.8.1. 
Histological Assessments ...................................................................................... 41 
6.8.2. 
Assessment of newly synthesized proteins ......................................................... 41 
6.8.3. 
Biological Assessments (Table 2) .......................................................................... 41 
Table 2 Biological assessments ...................................................................................................... 43 
6.8.4. 
Electrocardiogram ................................................................................................. 44 
6.8.5. 
MRE ....................................................................................................................... 44 
6.8.6. 
Fibroscan ............................................................................................................... 45 
6.8.7. 
Medical History ..................................................................................................... 45 


 
 
Protocol No: NTZ-218-1 
Version: V 15 APRIL 2020 
 
 
 
 
 
Page 14 
6.8.8. 
Physical Examination ............................................................................................ 45 
6.8.9. 
Vital Signs .............................................................................................................. 45 
6.8.10. 
Pregnancy Testing ................................................................................................. 46 
7. 
Adverse Events and Toxicity Management .......................................................................... 46 
7.1. 
Definitions of Adverse Events, Serious Adverse Events and Unexpected Events ...... 46 
7.1.1. 
Adverse Events ...................................................................................................... 46 
7.1.2. 
Clinical Laboratory Abnormalities and Other Abnormal Assessments as Adverse 
Events or Serious Adverse Events ........................................................................................ 47 
7.1.3. 
Serious Adverse Events ......................................................................................... 47 
7.1.4. 
Unexpected adverse event ................................................................................... 48 
7.2. 
Assessment of Adverse Events & Serious Adverse Events .......................................... 48 
7.2.1. 
Intensity assessment ............................................................................................ 48 
7.2.2. 
Relation to the study treatment .......................................................................... 49 
7.2.3. 
Action taken and outcome ................................................................................... 50 
7.3. 
Reporting ....................................................................................................................... 51 
7.3.1. 
Reporting an adverse event ................................................................................. 51 
7.3.2. 
Initial reports......................................................................................................... 51 
7.3.3. 
Follow-up reports ................................................................................................. 52 
7.3.4. 
Follow-up .............................................................................................................. 52 
7.4. 
Post Study Reporting Requirements ............................................................................ 53 
7.5. 
Special Situation Reports .............................................................................................. 53 
7.5.1. 
Pregnancy .............................................................................................................. 53 
7.5.2. 
Medication error ................................................................................................... 53 
7.5.3. 
Misuse ................................................................................................................... 54 
7.5.4. 
Overdose ............................................................................................................... 54 
7.5.5. 
Abuse ..................................................................................................................... 54 
8. 
Statistical Considerations ..................................................................................................... 54 
8.1. 
Analysis Endpoints ........................................................................................................ 54 
8.1.1. 
Primary Endpoint .................................................................................................. 54 
8.1.2. 
Secondary Endpoints ............................................................................................ 54 
8.2. 
Analysis Sets .................................................................................................................. 55 
8.3. 
Data Handling Conventions .......................................................................................... 55 


 
 
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8.4. 
Demographic Data and Baseline Characteristics ......................................................... 55 
8.5. 
Primary Safety Analysis ................................................................................................ 55 
8.5.1. 
Adverse Events ...................................................................................................... 56 
8.5.2. 
Laboratory Evaluations ......................................................................................... 56 
8.5.3. 
Other Safety Evaluations ...................................................................................... 56 
8.6. 
Secondary Efficacy Analyses ......................................................................................... 56 
8.6.1. 
FSR ......................................................................................................................... 56 
8.6.2. 
Liver stiffness ........................................................................................................ 57 
8.6.3. 
Exploratory Analysis ............................................................................................. 57 
8.6.4. 
Interim Analysis .................................................................................................... 57 
8.7. 
Sample Size ................................................................................................................... 57 
9. 
Responsibilities ..................................................................................................................... 57 
9.1. 
Investigator Responsibilities ........................................................................................ 58 
9.2. 
Good Clinical Practice ................................................................................................... 58 
9.3. 
Institutional Review Board Review & Approval .......................................................... 59 
9.4. 
Informed Consent ......................................................................................................... 59 
9.5. 
Confidentiality .............................................................................................................. 59 
9.6. 
Study Files and Retention of Records .......................................................................... 60 
9.7. 
Electronic Case Report Forms ....................................................................................... 61 
9.8. 
Investigational Medicinal Product Accountability and Return ................................... 62 
9.9. 
Inspections .................................................................................................................... 62 
9.10. Protocol Compliance ..................................................................................................... 62 
9.11. Protocol Modifications ................................................................................................. 62 
9.12. Study Reports ................................................................................................................ 63 
9.13. Study Termination ........................................................................................................ 63 
10. 
References ......................................................................................................................... 64 
10.1. Literature ....................................................................................................................... 64 
10.2. WHO Fact Sheet 312 ..................................................................................................... 67 
10.3. Studies on ClinicalTrials.gov ......................................................................................... 67 
 
 


 
 
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1. Introduction 
1.1. 
Background 
Non-alcoholic fatty liver disease (NAFLD) has become one of the most prominent forms 
of chronic liver disease worldwide, reflecting the epidemic of global obesity. Those with 
the progressive variant of NAFLD, non-alcoholic steatohepatitis (NASH), are at significantly 
increased risk of multisystem morbidity and mortality. However, there are currently no 
approved pharmacologic therapies for NASH (Konerman et al, 2018). The World Health 
Organization estimates the prevalence of obesity has more than doubled since 1980 with 
>600 million people (13%) having a body mass index (BMI)) ≥30 (Konerman et al, 2018). 
In this setting, the global prevalence of diabetes among adults has also increased from 
4.7% in 1980 to 8.5% as of 2014 (WHO Fact Sheet 312) . Prevalence rates of NAFLD among 
those with metabolic disease are notably higher.  Approximately a third of patients with 
hypertension, half of patients with dyslipidaemia, up to two-thirds of patients with type II 
diabetes, and  >90% of patients undergoing bariatric surgery had evidence of NAFLD (Leite 
et al, 2009; Fruci et al, 2013; Assy et al, 2000; Wu  et al, 2016; Sasaki et al, 2014 Subichin 
et al; 2015 ; Donati et al ,2004). 
The underlying pathophysiologic mechanisms that contribute to the development and 
progression of NAFLD and NASH are complex and include modulation of metabolic 
pathways, inflammatory cascades, and/or mechanisms impacting fibrosis.  During disease 
progression fibrosis may worsen, while features of steatohepatitis resolve or ‘‘burnout”.    
As a result, NASH and fibrosis need to be evaluated independently to ensure a beneficial 
impact on one parameter does not simultaneously result in a negative impact on another 
endpoint of interest, particularly given that individual treatments tend to focus on one 
primary mechanism of action (i.e. metabolic or NASH disease modifier vs. antifibrotic) ( 
Konerman et al, 2018). 


 
 
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Persistent and progressive fibrosis is a characteristic of many chronic liver diseases in 
which excessive accumulation of collagen-rich extracellular matrix is a key feature. 
Following liver injury, quiescent Hepatic Stellate Cells (HSC) undergo a process of 
activation that is characterized by a differentiation into (α-SMA)-positive contractile 
myofibroblasts which secrete high amounts of collagen and play a central role in the 
fibrogenic process (Friedman et al, 2008, Carpino et al, 2005). Nitazoxanide may be a 
potent antifibrotic agent that can potentially be used for the treatment of different fibrotic 
pathologies, including liver fibrosis. (Belanger et al;2017) 
1.2. 
General Information for Nitazoxanide 
1.2.1. Nitazoxanide (NTZ)  
Nitazoxanide (NTZ) is a broad-spectrum antiparasitic and broad-spectrum antiviral drug 
used for the treatment of various helminthic, protozoal, and viral infections.  It was 
approved in the United States by the Food and Drug Administration (Alinia®, Romark 
Laboratories) as an Orphan Drug for treatment of diarrhea caused by C. parvum and 
Giardia intestinalis in adults and children at least 12 months of age [Rossignol, 2014]. 
The approved Alinia dose for adults is 500 mg twice daily (BID) for three days.  
1.2.2. Clinical Safety & Toxicology 
Nitazoxanide is rapidly metabolized in the liver to an active metabolite tizoxanide (TZ) in 
vivo. TZ undergoes conjugation to form tizoxanide glucuronide. Nitazoxanide is not 
detectable in the serum following oral administration.  Peak concentrations in blood 
plasma of TZ and TZ glucuronide are seen between 1-4 hours.  NTZ is excreted in both 
Urine (~33%) and feces (~67%).   
The safety of ALINIA was evaluated in 2177 HIV-uninfected subjects 12 months of age 
and older who received ALINIA Tablets or ALINIA for Oral Suspension at the 
recommended dose for at least three days. In pooled controlled clinical trials involving 
536 HIV-uninfected subjects treated with ALINIA Tablets or ALINIA for Oral Suspension, 


 
 
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the most common adverse reactions were abdominal pain, headache, chromaturia and 
nausea (≥2%).[Alinia USPI] 
Safety data were analyzed separately for 280 HIV-uninfected subjects ≥12 years of age 
receiving Alinia at the recommended dose for at least 3 days in 5 placebo-controlled 
clinical trials and for 256 HIV-uninfected subjects 1 through 11 years of age in 7 
controlled clinical trials. There were no differences between the adverse reactions 
reported for Alinia-treated subjects based upon age [Alinia USPI]. 
Twenty-one clinical studies of NTZ were conducted in patients with chronic hepatitis C 
(14 studies: Rossignol et al, 2008; Rossignol et al, 2009; Bacon et al, 2010, Rossignol et 
al, 2010; Shiffman et al, 2010 ; Laufer et al, 2011; Kabil et al, 2011 ; Amorosa et al, 2013; 
Shebab et al, 2014; Macias et al, 2015; Kohla et al, 2016; Basu et al, 2013; 
NCT01185028; NCT01770483), hepatic encephalopathy in chronic liver failure (1 study: 
Basu et al, 2008), acquired immune deficiency syndrome (AIDS)-related 
cryptosporidiosis (3 studies: Rossignol et al, 1998; Rossignol et al, 2006; Zulu et al, 
2005), or Helicobacter pylori (2 studies: Abd-Elsalam et al, 2016; Shehata et al., 2017), 
and Clostridium difficile infections (1 study: Musher et al, 2009) in the literature. The 
dose of NTZ administered in 19 studies was 500 mg BID; in one study 1000 mg NTZ BID 
was administered, whereas in one compassionate-use study the NTZ dose administered 
ranged from 500 to 1500 mg BID. Most studies administered NTZ treatment for 48 to 60 
weeks, whereas the duration of treatment in the compassionate-use study ranged from 
1 day to 4 years.  
The studies identified from the literature review demonstrate the safety of NTZ in the 
treatment of chronic hepatitis C, hepatic encephalopathy in chronic liver failure, AIDS-
related cryptosporidiosis, persistent diarrhea in patients with AIDS, and Helicobacter 
pylori and Clostridium difficile infections.  
The adverse event (AE) profile of NTZ was predominantly GI in nature and included 
nausea, vomiting, diarrhea, abdominal pain, dyspepsia, constipation, flatulence, and 


 
 
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dysphagia. These GI AEs were generally mild in intensity and transient. Other AEs 
related to NTZ treatment were fatigue, rash, headache, myalgia, and arthralgia. When 
reported, no significant changes were observed in the electrocardiograms (ECGs), vital 
signs, and clinical laboratory parameters after administration with NTZ [Stockis et al, 
2002a; Stockis et al, 2002b]. A formal International Council on Harmonization (ICH) E14 
compliant thorough QT (TQT) study [Taubel et al, 2014] completed the cardiac safety 
evaluation and showed that neither a therapeutic single dose of 675 mg nor a 2700 mg 
(supra-therapeutic) dose of NTZ prolonged the QT interval in healthy male and female 
volunteers.   
The safety profile of NTZ reported in all these studies therefore provides supporting 
evidence for the potential use of NTZ for the treatment of liver fibrosis in patients with 
NASH.  
The following adverse reactions have been identified during post approval use of ALINIA 
as described in the Package Insert:  
• Gastrointestinal disorders: diarrhea, gastroesophageal reflux disease 
• Nervous System disorders: dizziness 
• Respiratory, thoracic and mediastinal disorders: dyspnea 
• Skin and subcutaneous tissue disorders: rash, urticaria 
1.2.3. Non clinical pharmacology 
Nonclinical pharmacology studies were conducted to determine whether NTZ and its 
main metabolites (TZ and TZ glucuronide [TZ glu]) exhibit anti-fibrotic properties in vitro, 
and in vivo. 
NTZ and its main metabolite, TZ, significantly (p<0.001 from 0.3 µM) inhibited, in a dose-
dependent manner, the expression of αSMA protein in TGF-β-induced human HSCs, but 
TZ glu had no anti-fibrotic properties. NTZ and TZ demonstrated good potency as the 
IC50 was in the micromolar range.  


 
 
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The CDAA diet-induced rodent model develops a fibrosing NASH within a relatively short 
period [Takeuchi-Yorimoto et al, 2013], which makes it a suitable model for studying the 
treatment of both NASH pathology and hepatic fibrosis. 
In studies to determine the effects of NTZ on liver fibrosis in C57BL/6J mice induced by a 
CDAA diet supplemented with 1% cholesterol, microscopic examination revealed that 
NTZ at doses of 11.4 mg/kg/day and 33.9 mg/kg/day tended to reduce, in a dose-
dependent manner, CDAA + 1% cholesterol diet-induced NASH. Although NTZ did not 
have a beneficial effect on hepatic steatosis and lobular inflammation grade, a tendency 
to reduce hepatocyte ballooning was observed, which resulted in a slight reduction in 
NASH prevalence.  
At 12 weeks, NTZ significantly prevented, in a dose-dependent manner, hepatic collagen 
storage induced by the CDAA + 1% cholesterol diet. However, upon histological 
examination, this beneficial effect was not reflected by a dose-dependent improvement 
of the fibrosis grade. A significant decrease of the fibrosis area was observed with NTZ at 
doses of 11.4 mg/kg/day (p<0.05) and 78.1 mg/kg/day (p<0.001) and correlated with 
the significant decrease of the hepatic collagen content.  
The CDAA + 1% cholesterol diet notably induces a significant augmentation in circulating 
markers of liver injury such as AST, ALT, and PIIINP, which is directly associated with 
fibrosis [Tanwar et al, 2013]. Moreover, the hepatic content is dramatically modified as 
illustrated by the significant increase of collagen. Mice treated with NTZ at 
78.1 mg/kg/day exhibited lower plasma levels of ALT (-17%, p>0.05), AST (-24%, p<0.05), 
and PIIINP (-25%, p<0.01). These data suggest a potential preventive action of NTZ on 
liver damage induced by the CDAA + 1% cholesterol diet in mice.  
The hepatic expression of fibrotic markers (collagens, MMP2), but also inflammatory 
markers (TNFα) were enhanced in the animals fed the CDAA + 1% cholesterol diet as 
compared with animals fed a CSAA control diet. Chronic administration of NTZ at 
78.1 mg/kg/day to mice fed a CDAA + 1% cholesterol diet led to the inhibition of the 


 
 
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Col1α1 (-17%, p>0.05), Col1α2 (-25%, p<0.05), MMP2 (-32%, p<0.05), and TNFα  
(-28%, p<0.01) gene expression.  
In the studies with CCl4-treated mice, NTZ significantly prevented hepatic fibrosis. This 
was shown at the dose of 95.3 mg/kg/day on the hepatic collagen content decrease in 
the first study [SSR_NTZ_PHA1_004], whereas no significant improvement was observed 
upon histological examination. Conversely, in the second study [SSR_NTZ_PHA1_005], 
no significant improvement of CCl4-induced hepatic collagen content was observed, 
whereas the histological evaluation of hepatic fibrosis revealed a significant effect of 
NTZ at 32 and 104 mg/kg/day. 
There was no significant beneficial effect of NTZ treatment on plasma markers of 
hepatotoxicity in CCl4-treated mice.  
These studies demonstrate the anti-fibrotic effect of NTZ, which suggests that NTZ may 
have potential as an anti-fibrotic drug. These studies also suggest that NTZ may show 
some beneficial therapeutic properties for NASH, such as a tendency to reduce 
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1.3. 
Rationale for This Study 
Based on the anti-fibrotic properties demonstrated in the animal models of fibrosis, this proof 
of concept clinical study aims at  evaluating NTZ in patients with non-alcoholic steatohepatitis 
(NASH) and  fibrosis stage 2 and 3.Although NTZ has been evaluated in liver disease 
populations (viral hepatitis C) up to 60 weeks, this is the first study evaluating NTZ treatment in 
a population with NASH induced stage 2 and 3 fibrosis.  The aim of this study is to evaluate the 
safety and tolerability of NTZ 500 mg BID after 24 weeks of treatment in this population. This 
proof of concept study will also evaluate the anti-fibrotic effect of NTZ as a secondary 
objective. 
The methods of evaluation of fibrosis will include an innovative method of metabolic labeling. 
This approach is based on the concept that liver status can be determined by measuring the 
ratio of newly synthesized/pre-existing proteins. The turn-over rate of newly synthesized 
collagen and proteins represents the hepatic fibrogenic disease activity.  Patients will be given 
“heavy water” to drink. Heavy water contains D20, deuterium being a stable isotope of 
hydrogen. Mass spectrometry is used to identify individual proteins and to quantify the ratio of 
labeled protein to total protein. The results are expressed as fractional synthesis rate of these 
proteins (FSR). This method has been previously published (Decaris et al, 2017). 
Other non-invasive methods will be used to evaluate the liver stiffness changes after NTZ 
treatment: Magnetic Resonance Elastography (MRE) and FibroScan®.  
2. 
Objectives 
2.1. Primary Objective 
 
The primary objective of the study is: 
• To evaluate the safety and tolerability of NTZ 500mg bid after 24 weeks of treatment in 
patients with NASH induced Stage 2 or Stage 3 fibrosis. 
2.2. 
Secondary Objectives: 
The secondary objectives of the study are: 


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• To determine the effect of daily oral administration of NTZ 500mg bid on de novo 
collagen synthesis through Fractional Synthesis Rate (FSR) of circulating plasma 
proteins in patients with NASH-induced Stage 2 or Stage 3 fibrosis. 
• To evaluate changes in liver stiffness as measured by FibroScan® and by Magnetic 
Resonance Elastography (MRE). 
• To assess the change from baseline in non-invasive markers of fibrosis: ELFTM test 
score, PIIINP, Hyaluronic acid, CK18, TIMP-1, YKL-40, Alpha 2 macroglobulin,, ,miRNA, 
ProC3, ProC6, FGF19, FGF21. 
• To assess changes in additional non-invasive tests: fibrosis scores: NAFLD fibrosis 
score, FIB-4 
3. 
Study Design 
3.1. 
Study Design 
This is a phase 2 proof of concept study to evaluate the efficacy and safety of open-label NTZ 
500mg bid in a NASH population with NASH induced Stage 2 or 3 fibrosis. 
The study will include a screening visit within 6 weeks of randomization (Day 1) followed by a 
24-week treatment period consisting of daily, BID, oral administration of NTZ and an end of 
treatment follow up period of 4 weeks.   
Patients will come to the research center and complete the following visits: 
• 
Screening Visit within 6 weeks of Randomization 
• 
Day -14 Deuterated Water Run-In 
o Day-14, Day-11, Day-7 and Day 1: sampling for labelling of Deuterated water 
during run-in 
• 
Repeat baseline labs for ALT, AST, total bilirubin, ALP, CPK collected at least 4 
weeks from initial screening.  
• 
Day 1 Randomization: start of treatment 
• 
Every 4 weeks after randomization until 20 weeks post randomization 


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• 
 22 weeks post randomization and Deuterated Water Administration Period:  
o 22 weeks plus 3 days 
o sampling for labelling of Deuterated water on Day 155, 158, 162, and Day 
169/24 weeks post randomization: End of Treatment 
• 
4 Week Post Treatment Follow Up 
3.2. 
Treatment Plan and Regimen 
NTZ will be administered in an open label fashion to participating subjects as 500mg bid, for 24 
weeks. 
4. 
Subject Population 
4.1. 
Number of Subjects & Subject Selection 
Twenty male and female subjects with histologically confirmed NASH and fibrosis stage 2 or 3 
will be enrolled in the study at a single site in the United States. 
4.2. 
Inclusion Criteria 
Patients must meet all of the following inclusion criteria to be eligible for enrollment in the 
trial:  
1. 
Males or females aged from 18 to 75 years inclusive the Screening Visit.  
2. 
Must provide signed written informed consent and agree to comply with the 
study protocol.  
3. Females participating in this study must be of non-childbearing potential or using 
highly efficient contraception for the full duration of the study.   Childbearing 
potential refers to those female subjects who have not had a hysterectomy, 
bilateral oophorectomy, or medically-documented ovarian failure. Female subjects 
are considered postmenopausal and not of child-bearing potential if they are >55 
years of age, or >50 years of age with >12 months of amenorrhea, or have serum 
follicle stimulating hormone (FSH) values >40 mIU/ml with >12 months of 
amenorrhea.  Highly efficient contraception is defined as the use of one of the 


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following methods of birth control in addition to a male partner using a condom 
(unless surgically sterile) from Screening to 90 days after the last dose of study 
medication: 
• hormone-containing contraceptive 
• intrauterine device 
• cervical cap or diaphragm with spermicidal agent 
4. 
 Histological confirmation of steatohepatitis on a diagnostic liver biopsy (biopsy 
obtained within 6 months prior to Screening or during the Screening Period) 
with at least 1 in each component of the NAS (steatosis scored 0-3, ballooning 
degeneration scored 0-2, and lobular inflammation scored 0-3).   
5. 
Fibrosis stage of 2 or 3, according to the NASH CRN fibrosis staging system on a 
diagnostic liver biopsy (biopsy obtained within 6 months prior to Screening or 
during the Screening Period).   
6. 
Two assessments of ALT, AST, Total bilirubin, ALP, CPK will be collected during 
screening at least 4 weeks apart. To be eligible the second value cannot be ≥2x 
the first value. 
4.3. 
Exclusion Criteria 
Patients who meet any of the following criteria will be excluded from entering the study:  
1. 
History of efficient bariatric surgery within 5 years prior to Screening, or 
planned bariatric surgery in the course of the study.  
2. 
Patients with HbA1c >10.0%. If abnormal at the first Screening Visit, the HbA1c 
measurement can be repeated. A repeated abnormal HbA1c (HbA1c >10.0%) 
leads to exclusion.  
3. 
Patients with a history of clinically significant acute cardiac event, or 
cerebrovascular event, within 6 months prior to Screening such as: stroke, 
transient ischemic attack, or coronary heart disease (angina pectoris, 
myocardial infarction, revascularization procedures).  
4. 
Weight loss of more than 10% within 6 months prior to Randomization.  
5. 
Patient with any history or presence of cirrhosis. 


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6. 
Current or recent history (within a year of screening) of significant alcohol 
consumption. For men, significant consumption is typically defined as higher 
than 30 g pure alcohol per day. For women, it is typically defined as higher than 
20 g pure alcohol per day.  
7. 
Current or history of other substance abuse within 1 year prior to screening. 
8. 
Pregnant or lactating females or females planning to become pregnant during 
the study period.  
9. 
Other well documented causes of chronic liver disease according to standard 
diagnostic procedures including, but not restricted to:  
a) Positive hepatitis B surface antigen (HBsAg)  
b) Positive HCV RNA, (tested for in case of known cured HCV infection, or positive 
HCV Ab at Screening)  
c) Suspicion of drug-induced liver disease  
d) Alcoholic liver disease  
e) Autoimmune hepatitis  
f) Wilson’s disease   
g) Primary biliary cirrhosis 
h) Primary sclerosing cholangitis  
i) Genetic homozygous hemochromatosis  
j) Known or suspected HCC  
k) History or planned liver transplant, or current MELD score >15.  
10. 
Patients who cannot be contacted in case of emergency.  
11. 
Known hypersensitivity to the investigation product or any of its formulation 
excipients.  
12. 
Patients who are taking warfarin or other highly plasma protein-bound drugs 
with narrow therapeutic indices. 
13. 
Patients who are currently participating in, plan to participate in, or have 
participated in an investigational drug trial or medical device trial containing 
active substance within 30 days or five half-lives, whichever is longer, prior to 


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Screening. Patients who have screened for, and failed to qualify, for another 
clinical trial are eligible for screening for the NTZ-218-1, even if the Screening 
Visit for the NTZ-218-1 study is less than 30 days from the failed study screening 
visit, provided that the subject received no investigational product within 30 
days of 5 half-lives of the investigational product prior to screening for NTZ-218-
1. 
14. 
Evidence of any other unstable or, untreated clinically significant 
immunological, endocrine, hematological, gastrointestinal, neurological, 
neoplastic, or psychiatric disease.  
15. 
Mental instability or incompetence, such that the validity of informed consent 
or ability to be compliant with the study is uncertain.  
16. 
 History of noncompliance with medical regimens, or patients who are 
considered to be unreliable. 
17. 
Positive anti-human immunodeficiency virus (HIV) antibody. 
18. 
AST and/or ALT >5 x upper limit of normal (ULN).  
19. 
Total bilirubin >1.3 mg/dL due to altered hepatic function. 
20. 
Direct bilirubin > ULN 
 Note: Gilbert Disease patients are allowed into the study.  
21. 
INR >1.2 in the absence of anticoagulant therapy.  
22. 
Platelet count <150,000/mm3 in the context of portal hypertension.  
23. 
Significant renal disease, including nephritic syndrome, chronic kidney disease 
(defined as patients with markers of kidney damage or eGFR of less than 60 
ml/min/1.73 m2). 
5. 
Investigational Medicinal Products 
Patients will receive 500 mg NTZ (ALINIA®) twice daily for 24 weeks. 
5.1. Description  


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NTZ will be supplied as 500 mg of ALINIA® (nitazoxanide) tablets, for oral use.  The tables are 
round, yellow, film-coated and debossed with ALINIA on one side and 500 on the other side. 
Each tablet contains 500 mg of nitazoxanide. 
For additional information see the Package Insert for ALINIA®. 
5.2. Packaging  
ALINIA® tablets are packaged in HDPE bottles of 12 tablets. Period boxes will be delivered 
every 4 weeks, each box containing 6 bottles. 
5.3. 
Labeling 
All labels for study drugs meet all applicable requirements of the US Food and Drug 
Administration (FDA) and the EU annex 13 of Good Manufacturing Practices: Manufacture of 
Investigational Medicinal Products (February 2010) and /or other local regulations, as 
applicable.  
Distribution of study drug will be performed according to the Good Distribution Practices.  
Product boxes for each period will be labeled with the protocol number, Sponsor’s name and 
address, description of contents, storage conditions, expiry date, dosage instructions, and any 
other applicable items required by national and regional guidelines/regulations. The label will 
contain the statements “Caution – new drug, limited by Federal Law to investigational use” or 
other similar/appropriate statements as well as the following instructions “Please return 
empty packaging and unused products to your doctor at your next visit.”  
5.4. 
Storage and Handling 
The tablets are to be stored at 20-25oC (68-77oF). Storage conditions are specified on the label. 
5.5. 
Dosage and Administration 
Patients will be instructed to take one tablet of 500mg NTZ mg orally, twice a day with water 
during a meal, approximately 12 hours apart.    
The study staff will administer the first dose of the medication at the research center.  Patients 
will self-administer the evening dose on Day 1 and twice a day thereafter, when not attending 


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a visit at the research center.  On scheduled follow up visit days while the patient is being 
treated with study drug, the patient will be directed to bring back all used and unused study 
medication containers. Compliance will be checked by the Investigator or designee during 
those visits and registered in the electronic Case Report Form (eCRF).  
If treatment is interrupted, whatever the cause, duration and reason of the interruption will be 
documented. 
5.6. 
Dose Adjustment 
Enrolled subjects who experience treatment emergent adverse events may have their dose 
adjusted, at the discretion of the Principal Investigator, in accordance with section 6.6.3. 
5.7. 
Prior and Concomitant Medications 
In a general manner, patients should be discouraged from starting any new medication 
without consulting the Investigator unless the new medication is required for emergency 
purpose.  In the same way, any qualitative or quantitative change in concomitant therapy 
should be avoided, when possible. In the event that it becomes necessary during the study, 
this should be recorded by the Investigator or designee in the eCRF (including concomitant 
medications taken within 6 months prior to Screening). This includes drugs used on a chronic 
as well as on an “as needed” basis. 
5.7.1.  Non-permitted medication 
The following medications are not allowed within the timeframe given:  
• Warfarin or other highly plasma protein-bound drugs with narrow therapeutic indices 
are not permitted from Day 1 until end of treatment 
5.7.2.  Permitted Medications 
Any medications other than those listed above are permitted. However, the dosage of a 
current medication for a chronic disease should remain unchanged as far as possible in order 
to reduce the risk of unknown drug-drug interactions.  
In the event that additional concomitant therapy becomes necessary during the study, this will 
be recorded by the Investigator in the eCRF. This includes drugs used on a chronic as well as on 


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an “as-needed” basis. Patients should be discouraged from starting any new medication 
without consulting the Investigator unless the new medication is required for emergency 
purpose. 
5.8. Study Drug Accountability 
The Investigator will acknowledge receipt for each study treatment on the day of receipt. A 
drug accountability record will be maintained by the person responsible for dispensing the trial 
medication to the patient.  
All partially used or unused treatments will be inventoried during and at the conclusion of the 
study.  
The Drug Distribution Center will organize the retrieval of all treatments (used or unused) and 
will proceed to their destruction only after written authorization and review of accountability 
and distribution records.  
6. 
Study Procedures 
The procedures performed at each visit are summarized in the Schedule of Events (Table 1).  
A patient may be seen at any time for reasons of safety.  
During each visit, safety evaluations will be done, and the patient will be queried in the form of 
an open question regarding new or continuing events.  
6.1. 
Duration of Study Participation 
The estimated duration of the study will be approximately 34 weeks inclusive of the screening 
period. 
6.2. 
Screening Assessment 
Screening Visit 
The following screening procedures will be performed for all potential patients at the 
Screening Visit and prior to Day 1:  
• 
Informed consent witnessed by the Investigator or designated person.  


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• 
Medical history & demographics.  
• 
Assessment of inclusion/exclusion criteria 
• 
Complete physical examination. 
• 
Record vital signs  
• 
Record height, weight, BMI  
• 
Check concomitant/prior medication (within 6 months prior to Screening)  
• 
12-lead ECG 
• 
Liver biopsy.  A historical liver biopsy with confirmed NASH and fibrosis is 
acceptable if performed within 6 months prior to the Screening Visit.  
• 
Adverse Events assessment  
• 
Blood samples will be collected for 
o 
Biochemistry 
o 
Hematology 
o 
Serology (HIV, HBs, HCV). In the event of a positive HCV Ab, HCV RNA 
can be tested for confirmation. In case of known cured HCV infection, HCV RNA 
can be tested directly at Screening.  
• 
Repeat baseline labs for ALT, AST, total bilirubin, ALP, CPK collected at least 4 
weeks from initial screening. 
• 
Urine samples will be collected for Urinalysis  
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
If needed, a retesting of any laboratory results may be performed during the screening 
window to determine the eligibility for the study.  
6.3. 
Day -14 Deuterated Water Run In 
Potential Subjects who complete all of the Screening Visit assessments and who 
continue to be eligible for the study, will return to the research center to complete the 
Deuterated Water Run In.  The visit is to be done on Day -14 + 3 days, provided that the 
subject completes 7 consecutive days of deuterated water administration.  The 
following procedures will be performed at the Day -14 Visit: 


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• Dispense deuterated water (D2O). Instruct patient to drink 50ml of deuterated 
water 3 times per day starting on Day-14 for 7 consecutive days (until day-8).  
• Blood sample for baseline labeling by D2O (kinetic biomarkers) (patients should 
be instructed to fast the evening prior to the visit to ensure a 10-hour fast) 
•  Adverse Events assessment  
• Record concomitant medication taken 
Patients will return at Day-11, Day-7 (+ 1 day) and Day 1 for blood sampling (labeled 
D2O) 
6.4. 
Treatment Period Assessments 
6.4.1. Day 1 Visit 
The following procedures will be performed at the Day 1 Visit prior to dose administration:  
• 
Review inclusion/exclusion criteria 
• 
Complete physical examination 
• 
Weight, BMI 
• 
Record vital signs  
• 
Record concomitant medication taken  
• 
Adverse Events assessment  
• 
MRE (may be done within + 5 days of the visit)  
• 
Fibroscan 
• 
Blood samples will be collected  
• 
Urine samples will be collected for Urinalysis  
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
• 
Dispense the study drug as directed 
• 
Review study drug administration instructions with subject  
• 
Administer 500 mg of NTZ with water & food 
6.4.2. Week 4 and Week 8 Visit + 3 days 


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The following procedures will be performed at these Visits:  
• 
Record concomitant medication taken  
• 
Adverse Events assessment  
• 
Blood samples will be collected  
• 
Urine samples will be collected for Urinalysis  
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
• 
Dispense the study drug as directed 
• 
Review study drug compliance and drug administration instructions with subject  
• 
Reconcile study drug administration using pill counts  
• 
Administer 500 mg of NTZ with water & food 
6.4.3. Week 12 Visit + 3 days 
The following procedures will be performed at the Visit:  
• 
Complete physical examination 
• 
Weight, BMI 
• 
Record vital signs  
• 
Record concomitant medication taken  
• 
Adverse Events assessment  
• 
MRE (may be done within + 5 days of the visit) 
• 
Blood samples will be collected  
• 
Urine samples will be collected for Urinalysis  
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
• 
Dispense the study drug as directed 
• 
Review study drug compliance and drug administration instructions with subject  
• 
Reconcile study drug administration using pill counts  
• 
Administer 500 mg of NTZ with water and food 
6.4.4. Week 16 and Week 20 visits ±3 days 
The following procedures will be performed at these Visits:  


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• 
Record concomitant medication taken  
• 
Adverse Events assessment  
• 
Blood samples will be collected  
• 
Urine samples will be collected for Urinalysis  
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
• 
Dispense the study drug as directed 
• 
Review study drug compliance and drug administration instructions with subject  
• 
Reconcile study drug administration using pill counts  
• 
Administer 500 mg of NTZ with water & food 
6.4.5. Week 22 Visit + 3 days (Day 155) 
The following procedures will be performed at the Visit:  
• 
Record concomitant medication taken  
• 
Adverse Events assessment  
• 
Blood samples will be collected for D2O labeling 
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]) 
• Dispense Deuterated water. Instruct patient to drink 50ml of deuterated water 
3 times per day starting on Day 155 for 7 consecutive days (until day 162).  
• Blood sample for kinetic biomarkers analysis (patients should be instructed to 
fast the evening prior to the visit to ensure a 10-hour fast) 
Patients will return for blood sampling for kinetic biomarker analysis at 22weeks/+ 3 days),  
Day 158, Day 162 (+ 1 day), and at day 169 (Week 24). 
6.4.6. Week 24 Visit +1 
The following procedures will be performed at the visit: 
• 
Complete physical examination 
• 
12-lead ECG 
• 
Record vital signs  
• 
Record weight, BMI  


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• 
Record concomitant medication taken  
• 
MRE (may be done within + 5 days of the visit) 
• 
Fibroscan  
• 
Adverse Events assessment  
• 
Blood samples will be collected  
• 
Urine samples will be collected for Urinalysis  
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
• 
Review study drug compliance and drug administration instructions with subject  
• 
Reconcile study drug administration using pill counts  
6.5. 
Follow Up Period 
6.5.1. 4-Week Post Treatment Follow up (EOS – End of study visit) ±3 days 
The following procedures will be performed at the visit: 
• Complete physical examination 
• Weight, BMI 
• Record vital signs  
• Record concomitant medication taken  
• Adverse Events assessment  
• Blood samples will be collected Urine samples will be collected for Urinalysis  
• Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
6.5.2. Early Termination (ET) 
Patients who have discontinued from the study after the first dose of study medication and for 
any reason will have the following procedures completed: 
• 
Complete physical examination 
• 
Record vital signs 
• 
Weight, BMI  
• 
ECG 
• 
Record concomitant medication taken  
• 
MRE (may be done within + 5 days of the visit) 


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• 
Fibroscan 
• 
Adverse Events assessment  
• 
Blood samples will be collected  
• 
Urine samples will be collected for Urinalysis  
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]) 
• 
Review study drug compliance with subject  
• 
Reconcile study drug administration using pill counts  
• 
Documentation if Early Termination is due to COVID-19 related AE or concerns. 
6.5.3. 
Unscheduled Visits 
An unscheduled visit is defined as any visit to the study unit outside of the protocol-evaluation 
time points where the patient is seen by study unit personnel, e.g., when follow-up 
assessments are required for safety reasons or when repeat measurements are required out of 
the screening period (either to confirm a measurement or in case of errors, measuring device 
failure, etc.).  
Unscheduled visits will be needed for patients who may require further follow-up due to 
safety. 
6.6. 
Missed or Partially Completed Visits due to COVID-19 
If a subject is unwilling or unable to complete regularly scheduled visit(s) due to COVID-19, the 
study team will document the following: 
• Was the visit partially completed (some procedures completed, while other protocol 
directed procedures were not completed)?  If so:   
o Clinical safety lab tests (hematology, coagulation, pregnancy test, and 
biochemistry), as required by the visit schedule, must be completed to dispense 
NTZ to the patient for continued safety monitoring.  
• Was any part of the visit performed via a virtual method (e.g. phone, or other 
technology)? These procedures must be clearly documented as being done “virtually” 
in the source documentation. 
• Description why the visit was partially completed or missed. 
• Any COVID-19 related symptoms defined below since the last visit, to include AE onset 
date, description and resolution date: 
o Fever  
o Cough 


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o Shortness of breath 
o Other symptom as defined by the Centers for Disease Control and Prevention. 
  
6.7. 
Assessment for Premature Discontinuation from Study 
Study medication may be discontinued in the following instances: 
• Intercurrent illness that would, in the judgment of the investigator, affect 
assessments of clinical status to a significant degree. Following resolution of 
intercurrent illness, the subject may resume study dosing at the discretion of the 
investigator. 
• Subject noncompliance  
• Investigator discretion  
• Study medication must be discontinued in the following instances:  
o 
Unacceptable toxicity,  or toxicity that, in the judgment of the 
investigator, compromises the ability to continue study-specific 
procedures or is considered to not be in the subject’s best interest  
o 
Subject request to discontinue for any reason  
o 
Pregnancy  
o 
Discontinuation of the study at the request of a regulatory agency or an 
institutional review board or independent ethics committee (IRB/IEC) 
Pre-specified stopping criteria are defined at the patient level and at the study level. These are 
described in the following sections 6.6.1., 6.6.2., and 6.6.3. In all cases, when the stopping 
criteria are met, the Data Safety Monitoring Board will perform causality assessment, and 
decide whether the study drug/study trial may be continued (See section 6.6.4.). 
6.6.1. 
Pre-specified Patient Discontinuation rules 
If a patient experiences a Grade IV Common Terminology Criteria for Adverse Event (CTCAE) or 
meets liver injury criteria as listed in section 6.6.3., the study drug should be interrupted. After 
causality assessment by the DSMB, if the AE or lab abnormality is considered unrelated to 
study drug, and the event has resolved or returned to baseline, then the study drug may be re-


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started. If the event reoccurs after restart of study drug, then the study drug will be 
permanently discontinued. 
6.6.2. 
Pre-specified Trial Discontinuation rules 
If one patient experiences a Grade V CTCAE, or 2 patients experience the same Grade IV 
CTCAE, or 3 patients experience the same Grade III CTCAE, or 4 patients experience the same 
Grade II CTCAE, the trial will be paused. The DSMB will assess causality of these adverse 
events; if the events are deemed unrelated to study drug, the trial will continue.  
6.6.3. 
Dose Adjustment 
Enrolled subjects who experience treatment emergent adverse events should be monitored 
closely and may have the NTZ dose adjusted at the Principal Investigator’s discretion.  
Enrolled subjects who experience a possible treatment emergent adverse event will be 
assessed for symptoms and further biochemical assessments as follows: 
• GI Adverse Events: the subject will be assess for abdominal pain, loss of 
appetite, nausea and vomiting.  Blood will be collected for amylase and 
lipase assessment to rule out pancreatitis.   
• Kidney Function Adverse Events: the subject will have blood samples 
collected for eGFR to rule out kidney dysfunction. 
• Musculoskeletal Adverse Events: the subject will be assessed for fatigue, 
muscle aches and pain. Blood samples will be collected for Creatine 
Kinase. 
The following procedure will be followed for dose adjustments: 
1. Patients will be asked to hold drug for 3 days 
2. After the 3-day drug hold, patients will be reassessed for adverse event symptoms.   
• If the AE symptoms have resolved following 3 days of drug hold, the patient will 
discontinue NTZ dosing permanently and will be followed per the schedule of 
assessments. 


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If the AE symptoms have not resolved following 3 days of drug hold, the patient will be 
restarted on a reduced dose of 500 mg NTZ, once a day for 7 days.  Patients will be evaluated 
for treatment emergent adverse events and consideration for resuming the dosage of 500mg 
NTZ twice a day.  PI may determine that the patient should remain at the 500 mg NTZ once a 
day dose following 7 days of reduced dose. In such scenarios, the patient will be monitored 
and consideration for resuming the dosage of 500 mg NTZ twice daily will be evaluated at each 
subsequent visit. 
6.6.4. 
Specific Liver Function Monitoring after Start of Treatment 
Liver function monitoring guidelines are detailed below. The cases will be reviewed by the 
DSMB, who will assess potential Drug Induced Liver Injury (DILI), and apply discontinuation 
rules as described in 6.6.1., 6.6.2., and in this section. 
Study drug interruption 
Study drug must be interrupted in the following cases:  
• ALT >8x ULN  (if ALT is normal at baseline) 
• ALT > 5x Baseline (if ALT is elevated at baseline) OR ALT>500 U/L 
• ALT >5x ULN (if ALT is normal at baseline) AND Total Bilirubin>2xULN 
• ALT >3x Baseline (if ALT is elevated at baseline) OR ALT>300 U/L AND total 
bilirubin>2xULN  
• ALT >5x ULN (if ALT is normal at baseline) AND liver-associated symptoms 
• ALT >3x Baseline (if ALT is elevated at baseline) OR ALT>300 U/L AND liver-associated 
symptoms 
In all these cases, close monitoring should be initiated, with repeat liver biochemistries within 
24-72 hours. Continue monitoring twice/week until liver biochemistries resolve, stabilize, or 
return within baseline values. If abnormalities stabilize and patient is asymptomatic, then 
monitoring should be performed once a week. History of symptoms, disease, concomitant 
medication, or other conditions such as alcohol consumption or alcohol hepatitis should be 
obtained for competing etiologies. The cases will be reviewed by the DSMB, who will 


Protocol No: NTZ-218-1 
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determine if the patient may restart study drug (if causality assessment is deemed to be not 
related to study drug). 
 
Management of liver biochemistries elevations 
In the following cases of liver enzyme increase, liver biochemistries including ALT, AST, Total 
bilirubin, ALP, should be repeated within 2-5 days. Follow up of symptoms should be carefully 
monitored. Frequency of monitoring should continue once/week until liver abnormalities 
resolve, stabilize, or return to values below the thresholds mentioned below: 
• ALT >5x ULN  (if ALT is normal at baseline) 
• ALT >3x Baseline (if ALT is elevated at baseline) OR ALT >300 U/L 
NOTE: Laboratory assessments should be done centrally. If the patient lives remotely, he/she 
can have laboratory tests performed locally, and the results communicated to the investigator 
promptly. 
 
6.6.5. 
Data Safety Monitoring Board 
An independent Data Safety Monitoring Board (DSMB) composed of 3 liver experts will review 
the safety of patients enrolled in the trial. The DSMB will perform a safety data review every 3 
months, the first review occurring 3 months after first patient randomized. A DSMB charter 
will define the role, responsibilities, rules, and tasks of the DSMB. 
The DSMB will ensure that the patient and trial discontinuation rules are respected and will 
determine the causality assessment of the events described in sections 6.6.1, 6.6.2., and 6.6.3., 
in order to make the recommendation for trial /patient discontinuation. If the events are 
deemed unrelated to the study drug by the DSMB, then the patient or the trial will continue. 
 
 
 


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6.8. 
Description of Assessments 
6.8.1. Histological Assessments 
All possible attempts should be made to acquire a liver biopsy specimen of at least 2.0 cm in 
length, from a 16 gauge needle to ensure accurate staging of fibrosis and other histological 
lesions.  A historical biopsy within 6 months of the Screening visit may be accepted as the 
Screening biopsy. The liver biopsy sample must be deemed adequate for evaluation by the 
pathologist for inclusion. Liver biopsies will be assessed by a local reader.  This assessment will 
include an assessment of the adequacy of the specimen as well as the fibrosis stage and a 
determination that the biopsy is consistent with NASH. If either the historical or screening liver 
biopsy is deemed unacceptable, it may be repeated. 
6.8.2. Assessment of newly synthesized proteins 
Kinetic biomarkers will be analyzed to evaluate the pharmacodynamic effect of NTZ on 
fibrogenesis. Blood samples analyses will be performed after deuterated water intake before 
and after 24 weeks of treatment. The assessment will involve the analysis of the change 
(absolute and relative) from baseline between the post-dose and pre-dose deuterated water 
loading periods. 
6.8.3. Biological Assessments (Table 2) 
Chemistry:  
HbA1c, fasting plasma glucose, insulin, HOMA-IR, fructosamine, alkaline phosphatase, ALT, 
AST, GGT, CPK, total and conjugated bilirubin, ferritin, creatinine, eGFR, uric acid, BUN, total 
cholesterol, HDL-C, LDL-C, triglycerides, albumin, lipase, amylase, electrolytes (sodium, 
potassium, chloride, calcium), MELD 
Hematology:  
Hematocrit (Hct), hemoglobin (Hb), platelet count, red blood cell count (RBC), white blood cell 
count (WBC) with differential (absolute and percentage) including lymphocytes, monocytes, 


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neutrophils, eosinophils, and basophils and, reticulocyte count and mean corpuscular volume 
(MCV).  
Inflammatory markers:  
hsCRP, fibrinogen, haptoglobin, resistin, TNF-α, TGF-β, IL-6, PAI-1 
Coagulation Panel:  
Prothrombin time (PT), and international normalized ratio (INR)  
Pregnancy Tests:  
Urine β-hCG (if positive, requires immediate confirmation with Serum β-hCG)  
Urinary Tests:  
Urinary dipstick analysis, urinary albumin, urinary creatinine, urinary ACR 
Additional Tests:  
Serology (HIV-1, HBsAg, HCVAb & HCV RNA (as needed)), serum cystatin C, troponin T, NT 
proBNP 
Fibrosis Biomarker tests:  
ELFTM test score, PIIINP, Hyaluronic acid, CK18, TIMP-1, YKL-40, Alpha 2 macroglobulin, miRNA, 
FGF19, FGF21, ProC3 and ProC6.  
A laboratory manual will be provided 
The manual will outline the collection and processing requirements for the laboratory. Blood 
sampling will be performed by trained personnel. Blood samples will be processed and shipped 
as outlined in the laboratory manual. Refer to the laboratory manual for exact amounts of 
blood required for each test.  
The option to retest during the study is left to the Investigator’s judgment.  
Additional serum and plasma samples will be maintained for future analysis 
 
 


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Table 2 Biological assessments 
 
Screening period 
 
 
Treatment period 
Visit 
SV 
 
S
V2 
SV3 
S
V
4 
V
1 
 
V
2 
V
3 
V4 
 
V5 
V6 
V7 
V8 
V9 
V10 
 
EOS 
 
ET 
Day 
-42 
-
14 
-11 
-7 
1 
29 
57 
85 
113 
141 
155 
158 
162 
169 
+28 
days 
 
Week 
-6 
-2 
 
 
0 
4 
8 
12 
16 
20 
22 
 
 
24 
28 
 
Labs - 
Haematology 
haemoglobin, 
haematocrit, RBC, 
WBC, differential 
count, platelet 
count, reticulocytes 
count, MCV) 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
 
 
X 
Coagulatio (PT, 
INR) 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Labs- Urinary 
Pregnancy tests 
X 
 
 
 
 
X 
X 
X 
X 
X 
 
 
 
X 
X 
 
X 
Labs – Serology 
HIV, HBs and HCV 
serology 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Labs – 
Biochemistry 
HbA1c, fasting 
plasma glucose, 
insulin, HOMA-IR, 
fructosamine, 
alkaline 
phosphatase, ALT, 
AST, GGT, CPK, 
total and conjugated 
bilirubin, ferritin, 
creatinine, eGFR, 
uric acid, BUN, 
total cholesterol, 
HDL-C, LDL-C, 
triglycerides, 
albumin, lipase, 
amylase, 
electrolytes 
(sodium, potassium, 
chloride, calcium), 
MELD 
X 
 
 
X1 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Inflammatory 
markers 
hsCRP, fibrinogen, 
haptoglobin, 
resistin,  TNF-α, 
TGF-β, IL-6, PAI-1 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 


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Screening period 
 
 
Treatment period 
Visit 
SV 
 
S
V2 
SV3 
S
V
4 
V
1 
 
V
2 
V
3 
V4 
 
V5 
V6 
V7 
V8 
V9 
V10 
 
EOS 
 
ET 
Day 
-42 
-
14 
-11 
-7 
1 
29 
57 
85 
113 
141 
155 
158 
162 
169 
+28 
days 
 
Biomarkers of 
fibrosis 
ELF Score, PIIINP, 
Hyaluronic acid, 
CK18, TIMP-1, 
YKL-40, Alpha 2 
macroglobulin,, 
,miRNA, ProC3, 
ProC6, FGF19, 
FGF21 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
NAFLD Fibrosis 
Score (NAS) & 
FIB-4 
 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
 
X 
Labs – Urinalysis 
dipstick  
Specific gravity, pH, 
protein, glucose, 
ketones, bilirubin, 
urobilinogen, blood, 
nitrite, leukocytes 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
 
 
 
X 
Safety markers 
Serum Cystatin C, 
troponin T, NT 
proBNP 
Urinary albumin, 
urinary creatinine, 
urinary ACR 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Labs -- FSR 
 
X 
X 
X 
X 
 
 
 
 
 
X 
X 
X 
X 
 
 
Sampling for 
additional 
parameters 
X 
 
 
 
X 
 
 
X 
 
X 
 
 
 
X 
X 
X 
 
6.8.4. Electrocardiogram 
Standard 12-lead electrocardiogram (ECG) assessments will be performed. The Investigator 
will review the ECGs for any clinically significant abnormalities to ensure subject safety. 
Abnormal ECG findings that are considered clinically significant by the Investigator and meet 
the definition of an AE should be reported and recorded in the AE eCRF page.  
6.8.5. MRE 


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Liver stiffness will be assessed by MRE. It is recommended that each subject’s radiological 
assessment is performed using the same procedure for each study visit.  Exploratory measures 
for MRI/MRE sequence development will be maintained for future analysis.  
6.8.6. Fibroscan 
Liver stiffness will be assessed by FibroScan®. It is required that each subject’s FibroScan® 
assessments be done with the same type of probe at each study visit.  
6.8.7. Medical History 
Medical history including details regarding illnesses and allergies, date(s) of onset, and 
whether condition(s) is currently ongoing, and medication history, including nicotine and 
alcohol use, will be collected on all subjects during screening. 
6.8.8. Physical Examination 
A complete physical examination should include source documentation of general appearance, 
and the following body systems: head, neck, and thyroid; eyes, ears, nose, throat, mouth, and 
tongue; chest (excluding breasts); respiratory; cardiovascular; lymph nodes; abdomen; skin, 
hair, nails; musculoskeletal and neurological.  
The patient’s weight will be measured under the same conditions at each visit where the 
procedure is required. Where possible, the scale for weight must be the same for a given 
patient throughout the visits. 
6.8.9. Vital Signs 
Blood pressure (mmHg) and pulse rate (beats per minute) will be measured at each visit using 
automated blood pressure equipment. 
Important points for clinical blood pressure measurement  
• 
The patient should be seated comfortably with the back supported and the 
upper arm bare without constrictive clothing. The legs should not be crossed. 
• 
The arm should be supported at heart level, and the bladder of the cuff should 
encircle at least 80% of the arm circumference.  


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• 
Neither the patient nor the observer should talk during the measurement.  
Systolic BP and diastolic BP will be measured after 5 minutes rest in the seating.  Where 
possible, the blood pressure equipment should be the same for a given patient throughout the 
visits. 
6.8.10. Pregnancy Testing 
Urinary pregnancy tests will be done on WOCBP.  
7. Adverse Events and Toxicity Management 
7.1. Definitions of Adverse Events, Serious Adverse Events and Unexpected Events 
7.1.1. Adverse Events 
An adverse event (AE) is any untoward medical occurrence in a clinical study subject 
administered a medicinal product, which does not necessarily have a causal relationship with 
the treatment. An AE can therefore be any unfavorable and/or unintended sign, symptom, or 
disease temporally associated with the use of a 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 specified procedures, overdose, drug abuse/misuse reports, 
or occupational exposure. Preexisting events that increase in severity or change in nature 
during or as a consequence of participation in the clinical study will also be considered AEs.  
An AE does not include the following:  
• 
Medical or surgical procedures such as surgery, endoscopy, tooth extraction, 
and transfusion. The condition that led to the procedure may be an adverse 
event and must be reported  
• 
Pre-existing diseases, conditions, or laboratory abnormalities present or 
detected before the Screening visit that do not worsen  
• 
Situations where an untoward medical occurrence has not occurred (eg., 
hospitalization for elective surgery, social and/or convenience admissions)  
• 
Overdose without clinical sequelae.   


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• 
Any medical condition or clinically significant laboratory abnormality with an 
onset date before the consent form is signed and not related to a protocol-
associated procedure is not an AE. It is considered to be pre-existing and should 
be documented on the medical history eCRF. 
7.1.2.  Clinical Laboratory Abnormalities and Other Abnormal Assessments as Adverse 
Events or Serious Adverse Events 
Laboratory abnormalities without clinical significance are not recorded as AEs or SAEs. 
However, laboratory abnormalities (eg, clinical chemistry, hematology, and urinalysis) that 
require medical or surgical intervention or lead to study drug interruption, modification, or 
discontinuation must be recorded as an AE, as well as an SAE, if applicable. In addition, 
laboratory or other abnormal assessments (eg, electrocardiogram, x-rays, vital signs) that are 
associated with signs and/or symptoms must be recorded as an AE or SAE if they meet the 
definition of an AE or SAE as described above.  If the laboratory abnormality is part of a 
syndrome, record the syndrome or diagnosis (eg, anemia), not the laboratory result (ie, 
decreased hemoglobin). 
7.1.3. Serious Adverse Events 
A serious adverse event (SAE) is defined as an event that, at any dose, results in the following:  
• 
Death  
• 
Life-threatening (Note: The term “life-threatening” in the definition of 
“serious” refers to an event in which the subject was at risk of death at the 
time of the event; it does not refer to an event that hypothetically might 
have caused death if it were more severe.) 
• 
In-patient hospitalization or prolongation of existing hospitalization 
• 
Persistent or significant disability/incapacity  
• 
A congenital anomaly/birth defect  
• 
A medically important event or reaction: such events may not be 
immediately life-threatening or result in death or hospitalization but may 
jeopardize the subject or may require intervention to prevent one of the 


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other outcomes constituting SAEs. Medical and scientific judgment must be 
exercised to determine whether such an event is a reportable under 
expedited reporting rules. 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 
hospitalization; and development of drug dependency or drug abuse. For the 
avoidance of doubt, infections resulting from contaminated medicinal 
product will be considered a medically important event and subject to 
expedited reporting requirements. 
7.1.4. Unexpected adverse event  
Expectedness is assessed by the Sponsor-Investigator. An unexpected AE is defined as an event 
that has a nature of severity or specificity that is not consistent with the applicable Package 
Insert or that is symptomatically and pathophysiologically related to a known toxicity but 
differs because of a greater severity or specificity.  
“Unexpected” refers to an event that has not been previously observed and reported rather 
than an event that has not been anticipated based on the properties of the drug. 
7.2. 
Assessment of Adverse Events & Serious Adverse Events 
The Investigator will establish whether or not any AE have occurred at each visit from the date 
of consent. The patient will be questioned in a general manner to determine specific 
symptoms without offering the patient any suggestion. The investigator or qualified sub-
investigator is responsible for assessing AEs and SAEs for causality and severity, and for final 
review and confirmation of accuracy of event information and assessments. 
7.2.1.  Intensity assessment  
The intensity of the AE will be graded as follows:  
• 
Mild: Awareness of signs or symptoms, but easily tolerated and are of 
minor irritant type causing no loss of time from normal activities. 


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Symptoms do not require therapy or a medical evaluation; signs and 
symptoms are transient.  
• 
Moderate: Events introduce a low level of inconvenience or concern to 
the participant and may interfere with daily activities, but are usually 
improved by simple therapeutic measures; moderate experiences may 
cause some interference with functioning. 
• 
Severe: Events interrupt the participant’s normal daily activities and 
generally require systemic drug therapy or other treatment; they are 
usually incapacitating. 
7.2.2.  Relation to the study treatment  
The Investigator will make a clinical and scientific judgment regarding whether or not the AE 
was related to study treatment. The Investigator will evaluate any changes in laboratory 
values, make a determination as to whether or not the change is clinically important, and 
whether or not the changes were related to study drug. However, even if the Investigator feels 
there is no relationship to the study drug, the AE or clinically significant laboratory abnormality 
must be recorded in the eCRF.  
The Investigator will record the relation to the study treatment according to the following 
causality terms:  
• 
Related: the AE follows a reasonable temporal sequence from the time 
of drug administration and it cannot be explained by the patient's 
clinical state or the study procedures/conditions. The AE abates upon 
discontinuation of the study drug and reappears when the study drug is 
introduced.  
• 
Possibly related: the AE follows a reasonable temporal sequence from 
the time of drug administration, but could have been produced by the 
patient's clinical state or the study procedures/conditions.  


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• 
Unlikely related: the temporal association between the AE and the study 
drug is such that the study drug is not likely to have any reasonable 
association with the AE. The relationship is not likely because of other 
plausible explanations.  
• 
Not related: the AE must definitely be caused by the patient's clinical 
state or the study procedure/conditions. A reasonable explanation must 
be given, e.g., no investigational product taken, preplanned elective 
medical intervention, or incompatible temporal relationship.  
• 
Not assessable: the report suggesting an adverse reaction cannot be 
judged because information is insufficient or contradictory and data 
cannot be supplemented or verified. 
7.2.3. 
 Action taken and outcome  
The Investigator will record the action taken with drug and outcome of the event for each AE 
according to the following:  
Action taken with investigational drug  
• 
Drug permanently withdrawn – in case a patient is permanently 
withdrawn from the study drug  
• 
Drug temporarily withdrawn – in case the study drug is temporarily 
withdrawn  
• 
Dose not changed – in case no action is taken regarding the study drug  
• 
Unknown  
• 
Not applicable – an AE started before initiation of treatment with study 
drug, the treatment had been completed prior to reaction/event, or the 
patient has died. 
Outcome  
• 
Recovered/resolved  


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• 
Recovering/resolving  
• 
Not recovered/not resolved  
• 
Recovered/resolved with sequelae  
• 
Fatal  
• 
Unknown.  
Note: In case of irreversible congenital anomalies the choice not recovered/not resolved 
should be used. “Fatal” should be used when death is possibly related to the reaction/event. 
7.3. Reporting 
7.3.1.  Reporting an adverse event  
All AEs regardless of seriousness or relationship to study drug, including those occurring during 
the Screening Period, are to be recorded on the corresponding page(s) of the eCRF and in the 
patient’s medical record from the ICF signature until study end for each patient. Whenever 
possible, symptoms should be grouped as a single syndrome or diagnosis. The Investigator 
should specify the date of onset, maximal intensity, action taken with respect to study drug, 
corrective therapy given, outcome, and his/her opinion as to whether there is a reasonable 
possibility that the AE was caused by the study drug.  
Adverse event reporting begins from signature of the patient ICF at the first Screening Visit and 
ends at study end for each patient. 
7.3.2. Initial reports 
All SAEs occurring from the time of informed consent until 30 days following the 
last administration of study drug must be reported to Medpace Clinical Safety 
within 24 hours of the knowledge of the occurrence (this refers to any adverse 
event that meets any of the aforementioned serious criteria). All SAEs that the 
Investigator considers related to study drug occurring after the 30-day follow-up 
period must be reported to the Sponsor. 
To report the SAE, complete the SAE form electronically in the electronic data 
capture (EDC) system for the study. When the form is completed, Medpace Safety 


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personnel will be notified electronically and will retrieve the form. If the event 
meets serious criteria and it is not possible to access the EDC system, send an 
email to Medpace Safety at medpace safetynotification@medpace.com or call the 
Medpace SAE hotline (phone number listed below), and fax the completed paper 
SAE form to Medpace (fax number listed below) within 24 hours of awareness. 
When the EDC system becomes available, the SAE information must be entered 
within 24 hours of the system becoming available.  
7.3.3. Follow-up reports 
The Investigator must continue to follow the patient until the SAE has subsided or 
until the condition becomes chronic in nature, stabilizes (in the case of persistent 
impairment), or the patient dies. 
Within 24 hours of receipt of follow-up information, the Investigator must update 
the SAE form electronically in the EDC system for the study and submit any 
supporting documentation (eg, patient discharge summary or autopsy reports) to 
Medpace Clinical Safety via fax or e-mail. If it is not possible to access the EDC 
system, refer to the procedures outlined above for initial reporting of SAEs. 
All SAEs independent of the circumstances or suspected cause must be reported on a SAE 
Form/captured in EDC. The SAE Form should include a clearly written narrative describing 
signs, symptoms, and treatment of the event, diagnostic procedures, as well as any relevant 
laboratory data and any sequelae, in order to allow a complete medical assessment of the case 
and independent determination of the possible causality.  
It is critical that the information provided on the initial or follow-up SAE Form matches the 
information recorded in the source documents and the eCRF for the same event.  
Any unexpected safety issue that changes the risk benefit analysis and is likely to have an 
impact on the patients who have participated in the trial will be reported as soon as possible 
to the Competent Authority(ies) concerned together with proposed actions. 
7.3.4. Follow-up  


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The Investigator should take all appropriate measures to ensure the safety of the patients, 
notably he/she should follow up the outcome of any AE until the return to normal or until 
stabilization of the patient’s condition.  
The patient must be followed up until clinical recovery is complete and laboratory results have 
returned to normal, or until progression has been stabilized. This information should be 
documented in the patient’s medical records.  
7.4. Post Study Reporting Requirements 
Any SAEs and deaths that occur within 30 days of the last dose of the study drug, regardless of 
causality, should be reported.  
Any SAE that is brought to the attention of the Investigator at any time after the reporting 
period and which is considered by him/her to be caused by the study drug within a reasonable 
possibility, should be reported.  
7.5. Special Situation Reports 
Special situations reports include pregnancy reports, reports of medication error, abuse, 
misuse or overdose, and reports associated with product complaints.  
7.5.1. Pregnancy  
In case of pregnancy, the Investigator will notify regulatory authorities and IRB within 24 hours 
of his/her knowledge of the pregnancy.  
Female patients must be instructed to discontinue the study drug immediately and inform the 
Investigator as soon as possible once they are aware of being pregnant or suspect that they 
are pregnant during the study or within 30 days of the last dose of the study drug.  
Female patients will be requested, as part of the general ICF, to provide informed consent to 
allow reasonable attempts to be made to obtain information on any possible medicinal 
product exposure to an embryo or fetus and to follow up on the outcome of the pregnancy.  
The Investigator will contact the patient at the expected time of delivery for follow-up.  
7.5.2. Medication error  


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Medication error is defined as an unintentional error in the prescribing, dispensing, or 
administration of a medicinal product while in the control of the healthcare professional, 
patient, or consumer. All medication errors will be documented in the eCRF and, in case of any 
potential risk to patient safety, would be reported as appropriate.  
7.5.3. Misuse  
This refers to situations where the medicinal product is intentionally and inappropriately used 
not in accordance with the authorized product information and will be reported in the eCRF. 
All misuse will be documented in the eCRF and, in case of any potential risk to patient safety, 
would be reported as appropriate. 
7.5.4. 
 Overdose  
This refers to the administration of a quantity of a medicinal product given per administration 
or cumulatively, which is above the maximum recommended dose according to the authorized 
product information. Clinical judgment should always be applied. Doses >4000 mg in one day 
are considered overdose. 
7.5.5. 
 Abuse  
This corresponds to the persistent or sporadic, intentional excessive use of a medicinal 
product, which is accompanied by harmful physical or psychological effects. 
8. 
Statistical Considerations 
This section is an overview of the principal elements of the statistical analysis for this study. 
Further details will be contained in a separate statistical analysis plan (SAP). 
8.1. 
Analysis Endpoints 
8.1.1. Primary Endpoint 
The primary endpoint is to assess the safety and tolerability of NTZ after 24 weeks of treatment 
8.1.2. Secondary Endpoints 
The secondary endpoints are: 


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• To assess the % change in FSR from baseline to end of treatment (24 weeks of 
treatment) 
• To assess the change in liver stiffness by MRE from baseline to 12 weeks and 24 weeks 
of treatment 
• To assess the change in liver stiffness by fibroscan from baseline to 24 weeks of 
treatment  
• To assess the change from baseline to 12 weeks and 24 weeks of treatment of fibrosis 
serum biomarkers and scores  
8.2. 
Analysis Sets 
The Full Analysis Set will consist of all subjects that meet the eligibility criteria and enroll into 
the study (Visit 1 Day 1). 
The Safety Set will consist of all enrolled subjects who receive at least one dose of study drug.  
8.3. 
Data Handling Conventions 
Continuous variables will be summarized with descriptive statistics (i.e., sample size, mean, 
standard deviation, minimum, maximum, median, and quintiles). Categorical variables will be 
summarized with descriptive statistics (i.e., sample size, frequency counts, and percentage). 
Statistical testing on change from baseline values will be performed using analysis of variance 
techniques with a term for baseline in the model. Statistical significance will be considered to 
have been reached at an alpha level of 0.05. 
Baseline value is measured on Day 1 of the treatment period before the administration of NTZ. 
8.4. 
Demographic Data and Baseline Characteristics 
Demographic and baseline characteristics will be descriptively summarized. Quantitative 
and/or categorical summaries will be presented for demographics and other baseline 
characteristics. Demographic and baseline characteristics will be summarized using the Full 
Analysis Set. 
8.5. 
Primary Safety Analysis 
Safety and tolerability will be assessed by the following parameters:  


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AEs including TEAEs and SAEs, laboratory evaluations, vital signs, ECGs, and physical 
examinations.  
8.5.1. Adverse Events 
Treatment-emergent adverse events (TEAEs) are defined as AEs that worsen or commence on 
or after the time of start of first study drug administration. AEs, including SAEs, will be coded 
using MedDRA. The number and percentage of subjects experiencing TEAE, including TEAEs 
leading to study drug discontinuation and SAEs, will be summarized for each system organ 
class (SOC) and Preferred Term. In addition, AEs will be tabulated according to severity, 
causality, and relation to the study drug.  
Individual data listings for AEs will be presented for each subject.  
8.5.2. Laboratory Evaluations 
Absolute and change from baseline values for laboratory parameters will be summarized by 
visit. In addition, shift from baseline tables will be presented for selected laboratory 
abnormalities.  
8.5.3. Other Safety Evaluations 
Observed values and change from baseline will be summarized descriptively for all vital sign 
parameters and ECG parameters by visit. Individual listings will be presented for each subject. 
Individual data listings for physical examination results will be presented for each subject. 
Safety data will be summarized using descriptive statistics. Further details will be provided in 
the SAP. 
 All safety analyses will be conducted using the Safety Population. 
8.6. 
Secondary Efficacy Analyses 
8.6.1. FSR 
Fractional Synthesis Rate of circulating plasma proteins will be measured at Baseline, and after 
24 weeks of treatment.  


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The change in FSR (absolute and relative) from baseline to end of treatment between the first 
and second deuterated water loading periods will be assessed to determine the effects of NTZ 
500mg BID on de novo collagen synthesis in the Full Analysis Set.  
Wilcoxon signed-rank test will be used for statistical inference. Estimated mean, standard error 
and 95% confidence interval of change in FSR (absolute and relative) change from baseline in 
FSR will be reported and evaluated at a significance level of 0.05. Exploratory analyses may 
also be performed to evaluate the association of individual exploratory biomarkers or 
combination of biomarkers with clinical measurements and other risk factors. 
Additional details will be provided in the SAP. 
8.6.2. Liver stiffness 
The change in liver stiffness from baseline at 24 weeks as measured by FibroScan, and at 12 
weeks and 24 weeks by MRE, the change in non-invasive markers of fibrosis from baseline to 
24 weeks, and the change in fibrosis scores from baseline to 12 weeks, to 24 weeks for the Full 
Analysis Set will be assessed. Summary statistics for the Full Analysis Set will be provided at all 
visits and the change from baseline will be provided. 
8.6.3. Exploratory Analysis 
Additional serum and plasma samples will be maintained for future analysis.  Exploratory 
measures for MRI/MRE sequence development will be maintained for future analysis.  
8.6.4. Interim Analysis 
 An interim analysis is not planned. 
8.7. 
Sample Size 
The sample size of this Phase 2 proof of concept study is based on clinical assessment. Twenty 
male and female subjects with histologically confirmed NASH and fibrosis stage 2 or 3) will 
provide sufficient information to assess the safety and tolerability of NTZ 500mg BID over 24 
weeks. 
9. 
Responsibilities 


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9.1. 
Investigator Responsibilities 
By signing this protocol, the Investigator agrees to conduct the study in an efficient and 
diligent manner and in conformance with this protocol; generally accepted standards of GCP; 
and all applicable local laws, rules and regulations relating to the conduct of the clinical study.  
The Investigator also agrees to allow monitoring, audits, IRB review and regulatory agency 
inspection of trial-related documents and procedures and provide for direct access to all study 
related source data and documents.  
The Investigator shall prepare and maintain complete and accurate study documentation in 
compliance with GCP standards and applicable local laws, rules and regulations; and, for each 
subject participating in the study, provide all data, and upon completion or termination of the 
clinical study submit any other reports to the Funder as required by this protocol or as 
otherwise required pursuant to any agreement with the Funder.  
Study documentation will be promptly and fully disclosed to regulatory authorities by the 
Investigator upon request and also shall be made available at the Investigator’s site upon 
request for inspection, copying, review and audit at reasonable times by representatives any 
regulatory agencies.   
Persons debarred from conducting or working on clinical studies by any court or regulatory 
agency will not be allowed to conduct or work on this study.   
9.2. 
Good Clinical Practice 
The investigator will ensure that this study is conducted in accordance with the principles of 
the Declaration of Helsinki (as amended in Edinburgh, Tokyo, Venice, Hong Kong, and South 
Africa), International Conference on Harmonisation (ICH) guidelines.  These standards are 
consistent with the European Union Clinical Trials Directive 2001/20/EC and Good Clinical 
Practice Directive 2005/28/EC. The investigator will ensure adherence to the basic principles of 
Good Clinical Practice, as outlined in 21CFR312, subpart D, “Responsibilities of Sponsors and 
Investigators,” 21CFR, part 50, 1998, and 21CFR, part56, 1998. The investigator and all 
applicable sub investigators will comply with 21CFR, Part54, 1998, providing documentation of 


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their financial interest or arrangements with any study funder, or proprietary interests in the 
investigational drug under study. This documentation must be provided prior to the 
investigator’s (and any sub investigator’s) participation in the study. The investigator and sub 
investigator agree to notify study funder of any change in reportable interests during the study 
and for 1 year following completion of the study. Study completion is defined as the date 
when the last subject completes the protocol-defined activities 
9.3. 
Institutional Review Board Review & Approval 
The investigator will submit this protocol, informed consent form, and any accompanying 
material to be provided to the subject (such as advertisements, subject information sheets, or 
descriptions of the study used to obtain informed consent) to an IRB/IEC.  The investigator will 
not begin any study subject activities until approval from the IRB/IEC has been documented 
and provided as a letter to the investigator. Before implementation, the investigator will 
submit to and receive documented approval from the IRB/IEC any modifications made to the 
protocol or any accompanying material to be provided to the subject after initial approval, 
with the exception of those necessary to reduce immediate risk to study subjects. 
9.4. 
Informed Consent 
The investigator is responsible for obtaining written informed consent from each individual 
participating in this study after adequate explanation of the aims, methods, objectives, and 
potential hazards of the study and before undertaking any study-related procedures. The 
investigator must use the most current IRB/IEC approved consent form for documenting 
written informed consent. Each informed consent will be appropriately signed and dated by 
the subject and the person conducting the consent discussion, and also by an impartial witness 
if required by local requirements. 
9.5. 
Confidentiality 
The investigator will assure that subjects’ anonymity will be strictly maintained and that their 
identities are protected from unauthorized parties. Only subject initials, date of birth, another 
unique identifier (as allowed by local law) and an identification code will be recorded on any 
form or biological sample submitted to the study funder, IRB/IEC or laboratory. Laboratory 


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specimens will be labeled in such a way as to protect subject identity while allowing the results 
to be recorded to the proper subject. Refer to specific laboratory instructions for further 
details. NOTE: The investigator will keep a screening log showing codes, names, and addresses 
for all subjects screened and for all subjects enrolled in the trial. Subject data will be processed 
in accordance with all applicable regulations. The investigator agrees that all information 
received from the study funder, including but not limited to the Package Insert, the IMP, and 
any other study information, remain the sole and exclusive property of the study funder during 
the conduct of the study and thereafter. This information is not to be disclosed to any third 
party (except employees or agents directly involved in the conduct of the study or as required 
by law) without prior written consent from the study funder.  The investigator further agrees 
to take all reasonable precautions to prevent the disclosure by any employee or agent of the 
study site to any third party or otherwise into the public domain. 
9.6. 
Study Files and Retention of Records 
The investigator will maintain adequate and accurate records to enable the conduct of the 
study to be fully documented and the study data to be subsequently verified. These 
documents should be classified into at least the following two categories: (1) investigator’s 
study master file, and (2) subject clinical source documents. The investigator’s study file will 
contain the protocol/amendments, CRF and query forms, IRB/IEC and governmental approval 
with correspondence, informed consent, drug records, staff curriculum vitae and authorization 
forms, and other appropriate documents and correspondence. The required source data 
should include sequential notes containing at least the following information for each subject:  
• 
Subject identification (name, date of birth, gender);  
• 
Documentation that subject meets eligibility criteria, i.e., history, physical 
examination, and confirmation of diagnosis (to support inclusion and exclusion 
criteria); 
• 
Documentation of the reason(s) a consented subject is not enrolled  
• 
Participation in study (including study number);  


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• 
Study discussed and date of informed consent;  
• 
Dates of all visits;  
• 
Documentation that protocol specific procedures were performed;  
• 
Results of efficacy parameters, as required by the protocol;  
• 
Start and end date (including dose regimen) of IMP, including dates of 
dispensing and return;  
• 
Record of all adverse events and other safety parameters (start and end date, 
and including causality and severity);  
• 
Concomitant medication (including start and end date, dose if relevant; dose 
changes); 
• 
Date of study completion and reason for early discontinuation, if it occurs.  
All clinical study documents must be retained by the investigator until at least 2years or 
according to local laws, whichever is longer, after the last approval of a marketing application 
in an ICH region (i.e., United States, Europe, or Japan) and until there are no pending or 
planned marketing applications in an ICH region; or, if no application is filed or if the 
application is not approved for such indication, until 2years after the investigation is 
discontinued and regulatory authorities have been notified. Investigators may be required to 
retain documents longer if specified by regulatory requirements, by local regulations, or by an 
agreement with study funder. The investigator must notify study funder before destroying any 
clinical study records. Should the investigator wish to assign the study records to another party 
or move them to another location, study funder must be notified in advance. If the 
investigator cannot provide for this archiving requirement at the study site for any or all of the 
documents, special arrangements must be made between the investigator and study funder to 
store these records securely away from the site so that they can be returned sealed to the 
investigator in case of an inspection. When source documents are required for the continued 
care of the subject, appropriate copies should be made for storage away from the site. 
9.7. 
Electronic Case Report Forms 


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For each subject consented, an eCRF will be completed by an authorized study staff member 
whose training for this function is documented according to study procedures. The eCRF will 
be completed in a timely manner. Original entries as well as any changes to data fields will be 
stored in the audit trail of the system. At the conclusion of the trial, a read-only archive copy of 
the data entered will be generated. This archive will be stored in accordance with the records 
retention requirements. 
9.8. 
Investigational Medicinal Product Accountability and Return 
The investigator or designee is responsible for ensuring adequate accountability of all used and 
unused study drug.  This includes acknowledgment of receipt of each shipment of study 
product (quantity and condition), subject dispensing records, and returned or destroyed study 
product. Dispensing records will document quantities received from study funder and 
quantities dispensed to subjects, including lot number, date dispensed, subject identifier 
number, subject initials, and the initials of the person dispensing the medication. Drug may be 
returned or destroyed on an ongoing basis during the study, if appropriate.  At the end of the 
study, following final drug inventory reconciliation, the study site will arrange for the return of 
unused investigational medicinal product supplies. All drug supplies and associated 
documentation will be periodically reviewed and verified by the study funder over the course 
of the study. 
9.9. 
Inspections 
The investigator understands that source documents for this trial should be made available to 
appropriately qualified personnel from study funder and its representatives, to IRBs/IECs, or to 
regulatory authority or health authority inspectors. 
9.10. Protocol Compliance 
The investigator is responsible for ensuring the study is conducted in accordance with the 
procedures and evaluations described in this protocol. 
9.11. Protocol Modifications 


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Protocol modifications, except those intended to reduce immediate risk to study subjects, may 
be made by the investigator in collaboration with the study funder. All protocol modifications 
must be submitted to the IRB/IEC in accordance with local requirements. Approval must be 
obtained before changes can be implemented. 
9.12. Study Reports 
A clinical study report (CSR) will be prepared and provided to the regulatory agency (ies). The 
Investigator will ensure that the report meets the standards set out in the ICH Guideline for 
Structure and Content of Clinical Study Reports (ICHE3). Note that an abbreviated report may 
be prepared in certain cases. After conclusion of the study, Investigators in this study may 
communicate, orally present, or publish in scientific journals or other scholarly media only 
after the following conditions have been met:  
• 
The results of the study in their entirety have been publicly disclosed by or with 
the consent of the study funder in an abstract, manuscript, or presentation form. 
9.13. Study Termination 
The Investigator reserves the right to terminate the study at any time. Should this be 
necessary, the investigator will arrange discontinuation procedures and notify the appropriate 
regulatory authorities, IRBs, IECs, and the Funder. In terminating the study, the investigator 
will assure that adequate consideration is given to the protection of the subjects’ interests. 


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10. References 
 
10.1. 
Literature  
Abd-Elsalam, S., Kobtan, A., El-Kalla, F., Elkhalawany, W., Nawasany, S.E., Saif, S.A., Yousef, M., 
Ali, L.A., Soliman, S., Mansour, L., et al. (2016). A 2-week Nitazoxanide-based quadruple 
treatment as a rescue therapy for Helicobacter pylori eradication: A single center experience. 
Medicine (Baltimore) 95, e3879. 
 
Alinia (nitazoxanide): US Prescribing Information. Tampa, FL. Romark, L.C., 2016 
 
Amorosa, V.K., Luetkemeyer, A., Kang, M., Johnson, V.A., Umbleja, T., Haas, D.W., Yesmin, S., 
Bardin, M.C., Chung, R.T., Alston-Smith, B., et al. (2013). Addition of nitazoxanide to PEG-IFN 
and ribavirin to improve HCV treatment response in HIV-1 and HCV genotype 1 coinfected 
persons naive to HCV therapy: results of the ACTG A5269 trial. HIV Clin Trials 14, 274-283. 
 
Assy, N., Kaita, K., Mymin, D., Levy, C., Rosser, B., and Minuk, G. (2000). Fatty infiltration of 
liver in hyperlipidemic patients. Dig Dis Sci 45, 1929-1934. 
 
Bacon, B.R., Shiffman, M.L., Lim, J.K., Berman, A., Rustgi, V.K., and Keeffe, E.B. (2010). A Phase 
II, Randomized, Double-Blind, Placebo-Controlled Study of Nitazoxanide Plus Peginterferon 
and Ribavirin in Naive Patients With Chronic Hepatitis C Genotype 1 Infection: Final Report. 
Gastroenterology 139, e18. 
 
Basu, P.P., Rayapudi, K., Shah, N.J., Krishnaswamy, N., and Brown, R.S. (2013). Effects of triple-
drug therapy with nitazoxanide, high-dose ribavirin and peginterferon-alpha-2a in patients 
with chronic hepatitis C. Hepatol Res 43, 589-595. 
 
Basu PP, Rayapudi K, Esteves J, Brown R. A pilot study utilizing nitazoxanide for hepatic 
encephalopathy in chronic liver failure. Abstract S151 (392) for the 59th Annual Meeting of the 
American Association for the Study of Liver Diseases. 31 Oct-4 Nov. 2008. p1742 
 
Belanger, C., Foucart, C., Negro, E., Dubernet, M., Hum, D.W., Staels, B., and Walczak, R. 
(2017). Drug repurposing screen identifies novel small molecule compounds with potent 
antifibrotic properties. Journal of Hepatology 66, S605. 
 
Carpino, G., Morini, S., Ginanni Corradini, S., Franchitto, A., Merli, M., Siciliano, M., Gentili, F., 
Onetti Muda, A., Berloco, P., Rossi, M., et al. (2005). Alpha-SMA expression in hepatic stellate 
cells and quantitative analysis of hepatic fibrosis in cirrhosis and in recurrent chronic hepatitis 
after liver transplantation. Dig Liver Dis 37, 349-356. 
 
Decaris, M.L., Li, K.W., Emson, C.L., Gatmaitan, M., Liu, S., Wang, Y., Nyangau, E., Colangelo, 
M., Angel, T.E., Beysen, C., et al. (2017). Identifying nonalcoholic fatty liver disease patients 


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with active fibrosis by measuring extracellular matrix remodeling rates in tissue and blood. 
Hepatology 65, 78-88. 
 
Donati, G., Stagni, B., Piscaglia, F., Venturoli, N., Morselli-Labate, A.M., Rasciti, L., and Bolondi, 
L. (2004). Increased prevalence of fatty liver in arterial hypertensive patients with normal liver 
enzymes: role of insulin resistance. Gut 53, 1020-1023. 
 
Friedman, S.L. (2008). Hepatic stellate cells: protean, multifunctional, and enigmatic cells of 
the liver. Physiol Rev 88, 125-172. 
 
Fruci, B., Giuliano, S., Mazza, A., Malaguarnera, R., and Belfiore, A. (2013). Nonalcoholic Fatty 
liver: a possible new target for type 2 diabetes prevention and treatment. Int J Mol Sci 14, 
22933-22966. 
 
Kabil, S.M., Soliman, M.S., Youssef, S.M., and Mounier, B.I. (2011). A controlled study of 
nitazoxanide (NTZ) 3 years after treatment of hepatitis C genotype 4. J Egypt Soc Parasitol 41, 
251-261. 
 
Kohla, M.A., El-Said, H., El-Fert, A., Ehsan, N., Ezzat, S., and Taha, H. (2016). Impact of 
nitazoxanide on sustained virologic response in Egyptian patients with chronic hepatitis C 
genotype 4: a double-blind placebo-controlled trial. Eur J Gastroenterol Hepatol 28, 42-47. 
 
Konerman, M.A., Jones, J.C., and Harrison, S.A. (2018). Pharmacotherapy for NASH: Current 
and emerging. J Hepatol 68, 362-375. 
 
Laufer, N., Abusamra, L., Bolcic, F., Gun, A., Rolon, M.J., Perez, H., Krolewiecki, A., Salomon, H., 
Quarleri, J., and Cahn, P. (2011). No reduction of HCV viral load in HIV patients co-infected with 
HCV genotype 1 during a 30 days course of nitazoxanide monotherapy. Antiviral Res 92, 497-
499. 
 
Leite, N.C., Salles, G.F., Araujo, A.L., Villela-Nogueira, C.A., and Cardoso, C.R. (2009). Prevalence 
and associated factors of non-alcoholic fatty liver disease in patients with type-2 diabetes 
mellitus. Liver Int 29, 113-119. 
 
Macias, J., Lopez-Cortes, L.F., Tellez, F., Recio, E., Ojeda-Burgos, G., Rios, M.J., Rivero-Juarez, 
A., Delgado, M., Rivas, J., and Pineda, J.A. (2015). Low Efficacy of Pegylated Interferon plus 
Ribavirin plus Nitazoxanide for HCV Genotype 4 and HIV Coinfection. PLoS One 10, e0143492. 
 
Musher, D.M., Logan, N., Bressler, A.M., Johnson, D.P., and Rossignol, J.F. (2009). Nitazoxanide 
versus vancomycin in Clostridium difficile infection: a randomized, double-blind study. Clin 
Infect Dis 48, e41-46. 
 


Protocol No: NTZ-218-1 
Version: 15 APRIL 2020 
 
 
66 
Rossignol, J.F. (2006). Nitazoxanide in the treatment of acquired immune deficiency syndrome-
related cryptosporidiosis: results of the United States compassionate use program in 365 
patients. Aliment Pharmacol Ther 24, 887-894. 
 
Rossignol, J.F. (2014). Nitazoxanide: a first-in-class broad-spectrum antiviral agent. Antiviral 
Res 110, 94-103. 
 
Rossignol, J.F., Elfert, A., El-Gohary, Y., and Keeffe, E.B. (2009). Improved virologic response in 
chronic hepatitis C genotype 4 treated with nitazoxanide, peginterferon, and ribavirin. 
Gastroenterology 136, 856-862. 
 
Rossignol, J.F., Elfert, A., and Keeffe, E.B. (2010). Treatment of chronic hepatitis C using a 4-
week lead-in with nitazoxanide before peginterferon plus nitazoxanide. J Clin Gastroenterol 
44, 504-509. 
 
Rossignol, J.F., Hidalgo, H., Feregrino, M., Higuera, F., Gomez, W.H., Romero, J.L., Padierna, J., 
Geyne, A., and Ayers, M.S. (1998). A double-'blind' placebo-controlled study of nitazoxanide in 
the treatment of cryptosporidial diarrhoea in AIDS patients in Mexico. Trans R Soc Trop Med 
Hyg 92, 663-666. 
 
Rossignol, J.F., Kabil, S.M., El-Gohary, Y., Elfert, A., and Keeffe, E.B. (2008). Clinical trial: 
randomized, double-blind, placebo-controlled study of nitazoxanide monotherapy for the 
treatment of patients with chronic hepatitis C genotype 4. Aliment Pharmacol Ther 28, 574-
580. 
 
Sasaki, A., Nitta, H., Otsuka, K., Umemura, A., Baba, S., Obuchi, T., and Wakabayashi, G. (2014). 
Bariatric surgery and non-alcoholic Fatty liver disease: current and potential future treatments. 
Front Endocrinol (Lausanne) 5, 164. 
 
Shehab, H.M., Elbaz, T.M., and Deraz, D.M. (2014). Nitazoxanide plus pegylated interferon and 
ribavirin in the treatment of genotype 4 chronic hepatitis C, a randomized controlled trial. Liver 
Int 34, 259-265. 
 
Shehata, M.A., Talaat, R., Soliman, S., Elmesseri, H., and Abd-Elsalam, S. (2017). Randomized 
controlled study of a novel triple nitazoxanide (NTZ)-containing therapeutic regimen versus the 
traditional regimen for eradication of Helicobacter pylori infection. Helicobacter 22. 
 
Shiffman, M.L., Ahmed, A., Jacobson, I.M., Pruitt, R.E., and Keeffe, E.B. (2010). 1188  Phase 2 
randomized, double-blind, placebo-controlled study of nitazoxanide with peginterferon alfa-2a 
and ribavirin in nonresponders (NR) with chronic hepatitis C genotype 1: final report. Journal 
of Hepatology 52, S461. 
 


Protocol No: NTZ-218-1 
Version: 15 APRIL 2020 
 
 
67 
Stockis, A., Allemon, A.M., De Bruyn, S., and Gengler, C. (2002a). Nitazoxanide 
pharmacokinetics and tolerability in man using single ascending oral doses. Int J Clin 
Pharmacol Ther 40, 213-220. 
 
Stockis, A., De Bruyn, S., Gengler, C., and Rosillon, D. (2002b). Nitazoxanide pharmacokinetics 
and tolerability in man during 7 days dosing with 0.5 g and 1 g b.i.d. Int J Clin Pharmacol Ther 
40, 221-227. 
 
Subichin, M., Clanton, J., Makuszewski, M., Bohon, A., Zografakis, J.G., and Dan, A. (2015). Liver 
disease in the morbidly obese: a review of 1000 consecutive patients undergoing weight loss 
surgery. Surg Obes Relat Dis 11, 137-141. 
 
Takeuchi-Yorimoto, A., Noto, T., Yamada, A., Miyamae, Y., Oishi, Y., and Matsumoto, M. 
(2013). Persistent fibrosis in the liver of choline-deficient and iron-supplemented L-amino acid-
defined diet-induced nonalcoholic steatohepatitis rat due to continuing oxidative stress after 
choline supplementation. Toxicol Appl Pharmacol 268, 264-277. 
 
Tanwar, S., Trembling, P.M., Guha, I.N., Parkes, J., Kaye, P., Burt, A.D., Ryder, S.D., Aithal, G.P., 
Day, C.P., and Rosenberg, W.M. (2013). Validation of terminal peptide of procollagen III for the 
detection and assessment of nonalcoholic steatohepatitis in patients with nonalcoholic fatty 
liver disease. Hepatology 57, 103-111. 
 
Taubel, J., Lorch, U., Rossignol, J.F., Ferber, G., and Camm, A.J. (2014). Analyzing the 
relationship of QT interval and exposure to nitazoxanide, a prospective candidate for influenza 
antiviral therapy--A formal TQT study. J Clin Pharmacol 54, 987-994. 
 
Wu, K.T., Kuo, P.L., Su, S.B., Chen, Y.Y., Yeh, M.L., Huang, C.I., Yang, J.F., Lin, C.I., Hsieh, M.H., 
Hsieh, M.Y., et al. (2016). Nonalcoholic fatty liver disease severity is associated with the ratios 
of total cholesterol and triglycerides to high-density lipoprotein cholesterol. J Clin Lipidol 10, 
420-425 e421. 
 
Zulu, I., Kelly, P., Njobvu, L., Sianongo, S., Kaonga, K., McDonald, V., Farthing, M., and Pollok, R. 
(2005). Nitazoxanide for persistent diarrhoea in Zambian acquired immune deficiency 
syndrome patients: a randomized-controlled trial. Aliment Pharmacol Ther 21, 757-763. 
 
10.2. 
WHO Fact Sheet 312 
World Health Organization. Media Centre- Diabetes. 
http://www.who.int/mediacentre/factsheets/fs312/en/  
 
10.3. 
Studies on ClinicalTrials.gov 


Protocol No: NTZ-218-1 
Version: 15 APRIL 2020 
 
 
68 
NCT01185028. A safety and tolerability study of nitazoxanide in HIV-HCV treatment failures. 
Last updated: March 21, 2018  
https://clinicaltrials.gov/ct2/show/results/NCT01185028?sect=X430156#othr 
NCT01770483. The role of nitazoxanide, interferon alfa and ribavirin in treatment of hepatitis C 
infected Type 2 diabetic patients (HEP-C-FM). Last updated: September 20, 2013 
https://clinicaltrials.gov/ct2/show/results/NCT01770483 
 
 


CRIO Envelope ID: 1119063 - Page: 1/2

Prot_002.pdf: 
Protocol 
 
A Monocentric, Open-Label, Proof of Concept Study to Evaluate the 
Safety and Efficacy of NTZ at 500mg Twice Daily on Collagen Turnover 
in Plasma in NASH Patients with Fibrosis Stage 2 or 3. 
 
 
NCT Number: 03656068 
Unique Protocol ID: NTZ-218-1 
Version Date : 02 March 2019 









































































































































Prot_004.pdf: 
Protocol 
 
A Monocentric, Open-Label, Proof of Concept Study to Evaluate the 
Safety and Efficacy of NTZ at 500mg Twice Daily on Collagen Turnover 
in Plasma in NASH Patients with Fibrosis Stage 2 or 3. 
 
 
NCT Number: 03656068 
Unique Protocol ID: NTZ-218-1 
Version Date : 26 February 2020 


 
 
Protocol No: NTZ-218-1 
Version: V 26February 2020 
 
 
 
 
 
Page 1 
 
 
 
 
 
 
 
 
 
A Monocentric, Open-Label, Proof of Concept Study to Evaluate the Safety 
and Efficacy of NTZ at 500mg Twice Daily on Collagen Turnover in Plasma in 
NASH Patients with Fibrosis Stage 2 or 3. 
  
 
 
 
Protocol Number: NTZ-218-1 
Version Date: 26 February 2020 
 


 
 
Protocol No: NTZ-218-1 
Version: V 26February 2020 
 
 
 
 
 
Page 2 
List of Abbreviations 
  
ACR 
 
Albumin to Creatinine Ratio 
AE             
Adverse Event  
ALT            
Alanine Aminotransferase/GPT   
AST            
Aspartate aminotransferase  
BMI            
Body Mass Index   
CDAA  
Choline-deficient L-amino Acid Defined Diet 
CFR 
 
Code of Federal Regulations 
CSR 
 
Clinical Study Report 
CPK            
Creatine phosphokinase  
CRF            
Case Report/Record Form  
CRN            
Clinical Research Network  
CTCAE  
Common Terminology Criteria for Adverse Events 
D2O 
 
Deuterated Water 
DILI 
 
Drug Induce Liver Injury 
DSMB  
Data Safety Monitoring Board 
GGT 
 
Gamma-glutamyltransferase 
eCRF           
Electronic case report form  
eGFR            
estimated Glomerular Filtration Rate   
EC            
Ethics Committee   
ECG            
Electrocardiogram  
ET             
Early Termination  
EOS 
 
End Of Study 
EU             
European Union  
FDA            
Food and Drug Administration  
FSR 
 
Fractional Synthesis Rate 
GCP            
Good Clinical Practice  
Hb             
Hemoglobin  
HBV           
Hepatitis B Virus  
HCG 
 
Human Chorionic Gonadotropin 
HCV           
Hepatitis C Virus  
HDPE  
High-density polyethylene 
HDL            
High Density Lipoprotein  
HIV            
Human Immunodeficiency Virus  
HOMA           Homeostasis Model Assessment  
HSC 
 
Hepatic Stellate Cell 
hs-CRP            High Sensitivity C-reactive protein  
ICH           
International Conference on Harmonization  
IEC 
 
Independent Ethics Committee 
IMP            
Investigational medical product 


 
 
Protocol No: NTZ-218-1 
Version: V 26February 2020 
 
 
 
 
 
Page 3 
INR 
 
International Normalized Ratio  
IRB            
Institutional Review Board  
LDL            
Low density lipoprotein 
MedDRA          Medical Dictionary for Regulatory Activity  
MRE 
 
Magnetic Resonance Elastography 
MRI            
Magnetic Resonance Imaging  
NAFLD            Non Alcoholic Fatty Liver Disease  
NAS            
NAFLD Activity Score  
NASH            
Non Alcoholic Steatohepatitis  
NTZ 
 
Nitazoxanide 
PI             
Principal Investigator  
PK             
Pharmacokinetic 
PT/INR            Prothrombin Time/International Normalized Ratio  
 
RNA            
Ribonucleic Acid  
SAE            
Serious Adverse Event 
SAP 
 
Statistical Analysis Plan  
TQT 
 
Thorough-QT study 
  
 
 
 


 
 
Protocol No: NTZ-218-1 
Version: V 26February 2020 
 
 
 
 
 
Page 4 
Study Synopsis 
Protocol Number: 
NTZ-218-1 
Study Title: 
A Monocentric, Open-Label, Proof of Concept Study to Evaluate the Safety 
and Efficacy of NTZ at 500mg Twice Daily on Collagen Turnover in Plasma in 
NASH Patients with Fibrosis Stage 2 or 3. 
Phase: 
Proof of Concept 
Study Drug: 
Nitazoxanide 
Indication: 
NASH induced fibrosis 
Principal Investigator & Sponsor 
Stephen Harrison, MD 
Pinnacle Clinical Research 
5109 Medical Drive, Suite 200 
San Antonio, TX 78229 
Study Duration: 
34 weeks 
Objectives: 
The primary objective of the study is: 
• 
To evaluate the safety and tolerability of NTZ 500mg bid after 24 
weeks of treatment in patients with NASH-induced stage 2 or 
stage 3 fibrosis  
 
 
The secondary objectives of the study are: 
• 
To determine the effect of oral administration of NTZ 500mg bid 
on de novo collagen synthesis through Fractional Synthesis Rate 
(FSR) of circulating plasma proteins in patients with NASH-
induced stage 2 or stage 3 fibrosis. 
• 
To assess changes in liver stiffness as measured by FibroScan® 
and by Magnetic Resonance Elastography (MRE). 
• 
To assess the change from baseline in non-invasive markers of 
fibrosis: ELFTM test score, PIIINP, Hyaluronic acid, CK18, TIMP-1, 
YKL-40, Alpha 2 macroglobulin,, ,miRNA, ProC3, ProC6, FGF19, 
FGF21. 
• 
To assess changes in additional non-invasive tests: fibrosis scores: 
NAFLD fibrosis score, FIB-4 
 
Study Design: 
This is a Phase 2 proof of concept study to evaluate the safety and efficacy 
of open-label NTZ 500mg bid in a NASH population with NASH induced 
Stage 2 or 3 fibrosis. 
 
The study will include a screening visit within 6 weeks of randomization 
(Day 1) followed by a 24-week treatment period consisting of daily, BID, 
oral administration of NTZ and an end of treatment follow up period   of 4 
weeks.   
Patients will come to the research center and complete the following visits: 
• 
Screening Visit within 6 weeks of Randomization 
• 
Day -14 Deuterated Water Run-In 
o 
Day-14, Day-11, Day-7 and Day 1: sampling for labeling 
of Deuterated water during run-in, 
• 
Repeat baseline labs for ALT, AST, total bilirubin, ALP, CPK 
collected at least 4 weeks from initial screening. 


 
 
Protocol No: NTZ-218-1 
Version: V 26February 2020 
 
 
 
 
 
Page 5 
• 
Day 1 randomization: start of treatment 
• 
Every 4 weeks after randomization until 20 weeks post 
randomization 
• 22 weeks post randomization and Deuterated Water       
Administration Period 
o 
22 weeks plus 3 days 
o 
sampling for labeling of Deuterated water on Day 155, 
Day 158, Day 162 and Day 169  
• 
24 weeks post randomization: End of Treatment 
• 
4 Week Post Treatment Follow Up 
Number of Sites: 
1 site  
Number of Subjects: 
20 evaluable subjects 
Drug Dosage / Frequency: 
500 mg NTZ BID 
Route of Administration: 
Oral Administration 
Inclusion Criteria: 
Patients must meet all of the following inclusion criteria to be eligible for 
enrollment in the trial:  
1. Males or females aged from 18 to 75 years inclusive at the Screening 
Visit.  
2. Must provide signed written informed consent and agree to comply 
with the study protocol.  
3. Females participating in this study must be of non-childbearing 
potential or using highly efficient contraception for the full duration of 
the study.  Childbearing potential refers to those female subjects who 
have not had a hysterectomy, bilateral oophorectomy, or medically-
documented ovarian failure. Female subjects are considered 
postmenopausal and not of child-bearing potential if they are >55 years 
of age, or >50 years of age with >12 months of amenorrhea or have 
serum follicle stimulating hormone (FSH) values >40 mIU/ml with >12 
months of amenorrhea.  Highly efficient contraception is defined as the 
use of one of the following methods of birth control in addition to a 
male partner using a condom (unless surgically sterile) from Screening 
to 90 days after the last dose of study medication: 
(1) hormone-containing contraceptive 
(2) intrauterine device 
(3) cervical cap or diaphragm with spermicidal agent 
4. Histological confirmation of steatohepatitis on a diagnostic liver biopsy 
(biopsy obtained within 6 months prior to Screening or during the 
Screening Period) with at least 1 in each component of the NAS 
(steatosis scored 0-3, ballooning degeneration scored 0-2, and lobular 
inflammation scored 0-3).   
5.    Fibrosis stage of 2 or 3, according to the NASH CRN fibrosis staging 
system on a diagnostic liver biopsy (biopsy obtained within 6 months 
prior to Screening or during the Screening Period).   
6. Two assessments of ALT, AST, Total bilirubin, ALP, CPK will be collected 
during screening at least 4 weeks apart. To be eligible the second value 
cannot be ≥2x the first value. 
Exclusion Criteria: 
Patients who meet any of the following criteria will be excluded from 
entering the study:  


 
 
Protocol No: NTZ-218-1 
Version: V 26February 2020 
 
 
 
 
 
Page 6 
1. History of efficient bariatric surgery within 5 years prior to Screening, 
or planned bariatric surgery in the course of the study.  
2. Patients with HbA1c >10.0%. If abnormal at the first Screening Visit, the 
HbA1c measurement can be repeated. A repeated abnormal HbA1c 
(HbA1c >10.0%) leads to exclusion.  
3. Patients with a history of clinically significant acute cardiac event, or 
cerebrovascular event within 6 months prior to Screening such as: 
stroke, transient ischemic attack, or coronary heart disease (angina 
pectoris, myocardial infarction, revascularization procedures).  
4. Weight loss of more than 10% within 6 months prior to Randomization.  
5. Patient with any history or presence of cirrhosis. 
6. Current or recent history (within a <1 year prior to screening ) of 
significant alcohol consumption. For men, significant consumption is 
typically defined as higher than 30 g pure alcohol per day. For women, 
it is typically defined as higher than 20 g pure alcohol per day.  
7. Current or history of other substance abuse within 1 year prior to 
screening. 
8. Pregnant or lactating females or females planning to become pregnant 
during the study period.  
9. Other well documented causes of chronic liver disease according to 
standard diagnostic procedures including, but not restricted to:  
a. Positive hepatitis B surface antigen (HBsAg)  
b. Positive HCV RNA, (tested for in case of known cured HCV 
infection, or positive HCV Ab at Screening)  
c. 
Suspicion of drug-induced liver disease  
d. Alcoholic liver disease  
e. Autoimmune hepatitis  
f. 
Wilson’s disease   
g. 
Primary biliary cirrhosis 
h. Primary sclerosing cholangitis  
i. 
Genetic homozygous hemochromatosis  
j. 
Known or suspected HCC  
k. 
History or planned liver transplant, or current MELD 
score >15.  
10. Patients who cannot be contacted in case of emergency.  
11. Known hypersensitivity to the investigational product or any of its 
formulation excipients.  
12. Patients who are taking warfarin or other highly plasma protein-bound 
drugs with narrow therapeutic indices. 
13. Patients who are currently participating in, plan to participate in, or 
have participated in an investigational drug trial or medical device trial 
containing active substance within 30 days or five half-lives, whichever 
is longer, prior to Screening. Patients who have screened for, and failed 
to qualify, for another clinical trial are eligible for screening for the NTZ-
218-1, even if the Screening Visit for the NTZ-218-1 study is less than 30 
days from the failed study screening visit, provided that the subject 
received no investigational product within 30 days of 5 half-lives of the 
investigational product prior to screening for NTZ-218-1. 


 
 
Protocol No: NTZ-218-1 
Version: V 26February 2020 
 
 
 
 
 
Page 7 
14. Evidence of any other unstable or, untreated clinically significant 
immunological, endocrine, hematological, gastrointestinal, 
neurological, neoplastic, or psychiatric disease.  
15. Mental instability or incompetence, such that the validity of informed 
consent or ability to be compliant with the study is uncertain.  
16.  History of noncompliance with medical regimens, or patients who are 
considered to be unreliable 
17. Positive anti-human immunodeficiency virus (HIV) antibody. 
18. AST and/or ALT >5 x upper limit of normal (ULN).  
19. Total bilirubin >1.3 mg/dL due to altered hepatic function.  
20. Direct bilirubin > ULN 
Note: Gilbert Disease patients are allowed into the study.  
21. INR >1.2 in the absence of anticoagulant therapy.  
22. Platelet count <150,000/mm3 in the   context of portal hypertension.  
23. Significant renal disease, including nephritic syndrome, chronic kidney 
disease (defined as patients with markers of kidney damage or eGFR of 
less than 60 ml/min/1.73 m2). 
 
Evaluation Criteria 
Primary Endpoint: 
To assess the safety and tolerability of NTZ after 24 weeks of treatment  
 
Secondary Endpoints: 
To assess the % change in FSR from baseline to end of treatment (24 weeks 
of treatment) 
To assess the change in liver stiffness by MRE from baseline to 12 weeks 
and 24 weeks of treatment 
To assess the change in liver stiffness by fibroscan from baseline to 24 
weeks of treatment  
To assess the change from baseline to 12 weeks and 24 weeks of treatment 
of fibrosis serum biomarkers and scores. 
 
 


 
 
Protocol No: NTZ-218-1 
Version: V 26February 2020 
 
 
 
 
 
Page 8 
TABLE 1: Schedule of events 
 
Screening period 
 
 
Treatment period 
Visit 
SV 
SV2 
SV3 
SV4 
V1 
V2 
V3 
V4 
V5 
V6 
V7 
V8 
V9 
V10 
EOS 
ET 
Day 
-42 
-14 
-11 
-7 
1 
29 
57 
85 
113 
141 
155 
158 
162 
169 
+28 
days 
 
Window 
 
+3 
+1 
+1 
+3 
+3 
+3 
+3 
+3 
+3 
+3 
+1 
+1 
+1 
+3 
 
Week 
-6 
-2 
 
 
0 
4 
8 
12 
16 
20 
22 
 
 
24 
28 
 
Informed 
Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical 
History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inclusion 
Exclusion 
Criteria 
X 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
Height 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Weight, BMI 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
Physical Exam 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
12-Lead ECG 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
X 
Vital Signs 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
Con Meds 
X 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
AEs 
X 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Liver Biopsy1 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
MRE 
 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
 
X 
Fibroscan 
 
 
 
 
X 
 
 
 
 
 
 
 
 
X 
 
X 
Dispense 
deuterated 
water 
 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
FSR 
 
X 
X 
X 
X 
 
 
 
 
 
X 
X 
X 
X 
 
 
Study Drug 
Dispense 
 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
Study Drug 
Compliance 
 
 
 
 
 
X 
X 
X 
X 
X 
 
 
 
X 
 
 
X 
 
 
 
1 Required only if there is no history of a liver biopsy within the past 6 months.  


 
 
Protocol No: NTZ-218-1 
Version: V 26February 2020 
 
 
 
 
 
Page 9 
TABLE 2: Biological assessments 
 
Screening period 
 
 
Treatment period 
Visit 
SV 
SV2 
SV3 
SV4 
V1 
V2 
V3 
V4 
V5 
V6 
V7 
V8 
V9 
V10 
EOS 
ET 
Day 
-42 
-14 
-11 
-7 
1 
29 
57 
85 
113 
141 
155 
158 
162 
169 
+28 
days 
 
Week 
-6 
-2 
 
 
0 
4 
8 
12 
16 
20 
22 
 
 
24 
28 
 
Labs - 
Haematology 
haemoglobin, 
haematocrit, RBC, 
WBC, differential 
count, platelet 
count, 
reticulocytes 
count, MCV 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Coagulation (PT 
(INR)) 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Labs- Urinary 
Pregnancy tests2 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
 
X 
Labs – Serology 
HIV, HBs and HCV 
serology 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
2 If Urine pregnancy test is positive, a serum β-hCG is required.  
 
 
 


 
 
Protocol No: NTZ-218-1 
Version: V 26February 2020 
 
 
 
 
 
Page 10 
 
Screening period 
 
 
Treatment period 
Visit 
SV 
SV2 
SV3 
SV4 
V1 
V2 
V3 
V4 
V5 
V6 
V7 
V8 
V9 
V10 
EOS 
ET 
Day 
-42 
-14 
-11 
-7 
1 
29 
57 
85 
113 
141 
155 
158 
162 
169 
+28 
days 
 
Labs – 
Biochemistry 
HbA1c, fasting 
plasma glucose, 
insulin, HOMA-IR, 
fructosamine, 
alkaline 
phosphatase, ALT, 
AST, GGT, CPK, 
total and 
conjugated 
bilirubin, ferritin, 
creatinine, eGFR, 
uric acid, BUN, 
total cholesterol, 
HDL-C, LDL-C, 
triglycerides, 
albumin, lipase, 
amylase, 
electrolytes 
(sodium, 
potassium, 
chloride, calcium), 
MELD 
X 
 
 
X3 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Inflammatory 
markers 
hsCRP, fibrinogen, 
haptoglobin, 
resistin,  TNF-, 
TGF-, IL-6, PAI-1 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
Biomarkers of 
fibrosis 
ELF Score, PIIINP, 
Hyaluronic acid, 
CK18, TIMP-1, YKL-
40, Alpha 2 
macroglobulin,, 
,miRNA, ProC3, 
ProC6, FGF19, 
FGF21 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
NAFLD Fibrosis 
Score (NFS) &  
FIB-4 
 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
 
X 


 
 
Protocol No: NTZ-218-1 
Version: V 26February 2020 
 
 
 
 
 
Page 11 
 
Screening period 
 
 
Treatment period 
Visit 
SV 
SV2 
SV3 
SV4 
V1 
V2 
V3 
V4 
V5 
V6 
V7 
V8 
V9 
V10 
EOS 
ET 
Day 
-42 
-14 
-11 
-7 
1 
29 
57 
85 
113 
141 
155 
158 
162 
169 
+28 
days 
 
Labs – Urinalysis 
dipstick  
Specific gravity, 
pH, protein, 
glucose, ketones, 
bilirubin, 
urobilinogen, 
blood, nitrite, 
leukocytes 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
 
X 
Safety markers 
Serum Cystatin C, 
troponin T, NT 
proBNP 
Urinary albumin, 
urinary creatinine, 
urinary ACR 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Labs -- FSR 
 
X 
X 
X 
X 
 
 
 
 
 
X 
X 
X 
X 
 
 
Sampling for 
additional 
parameters 
X 
 
 
 
X 
 
 
X 
 
X 
 
 
 
X 
X 
X 
 
3 ALT, AST, Total bilirubin, Alkaline phosphatase and CPK must be retested at least 4 weeks after initial 
screening labs. . If these values at SV4 are ≥2x Screening values SV, then the patient cannot be 
randomized. A repeat test may be performed if a screening value is considered an error. 
 
 


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Table of Contents 
1.
Introduction .......................................................................................................................... 17 
1.1. 
Background ................................................................................................................... 17 
1.2. 
General Information for Nitazoxanide ......................................................................... 18 
1.2.1. 
Nitazoxanide (NTZ) ............................................................................................... 18 
1.2.2. 
Clinical Safety & Toxicology .................................................................................. 18 
1.2.3. 
Non clinical pharmacology ................................................................................... 20 
1.3. 
Rationale for This Study................................................................................................ 23 
2.
Objectives .............................................................................................................................. 23 
2.1. 
Primary Objective ......................................................................................................... 23 
2.2. 
Secondary Objectives: .................................................................................................. 23 
3.
Study Design.......................................................................................................................... 24 
3.1. 
Study Design.................................................................................................................. 24 
3.2. 
Treatment Plan and Regimen ....................................................................................... 25 
4.
Subject Population ................................................................................................................ 25 
4.1. 
Number of Subjects & Subject Selection ..................................................................... 25 
4.2. 
Inclusion Criteria ........................................................................................................... 25 
4.3. 
Exclusion Criteria .......................................................................................................... 26 
5.
Investigational Medicinal Products ...................................................................................... 28 
5.1. 
Description .................................................................................................................... 28 
5.2. 
Packaging ...................................................................................................................... 29 
5.3. 
Labeling ......................................................................................................................... 29 
5.4. 
Storage and Handling ................................................................................................... 29 
5.5. 
Dosage and Administration .......................................................................................... 29 
5.6. 
Dose Adjustment .......................................................................................................... 30 
5.7. 
Prior and Concomitant Medications ............................................................................ 30 
5.7.1. 
Non-permitted medication ................................................................................... 30 
5.7.2. 
Permitted Medications ......................................................................................... 30 
5.8. 
Study Drug Accountability ............................................................................................ 31 
6.
Study Procedures .................................................................................................................. 31 


 
 
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6.1. 
Duration of Study Participation ................................................................................... 31 
6.2. 
Screening Assessment .................................................................................................. 31 
Screening Visit ....................................................................................................................... 31 
6.3. 
Day -14 Deuterated Water Run In ................................................................................ 32 
6.4. 
Treatment Period Assessments .................................................................................... 33 
6.4.1. 
Day 1 Visit.............................................................................................................. 33 
6.4.2. 
Week 4 and Week 8 Visit + 3 days ........................................................................ 33 
6.4.3. 
Week 12 Visit + 3 days .......................................................................................... 34 
6.4.4. 
Week 16 and Week 20 visits ±3 days ................................................................... 34 
6.4.5. 
Week 22 Visit + 3 days (Day 155) ......................................................................... 35 
6.4.6. 
Week 24 Visit +1 ................................................................................................... 35 
6.5. 
Follow Up Period ........................................................................................................... 36 
6.5.1. 
4-Week Post Treatment Follow up (EOS – End of study visit) ±3 days ............... 36 
6.5.2. 
Early Termination (ET) .......................................................................................... 36 
6.5.3. 
Unscheduled Visits ................................................................................................ 37 
6.6. 
Assessment for Premature Discontinuation from Study ............................................. 37 
6.6.1. 
Pre-specified Patient Discontinuation rules ........................................................ 38 
6.6.2. 
Pre-specified Trial Discontinuation rules ............................................................. 38 
6.6.3. 
Dose Adjustment .................................................................................................. 38 
6.6.4. 
Specific Liver Function Monitoring after Start of Treatment .............................. 39 
6.6.5. 
Data Safety Monitoring Board ............................................................................. 40 
6.7. 
Description of Assessments .......................................................................................... 42 
6.7.1. 
Histological Assessments ...................................................................................... 42 
6.7.2. 
Assessment of newly synthesized proteins ......................................................... 42 
6.7.3. 
Biological Assessments (Table 2) .......................................................................... 42 
Table 2 Biological assessments ...................................................................................................... 44 
6.7.4. 
Electrocardiogram ................................................................................................. 45 
6.7.5. 
MRE ....................................................................................................................... 45 
6.7.6. 
Fibroscan ............................................................................................................... 46 
6.7.7. 
Medical History ..................................................................................................... 46 
6.7.8. 
Physical Examination ............................................................................................ 46 


 
 
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6.7.9. 
Vital Signs .............................................................................................................. 46 
6.7.10. 
Pregnancy Testing ................................................................................................. 47 
7. 
Adverse Events and Toxicity Management .......................................................................... 47 
7.1. 
Definitions of Adverse Events, Serious Adverse Events and Unexpected Events ...... 47 
7.1.1. 
Adverse Events ...................................................................................................... 47 
7.1.2. 
Clinical Laboratory Abnormalities and Other Abnormal Assessments as Adverse 
Events or Serious Adverse Events ........................................................................................ 48 
7.1.3. 
Serious Adverse Events ......................................................................................... 48 
7.1.4. 
Unexpected adverse event ................................................................................... 49 
7.2. 
Assessment of Adverse Events & Serious Adverse Events .......................................... 49 
7.2.1. 
Intensity assessment ............................................................................................ 49 
7.2.2. 
Relation to the study treatment .......................................................................... 50 
7.2.3. 
Action taken and outcome ................................................................................... 51 
7.3. 
Reporting ....................................................................................................................... 52 
7.3.1. 
Reporting an adverse event ................................................................................. 52 
7.3.2. 
Initial reports......................................................................................................... 52 
7.3.3. 
Follow-up reports ................................................................................................. 53 
7.3.4. 
Follow-up .............................................................................................................. 53 
7.4. 
Post Study Reporting Requirements ............................................................................ 54 
7.5. 
Special Situation Reports .............................................................................................. 54 
7.5.1. 
Pregnancy .............................................................................................................. 54 
7.5.2. 
Medication error ................................................................................................... 54 
7.5.3. 
Misuse ................................................................................................................... 55 
7.5.4. 
Overdose ............................................................................................................... 55 
7.5.5. 
Abuse ..................................................................................................................... 55 
8. 
Statistical Considerations ..................................................................................................... 55 
8.1. 
Analysis Endpoints ........................................................................................................ 55 
8.1.1. 
Primary Endpoint .................................................................................................. 55 
8.1.2. 
Secondary Endpoints ............................................................................................ 55 
8.2. 
Analysis Sets .................................................................................................................. 56 
8.3. 
Data Handling Conventions .......................................................................................... 56 
8.4. 
Demographic Data and Baseline Characteristics ......................................................... 56 


 
 
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8.5. 
Primary Safety Analysis ................................................................................................ 56 
8.5.1. 
Adverse Events ...................................................................................................... 57 
8.5.2. 
Laboratory Evaluations ......................................................................................... 57 
8.5.3. 
Other Safety Evaluations ...................................................................................... 57 
8.6. 
Secondary Efficacy Analyses ......................................................................................... 57 
8.6.1. 
FSR ......................................................................................................................... 57 
8.6.2. 
Liver stiffness ........................................................................................................ 58 
8.6.3. 
Exploratory Analysis ............................................................................................. 58 
8.6.4. 
Interim Analysis .................................................................................................... 58 
8.7. 
Sample Size ................................................................................................................... 58 
9. 
Responsibilities ..................................................................................................................... 58 
9.1. 
Investigator Responsibilities ........................................................................................ 59 
9.2. 
Good Clinical Practice ................................................................................................... 59 
9.3. 
Institutional Review Board Review & Approval .......................................................... 60 
9.4. 
Informed Consent ......................................................................................................... 60 
9.5. 
Confidentiality .............................................................................................................. 60 
9.6. 
Study Files and Retention of Records .......................................................................... 61 
9.7. 
Electronic Case Report Forms ....................................................................................... 62 
9.8. 
Investigational Medicinal Product Accountability and Return ................................... 63 
9.9. 
Inspections .................................................................................................................... 63 
9.10. Protocol Compliance ..................................................................................................... 63 
9.11. Protocol Modifications ................................................................................................. 63 
9.12. Study Reports ................................................................................................................ 64 
9.13. Study Termination ........................................................................................................ 64 
10. 
References ......................................................................................................................... 65 
10.1. Literature ....................................................................................................................... 65 
10.2. WHO Fact Sheet 312 ..................................................................................................... 68 
10.3. Studies on ClinicalTrials.gov ......................................................................................... 68 
 
 
 


 
 
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1. Introduction 
1.1. 
Background 
Non-alcoholic fatty liver disease (NAFLD) has become one of the most prominent forms 
of chronic liver disease worldwide, reflecting the epidemic of global obesity. Those with 
the progressive variant of NAFLD, non-alcoholic steatohepatitis (NASH), are at significantly 
increased risk of multisystem morbidity and mortality. However, there are currently no 
approved pharmacologic therapies for NASH (Konerman et al, 2018). The World Health 
Organization estimates the prevalence of obesity has more than doubled since 1980 with 
>600 million people (13%) having a body mass index (BMI)) ≥30 (Konerman et al, 2018). 
In this setting, the global prevalence of diabetes among adults has also increased from 
4.7% in 1980 to 8.5% as of 2014 (WHO Fact Sheet 312) . Prevalence rates of NAFLD among 
those with metabolic disease are notably higher.  Approximately a third of patients with 
hypertension, half of patients with dyslipidaemia, up to two-thirds of patients with type II 
diabetes, and  >90% of patients undergoing bariatric surgery had evidence of NAFLD (Leite 
et al, 2009; Fruci et al, 2013; Assy et al, 2000; Wu  et al, 2016; Sasaki et al, 2014 Subichin 
et al; 2015 ; Donati et al ,2004). 
The underlying pathophysiologic mechanisms that contribute to the development and 
progression of NAFLD and NASH are complex and include modulation of metabolic 
pathways, inflammatory cascades, and/or mechanisms impacting fibrosis.  During disease 
progression fibrosis may worsen, while features of steatohepatitis resolve or ‘‘burnout”.    
As a result, NASH and fibrosis need to be evaluated independently to ensure a beneficial 
impact on one parameter does not simultaneously result in a negative impact on another 
endpoint of interest, particularly given that individual treatments tend to focus on one 
primary mechanism of action (i.e. metabolic or NASH disease modifier vs. antifibrotic) ( 
Konerman et al, 2018). 
Persistent and progressive fibrosis is a characteristic of many chronic liver diseases in 
which excessive accumulation of collagen-rich extracellular matrix is a key feature. 


 
 
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Following liver injury, quiescent Hepatic Stellate Cells (HSC) undergo a process of 
activation that is characterized by a differentiation into (α-SMA)-positive contractile 
myofibroblasts which secrete high amounts of collagen and play a central role in the 
fibrogenic process (Friedman et al, 2008, Carpino et al, 2005). Nitazoxanide may be a 
potent antifibrotic agent that can potentially be used for the treatment of different fibrotic 
pathologies, including liver fibrosis. (Belanger et al;2017) 
1.2. 
General Information for Nitazoxanide 
1.2.1. Nitazoxanide (NTZ)  
Nitazoxanide (NTZ) is a broad-spectrum antiparasitic and broad-spectrum antiviral drug 
used for the treatment of various helminthic, protozoal, and viral infections.  It was 
approved in the United States by the Food and Drug Administration (Alinia®, Romark 
Laboratories) as an Orphan Drug for treatment of diarrhea caused by C. parvum and 
Giardia intestinalis in adults and children at least 12 months of age [Rossignol, 2014]. 
The approved Alinia dose for adults is 500 mg twice daily (BID) for three days.  
1.2.2. Clinical Safety & Toxicology 
Nitazoxanide is rapidly metabolized in the liver to an active metabolite tizoxanide (TZ) in 
vivo. TZ undergoes conjugation to form tizoxanide glucuronide. Nitazoxanide is not 
detectable in the serum following oral administration.  Peak concentrations in blood 
plasma of TZ and TZ glucuronide are seen between 1-4 hours.  NTZ is excreted in both 
Urine (~33%) and feces (~67%).   
The safety of ALINIA was evaluated in 2177 HIV-uninfected subjects 12 months of age 
and older who received ALINIA Tablets or ALINIA for Oral Suspension at the 
recommended dose for at least three days. In pooled controlled clinical trials involving 
536 HIV-uninfected subjects treated with ALINIA Tablets or ALINIA for Oral Suspension, 
the most common adverse reactions were abdominal pain, headache, chromaturia and 
nausea (≥2%).[Alinia USPI] 


 
 
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Safety data were analyzed separately for 280 HIV-uninfected subjects ≥12 years of age 
receiving Alinia at the recommended dose for at least 3 days in 5 placebo-controlled 
clinical trials and for 256 HIV-uninfected subjects 1 through 11 years of age in 7 
controlled clinical trials. There were no differences between the adverse reactions 
reported for Alinia-treated subjects based upon age [Alinia USPI]. 
Twenty-one clinical studies of NTZ were conducted in patients with chronic hepatitis C 
(14 studies: Rossignol et al, 2008; Rossignol et al, 2009; Bacon et al, 2010, Rossignol et 
al, 2010; Shiffman et al, 2010 ; Laufer et al, 2011; Kabil et al, 2011 ; Amorosa et al, 2013; 
Shebab et al, 2014; Macias et al, 2015; Kohla et al, 2016; Basu et al, 2013; 
NCT01185028; NCT01770483), hepatic encephalopathy in chronic liver failure (1 study: 
Basu et al, 2008), acquired immune deficiency syndrome (AIDS)-related 
cryptosporidiosis (3 studies: Rossignol et al, 1998; Rossignol et al, 2006; Zulu et al, 
2005), or Helicobacter pylori (2 studies: Abd-Elsalam et al, 2016; Shehata et al., 2017), 
and Clostridium difficile infections (1 study: Musher et al, 2009) in the literature. The 
dose of NTZ administered in 19 studies was 500 mg BID; in one study 1000 mg NTZ BID 
was administered, whereas in one compassionate-use study the NTZ dose administered 
ranged from 500 to 1500 mg BID. Most studies administered NTZ treatment for 48 to 60 
weeks, whereas the duration of treatment in the compassionate-use study ranged from 
1 day to 4 years.  
The studies identified from the literature review demonstrate the safety of NTZ in the 
treatment of chronic hepatitis C, hepatic encephalopathy in chronic liver failure, AIDS-
related cryptosporidiosis, persistent diarrhea in patients with AIDS, and Helicobacter 
pylori and Clostridium difficile infections.  
The adverse event (AE) profile of NTZ was predominantly GI in nature and included 
nausea, vomiting, diarrhea, abdominal pain, dyspepsia, constipation, flatulence, and 
dysphagia. These GI AEs were generally mild in intensity and transient. Other AEs 
related to NTZ treatment were fatigue, rash, headache, myalgia, and arthralgia. When 


 
 
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reported, no significant changes were observed in the electrocardiograms (ECGs), vital 
signs, and clinical laboratory parameters after administration with NTZ [Stockis et al, 
2002a; Stockis et al, 2002b]. A formal International Council on Harmonization (ICH) E14 
compliant thorough QT (TQT) study [Taubel et al, 2014] completed the cardiac safety 
evaluation and showed that neither a therapeutic single dose of 675 mg nor a 2700 mg 
(supra-therapeutic) dose of NTZ prolonged the QT interval in healthy male and female 
volunteers.   
The safety profile of NTZ reported in all these studies therefore provides supporting 
evidence for the potential use of NTZ for the treatment of liver fibrosis in patients with 
NASH.  
The following adverse reactions have been identified during post approval use of ALINIA 
as described in the Package Insert:  
• Gastrointestinal disorders: diarrhea, gastroesophageal reflux disease 
• Nervous System disorders: dizziness 
• Respiratory, thoracic and mediastinal disorders: dyspnea 
• Skin and subcutaneous tissue disorders: rash, urticaria 
1.2.3. Non clinical pharmacology 
Nonclinical pharmacology studies were conducted to determine whether NTZ and its 
main metabolites (TZ and TZ glucuronide [TZ glu]) exhibit anti-fibrotic properties in vitro, 
and in vivo. 
NTZ and its main metabolite, TZ, significantly (p<0.001 from 0.3 µM) inhibited, in a dose-
dependent manner, the expression of αSMA protein in TGF-β-induced human HSCs, but 
TZ glu had no anti-fibrotic properties. NTZ and TZ demonstrated good potency as the 
IC50 was in the micromolar range.  


 
 
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The CDAA diet-induced rodent model develops a fibrosing NASH within a relatively short 
period [Takeuchi-Yorimoto et al, 2013], which makes it a suitable model for studying the 
treatment of both NASH pathology and hepatic fibrosis. 
In studies to determine the effects of NTZ on liver fibrosis in C57BL/6J mice induced by a 
CDAA diet supplemented with 1% cholesterol, microscopic examination revealed that 
NTZ at doses of 11.4 mg/kg/day and 33.9 mg/kg/day tended to reduce, in a dose-
dependent manner, CDAA + 1% cholesterol diet-induced NASH. Although NTZ did not 
have a beneficial effect on hepatic steatosis and lobular inflammation grade, a tendency 
to reduce hepatocyte ballooning was observed, which resulted in a slight reduction in 
NASH prevalence.  
At 12 weeks, NTZ significantly prevented, in a dose-dependent manner, hepatic collagen 
storage induced by the CDAA + 1% cholesterol diet. However, upon histological 
examination, this beneficial effect was not reflected by a dose-dependent improvement 
of the fibrosis grade. A significant decrease of the fibrosis area was observed with NTZ at 
doses of 11.4 mg/kg/day (p<0.05) and 78.1 mg/kg/day (p<0.001) and correlated with 
the significant decrease of the hepatic collagen content.  
The CDAA + 1% cholesterol diet notably induces a significant augmentation in circulating 
markers of liver injury such as AST, ALT, and PIIINP, which is directly associated with 
fibrosis [Tanwar et al, 2013]. Moreover, the hepatic content is dramatically modified as 
illustrated by the significant increase of collagen. Mice treated with NTZ at 
78.1 mg/kg/day exhibited lower plasma levels of ALT (-17%, p>0.05), AST (-24%, p<0.05), 
and PIIINP (-25%, p<0.01). These data suggest a potential preventive action of NTZ on 
liver damage induced by the CDAA + 1% cholesterol diet in mice.  
The hepatic expression of fibrotic markers (collagens, MMP2), but also inflammatory 
markers (TNFα) were enhanced in the animals fed the CDAA + 1% cholesterol diet as 
compared with animals fed a CSAA control diet. Chronic administration of NTZ at 
78.1 mg/kg/day to mice fed a CDAA + 1% cholesterol diet led to the inhibition of the 


 
 
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Col1α1 (-17%, p>0.05), Col1α2 (-25%, p<0.05), MMP2 (-32%, p<0.05), and TNFα  
(-28%, p<0.01) gene expression.  
In the studies with CCl4-treated mice, NTZ significantly prevented hepatic fibrosis. This 
was shown at the dose of 95.3 mg/kg/day on the hepatic collagen content decrease in 
the first study [SSR_NTZ_PHA1_004], whereas no significant improvement was observed 
upon histological examination. Conversely, in the second study [SSR_NTZ_PHA1_005], 
no significant improvement of CCl4-induced hepatic collagen content was observed, 
whereas the histological evaluation of hepatic fibrosis revealed a significant effect of 
NTZ at 32 and 104 mg/kg/day. 
There was no significant beneficial effect of NTZ treatment on plasma markers of 
hepatotoxicity in CCl4-treated mice.  
These studies demonstrate the anti-fibrotic effect of NTZ, which suggests that NTZ may 
have potential as an anti-fibrotic drug. These studies also suggest that NTZ may show 
some beneficial therapeutic properties for NASH, such as a tendency to reduce 
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1.3. 
Rationale for This Study 
Based on the anti-fibrotic properties demonstrated in the animal models of fibrosis, this proof 
of concept clinical study aims at  evaluating NTZ in patients with non-alcoholic steatohepatitis 
(NASH) and  fibrosis stage 2 and 3.Although NTZ has been evaluated in liver disease 
populations (viral hepatitis C) up to 60 weeks, this is the first study evaluating NTZ treatment in 
a population with NASH induced stage 2 and 3 fibrosis.  The aim of this study is to evaluate the 
safety and tolerability of NTZ 500 mg BID after 24 weeks of treatment in this population. This 
proof of concept study will also evaluate the anti-fibrotic effect of NTZ as a secondary 
objective. 
The methods of evaluation of fibrosis will include an innovative method of metabolic labeling. 
This approach is based on the concept that liver status can be determined by measuring the 
ratio of newly synthesized/pre-existing proteins. The turn-over rate of newly synthesized 
collagen and proteins represents the hepatic fibrogenic disease activity.  Patients will be given 
“heavy water” to drink. Heavy water contains D20, deuterium being a stable isotope of 
hydrogen. Mass spectrometry is used to identify individual proteins and to quantify the ratio of 
labeled protein to total protein. The results are expressed as fractional synthesis rate of these 
proteins (FSR). This method has been previously published (Decaris et al, 2017). 
Other non-invasive methods will be used to evaluate the liver stiffness changes after NTZ 
treatment: Magnetic Resonance Elastography (MRE) and FibroScan®.  
2. 
Objectives 
2.1. Primary Objective 
 
The primary objective of the study is: 
• To evaluate the safety and tolerability of NTZ 500mg bid after 24 weeks of treatment in 
patients with NASH induced Stage 2 or Stage 3 fibrosis. 
2.2. 
Secondary Objectives: 
The secondary objectives of the study are: 


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• To determine the effect of daily oral administration of NTZ 500mg bid on de novo 
collagen synthesis through Fractional Synthesis Rate (FSR) of circulating plasma 
proteins in patients with NASH-induced Stage 2 or Stage 3 fibrosis. 
• To evaluate changes in liver stiffness as measured by FibroScan® and by Magnetic 
Resonance Elastography (MRE). 
• To assess the change from baseline in non-invasive markers of fibrosis: ELFTM test 
score, PIIINP, Hyaluronic acid, CK18, TIMP-1, YKL-40, Alpha 2 macroglobulin,, ,miRNA, 
ProC3, ProC6, FGF19, FGF21. 
• To assess changes in additional non-invasive tests: fibrosis scores: NAFLD fibrosis 
score, FIB-4 
3. 
Study Design 
3.1. 
Study Design 
This is a phase 2 proof of concept study to evaluate the efficacy and safety of open-label NTZ 
500mg bid in a NASH population with NASH induced Stage 2 or 3 fibrosis. 
The study will include a screening visit within 6 weeks of randomization (Day 1) followed by a 
24-week treatment period consisting of daily, BID, oral administration of NTZ and an end of 
treatment follow up period of 4 weeks.   
Patients will come to the research center and complete the following visits: 
• 
Screening Visit within 6 weeks of Randomization 
• 
Day -14 Deuterated Water Run-In 
o Day-14, Day-11, Day-7 and Day 1: sampling for labelling of Deuterated water 
during run-in 
• 
Repeat baseline labs for ALT, AST, total bilirubin, ALP, CPK collected at least 4 
weeks from initial screening.  
• 
Day 1 Randomization: start of treatment 
• 
Every 4 weeks after randomization until 20 weeks post randomization 


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• 
 22 weeks post randomization and Deuterated Water Administration Period:  
o 22 weeks plus 3 days 
o sampling for labelling of Deuterated water on Day 155, 158, 162, and Day 
169/24 weeks post randomization: End of Treatment 
• 
4 Week Post Treatment Follow Up 
3.2. 
Treatment Plan and Regimen 
NTZ will be administered in an open label fashion to participating subjects as 500mg bid, for 24 
weeks. 
4. 
Subject Population 
4.1. 
Number of Subjects & Subject Selection 
Twenty male and female subjects with histologically confirmed NASH and fibrosis stage 2 or 3 
will be enrolled in the study at a single site in the United States. 
4.2. 
Inclusion Criteria 
Patients must meet all of the following inclusion criteria to be eligible for enrollment in the 
trial:  
1. 
Males or females aged from 18 to 75 years inclusive the Screening Visit.  
2. 
Must provide signed written informed consent and agree to comply with the 
study protocol.  
3. Females participating in this study must be of non-childbearing potential or using 
highly efficient contraception for the full duration of the study.   Childbearing 
potential refers to those female subjects who have not had a hysterectomy, 
bilateral oophorectomy, or medically-documented ovarian failure. Female subjects 
are considered postmenopausal and not of child-bearing potential if they are >55 
years of age, or >50 years of age with >12 months of amenorrhea, or have serum 
follicle stimulating hormone (FSH) values >40 mIU/ml with >12 months of 
amenorrhea.  Highly efficient contraception is defined as the use of one of the 


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following methods of birth control in addition to a male partner using a condom 
(unless surgically sterile) from Screening to 90 days after the last dose of study 
medication: 
• hormone-containing contraceptive 
• intrauterine device 
• cervical cap or diaphragm with spermicidal agent 
4. 
 Histological confirmation of steatohepatitis on a diagnostic liver biopsy (biopsy 
obtained within 6 months prior to Screening or during the Screening Period) 
with at least 1 in each component of the NAS (steatosis scored 0-3, ballooning 
degeneration scored 0-2, and lobular inflammation scored 0-3).   
5. 
Fibrosis stage of 2 or 3, according to the NASH CRN fibrosis staging system on a 
diagnostic liver biopsy (biopsy obtained within 6 months prior to Screening or 
during the Screening Period).   
6. 
Two assessments of ALT, AST, Total bilirubin, ALP, CPK will be collected during 
screening at least 4 weeks apart. To be eligible the second value cannot be ≥2x 
the first value. 
4.3. 
Exclusion Criteria 
Patients who meet any of the following criteria will be excluded from entering the study:  
1. 
History of efficient bariatric surgery within 5 years prior to Screening, or 
planned bariatric surgery in the course of the study.  
2. 
Patients with HbA1c >10.0%. If abnormal at the first Screening Visit, the HbA1c 
measurement can be repeated. A repeated abnormal HbA1c (HbA1c >10.0%) 
leads to exclusion.  
3. 
Patients with a history of clinically significant acute cardiac event, or 
cerebrovascular event, within 6 months prior to Screening such as: stroke, 
transient ischemic attack, or coronary heart disease (angina pectoris, 
myocardial infarction, revascularization procedures).  
4. 
Weight loss of more than 10% within 6 months prior to Randomization.  
5. 
Patient with any history or presence of cirrhosis. 


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6. 
Current or recent history (within a year of screening) of significant alcohol 
consumption. For men, significant consumption is typically defined as higher 
than 30 g pure alcohol per day. For women, it is typically defined as higher than 
20 g pure alcohol per day.  
7. 
Current or history of other substance abuse within 1 year prior to screening. 
8. 
Pregnant or lactating females or females planning to become pregnant during 
the study period.  
9. 
Other well documented causes of chronic liver disease according to standard 
diagnostic procedures including, but not restricted to:  
a) Positive hepatitis B surface antigen (HBsAg)  
b) Positive HCV RNA, (tested for in case of known cured HCV infection, or positive 
HCV Ab at Screening)  
c) Suspicion of drug-induced liver disease  
d) Alcoholic liver disease  
e) Autoimmune hepatitis  
f) Wilson’s disease   
g) Primary biliary cirrhosis 
h) Primary sclerosing cholangitis  
i) Genetic homozygous hemochromatosis  
j) Known or suspected HCC  
k) History or planned liver transplant, or current MELD score >15.  
10. 
Patients who cannot be contacted in case of emergency.  
11. 
Known hypersensitivity to the investigation product or any of its formulation 
excipients.  
12. 
Patients who are taking warfarin or other highly plasma protein-bound drugs 
with narrow therapeutic indices. 
13. 
Patients who are currently participating in, plan to participate in, or have 
participated in an investigational drug trial or medical device trial containing 
active substance within 30 days or five half-lives, whichever is longer, prior to 


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Screening. Patients who have screened for, and failed to qualify, for another 
clinical trial are eligible for screening for the NTZ-218-1, even if the Screening 
Visit for the NTZ-218-1 study is less than 30 days from the failed study screening 
visit, provided that the subject received no investigational product within 30 
days of 5 half-lives of the investigational product prior to screening for NTZ-218-
1. 
14. 
Evidence of any other unstable or, untreated clinically significant 
immunological, endocrine, hematological, gastrointestinal, neurological, 
neoplastic, or psychiatric disease.  
15. 
Mental instability or incompetence, such that the validity of informed consent 
or ability to be compliant with the study is uncertain.  
16. 
 History of noncompliance with medical regimens, or patients who are 
considered to be unreliable. 
17. 
Positive anti-human immunodeficiency virus (HIV) antibody. 
18. 
AST and/or ALT >5 x upper limit of normal (ULN).  
19. 
Total bilirubin >1.3 mg/dL due to altered hepatic function. 
20. 
Direct bilirubin > ULN 
 Note: Gilbert Disease patients are allowed into the study.  
21. 
INR >1.2 in the absence of anticoagulant therapy.  
22. 
Platelet count <150,000/mm3 in the context of portal hypertension.  
23. 
Significant renal disease, including nephritic syndrome, chronic kidney disease 
(defined as patients with markers of kidney damage or eGFR of less than 60 
ml/min/1.73 m2). 
5. 
Investigational Medicinal Products 
Patients will receive 500 mg NTZ (ALINIA®) twice daily for 24 weeks. 
5.1. Description  


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NTZ will be supplied as 500 mg of ALINIA® (nitazoxanide) tablets, for oral use.  The tables are 
round, yellow, film-coated and debossed with ALINIA on one side and 500 on the other side. 
Each tablet contains 500 mg of nitazoxanide. 
For additional information see the Package Insert for ALINIA®. 
5.2. Packaging  
ALINIA® tablets are packaged in HDPE bottles of 12 tablets. Period boxes will be delivered 
every 4 weeks, each box containing 6 bottles. 
5.3. 
Labeling 
All labels for study drugs meet all applicable requirements of the US Food and Drug 
Administration (FDA) and the EU annex 13 of Good Manufacturing Practices: Manufacture of 
Investigational Medicinal Products (February 2010) and /or other local regulations, as 
applicable.  
Distribution of study drug will be performed according to the Good Distribution Practices.  
Product boxes for each period will be labeled with the protocol number, Sponsor’s name and 
address, description of contents, storage conditions, expiry date, dosage instructions, and any 
other applicable items required by national and regional guidelines/regulations. The label will 
contain the statements “Caution – new drug, limited by Federal Law to investigational use” or 
other similar/appropriate statements as well as the following instructions “Please return 
empty packaging and unused products to your doctor at your next visit.”  
5.4. 
Storage and Handling 
The tablets are to be stored at 20-25oC (68-77oF). Storage conditions are specified on the label. 
5.5. 
Dosage and Administration 
Patients will be instructed to take one tablet of 500mg NTZ mg orally, twice a day with water 
during a meal, approximately 12 hours apart.    
The study staff will administer the first dose of the medication at the research center.  Patients 
will self-administer the evening dose on Day 1 and twice a day thereafter, when not attending 


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a visit at the research center.  On scheduled follow up visit days while the patient is being 
treated with study drug, the patient will be directed to bring back all used and unused study 
medication containers. Compliance will be checked by the Investigator or designee during 
those visits and registered in the electronic Case Report Form (eCRF).  
If treatment is interrupted, whatever the cause, duration and reason of the interruption will be 
documented. 
5.6. 
Dose Adjustment 
Enrolled subjects who experience treatment emergent adverse events may have their dose 
adjusted, at the discretion of the Principal Investigator, in accordance with section 6.6.3. 
5.7. 
Prior and Concomitant Medications 
In a general manner, patients should be discouraged from starting any new medication 
without consulting the Investigator unless the new medication is required for emergency 
purpose.  In the same way, any qualitative or quantitative change in concomitant therapy 
should be avoided, when possible. In the event that it becomes necessary during the study, 
this should be recorded by the Investigator or designee in the eCRF (including concomitant 
medications taken within 6 months prior to Screening). This includes drugs used on a chronic 
as well as on an “as needed” basis. 
5.7.1.  Non-permitted medication 
The following medications are not allowed within the timeframe given:  
• Warfarin or other highly plasma protein-bound drugs with narrow therapeutic indices 
are not permitted from Day 1 until end of treatment 
5.7.2.  Permitted Medications 
Any medications other than those listed above are permitted. However, the dosage of a 
current medication for a chronic disease should remain unchanged as far as possible in order 
to reduce the risk of unknown drug-drug interactions.  
In the event that additional concomitant therapy becomes necessary during the study, this will 
be recorded by the Investigator in the eCRF. This includes drugs used on a chronic as well as on 


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an “as-needed” basis. Patients should be discouraged from starting any new medication 
without consulting the Investigator unless the new medication is required for emergency 
purpose. 
5.8. Study Drug Accountability 
The Investigator will acknowledge receipt for each study treatment on the day of receipt. A 
drug accountability record will be maintained by the person responsible for dispensing the trial 
medication to the patient.  
All partially used or unused treatments will be inventoried during and at the conclusion of the 
study.  
The Drug Distribution Center will organize the retrieval of all treatments (used or unused) and 
will proceed to their destruction only after written authorization and review of accountability 
and distribution records.  
6. 
Study Procedures 
The procedures performed at each visit are summarized in the Schedule of Events (Table 1).  
A patient may be seen at any time for reasons of safety.  
During each visit, safety evaluations will be done, and the patient will be queried in the form of 
an open question regarding new or continuing events.  
6.1. 
Duration of Study Participation 
The estimated duration of the study will be approximately 34 weeks inclusive of the screening 
period. 
6.2. 
Screening Assessment 
Screening Visit 
The following screening procedures will be performed for all potential patients at the 
Screening Visit and prior to Day 1:  
• 
Informed consent witnessed by the Investigator or designated person.  


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• 
Medical history & demographics.  
• 
Assessment of inclusion/exclusion criteria 
• 
Complete physical examination. 
• 
Record vital signs  
• 
Record height, weight, BMI  
• 
Check concomitant/prior medication (within 6 months prior to Screening)  
• 
12-lead ECG 
• 
Liver biopsy.  A historical liver biopsy with confirmed NASH and fibrosis is 
acceptable if performed within 6 months prior to the Screening Visit.  
• 
Adverse Events assessment  
• 
Blood samples will be collected for 
o 
Biochemistry 
o 
Hematology 
o 
Serology (HIV, HBs, HCV). In the event of a positive HCV Ab, HCV RNA 
can be tested for confirmation. In case of known cured HCV infection, HCV RNA 
can be tested directly at Screening.  
• 
Repeat baseline labs for ALT, AST, total bilirubin, ALP, CPK collected at least 4 
weeks from initial screening. 
• 
Urine samples will be collected for Urinalysis  
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
If needed, a retesting of any laboratory results may be performed during the screening 
window to determine the eligibility for the study.  
6.3. 
Day -14 Deuterated Water Run In 
Potential Subjects who complete all of the Screening Visit assessments and who 
continue to be eligible for the study, will return to the research center to complete the 
Deuterated Water Run In.  The visit is to be done on Day -14 + 3 days, provided that the 
subject completes 7 consecutive days of deuterated water administration.  The 
following procedures will be performed at the Day -14 Visit: 


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• Dispense deuterated water (D2O). Instruct patient to drink 50ml of deuterated 
water 3 times per day starting on Day-14 for 7 consecutive days (until day-8).  
• Blood sample for baseline labeling by D2O (kinetic biomarkers) (patients should 
be instructed to fast the evening prior to the visit to ensure a 10-hour fast) 
•  Adverse Events assessment  
• Record concomitant medication taken 
Patients will return at Day-11, Day-7 (+ 1 day) and Day 1 for blood sampling (labeled 
D2O) 
6.4. 
Treatment Period Assessments 
6.4.1. Day 1 Visit 
The following procedures will be performed at the Day 1 Visit prior to dose administration:  
• 
Review inclusion/exclusion criteria 
• 
Complete physical examination 
• 
Weight, BMI 
• 
Record vital signs  
• 
Record concomitant medication taken  
• 
Adverse Events assessment  
• 
MRE (may be done within + 5 days of the visit)  
• 
Fibroscan 
• 
Blood samples will be collected  
• 
Urine samples will be collected for Urinalysis  
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
• 
Dispense the study drug as directed 
• 
Review study drug administration instructions with subject  
• 
Administer 500 mg of NTZ with water & food 
6.4.2. Week 4 and Week 8 Visit + 3 days 


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The following procedures will be performed at these Visits:  
• 
Record concomitant medication taken  
• 
Adverse Events assessment  
• 
Blood samples will be collected  
• 
Urine samples will be collected for Urinalysis  
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
• 
Dispense the study drug as directed 
• 
Review study drug compliance and drug administration instructions with subject  
• 
Reconcile study drug administration using pill counts  
• 
Administer 500 mg of NTZ with water & food 
6.4.3. Week 12 Visit + 3 days 
The following procedures will be performed at the Visit:  
• 
Complete physical examination 
• 
Weight, BMI 
• 
Record vital signs  
• 
Record concomitant medication taken  
• 
Adverse Events assessment  
• 
MRE (may be done within + 5 days of the visit) 
• 
Blood samples will be collected  
• 
Urine samples will be collected for Urinalysis  
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
• 
Dispense the study drug as directed 
• 
Review study drug compliance and drug administration instructions with subject  
• 
Reconcile study drug administration using pill counts  
• 
Administer 500 mg of NTZ with water and food 
6.4.4. Week 16 and Week 20 visits ±3 days 
The following procedures will be performed at these Visits:  


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• 
Record concomitant medication taken  
• 
Adverse Events assessment  
• 
Blood samples will be collected  
• 
Urine samples will be collected for Urinalysis  
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
• 
Dispense the study drug as directed 
• 
Review study drug compliance and drug administration instructions with subject  
• 
Reconcile study drug administration using pill counts  
• 
Administer 500 mg of NTZ with water & food 
6.4.5. Week 22 Visit + 3 days (Day 155) 
The following procedures will be performed at the Visit:  
• 
Record concomitant medication taken  
• 
Adverse Events assessment  
• 
Blood samples will be collected for D2O labeling 
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]) 
• Dispense Deuterated water. Instruct patient to drink 50ml of deuterated water 
3 times per day starting on Day 155 for 7 consecutive days (until day 162).  
• Blood sample for kinetic biomarkers analysis (patients should be instructed to 
fast the evening prior to the visit to ensure a 10-hour fast) 
Patients will return for blood sampling for kinetic biomarker analysis at 22weeks/+ 3 days),  
Day 158, Day 162 (+ 1 day), and at day 169 (Week 24). 
6.4.6. Week 24 Visit +1 
The following procedures will be performed at the visit: 
• 
Complete physical examination 
• 
12-lead ECG 
• 
Record vital signs  
• 
Record weight, BMI  


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• 
Record concomitant medication taken  
• 
MRE (may be done within + 5 days of the visit) 
• 
Fibroscan  
• 
Adverse Events assessment  
• 
Blood samples will be collected  
• 
Urine samples will be collected for Urinalysis  
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
• 
Review study drug compliance and drug administration instructions with subject  
• 
Reconcile study drug administration using pill counts  
6.5. 
Follow Up Period 
6.5.1. 4-Week Post Treatment Follow up (EOS – End of study visit) ±3 days 
The following procedures will be performed at the visit: 
• Complete physical examination 
• Weight, BMI 
• Record vital signs  
• Record concomitant medication taken  
• Adverse Events assessment  
• Blood samples will be collected Urine samples will be collected for Urinalysis  
• Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
6.5.2. Early Termination (ET) 
Patients who have discontinued from the study after the first dose of study medication and for 
any reason will have the following procedures completed: 
• 
Complete physical examination 
• 
Record vital signs 
• 
Weight, BMI  
• 
ECG 
• 
Record concomitant medication taken  
• 
MRE (may be done within + 5 days of the visit) 


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• 
Fibroscan 
• 
Adverse Events assessment  
• 
Blood samples will be collected  
• 
Urine samples will be collected for Urinalysis  
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]) 
• 
Review study drug compliance with subject  
• 
Reconcile study drug administration using pill counts  
6.5.3. 
Unscheduled Visits 
An unscheduled visit is defined as any visit to the study unit outside of the protocol-evaluation 
time points where the patient is seen by study unit personnel, e.g., when follow-up 
assessments are required for safety reasons or when repeat measurements are required out of 
the screening period (either to confirm a measurement or in case of errors, measuring device 
failure, etc.).  
Unscheduled visits will be needed for patients who may require further follow-up due to 
safety. 
6.6. 
Assessment for Premature Discontinuation from Study 
Study medication may be discontinued in the following instances: 
• Intercurrent illness that would, in the judgment of the investigator, affect 
assessments of clinical status to a significant degree. Following resolution of 
intercurrent illness, the subject may resume study dosing at the discretion of the 
investigator. 
• Subject noncompliance  
• Investigator discretion  
• Study medication must be discontinued in the following instances:  
o 
Unacceptable toxicity,  or toxicity that, in the judgment of the 
investigator, compromises the ability to continue study-specific 
procedures or is considered to not be in the subject’s best interest  
o 
Subject request to discontinue for any reason  


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38 
o 
Pregnancy  
o 
Discontinuation of the study at the request of a regulatory agency or an 
institutional review board or independent ethics committee (IRB/IEC) 
Pre-specified stopping criteria are defined at the patient level and at the study level. These are 
described in the following sections 6.6.1., 6.6.2., and 6.6.3. In all cases, when the stopping 
criteria are met, the Data Safety Monitoring Board will perform causality assessment, and 
decide whether the study drug/study trial may be continued (See section 6.6.4.). 
 
6.6.1. 
Pre-specified Patient Discontinuation rules 
If a patient experiences a Grade IV Common Terminology Criteria for Adverse Event (CTCAE) or 
meets liver injury criteria as listed in section 6.6.3., the study drug should be interrupted. After 
causality assessment by the DSMB, if the AE or lab abnormality is considered unrelated to 
study drug, and the event has resolved or returned to baseline, then the study drug may be re-
started. If the event reoccurs after restart of study drug, then the study drug will be 
permanently discontinued. 
6.6.2. 
Pre-specified Trial Discontinuation rules 
If one patient experiences a Grade V CTCAE, or 2 patients experience the same Grade IV 
CTCAE, or 3 patients experience the same Grade III CTCAE, or 4 patients experience the same 
Grade II CTCAE, the trial will be paused. The DSMB will assess causality of these adverse 
events; if the events are deemed unrelated to study drug, the trial will continue.  
6.6.3. 
Dose Adjustment 
Enrolled subjects who experience treatment emergent adverse events should be monitored 
closely and may have the NTZ dose adjusted at the Principal Investigator’s discretion.  
Enrolled subjects who experience a possible treatment emergent adverse event will be 
assessed for symptoms and further biochemical assessments as follows: 
• GI Adverse Events: the subject will be assess for abdominal pain, loss of 
appetite, nausea and vomiting.  Blood will be collected for amylase and 
lipase assessment to rule out pancreatitis.   


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• Kidney Function Adverse Events: the subject will have blood samples 
collected for eGFR to rule out kidney dysfuction. 
• Musculoskeletal Adverse Events: the subject will be assessed for fatigue, 
muscle aches and pain. Blood samples will be collected for Creatinine 
Kinase. 
The following procedure will be followed for dose adjustments: 
1. Patients will be asked to hold drug for 3 days 
2. After the 3-day drug hold, patients will be reassessed for adverse event symptoms.   
• If the AE symptoms have resolved following 3 days of drug hold, the patient will 
discontinue NTZ dosing permanently and will be followed per the schedule of 
assessments. 
If the AE symptoms have not resolved following 3 days of drug hold, the patient will be 
restarted on a reduced dose of 50 mg NTZ, once a day for 7 days.  Patients will be evaluated 
for treatment emergent adverse events and consideration for resuming the dosage of 500mg 
NTZ twice a day.  PI may determine that the patient should remain at the 500 mg NTZ once a 
day dose following 7 days of reduced dose. In such scenarios, the patient will be monitored 
and consideration for resuming the dosage of 500 mg NTZ twice daily will be evaluated at 
each subsequent visit. 
6.6.4. 
Specific Liver Function Monitoring after Start of Treatment 
Liver function monitoring guidelines are detailed below. The cases will be reviewed by the 
DSMB, who will assess potential Drug Induced Liver Injury (DILI), and apply discontinuation 
rules as described in 6.6.1., 6.6.2., and in this section. 
Study drug interruption 
Study drug must be interrupted in the following cases:  
• ALT >8x ULN  (if ALT is normal at baseline) 
• ALT > 5x Baseline (if ALT is elevated at baseline) OR ALT>500 U/L 
• ALT >5x ULN (if ALT is normal at baseline) AND Total Bilirubin>2xULN 
• ALT >3x Baseline (if ALT is elevated at baseline) OR ALT>300 U/L AND total 
bilirubin>2xULN  
• ALT >5x ULN (if ALT is normal at baseline) AND liver-associated symptoms 


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• ALT >3x Baseline (if ALT is elevated at baseline) OR ALT>300 U/L AND liver-associated 
symptoms 
In all these cases, close monitoring should be initiated, with repeat liver biochemistries within 
24-72 hours. Continue monitoring twice/week until liver biochemistries resolve, stabilize, or 
return within baseline values. If abnormalities stabilize and patient is asymptomatic, then 
monitoring should be performed once a week. History of symptoms, disease, concomitant 
medication, or other conditions such as alcohol consumption or alcohol hepatitis should be 
obtained for competing etiologies. The cases will be reviewed by the DSMB, who will 
determine if the patient may restart study drug (if causality assessment is deemed to be not 
related to study drug). 
 
Management of liver biochemistries elevations 
In the following cases of liver enzyme increase, liver biochemistries including ALT, AST, Total 
bilirubin, ALP, should be repeated within 2-5 days. Follow up of symptoms should be carefully 
monitored. Frequency of monitoring should continue once/week until liver abnormalities 
resolve, stabilize, or return to values below the thresholds mentioned below: 
• ALT >5x ULN  (if ALT is normal at baseline) 
• ALT >3x Baseline (if ALT is elevated at baseline) OR ALT >300 U/L 
NOTE: Laboratory assessments should be done centrally. If the patient lives remotely, he/she 
can have laboratory tests performed locally, and the results communicated to the investigator 
promptly. 
 
6.6.5. 
Data Safety Monitoring Board 
An independent Data Safety Monitoring Board (DSMB) composed of 3 liver experts will review 
the safety of patients enrolled in the trial. The DSMB will perform a safety data review every 3 
months, the first review occurring 3 months after first patient randomized. A DSMB charter 
will define the role, responsibilities, rules, and tasks of the DSMB. 


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The DSMB will ensure that the patient and trial discontinuation rules are respected and will 
determine the causality assessment of the events described in sections 6.6.1, 6.6.2., and 6.6.3., 
in order to make the recommendation for trial /patient discontinuation. If the events are 
deemed unrelated to the study drug by the DSMB, then the patient or the trial will continue. 
 
 
 


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42 
  
 
6.7. 
Description of Assessments 
6.7.1. Histological Assessments 
All possible attempts should be made to acquire a liver biopsy specimen of at least 2.0 cm in 
length, from a 16 gauge needle to ensure accurate staging of fibrosis and other histological 
lesions.  A historical biopsy within 6 months of the Screening visit may be accepted as the 
Screening biopsy. The liver biopsy sample must be deemed adequate for evaluation by the 
pathologist for inclusion. Liver biopsies will be assessed by a local reader.  This assessment will 
include an assessment of the adequacy of the specimen as well as the fibrosis stage and a 
determination that the biopsy is consistent with NASH. If either the historical or screening liver 
biopsy is deemed unacceptable, it may be repeated. 
6.7.2. Assessment of newly synthesized proteins 
Kinetic biomarkers will be analyzed to evaluate the pharmacodynamic effect of NTZ on 
fibrogenesis. Blood samples analyses will be performed after deuterated water intake before 
and after 24 weeks of treatment. The assessment will involve the analysis of the change 
(absolute and relative) from baseline between the post-dose and pre-dose deuterated water 
loading periods. 
6.7.3. Biological Assessments (Table 2) 
Chemistry:  
HbA1c, fasting plasma glucose, insulin, HOMA-IR, fructosamine, alkaline phosphatase, ALT, 
AST, GGT, CPK, total and conjugated bilirubin, ferritin, creatinine, eGFR, uric acid, BUN, total 
cholesterol, HDL-C, LDL-C, triglycerides, albumin, lipase, amylase, electrolytes (sodium, 
potassium, chloride, calcium), MELD 
Hematology:  
Hematocrit (Hct), hemoglobin (Hb), platelet count, red blood cell count (RBC), white blood cell 
count (WBC) with differential (absolute and percentage) including lymphocytes, monocytes, 


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neutrophils, eosinophils, and basophils and, reticulocyte count and mean corpuscular volume 
(MCV).  
Inflammatory markers:  
hsCRP, fibrinogen, haptoglobin, resistin, TNF-α, TGF-β, IL-6, PAI-1 
Coagulation Panel:  
Prothrombin time (PT), and international normalized ratio (INR)  
Pregnancy Tests:  
Urine β-hCG (if positive, requires immediate confirmation with Serum β-hCG)  
Urinary Tests:  
Urinary dipstick analysis, urinary albumin, urinary creatinine, urinary ACR 
Additional Tests:  
Serology (HIV-1, HBsAg, HCVAb & HCV RNA (as needed)), serum cystatin C, troponin T, NT 
proBNP 
Fibrosis Biomarker tests:  
ELFTM test score, PIIINP, Hyaluronic acid, CK18, TIMP-1, YKL-40, Alpha 2 macroglobulin, miRNA, 
FGF19, FGF21, ProC3 and ProC6.  
A laboratory manual will be provided 
The manual will outline the collection and processing requirements for the laboratory. Blood 
sampling will be performed by trained personnel. Blood samples will be processed and shipped 
as outlined in the laboratory manual. Refer to the laboratory manual for exact amounts of 
blood required for each test.  
The option to retest during the study is left to the Investigator’s judgment.  
Additional serum and plasma samples will be maintained for future analysis 
 
 


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Table 2 Biological assessments 
 
Screening period 
 
 
Treatment period 
Visit 
SV 
 
S
V2 
SV3 
S
V
4 
V
1 
 
V
2 
V
3 
V4 
 
V5 
V6 
V7 
V8 
V9 
V10 
 
EOS 
 
ET 
Day 
-42 
-
14 
-11 
-7 
1 
29 
57 
85 
113 
141 
155 
158 
162 
169 
+28 
days 
 
Week 
-6 
-2 
 
 
0 
4 
8 
12 
16 
20 
22 
 
 
24 
28 
 
Labs - 
Haematology 
haemoglobin, 
haematocrit, RBC, 
WBC, differential 
count, platelet 
count, reticulocytes 
count, MCV) 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
 
 
X 
Coagulatio (PT, 
INR) 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Labs- Urinary 
Pregnancy tests 
X 
 
 
 
 
X 
X 
X 
X 
X 
 
 
 
X 
X 
 
X 
Labs – Serology 
HIV, HBs and HCV 
serology 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Labs – 
Biochemistry 
HbA1c, fasting 
plasma glucose, 
insulin, HOMA-IR, 
fructosamine, 
alkaline 
phosphatase, ALT, 
AST, GGT, CPK, 
total and conjugated 
bilirubin, ferritin, 
creatinine, eGFR, 
uric acid, BUN, 
total cholesterol, 
HDL-C, LDL-C, 
triglycerides, 
albumin, lipase, 
amylase, 
electrolytes 
(sodium, potassium, 
chloride, calcium), 
MELD 
X 
 
 
X1 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Inflammatory 
markers 
hsCRP, fibrinogen, 
haptoglobin, 
resistin,  TNF-, 
TGF-, IL-6, PAI-1 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 


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45 
 
Screening period 
 
 
Treatment period 
Visit 
SV 
 
S
V2 
SV3 
S
V
4 
V
1 
 
V
2 
V
3 
V4 
 
V5 
V6 
V7 
V8 
V9 
V10 
 
EOS 
 
ET 
Day 
-42 
-
14 
-11 
-7 
1 
29 
57 
85 
113 
141 
155 
158 
162 
169 
+28 
days 
 
Biomarkers of 
fibrosis 
ELF Score, PIIINP, 
Hyaluronic acid, 
CK18, TIMP-1, 
YKL-40, Alpha 2 
macroglobulin,, 
,miRNA, ProC3, 
ProC6, FGF19, 
FGF21 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
NAFLD Fibrosis 
Score (NAS) & 
FIB-4 
 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
 
X 
Labs – Urinalysis 
dipstick  
Specific gravity, pH, 
protein, glucose, 
ketones, bilirubin, 
urobilinogen, blood, 
nitrite, leukocytes 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
 
 
 
X 
Safety markers 
Serum Cystatin C, 
troponin T, NT 
proBNP 
Urinary albumin, 
urinary creatinine, 
urinary ACR 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Labs -- FSR 
 
X 
X 
X 
X 
 
 
 
 
 
X 
X 
X 
X 
 
 
Sampling for 
additional 
parameters 
X 
 
 
 
X 
 
 
X 
 
X 
 
 
 
X 
X 
X 
 
6.7.4. Electrocardiogram 
Standard 12-lead electrocardiogram (ECG) assessments will be performed. The Investigator 
will review the ECGs for any clinically significant abnormalities to ensure subject safety. 
Abnormal ECG findings that are considered clinically significant by the Investigator and meet 
the definition of an AE should be reported and recorded in the AE eCRF page.  
6.7.5. MRE 


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46 
Liver stiffness will be assessed by MRE. It is recommended that each subject’s radiological 
assessment is performed using the same procedure for each study visit.  Exploratory measures 
for MRI/MRE sequence development will be maintained for future analysis.  
6.7.6. Fibroscan 
Liver stiffness will be assessed by FibroScan®. It is required that each subject’s FibroScan® 
assessments be done with the same type of probe at each study visit.  
6.7.7. Medical History 
Medical history including details regarding illnesses and allergies, date(s) of onset, and 
whether condition(s) is currently ongoing, and medication history, including nicotine and 
alcohol use, will be collected on all subjects during screening. 
6.7.8. Physical Examination 
A complete physical examination should include source documentation of general appearance, 
and the following body systems: head, neck, and thyroid; eyes, ears, nose, throat, mouth, and 
tongue; chest (excluding breasts); respiratory; cardiovascular; lymph nodes; abdomen; skin, 
hair, nails; musculoskeletal and neurological.  
The patient’s weight will be measured under the same conditions at each visit where the 
procedure is required. Where possible, the scale for weight must be the same for a given 
patient throughout the visits. 
6.7.9. Vital Signs 
Blood pressure (mmHg) and pulse rate (beats per minute) will be measured at each visit using 
automated blood pressure equipment. 
Important points for clinical blood pressure measurement  
• 
The patient should be seated comfortably with the back supported and the 
upper arm bare without constrictive clothing. The legs should not be crossed. 
• 
The arm should be supported at heart level, and the bladder of the cuff should 
encircle at least 80% of the arm circumference.  


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• 
Neither the patient nor the observer should talk during the measurement.  
Systolic BP and diastolic BP will be measured after 5 minutes rest in the seating.  Where 
possible, the blood pressure equipment should be the same for a given patient throughout the 
visits. 
6.7.10. Pregnancy Testing 
Urinary pregnancy tests will be done on WOCBP.  
7. Adverse Events and Toxicity Management 
7.1. Definitions of Adverse Events, Serious Adverse Events and Unexpected Events 
7.1.1. Adverse Events 
An adverse event (AE) is any untoward medical occurrence in a clinical study subject 
administered a medicinal product, which does not necessarily have a causal relationship with 
the treatment. An AE can therefore be any unfavorable and/or unintended sign, symptom, or 
disease temporally associated with the use of a 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 specified procedures, overdose, drug abuse/misuse reports, 
or occupational exposure. Preexisting events that increase in severity or change in nature 
during or as a consequence of participation in the clinical study will also be considered AEs.  
An AE does not include the following:  
• 
Medical or surgical procedures such as surgery, endoscopy, tooth extraction, 
and transfusion. The condition that led to the procedure may be an adverse 
event and must be reported  
• 
Pre-existing diseases, conditions, or laboratory abnormalities present or 
detected before the Screening visit that do not worsen  
• 
Situations where an untoward medical occurrence has not occurred (eg., 
hospitalization for elective surgery, social and/or convenience admissions)  
• 
Overdose without clinical sequelae.   


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• 
Any medical condition or clinically significant laboratory abnormality with an 
onset date before the consent form is signed and not related to a protocol-
associated procedure is not an AE. It is considered to be pre-existing and should 
be documented on the medical history eCRF. 
7.1.2.  Clinical Laboratory Abnormalities and Other Abnormal Assessments as Adverse 
Events or Serious Adverse Events 
Laboratory abnormalities without clinical significance are not recorded as AEs or SAEs. 
However, laboratory abnormalities (eg, clinical chemistry, hematology, and urinalysis) that 
require medical or surgical intervention or lead to study drug interruption, modification, or 
discontinuation must be recorded as an AE, as well as an SAE, if applicable. In addition, 
laboratory or other abnormal assessments (eg, electrocardiogram, x-rays, vital signs) that are 
associated with signs and/or symptoms must be recorded as an AE or SAE if they meet the 
definition of an AE or SAE as described above.  If the laboratory abnormality is part of a 
syndrome, record the syndrome or diagnosis (eg, anemia), not the laboratory result (ie, 
decreased hemoglobin). 
7.1.3. Serious Adverse Events 
A serious adverse event (SAE) is defined as an event that, at any dose, results in the following:  
• 
Death  
• 
Life-threatening (Note: The term “life-threatening” in the definition of 
“serious” refers to an event in which the subject was at risk of death at the 
time of the event; it does not refer to an event that hypothetically might 
have caused death if it were more severe.) 
• 
In-patient hospitalization or prolongation of existing hospitalization 
• 
Persistent or significant disability/incapacity  
• 
A congenital anomaly/birth defect  
• 
A medically important event or reaction: such events may not be 
immediately life-threatening or result in death or hospitalization but may 
jeopardize the subject or may require intervention to prevent one of the 


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other outcomes constituting SAEs. Medical and scientific judgment must be 
exercised to determine whether such an event is a reportable under 
expedited reporting rules. 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 
hospitalization; and development of drug dependency or drug abuse. For the 
avoidance of doubt, infections resulting from contaminated medicinal 
product will be considered a medically important event and subject to 
expedited reporting requirements. 
7.1.4. Unexpected adverse event  
Expectedness is assessed by the Sponsor-Investigator. An unexpected AE is defined as an event 
that has a nature of severity or specificity that is not consistent with the applicable Package 
Insert or that is symptomatically and pathophysiologically related to a known toxicity but 
differs because of a greater severity or specificity.  
“Unexpected” refers to an event that has not been previously observed and reported rather 
than an event that has not been anticipated based on the properties of the drug. 
7.2. 
Assessment of Adverse Events & Serious Adverse Events 
The Investigator will establish whether or not any AE have occurred at each visit from the date 
of consent. The patient will be questioned in a general manner to determine specific 
symptoms without offering the patient any suggestion. The investigator or qualified sub-
investigator is responsible for assessing AEs and SAEs for causality and severity, and for final 
review and confirmation of accuracy of event information and assessments. 
7.2.1.  Intensity assessment  
The intensity of the AE will be graded as follows:  
• 
Mild: Awareness of signs or symptoms, but easily tolerated and are of 
minor irritant type causing no loss of time from normal activities. 


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Symptoms do not require therapy or a medical evaluation; signs and 
symptoms are transient.  
• 
Moderate: Events introduce a low level of inconvenience or concern to 
the participant and may interfere with daily activities, but are usually 
improved by simple therapeutic measures; moderate experiences may 
cause some interference with functioning. 
• 
Severe: Events interrupt the participant’s normal daily activities and 
generally require systemic drug therapy or other treatment; they are 
usually incapacitating. 
7.2.2.  Relation to the study treatment  
The Investigator will make a clinical and scientific judgment regarding whether or not the AE 
was related to study treatment. The Investigator will evaluate any changes in laboratory 
values, make a determination as to whether or not the change is clinically important, and 
whether or not the changes were related to study drug. However, even if the Investigator feels 
there is no relationship to the study drug, the AE or clinically significant laboratory abnormality 
must be recorded in the eCRF.  
The Investigator will record the relation to the study treatment according to the following 
causality terms:  
• 
Related: the AE follows a reasonable temporal sequence from the time 
of drug administration and it cannot be explained by the patient's 
clinical state or the study procedures/conditions. The AE abates upon 
discontinuation of the study drug and reappears when the study drug is 
introduced.  
• 
Possibly related: the AE follows a reasonable temporal sequence from 
the time of drug administration, but could have been produced by the 
patient's clinical state or the study procedures/conditions.  


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• 
Unlikely related: the temporal association between the AE and the study 
drug is such that the study drug is not likely to have any reasonable 
association with the AE. The relationship is not likely because of other 
plausible explanations.  
• 
Not related: the AE must definitely be caused by the patient's clinical 
state or the study procedure/conditions. A reasonable explanation must 
be given, e.g., no investigational product taken, preplanned elective 
medical intervention, or incompatible temporal relationship.  
• 
Not assessable: the report suggesting an adverse reaction cannot be 
judged because information is insufficient or contradictory and data 
cannot be supplemented or verified. 
7.2.3. 
 Action taken and outcome  
The Investigator will record the action taken with drug and outcome of the event for each AE 
according to the following:  
Action taken with investigational drug  
• 
Drug permanently withdrawn – in case a patient is permanently 
withdrawn from the study drug  
• 
Drug temporarily withdrawn – in case the study drug is temporarily 
withdrawn  
• 
Dose not changed – in case no action is taken regarding the study drug  
• 
Unknown  
• 
Not applicable – an AE started before initiation of treatment with study 
drug, the treatment had been completed prior to reaction/event, or the 
patient has died. 
Outcome  
• 
Recovered/resolved  


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• 
Recovering/resolving  
• 
Not recovered/not resolved  
• 
Recovered/resolved with sequelae  
• 
Fatal  
• 
Unknown.  
Note: In case of irreversible congenital anomalies the choice not recovered/not resolved 
should be used. “Fatal” should be used when death is possibly related to the reaction/event. 
7.3. Reporting 
7.3.1.  Reporting an adverse event  
All AEs regardless of seriousness or relationship to study drug, including those occurring during 
the Screening Period, are to be recorded on the corresponding page(s) of the eCRF and in the 
patient’s medical record from the ICF signature until study end for each patient. Whenever 
possible, symptoms should be grouped as a single syndrome or diagnosis. The Investigator 
should specify the date of onset, maximal intensity, action taken with respect to study drug, 
corrective therapy given, outcome, and his/her opinion as to whether there is a reasonable 
possibility that the AE was caused by the study drug.  
Adverse event reporting begins from signature of the patient ICF at the first Screening Visit and 
ends at study end for each patient. 
7.3.2. Initial reports 
All SAEs occurring from the time of informed consent until 30 days following the 
last administration of study drug must be reported to Medpace Clinical Safety 
within 24 hours of the knowledge of the occurrence (this refers to any adverse 
event that meets any of the aforementioned serious criteria). All SAEs that the 
Investigator considers related to study drug occurring after the 30-day follow-up 
period must be reported to the Sponsor. 
To report the SAE, complete the SAE form electronically in the electronic data 
capture (EDC) system for the study. When the form is completed, Medpace Safety 


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personnel will be notified electronically and will retrieve the form. If the event 
meets serious criteria and it is not possible to access the EDC system, send an 
email to Medpace Safety at medpace safetynotification@medpace.com or call the 
Medpace SAE hotline (phone number listed below), and fax the completed paper 
SAE form to Medpace (fax number listed below) within 24 hours of awareness. 
When the EDC system becomes available, the SAE information must be entered 
within 24 hours of the system becoming available.  
7.3.3. Follow-up reports 
The Investigator must continue to follow the patient until the SAE has subsided or 
until the condition becomes chronic in nature, stabilizes (in the case of persistent 
impairment), or the patient dies. 
Within 24 hours of receipt of follow-up information, the Investigator must update 
the SAE form electronically in the EDC system for the study and submit any 
supporting documentation (eg, patient discharge summary or autopsy reports) to 
Medpace Clinical Safety via fax or e-mail. If it is not possible to access the EDC 
system, refer to the procedures outlined above for initial reporting of SAEs. 
All SAEs independent of the circumstances or suspected cause must be reported on a SAE 
Form/captured in EDC. The SAE Form should include a clearly written narrative describing 
signs, symptoms, and treatment of the event, diagnostic procedures, as well as any relevant 
laboratory data and any sequelae, in order to allow a complete medical assessment of the case 
and independent determination of the possible causality.  
It is critical that the information provided on the initial or follow-up SAE Form matches the 
information recorded in the source documents and the eCRF for the same event.  
Any unexpected safety issue that changes the risk benefit analysis and is likely to have an 
impact on the patients who have participated in the trial will be reported as soon as possible 
to the Competent Authority(ies) concerned together with proposed actions. 
7.3.4. Follow-up  


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The Investigator should take all appropriate measures to ensure the safety of the patients, 
notably he/she should follow up the outcome of any AE until the return to normal or until 
stabilization of the patient’s condition.  
The patient must be followed up until clinical recovery is complete and laboratory results have 
returned to normal, or until progression has been stabilized. This information should be 
documented in the patient’s medical records.  
7.4. Post Study Reporting Requirements 
Any SAEs and deaths that occur within 30 days of the last dose of the study drug, regardless of 
causality, should be reported.  
Any SAE that is brought to the attention of the Investigator at any time after the reporting 
period and which is considered by him/her to be caused by the study drug within a reasonable 
possibility, should be reported.  
7.5. Special Situation Reports 
Special situations reports include pregnancy reports, reports of medication error, abuse, 
misuse or overdose, and reports associated with product complaints.  
7.5.1. Pregnancy  
In case of pregnancy, the Investigator will notify regulatory authorities and IRB within 24 hours 
of his/her knowledge of the pregnancy.  
Female patients must be instructed to discontinue the study drug immediately and inform the 
Investigator as soon as possible once they are aware of being pregnant or suspect that they 
are pregnant during the study or within 30 days of the last dose of the study drug.  
Female patients will be requested, as part of the general ICF, to provide informed consent to 
allow reasonable attempts to be made to obtain information on any possible medicinal 
product exposure to an embryo or fetus and to follow up on the outcome of the pregnancy.  
The Investigator will contact the patient at the expected time of delivery for follow-up.  
7.5.2. Medication error  


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Medication error is defined as an unintentional error in the prescribing, dispensing, or 
administration of a medicinal product while in the control of the healthcare professional, 
patient, or consumer. All medication errors will be documented in the eCRF and, in case of any 
potential risk to patient safety, would be reported as appropriate.  
7.5.3. Misuse  
This refers to situations where the medicinal product is intentionally and inappropriately used 
not in accordance with the authorized product information and will be reported in the eCRF. 
All misuse will be documented in the eCRF and, in case of any potential risk to patient safety, 
would be reported as appropriate. 
7.5.4. 
 Overdose  
This refers to the administration of a quantity of a medicinal product given per administration 
or cumulatively, which is above the maximum recommended dose according to the authorized 
product information. Clinical judgment should always be applied. Doses >4000 mg in one day 
are considered overdose. 
7.5.5. 
 Abuse  
This corresponds to the persistent or sporadic, intentional excessive use of a medicinal 
product, which is accompanied by harmful physical or psychological effects. 
8. 
Statistical Considerations 
This section is an overview of the principal elements of the statistical analysis for this study. 
Further details will be contained in a separate statistical analysis plan (SAP). 
8.1. 
Analysis Endpoints 
8.1.1. Primary Endpoint 
The primary endpoint is to assess the safety and tolerability of NTZ after 24 weeks of treatment 
8.1.2. Secondary Endpoints 
The secondary endpoints are: 


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• To assess the % change in FSR from baseline to end of treatment (24 weeks of 
treatment) 
• To assess the change in liver stiffness by MRE from baseline to 12 weeks and 24 weeks 
of treatment 
• To assess the change in liver stiffness by fibroscan from baseline to 24 weeks of 
treatment  
• To assess the change from baseline to 12 weeks and 24 weeks of treatment of fibrosis 
serum biomarkers and scores  
8.2. 
Analysis Sets 
The Full Analysis Set will consist of all subjects that meet the eligibility criteria and enroll into 
the study (Visit 1 Day 1). 
The Safety Set will consist of all enrolled subjects who receive at least one dose of study drug.  
8.3. 
Data Handling Conventions 
Continuous variables will be summarized with descriptive statistics (i.e., sample size, mean, 
standard deviation, minimum, maximum, median, and quintiles). Categorical variables will be 
summarized with descriptive statistics (i.e., sample size, frequency counts, and percentage). 
Statistical testing on change from baseline values will be performed using analysis of variance 
techniques with a term for baseline in the model. Statistical significance will be considered to 
have been reached at an alpha level of 0.05. 
Baseline value is measured on Day 1 of the treatment period before the administration of NTZ. 
8.4. 
Demographic Data and Baseline Characteristics 
Demographic and baseline characteristics will be descriptively summarized. Quantitative 
and/or categorical summaries will be presented for demographics and other baseline 
characteristics. Demographic and baseline characteristics will be summarized using the Full 
Analysis Set. 
8.5. 
Primary Safety Analysis 
Safety and tolerability will be assessed by the following parameters:  


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AEs including TEAEs and SAEs, laboratory evaluations, vital signs, ECGs, and physical 
examinations.  
8.5.1. Adverse Events 
Treatment-emergent adverse events (TEAEs) are defined as AEs that worsen or commence on 
or after the time of start of first study drug administration. AEs, including SAEs, will be coded 
using MedDRA. The number and percentage of subjects experiencing TEAE, including TEAEs 
leading to study drug discontinuation and SAEs, will be summarized for each system organ 
class (SOC) and Preferred Term. In addition, AEs will be tabulated according to severity, 
causality, and relation to the study drug.  
Individual data listings for AEs will be presented for each subject.  
8.5.2. Laboratory Evaluations 
Absolute and change from baseline values for laboratory parameters will be summarized by 
visit. In addition, shift from baseline tables will be presented for selected laboratory 
abnormalities.  
8.5.3. Other Safety Evaluations 
Observed values and change from baseline will be summarized descriptively for all vital sign 
parameters and ECG parameters by visit. Individual listings will be presented for each subject. 
Individual data listings for physical examination results will be presented for each subject. 
Safety data will be summarized using descriptive statistics. Further details will be provided in 
the SAP. 
 All safety analyses will be conducted using the Safety Population. 
8.6. 
Secondary Efficacy Analyses 
8.6.1. FSR 
Fractional Synthesis Rate of circulating plasma proteins will be measured at Baseline, and after 
24 weeks of treatment.  


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The change in FSR (absolute and relative) from baseline to end of treatment between the first 
and second deuterated water loading periods will be assessed to determine the effects of NTZ 
500mg BID on de novo collagen synthesis in the Full Analysis Set.  
Wilcoxon signed-rank test will be used for statistical inference. Estimated mean, standard error 
and 95% confidence interval of change in FSR (absolute and relative) change from baseline in 
FSR will be reported and evaluated at a significance level of 0.05. Exploratory analyses may 
also be performed to evaluate the association of individual exploratory biomarkers or 
combination of biomarkers with clinical measurements and other risk factors. 
Additional details will be provided in the SAP. 
8.6.2. Liver stiffness 
The change in liver stiffness from baseline at 24 weeks as measured by FibroScan, and at 12 
weeks and 24 weeks by MRE, the change in non-invasive markers of fibrosis from baseline to 
24 weeks, and the change in fibrosis scores from baseline to 12 weeks, to 24 weeks for the Full 
Analysis Set will be assessed. Summary statistics for the Full Analysis Set will be provided at all 
visits and the change from baseline will be provided. 
8.6.3. Exploratory Analysis 
Additional serum and plasma samples will be maintained for future analysis.  Exploratory 
measures for MRI/MRE sequence development will be maintained for future analysis.  
8.6.4. Interim Analysis 
 An interim analysis is not planned. 
8.7. 
Sample Size 
The sample size of this Phase 2 proof of concept study is based on clinical assessment. Twenty 
male and female subjects with histologically confirmed NASH and fibrosis stage 2 or 3) will 
provide sufficient information to assess the safety and tolerability of NTZ 500mg BID over 24 
weeks. 
9. 
Responsibilities 


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9.1. 
Investigator Responsibilities 
By signing this protocol, the Investigator agrees to conduct the study in an efficient and 
diligent manner and in conformance with this protocol; generally accepted standards of GCP; 
and all applicable local laws, rules and regulations relating to the conduct of the clinical study.  
The Investigator also agrees to allow monitoring, audits, IRB review and regulatory agency 
inspection of trial-related documents and procedures and provide for direct access to all study 
related source data and documents.  
The Investigator shall prepare and maintain complete and accurate study documentation in 
compliance with GCP standards and applicable local laws, rules and regulations; and, for each 
subject participating in the study, provide all data, and upon completion or termination of the 
clinical study submit any other reports to the Funder as required by this protocol or as 
otherwise required pursuant to any agreement with the Funder.  
Study documentation will be promptly and fully disclosed to regulatory authorities by the 
Investigator upon request and also shall be made available at the Investigator’s site upon 
request for inspection, copying, review and audit at reasonable times by representatives any 
regulatory agencies.   
Persons debarred from conducting or working on clinical studies by any court or regulatory 
agency will not be allowed to conduct or work on this study.   
9.2. 
Good Clinical Practice 
The investigator will ensure that this study is conducted in accordance with the principles of 
the Declaration of Helsinki (as amended in Edinburgh, Tokyo, Venice, Hong Kong, and South 
Africa), International Conference on Harmonisation (ICH) guidelines.  These standards are 
consistent with the European Union Clinical Trials Directive 2001/20/EC and Good Clinical 
Practice Directive 2005/28/EC. The investigator will ensure adherence to the basic principles of 
Good Clinical Practice, as outlined in 21CFR312, subpart D, “Responsibilities of Sponsors and 
Investigators,” 21CFR, part50, 1998, and 21CFR, part56, 1998. The investigator and all 
applicable sub investigators will comply with 21CFR, Part54, 1998, providing documentation of 


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their financial interest or arrangements with any study funder, or proprietary interests in the 
investigational drug under study. This documentation must be provided prior to the 
investigator’s (and any sub investigator’s) participation in the study. The investigator and sub 
investigator agree to notify study funder of any change in reportable interests during the study 
and for 1 year following completion of the study. Study completion is defined as the date 
when the last subject completes the protocol-defined activities 
9.3. 
Institutional Review Board Review & Approval 
The investigator will submit this protocol, informed consent form, and any accompanying 
material to be provided to the subject (such as advertisements, subject information sheets, or 
descriptions of the study used to obtain informed consent) to an IRB/IEC.  The investigator will 
not begin any study subject activities until approval from the IRB/IEC has been documented 
and provided as a letter to the investigator. Before implementation, the investigator will 
submit to and receive documented approval from the IRB/IEC any modifications made to the 
protocol or any accompanying material to be provided to the subject after initial approval, 
with the exception of those necessary to reduce immediate risk to study subjects. 
9.4. 
Informed Consent 
The investigator is responsible for obtaining written informed consent from each individual 
participating in this study after adequate explanation of the aims, methods, objectives, and 
potential hazards of the study and before undertaking any study-related procedures. The 
investigator must use the most current IRB/IEC approved consent form for documenting 
written informed consent. Each informed consent will be appropriately signed and dated by 
the subject and the person conducting the consent discussion, and also by an impartial witness 
if required by local requirements. 
9.5. 
Confidentiality 
The investigator will assure that subjects’ anonymity will be strictly maintained and that their 
identities are protected from unauthorized parties. Only subject initials, date of birth, another 
unique identifier (as allowed by local law) and an identification code will be recorded on any 
form or biological sample submitted to the study funder, IRB/IEC or laboratory. Laboratory 


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specimens will be labeled in such a way as to protect subject identity while allowing the results 
to be recorded to the proper subject. Refer to specific laboratory instructions for further 
details. NOTE: The investigator will keep a screening log showing codes, names, and addresses 
for all subjects screened and for all subjects enrolled in the trial. Subject data will be processed 
in accordance with all applicable regulations. The investigator agrees that all information 
received from the study funder, including but not limited to the Package Insert, the IMP, and 
any other study information, remain the sole and exclusive property of the study funder during 
the conduct of the study and thereafter. This information is not to be disclosed to any third 
party (except employees or agents directly involved in the conduct of the study or as required 
by law) without prior written consent from the study funder.  The investigator further agrees 
to take all reasonable precautions to prevent the disclosure by any employee or agent of the 
study site to any third party or otherwise into the public domain. 
9.6. 
Study Files and Retention of Records 
The investigator will maintain adequate and accurate records to enable the conduct of the 
study to be fully documented and the study data to be subsequently verified. These 
documents should be classified into at least the following two categories: (1) investigator’s 
study master file, and (2) subject clinical source documents. The investigator’s study file will 
contain the protocol/amendments, CRF and query forms, IRB/IEC and governmental approval 
with correspondence, informed consent, drug records, staff curriculum vitae and authorization 
forms, and other appropriate documents and correspondence. The required source data 
should include sequential notes containing at least the following information for each subject:  
• 
Subject identification (name, date of birth, gender);  
• 
Documentation that subject meets eligibility criteria, i.e., history, physical 
examination, and confirmation of diagnosis (to support inclusion and exclusion 
criteria); 
• 
Documentation of the reason(s) a consented subject is not enrolled  
• 
Participation in study (including study number);  


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• 
Study discussed and date of informed consent;  
• 
Dates of all visits;  
• 
Documentation that protocol specific procedures were performed;  
• 
Results of efficacy parameters, as required by the protocol;  
• 
Start and end date (including dose regimen) of IMP, including dates of 
dispensing and return;  
• 
Record of all adverse events and other safety parameters (start and end date, 
and including causality and severity);  
• 
Concomitant medication (including start and end date, dose if relevant; dose 
changes); 
• 
Date of study completion and reason for early discontinuation, if it occurs.  
All clinical study documents must be retained by the investigator until at least 2years or 
according to local laws, whichever is longer, after the last approval of a marketing application 
in an ICH region (i.e., United States, Europe, or Japan) and until there are no pending or 
planned marketing applications in an ICH region; or, if no application is filed or if the 
application is not approved for such indication, until 2years after the investigation is 
discontinued and regulatory authorities have been notified. Investigators may be required to 
retain documents longer if specified by regulatory requirements, by local regulations, or by an 
agreement with study funder. The investigator must notify study funder before destroying any 
clinical study records. Should the investigator wish to assign the study records to another party 
or move them to another location, study funder must be notified in advance. If the 
investigator cannot provide for this archiving requirement at the study site for any or all of the 
documents, special arrangements must be made between the investigator and study funder to 
store these records securely away from the site so that they can be returned sealed to the 
investigator in case of an inspection. When source documents are required for the continued 
care of the subject, appropriate copies should be made for storage away from the site. 
9.7. 
Electronic Case Report Forms 


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For each subject consented, an eCRF will be completed by an authorized study staff member 
whose training for this function is documented according to study procedures. The eCRF will 
be completed in a timely manner. Original entries as well as any changes to data fields will be 
stored in the audit trail of the system. At the conclusion of the trial, a read-only archive copy of 
the data entered will be generated. This archive will be stored in accordance with the records 
retention requirements. 
9.8. 
Investigational Medicinal Product Accountability and Return 
The investigator or designee is responsible for ensuring adequate accountability of all used and 
unused study drug.  This includes acknowledgment of receipt of each shipment of study 
product (quantity and condition), subject dispensing records, and returned or destroyed study 
product. Dispensing records will document quantities received from study funder and 
quantities dispensed to subjects, including lot number, date dispensed, subject identifier 
number, subject initials, and the initials of the person dispensing the medication. Drug may be 
returned or destroyed on an ongoing basis during the study, if appropriate.  At the end of the 
study, following final drug inventory reconciliation, the study site will arrange for the return of 
unused investigational medicinal product supplies. All drug supplies and associated 
documentation will be periodically reviewed and verified by the study funder over the course 
of the study. 
9.9. 
Inspections 
The investigator understands that source documents for this trial should be made available to 
appropriately qualified personnel from study funder and its representatives, to IRBs/IECs, or to 
regulatory authority or health authority inspectors. 
9.10. Protocol Compliance 
The investigator is responsible for ensuring the study is conducted in accordance with the 
procedures and evaluations described in this protocol. 
9.11. Protocol Modifications 


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Protocol modifications, except those intended to reduce immediate risk to study subjects, may 
be made by the investigator in collaboration with the study funder. All protocol modifications 
must be submitted to the IRB/IEC in accordance with local requirements. Approval must be 
obtained before changes can be implemented. 
9.12. Study Reports 
A clinical study report (CSR) will be prepared and provided to the regulatory agency (ies). The 
Investigator will ensure that the report meets the standards set out in the ICH Guideline for 
Structure and Content of Clinical Study Reports (ICHE3). Note that an abbreviated report may 
be prepared in certain cases. After conclusion of the study, Investigators in this study may 
communicate, orally present, or publish in scientific journals or other scholarly media only 
after the following conditions have been met:  
• 
The results of the study in their entirety have been publicly disclosed by or with 
the consent of the study funder in an abstract, manuscript, or presentation form. 
9.13. Study Termination 
The Investigator reserves the right to terminate the study at any time. Should this be 
necessary, the investigator will arrange discontinuation procedures and notify the appropriate 
regulatory authorities, IRBs, IECs, and the Funder. In terminating the study, the investigator 
will assure that adequate consideration is given to the protection of the subjects’ interests. 


Protocol No: NTZ-218-1 
Version: 26February 2020 
 
 
65 
10. References 
 
10.1. 
Literature  
Abd-Elsalam, S., Kobtan, A., El-Kalla, F., Elkhalawany, W., Nawasany, S.E., Saif, S.A., Yousef, M., 
Ali, L.A., Soliman, S., Mansour, L., et al. (2016). A 2-week Nitazoxanide-based quadruple 
treatment as a rescue therapy for Helicobacter pylori eradication: A single center experience. 
Medicine (Baltimore) 95, e3879. 
 
Alinia (nitazoxanide): US Prescribing Information. Tampa, FL. Romark, L.C., 2016 
 
Amorosa, V.K., Luetkemeyer, A., Kang, M., Johnson, V.A., Umbleja, T., Haas, D.W., Yesmin, S., 
Bardin, M.C., Chung, R.T., Alston-Smith, B., et al. (2013). Addition of nitazoxanide to PEG-IFN 
and ribavirin to improve HCV treatment response in HIV-1 and HCV genotype 1 coinfected 
persons naive to HCV therapy: results of the ACTG A5269 trial. HIV Clin Trials 14, 274-283. 
 
Assy, N., Kaita, K., Mymin, D., Levy, C., Rosser, B., and Minuk, G. (2000). Fatty infiltration of 
liver in hyperlipidemic patients. Dig Dis Sci 45, 1929-1934. 
 
Bacon, B.R., Shiffman, M.L., Lim, J.K., Berman, A., Rustgi, V.K., and Keeffe, E.B. (2010). A Phase 
II, Randomized, Double-Blind, Placebo-Controlled Study of Nitazoxanide Plus Peginterferon 
and Ribavirin in Naive Patients With Chronic Hepatitis C Genotype 1 Infection: Final Report. 
Gastroenterology 139, e18. 
 
Basu, P.P., Rayapudi, K., Shah, N.J., Krishnaswamy, N., and Brown, R.S. (2013). Effects of triple-
drug therapy with nitazoxanide, high-dose ribavirin and peginterferon-alpha-2a in patients 
with chronic hepatitis C. Hepatol Res 43, 589-595. 
 
Basu PP, Rayapudi K, Esteves J, Brown R. A pilot study utilizing nitazoxanide for hepatic 
encephalopathy in chronic liver failure. Abstract S151 (392) for the 59th Annual Meeting of the 
American Association for the Study of Liver Diseases. 31 Oct-4 Nov. 2008. p1742 
 
Belanger, C., Foucart, C., Negro, E., Dubernet, M., Hum, D.W., Staels, B., and Walczak, R. 
(2017). Drug repurposing screen identifies novel small molecule compounds with potent 
antifibrotic properties. Journal of Hepatology 66, S605. 
 
Carpino, G., Morini, S., Ginanni Corradini, S., Franchitto, A., Merli, M., Siciliano, M., Gentili, F., 
Onetti Muda, A., Berloco, P., Rossi, M., et al. (2005). Alpha-SMA expression in hepatic stellate 
cells and quantitative analysis of hepatic fibrosis in cirrhosis and in recurrent chronic hepatitis 
after liver transplantation. Dig Liver Dis 37, 349-356. 
 
Decaris, M.L., Li, K.W., Emson, C.L., Gatmaitan, M., Liu, S., Wang, Y., Nyangau, E., Colangelo, 
M., Angel, T.E., Beysen, C., et al. (2017). Identifying nonalcoholic fatty liver disease patients 


Protocol No: NTZ-218-1 
Version: 26February 2020 
 
 
66 
with active fibrosis by measuring extracellular matrix remodeling rates in tissue and blood. 
Hepatology 65, 78-88. 
 
Donati, G., Stagni, B., Piscaglia, F., Venturoli, N., Morselli-Labate, A.M., Rasciti, L., and Bolondi, 
L. (2004). Increased prevalence of fatty liver in arterial hypertensive patients with normal liver 
enzymes: role of insulin resistance. Gut 53, 1020-1023. 
 
Friedman, S.L. (2008). Hepatic stellate cells: protean, multifunctional, and enigmatic cells of 
the liver. Physiol Rev 88, 125-172. 
 
Fruci, B., Giuliano, S., Mazza, A., Malaguarnera, R., and Belfiore, A. (2013). Nonalcoholic Fatty 
liver: a possible new target for type 2 diabetes prevention and treatment. Int J Mol Sci 14, 
22933-22966. 
 
Kabil, S.M., Soliman, M.S., Youssef, S.M., and Mounier, B.I. (2011). A controlled study of 
nitazoxanide (NTZ) 3 years after treatment of hepatitis C genotype 4. J Egypt Soc Parasitol 41, 
251-261. 
 
Kohla, M.A., El-Said, H., El-Fert, A., Ehsan, N., Ezzat, S., and Taha, H. (2016). Impact of 
nitazoxanide on sustained virologic response in Egyptian patients with chronic hepatitis C 
genotype 4: a double-blind placebo-controlled trial. Eur J Gastroenterol Hepatol 28, 42-47. 
 
Konerman, M.A., Jones, J.C., and Harrison, S.A. (2018). Pharmacotherapy for NASH: Current 
and emerging. J Hepatol 68, 362-375. 
 
Laufer, N., Abusamra, L., Bolcic, F., Gun, A., Rolon, M.J., Perez, H., Krolewiecki, A., Salomon, H., 
Quarleri, J., and Cahn, P. (2011). No reduction of HCV viral load in HIV patients co-infected with 
HCV genotype 1 during a 30 days course of nitazoxanide monotherapy. Antiviral Res 92, 497-
499. 
 
Leite, N.C., Salles, G.F., Araujo, A.L., Villela-Nogueira, C.A., and Cardoso, C.R. (2009). Prevalence 
and associated factors of non-alcoholic fatty liver disease in patients with type-2 diabetes 
mellitus. Liver Int 29, 113-119. 
 
Macias, J., Lopez-Cortes, L.F., Tellez, F., Recio, E., Ojeda-Burgos, G., Rios, M.J., Rivero-Juarez, 
A., Delgado, M., Rivas, J., and Pineda, J.A. (2015). Low Efficacy of Pegylated Interferon plus 
Ribavirin plus Nitazoxanide for HCV Genotype 4 and HIV Coinfection. PLoS One 10, e0143492. 
 
Musher, D.M., Logan, N., Bressler, A.M., Johnson, D.P., and Rossignol, J.F. (2009). Nitazoxanide 
versus vancomycin in Clostridium difficile infection: a randomized, double-blind study. Clin 
Infect Dis 48, e41-46. 
 


Protocol No: NTZ-218-1 
Version: 26February 2020 
 
 
67 
Rossignol, J.F. (2006). Nitazoxanide in the treatment of acquired immune deficiency syndrome-
related cryptosporidiosis: results of the United States compassionate use program in 365 
patients. Aliment Pharmacol Ther 24, 887-894. 
 
Rossignol, J.F. (2014). Nitazoxanide: a first-in-class broad-spectrum antiviral agent. Antiviral 
Res 110, 94-103. 
 
Rossignol, J.F., Elfert, A., El-Gohary, Y., and Keeffe, E.B. (2009). Improved virologic response in 
chronic hepatitis C genotype 4 treated with nitazoxanide, peginterferon, and ribavirin. 
Gastroenterology 136, 856-862. 
 
Rossignol, J.F., Elfert, A., and Keeffe, E.B. (2010). Treatment of chronic hepatitis C using a 4-
week lead-in with nitazoxanide before peginterferon plus nitazoxanide. J Clin Gastroenterol 
44, 504-509. 
 
Rossignol, J.F., Hidalgo, H., Feregrino, M., Higuera, F., Gomez, W.H., Romero, J.L., Padierna, J., 
Geyne, A., and Ayers, M.S. (1998). A double-'blind' placebo-controlled study of nitazoxanide in 
the treatment of cryptosporidial diarrhoea in AIDS patients in Mexico. Trans R Soc Trop Med 
Hyg 92, 663-666. 
 
Rossignol, J.F., Kabil, S.M., El-Gohary, Y., Elfert, A., and Keeffe, E.B. (2008). Clinical trial: 
randomized, double-blind, placebo-controlled study of nitazoxanide monotherapy for the 
treatment of patients with chronic hepatitis C genotype 4. Aliment Pharmacol Ther 28, 574-
580. 
 
Sasaki, A., Nitta, H., Otsuka, K., Umemura, A., Baba, S., Obuchi, T., and Wakabayashi, G. (2014). 
Bariatric surgery and non-alcoholic Fatty liver disease: current and potential future treatments. 
Front Endocrinol (Lausanne) 5, 164. 
 
Shehab, H.M., Elbaz, T.M., and Deraz, D.M. (2014). Nitazoxanide plus pegylated interferon and 
ribavirin in the treatment of genotype 4 chronic hepatitis C, a randomized controlled trial. Liver 
Int 34, 259-265. 
 
Shehata, M.A., Talaat, R., Soliman, S., Elmesseri, H., and Abd-Elsalam, S. (2017). Randomized 
controlled study of a novel triple nitazoxanide (NTZ)-containing therapeutic regimen versus the 
traditional regimen for eradication of Helicobacter pylori infection. Helicobacter 22. 
 
Shiffman, M.L., Ahmed, A., Jacobson, I.M., Pruitt, R.E., and Keeffe, E.B. (2010). 1188  Phase 2 
randomized, double-blind, placebo-controlled study of nitazoxanide with peginterferon alfa-2a 
and ribavirin in nonresponders (NR) with chronic hepatitis C genotype 1: final report. Journal 
of Hepatology 52, S461. 
 


Protocol No: NTZ-218-1 
Version: 26February 2020 
 
 
68 
Stockis, A., Allemon, A.M., De Bruyn, S., and Gengler, C. (2002a). Nitazoxanide 
pharmacokinetics and tolerability in man using single ascending oral doses. Int J Clin 
Pharmacol Ther 40, 213-220. 
 
Stockis, A., De Bruyn, S., Gengler, C., and Rosillon, D. (2002b). Nitazoxanide pharmacokinetics 
and tolerability in man during 7 days dosing with 0.5 g and 1 g b.i.d. Int J Clin Pharmacol Ther 
40, 221-227. 
 
Subichin, M., Clanton, J., Makuszewski, M., Bohon, A., Zografakis, J.G., and Dan, A. (2015). Liver 
disease in the morbidly obese: a review of 1000 consecutive patients undergoing weight loss 
surgery. Surg Obes Relat Dis 11, 137-141. 
 
Takeuchi-Yorimoto, A., Noto, T., Yamada, A., Miyamae, Y., Oishi, Y., and Matsumoto, M. 
(2013). Persistent fibrosis in the liver of choline-deficient and iron-supplemented L-amino acid-
defined diet-induced nonalcoholic steatohepatitis rat due to continuing oxidative stress after 
choline supplementation. Toxicol Appl Pharmacol 268, 264-277. 
 
Tanwar, S., Trembling, P.M., Guha, I.N., Parkes, J., Kaye, P., Burt, A.D., Ryder, S.D., Aithal, G.P., 
Day, C.P., and Rosenberg, W.M. (2013). Validation of terminal peptide of procollagen III for the 
detection and assessment of nonalcoholic steatohepatitis in patients with nonalcoholic fatty 
liver disease. Hepatology 57, 103-111. 
 
Taubel, J., Lorch, U., Rossignol, J.F., Ferber, G., and Camm, A.J. (2014). Analyzing the 
relationship of QT interval and exposure to nitazoxanide, a prospective candidate for influenza 
antiviral therapy--A formal TQT study. J Clin Pharmacol 54, 987-994. 
 
Wu, K.T., Kuo, P.L., Su, S.B., Chen, Y.Y., Yeh, M.L., Huang, C.I., Yang, J.F., Lin, C.I., Hsieh, M.H., 
Hsieh, M.Y., et al. (2016). Nonalcoholic fatty liver disease severity is associated with the ratios 
of total cholesterol and triglycerides to high-density lipoprotein cholesterol. J Clin Lipidol 10, 
420-425 e421. 
 
Zulu, I., Kelly, P., Njobvu, L., Sianongo, S., Kaonga, K., McDonald, V., Farthing, M., and Pollok, R. 
(2005). Nitazoxanide for persistent diarrhoea in Zambian acquired immune deficiency 
syndrome patients: a randomized-controlled trial. Aliment Pharmacol Ther 21, 757-763. 
 
10.2. 
WHO Fact Sheet 312 
World Health Organization. Media Centre- Diabetes. 
http://www.who.int/mediacentre/factsheets/fs312/en/  
 
10.3. 
Studies on ClinicalTrials.gov 


Protocol No: NTZ-218-1 
Version: 26February 2020 
 
 
69 
NCT01185028. A safety and tolerability study of nitazoxanide in HIV-HCV treatment failures. 
Last updated: March 21, 2018  
https://clinicaltrials.gov/ct2/show/results/NCT01185028?sect=X430156#othr 
NCT01770483. The role of nitazoxanide, interferon alfa and ribavirin in treatment of hepatitis C 
infected Type 2 diabetic patients (HEP-C-FM). Last updated: September 20, 2013 
https://clinicaltrials.gov/ct2/show/results/NCT01770483 
 
 


CRIO Envelope ID: 1119066 - Page: 1/2

Prot_001.pdf: 
Protocol 
 
A Monocentric, Open-Label, Proof of Concept Study to Evaluate the 
Safety and Efficacy of NTZ at 500mg Twice Daily on Collagen Turnover 
in Plasma in NASH Patients with Fibrosis Stage 2 or 3. 
 
 
NCT Number: 03656068 
Unique Protocol ID: NTZ-218-1 
Version Date : 21 October 2018 


 
 
Protocol No: NTZ-218-1 
Version: V21October18 
 
 
 
 
 
Page 1 
 
 
 
 
 
 
 
 
 
A Monocentric, Open-Label, Proof of Concept Study to Evaluate the Safety 
and Efficacy of NTZ at 500mg Twice Daily on Collagen Turnover in Plasma in 
NASH Patients with Fibrosis Stage 2 or 3. 
  
 
 
 
Protocol Number: NTZ-218-1 
Version Date: 15October2018 
 


 
 
Protocol No: NTZ-218-1 
Version: V21October18 
 
 
 
 
 
Page 2 
List of Abbreviations 
  
ACR 
 
Albumin to Creatinine Ratio 
AE             
Adverse Event  
ALT            
Alanine Aminotransferase/GPT   
AST            
Aspartate aminotransferase  
BMI            
Body Mass Index   
CDAA  
Choline-deficient L-amino Acid Defined Diet 
CFR 
 
Code of Federal Regulations 
CSR 
 
Clinical Study Report 
CPK            
Creatine phosphokinase  
CRF            
Case Report/Record Form  
CRN            
Clinical Research Network  
CTCAE  
Common Terminology Criteria for Adverse Events 
D2O 
 
Deuterated Water 
DILI 
 
Drug Induce Liver Injury 
DSMB  
Data Safety Monitoring Board 
GGT 
 
Gamma-glutamyltransferase 
eCRF           
Electronic case report form  
eGFR            
estimated Glomerular Filtration Rate   
EC            
Ethics Committee   
ECG            
Electrocardiogram  
ET             
Early Termination  
EOS 
 
End Of Study 
EU             
European Union  
FDA            
Food and Drug Administration  
FSR 
 
Fractional Synthesis Rate 
GCP            
Good Clinical Practice  
Hb             
Hemoglobin  
HBV           
Hepatitis B Virus  
HCG 
 
Human Chorionic Gonadotropin 
HCV           
Hepatitis C Virus  
HDPE  
High-density polyethylene 
HDL            
High Density Lipoprotein  
HIV            
Human Immunodeficiency Virus  
HOMA           Homeostasis Model Assessment  
HSC 
 
Hepatic Stellate Cell 
hs-CRP            High Sensitivity C-reactive protein  
ICH           
International Conference on Harmonization  
IEC 
 
Independent Ethics Committee 
IMP            
Investigational medical product 


 
 
Protocol No: NTZ-218-1 
Version: V21October18 
 
 
 
 
 
Page 3 
INR 
 
International Normalized Ratio  
IRB            
Institutional Review Board  
LDL            
Low density lipoprotein 
MedDRA          Medical Dictionary for Regulatory Activity  
MRE 
 
Magnetic Resonance Elastography 
MRI            
Magnetic Resonance Imaging  
NAFLD            Non Alcoholic Fatty Liver Disease  
NAS            
NAFLD Activity Score  
NASH            
Non Alcoholic Steatohepatitis  
NTZ 
 
Nitazoxanide 
PI             
Principle Investigator  
PK             
Pharmacokinetic 
PT/INR            Prothrombin Time/International Normalized Ratio  
 
RNA            
Ribonucleic Acid  
SAB            
Safety Advisory Board   
SAE            
Serious Adverse Event 
SAP 
 
Statistical Analysis Plan  
TQT 
 
Thorough-QT study 
  
 
 
 


 
 
Protocol No: NTZ-218-1 
Version: V21October18 
 
 
 
 
 
Page 4 
Study Synopsis 
Protocol Number: 
NTZ-218-1 
Study Title: 
A Monocentric, Open-Label, Proof of Concept Study to Evaluate the Safety 
and Efficacy of NTZ at 500mg Twice Daily on Collagen Turnover in Plasma in 
NASH Patients with Fibrosis Stage 2 or 3. 
Phase: 
Proof of Concept 
Study Drug: 
Nitazoxanide 
Indication: 
NASH induced fibrosis 
Principal Investigator & Sponsor 
Stephen Harrison, MD 
Pinnacle Clinical Research 
12950 Toepperwein Road 
Live Oak, TX 78233 
Study Duration: 
34 weeks 
Objectives: 
The primary objective of the study is: 
 
To evaluate the safety and tolerability of NTZ 500mg bid after 24 
weeks of treatment in patients with NASH-induced stage 2 or 
stage 3 fibrosis  
 
 
The secondary objectives of the study are: 
 
To determine the effect of oral administration of NTZ 500mg bid 
on de novo collagen synthesis through Fractional Synthesis Rate 
(FSR) of circulating plasma proteins in patients with NASH-
induced stage 2 or stage 3 fibrosis . 
 
To assess changes in liver stiffness as measured by FibroScan® 
and by Magnetic Resonance Elastography (MRE). 
 
To assess the change from baseline in non-invasive markers of 
fibrosis 
 
To assess changes in Fibrosis scores: ELFTM test score, Fibrotest, 
Fibrometer, hepascore, NAFLD fibrosis score, FIB-4 
 
Study Design: 
This is a Phase 2 proof of concept study to evaluate the safety and efficacy 
of open-label NTZ 500mg bid in a NASH population with NASH induced 
Stage 2 or 3 fibrosis. 
 
The study will include a screening visit within 6 weeks of randomization 
(Day 1) followed by a 24-week treatment period consisting of daily, BID, 
oral administration of NTZ and an end of treatment follow up period   of 4 
weeks.   
Patients will come to the research center and complete the following visits: 
• 
Screening Visit within 6 weeks of Randomization 
• 
Day -14 Deuterated Water Run-In 
o 
Day-14, Day-11, Day-7 and Day 1: sampling for labeling 
of Deuterated water during run-in, 
 
Repeat baseline labs for ALT, AST, total bilirubin, ALP, CPK 
collected at least 4 weeks from initial screening. 
• 
Day 1 randomization : start of treatment 


 
 
Protocol No: NTZ-218-1 
Version: V21October18 
 
 
 
 
 
Page 5 
• 
Every 4 weeks after randomization until 20 weeks post 
randomization 
• 
22 weeks post randomization and Deuterated Water 
Administration Period 
o 
22 weeks plus 3 days, plus 7 days, plus 14 days for 
sampling for kinetic biomarker analyses 
• 
24 weeks post randomization : End of Treatment 
• 
4 Week Post Treatment Follow Up 
Number of Sites: 
1 site  
Number of Subjects: 
20 evaluable subjects 
Drug Dosage / Frequency: 
500 mg NTZ BID 
Route of Administration: 
Oral Administration 
Inclusion Criteria: 
Patients must meet all of the following inclusion criteria to be eligible for 
enrollment in the trial:  
1. Males or females aged from 18 to 75 years inclusive at the Screening 
Visit.  
2. Must provide signed written informed consent and agree to comply 
with the study protocol.  
3. Females participating in this study must be of non-childbearing 
potential or using highly efficient contraception for the full duration of 
the study.  Childbearing potential refers to those female subjects who 
have not had a hysterectomy, bilateral oophorectomy, or medically-
documented ovarian failure. Female subjects are considered 
postmenopausal and not of child-bearing potential if they are >55 years 
of age, or >50 years of age with >12 months of amenorrhea, or have 
serum follicle stimulating hormone (FSH) values >40 mIU/ml with >12 
months of amenorrhea.  Highly efficient contraception is defined as the 
use of one of the following methods of birth control in addition to a 
male partner using a condom (unless surgically sterile) from Screening 
to 90 days after the last dose of study medication: 
(1) hormone-containing contraceptive 
(2) intrauterine device 
(3) cervical cap or diaphragm with spermicidal agent 
4. Histological confirmation of steatohepatitis on a diagnostic liver biopsy 
(biopsy obtained within 6 months prior to Screening or during the 
Screening Period) with at least 1 in each component of the NAS 
(steatosis scored 0-3, ballooning degeneration scored 0-2, and lobular 
inflammation scored 0-3).   
5.    Fibrosis stage of 2 or 3, according to the NASH CRN fibrosis staging 
system on a diagnostic liver biopsy (biopsy obtained within 6 months 
prior to Screening or during the Screening Period).   
6. Two assessments of ALT, AST, Total bilirubin, ALP, CPK will be collected 
during screening at least 4 weeks apart. To be eligible the second value 
cannot be ≥2x the first value. 
Exclusion Criteria: 
Patients who meet any of the following criteria will be excluded from 
entering the study:  
1. History of efficient bariatric surgery within 5 years prior to Screening, 
or planned bariatric surgery in the course of the study.  


 
 
Protocol No: NTZ-218-1 
Version: V21October18 
 
 
 
 
 
Page 6 
2. Patients with HbA1c >10.0%. If abnormal at the first Screening Visit, the 
HbA1c measurement can be repeated. A repeated abnormal HbA1c 
(HbA1c >10.0%) leads to exclusion.  
3. Patients with a history of clinically significant acute cardiac event within 
6 months prior to Screening such as: stroke, transient ischemic attack, 
or coronary heart disease (angina pectoris, myocardial infarction, 
revascularization procedures).  
4. Weight loss of more than 10% within 6 months prior to Randomization.  
5. Patient with any history or presence of cirrhosis. 
6. Current or recent history (<1 year) of significant alcohol consumption. 
For men, significant consumption is typically defined as higher than 30 
g pure alcohol per day. For women, it is typically defined as higher than 
20 g pure alcohol per day.  
7. Current or history of other substance abuse within 1 year prior to 
screening. 
8. Pregnant or lactating females or females planning to become pregnant 
during the study period.  
9. Other well documented causes of chronic liver disease according to 
standard diagnostic procedures including, but not restricted to:  
a. Positive hepatitis B surface antigen (HBsAg)  
b. Positive HCV RNA, (tested for in case of known cured HCV 
infection, or positive HCV Ab at Screening)  
c. 
Suspicion of drug-induced liver disease  
d. Alcoholic liver disease  
e. Autoimmune hepatitis  
f. 
Wilson’s disease   
g. 
Primary biliary cirrhosis, primary sclerosing cholangitis  
h. Genetic homozygous hemochromatosis  
i. 
Known or suspected HCC  
j. 
History or planned liver transplant, or current MELD 
score >15.  
10. Patients who cannot be contacted in case of emergency.  
11. Known hypersensitivity to the investigational product or any of its 
formulation excipients.  
12. Patients who are taking warfarin or other highly plasma protein-bound 
drugs with narrow therapeutic indices. 
13. Patients who are currently participating in, plan to participate in, or 
have participated in an investigational drug trial or medical device trial 
containing active substance within 30 days or five half-lives, whichever 
is longer, prior to Screening.  
14. Evidence of any other unstable or, untreated clinically significant 
immunological, endocrine, hematological, gastrointestinal, 
neurological, neoplastic, or psychiatric disease.  
15. Mental instability or incompetence, such that the validity of informed 
consent or ability to be compliant with the study is uncertain.  
16.  History of noncompliance with medical regimens, or patients who are 
considered to be unreliable 
17. Positive anti-human immunodeficiency virus (HIV) antibody. 
18. AST and/or ALT >5 x upper limit of normal (ULN).  
19. Total bilirubin >1.3 mg/dL due to altered hepatic function.  


 
 
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Page 7 
20. Direct bilirubin > ULN 
Note: Gilbert Disease patients are allowed into the study.  
21. INR >1.2 in the absence of anticoagulant therapy.  
22. Platelet count <150,000/mm3 in the   context of portal hypertension.  
23. Significant renal disease, including nephritic syndrome, chronic kidney 
disease (defined as patients with markers of kidney damage or eGFR of 
less than 60 ml/min/1.73 m2). 
 
Evaluation Criteria 
Primary Endpoint: 
To assess the safety and tolerability of NTZ after 24 weeks of treatment  
 
Secondary Endpoints: 
To assess the % change in FSR from baseline to end of treatment (24 weeks 
of treatment) 
To assess the change in liver stiffness by MRE from baseline to 12 weeks 
and 24 weeks of treatment 
To assess the change in liver stiffness by fibroscan from baseline to 24 
weeks of treatment  
To assess the change from baseline to 12 weeks and 24 weeks of treatment 
of fibrosis serum biomarkers and scores. 
 
 


 
 
Protocol No: NTZ-218-1 
Version: V21October18 
 
 
 
 
 
Page 8 
TABLE 1 : Schedule of events 
 
Screening period 
 
 
Treatment period 
Visit 
SV 
SV2 
SV3 
SV4 
V1 
V2 
V3 
V4 
V5 
V6 
V7 
V8 
V9 
V10 
EOS 
ET 
Day 
-42 
-14 
-11 
-7 
1 
29 
57 
85 
113 
141 
155 
158 
162 
169 
+28 
days 
 
Window 
 
+1 
+1 
+1 
+3 
+3 
+3 
+3 
+3 
+3 
+1 
+1 
+1 
+1 
+3 
 
Week 
-6 
-2 
 
 
0 
4 
8 
12 
16 
20 
22 
 
 
24 
28 
 
Informed 
Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical 
History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inclusion 
Exclusion 
Criteria 
X 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
Height 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Weight, BMI 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
Physical Exam 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
12-Lead ECG 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
X 
Vital Signs 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
Con Meds 
X 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
AEs 
X 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Liver Biopsy1 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
MRE 
 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
 
X 
Fibroscan 
 
 
 
 
X 
 
 
 
 
 
 
 
 
X 
 
X 
Dispense 
deuterated 
water 
 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
FSR 
 
X 
X 
X 
X 
 
 
 
 
 
X 
X 
X 
X 
 
 
Study Drug 
Dispense 
 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
Study Drug 
Compliance 
 
 
 
 
 
X 
X 
X 
X 
X 
 
 
 
X 
 
 
X 
 
 
                                                          
1 Required only if there is no history of a liver biopsy within the past 6 months.  


 
 
Protocol No: NTZ-218-1 
Version: V21October18 
 
 
 
 
 
Page 9 
TABLE 2 : Biological assessments 
 
Screening period 
 
 
Treatment period 
Visit 
SV 
SV2 
SV3 
SV4 
V1 
V2 
V3 
V4 
V5 
V6 
V7 
V8 
V9 
V10 
EOS 
ET 
Day 
-42 
-14 
-11 
-7 
1 
29 
57 
85 
113 
141 
155 
158 
162 
169 
+28 
days 
 
Week 
-6 
-2 
 
 
0 
4 
8 
12 
16 
20 
22 
 
 
24 
28 
 
Labs - 
Haematology 
haemoglobin, 
haematocrit, RBC, 
WBC, differential 
count, platelet 
count, 
reticulocytes 
count, MCV 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Coagulation (PT 
(INR)) 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Labs- Urinary 
Pregnancy tests2 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
 
X 
Labs – Serology 
HIV, HBs and HCV 
serology 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
                                                          
2 If Urine pregnancy test is positive, a serum β-hCG is required.  
 
 
 


 
 
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Screening period 
 
 
Treatment period 
Visit 
SV 
SV2 
SV3 
SV4 
V1 
V2 
V3 
V4 
V5 
V6 
V7 
V8 
V9 
V10 
EOS 
ET 
Day 
-42 
-14 
-11 
-7 
1 
29 
57 
85 
113 
141 
155 
158 
162 
169 
+28 
days 
 
Labs – 
Biochemistry 
HbA1c, fasting 
plasma glucose, 
insulin, HOMA-IR, 
fructosamine, 
alkaline 
phosphatase, ALT, 
AST, GGT, CPK, 
total and 
conjugated 
bilirubin, ferritin, 
creatinine, eGFR, 
uric acid, BUN, 
total cholesterol, 
HDL-C, LDL-C, 
triglycerides, 
albumin, lipase, 
amylase, 
electrolytes 
(sodium, 
potassium, 
chloride, calcium), 
MELD 
X 
 
 
X3 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Inflammatory 
markers 
hsCRP, fibrinogen, 
haptoglobin, 
resistin,  TNF-, 
TGF-, IL-6, PAI-1 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
Biomarkers of 
fibrosis 
ELF Score, PIIINP, 
Hyaluronic acid, 
CK18, TIMP-1, YKL-
40, Alpha 2 
macroglobulin,, 
,miRNA, ProC3, 
ProC6, FGF19, 
FGF21 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
Labs – Urinalysis 
dipstick  
Specific gravity, 
pH, protein, 
glucose, ketones, 
bilirubin, 
urobilinogen, 
blood, nitrite, 
leukocytes 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
 
X 


 
 
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Screening period 
 
 
Treatment period 
Visit 
SV 
SV2 
SV3 
SV4 
V1 
V2 
V3 
V4 
V5 
V6 
V7 
V8 
V9 
V10 
EOS 
ET 
Day 
-42 
-14 
-11 
-7 
1 
29 
57 
85 
113 
141 
155 
158 
162 
169 
+28 
days 
 
Safety markers 
Serum Cystatin C, 
troponin T, NT 
proBNP 
Urinary albumin, 
urinary creatinine, 
urinary ACR 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Labs -- FSR 
 
X 
X 
X 
X 
 
 
 
 
 
X 
X 
X 
X 
 
 
Sampling for 
additional 
parameters 
X 
 
 
 
X 
 
 
X 
 
X 
 
 
 
X 
X 
X 
 
3 ALT, AST, Total bilirubin, Alkaline phosphatase and CPK must be retested at least 4 weeks after initial 
screening labs. . If these values at SV4 are ≥2x Screening values SV, then the patient cannot be 
randomized. A repeat test may be performed if a screening value is considered an error. 
 
 


 
 
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Table of Contents 
1. 
Introduction .......................................................................................................................... 16 
1.1. 
Background ................................................................................................................... 16 
1.2. 
General Information for Nitazoxanide ......................................................................... 17 
1.2.1. 
Nitazoxanide (NTZ) ............................................................................................... 17 
1.2.2. 
Clinical Safety & Toxicology .................................................................................. 17 
1.2.3. 
Non clinical pharmacology ................................................................................... 19 
1.3. 
Rationale for This Study................................................................................................ 22 
2. 
Objectives .............................................................................................................................. 22 
2.1. 
Primary Objective ......................................................................................................... 22 
2.2. 
Secondary Objectives: .................................................................................................. 22 
3. 
Study Design.......................................................................................................................... 23 
3.1. 
Study Design.................................................................................................................. 23 
3.2. 
Treatment Plan and Regimen ....................................................................................... 24 
4. 
Subject Population ................................................................................................................ 24 
4.1. 
Number of Subjects & Subject Selection ..................................................................... 24 
4.2. 
Inclusion Criteria ........................................................................................................... 24 
4.3. 
Exclusion Criteria .......................................................................................................... 25 
5. 
Investigational Medicinal Products ...................................................................................... 27 
5.1. 
Description .................................................................................................................... 27 
5.2. 
Packaging ...................................................................................................................... 27 
5.3. 
Labeling ......................................................................................................................... 28 
5.4. 
Storage and Handling ................................................................................................... 28 
5.5. 
Dosage and Administration .......................................................................................... 28 
5.6. 
Prior and Concomitant Medications ............................................................................ 28 
5.6.1. 
Non-permitted medication ................................................................................... 29 
5.6.2. 
Permitted Medications ......................................................................................... 29 
5.7. 
Study Drug Accountability ............................................................................................ 29 
6. 
Study Procedures .................................................................................................................. 30 
6.1. 
Duration of Study Participation ................................................................................... 30 


 
 
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6.2. 
Screening Assessment .................................................................................................. 30 
Screening Visit ....................................................................................................................... 30 
6.3. 
Day -14 Deuterated Water Run In ................................................................................ 31 
6.4. 
Treatment Period Assessments .................................................................................... 31 
6.4.1. 
Day 1 Visit.............................................................................................................. 31 
6.4.2. 
Week 4 and Week 8 Visit + 3 days ........................................................................ 32 
6.4.3. 
Week 12 Visit + 3 days .......................................................................................... 32 
6.4.4. 
Week 16 and Week 20 visits ±3 days ................................................................... 33 
6.4.5. 
Week 22 Visit + 3 days (Day 155) ......................................................................... 33 
6.4.6. 
Week 24 Visit +3 ................................................................................................... 34 
6.5. 
Follow Up Period ........................................................................................................... 34 
6.5.1. 
4-Week Post Treatment Follow up (EOS – End of study visit) ±3 days ............... 34 
6.5.2. 
Early Termination (ET) .......................................................................................... 35 
6.5.3. 
Unscheduled Visits ................................................................................................ 35 
6.6. 
Assessment for Premature Discontinuation from Study ............................................. 36 
6.6.1. 
Pre-specified Patient Discontinuation rules ........................................................ 36 
6.6.2. 
Pre-specified Trial Discontinuation rules ............................................................. 37 
6.6.3. 
Specific Liver Function Monitoring after Start of Treatment .............................. 37 
6.6.4. 
Data Safety Monitoring Board ............................................................................. 38 
6.7. 
Description of Assessments .......................................................................................... 39 
6.7.1. 
Histological Assessments ...................................................................................... 39 
6.7.2. 
Assessment of newly synthesized proteins ......................................................... 39 
6.7.3. 
Biological Assessments (Table 2) .......................................................................... 39 
Table 2 Biological assessments ...................................................................................................... 41 
6.7.4. 
Electrocardiogram ................................................................................................. 42 
6.7.5. 
MRE ....................................................................................................................... 42 
6.7.6. 
Fibroscan ............................................................................................................... 43 
6.7.7. 
Medical History ..................................................................................................... 43 
6.7.8. 
Physical Examination ............................................................................................ 43 
6.7.9. 
Vital Signs .............................................................................................................. 43 
6.7.10. 
Pregnancy Testing ................................................................................................. 44 


 
 
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7. 
Adverse Events and Toxicity Management .......................................................................... 44 
7.1. 
Definitions of Adverse Events, Serious Adverse Events and Unexpected Events ...... 44 
7.1.1. 
Adverse Events ...................................................................................................... 44 
7.1.2. 
Clinical Laboratory Abnormalities and Other Abnormal Assessments as Adverse 
Events or Serious Adverse Events ........................................................................................ 45 
7.1.3. 
Serious Adverse Events ......................................................................................... 45 
7.1.4. 
Unexpected adverse event ................................................................................... 46 
7.2. 
Assessment of Adverse Events & Serious Adverse Events .......................................... 46 
7.2.1. 
Intensity assessment ............................................................................................ 46 
7.2.2. 
Relation to the study treatment .......................................................................... 47 
7.2.3. 
Action taken and outcome ................................................................................... 48 
7.3. 
Reporting ....................................................................................................................... 49 
7.3.1. 
Reporting an adverse event ................................................................................. 49 
7.3.2. 
Initial reports......................................................................................................... 49 
7.3.3. 
Follow-up reports ................................................................................................. 50 
7.3.4. 
Follow-up .............................................................................................................. 50 
7.4. 
Post Study Reporting Requirements ............................................................................ 51 
7.5. 
Special Situation Reports .............................................................................................. 51 
7.5.1. 
Pregnancy .............................................................................................................. 51 
7.5.2. 
Medication error ................................................................................................... 51 
7.5.3. 
Misuse ................................................................................................................... 52 
7.5.4. 
Overdose ............................................................................................................... 52 
7.5.5. 
Abuse ..................................................................................................................... 52 
8. 
Statistical Considerations ..................................................................................................... 52 
8.1. 
Analysis Endpoints ........................................................................................................ 52 
8.1.1. 
Primary Endpoint .................................................................................................. 52 
8.1.2. 
Secondary Endpoints ............................................................................................ 52 
8.2. 
Analysis Sets .................................................................................................................. 53 
8.3. 
Data Handling Conventions .......................................................................................... 53 
8.4. 
Demographic Data and Baseline Characteristics ......................................................... 53 
8.5. 
Primary Safety Analysis ................................................................................................ 53 
8.5.1. 
Adverse Events ...................................................................................................... 54 


 
 
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8.5.2. 
Laboratory Evaluations ......................................................................................... 54 
8.5.3. 
Other Safety Evaluations ...................................................................................... 54 
8.6. 
Secondary Efficacy Analyses ......................................................................................... 54 
8.6.1. 
FSR ......................................................................................................................... 54 
8.6.2. 
Liver stiffness ........................................................................................................ 55 
8.6.3. 
Exploratory Analysis ............................................................................................. 55 
8.6.4. 
Interim Analysis .................................................................................................... 55 
8.7. 
Sample Size ................................................................................................................... 55 
9. 
Responsibilities ..................................................................................................................... 55 
9.1. 
Investigator Responsibilities ........................................................................................ 55 
9.2. 
Good Clinical Practice ................................................................................................... 56 
9.3. 
Institutional Review Board Review & Approval .......................................................... 57 
9.4. 
Informed Consent ......................................................................................................... 57 
9.5. 
Confidentiality .............................................................................................................. 57 
9.6. 
Study Files and Retention of Records .......................................................................... 58 
9.7. 
Electronic Case Report Forms ....................................................................................... 59 
9.8. 
Investigational Medicinal Product Accountability and Return ................................... 60 
9.9. 
Inspections .................................................................................................................... 60 
9.10. Protocol Compliance ..................................................................................................... 60 
9.11. Protocol Modifications ................................................................................................. 60 
9.12. Study Reports ................................................................................................................ 61 
9.13. Study Termination ........................................................................................................ 61 
10. 
References ......................................................................................................................... 62 
10.1. Literature ....................................................................................................................... 62 
10.2. WHO Fact Sheet 312 ..................................................................................................... 65 
10.3. Studies on ClinicalTrials.gov ......................................................................................... 65 
 
 
 
 
 


 
 
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1. Introduction 
1.1. 
Background 
Non-alcoholic fatty liver disease (NAFLD) has become one of the most prominent forms 
of chronic liver disease worldwide, reflecting the epidemic of global obesity. Those with 
the progressive variant of NAFLD, non-alcoholic steatohepatitis (NASH), are at significantly 
increased risk of multisystem morbidity and mortality. However, there are currently no 
approved pharmacologic therapies for NASH (Konerman et al, 2018). The World Health 
Organization estimates the prevalence of obesity has more than doubled since 1980 with 
>600 million people (13%) having a body mass index (BMI)) ≥30 (Konerman et al, 2018). 
In this setting, the global prevalence of diabetes among adults has also increased from 
4.7% in 1980 to 8.5% as of 2014 (WHO Fact Sheet 312) . Prevalence rates of NAFLD among 
those with metabolic disease are notably higher.  Approximately a third of patients with 
hypertension, half of patients with dyslipidaemia, up to two-thirds of patients with type II 
diabetes, and  >90% of patients undergoing bariatric surgery had evidence of NAFLD .( 
Leite et al, 2009; Fruci et al, 2013; Assy et al, 2000; Wu  et al, 2016; Sasaki et al, 2014 
Subichin et al; 2015 ; Donati et al ,2004). 
The underlying pathophysiologic mechanisms that contribute to the development and 
progression of NAFLD and NASH are complex and include modulation of metabolic 
pathways, inflammatory cascades, and/or mechanisms impacting fibrosis.  During disease 
progression fibrosis may worsen, while features of steatohepatitis resolve or ‘‘burnout”.    
As a result, NASH and fibrosis need to be evaluated independently to ensure a beneficial 
impact on one parameter does not simultaneously result in a negative impact on another 
endpoint of interest, particularly given that individual treatments tend to focus on one 
primary mechanism of action (i.e. metabolic or NASH disease modifier vs. antifibrotic) ( 
Konerman et al, 2018). 
Persistent and progressive fibrosis is a characteristic of many chronic liver diseases in 
which excessive accumulation of collagen-rich extracellular matrix is a key feature. 


 
 
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Following liver injury, quiescent Hepatic Stellate Cells (HSC) undergo a process of 
activation that is characterized by a differentiation into (α-SMA)-positive contractile 
myofibroblasts which secrete high amounts of collagen and play a central role in the 
fibrogenic process (Friedman et al, 2008, Carpino et al, 2005). Nitazoxanide may be a 
potent antifibrotic agent that can potentially be used for the treatment of different fibrotic 
pathologies, including liver fibrosis. (Belanger et al;2017) 
1.2. 
General Information for Nitazoxanide 
1.2.1. Nitazoxanide (NTZ)  
Nitazoxanide (NTZ) is a broad-spectrum antiparasitic and broad-spectrum antiviral drug 
used for the treatment of various helminthic, protozoal, and viral infections.  It was 
approved in the United States by the Food and Drug Administration (Alinia®, Romark 
Laboratories) as an Orphan Drug for treatment of diarrhea caused by C. parvum and 
Giardia intestinalis in adults and children at least 12 months of age [Rossignol, 2014]. 
The approved Alinia dose for adults is 500 mg twice daily (BID) for three days.  
1.2.2. Clinical Safety & Toxicology 
Nitazoxanide is rapidly metabolized in the liver to an active metabolite tizoxanide (TZ) in 
vivo. TZ undergoes conjugation to form tizoxanide glucuronide. Nitazoxanide is not 
detectable in the serum following oral administration.  Peak concentrations in blood 
plasma of TZ and TZ glucuronide are seen between 1-4 hours.  NTZ is excreted in both 
Urine (~33%) and feces (~67%).   
The safety of ALINIA was evaluated in 2177 HIV-uninfected subjects 12 months of age 
and older who received ALINIA Tablets or ALINIA for Oral Suspension at the 
recommended dose for at least three days. In pooled controlled clinical trials involving 
536 HIV-uninfected subjects treated with ALINIA Tablets or ALINIA for Oral Suspension, 
the most common adverse reactions were abdominal pain, headache, chromaturia and 
nausea (≥2%).[Alinia USPI] 


 
 
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Safety data were analyzed separately for 280 HIV-uninfected subjects ≥12 years of age 
receiving Alinia at the recommended dose for at least 3 days in 5 placebo-controlled 
clinical trials and for 256 HIV-uninfected subjects 1 through 11 years of age in 7 
controlled clinical trials. There were no differences between the adverse reactions 
reported for Alinia-treated subjects based upon age [Alinia USPI]. 
Twenty-one clinical studies of NTZ were conducted in patients with chronic hepatitis C 
(14 studies: Rossignol et al, 2008; Rossignol et al, 2009; Bacon et al, 2010, Rossignol et 
al, 2010; Shiffman et al, 2010 ; Laufer et al, 2011; Kabil et al, 2011 ; Amorosa et al, 2013; 
Shebab et al, 2014; Macias et al, 2015; Kohla et al, 2016; Basu et al, 2013; 
NCT01185028; NCT01770483), hepatic encephalopathy in chronic liver failure (1 study: 
Basu et al, 2008), acquired immune deficiency syndrome (AIDS)-related 
cryptosporidiosis (3 studies: Rossignol et al, 1998; Rossignol et al, 2006; Zulu et al, 
2005), or Helicobacter pylori (2 studies: Abd-Elsalam et al, 2016; Shehata et al., 2017), 
and Clostridium difficile infections (1 study: Musher et al, 2009) in the literature. The 
dose of NTZ administered in 19 studies was 500 mg BID; in one study 1000 mg NTZ BID 
was administered, whereas in one compassionate-use study the NTZ dose administered 
ranged from 500 to 1500 mg BID. Most studies administered NTZ treatment for 48 to 60 
weeks, whereas the duration of treatment in the compassionate-use study ranged from 
1 day to 4 years.  
The studies identified from the literature review demonstrate the safety of NTZ in the 
treatment of chronic hepatitis C, hepatic encephalopathy in chronic liver failure, AIDS-
related cryptosporidiosis, persistent diarrhea in patients with AIDS, and Helicobacter 
pylori and Clostridium difficile infections.  
The adverse event (AE) profile of NTZ was predominantly GI in nature and included 
nausea, vomiting, diarrhea, abdominal pain, dyspepsia, constipation, flatulence, and 
dysphagia. These GI AEs were generally mild in intensity and transient. Other AEs 
related to NTZ treatment were fatigue, rash, headache, myalgia, and arthralgia. When 


 
 
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reported, no significant changes were observed in the electrocardiograms (ECGs), vital 
signs, and clinical laboratory parameters after administration with NTZ [Stockis et al, 
2002a; Stockis et al, 2002b]. A formal International Council on Harmonization (ICH) E14 
compliant thorough QT (TQT) study [Taubel et al, 2014] completed the cardiac safety 
evaluation and showed that neither a therapeutic single dose of 675 mg nor a 2700 mg 
(supra-therapeutic) dose of NTZ prolonged the QT interval in healthy male and female 
volunteers.   
The safety profile of NTZ reported in all these studies therefore provides supporting 
evidence for the potential use of NTZ for the treatment of liver fibrosis in patients with 
NASH.  
The following adverse reactions have been identified during post approval use of ALINIA 
as described in the Package Insert:  
 Gastrointestinal disorders: diarrhea, gastroesophageal reflux disease 
 Nervous System disorders: dizziness 
 Respiratory, thoracic and mediastinal disorders: dyspnea 
 Skin and subcutaneous tissue disorders: rash, urticaria 
1.2.3. Non clinical pharmacology 
Nonclinical pharmacology studies were conducted to determine whether NTZ and its 
main metabolites (TZ and TZ glucuronide [TZ glu]) exhibit anti-fibrotic properties in vitro, 
and in vivo. 
NTZ and its main metabolite, TZ, significantly (p<0.001 from 0.3 µM) inhibited, in a dose-
dependent manner, the expression of αSMA protein in TGF-β-induced human HSCs, but 
TZ glu had no anti-fibrotic properties. NTZ and TZ demonstrated good potency as the 
IC50 was in the micromolar range.  


 
 
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The CDAA diet-induced rodent model develops a fibrosing NASH within a relatively short 
period [Takeuchi-Yorimoto et al, 2013], which makes it a suitable model for studying the 
treatment of both NASH pathology and hepatic fibrosis. 
In studies to determine the effects of NTZ on liver fibrosis in C57BL/6J mice induced by a 
CDAA diet supplemented with 1% cholesterol, microscopic examination revealed that 
NTZ at doses of 11.4 mg/kg/day and 33.9 mg/kg/day tended to reduce, in a dose-
dependent manner, CDAA + 1% cholesterol diet-induced NASH. Although NTZ did not 
have a beneficial effect on hepatic steatosis and lobular inflammation grade, a tendency 
to reduce hepatocyte ballooning was observed, which resulted in a slight reduction in 
NASH prevalence.  
At 12 weeks, NTZ significantly prevented, in a dose-dependent manner, hepatic collagen 
storage induced by the CDAA + 1% cholesterol diet. However, upon histological 
examination, this beneficial effect was not reflected by a dose-dependent improvement 
of the fibrosis grade. A significant decrease of the fibrosis area was observed with NTZ at 
doses of 11.4 mg/kg/day (p<0.05) and 78.1 mg/kg/day (p<0.001) and correlated with 
the significant decrease of the hepatic collagen content.  
The CDAA + 1% cholesterol diet notably induces a significant augmentation in circulating 
markers of liver injury such as AST, ALT, and PIIINP, which is directly associated with 
fibrosis [Tanwar et al, 2013]. Moreover, the hepatic content is dramatically modified as 
illustrated by the significant increase of collagen. Mice treated with NTZ at 
78.1 mg/kg/day exhibited lower plasma levels of ALT (-17%, p>0.05), AST (-24%, p<0.05), 
and PIIINP (-25%, p<0.01). These data suggest a potential preventive action of NTZ on 
liver damage induced by the CDAA + 1% cholesterol diet in mice.  
The hepatic expression of fibrotic markers (collagens, MMP2), but also inflammatory 
markers (TNFα) were enhanced in the animals fed the CDAA + 1% cholesterol diet as 
compared with animals fed a CSAA control diet. Chronic administration of NTZ at 
78.1 mg/kg/day to mice fed a CDAA + 1% cholesterol diet led to the inhibition of the 


 
 
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Col1α1 (-17%, p>0.05), Col1α2 (-25%, p<0.05), MMP2 (-32%, p<0.05), and TNFα  
(-28%, p<0.01) gene expression.  
In the studies with CCl4-treated mice, NTZ significantly prevented hepatic fibrosis. This 
was shown at the dose of 95.3 mg/kg/day on the hepatic collagen content decrease in 
the first study [SSR_NTZ_PHA1_004], whereas no significant improvement was observed 
upon histological examination. Conversely, in the second study [SSR_NTZ_PHA1_005], 
no significant improvement of CCl4-induced hepatic collagen content was observed, 
whereas the histological evaluation of hepatic fibrosis revealed a significant effect of 
NTZ at 32 and 104 mg/kg/day. 
There was no significant beneficial effect of NTZ treatment on plasma markers of 
hepatotoxicity in CCl4-treated mice.  
These studies demonstrate the anti-fibrotic effect of NTZ, which suggests that NTZ may 
have potential as an anti-fibrotic drug. These studies also suggest that NTZ may show 
some beneficial therapeutic properties for NASH, such as a tendency to reduce 
hepatocyte ballooning. 


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22 
1.3.
Rationale for This Study 
Based on the anti-fibrotic properties demonstrated in the animal models of fibrosis, this proof 
of concept clinical study aims at  evaluating NTZ in patients with non-alcoholic steatohepatitis 
(NASH) and  fibrosis stage 2 and 3.Although NTZ has been evaluated in liver disease 
populations (viral hepatitis C) up to 60 weeks, this is the first study evaluating NTZ treatment in 
a population with NASH induced stage 2 and 3 fibrosis.  The aim of this study is to evaluate the 
safety and tolerability of NTZ 500 mg BID after 24 weeks of treatment in this population. This 
proof of concept study will also evaluate the anti-fibrotic effect of NTZ as a secondary 
objective. 
The methods of evaluation of fibrosis will include an innovative method of metabolic labeling. 
This approach is based on the concept that liver status can be determined by measuring the 
ratio of newly synthesized/pre-existing proteins. The turn-over rate of newly synthesized 
collagen and proteins represents the hepatic fibrogenic disease activity.  Patients will be given 
“heavy water” to drink. Heavy water contains D20, deuterium being a stable isotope of 
hydrogen. Mass spectrometry is used to identify individual proteins and to quantify the ratio of 
labeled protein to total protein. The results are expressed as fractional synthesis rate of these 
proteins (FSR). This method has been previously published (Decaris et al, 2017). 
Other non-invasive methods will be used to evaluate the liver stiffness changes after NTZ 
treatment: Magnetic Resonance Elastography (MRE) and FibroScan®.  
2.
Objectives 
2.1.Primary Objective 
 
The primary objective of the study is: 
x To evaluate the safety and tolerability of NTZ 500mg bid after 24 weeks of treatment in 
patients with NASH induced Stage 2 or Stage 3 fibrosis. 
2.2.
Secondary Objectives: 
The secondary objectives of the study are: 


Protocol No: NTZ-218-1 
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23 
x
To determine the effect of daily oral administration of NTZ 500mg bid on de novo 
collagen synthesis through Fractional Synthesis Rate (FSR) of circulating plasma 
proteins in patients with NASH-induced Stage 2 or Stage 3 fibrosis. 
x To evaluate changes in liver stiffness as measured by FibroScan® and by Magnetic 
Resonance Elastography (MRE). 
x To assess the change from baseline in non-invasive markers of fibrosis  
x To assess changes in fibrosis scores: ELFTM test score, Fibrotest, Fibrometer, hepascore, 
NAFLD fibrosis score, FIB-4 
3.
Study Design 
3.1.
Study Design 
This is a phase 2 proof of concept study to evaluate the efficacy and safety of open-label NTZ 
500mg bid in a NASH population with NASH induced Stage 2 or 3 fibrosis. 
The study will include a screening visit within 6 weeks of randomization (Day 1) followed by a 
24-week treatment period consisting of daily, BID, oral administration of NTZ and an end of 
treatment follow up period of 4 weeks.   
Patients will come to the research center and complete the following visits: 
x
Screening Visit within 6 weeks of Randomization 
x
Day -14 Deuterated Water Run-In 
o Day-14, Day-11, Day-7 and Day 1: sampling for labelling of Deuterated water 
during run-in 
x
Repeat baseline labs for ALT, AST, total bilirubin, ALP, CPK collected at least 4 
weeks from initial screening.  
x
Day 1 Randomization : start of treatment 
x
Every 4 weeks after randomization until 20 weeks post randomization 
x
 22 weeks post randomization and Deuterated Water Administration Period: 


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24 
o 22 weeks plus 3 days, plus 7 days, plus 14 days for sampling for kinetic 
biomarker analysis 
x
24 weeks post randomization : End of Treatment 
x
4 Week Post Treatment Follow Up 
3.2.
Treatment Plan and Regimen 
NTZ will be administered in an open label fashion to participating subjects as 500mg bid, for 24 
weeks. 
4.
Subject Population 
4.1.
Number of Subjects & Subject Selection 
Twenty male and female subjects with histologically confirmed NASH and fibrosis stage 2 or 3 
will be enrolled in the study at a single site in the United States. 
4.2.
Inclusion Criteria 
Patients must meet all of the following inclusion criteria to be eligible for enrollment in the 
trial:  
1.
Males or females aged from 18 to 75 years inclusive the Screening Visit.  
2.
Must provide signed written informed consent and agree to comply with the 
study protocol.  
3. Females participating in this study must be of non-childbearing potential or using 
highly efficient contraception for the full duration of the study.   Childbearing 
potential refers to those female subjects who have not had a hysterectomy, 
bilateral oophorectomy, or medically-documented ovarian failure. Female subjects 
are considered postmenopausal and not of child-bearing potential if they are >55 
years of age, or >50 years of age with >12 months of amenorrhea, or have serum 
follicle stimulating hormone (FSH) values >40 mIU/ml with >12 months of 
amenorrhea.  Highly efficient contraception is defined as the use of one of the 
following methods of birth control in addition to a male partner using a condom 


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25 
(unless surgically sterile) from Screening to 90 days after the last dose of study 
medication: 
x hormone-containing contraceptive 
x intrauterine device 
x cervical cap or diaphragm with spermicidal agent 
4.
 Histological confirmation of steatohepatitis on a diagnostic liver biopsy (biopsy 
obtained within 6 months prior to Screening or during the Screening Period) 
with at least 1 in each component of the NAS (steatosis scored 0-3, ballooning 
degeneration scored 0-2, and lobular inflammation scored 0-3).   
5.
Fibrosis stage of 2 or 3, according to the NASH CRN fibrosis staging system on a 
diagnostic liver biopsy (biopsy obtained within 6 months prior to Screening or 
during the Screening Period).   
6.
Two assessments of ALT, AST, Total bilirubin, ALP, CPK will be collected during 
screening at least 4 weeks apart. To be eligible the second value cannot be ≥2x 
the first value. 
4.3.
Exclusion Criteria 
Patients who meet any of the following criteria will be excluded from entering the study:  
1.
History of efficient bariatric surgery within 5 years prior to Screening, or 
planned bariatric surgery in the course of the study.  
2.
Patients with HbA1c >10.0%. If abnormal at the first Screening Visit, the HbA1c 
measurement can be repeated. A repeated abnormal HbA1c (HbA1c >10.0%) 
leads to exclusion.  
3.
Patients with a history of clinically significant acute cardiac event within 6 
months prior to Screening such as: stroke, transient ischemic attack, or 
coronary heart disease (angina pectoris, myocardial infarction, revascularization 
procedures).  
4.
Weight loss of more than 10% within 6 months prior to Randomization.  
5.
Patient with any history or presence of cirrhosis. 


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6.
Current or recent history (<1 year) of significant alcohol consumption. For men, 
significant consumption is typically defined as higher than 30 g pure alcohol per 
day. For women, it is typically defined as higher than 20 g pure alcohol per day.  
7.
Current or history of other substance abuse within 1 year prior to screening. 
8.
Pregnant or lactating females or females planning to become pregnant during 
the study period.  
9.
Other well documented causes of chronic liver disease according to standard 
diagnostic procedures including, but not restricted to:  
a) Positive hepatitis B surface antigen (HBsAg)  
b) Positive HCV RNA, (tested for in case of known cured HCV infection, or positive 
HCV Ab at Screening)  
c) Suspicion of drug-induced liver disease  
d) Alcoholic liver disease  
e) Autoimmune hepatitis  
f) Wilson’s disease   
g) Primary biliary cirrhosis, primary sclerosing cholangitis  
h) Genetic homozygous hemochromatosis  
i) Known or suspected HCC  
j) History or planned liver transplant, or current MELD score >15.  
10.
Patients who cannot be contacted in case of emergency.  
11.
Known hypersensitivity to the investigation product or any of its formulation 
excipients.  
12.
Patients who are taking warfarin or other highly plasma protein-bound drugs 
with narrow therapeutic indices. 
13.
Patients who are currently participating in, plan to participate in, or have 
participated in an investigational drug trial or medical device trial containing 
active substance within 30 days or five half-lives, whichever is longer, prior to 
Screening.  


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14.
Evidence of any other unstable or, untreated clinically significant 
immunological, endocrine, hematological, gastrointestinal, neurological, 
neoplastic, or psychiatric disease.  
15.
Mental instability or incompetence, such that the validity of informed consent 
or ability to be compliant with the study is uncertain.  
16.
 History of noncompliance with medical regimens, or patients who are 
considered to be unreliable. 
17.
Positive anti-human immunodeficiency virus (HIV) antibody. 
18.
AST and/or ALT >5 x upper limit of normal (ULN).  
19.
Total bilirubin >1.3 mg/dL due to altered hepatic function. 
20.
Direct bilirubin > ULN 
 Note: Gilbert Disease patients are allowed into the study.  
21.
INR >1.2 in the absence of anticoagulant therapy.  
22.
Platelet count <150,000/mm3 in the context of portal hypertension.  
23.
Significant renal disease, including nephritic syndrome, chronic kidney disease 
(defined as patients with markers of kidney damage or eGFR of less than 60 
ml/min/1.73 m2). 
5.
Investigational Medicinal Products 
Patients will receive 500 mg NTZ (ALINIA®) twice daily for 24 weeks. 
5.1. Description  
NTZ will be supplied as 500 mg of ALINIA® (nitazoxanide) tablets, for oral use.  The tables are 
round, yellow, film-coated and debossed with ALINIA on one side and 500 on the other side. 
Each tablet contains 500 mg of nitazoxanide. 
For additional information see the Package Insert for ALINIA®. 
5.2. Packaging  
ALINIA® tablets are packaged in HDPE bottles of 12 tablets. Period boxes will be delivered 
every 4 weeks, each box containing 6 bottles. 


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5.3.
Labeling 
All labels for study drugs meet all applicable requirements of the US Food and Drug 
Administration (FDA) and the EU annex 13 of Good Manufacturing Practices: Manufacture of 
Investigational Medicinal Products (February 2010) and /or other local regulations, as 
applicable.  
Distribution of study drug will be performed according to the Good Distribution Practices.  
Product boxes for each period will be labeled with the protocol number, Sponsor’s name and 
address, description of contents, storage conditions, expiry date, dosage instructions, and any 
other applicable items required by national and regional guidelines/regulations. The label will 
contain the statements “Caution – new drug, limited by Federal Law to investigational use” or 
other similar/appropriate statements as well as the following instructions “Please return 
empty packaging and unused products to your doctor at your next visit.”  
5.4.
Storage and Handling 
The tablets are to be stored at 20-25oC (68-77oF). Storage conditions are specified on the label. 
5.5.
Dosage and Administration 
Patients will be instructed to take one tablet of 500mg NTZ mg orally, twice a day with water 
during a meal, approximately 12 hours apart.    
The study staff will administer the first dose of the medication at the research center.  Patients 
will self-administer the evening dose on Day 1 and twice a day thereafter, when not attending 
a visit at the research center.  On scheduled follow up visit days while the patient is being 
treated with study drug, the patient will be directed to bring back all used and unused study 
medication containers. Compliance will be checked by the Investigator or designee during 
those visits and registered in the electronic Case Report Form (eCRF).  
If treatment is interrupted, whatever the cause, duration and reason of the interruption will be 
documented. 
5.6.
Prior and Concomitant Medications 


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In a general manner, patients should be discouraged from starting any new medication 
without consulting the Investigator unless the new medication is required for emergency 
purpose.  In the same way, any qualitative or quantitative change in concomitant therapy 
should be avoided, when possible. In the event that it becomes necessary during the study, 
this should be recorded by the Investigator or designee in the eCRF (including concomitant 
medications taken within 6 months prior to Screening). This includes drugs used on a chronic 
as well as on an “as needed” basis. 
5.6.1.  Non-permitted medication 
The following medications are not allowed within the timeframe given:  
x Warfarin or other highly plasma protein-bound drugs with narrow therapeutic indices 
are not permitted from Day 1 until end of treatment 
5.6.2.  Permitted Medications 
Any medications other than those listed above are permitted. However, the dosage of a 
current medication for a chronic disease should remain unchanged as far as possible in order 
to reduce the risk of unknown drug-drug interactions.  
In the event that additional concomitant therapy becomes necessary during the study, this will 
be recorded by the Investigator in the eCRF. This includes drugs used on a chronic as well as on 
an “as-needed” basis. Patients should be discouraged from starting any new medication 
without consulting the Investigator unless the new medication is required for emergency 
purpose. 
5.7. Study Drug Accountability 
The Investigator will acknowledge receipt for each study treatment on the day of receipt. A 
drug accountability record will be maintained by the person responsible for dispensing the trial 
medication to the patient.  
All partially used or unused treatments will be inventoried during and at the conclusion of the 
study.  


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The Drug Distribution Center will organize the retrieval of all treatments (used or unused) and 
will proceed to their destruction only after written authorization and review of accountability 
and distribution records.  
6.
Study Procedures 
The procedures performed at each visit are summarized in the Schedule of Events (Table 1).  
A patient may be seen at any time for reasons of safety.  
During each visit, safety evaluations will be done, and the patient will be queried in the form of 
an open question regarding new or continuing events.  
6.1.
Duration of Study Participation 
The estimated duration of the study will be approximately 34 weeks inclusive of the screening 
period. 
6.2.
Screening Assessment 
Screening Visit 
The following screening procedures will be performed for all potential patients at the 
Screening Visit and prior to Day 1:  
x
Informed consent witnessed by the Investigator or designated person.  
x
Medical history & demographics.  
x
Assessment of inclusion/exclusion criteria 
x
Complete physical examination. 
x
Record vital signs  
x
Record height, weight, BMI  
x
Check concomitant/prior medication (within 6 months prior to Screening)  
x
12-lead ECG 
x
Liver biopsy.  A historical liver biopsy with confirmed NASH and fibrosis is 
acceptable if performed within 6 months prior to the Screening Visit.  
x
Adverse Events assessment  


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x
Blood samples will be collected for 
o
Biochemistry 
o
Hematology 
o
Serology (HIV, HBs, HCV). In the event of a positive HCV Ab, HCV RNA 
can be tested for confirmation. In case of known cured HCV infection, HCV RNA 
can be tested directly at Screening.  
x
Repeat baseline labs for ALT, AST, total bilirubin, ALP, CPK collected at least 4 
weeks from initial screening. 
x
Urine samples will be collected for Urinalysis  
x
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
If needed, a retesting of any laboratory results may be performed during the screening 
window to determine the eligibility for the study.  
6.3.
Day -14 Deuterated Water Run In 
Potential Subjects who complete all of the Screening Visit assessments and who 
continue to be eligible for the study, will return to the research center to complete the 
Deuterated Water Run In.  The visit is to be done on Day -14 + 3 days, provided that the 
subject completes 7 consecutive days of deuterated water administration.  The 
following procedures will be performed at the Day -14 Visit: 
x Dispense deuterated water (D2O). Instruct patient to drink 50ml of deuterated 
water 3 times per day starting on Day-14 for 7 consecutive days (until day-8).  
x Blood sample for baseline labeling by D2O (kinetic biomarkers) (patients should 
be instructed to fast the evening prior to the visit to ensure a 10-hour fast) 
x  Adverse Events assessment  
x Record concomitant medication taken 
Patients will return at Day-11, Day-7 and Day 1 for blood sampling (labeled D2O) 
6.4.
Treatment Period Assessments 
6.4.1. Day 1 Visit 


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The following procedures will be performed at the Day 1 Visit prior to dose administration:  
x
Review inclusion/exclusion criteria 
x
Complete physical examination 
x
Weight, BMI 
x
Record vital signs  
x
Record concomitant medication taken  
x
Adverse Events assessment  
x
MRE (may be done within + 5 days of the visit)  
x
Fibroscan 
x
Blood samples will be collected  
x
Urine samples will be collected for Urinalysis  
x
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
x
Dispense the study drug as directed 
x
Review study drug administration instructions with subject  
x
Administer 500 mg of NTZ with water & food 
6.4.2. Week 4 and Week 8 Visit + 3 days 
The following procedures will be performed at these Visits:  
x
Record concomitant medication taken  
x
Adverse Events assessment  
x
Blood samples will be collected  
x
Urine samples will be collected for Urinalysis  
x
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
x
Dispense the study drug as directed 
x
Review study drug compliance and drug administration instructions with subject  
x
Reconcile study drug administration using pill counts  
x
Administer 500 mg of NTZ with water & food 
6.4.3. Week 12 Visit + 3 days 


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The following procedures will be performed at the Visit:  
x
Complete physical examination 
x
Weight, BMI 
x
Record vital signs  
x
Record concomitant medication taken  
x
Adverse Events assessment  
x
MRE(may be done within + 5 days of the visit) 
x
Blood samples will be collected  
x
Urine samples will be collected for Urinalysis  
x
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
x
Dispense the study drug as directed 
x
Review study drug compliance and drug administration instructions with subject  
x
Reconcile study drug administration using pill counts  
x
Administer 500 mg of NTZ with water and food 
6.4.4. Week 16 and Week 20 visits ±3 days 
The following procedures will be performed at these Visits:  
x
Record concomitant medication taken  
x
Adverse Events assessment  
x
Blood samples will be collected  
x
Urine samples will be collected for Urinalysis  
x
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
x
Dispense the study drug as directed 
x
Review study drug compliance and drug administration instructions with subject  
x
Reconcile study drug administration using pill counts  
x
Administer 500 mg of NTZ with water & food 
6.4.5. Week 22 Visit + 3 days (Day 155) 
The following procedures will be performed at the Visit:  


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34 
x
Record concomitant medication taken  
x
Adverse Events assessment  
x
Blood samples will be collected for D2O labeling 
x
Urinary pregnancy test (for women of childbearing potential only [WOCBP]) 
x Dispense Deuterated water. Instruct patient to drink 50ml of deuterated water 
3 times per day starting on Day 155 for 7 consecutive days (until day 162).  
x Blood sample for kinetic biomarkers analysis (patients should be instructed to 
fast the evening prior to the visit to ensure a 10-hour fast) 
Patients will return for blood sampling for kinetic biomarker analysis at 22 weeks plus 3 
days (Day 158), at Day 162, and at day 169 (Week 24). 
6.4.6. Week 24 Visit +3 
The following procedures will be performed at the visit: 
x
Complete physical examination 
x
12-lead ECG 
x
Record vital signs  
x
Record weight, BMI  
x
Record concomitant medication taken  
x
MRE (may be done within + 5 days of the visit) 
x
Fibroscan  
x
Adverse Events assessment  
x
Blood samples will be collected Urine samples will be collected for Urinalysis  
x
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
x
Review study drug compliance and drug administration instructions with subject  
x
Reconcile study drug administration using pill counts  
6.5.
Follow Up Period 
6.5.1. 4-Week Post Treatment Follow up (EOS – End of study visit) ±3 days 
The following procedures will be performed at the visit: 
x Complete physical examination 


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x Weight, BMI 
x Record vital signs  
x Record concomitant medication taken  
x Adverse Events assessment  
x Blood samples will be collected Urine samples will be collected for Urinalysis  
x Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
6.5.2. Early Termination (ET) 
Patients who have discontinued from the study after the first dose of study medication and for 
any reason will have the following procedures completed: 
x
Complete physical examination 
x
Record vital signs 
x
Weight, BMI  
x
ECG 
x
Record concomitant medication taken  
x
MRE (may be done within + 5 days of the visit) 
x
Fibroscan 
x
Adverse Events assessment  
x
Blood samples will be collected  
x
Urine samples will be collected for Urinalysis  
x
Urinary pregnancy test (for women of childbearing potential only [WOCBP]) 
x
Review study drug compliance with subject  
x
Reconcile study drug administration using pill counts  
6.5.3.
Unscheduled Visits 
An unscheduled visit is defined as any visit to the study unit outside of the protocol-evaluation 
time points where the patient is seen by study unit personnel, e.g., when follow-up 
assessments are required for safety reasons or when repeat measurements are required out of 
the screening period (either to confirm a measurement or in case of errors, measuring device 
failure, etc.).  


Protocol No: NTZ-218-1 
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36 
Unscheduled visits will be needed for patients who may require further follow-up due to 
safety. 
6.6.
Assessment for Premature Discontinuation from Study 
Study medication may be discontinued in the following instances: 
x Intercurrent illness that would, in the judgment of the investigator, affect 
assessments of clinical status to a significant degree. Following resolution of 
intercurrent illness, the subject may resume study dosing at the discretion of the 
investigator. 
x Subject noncompliance  
x Investigator discretion  
x Study medication must be discontinued in the following instances:  
o
Unacceptable toxicity,  or toxicity that, in the judgment of the 
investigator, compromises the ability to continue study-specific 
procedures or is considered to not be in the subject’s best interest  
o
Subject request to discontinue for any reason  
o
Pregnancy  
o
Discontinuation of the study at the request of a regulatory agency or an 
institutional review board or independent ethics committee (IRB/IEC) 
Pre-specified stopping criteria are defined at the patient level and at the study level. These are 
described in the following sections 6.6.1., 6.6.2., and 6.6.3. In all cases, when the stopping 
criteria are met, the Data Safety Monitoring Board will perform causality assessment, and 
decide whether the study drug/study trial may be continued (See section 6.6.4.). 
 
6.6.1.
Pre-specified Patient Discontinuation rules 
If a patient experiences a Grade IV Common Terminology Criteria for Adverse Event (CTCAE) or 
meets liver injury criteria as listed in section 6.6.3., the study drug should be interrupted. After 
causality assessment by the DSMB, if the AE or lab abnormality is considered unrelated to 


Protocol No: NTZ-218-1 
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37 
study drug, and the event has resolved or returned to baseline, then the study drug may be re-
started. If the event reoccurs after restart of study drug, then the study drug will be 
permanently discontinued. 
6.6.2.
Pre-specified Trial Discontinuation rules 
If one patient experiences a Grade V CTCAE, or 2 patients experience the same Grade IV 
CTCAE, or 3 patients experience the same Grade III CTCAE, or 4 patients experience the same 
Grade II CTCAE, the trial will be paused. The DSMB will assess causality of these adverse 
events; if the events are deemed unrelated to study drug, the trial will continue.  
6.6.3.
Specific Liver Function Monitoring after Start of Treatment 
Liver function monitoring guidelines are detailed below. The cases will be reviewed by the 
DSMB, who will assess potential Drug Induced Liver Injury (DILI), and apply discontinuation 
rules as described in 6.6.1., 6.6.2., and in this section. 
Study drug interruption 
Study drug must be interrupted in the following cases:  
x ALT >8x ULN  (if ALT is normal at baseline) 
x ALT > 5x Baseline (if ALT is elevated at baseline) OR ALT>500 U/L 
x ALT >5x ULN (if ALT is normal at baseline) AND Total Bilirubin>2xULN 
x ALT >3x Baseline (if ALT is elevated at baseline) OR ALT>300 U/L AND total 
bilirubin>2xULN  
x ALT >5x ULN (if ALT is normal at baseline) AND liver-associated symptoms 
x ALT >3x Baseline (if ALT is elevated at baseline) OR ALT>300 U/L AND liver-associated 
symptoms 
In all these cases, close monitoring should be initiated, with repeat liver biochemistries within 
24-72 hours. Continue monitoring twice/week until liver biochemistries resolve, stabilize, or 
return within baseline values. If abnormalities stabilize and patient is asymptomatic, then 
monitoring should be performed once a week. History of symptoms, disease, concomitant 
medication, or other conditions such as alcohol consumption or alcohol hepatitis should be 


Protocol No: NTZ-218-1 
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38 
obtained for competing etiologies. The cases will be reviewed by the DSMB, who will 
determine if the patient may restart study drug (if causality assessment is deemed to be not 
related to study drug). 
Management of liver biochemistries elevations 
In the following cases of liver enzyme increase, liver biochemistries including ALT, AST, Total 
bilirubin, ALP, should be repeated within 2-5 days. Follow up of symptoms should be carefully 
monitored. Frequency of monitoring should continue once/week until liver abnormalities 
resolve, stabilize, or return to values below the thresholds mentioned below: 
x ALT >5x ULN  (if ALT is normal at baseline) 
x ALT >3x Baseline (if ALT is elevated at baseline) OR ALT >300 U/L 
NOTE: Laboratory assessments should be done centrally. If the patient lives remotely, he/she 
can have laboratory tests performed locally, and the results communicated to the investigator 
promptly. 
 
6.6.4.
Data Safety Monitoring Board 
An independent Data Safety Monitoring Board (DSMB) composed of 3 liver experts will review 
the safety of patients enrolled in the trial. The DSMB will perform a safety data review every 3 
months, the first review occurring 3 months after first patient randomized. A DSMB charter 
will define the role, responsibilities, rules, and tasks of the DSMB. 
The DSMB will ensure that the patient and trial discontinuation rules are respected and will 
determine the causality assessment of the events described in sections 6.6.1, 6.6.2., and 6.6.3., 
in order to make the recommendation for trial /patient discontinuation. If the events are 
deemed unrelated to the study drug by the DSMB, then the patient or the trial will continue. 
 
 
 


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39 
  
 
6.7.
Description of Assessments 
6.7.1. Histological Assessments 
All possible attempts should be made to acquire a liver biopsy specimen of at least 2.0 cm in 
length, from a 16 gauge needle to ensure accurate staging of fibrosis and other histological 
lesions.  A historical biopsy within 6 months of the Screening visit may be accepted as the 
Screening biopsy. The liver biopsy sample must be deemed adequate for evaluation by the 
pathologist for inclusion. Liver biopsies will be assessed by a local reader.  This assessment will 
include an assessment of the adequacy of the specimen as well as the fibrosis stage and a 
determination that the biopsy is consistent with NASH. If either the historical or screening liver 
biopsy is deemed unacceptable, it may be repeated. 
6.7.2. Assessment of newly synthesized proteins 
Kinetic biomarkers will be analyzed to evaluate the pharmacodynamic effect of NTZ on 
fibrogenesis. Blood samples analyses will be performed after deuterated water intake before 
and after 24 weeks of treatment. The assessment will involve the analysis of the change 
(absolute and relative) from baseline between the post-dose and pre-dose deuterated water 
loading periods. 
6.7.3. Biological Assessments (Table 2) 
Chemistry:  
HbA1c, fasting plasma glucose, insulin, HOMA-IR, fructosamine, alkaline phosphatase, ALT, 
AST, GGT, CPK, total and conjugated bilirubin, ferritin, creatinine, eGFR, uric acid, BUN, total 
cholesterol, HDL-C, LDL-C, triglycerides, albumin, lipase, amylase, electrolytes (sodium, 
potassium, chloride, calcium), MELD 
Hematology:  
Hematocrit (Hct), hemoglobin (Hb), platelet count, red blood cell count (RBC), white blood cell 
count (WBC) with differential (absolute and percentage) including lymphocytes, monocytes, 


Protocol No: NTZ-218-1 
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40 
neutrophils, eosinophils, and basophils and, reticulocyte count and mean corpuscular volume 
(MCV).  
Inflammatory markers:  
hsCRP, fibrinogen, haptoglobin, resistin, TNF-α, TGF-β, IL-6, PAI-1 
Coagulation Panel:  
Prothrombin time (PT), and international normalized ratio (INR)  
Pregnancy Tests:  
Urine β-hCG (if positive, requires immediate confirmation with Serum β-hCG)  
Urinary Tests:  
Urinary dipstick analysis, urinary albumin, urinary creatinine, urinary ACR 
Additional Tests:  
Serology (HIV-1, HBsAg, HCVAb & HCV RNA (as needed)), serum cystatin C, troponin T, NT 
proBNP 
Fibrosis Biomarker tests:  
ELFTM test score, PIIINP, Hyaluronic acid, CK18, TIMP-1, YKL-40, Alpha 2 macroglobulin, miRNA, 
FGF19, FGF21, ProC3 and ProC6.  
A laboratory manual will be provided 
The manual will outline the collection and processing requirements for the laboratory. Blood 
sampling will be performed by trained personnel. Blood samples will be processed and shipped 
as outlined in the laboratory manual. Refer to the laboratory manual for exact amounts of 
blood required for each test.  
The option to retest during the study is left to the Investigator’s judgment.  
Additional serum and plasma samples will be maintained for future analysis 
 
 


Protocol No: NTZ-218-1 
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41 
Table 2 Biological assessments 
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Protocol No: NTZ-218-1 
Version: 21October18 
 
 
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6.7.4. Electrocardiogram 
Standard 12-lead electrocardiogram (ECG) assessments will be performed. The Investigator 
will review the ECGs for any clinically significant abnormalities to ensure subject safety. 
Abnormal ECG findings that are considered clinically significant by the Investigator and meet 
the definition of an AE should be reported and recorded in the AE eCRF page.  
6.7.5. MRE 
Liver stiffness will be assessed by MRE. It is recommended that each subject’s radiological 
assessment is performed using the same procedure for each study visit.  Exploratory measures 
for MRI/MRE sequence development will be maintained for future analysis.  


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6.7.6. Fibroscan 
Liver stiffness will be assessed by FibroScan®. It is required that each subject’s FibroScan® 
assessments be done with the same type of probe at each study visit.  
6.7.7. Medical History 
Medical history including details regarding illnesses and allergies, date(s) of onset, and 
whether condition(s) is currently ongoing, and medication history, including nicotine and 
alcohol use, will be collected on all subjects during screening. 
6.7.8. Physical Examination 
A complete physical examination should include source documentation of general appearance, 
and the following body systems: head, neck, and thyroid; eyes, ears, nose, throat, mouth, and 
tongue; chest (excluding breasts); respiratory; cardiovascular; lymph nodes; abdomen; skin, 
hair, nails; musculoskeletal and neurological.  
The patient’s weight and waist circumference will be measured under the same conditions at 
each visit where the procedure is required. Where possible, the scale for weight must be the 
same for a given patient throughout the visits. 
6.7.9. Vital Signs 
Blood pressure (mmHg) and pulse rate (beats per minute) will be measured at each visit using 
automated blood pressure equipment. 
Important points for clinical blood pressure measurement  
•
The patient should be seated comfortably with the back supported and the 
upper arm bare without constrictive clothing. The legs should not be crossed. 
•
The arm should be supported at heart level, and the bladder of the cuff should 
encircle at least 80% of the arm circumference.  
•
Neither the patient nor the observer should talk during the measurement.  


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Systolic BP and diastolic BP will be measured after 5 minutes rest in the seating.  Where 
possible, the blood pressure equipment should be the same for a given patient throughout the 
visits. 
6.7.10.Pregnancy Testing 
Urinary pregnancy tests will be done on WOCBP.  
7. Adverse Events and Toxicity Management 
7.1. Definitions of Adverse Events, Serious Adverse Events and Unexpected Events 
7.1.1. Adverse Events 
An adverse event (AE) is any untoward medical occurrence in a clinical study subject 
administered a medicinal product, which does not necessarily have a causal relationship with 
the treatment. An AE can therefore be any unfavorable and/or unintended sign, symptom, or 
disease temporally associated with the use of a 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 specified procedures, overdose, drug abuse/misuse reports, 
or occupational exposure. Preexisting events that increase in severity or change in nature 
during or as a consequence of participation in the clinical study will also be considered AEs.  
An AE does not include the following:  
x
Medical or surgical procedures such as surgery, endoscopy, tooth extraction, 
and transfusion. The condition that led to the procedure may be an adverse 
event and must be reported  
x
Pre-existing diseases, conditions, or laboratory abnormalities present or 
detected before the Screening visit that do not worsen  
x
Situations where an untoward medical occurrence has not occurred (eg., 
hospitalization for elective surgery, social and/or convenience admissions)  
x
Overdose without clinical sequelae.   
x
Any medical condition or clinically significant laboratory abnormality with an 
onset date before the consent form is signed and not related to a protocol-


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associated procedure is not an AE. It is considered to be pre-existing and should 
be documented on the medical history eCRF. 
7.1.2.  Clinical Laboratory Abnormalities and Other Abnormal Assessments as Adverse 
Events or Serious Adverse Events 
Laboratory abnormalities without clinical significance are not recorded as AEs or SAEs. 
However, laboratory abnormalities (eg, clinical chemistry, hematology, and urinalysis) that 
require medical or surgical intervention or lead to study drug interruption, modification, or 
discontinuation must be recorded as an AE, as well as an SAE, if applicable. In addition, 
laboratory or other abnormal assessments (eg, electrocardiogram, x-rays, vital signs) that are 
associated with signs and/or symptoms must be recorded as an AE or SAE if they meet the 
definition of an AE or SAE as described above.  If the laboratory abnormality is part of a 
syndrome, record the syndrome or diagnosis (eg, anemia), not the laboratory result (ie, 
decreased hemoglobin). 
7.1.3. Serious Adverse Events 
A serious adverse event (SAE) is defined as an event that, at any dose, results in the following:  
x
Death  
x
Life-threatening (Note: The term “life-threatening” in the definition of 
“serious” refers to an event in which the subject was at risk of death at the 
time of the event; it does not refer to an event that hypothetically might 
have caused death if it were more severe.) 
x
In-patient hospitalization or prolongation of existing hospitalization 
x
Persistent or significant disability/incapacity  
x
A congenital anomaly/birth defect  
x
A medically important event or reaction: such events may not be 
immediately life-threatening or result in death or hospitalization but may 
jeopardize the subject or may require intervention to prevent one of the 
other outcomes constituting SAEs. Medical and scientific judgment must be 
exercised to determine whether such an event is a reportable under 


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expedited reporting rules. 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 
hospitalization; and development of drug dependency or drug abuse. For the 
avoidance of doubt, infections resulting from contaminated medicinal 
product will be considered a medically important event and subject to 
expedited reporting requirements. 
7.1.4. Unexpected adverse event  
Expectedness is assessed by the Sponsor-Investigator. An unexpected AE is defined as an event 
that has a nature of severity or specificity that is not consistent with the applicable Investigator 
Brochure or that is symptomatically and pathophysiologically related to a known toxicity but 
differs because of a greater severity or specificity.  
“Unexpected” refers to an event that has not been previously observed and reported rather 
than an event that has not been anticipated based on the properties of the drug. 
7.2.
Assessment of Adverse Events & Serious Adverse Events 
The Investigator will establish whether or not any AE have occurred at each visit from the date 
of consent. The patient will be questioned in a general manner to determine specific 
symptoms without offering the patient any suggestion. The investigator or qualified sub-
investigator is responsible for assessing AEs and SAEs for causality and severity, and for final 
review and confirmation of accuracy of event information and assessments. 
7.2.1.  Intensity assessment  
The intensity of the AE will be graded as follows:  
•
Mild: Awareness of signs or symptoms, but easily tolerated and are of 
minor irritant type causing no loss of time from normal activities. 
Symptoms do not require therapy or a medical evaluation; signs and 
symptoms are transient.  


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•
Moderate: Events introduce a low level of inconvenience or concern to 
the participant and may interfere with daily activities, but are usually 
improved by simple therapeutic measures; moderate experiences may 
cause some interference with functioning. 
•
Severe: Events interrupt the participant’s normal daily activities and 
generally require systemic drug therapy or other treatment; they are 
usually incapacitating. 
7.2.2.  Relation to the study treatment  
The Investigator will make a clinical and scientific judgment regarding whether or not the AE 
was related to study treatment. The Investigator will evaluate any changes in laboratory 
values, make a determination as to whether or not the change is clinically important, and 
whether or not the changes were related to study drug. However, even if the Investigator feels 
there is no relationship to the study drug, the AE or clinically significant laboratory abnormality 
must be recorded in the eCRF.  
The Investigator will record the relation to the study treatment according to the following 
causality terms:  
•
Related: the AE follows a reasonable temporal sequence from the time 
of drug administration and it cannot be explained by the patient's 
clinical state or the study procedures/conditions. The AE abates upon 
discontinuation of the study drug and reappears when the study drug is 
introduced.  
•
Possibly related: the AE follows a reasonable temporal sequence from 
the time of drug administration, but could have been produced by the 
patient's clinical state or the study procedures/conditions.  
•
Unlikely related: the temporal association between the AE and the study 
drug is such that the study drug is not likely to have any reasonable 


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association with the AE. The relationship is not likely because of other 
plausible explanations.  
•
Not related: the AE must definitely be caused by the patient's clinical 
state or the study procedure/conditions. A reasonable explanation must 
be given, e.g., no investigational product taken, preplanned elective 
medical intervention, or incompatible temporal relationship.  
•
Not assessable: the report suggesting an adverse reaction cannot be 
judged because information is insufficient or contradictory and data 
cannot be supplemented or verified. 
7.2.3.
 Action taken and outcome  
The Investigator will record the action taken with drug and outcome of the event for each AE 
according to the following:  
Action taken with investigational drug  
•
Drug permanently withdrawn – in case a patient is permanently 
withdrawn from the study drug  
•
Drug temporarily withdrawn – in case the study drug is temporarily 
withdrawn  
•
Dose not changed – in case no action is taken regarding the study drug  
•
Unknown  
•
Not applicable – an AE started before initiation of treatment with study 
drug, the treatment had been completed prior to reaction/event, or the 
patient has died. 
Outcome  
•
Recovered/resolved  
•
Recovering/resolving  
•
Not recovered/not resolved  


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•
Recovered/resolved with sequelae  
•
Fatal  
•
Unknown.  
Note: In case of irreversible congenital anomalies the choice not recovered/not resolved 
should be used. “Fatal” should be used when death is possibly related to the reaction/event. 
7.3. Reporting 
7.3.1.  Reporting an adverse event  
All AEs regardless of seriousness or relationship to study drug, including those occurring during 
the Screening Period, are to be recorded on the corresponding page(s) of the eCRF and in the 
patient’s medical record from the ICF signature until study end for each patient. Whenever 
possible, symptoms should be grouped as a single syndrome or diagnosis. The Investigator 
should specify the date of onset, maximal intensity, action taken with respect to study drug, 
corrective therapy given, outcome, and his/her opinion as to whether there is a reasonable 
possibility that the AE was caused by the study drug.  
Adverse event reporting begins from signature of the patient ICF at the first Screening Visit and 
ends at study end for each patient. 
7.3.2. Initial reports 
All SAEs occurring from the time of informed consent until 30 days following the 
last administration of study drug must be reported to Medpace Clinical Safety 
within 24 hours of the knowledge of the occurrence (this refers to any adverse 
event that meets any of the aforementioned serious criteria). All SAEs that the 
Investigator considers related to study drug occurring after the 30-day follow-up 
period must be reported to the Sponsor. 
To report the SAE, complete the SAE form electronically in the electronic data 
capture (EDC) system for the study. When the form is completed, Medpace Safety 
personnel will be notified electronically and will retrieve the form. If the event 
meets serious criteria and it is not possible to access the EDC system, send an 


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email to Medpace Safety at medpace safetynotification@medpace.com or call the 
Medpace SAE hotline (phone number listed below), and fax the completed paper 
SAE form to Medpace (fax number listed below) within 24 hours of awareness. 
When the EDC system becomes available, the SAE information must be entered 
within 24 hours of the system becoming available.  
7.3.3. Follow-up reports 
The Investigator must continue to follow the patient until the SAE has subsided or 
until the condition becomes chronic in nature, stabilizes (in the case of persistent 
impairment), or the patient dies. 
Within 24 hours of receipt of follow-up information, the Investigator must update 
the SAE form electronically in the EDC system for the study and submit any 
supporting documentation (eg, patient discharge summary or autopsy reports) to 
Medpace Clinical Safety via fax or e-mail. If it is not possible to access the EDC 
system, refer to the procedures outlined above for initial reporting of SAEs. 
All SAEs independent of the circumstances or suspected cause must be reported on a SAE 
Form/captured in EDC. The SAE Form should include a clearly written narrative describing 
signs, symptoms, and treatment of the event, diagnostic procedures, as well as any relevant 
laboratory data and any sequelae, in order to allow a complete medical assessment of the case 
and independent determination of the possible causality.  
It is critical that the information provided on the initial or follow-up SAE Form matches the 
information recorded in the source documents and the eCRF for the same event.  
Any unexpected safety issue that changes the risk benefit analysis and is likely to have an 
impact on the patients who have participated in the trial will be reported as soon as possible 
to the Competent Authority(ies) concerned together with proposed actions. 
7.3.4. Follow-up  
The Investigator should take all appropriate measures to ensure the safety of the patients, 
notably he/she should follow up the outcome of any AE until the return to normal or until 
stabilization of the patient’s condition.  


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The patient must be followed up until clinical recovery is complete and laboratory results have 
returned to normal, or until progression has been stabilized. This information should be 
documented in the patient’s medical records.  
7.4. Post Study Reporting Requirements 
Any SAEs and deaths that occur within 30 days of the last dose of the study drug, regardless of 
causality, should be reported.  
Any SAE that is brought to the attention of the Investigator at any time after the reporting 
period and which is considered by him/her to be caused by the study drug within a reasonable 
possibility, should be reported.  
7.5. Special Situation Reports 
Special situations reports include pregnancy reports, reports of medication error, abuse, 
misuse or overdose, and reports associated with product complaints.  
7.5.1. Pregnancy  
In case of pregnancy, the Investigator will notify regulatory authorities and IRB within 24 hours 
of his/her knowledge of the pregnancy.  
Female patients must be instructed to discontinue the study drug immediately and inform the 
Investigator as soon as possible once they are aware of being pregnant or suspect that they 
are pregnant during the study or within 30 days of the last dose of the study drug.  
Female patients will be requested, as part of the general ICF, to provide informed consent to 
allow reasonable attempts to be made to obtain information on any possible medicinal 
product exposure to an embryo or fetus and to follow up on the outcome of the pregnancy.  
The Investigator will contact the patient at the expected time of delivery for follow-up.  
7.5.2. Medication error  
Medication error is defined as an unintentional error in the prescribing, dispensing, or 
administration of a medicinal product while in the control of the healthcare professional, 


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patient, or consumer. All medication errors will be documented in the eCRF and, in case of any 
potential risk to patient safety, would be reported as appropriate.  
7.5.3. Misuse  
This refers to situations where the medicinal product is intentionally and inappropriately used 
not in accordance with the authorized product information and will be reported in the eCRF. 
All misuse will be documented in the eCRF and, in case of any potential risk to patient safety, 
would be reported as appropriate. 
7.5.4.
 Overdose  
This refers to the administration of a quantity of a medicinal product given per administration 
or cumulatively, which is above the maximum recommended dose according to the authorized 
product information. Clinical judgment should always be applied. Doses >4000 mg in one day 
are considered overdose. 
7.5.5.
 Abuse  
This corresponds to the persistent or sporadic, intentional excessive use of a medicinal 
product, which is accompanied by harmful physical or psychological effects. 
8.
Statistical Considerations 
This section is an overview of the principal elements of the statistical analysis for this study. 
Further details will be contained in a separate statistical analysis plan (SAP). 
8.1.
Analysis Endpoints 
8.1.1. Primary Endpoint 
The primary endpoint is to assess the safety and tolerability of NTZ after 24 weeks of treatment 
8.1.2. Secondary Endpoints 
The secondary endpoints are: 
x To assess the % change in FSR from baseline to end of treatment (24 weeks of 
treatment) 


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x
To assess the change in liver stiffness by MRE from baseline to 12 weeks and 24 weeks 
of treatment 
x To assess the change in liver stiffness by fibroscan from baseline to 24 weeks of 
treatment  
x To assess the change from baseline to 12 weeks and 24 weeks of treatment of fibrosis 
serum biomarkers and scores  
8.2.
Analysis Sets 
The Full Analysis Set will consist of all subjects that meet the eligibility criteria and enroll into 
the study (Visit 1 Day 1). 
The Safety Set will consist of all enrolled subjects who receive at least one dose of study drug.  
8.3.
Data Handling Conventions 
Continuous variables will be summarized with descriptive statistics (i.e., sample size, mean, 
standard deviation, minimum, maximum, median, and quintiles). Categorical variables will be 
summarized with descriptive statistics (i.e., sample size, frequency counts, and percentage). 
Statistical testing on change from baseline values will be performed using analysis of variance 
techniques with a term for baseline in the model. Statistical significance will be considered to 
have been reached at an alpha level of 0.05. 
Baseline value is measured on Day 1 of the treatment period before the administration of NTZ. 
8.4.
Demographic Data and Baseline Characteristics 
Demographic and baseline characteristics will be descriptively summarized. Quantitative 
and/or categorical summaries will be presented for demographics and other baseline 
characteristics. Demographic and baseline characteristics will be summarized using the Full 
Analysis Set. 
8.5.
Primary Safety Analysis 
Safety and tolerability will be assessed by the following parameters:  
AEs including TEAEs and SAEs, laboratory evaluations, vital signs, ECGs, and physical 
examinations.  


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8.5.1. Adverse Events 
Treatment-emergent adverse events (TEAEs) are defined as AEs that worsen or commence on 
or after the time of start of first study drug administration. AEs, including SAEs, will be coded 
using MedDRA. The number and percentage of subjects experiencing TEAE, including TEAEs 
leading to study drug discontinuation and SAEs, will be summarized for each system organ 
class (SOC) and Preferred Term. In addition, AEs will be tabulated according to severity, 
causality, and relation to the study drug.  
Individual data listings for AEs will be presented for each subject.  
8.5.2. Laboratory Evaluations 
Absolute and change from baseline values for laboratory parameters will be summarized by 
visit. In addition, shift from baseline tables will be presented for selected laboratory 
abnormalities.  
8.5.3. Other Safety Evaluations 
Observed values and change from baseline will be summarized descriptively for all vital sign 
parameters and ECG parameters by visit. Individual listings will be presented for each subject. 
Individual data listings for physical examination results will be presented for each subject. 
Safety data will be summarized using descriptive statistics. Further details will be provided in 
the SAP. 
 All safety analyses will be conducted using the Safety Population. 
8.6.
Secondary Efficacy Analyses 
8.6.1. FSR 
Fractional Synthesis Rate of circulating plasma proteins will be measured at Baseline, and after 
24 weeks of treatment.  
The change in FSR (absolute and relative) from baseline to end of treatment between the first 
and second deuterated water loading periods will be assessed to determine the effects of NTZ 
500mg BID on de novo collagen synthesis in the Full Analysis Set.  


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Wilcoxon signed-rank test will be used for statistical inference. Estimated mean, standard error 
and 95% confidence interval of change in FSR (absolute and relative) change from baseline in 
FSR will be reported and evaluated at a significance level of 0.05. Exploratory analyses may 
also be performed to evaluate the association of individual exploratory biomarkers or 
combination of biomarkers with clinical measurements and other risk factors. 
Additional details will be provided in the SAP. 
8.6.2. Liver stiffness 
The change in liver stiffness from baseline at 24 weeks as measured by FibroScan, and at 12 
weeks and 24 weeks by MRE, the change in non-invasive markers of fibrosis from baseline to 
24 weeks, and the change in fibrosis scores from baseline to 24 weeks for the Full Analysis Set 
will be assessed. Summary statistics for the Full Analysis Set will be provided at all visits and 
the change from baseline will be provided. 
8.6.3. Exploratory Analysis 
Additional serum and plasma samples will be maintained for future analysis.  Exploratory 
measures for MRI/MRE sequence development will be maintained for future analysis.  
8.6.4. Interim Analysis 
 An interim analysis is not planned. 
8.7.
Sample Size 
The sample size of this Phase 2 proof of concept study is based on clinical assessment. Twenty 
male and female subjects with histologically confirmed NASH and fibrosis stage 3 or 4 
(compensated cirrhosis only) will provide sufficient information to assess the safety and 
tolerability of NTZ 500mg BID over 24 weeks. 
9.
Responsibilities 
9.1.
Investigator Responsibilities 


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By signing this protocol, the Investigator agrees to conduct the study in an efficient and 
diligent manner and in conformance with this protocol; generally accepted standards of GCP; 
and all applicable local laws, rules and regulations relating to the conduct of the clinical study.  
The Investigator also agrees to allow monitoring, audits, IRB review and regulatory agency 
inspection of trial-related documents and procedures and provide for direct access to all study 
related source data and documents.  
The Investigator shall prepare and maintain complete and accurate study documentation in 
compliance with GCP standards and applicable local laws, rules and regulations; and, for each 
subject participating in the study, provide all data, and upon completion or termination of the 
clinical study submit any other reports to the Fundor as required by this protocol or as 
otherwise required pursuant to any agreement with the Fundor.  
Study documentation will be promptly and fully disclosed to regulatory authorities by the 
Investigator upon request and also shall be made available at the Investigator’s site upon 
request for inspection, copying, review and audit at reasonable times by representatives any 
regulatory agencies.   
Persons debarred from conducting or working on clinical studies by any court or regulatory 
agency will not be allowed to conduct or work on this study.   
9.2.
Good Clinical Practice 
The investigator will ensure that this study is conducted in accordance with the principles of 
the Declaration of Helsinki (as amended in Edinburgh, Tokyo, Venice, Hong Kong, and South 
Africa), International Conference on Harmonisation (ICH) guidelines.  These standards are 
consistent with the European Union Clinical Trials Directive 2001/20/EC and Good Clinical 
Practice Directive 2005/28/EC. The investigator will ensure adherence to the basic principles of 
Good Clinical Practice, as outlined in 21CFR312, subpart D, “Responsibilities of Sponsors and 
Investigators,” 21CFR, part50, 1998, and 21CFR, part56, 1998. The investigator and all 
applicable sub investigators will comply with 21CFR, Part54, 1998, providing documentation of 
their financial interest or arrangements with any study fundor, or proprietary interests in the 
investigational drug under study. This documentation must be provided prior to the 


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investigator’s (and any sub investigator’s) participation in the study. The investigator and sub 
investigator agree to notify study fundor of any change in reportable interests during the study 
and for 1 year following completion of the study. Study completion is defined as the date 
when the last subject completes the protocol-defined activities 
9.3.
Institutional Review Board Review & Approval 
The investigator will submit this protocol, informed consent form, and any accompanying 
material to be provided to the subject (such as advertisements, subject information sheets, or 
descriptions of the study used to obtain informed consent) to an IRB/IEC.  The investigator will 
not begin any study subject activities until approval from the IRB/IEC has been documented 
and provided as a letter to the investigator. Before implementation, the investigator will 
submit to and receive documented approval from the IRB/IEC any modifications made to the 
protocol or any accompanying material to be provided to the subject after initial approval, 
with the exception of those necessary to reduce immediate risk to study subjects. 
9.4.
Informed Consent 
The investigator is responsible for obtaining written informed consent from each individual 
participating in this study after adequate explanation of the aims, methods, objectives, and 
potential hazards of the study and before undertaking any study-related procedures. The 
investigator must use the most current IRB/IEC approved consent form for documenting 
written informed consent. Each informed consent will be appropriately signed and dated by 
the subject and the person conducting the consent discussion, and also by an impartial witness 
if required by local requirements. 
9.5.
Confidentiality 
The investigator will assure that subjects’ anonymity will be strictly maintained and that their 
identities are protected from unauthorized parties. Only subject initials, date of birth, another 
unique identifier (as allowed by local law) and an identification code will be recorded on any 
form or biological sample submitted to the study funder, IRB/IEC or laboratory. Laboratory 
specimens will be labeled in such a way as to protect subject identity while allowing the results 
to be recorded to the proper subject. Refer to specific laboratory instructions for further 


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details. NOTE: The investigator will keep a screening log showing codes, names, and addresses 
for all subjects screened and for all subjects enrolled in the trial. Subject data will be processed 
in accordance with all applicable regulations. The investigator agrees that all information 
received from the study funder, including but not limited to the investigator brochure, the 
IMP, and any other study information, remain the sole and exclusive property of the study 
funder during the conduct of the study and thereafter. This information is not to be disclosed 
to any third party (except employees or agents directly involved in the conduct of the study or 
as required by law) without prior written consent from the study funder.  The investigator 
further agrees to take all reasonable precautions to prevent the disclosure by any employee or 
agent of the study site to any third party or otherwise into the public domain. 
9.6.
Study Files and Retention of Records 
The investigator will maintain adequate and accurate records to enable the conduct of the 
study to be fully documented and the study data to be subsequently verified. These 
documents should be classified into at least the following two categories: (1) investigator’s 
study master file, and (2) subject clinical source documents. The investigator’s study file will 
contain the protocol/amendments, CRF and query forms, IRB/IEC and governmental approval 
with correspondence, informed consent, drug records, staff curriculum vitae and authorization 
forms, and other appropriate documents and correspondence. The required source data 
should include sequential notes containing at least the following information for each subject:  
x
Subject identification (name, date of birth, gender);  
x
Documentation that subject meets eligibility criteria, i.e., history, physical 
examination, and confirmation of diagnosis (to support inclusion and exclusion 
criteria); 
x
Documentation of the reason(s) a consented subject is not enrolled  
x
Participation in study (including study number);  
x
Study discussed and date of informed consent;  
x
Dates of all visits;  


Protocol No: NTZ-218-1 
Version: 21October18 
 
 
59 
x
Documentation that protocol specific procedures were performed;  
x
Results of efficacy parameters, as required by the protocol;  
x
Start and end date (including dose regimen) of IMP, including dates of 
dispensing and return;  
x
Record of all adverse events and other safety parameters (start and end date, 
and including causality and severity);  
x
Concomitant medication (including start and end date, dose if relevant; dose 
changes); 
x
Date of study completion and reason for early discontinuation, if it occurs.  
All clinical study documents must be retained by the investigator until at least 2years or 
according to local laws, whichever is longer, after the last approval of a marketing application 
in an ICH region (i.e., United States, Europe, or Japan) and until there are no pending or 
planned marketing applications in an ICH region; or, if no application is filed or if the 
application is not approved for such indication, until 2years after the investigation is 
discontinued and regulatory authorities have been notified. Investigators may be required to 
retain documents longer if specified by regulatory requirements, by local regulations, or by an 
agreement with study funder. The investigator must notify study funder before destroying any 
clinical study records. Should the investigator wish to assign the study records to another party 
or move them to another location, study funder must be notified in advance. If the 
investigator cannot provide for this archiving requirement at the study site for any or all of the 
documents, special arrangements must be made between the investigator and study funder to 
store these records securely away from the site so that they can be returned sealed to the 
investigator in case of an inspection. When source documents are required for the continued 
care of the subject, appropriate copies should be made for storage away from the site. 
9.7.
Electronic Case Report Forms 
For each subject consented, an eCRF will be completed by an authorized study staff member 
whose training for this function is documented according to study procedures. The eCRF will 


Protocol No: NTZ-218-1 
Version: 21October18 
 
 
60 
be completed in a timely manner. Original entries as well as any changes to data fields will be 
stored in the audit trail of the system. At the conclusion of the trial, a read-only archive copy of 
the data entered will be generated. This archive will be stored in accordance with the records 
retention requirements. 
9.8.
Investigational Medicinal Product Accountability and Return 
The investigator or designee is responsible for ensuring adequate accountability of all used and 
unused study drug.  This includes acknowledgment of receipt of each shipment of study 
product (quantity and condition), subject dispensing records, and returned or destroyed study 
product. Dispensing records will document quantities received from study funder and 
quantities dispensed to subjects, including lot number, date dispensed, subject identifier 
number, subject initials, and the initials of the person dispensing the medication. Drug may be 
returned or destroyed on an ongoing basis during the study, if appropriate.  At the end of the 
study, following final drug inventory reconciliation, the study site will arrange for the return of 
unused investigational medicinal product supplies. All drug supplies and associated 
documentation will be periodically reviewed and verified by the study funder over the course 
of the study. 
9.9.
Inspections 
The investigator understands that source documents for this trial should be made available to 
appropriately qualified personnel from study funder and its representatives, to IRBs/IECs, or to 
regulatory authority or health authority inspectors. 
9.10. Protocol Compliance 
The investigator is responsible for ensuring the study is conducted in accordance with the 
procedures and evaluations described in this protocol. 
9.11. Protocol Modifications 
Protocol modifications, except those intended to reduce immediate risk to study subjects, may 
be made by the investigator in collaboration with the study funder. All protocol modifications 


Protocol No: NTZ-218-1 
Version: 21October18 
 
 
61 
must be submitted to the IRB/IEC in accordance with local requirements. Approval must be 
obtained before changes can be implemented. 
9.12. Study Reports 
A clinical study report (CSR) will be prepared and provided to the regulatory agency (ies). The 
Investigator will ensure that the report meets the standards set out in the ICH Guideline for 
Structure and Content of Clinical Study Reports (ICHE3). Note that an abbreviated report may 
be prepared in certain cases. After conclusion of the study, Investigators in this study may 
communicate, orally present, or publish in scientific journals or other scholarly media only 
after the following conditions have been met:  
x
The results of the study in their entirety have been publicly disclosed by or with 
the consent of the study funder in an abstract, manuscript, or presentation form. 
9.13. Study Termination 
The Investigator reserves the right to terminate the study at any time. Should this be 
necessary, the investigator will arrange discontinuation procedures and notify the appropriate 
regulatory authorities, IRBs, IECs, and the Fundor. In terminating the study, the investigator 
will assure that adequate consideration is given to the protection of the subjects’ interests. 


Protocol No: NTZ-218-1 
Version: 21October18 
 
 
62 
10.References 

10.1.
Literature  
Abd-Elsalam, S., Kobtan, A., El-Kalla, F., Elkhalawany, W., Nawasany, S.E., Saif, S.A., Yousef, M., 
Ali, L.A., Soliman, S., Mansour, L., et al. (2016). A 2-week Nitazoxanide-based quadruple 
treatment as a rescue therapy for Helicobacter pylori eradication: A single center experience. 
Medicine (Baltimore) 95, e3879. 
 
Alinia (nitazoxanide): US Prescribing Information. Tampa, FL. Romark, L.C., 2016 
 
Amorosa, V.K., Luetkemeyer, A., Kang, M., Johnson, V.A., Umbleja, T., Haas, D.W., Yesmin, S., 
Bardin, M.C., Chung, R.T., Alston-Smith, B., et al. (2013). Addition of nitazoxanide to PEG-IFN 
and ribavirin to improve HCV treatment response in HIV-1 and HCV genotype 1 coinfected 
persons naive to HCV therapy: results of the ACTG A5269 trial. HIV Clin Trials 14, 274-283. 
 
Assy, N., Kaita, K., Mymin, D., Levy, C., Rosser, B., and Minuk, G. (2000). Fatty infiltration of 
liver in hyperlipidemic patients. Dig Dis Sci 45, 1929-1934. 
 
Bacon, B.R., Shiffman, M.L., Lim, J.K., Berman, A., Rustgi, V.K., and Keeffe, E.B. (2010). A Phase 
II, Randomized, Double-Blind, Placebo-Controlled Study of Nitazoxanide Plus Peginterferon 
and Ribavirin in Naive Patients With Chronic Hepatitis C Genotype 1 Infection: Final Report. 
Gastroenterology 139, e18. 
 
Basu, P.P., Rayapudi, K., Shah, N.J., Krishnaswamy, N., and Brown, R.S. (2013). Effects of triple-
drug therapy with nitazoxanide, high-dose ribavirin and peginterferon-alpha-2a in patients 
with chronic hepatitis C. Hepatol Res 43, 589-595. 
 
Basu PP, Rayapudi K, Esteves J, Brown R. A pilot study utilizing nitazoxanide for hepatic 
encephalopathy in chronic liver failure. Abstract S151 (392) for the 59th Annual Meeting of the 
American Association for the Study of Liver Diseases. 31 Oct-4 Nov. 2008. p1742 
 
Belanger, C., Foucart, C., Negro, E., Dubernet, M., Hum, D.W., Staels, B., and Walczak, R. 
(2017). Drug repurposing screen identifies novel small molecule compounds with potent 
antifibrotic properties. Journal of Hepatology 66, S605. 
 
Carpino, G., Morini, S., Ginanni Corradini, S., Franchitto, A., Merli, M., Siciliano, M., Gentili, F., 
Onetti Muda, A., Berloco, P., Rossi, M., et al. (2005). Alpha-SMA expression in hepatic stellate 
cells and quantitative analysis of hepatic fibrosis in cirrhosis and in recurrent chronic hepatitis 
after liver transplantation. Dig Liver Dis 37, 349-356. 
 
Decaris, M.L., Li, K.W., Emson, C.L., Gatmaitan, M., Liu, S., Wang, Y., Nyangau, E., Colangelo, 
M., Angel, T.E., Beysen, C., et al. (2017). Identifying nonalcoholic fatty liver disease patients 


Protocol No: NTZ-218-1 
Version: 21October18 
 
 
63 
with active fibrosis by measuring extracellular matrix remodeling rates in tissue and blood. 
Hepatology 65, 78-88. 
 
Donati, G., Stagni, B., Piscaglia, F., Venturoli, N., Morselli-Labate, A.M., Rasciti, L., and Bolondi, 
L. (2004). Increased prevalence of fatty liver in arterial hypertensive patients with normal liver 
enzymes: role of insulin resistance. Gut 53, 1020-1023. 
 
Friedman, S.L. (2008). Hepatic stellate cells: protean, multifunctional, and enigmatic cells of 
the liver. Physiol Rev 88, 125-172. 
 
Fruci, B., Giuliano, S., Mazza, A., Malaguarnera, R., and Belfiore, A. (2013). Nonalcoholic Fatty 
liver: a possible new target for type 2 diabetes prevention and treatment. Int J Mol Sci 14, 
22933-22966. 
 
Kabil, S.M., Soliman, M.S., Youssef, S.M., and Mounier, B.I. (2011). A controlled study of 
nitazoxanide (NTZ) 3 years after treatment of hepatitis C genotype 4. J Egypt Soc Parasitol 41, 
251-261. 
 
Kohla, M.A., El-Said, H., El-Fert, A., Ehsan, N., Ezzat, S., and Taha, H. (2016). Impact of 
nitazoxanide on sustained virologic response in Egyptian patients with chronic hepatitis C 
genotype 4: a double-blind placebo-controlled trial. Eur J Gastroenterol Hepatol 28, 42-47. 
 
Konerman, M.A., Jones, J.C., and Harrison, S.A. (2018). Pharmacotherapy for NASH: Current 
and emerging. J Hepatol 68, 362-375. 
 
Laufer, N., Abusamra, L., Bolcic, F., Gun, A., Rolon, M.J., Perez, H., Krolewiecki, A., Salomon, H., 
Quarleri, J., and Cahn, P. (2011). No reduction of HCV viral load in HIV patients co-infected with 
HCV genotype 1 during a 30 days course of nitazoxanide monotherapy. Antiviral Res 92, 497-
499. 
 
Leite, N.C., Salles, G.F., Araujo, A.L., Villela-Nogueira, C.A., and Cardoso, C.R. (2009). Prevalence 
and associated factors of non-alcoholic fatty liver disease in patients with type-2 diabetes 
mellitus. Liver Int 29, 113-119. 
 
Macias, J., Lopez-Cortes, L.F., Tellez, F., Recio, E., Ojeda-Burgos, G., Rios, M.J., Rivero-Juarez, 
A., Delgado, M., Rivas, J., and Pineda, J.A. (2015). Low Efficacy of Pegylated Interferon plus 
Ribavirin plus Nitazoxanide for HCV Genotype 4 and HIV Coinfection. PLoS One 10, e0143492. 
 
Musher, D.M., Logan, N., Bressler, A.M., Johnson, D.P., and Rossignol, J.F. (2009). Nitazoxanide 
versus vancomycin in Clostridium difficile infection: a randomized, double-blind study. Clin 
Infect Dis 48, e41-46. 
 


Protocol No: NTZ-218-1 
Version: 21October18 
 
 
64 
Rossignol, J.F. (2006). Nitazoxanide in the treatment of acquired immune deficiency syndrome-
related cryptosporidiosis: results of the United States compassionate use program in 365 
patients. Aliment Pharmacol Ther 24, 887-894. 
 
Rossignol, J.F. (2014). Nitazoxanide: a first-in-class broad-spectrum antiviral agent. Antiviral 
Res 110, 94-103. 
 
Rossignol, J.F., Elfert, A., El-Gohary, Y., and Keeffe, E.B. (2009). Improved virologic response in 
chronic hepatitis C genotype 4 treated with nitazoxanide, peginterferon, and ribavirin. 
Gastroenterology 136, 856-862. 
 
Rossignol, J.F., Elfert, A., and Keeffe, E.B. (2010). Treatment of chronic hepatitis C using a 4-
week lead-in with nitazoxanide before peginterferon plus nitazoxanide. J Clin Gastroenterol 
44, 504-509. 
 
Rossignol, J.F., Hidalgo, H., Feregrino, M., Higuera, F., Gomez, W.H., Romero, J.L., Padierna, J., 
Geyne, A., and Ayers, M.S. (1998). A double-'blind' placebo-controlled study of nitazoxanide in 
the treatment of cryptosporidial diarrhoea in AIDS patients in Mexico. Trans R Soc Trop Med 
Hyg 92, 663-666. 
 
Rossignol, J.F., Kabil, S.M., El-Gohary, Y., Elfert, A., and Keeffe, E.B. (2008). Clinical trial: 
randomized, double-blind, placebo-controlled study of nitazoxanide monotherapy for the 
treatment of patients with chronic hepatitis C genotype 4. Aliment Pharmacol Ther 28, 574-
580. 
 
Sasaki, A., Nitta, H., Otsuka, K., Umemura, A., Baba, S., Obuchi, T., and Wakabayashi, G. (2014). 
Bariatric surgery and non-alcoholic Fatty liver disease: current and potential future treatments. 
Front Endocrinol (Lausanne) 5, 164. 
 
Shehab, H.M., Elbaz, T.M., and Deraz, D.M. (2014). Nitazoxanide plus pegylated interferon and 
ribavirin in the treatment of genotype 4 chronic hepatitis C, a randomized controlled trial. Liver 
Int 34, 259-265. 
 
Shehata, M.A., Talaat, R., Soliman, S., Elmesseri, H., and Abd-Elsalam, S. (2017). Randomized 
controlled study of a novel triple nitazoxanide (NTZ)-containing therapeutic regimen versus the 
traditional regimen for eradication of Helicobacter pylori infection. Helicobacter 22. 
 
Shiffman, M.L., Ahmed, A., Jacobson, I.M., Pruitt, R.E., and Keeffe, E.B. (2010). 1188  Phase 2 
randomized, double-blind, placebo-controlled study of nitazoxanide with peginterferon alfa-2a 
and ribavirin in nonresponders (NR) with chronic hepatitis C genotype 1: final report. Journal 
of Hepatology 52, S461. 
 


Protocol No: NTZ-218-1 
Version: 21October18 
 
 
65 
Stockis, A., Allemon, A.M., De Bruyn, S., and Gengler, C. (2002a). Nitazoxanide 
pharmacokinetics and tolerability in man using single ascending oral doses. Int J Clin 
Pharmacol Ther 40, 213-220. 
 
Stockis, A., De Bruyn, S., Gengler, C., and Rosillon, D. (2002b). Nitazoxanide pharmacokinetics 
and tolerability in man during 7 days dosing with 0.5 g and 1 g b.i.d. Int J Clin Pharmacol Ther 
40, 221-227. 
 
Subichin, M., Clanton, J., Makuszewski, M., Bohon, A., Zografakis, J.G., and Dan, A. (2015). Liver 
disease in the morbidly obese: a review of 1000 consecutive patients undergoing weight loss 
surgery. Surg Obes Relat Dis 11, 137-141. 
 
Takeuchi-Yorimoto, A., Noto, T., Yamada, A., Miyamae, Y., Oishi, Y., and Matsumoto, M. 
(2013). Persistent fibrosis in the liver of choline-deficient and iron-supplemented L-amino acid-
defined diet-induced nonalcoholic steatohepatitis rat due to continuing oxidative stress after 
choline supplementation. Toxicol Appl Pharmacol 268, 264-277. 
 
Tanwar, S., Trembling, P.M., Guha, I.N., Parkes, J., Kaye, P., Burt, A.D., Ryder, S.D., Aithal, G.P., 
Day, C.P., and Rosenberg, W.M. (2013). Validation of terminal peptide of procollagen III for the 
detection and assessment of nonalcoholic steatohepatitis in patients with nonalcoholic fatty 
liver disease. Hepatology 57, 103-111. 
 
Taubel, J., Lorch, U., Rossignol, J.F., Ferber, G., and Camm, A.J. (2014). Analyzing the 
relationship of QT interval and exposure to nitazoxanide, a prospective candidate for influenza 
antiviral therapy--A formal TQT study. J Clin Pharmacol 54, 987-994. 
 
Wu, K.T., Kuo, P.L., Su, S.B., Chen, Y.Y., Yeh, M.L., Huang, C.I., Yang, J.F., Lin, C.I., Hsieh, M.H., 
Hsieh, M.Y., et al. (2016). Nonalcoholic fatty liver disease severity is associated with the ratios 
of total cholesterol and triglycerides to high-density lipoprotein cholesterol. J Clin Lipidol 10, 
420-425 e421. 
 
Zulu, I., Kelly, P., Njobvu, L., Sianongo, S., Kaonga, K., McDonald, V., Farthing, M., and Pollok, R. 
(2005). Nitazoxanide for persistent diarrhoea in Zambian acquired immune deficiency 
syndrome patients: a randomized-controlled trial. Aliment Pharmacol Ther 21, 757-763. 
 
10.2.
WHO Fact Sheet 312 
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KWWSZZZZKRLQWPHGLDFHQWUHIDFWVKHHWVIVHQ
 
10.3.
Studies on ClinicalTrials.gov 


Protocol No: NTZ-218-1 
Version: 21October18 
 
 
66 
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SAP_007.pdf: 
Statistical Analysis Plan 
 
A Monocentric, Open-Label, Proof of Concept Study to Evaluate the 
Safety and Efficacy of NTZ at 500mg Twice Daily on Collagen Turnover 
in Plasma in NASH Patients with Fibrosis Stage 2 or 3. 
 
 
NCT Number: 03656068 
Unique Protocol ID: NTZ-218-1 
Version Date : 09 April 2020 


STATISTICAL ANALYSIS PLAN 
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Pinnacle Clinical Research 
CONFIDENTIAL 
Statistical Analysis Plan 
Protocol No: NTZ-218-1 
 
Version 1.1 / 09-APR-2020 
Page 2 of 15 
 
SIGNATURE PAGE 
Protocol Title: 
A Monocentric, Open-Label, Proof of Concept Study to 
Evaluate the Safety and Efficacy of NTZ at 500 mg Twice 
Daily on Collagen Turnover in Plasma in NASH Patients 
with Fibrosis Stage 2 or 3. 
Protocol Number: 
NTZ-218-1 
We, the undersigned, have reviewed and approved this Statistical Analysis Plan: 
 
  
Signature 
 Date 
 
  
 
  
Miriam Zangmeister, MS 
Project Biostatistician 
Medpace, Inc. 
 DD-MMM-YYYY 
 
  
 
  
Phillippa Miranda, MD 
Medical Monitor 
Medpace, Inc. 
 DD-MMM-YYYY 
 
  
 
  
Stephen Harrison, MD 
Principal Investigator and Sponsor 
Pinnacle Clinical Research 
 DD-MMM-YYYY 
 
 


Pinnacle Clinical Research 
CONFIDENTIAL 
Statistical Analysis Plan 
Protocol No: NTZ-218-1 
 
Version 1.1 / 09-APR-2020 
Page 3 of 15 
 
VERSION HISTORY 
Version 
Version Date 
Description 
1.0 
30-APR-2019 
Original signed version 
1.1 
09-APR-2020 
Add clarification for baseline definition. 
 
 


Pinnacle Clinical Research 
CONFIDENTIAL 
Statistical Analysis Plan 
Protocol No: NTZ-218-1 
 
Version 1.1 / 09-APR-2020 
Page 4 of 15 
 
TABLE OF CONTENTS 
1 
Introduction ......................................................................................................................... 7 
2 
Study Overview ................................................................................................................... 7 
2.1 
Study Objectives and Endpoints .................................................................................. 7 
2.2 
Study Design ............................................................................................................... 8 
2.2.1 
Overview ............................................................................................................... 8 
2.2.2 
Randomization and Blinding ................................................................................. 8 
2.2.3 
Study Drug ............................................................................................................ 8 
2.2.4 
Sample Size Determination ................................................................................... 8 
3 
Statistical Methodology ....................................................................................................... 9 
3.1 
General Considerations ............................................................................................... 9 
3.1.1 
Analysis Day ......................................................................................................... 9 
3.1.2 
Analysis Visits ....................................................................................................... 9 
3.1.3 
Definition of Baseline ............................................................................................ 9 
3.1.4 
Summary Statistics ............................................................................................... 9 
3.1.5 
Hypothesis Testing ............................................................................................... 9 
3.1.6 
Handling of Dropouts and Missing Data ................................................................ 9 
3.2 
Analysis Populations .................................................................................................... 9 
3.2.1 
Full Analysis Set ................................................................................................... 9 
3.2.2 
Safety Analysis Set ..............................................................................................10 
3.3 
Subject Data and Study Conduct ................................................................................10 
3.3.1 
Subject Disposition and Analysis Populations ......................................................10 
3.3.2 
Protocol Deviations ..............................................................................................10 
3.3.3 
Demographic and Baseline Characteristics ..........................................................10 
3.3.4 
Medical History ....................................................................................................10 
3.3.5 
Concomitant Medications .....................................................................................10 
3.3.6 
Study Drug Exposure and Compliance ................................................................11 
3.4 
Efficacy Assessments .................................................................................................11 
3.4.1 
Fractional Synthesis Rate ....................................................................................11 
3.4.2 
Non-invasive Markers of Fibrosis .........................................................................12 
3.4.3 
Fibrosis Scores ....................................................................................................12 
3.4.4 
Exploratory Analysis ............................................................................................12 
3.5 
Safety Assessments ...................................................................................................13 
3.5.1 
Adverse Events ....................................................................................................13 
3.5.2 
Clinical Laboratory Tests .....................................................................................13 
3.5.3 
Vital Signs ............................................................................................................14 
3.5.4 
Electrocardiograms ..............................................................................................14 
3.5.5 
Physical Examinations .........................................................................................14 
3.5.6 
Other Safety Assessments ...................................................................................14 
4 
Data Safety Monitoring Board ............................................................................................14 


Pinnacle Clinical Research 
CONFIDENTIAL 
Statistical Analysis Plan 
Protocol No: NTZ-218-1 
 
Version 1.1 / 09-APR-2020 
Page 5 of 15 
 
5 
Analysis Timing ..................................................................................................................14 
5.1 
Interim Analysis ..........................................................................................................14 
5.2 
Pre-Final Analysis .......................................................................................................14 
5.3 
Final Analysis ..............................................................................................................14 
6 
Changes from Protocol-Specified Statistical Analyses .......................................................15 
7 
Programming Specifications ..............................................................................................15 
 
 
 


Pinnacle Clinical Research 
CONFIDENTIAL 
Statistical Analysis Plan 
Protocol No: NTZ-218-1 
 
Version 1.1 / 09-APR-2020 
Page 6 of 15 
 
LIST OF ABBREVIATIONS 
Abbreviation 
Definition 
ADaM 
Analysis Data Model 
AE 
Adverse event 
ALT 
Alanine Aminotransferase 
AST 
Aspartate aminotransferase 
ATC 
Anatomical therapeutic chemical 
BID 
Twice daily 
BMI 
Body mass index 
CI 
Confidence interval 
CK 
Creatine Kinase 
CRF 
Case report form 
CSR 
Clinical Study Report 
DSMB 
Data Safety Monitoring Board 
ECG 
Electrocardiogram 
EOS 
End of study 
FSR 
Fractional Synthesis Rate 
MedDRA 
Medical Dictionary for Regulatory Activities 
MRE 
Magnetic Resonance Elastrography 
MRI 
Magnetic resonance imaging 
NAFLD 
Non alcoholic fatty liver disease 
NASH 
Non alcoholic steatohepatitis 
Q1 
First quartile 
Q3 
Third quartile 
SAE 
Serious adverse event 
SAP 
Statistical Analysis Plan 
SDTM 
Study Data Tabulation Model 
TEAE 
Treatment-emergent adverse event 
TFL 
Table, figure, and listing 
ULN 
Upper limit of normal 
WHO 
World Health Organization 
 
 
 


Pinnacle Clinical Research 
CONFIDENTIAL 
Statistical Analysis Plan 
Protocol No: NTZ-218-1 
 
Version 1.1 / 09-APR-2020 
Page 7 of 15 
 
1 INTRODUCTION 
The purpose of this Statistical Analysis Plan (SAP) is to provide a description of the statistical 
methods to be implemented for the analysis of data from the study with protocol number 
NTZ-218-1.  The SAP will be finalized prior to database lock.  Any deviations from the SAP after 
database lock will be documented in the final Clinical Study Report (CSR). 
2 STUDY OVERVIEW 
2.1 Study Objectives and Endpoints 
Primary 
Objective 
Endpoint 
To evaluate the safety and tolerability of 
NTZ 500 mg twice daily (BID) after 24 weeks 
of treatment in patients with non alcoholic 
steatohepatitis (NASH)-induced stage 2 or 
stage 3 fibrosis. 
Adverse events (AEs) including treatment-
emergent AEs (TEAEs) and serious AEs 
(SAEs), laboratory evaluations, vital signs, 
electrocardiograms (ECGs), and physical 
examinations.   
Secondary 
Objective 
Endpoint 
To determine the effect of oral administration 
of NTZ 500 mg BID on de novo collagen 
synthesis through Fractional Synthesis Rate 
(FSR) of circulating plasma proteins in 
patients with NASH induced stage 2 or stage 
3 fibrosis. 
Percent change in FSR from baseline to end 
of treatment (24 weeks of treatment). 
To assess changes in liver stiffness as 
measured by FibroScan® and by Magnetic 
Resonance Elastography (MRE). 
Observed change in liver stiffness by 
FibroScan from baseline to 24 weeks of 
treatment 
Observed change in liver stiffness by MRE 
from baseline to 12 weeks and 24 weeks of 
treatment. 
To assess the change from baseline in non-
invasive markers of fibrosis: ELFTM test 
score, PIIINP, Hyaluronic acid, CK18, 
TIMP-1, YKL-40, Alpha 2 macroglobulin, 
miRNA, ProC3, ProC6, FGF19, and FGF21. 
Observed change in non-invasive biomarkers 
of fibrosis from baseline to 12 weeks and 24 
weeks of treatment. 
To assess changes in additional non-
invasive tests: fibrosis scores: non alcoholic 
fatty liver disease (NAFLD) fibrosis score 
and FIB-4. 
Observed change in fibrosis scores from 
baseline to 12 weeks and 24 weeks of 
treatment. 


Pinnacle Clinical Research 
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Protocol No: NTZ-218-1 
 
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2.2 Study Design 
2.2.1 Overview 
This is a Phase 2 proof of concept study to evaluate the safety and efficacy of open-label 
NTZ 500 mg BID in a NASH population with NASH induced Stage 2 or 3 fibrosis. 
The study will include a screening visit within 6 weeks of randomization (i.e. Day 1) followed by 
a 24-week treatment period consisting of daily, BID, oral administration of NTZ and an end of 
treatment follow up period of 4 weeks.   
Patients will come to the research center and complete the following visits: 
• 
Screening Visit within 6 weeks of Randomization (i.e. Day 1) 
• 
Day -14 Deuterated Water Run-In 
o Day-14, Day-11, Day-7 and Day 1: sampling for labeling of Deuterated water 
during run-in, 
• 
Repeat baseline labs for alanine aminotransferase (ALT), aspartate aminotransferase 
(AST), total bilirubin, alkaline phosphatase, creatine kinase (CK) collected at least 4 
weeks from initial screening. 
• 
Day 1 randomization: start of treatment  
• 
Every 4 weeks after randomization until 20 weeks post randomization 
• 
22 weeks post enrollment and Deuterated Water Administration Period 
o 22 weeks plus 3 days 
o Sampling for labeling of Deuterated Water on Day 155, Day 158, Day 162, and 
Day 169 
• 
24 weeks post randomization: End of Treatment 
• 
Week Post Treatment Follow Up 
2.2.2 Randomization and Blinding 
There is only one treatment group; there is no randomization. Any use of “randomization” refers 
to enrollment Day 1 (i.e. subject passed screening and will enter the treatment period). This is 
an open-label study; there is no blinding. 
2.2.3 Study Drug 
Patients will receive 500 mg NTZ (ALINIA®) twice daily for 24 weeks. NTZ will be supplied as 
500 mg of ALINIA (nitazoxanide) tablets, for oral use.  The tables are round, yellow, film-coated 
and debossed with ALINIA on one side and 500 on the other side. Each tablet contains 500 mg 
of nitazoxanide. 
2.2.4 Sample Size Determination 
The sample size of this Phase 2 proof of concept study is based on clinical assessment. Twenty 
male and female subjects with histologically confirmed NASH and fibrosis stage 2 or 3 will 


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provide sufficient information to assess the safety and tolerability of NTZ 500mg BID over 24 
weeks. 
3 STATISTICAL METHODOLOGY 
3.1 General Considerations 
3.1.1 Analysis Day 
Analysis day will be calculated from the date of first dose of study drug.  The day of the first 
dose of study drug will be Day 1, and the day immediately before Day 1 will be Day -1.  There 
will be no Day 0.  
3.1.2 Analysis Visits 
Scheduled visits will be assigned to analysis visits as recorded on the case report form (CRF). 
Where applicable, early termination visits will be summarized with Visit 10 Week 24 (i.e. Visit 10 
Week 24/Early Termination).  
3.1.3 Definition of Baseline  
For FibroScan and MRE, baseline is defined as the last measurement on or prior to the day of 
the first dose of study drug. In other words, time is not taken into consideration. 
Otherwise, baseline is defined as the last measurement prior to the first dose of study drug 
(generally should be the Day 1 pre-dose assessment). In other words, time is taken into 
consideration. 
3.1.4 Summary Statistics 
Categorical data will generally be summarized with counts and percentages of subjects.  The 
denominator used for the percentage calculation will be clearly defined in the table.  Continuous 
data will generally be summarized with descriptive statistics including n (number of non-missing 
values), mean, standard deviation, first quartile (Q1), median, third quartile (Q3), minimum, and 
maximum. 
3.1.5 Hypothesis Testing 
This is a proof of concept study to assess the safety and tolerability of NTZ 500 mg BID over 24 
weeks. There will be no formal hypothesis testing. 
3.1.6 Handling of Dropouts and Missing Data 
All data will be analyzed as observed. Dropouts and missing data will not be imputed.    
3.2 Analysis Populations 
3.2.1 Full Analysis Set 
The Full Analysis Set consists of all subjects that meet the eligibility criteria and enroll into the 
study (Visit 1 Day 1). 
The Full Analysis Set will be the primary population for all efficacy analyses. Only subjects with 
results for the specified efficacy parameter will be included in each analysis.  


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3.2.2 Safety Analysis Set 
The Safety Analysis Set consists of all enrolled subjects who receive at least one dose of study 
drug. 
The Safety Analysis Set will be the primary population for all safety analyses. 
3.3 Subject Data and Study Conduct 
3.3.1 Subject Disposition and Analysis Populations 
Counts and percentages of subjects who were screened (signed informed consent), 
discontinued early during screening (screen failures), and enrolled (Full Analysis Set) will be 
summarized in total based on all screened subjects.  Reasons for early discontinuation will also 
be summarized. 
Counts and percentages of subjects in the Full Analysis Set, Safety Analysis Set, discontinued 
early from the study, and completed the study will be summarized based on the Full Analysis 
Set.  Reasons for early discontinuation will also be summarized. 
3.3.2 Protocol Deviations 
Protocol deviations will be logged in the Medpace ClinTrak system. All protocol deviations will 
be listed. 
3.3.3 Demographic and Baseline Characteristics 
The following demographic and baseline characteristics will be summarized based on the Full 
Analysis Set: 
• 
Age (years) at informed consent and age categories (<65 years, ≥65 years),  
• 
Sex,  
• 
Race,  
• 
Ethnicity,  
• 
Height (cm) at Screening,  
• 
Baseline weight (kg), and 
• 
Baseline body mass index (BMI) (kg/m2). 
3.3.4 Medical History 
All medical history will be listed. 
3.3.5 Concomitant Medications 
Prior and concomitant medications will be coded to anatomical therapeutic chemical (ATC) 
class and preferred term using the World Health Organization (WHO) Drug Dictionary version 
Global B3 September 2018.  For summary purposes, medications will be considered prior 
medications if they stopped prior to the first dose of study drug and concomitant medications if 
they were taken at any time after the first dose of study drug (i.e. started prior to the first dose of 
study drug and were ongoing at the time of the first dose of study drug, or started after the first 
dose of study drug). Medications cannot be counted as both prior and concomitant. 
If a medication has incomplete start or stop dates, dates will not be imputed to determine 
whether a medication should be considered prior or concomitant. If the date is missing but it is 


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known that the medication was taken within the same month as the first dose of study drug, it 
will be considered concomitant. If the date and month are missing but it is known that the 
medication was taken within the same year as the first dose of study drug, it will be considered 
concomitant. 
Counts and percentages of subjects taking prior and concomitant medications by ATC class and 
preferred term will be summarized based on the Safety Analysis Set.   
3.3.6 Study Drug Exposure and Compliance 
Days of exposure to study drug will be calculated as date of last dose of study drug as recorded 
on the end of study form – date of first dose of study drug + 1.  Note that the exposure 
calculation is intended to describe the length of time a subject was exposed to study drug and 
therefore does not take study drug interruptions into account.  Days of exposure to study drug 
will be summarized based on the Safety Analysis Set with descriptive statistics and with counts 
and percentages of subjects with exposure in the following categories: 
• 
<4 weeks (<29 days) 
• 
4 to <8 weeks (29 – 56 days) 
• 
8 to <12 weeks (57 – 84 days) 
• 
12 to <16 weeks (85 – 112 days) 
• 
16 to <20 weeks (113 – 140 days) 
• 
20 to <24 weeks (141 – 168 days) 
• 
≥24 weeks (≥169 days) 
Percent compliance to the study drug regimen will be calculated as 100 x number of actual 
capsules taken / number of expected capsules taken.  The number of actual capsules taken will 
be calculated as number of capsules dispensed – number of capsules returned.  If study drug is 
not returned, the number of capsules returned will be considered 0 for the compliance 
calculation, i.e. it will be assumed that the subject took all capsules.  The number of capsules 
expected will be calculated as 2 x days of exposure.  Percent compliance to the study drug 
regimen will be summarized based on the Safety Analysis Set with descriptive statistics and 
with counts and percentages of subjects with compliance in the following categories: 
• 
<80% 
• 
80-120% 
• 
>120% 
3.4 Efficacy Assessments 
3.4.1 Fractional Synthesis Rate 
FSR of circulating plasma proteins will be measured at baseline and after 24 weeks of 
treatment.  The observed change and percent change in FSR from baseline to Visit 10 Week 24 
between the first and second deuterated water loading periods will be assessed to determine 
the effects of NTZ 500 mg BID on de novo collagen synthesis in the Full Analysis Set.   


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Wilcoxon signed-rank test will be used for statistical inference. Summary statistics of change in 
FSR (observed change and percent change) from baseline in FSR will be reported and 
evaluated at a significance level of 0.05. The p-value from Wilcoxon signed rank test will be 
reported. Only subjects with FSR at both baseline and Visit 10 Week 24 will be included in 
analysis.Liver Stiffness 
FibroScan 
Summary statistics for liver stiffness as measured by FibroScan will be provided at baseline and 
Visit 10 Week 24/Early Termination, including the change from baseline, based on the Full 
Analysis Set. 
Student’s T-test will be used for statistical inference. Summary statistics of change in liver 
stiffness as measured by FibroScan from baseline in FSR will be reported and evaluated at a 
significance level of 0.05. The 95% confidence interval (CI) and p-value from Student’s T-test 
will be reported. Only subjects with FibroScan at both baseline and Visit 10 Week 24/Early 
Termination will be included in analysis.  
MRE 
Summary statistics for liver stiffness as measured by MRE will be provided at baseline, Visit 4 
Week 12, and Visit 10 Week 24/Early Termination, including the changes from baseline, based 
on the Full Analysis Set. Student’s T-test will be used for statistical inference in the same 
manner as for FibroScan. 
3.4.2 Non-invasive Markers of Fibrosis 
Biomarkers of fibrosis include ELF test score, PIIINP, Hyaluronic acid, CK18, TIMP-1, YKL-40, 
Alpha 2 macroglobulin, miRNA, ProC3, ProC6, FGF19, and FGF21. 
Summary statistics for biomarkers will be provided at baseline, Visit 4 Week 12, Visit 10 Week 
24/Early Termination, and at end of study (EOS) Week 28, including the changes from baseline, 
based on the Full Analysis Set. Student’s T-test will be used for statistical inference in the same 
manner as for FibroScan. 
3.4.3 Fibrosis Scores 
NAFLD Fibrosis Score 
Summary statistics for NAFLD fibrosis score will be provided at baseline, Visit 4 Week 12, and 
Visit 10 Week 24/Early Termination, including changes from baseline, based on the Full 
Analysis Set. Student’s T-test will be used for statistical inference in the same manner as for 
FibroScan. 
FIB-4 
Summary statistics for FIB-4 will be provided at baseline, Visit 4 Week 12, and Visit 10 Week 
24/Early Termination, including changes from baseline, based on the Full Analysis Set. 
Student’s T-test will be used for statistical inference in the same manner as for FibroScan. 
3.4.4 Exploratory Analysis 
Additional serum and plasma samples will be maintained for future analysis.  Exploratory 
measures for magnetic resonance imaging (MRI)/MRE sequence development will be 
maintained for future analysis. 


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Exploratory scatter plots will be provided for FSR vs. ProC3, FSR vs. liver stiffness as measured 
by MRE, and FSR vs. ELF test score, including the Pearson Correlation Coefficients and p-
values. Additional exploratory analyses may also be performed to evaluate the association of 
individual exploratory biomarkers or combination of biomarkers with clinical measurements and 
other risk factors.   
3.5 Safety Assessments 
3.5.1 Adverse Events 
All AEs regardless of seriousness or relationship to study drug, including those occurring during 
the Screening Period, are to be recorded on the corresponding page(s) of the CRF and in the 
patient’s medical record from the informed consent form signature until study end for each 
patient. All AEs will be coded to system organ class and preferred term using Medical Dictionary 
for Regulatory Activities (MedDRA) version 21.0.   
TEAEs are defined as AEs that worsen in severity or relationship to study drug or commence on 
or after the time of start of first study drug administration. This may include AEs that start during 
follow-up after the last dose of study drug. 
Study drug related AEs are defined as AEs that are possibly related or related. 
An overview of AEs based on the Safety Analysis Set will be provided including counts and 
percentages of subjects with the following: 
• 
Any TEAEs (overall and by maximum severity) 
• 
Any study drug related TEAEs (overall and by maximum severity) 
• 
Any SAEs 
• 
Any study drug related SAEs 
• 
Any deaths due to AEs 
• 
Any AEs leading to withdrawal from study or study drug 
• 
Any study drug related AEs leading to withdrawal from study or study drug 
Counts and percentages of subjects will also be presented by system organ class and preferred 
term for TEAEs and study drug related TEAEs. In addition, summaries will be provided by 
system organ class, preferred term, and maximum severity. 
Listings will be presented specifically for SAEs and TEAEs leading to withdrawal from study or 
study drug. 
3.5.2 Clinical Laboratory Tests 
Blood and urine samples for chemistry, hematology, coagulation, urinalysis, and other safety 
markers will be processed by a central laboratory. Values and observed changes from baseline 
will be summarized at each scheduled visit (Visit 2 Week 4, Visit 3 Week 8, Visit 4 Week 12, 
Visit 5 Week 16, Visit 6 Week 20, Visit 10 Week 24/Early Termination, and EOS Week 28) by 
laboratory test based on the Safety Analysis Set. 
Shift tables from baseline to maximum post-baseline value (including consideration of 
unscheduled visits) will be presented for ALT and AST (≤1x upper limit of normal [ULN], 


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Protocol No: NTZ-218-1 
 
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>1xULN to ≤3xULN, >3xULN to ≤5xULN, >5xULN), CK (≤1xULN, >1xULN to ≤2.5xULN, 
>2.5xULN to ≤5xULN, >5xULN), and total bilirubin (≤1xULN, >1xULN to ≤1.5xULN, >1.5xULN to 
≤2xULN, >2xULN). 
3.5.3 Vital Signs 
Values and observed changes from baseline will be summarized at baseline and each 
scheduled visit (Visit 4 Week 12, Visit 10 Week 24/Early Termination, and EOS Week 28) for 
vital signs (systolic blood pressure, diastolic blood pressure, and heart rate), weight, and BMI 
based on the Safety Analysis Set. 
3.5.4 Electrocardiograms 
Values at baseline and Visit 10 Week 24/Early Termination, including the observed change from 
baseline, for 12-lead ECG parameters (heart rate, PR interval, QRS duration, QT interval, QTc 
interval, QTcF interval, and RR interval) will be summarized based on the Safety Analysis Set. 
The overall interpretations (normal, abnormal not clinically significant, or abnormal clinically 
significant) will be summarized by visit. 
3.5.5 Physical Examinations 
Physical examination findings (normal, abnormal not clinically significant, or abnormal clinically 
significant) at baseline, Visit 4 Week 12, Visit 10 Week 24/Early Termination, and EOS Week 28 
will be summarized by body system based on the Safety Analysis Set.  
3.5.6 Other Safety Assessments 
Other safety assessments such as pregnancy tests will be listed. 
4 DATA SAFETY MONITORING BOARD 
An independent Data Safety Monitoring Board (DSMB) composed of 3 liver experts will review 
the safety of patients enrolled in the trial. The DSMB will perform a safety data review every 3 
months, the first review occurring 3 months after first patient randomized. A DSMB charter will 
define the role, responsibilities, rules, and tasks of the DSMB. 
5 ANALYSIS TIMING 
5.1 Interim Analysis 
No interim analysis is planned.   
5.2 Pre-Final Analysis 
After the database is locked, the pre-final analysis will be generated.  Pre-final tables, figures, 
and listings (TFLs) will be provided approximately 3 weeks after database lock.   
5.3 Final Analysis 
After all comments on the pre-final analysis have been resolved and the study analysis 
database is declared final, the final analysis will be generated.  Final TFLs will be provided 
approximately 1 week after the study database is declared final.  If there were no changes to the 
pre-final analysis or the study database, the pre-final TFLs may be considered final.  In addition 


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Protocol No: NTZ-218-1 
 
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to TFLs, Study Data Tabulation Model (SDTM) data and Analysis Data Model (ADaM) data 
along with associated files will be provided.   
6 CHANGES FROM PROTOCOL-SPECIFIED STATISTICAL ANALYSES 
There are no changes from the protocol-specified statistical analysis. 
7 PROGRAMMING SPECIFICATIONS 
Analyses will be performed using SAS® version 9.4.  All available data will be presented in 
subject data listings which will be sorted by subject and visit date as applicable.  Detailed 
Programming Specifications will be provided in a separate document. 
 

Prot_000.pdf: 
Protocol 
 
A Monocentric, Open-Label, Proof of Concept Study to Evaluate the 
Safety and Efficacy of NTZ at 500mg Twice Daily on Collagen Turnover 
in Plasma in NASH Patients with Fibrosis Stage 2 or 3. 
 
 
NCT Number: 03656068 
Unique Protocol ID: NTZ-218-1 
Version Date : 27 August 2018 


 
 
Protocol No: NTZ-218-1 
Version: V27August18 
 
 
 
 
 
Page 1 
 
 
 
 
 
 
 
 
 
A Monocentric, Open-Label, Proof of Concept Study to Evaluate the Safety 
and Efficacy of NTZ at 500mg Twice Daily on Collagen Turnover in Plasma in 
NASH Patients with Fibrosis Stage 3 or 4 (compensated cirrhosis). 
  
 
 
 
Protocol Number: NTZ-218-1 
Version Date: 27AUGUST2018 
 


 
 
Protocol No: NTZ-218-1 
Version: V27August18 
 
 
 
 
 
Page 2 
List of Abbreviations 
  
ACR 
 
Albumin to Creatinine Ratio 
AE             
Adverse Event  
ALT            
Alanine Aminotransferase/GPT   
AST            
Aspartate aminotransferase  
BMI            
Body Mass Index   
CDAA  
Choline-deficient L-amino Acid Defined Diet 
CFR 
 
Code of Federal Regulations 
CSR 
 
Clinical Study Report 
CPK            
Creatine phosphokinase  
CRF            
Case Report/Record Form  
CRN            
Clinical Research Network  
D2O 
 
Deuterated Water 
GGT 
 
Gamma-glutamyltransferase 
eCRF           
Electronic case report form  
eGFR            
estimated Glomerular Filtration Rate   
EC            
Ethics Committee   
ECG            
Electrocardiogram  
ET             
Early Termination  
EOS 
 
End Of Study 
EU             
European Union  
FDA            
Food and Drug Administration  
FSR 
 
Fractional Synthesis Rate 
GCP            
Good Clinical Practice  
 
Hb             
Hemoglobin  
HBV           
Hepatitis B Virus  
HCG 
 
Human Chorionic Gonadotropin 
HCV           
Hepatitis C Virus  
HDPE  
High-density polyethylene 
HDL            
High Density Lipoprotein  
HIV            
Human Immunodeficiency Virus  
HOMA           Homeostasis Model Assessment  
HSC 
 
Hepatic Stellate Cell 
hs-CRP            High Sensitivity C-reactive protein  
ICH           
International Conference on Harmonization  
IEC 
 
Independent Ethics Committee 
IMP            
Investigational medical product  
IRB            
Institutional Review Board  
LDL            
Low density lipoprotein 


 
 
Protocol No: NTZ-218-1 
Version: V27August18 
 
 
 
 
 
Page 3 
MedDRA          Medical Dictionary for Regulatory Activity  
MRE 
 
Magnetic Resonance Elastography 
MRI            
Magnetic Resonance Imaging  
NAFLD            Non Alcoholic Fatty Liver Disease  
NAS            
NAFLD Activity Score  
NASH            
Non Alcoholic Steatohepatitis  
NTZ 
 
Nitazoxanide 
PI             
Principle Investigator  
PK             
Pharmacokinetic 
PT/INR            Prothrombin Time/International Normalized Ratio  
 
RNA            
Ribonucleic Acid  
SAB            
Safety Advisory Board   
SAE            
Serious Adverse Event 
SAP 
 
Statistical Analysis Plan  
TQT 
 
Thorough-QT study 
  
 
 
 


 
 
Protocol No: NTZ-218-1 
Version: V27August18 
 
 
 
 
 
Page 4 
Study Synopsis 
Protocol Number: 
NTZ-218-1 
Study Title: 
A Monocentric, Open-Label, Proof of Concept Study to Evaluate the Safety 
and Efficacy of NTZ at 500mg Twice Daily on Collagen Turnover in Plasma in 
NASH Patients with Fibrosis Stage 3 or 4 (compensated cirrhosis). 
Phase: 
Proof of Concept 
Study Drug: 
Nitazoxanide 
Indication: 
NASH induced fibrosis 
Principal Investigator & Sponsor 
Stephen Harrison, MD 
Pinnacle Clinical Research 
12950 Toepperwein Road 
Live Oak, TX 78233 
Study Duration: 
34 weeks 
Objectives: 
The primary objective of the study is: 
• 
To evaluate the safety and tolerability of NTZ 500mg bid after 24 
weeks of treatment in patients with NASH-induced stage 3 or 
Stage 4 fibrosis (compensated cirrhosis) 
 
 
The secondary objectives of the study are: 
• 
To determine the effect of oral administration of NTZ 500mg bid 
on de novo collagen synthesis through Fractional Synthesis Rate 
(FSR) of circulating plasma proteins in patients with NASH-
induced stage 3 or stage 4 fibrosis (compensated cirrhosis). 
• 
To assess changes in liver stiffness as measured by FibroScan® 
and by Magnetic Resonance Elastography (MRE). 
• 
To assess the change from baseline in non-invasive markers of 
fibrosis 
• 
To assess changes in Fibrosis scores: ELFTM test score, Fibrotest, 
Fibrometer, hepascore, NAFLD fibrosis score, FIB-4 
 
Study Design: 
This is a Phase 2 proof of concept study to evaluate the safety and efficacy 
of open-label NTZ 500mg bid in a NASH population with NASH induced 
Stage 3 or 4 fibrosis (compensated cirrhosis). 
 
The study will include a screening visit within 6 weeks of randomization 
(Day 1) followed by a 24-week treatment period consisting of daily, BID, 
oral administration of NTZ and an end of treatment follow up period   of 4 
weeks.   
Patients will come to the research center and complete the following visits: 
• 
Screening Visit within 6 weeks of Randomization 
• 
Day -14 Deuterated Water Run-In 
o 
Day-14, Day-11, Day-7 and Day 1: sampling for labeling 
of Deuterated water during run-in 
• 
Day 1 randomization : start of treatment 
• 
Every 4 weeks after randomization until 20 weeks post 
randomization 


 
 
Protocol No: NTZ-218-1 
Version: V27August18 
 
 
 
 
 
Page 5 
• 
22 weeks post randomization and Deuterated Water 
Administration Period 
o 
22 weeks plus 3 days, plus 7 days, plus 14 days for 
sampling for kinetic biomarker analyses 
• 
24 weeks post randomization : End of Treatment 
• 
4 Week Post Treatment Follow Up 
Number of Sites: 
1 site  
Number of Subjects: 
20 evaluable subjects 
Drug Dosage / Frequency: 
500 mg NTZ BID 
Route of Administration: 
Oral Administration 
Inclusion Criteria: 
Patients must meet all of the following inclusion criteria to be eligible for 
enrollment in the trial:  
1. Males or females aged from 18 to 75 years inclusive at the Screening 
Visit.  
2. Must provide signed written informed consent and agree to comply 
with the study protocol.  
3. Females participating in this study must be of non-childbearing 
potential or using highly efficient contraception for the full duration of 
the study.  Highly efficient contraception is defined as the use of one of 
the following methods of birth control in addition to a male partner 
using a condom (unless surgically sterile) from Screening to 90 days 
after the last dose of study medication: 
(1) hormone-containing contraceptive 
(2) intrauterine device 
(3) cervical cap or diaphragm with spermicidal agent 
4. Histological confirmation of steatohepatitis on a diagnostic liver biopsy 
(biopsy obtained within 6 months prior to Screening or during the 
Screening Period) with at least 1 in each component of the NAS 
(steatosis scored 0-3, ballooning degeneration scored 0-2, and lobular 
inflammation scored 0-3).   
5.    Fibrosis stage of 3 or 4, according to the NASH CRN fibrosis staging 
system on a diagnostic liver biopsy (biopsy obtained within 6 months 
prior to Screening or during the Screening Period).  For patients with 
fibrosis stage 4, only patients who have no current or historical 
evidence of decompensation of cirrhosis. 
Exclusion Criteria: 
Patients who meet any of the following criteria will be excluded from 
entering the study:  
1. History of efficient bariatric surgery within 5 years prior to Screening, 
or planned bariatric surgery in the course of the study.  
2. Patients with HbA1c >10.0%. If abnormal at the first Screening Visit, the 
HbA1c measurement can be repeated. A repeated abnormal HbA1c 
(HbA1c >10.0%) leads to exclusion.  
3. Patients with a history of clinically significant acute cardiac event within 
6 months prior to Screening such as: stroke, transient ischemic attack, 
or coronary heart disease (angina pectoris, myocardial infarction, 
revascularization procedures).  
4. Weight loss of more than 10% within 6 months prior to Randomization.  
5. Patient with any history or presence of decompensated cirrhosis. 


 
 
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Page 6 
6. Current or recent history (<1 year) of significant alcohol consumption. 
For men, significant consumption is typically defined as higher than 30 
g pure alcohol per day. For women, it is typically defined as higher than 
20 g pure alcohol per day.  
7. Current or history of other substance abuse within 1 year prior to 
screening. 
8. Pregnant or lactating females or females planning to become pregnant 
during the study period.  
9. Other well documented causes of chronic liver disease according to 
standard diagnostic procedures including, but not restricted to:  
a. Positive hepatitis B surface antigen (HBsAg)  
b. Positive HCV RNA, (tested for in case of known cured HCV 
infection, or positive HCV Ab at Screening)  
c. 
Suspicion of drug-induced liver disease  
d. Alcoholic liver disease  
e. Autoimmune hepatitis  
f. 
Wilson’s disease   
g. 
Primary biliary cirrhosis, primary sclerosing cholangitis  
h. Genetic homozygous hemochromatosis  
i. 
Known or suspected HCC  
j. 
History or planned liver transplant, or current MELD 
score >15.  
10. Patients who cannot be contacted in case of emergency.  
11. Known hypersensitivity to the investigational product or any of its 
formulation excipients.  
12. Patients who are taking warfarin or other highly plasma protein-bound 
drugs with narrow therapeutic indices. 
13. Patients who are currently participating in, plan to participate in, or 
have participated in an investigational drug trial or medical device trial 
containing active substance within 30 days or five half-lives, whichever 
is longer, prior to Screening.  
14. Evidence of any other unstable or, untreated clinically significant 
immunological, endocrine, hematological, gastrointestinal, 
neurological, neoplastic, or psychiatric disease.  
15. Mental instability or incompetence, such that the validity of informed 
consent or ability to be compliant with the study is uncertain.  
16.  History of noncompliance with medical regimens, or patients who are 
considered to be unreliable 
17. Positive anti-human immunodeficiency virus (HIV) antibody. 
18. AST and/or ALT >10 x upper limit of normal (ULN).  
19. Conjugated bilirubin >2.0 mg/dL due to altered hepatic function. Note: 
Gilbert Disease patients are allowed into the study.  
20. INR >1.50 due to altered hepatic function.  
21. Platelet count <100,000/mm3 due to portal hypertension.  
22. Significant renal disease, including nephritic syndrome, chronic kidney 
disease (defined as patients with markers of kidney damage or eGFR of 
less than 60 ml/min/1.73 m2). 
Evaluation Criteria 
Primary Endpoint: 
To assess the safety and tolerability of NTZ after 24 weeks of treatment  
 


 
 
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Secondary Endpoints: 
To assess the % change in FSR from baseline to end of treatment (24 weeks 
of treatment) 
To assess the change in liver stiffness by MRE from baseline to 12 weeks 
and 24 weeks of treatment 
To assess the change in liver stiffness by fibroscan from baseline to 24 
weeks of treatment  
To assess the change from baseline to 12 weeks and 24 weeks of treatment 
of fibrosis serum biomarkers and scores. 
 
 


 
 
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Version: V27August18 
 
 
 
 
 
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TABLE 1 : Schedule of events 
 
Screening period 
 
 
Treatment period 
Visit 
SV 
SV2 
SV3 
SV4 
V1 
V2 
V3 
V4 
V5 
V6 
V7 
V8 
V9 
V10 
EOS 
ET 
Day 
-42 
-14 
-11 
-7 
1 
29 
57 
85 
113 
141 
155 
158 
162 
169 
+28 
days 
 
Window 
 
+1 
+1 
+1 
+3 
+3 
+3 
+3 
+3 
+3 
+1 
+1 
+1 
+1 
+3 
 
Week 
-6 
-2 
 
 
0 
4 
8 
12 
16 
20 
22 
 
 
24 
28 
 
Informed 
Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical 
History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inclusion 
Exclusion 
Criteria 
X 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
Height 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Weight, BMI 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
Physical Exam 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
12-Lead ECG 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
X 
Vital Signs 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
Con Meds 
X 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
AEs 
X 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Liver Biopsy1 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
MRE 
 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
 
X 
Fibroscan 
 
 
 
 
X 
 
 
 
 
 
 
 
 
X 
 
X 
Dispense 
deuterated 
water 
 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
FSR 
 
X 
X 
X 
X 
 
 
 
 
 
X 
X 
X 
X 
 
 
Study Drug 
Dispense 
 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
Study Drug 
Compliance 
 
 
 
 
 
X 
X 
X 
X 
X 
 
 
 
X 
 
 
X 
 
 
 
1 Required only if there is no history of a liver biopsy within the past 6 months.  


 
 
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TABLE 2 : Biological assessments 
 
Screening period 
 
 
Treatment period 
Visit 
SV 
SV2 
SV3 
SV4 
V1 
V2 
V3 
V4 
V5 
V6 
V7 
V8 
V9 
V10 
EOS 
ET 
Day 
-42 
-14 
-11 
-7 
1 
29 
57 
85 
113 
141 
155 
158 
162 
169 
+28 
days 
 
Week 
-6 
-2 
 
 
0 
4 
8 
12 
16 
20 
22 
 
 
24 
28 
 
Labs - 
Haematology 
haemoglobin, 
haematocrit, RBC, 
WBC, differential 
count, platelet 
count, 
reticulocytes 
count, MCV 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Coagulation (PT 
(INR)) 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Labs- Urinary 
Pregnancy tests2 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
 
X 
Labs – Serology 
HIV, HBs and HCV 
serology 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Labs – 
Biochemistry 
HbA1c, fasting 
plasma glucose, 
insulin, HOMA-IR, 
fructosamine, 
alkaline 
phosphatase, ALT, 
AST, GGT, CPK, 
total and 
conjugated 
bilirubin, ferritin, 
creatinine, eGFR, 
uric acid, BUN, 
total cholesterol, 
HDL-C, LDL-C, 
triglycerides, 
albumin, lipase, 
amylase, 
electrolytes 
(sodium, 
potassium, 
chloride, calcium), 
MELD 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
 
2 If Urine pregnancy test is positive, a serum β-hCG is required.  


 
 
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Screening period 
 
 
Treatment period 
Visit 
SV 
SV2 
SV3 
SV4 
V1 
V2 
V3 
V4 
V5 
V6 
V7 
V8 
V9 
V10 
EOS 
ET 
Day 
-42 
-14 
-11 
-7 
1 
29 
57 
85 
113 
141 
155 
158 
162 
169 
+28 
days 
 
Inflammatory 
markers 
hsCRP, fibrinogen, 
haptoglobin, 
resistin,  TNF-, 
TGF-, IL-6, PAI-1 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
Biomarkers of 
fibrosis 
ELF Score, PIIINP, 
Hyaluronic acid, 
CK18, TIMP-1, YKL-
40, Alpha 2 
macroglobulin,, 
,miRNA, ProC3, 
ProC6, FGF19, 
FGF21 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
Labs – Urinalysis 
dipstick  
Specific gravity, 
pH, protein, 
glucose, ketones, 
bilirubin, 
urobilinogen, 
blood, nitrite, 
leukocytes 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
 
X 
Safety markers 
Serum Cystatin C, 
troponin T, NT 
proBNP 
Urinary albumin, 
urinary creatinine, 
urinary ACR 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Labs -- FSR 
 
X 
X 
X 
X 
 
 
 
 
 
X 
X 
X 
X 
 
 
Sampling for 
additional 
parameters 
X 
 
 
 
X 
 
 
X 
 
X 
 
 
 
X 
X 
X 
 
 
 


 
 
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Table of Contents 
1. 
Introduction .......................................................................................................................... 15 
1.1. 
Background ................................................................................................................... 15 
1.2. 
General Information for Nitazoxanide ......................................................................... 16 
1.2.1. 
Nitazoxanide (NTZ) ............................................................................................... 16 
1.2.2. 
Clinical Safety & Toxicology .................................................................................. 16 
1.2.3. 
Non clinical pharmacology ................................................................................... 18 
1.3. 
Rationale for This Study................................................................................................ 21 
2. 
Objectives .............................................................................................................................. 21 
2.1. 
Primary Objective ......................................................................................................... 21 
2.2. 
Secondary Objectives: .................................................................................................. 21 
3. 
Study Design.......................................................................................................................... 22 
3.1. 
Study Design.................................................................................................................. 22 
3.2. 
Treatment Plan and Regimen ....................................................................................... 23 
4. 
Subject Population ................................................................................................................ 23 
4.1. 
Number of Subjects & Subject Selection ..................................................................... 23 
4.2. 
Inclusion Criteria ........................................................................................................... 23 
4.3. 
Exclusion Criteria .......................................................................................................... 24 
5. 
Investigational Medicinal Products ...................................................................................... 26 
5.1. 
Description .................................................................................................................... 26 
5.2. 
Packaging ...................................................................................................................... 26 
5.3. 
Labeling ......................................................................................................................... 26 
5.4. 
Storage and Handling ................................................................................................... 27 
5.5. 
Dosage and Administration .......................................................................................... 27 
5.6. 
Prior and Concomitant Medications ............................................................................ 27 
5.6.1. 
Non-permitted medication ................................................................................... 27 
5.6.2. 
Permitted Medications ......................................................................................... 28 
5.7. 
Study Drug Accountability ............................................................................................ 28 
6. 
Study Procedures .................................................................................................................. 28 
6.1. 
Duration of Study Participation ................................................................................... 28 


 
 
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6.2. 
Screening Assessment .................................................................................................. 29 
6.2.1. 
Screening Visit ....................................................................................................... 29 
6.3. 
Day -14 Deuterated Water Run In ................................................................................ 30 
6.4. 
Treatment Period Assessments .................................................................................... 30 
6.4.1. 
Day 1 Visit.............................................................................................................. 30 
6.4.2. 
Week 4 and Week 8 Visit + 3 days ........................................................................ 31 
6.4.3. 
Week 12 Visit + 3 days .......................................................................................... 31 
6.4.4. 
Week 16 and Week 20 visits ±3 days ................................................................... 32 
6.4.5. 
Week 22 Visit + 3 days (Day 155) ......................................................................... 32 
6.4.6. 
Week 24 Visit +3 ................................................................................................... 32 
6.5. 
Follow Up Period ........................................................................................................... 33 
6.5.1. 
4-Week Post Treatment Follow up (EOS – End of study visit) ±3 days ............... 33 
6.5.2. 
Early Termination (ET) .......................................................................................... 33 
6.5.3. 
Unscheduled Visits ................................................................................................ 34 
6.6. 
Assessment for Premature Discontinuation from Study ............................................. 34 
6.7. 
Description of Assessments .......................................................................................... 36 
6.7.1. 
Histological Assessments ...................................................................................... 36 
6.7.2. 
Assessment of newly synthesized proteins ......................................................... 36 
6.7.3. 
Biological Assessments (Table 2) .......................................................................... 36 
6.7.4. 
Electrocardiogram ................................................................................................. 39 
6.7.5. 
MRE ....................................................................................................................... 39 
6.7.6. 
Fibroscan ............................................................................................................... 40 
6.7.7. 
Medical History ..................................................................................................... 40 
6.7.8. 
Physical Examination ............................................................................................ 40 
6.7.9. 
Vital Signs .............................................................................................................. 40 
6.7.10. 
Pregnancy Testing ................................................................................................. 41 
7. 
Adverse Events and Toxicity Management .......................................................................... 41 
7.1. 
Definitions of Adverse Events, Serious Adverse Events and Unexpected Events ...... 41 
7.1.1. 
Adverse Events ...................................................................................................... 41 
7.1.2. 
Clinical Laboratory Abnormalities and Other Abnormal Assessments as Adverse 
Events or Serious Adverse Events ........................................................................................ 42 
7.1.3. 
Serious Adverse Events ......................................................................................... 42 


 
 
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7.1.4. 
Unexpected adverse event ................................................................................... 43 
7.2. 
Assessment of Adverse Events & Serious Adverse Events .......................................... 43 
7.2.1. 
Intensity assessment ............................................................................................ 43 
7.2.2. 
Relation to the study treatment .......................................................................... 44 
7.2.3. 
Action taken and outcome ................................................................................... 45 
7.3. 
Reporting ....................................................................................................................... 46 
7.3.1. 
Reporting an adverse event ................................................................................. 46 
7.3.2. 
Initial reports......................................................................................................... 46 
7.3.3. 
Follow-up reports ................................................................................................. 47 
7.3.4. 
Follow-up .............................................................................................................. 47 
7.4. 
Post Study Reporting Requirements ............................................................................ 48 
7.5. 
Special Situation Reports .............................................................................................. 48 
7.5.1. 
Pregnancy .............................................................................................................. 48 
7.5.2. 
Medication error ................................................................................................... 48 
7.5.3. 
Misuse ................................................................................................................... 49 
7.5.4. 
Overdose ............................................................................................................... 49 
7.5.5. 
Abuse ..................................................................................................................... 49 
8. 
Statistical Considerations ..................................................................................................... 49 
8.1. 
Analysis Endpoints ........................................................................................................ 49 
8.1.1. 
Primary Endpoint .................................................................................................. 49 
8.1.2. 
Secondary Endpoints ............................................................................................ 49 
8.2. 
Analysis Sets .................................................................................................................. 50 
8.3. 
Data Handling Conventions .......................................................................................... 50 
8.4. 
Demographic Data and Baseline Characteristics ......................................................... 50 
8.5. 
Primary Safety Analysis ................................................................................................ 50 
8.5.1. 
Adverse Events ...................................................................................................... 50 
8.5.2. 
Laboratory Evaluations ......................................................................................... 51 
8.5.3. 
Other Safety Evaluations ...................................................................................... 51 
8.6. 
Secondary Efficacy Analyses ......................................................................................... 51 
8.6.1. 
FSR ......................................................................................................................... 51 
8.6.2. 
Liver stiffness ........................................................................................................ 52 


 
 
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8.6.3. 
Exploratory Analysis ............................................................................................. 52 
8.6.4. 
Interim Analysis .................................................................................................... 52 
8.7. 
Sample Size ................................................................................................................... 52 
9. 
Responsibilities ..................................................................................................................... 52 
9.1. 
Investigator Responsibilities ........................................................................................ 52 
9.2. 
Good Clinical Practice ................................................................................................... 53 
9.3. 
Institutional Review Board Review & Approval .......................................................... 54 
9.4. 
Informed Consent ......................................................................................................... 54 
9.5. 
Confidentiality .............................................................................................................. 54 
9.6. 
Study Files and Retention of Records .......................................................................... 55 
9.7. 
Electronic Case Report Forms ....................................................................................... 56 
9.8. 
Investigational Medicinal Product Accountability and Return ................................... 57 
9.9. 
Inspections .................................................................................................................... 57 
9.10. Protocol Compliance ..................................................................................................... 57 
9.11. Protocol Modifications ................................................................................................. 57 
9.12. Study Reports ................................................................................................................ 58 
9.13. Study Termination ........................................................................................................ 58 
10. 
References ......................................................................................................................... 59 
10.1. Literature ....................................................................................................................... 59 
10.2. WHO Fact Sheet 312 ..................................................................................................... 62 
10.3. Studies on ClinicalTrials.gov ......................................................................................... 62 
 
 
 
 
 


 
 
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1. Introduction 
1.1. 
Background 
Non-alcoholic fatty liver disease (NAFLD) has become one of the most prominent forms 
of chronic liver disease worldwide, reflecting the epidemic of global obesity. Those with 
the progressive variant of NAFLD, non-alcoholic steatohepatitis (NASH), are at significantly 
increased risk of multisystem morbidity and mortality. However, there are currently no 
approved pharmacologic therapies for NASH (Konerman et al, 2018). The World Health 
Organization estimates the prevalence of obesity has more than doubled since 1980 with 
>600 million people (13%) having a body mass index (BMI)) ≥30 (Konerman et al, 2018). 
In this setting, the global prevalence of diabetes among adults has also increased from 
4.7% in 1980 to 8.5% as of 2014 (WHO Fact Sheet 312) . Prevalence rates of NAFLD among 
those with metabolic disease are notably higher.  Approximately a third of patients with 
hypertension, half of patients with dyslipidaemia, up to two-thirds of patients with type II 
diabetes, and  >90% of patients undergoing bariatric surgery had evidence of NAFLD .( 
Leite et al, 2009; Fruci et al, 2013; Assy et al, 2000; Wu  et al, 2016; Sasaki et al, 2014 
Subichin et al; 2015 ; Donati et al ,2004). 
The underlying pathophysiologic mechanisms that contribute to the development and 
progression of NAFLD and NASH are complex and include modulation of metabolic 
pathways, inflammatory cascades, and/or mechanisms impacting fibrosis.  During disease 
progression fibrosis may worsen, while features of steatohepatitis resolve or ‘‘burnout”.    
As a result, NASH and fibrosis need to be evaluated independently to ensure a beneficial 
impact on one parameter does not simultaneously result in a negative impact on another 
endpoint of interest, particularly given that individual treatments tend to focus on one 
primary mechanism of action (i.e. metabolic or NASH disease modifier vs. antifibrotic) ( 
Konerman et al, 2018). 
Persistent and progressive fibrosis is a characteristic of many chronic liver diseases in 
which excessive accumulation of collagen-rich extracellular matrix is a key feature. 


 
 
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Following liver injury, quiescent Hepatic Stellate Cells (HSC) undergo a process of 
activation that is characterized by a differentiation into (α-SMA)-positive contractile 
myofibroblasts which secrete high amounts of collagen and play a central role in the 
fibrogenic process (Friedman et al, 2008, Carpino et al, 2005). Nitazoxanide may be a 
potent antifibrotic agent that can potentially be used for the treatment of different fibrotic 
pathologies, including liver fibrosis. (Belanger et al;2017) 
1.2. 
General Information for Nitazoxanide 
1.2.1. Nitazoxanide (NTZ)  
Nitazoxanide (NTZ) is a broad-spectrum antiparasitic and broad-spectrum antiviral drug 
used for the treatment of various helminthic, protozoal, and viral infections.  It was 
approved in the United States by the Food and Drug Administration (Alinia®, Romark 
Laboratories) as an Orphan Drug for treatment of diarrhea caused by C. parvum and 
Giardia intestinalis in adults and children at least 12 months of age [Rossignol, 2014]. 
The approved Alinia dose for adults is 500 mg twice daily (BID) for three days.  
1.2.2. Clinical Safety & Toxicology 
Nitazoxanide is rapidly metabolized in the liver to an active metabolite tizoxanide (TZ) in 
vivo. TZ undergoes conjugation to form tizoxanide glucuronide. Nitazoxanide is not 
detectable in the serum following oral administration.  Peak concentrations in blood 
plasma of TZ and TZ glucuronide are seen between 1-4 hours.  NTZ is excreted in both 
Urine (~33%) and feces (~67%).   
The safety of ALINIA was evaluated in 2177 HIV-uninfected subjects 12 months of age 
and older who received ALINIA Tablets or ALINIA for Oral Suspension at the 
recommended dose for at least three days. In pooled controlled clinical trials involving 
536 HIV-uninfected subjects treated with ALINIA Tablets or ALINIA for Oral Suspension, 
the most common adverse reactions were abdominal pain, headache, chromaturia and 
nausea (≥2%).[Alinia USPI] 


 
 
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Safety data were analyzed separately for 280 HIV-uninfected subjects ≥12 years of age 
receiving Alinia at the recommended dose for at least 3 days in 5 placebo-controlled 
clinical trials and for 256 HIV-uninfected subjects 1 through 11 years of age in 7 
controlled clinical trials. There were no differences between the adverse reactions 
reported for Alinia-treated subjects based upon age [Alinia USPI]. 
Twenty-one clinical studies of NTZ were conducted in patients with chronic hepatitis C 
((14 studies: Rossignol et al, 2008; Rossignol et al, 2009; Bacon et al, 2010, Rossignol et 
al, 2010; Shiffman et al, 2010 ; Laufer et al, 2011; Kabil et al, 2011 ; Amorosa et al, 2013; 
Shebab et al, 2014; Macias et al, 2015; Kohla et al, 2016; Basu et al, 2013; 
NCT01185028; NCT01770483)), hepatic encephalopathy in chronic liver failure (1 study: Basu 
et al, 2008), acquired immune deficiency syndrome (AIDS)-related cryptosporidiosis (3 studies: 
Rossignol et al, 1998; Rossignol et al, 2006; Zulu et al, 2005), or Helicobacter pylori (2 
studies: Abd-Elsalam et al, 2016; Shehata et al., 2017), and Clostridium difficile infections (1 
study: Musher et al, 2009) in the literature. The dose of NTZ administered in 19 studies was 
500 mg BID; in one study 1000 mg NTZ BID was administered, whereas in one compassionate-
use study the NTZ dose administered ranged from 500 to 1500 mg BID. Most studies 
administered NTZ treatment for 48 to 60 weeks, whereas the duration of treatment in the 
compassionate-use study ranged from 1 day to 4 years.  
The studies identified from the literature review demonstrate the safety of NTZ in the 
treatment of chronic hepatitis C, hepatic encephalopathy in chronic liver failure, AIDS-
related cryptosporidiosis, persistent diarrhea in patients with AIDS, and Helicobacter 
pylori and Clostridium difficile infections.  
The adverse event (AE) profile of NTZ was predominantly GI in nature and included 
nausea, vomiting, diarrhea, abdominal pain, dyspepsia, constipation, flatulence, and 
dysphagia. These GI AEs were generally mild in intensity and transient. Other AEs 
related to NTZ treatment were fatigue, rash, headache, myalgia, and arthralgia. When 
reported, no significant changes were observed in the electrocardiograms (ECGs), vital 


 
 
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signs, and clinical laboratory parameters after administration with NTZ [Stockis et al, 
2002a; Stockis et al, 2002b]. A formal International Council on Harmonization (ICH) E14 
compliant thorough QT (TQT) study [Taubel et al, 2014] completed the cardiac safety 
evaluation and showed that neither a therapeutic single dose of 675 mg nor a 2700 mg 
(supra-therapeutic) dose of NTZ prolonged the QT interval in healthy male and female 
volunteers.   
The safety profile of NTZ reported in all these studies therefore provides supporting 
evidence for the potential use of NTZ for the treatment of liver fibrosis in patients with 
NASH.  
The following adverse reactions have been identified during post approval use of ALINIA 
as described in the Package Insert:  
• Gastrointestinal disorders: diarrhea, gastroesophageal reflux disease 
• Nervous System disorders: dizziness 
• Respiratory, thoracic and mediastinal disorders: dyspnea 
• Skin and subcutaneous tissue disorders: rash, urticaria 
1.2.3. Non clinical pharmacology 
Nonclinical pharmacology studies were conducted to determine whether NTZ and its 
main metabolites (TZ and TZ glucuronide [TZ glu]) exhibit anti-fibrotic properties in vitro, 
and in vivo. 
NTZ and its main metabolite, TZ, significantly (p<0.001 from 0.3 µM) inhibited, in a dose-
dependent manner, the expression of αSMA protein in TGF-β-induced human HSCs, but 
TZ glu had no anti-fibrotic properties. NTZ and TZ demonstrated good potency as the 
IC50 was in the micromolar range.  
The CDAA diet-induced rodent model develops a fibrosing NASH within a relatively short 
period [Takeuchi-Yorimoto et al, 2013], which makes it a suitable model for studying the 
treatment of both NASH pathology and hepatic fibrosis. 


 
 
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In studies to determine the effects of NTZ on liver fibrosis in C57BL/6J mice induced by a 
CDAA diet supplemented with 1% cholesterol, microscopic examination revealed that 
NTZ at doses of 11.4 mg/kg/day and 33.9 mg/kg/day tended to reduce, in a dose-
dependent manner, CDAA + 1% cholesterol diet-induced NASH. Although NTZ did not 
have a beneficial effect on hepatic steatosis and lobular inflammation grade, a tendency 
to reduce hepatocyte ballooning was observed, which resulted in a slight reduction in 
NASH prevalence.  
At 12 weeks, NTZ significantly prevented, in a dose-dependent manner, hepatic collagen 
storage induced by the CDAA + 1% cholesterol diet. However, upon histological 
examination, this beneficial effect was not reflected by a dose-dependent improvement 
of the fibrosis grade. A significant decrease of the fibrosis area was observed with NTZ at 
doses of 11.4 mg/kg/day (p<0.05) and 78.1 mg/kg/day (p<0.001) and correlated with 
the significant decrease of the hepatic collagen content.  
The CDAA + 1% cholesterol diet notably induces a significant augmentation in circulating 
markers of liver injury such as AST, ALT, and PIIINP, which is directly associated with 
fibrosis [Tanwar et al, 2013]. Moreover, the hepatic content is dramatically modified as 
illustrated by the significant increase of collagen. Mice treated with NTZ at 
78.1 mg/kg/day exhibited lower plasma levels of ALT (-17%, p>0.05), AST (-24%, p<0.05), 
and PIIINP (-25%, p<0.01). These data suggest a potential preventive action of NTZ on 
liver damage induced by the CDAA + 1% cholesterol diet in mice.  
The hepatic expression of fibrotic markers (collagens, MMP2), but also inflammatory 
markers (TNFα) were enhanced in the animals fed the CDAA + 1% cholesterol diet as 
compared with animals fed a CSAA control diet. Chronic administration of NTZ at 
78.1 mg/kg/day to mice fed a CDAA + 1% cholesterol diet led to the inhibition of the 
Col1α1 (-17%, p>0.05), Col1α2 (-25%, p<0.05), MMP2 (-32%, p<0.05), and TNFα  
(-28%, p<0.01) gene expression.  


 
 
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In the studies with CCl4-treated mice, NTZ significantly prevented hepatic fibrosis. This 
was shown at the dose of 95.3 mg/kg/day on the hepatic collagen content decrease in 
the first study [SSR_NTZ_PHA1_004], whereas no significant improvement was observed 
upon histological examination. Conversely, in the second study [SSR_NTZ_PHA1_005], 
no significant improvement of CCl4-induced hepatic collagen content was observed, 
whereas the histological evaluation of hepatic fibrosis revealed a significant effect of 
NTZ at 32 and 104 mg/kg/day. 
There was no significant beneficial effect of NTZ treatment on plasma markers of 
hepatotoxicity in CCl4-treated mice.  
These studies demonstrate the anti-fibrotic effect of NTZ, which suggests that NTZ may 
have potential as an anti-fibrotic drug. These studies also suggest that NTZ may show 
some beneficial therapeutic properties for NASH, such as a tendency to reduce 
hepatocyte ballooning. 


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21 
1.3. 
Rationale for This Study 
Based on the anti-fibrotic properties demonstrated in the animal models of fibrosis, this proof 
of concept clinical study aims at  evaluating NTZ in patients with non-alcoholic steatohepatitis 
(NASH) and  fibrosis stage 3 and 4 (compensated cirrhosis). Although NTZ has been evaluated 
in liver disease populations (viral hepatitis C) up to 60 weeks, this is the first study evaluating 
NTZ treatment in a population with NASH induced stage 3 and 4 fibrosis.  The aim of this study 
is  to evaluate the safety and tolerability of NTZ 500 mg BID after 24 weeks of treatment in this 
population. This proof of concept study will also evaluate the anti-fibrotic effect of NTZ as a 
secondary objective. 
The methods of evaluation of fibrosis will include an innovative method of metabolic labeling. 
This approach is based on the concept that liver status can be determined by measuring the 
ratio of newly synthesized/pre-existing proteins. The turn-over rate of newly synthesized 
collagen and proteins represents the hepatic fibrogenic disease activity.  Patients will be given 
“heavy water” to drink. Heavy water contains D20, deuterium being a stable isotope of 
hydrogen. Mass spectrometry is used to identify individual proteins and to quantify the ratio of 
labeled protein to total protein. The results are expressed as fractional synthesis rate of these 
proteins (FSR). This method has been previously published (Decaris et al, 2017). 
Other non-invasive methods will be used to evaluate the liver stiffness changes after NTZ 
treatment: Magnetic Resonance Elastography (MRE) and FibroScan®.  
2. 
Objectives 
2.1. 
Primary Objective 
 
The primary objective of the study is: 
• To evaluate the safety and tolerability of NTZ 500mg bid after 24 weeks of treatment in 
patients with NASH induced Stage 3 or Stage 4 fibrosis (compensated cirrhosis).  
2.2. 
Secondary Objectives: 
The secondary objectives of the study are: 


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22 
• To determine the effect of daily oral administration of NTZ 500mg bid on de novo 
collagen synthesis through Fractional Synthesis Rate (FSR) of circulating plasma 
proteins in patients with NASH-induced Stage 3 or Stage 4 fibrosis (compensated 
cirrhosis). 
• To evaluate changes in liver stiffness as measured by FibroScan® and by Magnetic 
Resonance Elastography (MRE). 
• To assess the change from baseline in non-invasive markers of fibrosis  
• To assess changes in fibrosis scores: ELFTM test score, Fibrotest, Fibrometer, hepascore, 
NAFLD fibrosis score, FIB-4 
3. 
Study Design 
3.1. 
Study Design 
This is a phase 2 proof of concept study to evaluate the efficacy and safety of open-label NTZ 
500mg bid in a NASH population with NASH induced Stage 3 or 4 fibrosis (compensated 
cirrhosis). 
The study will include a screening visit within 6 weeks of randomization (Day 1) followed by a 
24-week treatment period consisting of daily, BID, oral administration of NTZ and an end of 
treatment follow up period of 4 weeks.   
Patients will come to the research center and complete the following visits: 
• 
Screening Visit within 6 weeks of Randomization 
• 
Day -14 Deuterated Water Run-In 
o Day-14, Day-11, Day-7 and Day 1: sampling for labelling of Deuterated water 
during run-in 
• 
Day 1 Randomization : start of treatment 
• 
Every 4 weeks after randomization until 20 weeks post randomization 
• 
 22 weeks post randomization and Deuterated Water Administration Period: 


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23 
o 22 weeks plus 3 days, plus 7 days, plus 14 days for sampling for kinetic 
biomarker analysis 
• 
24 weeks post randomization : End of Treatment 
• 
4 Week Post Treatment Follow Up 
3.2. 
Treatment Plan and Regimen 
NTZ will be administered in an open label fashion to participating subjects as 500mg bid, for 24 
weeks. 
4. 
Subject Population 
4.1. 
Number of Subjects & Subject Selection 
Twenty male and female subjects with histologically confirmed NASH and fibrosis stage 3 or 4 
(compensated cirrhosis only) will be enrolled in the study at a single site in the United States. 
4.2. 
Inclusion Criteria 
Patients must meet all of the following inclusion criteria to be eligible for enrollment in the 
trial:  
1. 
Males or females aged from 18 to 75 years inclusive the Screening Visit.  
2. 
Must provide signed written informed consent and agree to comply with the 
study protocol.  
3. 
Females participating in this study must be of non-childbearing potential or 
using highly efficient contraception for the full duration of the study.   Highly 
efficient contraception is defined as the use of one of the following methods of 
birth control in addition to a male partner using a condom (unless surgically 
sterile) from Screening to 90 days after the last dose of study medication: 
• hormone-containing contraceptive 
• intrauterine device 
• cervical cap or diaphragm with spermicidal agent 


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24 
4. 
 Histological confirmation of steatohepatitis on a diagnostic liver biopsy (biopsy 
obtained within 6 months prior to Screening or during the Screening Period) 
with at least 1 in each component of the NAS (steatosis scored 0-3, ballooning 
degeneration scored 0-2, and lobular inflammation scored 0-3).   
5. 
Fibrosis stage of 3 or 4, according to the NASH CRN fibrosis staging system on a 
diagnostic liver biopsy (biopsy obtained within 6 months prior to Screening or 
during the Screening Period).  For patients with fibrosis stage 4, only patients 
who have no current or historical evidence of decompensation of cirrhosis.  
4.3. 
Exclusion Criteria 
Patients who meet any of the following criteria will be excluded from entering the study:  
1. 
History of efficient bariatric surgery within 5 years prior to Screening, or 
planned bariatric surgery in the course of the study.  
2. 
Patients with HbA1c >10.0%. If abnormal at the first Screening Visit, the HbA1c 
measurement can be repeated. A repeated abnormal HbA1c (HbA1c >10.0%) 
leads to exclusion.  
3. 
Patients with a history of clinically significant acute cardiac event within 6 
months prior to Screening such as: stroke, transient ischemic attack, or 
coronary heart disease (angina pectoris, myocardial infarction, revascularization 
procedures).  
4. 
Weight loss of more than 10% within 6 months prior to Randomization.  
5. 
Patient with any history or presence of decompensated cirrhosis. 
6. 
Current or recent history (<1 year) of significant alcohol consumption. For men, 
significant consumption is typically defined as higher than 30 g pure alcohol per 
day. For women, it is typically defined as higher than 20 g pure alcohol per day.  
7. 
Current or history of other substance abuse within 1 year prior to screening. 
8. 
Pregnant or lactating females or females planning to become pregnant during 
the study period.  
9. 
Other well documented causes of chronic liver disease according to standard 
diagnostic procedures including, but not restricted to:  


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a) Positive hepatitis B surface antigen (HBsAg)  
b) Positive HCV RNA, (tested for in case of known cured HCV infection, or positive 
HCV Ab at Screening)  
c) Suspicion of drug-induced liver disease  
d) Alcoholic liver disease  
e) Autoimmune hepatitis  
f) Wilson’s disease   
g) Primary biliary cirrhosis, primary sclerosing cholangitis  
h) Genetic homozygous hemochromatosis  
i) Known or suspected HCC  
j) History or planned liver transplant, or current MELD score >15.  
10. 
Patients who cannot be contacted in case of emergency.  
11. 
Known hypersensitivity to the investigation product or any of its formulation 
excipients.  
12. 
Patients who are taking warfarin or other highly plasma protein-bound drugs 
with narrow therapeutic indices. 
13. 
Patients who are currently participating in, plan to participate in, or have 
participated in an investigational drug trial or medical device trial containing 
active substance within 30 days or five half-lives, whichever is longer, prior to 
Screening.  
14. 
Evidence of any other unstable or, untreated clinically significant 
immunological, endocrine, hematological, gastrointestinal, neurological, 
neoplastic, or psychiatric disease.  
15. 
Mental instability or incompetence, such that the validity of informed consent 
or ability to be compliant with the study is uncertain.  
16. 
 History of noncompliance with medical regimens, or patients who are 
considered to be unreliable. 
17. 
Positive anti-human immunodeficiency virus (HIV) antibody. 
18. 
AST and/or ALT >10 x upper limit of normal (ULN).  


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26 
19. 
Conjugated bilirubin >2.0 mg/dL due to altered hepatic function. Note: Gilbert 
Disease patients are allowed into the study.  
20. 
INR >1.50 due to altered hepatic function.  
21. 
Platelet count <100,000/mm3 due to portal hypertension.  
22. 
Significant renal disease, including nephritic syndrome, chronic kidney disease 
(defined as patients with markers of kidney damage or eGFR of less than 60 
ml/min/1.73 m2). 
5. 
Investigational Medicinal Products 
Patients will receive 500 mg NTZ (ALINIA®) twice daily for 24 weeks. 
5.1. Description  
NTZ will be supplied as 500 mg of ALINIA® (nitazoxanide) tablets, for oral use.  The tables are 
round, yellow, film-coated and debossed with ALINIA on one side and 500 on the other side. 
Each tablet contains 500 mg of nitazoxanide. 
For additional information see the Package Insert for ALINIA®. 
5.2. Packaging  
ALINIA® tablets are packaged in HDPE bottles of 12 tablets. Period boxes will be delivered 
every 4 weeks, each box containing 6 bottles. 
5.3. 
Labeling 
All labels for study drugs meet all applicable requirements of the US Food and Drug 
Administration (FDA) and the EU annex 13 of Good Manufacturing Practices: Manufacture of 
Investigational Medicinal Products (February 2010) and /or other local regulations, as 
applicable.  
Distribution of study drug will be performed according to the Good Distribution Practices.  
Product boxes for each period will be labeled with the protocol number, Sponsor’s name and 
address, description of contents, storage conditions, expiry date, dosage instructions, and any 
other applicable items required by national and regional guidelines/regulations. The label will 
contain the statements “Caution – new drug, limited by Federal Law to investigational use” or 


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other similar/appropriate statements as well as the following instructions “Please return 
empty packaging and unused products to your doctor at your next visit.”  
5.4. 
Storage and Handling 
The tablets are to be stored at 20-25oC (68-77oF). Storage conditions are specified on the label. 
5.5. 
Dosage and Administration 
Patients will be instructed to take one tablet of 500mg NTZ mg orally, twice a day with water 
during a meal, approximately 12 hours apart.    
The study staff will administer the first dose of the medication at the research center.  Patients 
will self-administer the evening dose on Day 1 and twice a day thereafter, when not attending 
a visit at the research center.  On scheduled follow up visit days while the patient is being 
treated with study drug, the patient will be directed to bring back all used and unused study 
medication containers. Compliance will be checked by the Investigator or designee during 
those visits and registered in the electronic Case Report Form (eCRF).  
If treatment is interrupted, whatever the cause, duration and reason of the interruption will be 
documented. 
5.6. 
Prior and Concomitant Medications 
In a general manner, patients should be discouraged from starting any new medication 
without consulting the Investigator unless the new medication is required for emergency 
purpose.  In the same way, any qualitative or quantitative change in concomitant therapy 
should be avoided, when possible. In the event that it becomes necessary during the study, 
this should be recorded by the Investigator or designee in the eCRF (including concomitant 
medications taken within 6 months prior to Screening). This includes drugs used on a chronic 
as well as on an “as needed” basis. 
5.6.1.  Non-permitted medication 
The following medications are not allowed within the timeframe given:  


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• Warfarin or other highly plasma protein-bound drugs with narrow therapeutic indices 
are not permitted from Day 1 until end of treatment 
5.6.2.  Permitted Medications 
Any medications other than those listed above are permitted. However, the dosage of a 
current medication for a chronic disease should remain unchanged as far as possible in order 
to reduce the risk of unknown drug-drug interactions.  
In the event that additional concomitant therapy becomes necessary during the study, this will 
be recorded by the Investigator in the eCRF. This includes drugs used on a chronic as well as on 
an “as-needed” basis. Patients should be discouraged from starting any new medication 
without consulting the Investigator unless the new medication is required for emergency 
purpose. 
5.7. Study Drug Accountability 
The Investigator will acknowledge receipt for each study treatment on the day of receipt. A 
drug accountability record will be maintained by the person responsible for dispensing the trial 
medication to the patient.  
All partially used or unused treatments will be inventoried during and at the conclusion of the 
study.  
The Drug Distribution Center will organize the retrieval of all treatments (used or unused) and 
will proceed to their destruction only after written authorization and review of accountability 
and distribution records.  
6. 
Study Procedures 
The procedures performed at each visit are summarized in the Schedule of Events (Table 1).  
A patient may be seen at any time for reasons of safety.  
During each visit, safety evaluations will be done, and the patient will be queried in the form of 
an open question regarding new or continuing events.  
6.1. Duration of Study Participation 


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The estimated duration of the study will be approximately 34 weeks inclusive of the screening 
period. 
6.2. 
Screening Assessment 
6.2.1. Screening Visit 
The following screening procedures will be performed for all potential patients at the 
Screening Visit and prior to Day 1:  
• 
Informed consent witnessed by the Investigator or designated person.  
• 
Medical history & demographics.  
• 
Assessment of inclusion/exclusion criteria 
• 
Complete physical examination. 
• 
Record vital signs  
• 
Record height, weight, BMI  
• 
Check concomitant/prior medication (within 6 months prior to Screening)  
• 
12-lead ECG 
• 
Liver biopsy.  A historical liver biopsy with confirmed NASH and fibrosis is 
acceptable if performed within 6 months prior to the Screening Visit.  
• 
Adverse Events assessment  
• 
Blood samples will be collected for 
o 
Biochemistry 
o 
Hematology 
o 
Serology (HIV, HBs, HCV). In the event of a positive HCV Ab, HCV RNA 
can be tested for confirmation. In case of known cured HCV infection, HCV RNA 
can be tested directly at Screening.  
• 
Urine samples will be collected for Urinalysis  
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
If needed, a retesting of any laboratory results may be performed during the screening 
window to determine the eligibility for the study.  


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6.3. 
Day -14 Deuterated Water Run In 
Potential Subjects who complete all of the Screening Visit assessments and who 
continue to be eligible for the study, will return to the research center to complete the 
Deuterated Water Run In.  The visit is to be done on Day -14 + 3 days, provided that the 
subject completes 7 consecutive days of deuterated water administration.  The 
following procedures will be performed at the Day -14 Visit: 
• Dispense deuterated water (D2O). Instruct patient to drink 50ml of deuterated 
water 3 times per day starting on Day-14 for 7 consecutive days (until day-8).  
• Blood sample for baseline labeling by D2O (kinetic biomarkers) (patients should 
be instructed to fast the evening prior to the visit to ensure a 10-hour fast) 
•  Adverse Events assessment  
• Record concomitant medication taken 
Patients will return at Day-11, Day-7 and Day 1 for blood sampling (labeled D2O) 
6.4. 
Treatment Period Assessments 
6.4.1. Day 1 Visit 
The following procedures will be performed at the Day 1 Visit prior to dose administration:  
• 
Review inclusion/exclusion criteria 
• 
Complete physical examination 
• 
Weight, BMI 
• 
Record vital signs  
• 
Record concomitant medication taken  
• 
Adverse Events assessment  
• 
MRE (may be done within + 5 days of the visit)  
• 
Fibroscan 
• 
Blood samples will be collected  
• 
Urine samples will be collected for Urinalysis  


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• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
• 
Dispense the study drug as directed 
• 
Review study drug administration instructions with subject  
• 
Administer 500 mg of NTZ with water & food 
6.4.2. Week 4 and Week 8 Visit + 3 days 
The following procedures will be performed at these Visits:  
• 
Record concomitant medication taken  
• 
Adverse Events assessment  
• 
Blood samples will be collected  
• 
Urine samples will be collected for Urinalysis  
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
• 
Dispense the study drug as directed 
• 
Review study drug compliance and drug administration instructions with subject  
• 
Reconcile study drug administration using pill counts  
• 
Administer 500 mg of NTZ with water & food 
6.4.3. Week 12 Visit + 3 days 
The following procedures will be performed at the Visit:  
• 
Complete physical examination 
• 
Weight, BMI 
• 
Record vital signs  
• 
Record concomitant medication taken  
• 
Adverse Events assessment  
• 
MRE(may be done within + 5 days of the visit) 
• 
Blood samples will be collected  
• 
Urine samples will be collected for Urinalysis  
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
• 
Dispense the study drug as directed 


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32 
• 
Review study drug compliance and drug administration instructions with subject  
• 
Reconcile study drug administration using pill counts  
• 
Administer 500 mg of NTZ with water and food 
6.4.4. Week 16 and Week 20 visits ±3 days 
The following procedures will be performed at these Visits:  
• 
Record concomitant medication taken  
• 
Adverse Events assessment  
• 
Blood samples will be collected  
• 
Urine samples will be collected for Urinalysis  
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
• 
Dispense the study drug as directed 
• 
Review study drug compliance and drug administration instructions with subject  
• 
Reconcile study drug administration using pill counts  
• 
Administer 500 mg of NTZ with water & food 
6.4.5. Week 22 Visit + 3 days (Day 155) 
The following procedures will be performed at the Visit:  
• 
Record concomitant medication taken  
• 
Adverse Events assessment  
• 
Blood samples will be collected for D2O labeling 
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]) 
• Dispense Deuterated water. Instruct patient to drink 50ml of deuterated water 
3 times per day starting on Day 155 for 7 consecutive days (until day 162).  
• Blood sample for kinetic biomarkers analysis (patients should be instructed to 
fast the evening prior to the visit to ensure a 10-hour fast) 
Patients will return for blood sampling for kinetic biomarker analysis at 22 weeks plus 3 
days (Day 158), at Day 162, and at day 169 (Week 24). 
6.4.6. Week 24 Visit +3 


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33 
The following procedures will be performed at the visit: 
• 
Complete physical examination 
• 
12-lead ECG 
• 
Record vital signs  
• 
Record weight, BMI  
• 
Record concomitant medication taken  
• 
MRE (may be done within + 5 days of the visit) 
• 
Fibroscan  
• 
Adverse Events assessment  
• 
Blood samples will be collected prior to morning dose of study medication  
• 
Urine samples will be collected for Urinalysis  
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
• 
Review study drug compliance and drug administration instructions with subject  
• 
Reconcile study drug administration using pill counts  
6.5. 
Follow Up Period 
6.5.1. 4-Week Post Treatment Follow up (EOS – End of study visit) ±3 days 
The following procedures will be performed at the visit: 
• Complete physical examination 
• Weight, BMI 
• Record vital signs  
• Record concomitant medication taken  
• Adverse Events assessment  
• Blood samples will be collected prior to morning dose of study medication  
• Urine samples will be collected for Urinalysis  
• Urinary pregnancy test (for women of childbearing potential only [WOCBP]).  
6.5.2. Early Termination (ET) 
Patients who have discontinued from the study after the first dose of study medication and for 
any reason will have the following procedures completed: 


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• 
Complete physical examination 
• 
Record vital signs 
• 
Weight, BMI  
• 
ECG 
• 
Record concomitant medication taken  
• 
MRE (may be done within + 5 days of the visit) 
• 
Fibroscan 
• 
Adverse Events assessment  
• 
Blood samples will be collected prior to morning dose of study medication  
• 
Urine samples will be collected for Urinalysis  
• 
Urinary pregnancy test (for women of childbearing potential only [WOCBP]) 
• 
Review study drug compliance with subject  
• 
Reconcile study drug administration using pill counts  
6.5.3. 
Unscheduled Visits 
An unscheduled visit is defined as any visit to the study unit outside of the protocol-evaluation 
time points where the patient is seen by study unit personnel, e.g., when follow-up 
assessments are required for safety reasons or when repeat measurements are required out of 
the screening period (either to confirm a measurement or in case of errors, measuring device 
failure, etc.).  
Unscheduled visits will be needed for patients who may require further follow-up due to 
safety. 
6.6. 
Assessment for Premature Discontinuation from Study 
Study medication may be discontinued in the following instances: 
• Intercurrent illness that would, in the judgment of the investigator, affect 
assessments of clinical status to a significant degree. Following resolution of 
intercurrent illness, the subject may resume study dosing at the discretion of the 
investigator. 
• Subject noncompliance  


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35 
• Investigator discretion  
• Study medication must be discontinued in the following instances:  
o 
Unacceptable toxicity,  or toxicity that, in the judgment of the 
investigator, compromises the ability to continue study-specific 
procedures or is considered to not be in the subject’s best interest  
o 
Subject request to discontinue for any reason  
o 
Pregnancy  
o 
Discontinuation of the study at the request of a regulatory agency or an 
institutional review board or independent ethics committee (IRB/IEC) 
 
 


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36 
  
 
6.7. 
Description of Assessments 
6.7.1. Histological Assessments 
All possible attempts should be made to acquire a liver biopsy specimen of at least 2.0 cm in 
length, from a 16 gauge needle to ensure accurate staging of fibrosis and other histological 
lesions.  A historical biopsy within 6 months of the Screening visit may be accepted as the 
Screening biopsy. The liver biopsy sample must be deemed adequate for evaluation by the 
pathologist for inclusion. Liver biopsies will be assessed by a local reader.  This assessment will 
include an assessment of the adequacy of the specimen as well as the fibrosis stage and a 
determination that the biopsy is consistent with NASH. If either the historical or screening liver 
biopsy is deemed unacceptable, it may be repeated. 
6.7.2. Assessment of newly synthesized proteins 
Kinetic biomarkers will be analyzed to evaluate the pharmacodynamic effect of NTZ on 
fibrogenesis. Blood samples analyses will be performed after deuterated water intake before 
and after 24 weeks of treatment. The assessment will involve the analysis of the change 
(absolute and relative) from baseline between the post-dose and pre-dose deuterated water 
loading periods. 
6.7.3. Biological Assessments (Table 2) 
Chemistry:  
HbA1c, fasting plasma glucose, insulin, HOMA-IR, fructosamine, alkaline phosphatase, ALT, 
AST, GGT, CPK, total and conjugated bilirubin, ferritin, creatinine, eGFR, uric acid, BUN, total 
cholesterol, HDL-C, LDL-C, triglycerides, albumin, lipase, amylase, electrolytes (sodium, 
potassium, chloride, calcium), MELD 
Hematology:  
Hematocrit (Hct), hemoglobin (Hb), platelet count, red blood cell count (RBC), white blood cell 
count (WBC) with differential (absolute and percentage) including lymphocytes, monocytes, 


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37 
neutrophils, eosinophils, and basophils and, reticulocyte count and mean corpuscular volume 
(MCV).  
Inflammatory markers:  
hsCRP, fibrinogen, haptoglobin, resistin, TNF-α, TGF-β, IL-6, PAI-1 
Coagulation Panel:  
Prothrombin time (PT), and international normalized ratio (INR)  
Pregnancy Tests:  
Urine β-hCG (if positive, requires immediate confirmation with Serum β-hCG)  
Urinary Tests:  
Urinary dipstick analysis, urinary albumin, urinary creatinine, urinary ACR 
Additional Tests:  
Serology (HIV-1, HBsAg, HCVAb & HCV RNA (as needed), serum cystatin C, troponin T, NT 
proBNP 
Fibrosis Biomarker tests:  
ELFTM test score, PIIINP, Hyaluronic acid, CK18, TIMP-1, YKL-40, Alpha 2 macroglobulin, miRNA, 
FGF19, FGF21, ProC3 and ProC6.  
A laboratory manual will be provided 
The manual will outline the collection and processing requirements for the laboratory. Blood 
sampling will be performed by trained personnel. Blood samples will be processed and shipped 
as outlined in the laboratory manual. Refer to the laboratory manual for exact amounts of 
blood required for each test.  
The option to retest during the study is left to the Investigator’s judgment.  
Additional serum and plasma samples will be maintained for future analysis 
 
 


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Table 2 Biological assessments 
 
Screening period 
 
 
Treatment period 
Visit 
SV 
 
S
V2 
SV3 
S
V
4 
V
1 
 
V
2 
V
3 
V4 
 
V5 
V6 
V7 
V8 
V9 
V10 
 
EOS 
 
ET 
Day 
-42 
-
14 
-11 
-7 
1 
29 
57 
85 
113 
141 
155 
158 
162 
169 
+28 
days 
 
Week 
-6 
-2 
 
 
0 
4 
8 
12 
16 
20 
22 
 
 
24 
28 
 
Labs - 
Haematology 
haemoglobin, 
haematocrit, RBC, 
WBC, differential 
count, platelet 
count, reticulocytes 
count, MCV) 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
 
 
X 
Coagulatio (PT, 
INR) 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Labs- Urinary 
Pregnancy tests3 
X 
 
 
 
 
X 
X 
X 
X 
X 
 
 
 
X 
X 
 
X 
Labs – Serology 
HIV, HBs and HCV 
serology 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Labs – 
Biochemistry 
HbA1c, fasting 
plasma glucose, 
insulin, HOMA-IR, 
fructosamine, 
alkaline 
phosphatase, ALT, 
AST, GGT, CPK, 
total and conjugated 
bilirubin, ferritin, 
creatinine, eGFR, 
uric acid, BUN, 
total cholesterol, 
HDL-C, LDL-C, 
triglycerides, 
albumin, lipase, 
amylase, 
electrolytes 
(sodium, potassium, 
chloride, calcium), 
MELD 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Inflammatory 
markers 
hsCRP, fibrinogen, 
haptoglobin, 
resistin,  TNF-, 
TGF-, IL-6, PAI-1 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
 
 


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39 
 
Screening period 
 
 
Treatment period 
Visit 
SV 
 
S
V2 
SV3 
S
V
4 
V
1 
 
V
2 
V
3 
V4 
 
V5 
V6 
V7 
V8 
V9 
V10 
 
EOS 
 
ET 
Day 
-42 
-
14 
-11 
-7 
1 
29 
57 
85 
113 
141 
155 
158 
162 
169 
+28 
days 
 
Biomarkers of 
fibrosis 
ELF Score, PIIINP, 
Hyaluronic acid, 
CK18, TIMP-1, 
YKL-40, Alpha 2 
macroglobulin,, 
,miRNA, ProC3, 
ProC6, FGF19, 
FGF21 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
X 
X 
Labs – Urinalysis 
dipstick  
Specific gravity, pH, 
protein, glucose, 
ketones, bilirubin, 
urobilinogen, blood, 
nitrite, leukocytes 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
 
 
 
X 
Safety markers 
Serum Cystatin C, 
troponin T, NT 
proBNP 
Urinary albumin, 
urinary creatinine, 
urinary ACR 
X 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
X 
X 
X 
Labs -- FSR 
 
X 
X 
X 
X 
 
 
 
 
 
X 
X 
X 
X 
 
 
Sampling for 
additional 
parameters 
X 
 
 
 
X 
 
 
X 
 
X 
 
 
 
X 
X 
X 
 
6.7.4. Electrocardiogram 
Standard 12-lead electrocardiogram (ECG) assessments will be performed. The Investigator 
will review the ECGs for any clinically significant abnormalities to ensure subject safety. 
Abnormal ECG findings that are considered clinically significant by the Investigator and meet 
the definition of an AE should be reported and recorded in the AE eCRF page.  
6.7.5. MRE 
Liver stiffness will be assessed by MRE. It is recommended that each subject’s radiological 
assessment is performed using the same procedure for each study visit.  Exploratory measures 
for MRI/MRE sequence development will be maintained for future analysis.  


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6.7.6. Fibroscan 
Liver stiffness will be assessed by FibroScan®. It is required that each subject’s FibroScan® 
assessments be done with the same type of probe at each study visit.  
6.7.7. Medical History 
Medical history including details regarding illnesses and allergies, date(s) of onset, and 
whether condition(s) is currently ongoing, and medication history, including nicotine and 
alcohol use, will be collected on all subjects during screening. 
6.7.8. Physical Examination 
A complete physical examination should include source documentation of general appearance, 
and the following body systems: head, neck, and thyroid; eyes, ears, nose, throat, mouth, and 
tongue; chest (excluding breasts); respiratory; cardiovascular; lymph nodes; abdomen; skin, 
hair, nails; musculoskeletal and neurological.  
The patient’s weight and waist circumference will be measured under the same conditions at 
each visit where the procedure is required. Where possible, the scale for weight must be the 
same for a given patient throughout the visits. 
6.7.9. Vital Signs 
Blood pressure (mmHg) and pulse rate (beats per minute) will be measured at each visit using 
automated blood pressure equipment. 
Important points for clinical blood pressure measurement  
• 
The patient should be seated comfortably with the back supported and the 
upper arm bare without constrictive clothing. The legs should not be crossed. 
• 
The arm should be supported at heart level, and the bladder of the cuff should 
encircle at least 80% of the arm circumference.  
• 
Neither the patient nor the observer should talk during the measurement.  


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41 
Systolic BP and diastolic BP will be measured after 5 minutes rest in the seating.  Where 
possible, the blood pressure equipment should be the same for a given patient throughout the 
visits. 
6.7.10. Pregnancy Testing 
Urinary pregnancy tests will be done on WOCBP.  
7. Adverse Events and Toxicity Management 
7.1. Definitions of Adverse Events, Serious Adverse Events and Unexpected Events 
7.1.1. Adverse Events 
An adverse event (AE) is any untoward medical occurrence in a clinical study subject 
administered a medicinal product, which does not necessarily have a causal relationship with 
the treatment. An AE can therefore be any unfavorable and/or unintended sign, symptom, or 
disease temporally associated with the use of a 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 specified procedures, overdose, drug abuse/misuse reports, 
or occupational exposure. Preexisting events that increase in severity or change in nature 
during or as a consequence of participation in the clinical study will also be considered AEs.  
An AE does not include the following:  
• 
Medical or surgical procedures such as surgery, endoscopy, tooth extraction, 
and transfusion. The condition that led to the procedure may be an adverse 
event and must be reported  
• 
Pre-existing diseases, conditions, or laboratory abnormalities present or 
detected before the Screening visit that do not worsen  
• 
Situations where an untoward medical occurrence has not occurred (eg., 
hospitalization for elective surgery, social and/or convenience admissions)  
• 
Overdose without clinical sequelae  
• 
Any medical condition or clinically significant laboratory abnormality with an 
onset date before the consent form is signed and not related to a protocol-


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associated procedure is not an AE. It is considered to be pre-existing and should 
be documented on the medical history eCRF. 
7.1.2.  Clinical Laboratory Abnormalities and Other Abnormal Assessments as Adverse 
Events or Serious Adverse Events 
Laboratory abnormalities without clinical significance are not recorded as AEs or SAEs. 
However, laboratory abnormalities (eg, clinical chemistry, hematology, and urinalysis) that 
require medical or surgical intervention or lead to study drug interruption, modification, or 
discontinuation must be recorded as an AE, as well as an SAE, if applicable. In addition, 
laboratory or other abnormal assessments (eg, electrocardiogram, x-rays, vital signs) that are 
associated with signs and/or symptoms must be recorded as an AE or SAE if they meet the 
definition of an AE or SAE as described above.  If the laboratory abnormality is part of a 
syndrome, record the syndrome or diagnosis (eg, anemia), not the laboratory result (ie, 
decreased hemoglobin). 
7.1.3. Serious Adverse Events 
A serious adverse event (SAE) is defined as an event that, at any dose, results in the following:  
• 
Death  
• 
Life-threatening (Note: The term “life-threatening” in the definition of 
“serious” refers to an event in which the subject was at risk of death at the 
time of the event; it does not refer to an event that hypothetically might 
have caused death if it were more severe.) 
• 
In-patient hospitalization or prolongation of existing hospitalization 
• 
Persistent or significant disability/incapacity  
• 
A congenital anomaly/birth defect  
• 
A medically important event or reaction: such events may not be 
immediately life-threatening or result in death or hospitalization but may 
jeopardize the subject or may require intervention to prevent one of the 
other outcomes constituting SAEs. Medical and scientific judgment must be 
exercised to determine whether such an event is a reportable under 


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expedited reporting rules. 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 
hospitalization; and development of drug dependency or drug abuse. For the 
avoidance of doubt, infections resulting from contaminated medicinal 
product will be considered a medically important event and subject to 
expedited reporting requirements. 
7.1.4. Unexpected adverse event  
Expectedness is assessed by the Sponsor-Investigator. An unexpected AE is defined as an event 
that has a nature of severity or specificity that is not consistent with the applicable Investigator 
Brochure or that is symptomatically and pathophysiologically related to a known toxicity but 
differs because of a greater severity or specificity.  
“Unexpected” refers to an event that has not been previously observed and reported rather 
than an event that has not been anticipated based on the properties of the drug. 
7.2. 
Assessment of Adverse Events & Serious Adverse Events 
The Investigator will establish whether or not any AE have occurred at each visit from the date 
of consent. The patient will be questioned in a general manner to determine specific 
symptoms without offering the patient any suggestion. The investigator or qualified sub-
investigator is responsible for assessing AEs and SAEs for causality and severity, and for final 
review and confirmation of accuracy of event information and assessments. 
7.2.1.  Intensity assessment  
The intensity of the AE will be graded as follows:  
• 
Mild: Awareness of signs or symptoms, but easily tolerated and are of 
minor irritant type causing no loss of time from normal activities. 
Symptoms do not require therapy or a medical evaluation; signs and 
symptoms are transient.  


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• 
Moderate: Events introduce a low level of inconvenience or concern to 
the participant and may interfere with daily activities, but are usually 
improved by simple therapeutic measures; moderate experiences may 
cause some interference with functioning. 
• 
Severe: Events interrupt the participant’s normal daily activities and 
generally require systemic drug therapy or other treatment; they are 
usually incapacitating. 
7.2.2.  Relation to the study treatment  
The Investigator will make a clinical and scientific judgment regarding whether or not the AE 
was related to study treatment. The Investigator will evaluate any changes in laboratory 
values, make a determination as to whether or not the change is clinically important, and 
whether or not the changes were related to study drug. However, even if the Investigator feels 
there is no relationship to the study drug, the AE or clinically significant laboratory abnormality 
must be recorded in the eCRF.  
The Investigator will record the relation to the study treatment according to the following 
causality terms:  
• 
Related: the AE follows a reasonable temporal sequence from the time 
of drug administration and it cannot be explained by the patient's 
clinical state or the study procedures/conditions. The AE abates upon 
discontinuation of the study drug and reappears when the study drug is 
introduced.  
• 
Possibly related: the AE follows a reasonable temporal sequence from 
the time of drug administration, but could have been produced by the 
patient's clinical state or the study procedures/conditions.  
• 
Unlikely related: the temporal association between the AE and the study 
drug is such that the study drug is not likely to have any reasonable 


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association with the AE. The relationship is not likely because of other 
plausible explanations.  
• 
Not related: the AE must definitely be caused by the patient's clinical 
state or the study procedure/conditions. A reasonable explanation must 
be given, e.g., no investigational product taken, preplanned elective 
medical intervention, or incompatible temporal relationship.  
• 
Not assessable: the report suggesting an adverse reaction cannot be 
judged because information is insufficient or contradictory and data 
cannot be supplemented or verified. 
7.2.3. 
 Action taken and outcome  
The Investigator will record the action taken with drug and outcome of the event for each AE 
according to the following:  
Action taken with investigational drug  
• 
Drug permanently withdrawn – in case a patient is permanently 
withdrawn from the study drug  
• 
Drug temporarily withdrawn – in case the study drug is temporarily 
withdrawn  
• 
Dose not changed – in case no action is taken regarding the study drug  
• 
Unknown  
• 
Not applicable – an AE started before initiation of treatment with study 
drug, the treatment had been completed prior to reaction/event, or the 
patient has died. 
Outcome  
• 
Recovered/resolved  
• 
Recovering/resolving  
• 
Not recovered/not resolved  


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• 
Recovered/resolved with sequelae  
• 
Fatal  
• 
Unknown.  
Note: In case of irreversible congenital anomalies the choice not recovered/not resolved 
should be used. “Fatal” should be used when death is possibly related to the reaction/event. 
7.3. Reporting 
7.3.1.  Reporting an adverse event  
All AEs regardless of seriousness or relationship to study drug, including those occurring during 
the Screening Period, are to be recorded on the corresponding page(s) of the eCRF and in the 
patient’s medical record from the ICF signature until study end for each patient. Whenever 
possible, symptoms should be grouped as a single syndrome or diagnosis. The Investigator 
should specify the date of onset, maximal intensity, action taken with respect to study drug, 
corrective therapy given, outcome, and his/her opinion as to whether there is a reasonable 
possibility that the AE was caused by the study drug.  
Adverse event reporting begins from signature of the patient ICF at the first Screening Visit and 
ends at study end for each patient. 
7.3.2. Initial reports 
All SAEs occurring from the time of informed consent until 30 days following the 
last administration of study drug must be reported to Medpace Clinical Safety 
within 24 hours of the knowledge of the occurrence (this refers to any adverse 
event that meets any of the aforementioned serious criteria). All SAEs that the 
Investigator considers related to study drug occurring after the 30-day follow-up 
period must be reported to the Sponsor. 
To report the SAE, complete the SAE form electronically in the electronic data 
capture (EDC) system for the study. When the form is completed, Medpace Safety 
personnel will be notified electronically and will retrieve the form. If the event 
meets serious criteria and it is not possible to access the EDC system, send an 


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email to Medpace Safety at medpace safetynotification@medpace.com or call the 
Medpace SAE hotline (phone number listed below), and fax the completed paper 
SAE form to Medpace (fax number listed below) within 24 hours of awareness. 
When the EDC system becomes available, the SAE information must be entered 
within 24 hours of the system becoming available.  
7.3.3. Follow-up reports 
The Investigator must continue to follow the patient until the SAE has subsided or 
until the condition becomes chronic in nature, stabilizes (in the case of persistent 
impairment), or the patient dies. 
Within 24 hours of receipt of follow-up information, the Investigator must update 
the SAE form electronically in the EDC system for the study and submit any 
supporting documentation (eg, patient discharge summary or autopsy reports) to 
Medpace Clinical Safety via fax or e-mail. If it is not possible to access the EDC 
system, refer to the procedures outlined above for initial reporting of SAEs. 
All SAEs independent of the circumstances or suspected cause must be reported on a SAE 
Form/captured in EDC. The SAE Form should include a clearly written narrative describing 
signs, symptoms, and treatment of the event, diagnostic procedures, as well as any relevant 
laboratory data and any sequelae, in order to allow a complete medical assessment of the case 
and independent determination of the possible causality.  
It is critical that the information provided on the initial or follow-up SAE Form matches the 
information recorded in the source documents and the eCRF for the same event.  
Any unexpected safety issue that changes the risk benefit analysis and is likely to have an 
impact on the patients who have participated in the trial will be reported as soon as possible 
to the Competent Authority(ies) concerned together with proposed actions. 
7.3.4. Follow-up  
The Investigator should take all appropriate measures to ensure the safety of the patients, 
notably he/she should follow up the outcome of any AE until the return to normal or until 
stabilization of the patient’s condition.  


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The patient must be followed up until clinical recovery is complete and laboratory results have 
returned to normal, or until progression has been stabilized. This information should be 
documented in the patient’s medical records.  
7.4. Post Study Reporting Requirements 
Any SAEs and deaths that occur within 30 days of the last dose of the study drug, regardless of 
causality, should be reported.  
Any SAE that is brought to the attention of the Investigator at any time after the reporting 
period and which is considered by him/her to be caused by the study drug within a reasonable 
possibility, should be reported.  
7.5. Special Situation Reports 
Special situations reports include pregnancy reports, reports of medication error, abuse, 
misuse or overdose, and reports associated with product complaints.  
7.5.1. Pregnancy  
In case of pregnancy the Investigator will notify regulatory authorities and IRB within 24 hours 
of his/her knowledge of the pregnancy.  
Female patients must be instructed to discontinue the study drug immediately and inform the 
Investigator as soon as possible once they are aware of being pregnant or suspect that they 
are pregnant during the study or within 30 days of the last dose of the study drug.  
Female patients will be requested, as part of the general ICF, to provide informed consent to 
allow reasonable attempts to be made to obtain information on any possible medicinal 
product exposure to an embryo or fetus and to follow up on the outcome of the pregnancy.  
The Investigator will contact the patient at the expected time of delivery for follow-up.  
7.5.2. Medication error  
Medication error is defined as an unintentional error in the prescribing, dispensing, or 
administration of a medicinal product while in the control of the healthcare professional, 


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patient, or consumer. All medication errors will be documented in the eCRF and, in case of any 
potential risk to patient safety, would be reported as appropriate.  
7.5.3. Misuse  
This refers to situations where the medicinal product is intentionally and inappropriately used 
not in accordance with the authorized product information and will be reported in the eCRF. 
All misuse will be documented in the eCRF and, in case of any potential risk to patient safety, 
would be reported as appropriate. 
7.5.4. 
 Overdose  
This refers to the administration of a quantity of a medicinal product given per administration 
or cumulatively, which is above the maximum recommended dose according to the authorized 
product information. Clinical judgment should always be applied.  
7.5.5. 
 Abuse  
This corresponds to the persistent or sporadic, intentional excessive use of a medicinal 
product, which is accompanied by harmful physical or psychological effects. 
8. 
Statistical Considerations 
This section is an overview of the principal elements of the statistical analysis for this study. 
Further details will be contained in a separate statistical analysis plan (SAP). 
8.1. 
Analysis Endpoints 
8.1.1. Primary Endpoint 
The primary endpoint is to assess the safety and tolerability of NTZ after 24 weeks of treatment 
8.1.2. Secondary Endpoints 
The secondary endpoints are: 
• To assess the % change in FSR from baseline to end of treatment (24 weeks of 
treatment) 
• To assess the change in liver stiffness by MRE from baseline to 12 weeks and 24 weeks 
of treatment 


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• To assess the change in liver stiffness by fibroscan from baseline to 24 weeks of 
treatment  
• To assess the change from baseline to 12 weeks and 24 weeks of treatment of fibrosis 
serum biomarkers and scores  
8.2. 
Analysis Sets 
The Full Analysis Set will consist of all subjects that meet the eligibility criteria and enroll into 
the study (Visit 1 Day 1). 
The Safety Set will consist of all enrolled subjects who receive at least one dose of study drug.  
8.3. 
Data Handling Conventions 
Continuous variables will be summarized with descriptive statistics (i.e., sample size, mean, 
standard deviation, minimum, maximum, median, and quintiles). Categorical variables will be 
summarized with descriptive statistics (i.e., sample size, frequency counts, and percentage). 
Statistical testing on change from baseline values will be performed using analysis of variance 
techniques with a term for baseline in the model. Statistical significance will be considered to 
have been reached at an alpha level of 0.05. 
Baseline value is measured on Day 1 of the treatment period before the administration of NTZ. 
8.4. 
Demographic Data and Baseline Characteristics 
Demographic and baseline characteristics will be descriptively summarized. Quantitative 
and/or categorical summaries will be presented for demographics and other baseline 
characteristics. Demographic and baseline characteristics will be summarized using the Full 
Analysis Set. 
8.5. 
Primary Safety Analysis 
Safety and tolerability will be assessed by the following parameters:  
AEs including TEAEs and SAEs, laboratory evaluations, vital signs, ECGs, and physical 
examinations.  
8.5.1. Adverse Events 


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Treatment-emergent adverse events (TEAEs) are defined as AEs that worsen or commence on 
or after the time of start of first study drug administration. AEs, including SAEs, will be coded 
using MedDRA. The number and percentage of subjects experiencing TEAE, including TEAEs 
leading to study drug discontinuation and SAEs, will be summarized for each system organ 
class (SOC) and Preferred Term by treatment group. In addition, AEs will be tabulated 
according to severity, causality, and relation to the study drug.  
Individual data listings for AEs will be presented for each subject.  
8.5.2. Laboratory Evaluations 
Absolute and change from baseline values for laboratory parameters will be summarized by 
visit. In addition, shift from baseline tables will be presented for selected laboratory 
abnormalities.  
8.5.3. Other Safety Evaluations 
Observed values and change from baseline will be summarized descriptively for all vital sign 
parameters and ECG parameters by visit. Individual listings will be presented for each subject. 
Individual data listings for physical examination results will be presented for each subject. 
Safety data will be summarized using descriptive statistics. Further details will be provided in 
the SAP. 
 All safety analyses will be conducted using the Safety Population. 
8.6. 
Secondary Efficacy Analyses 
8.6.1. FSR 
Fractional Synthesis Rate of circulating plasma proteins will be measured at Baseline, and after 
24 weeks of treatment.  
The change in FSR (absolute and relative) from baseline to end of treatment between the first 
and second deuterated water loading periods will be assessed to determine the effects of NTZ 
500mg BID on de novo collagen synthesis in the Full Analysis Set.  


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Wilcoxon signed-rank test will be used for statistical inference. Estimated mean, standard error 
and 95% confidence interval of change in FSR (absolute and relative) percentage change from 
baseline in FSR will be reported and evaluated at a significance level of 0.05. Exploratory 
analyses may also be performed to evaluate the association of individual exploratory 
biomarkers or combination of biomarkers with clinical measurements and other risk factors. 
Additional details will be provided in the SAP. 
8.6.2. Liver stiffness 
The change in liver stiffness from baseline at 24 weeks as measured by FibroScan, and at 12 
weeks and 24 weeks by MRE, the change in non-invasive markers of fibrosis from baseline to 
24 weeks, and the change in fibrosis scores from baseline to 24 weeks for the Full Analysis Set 
will be assessed. Summary statistics for the Full Analysis Set will be provided at all visits and 
the change from baseline will be provided. 
8.6.3. Exploratory Analysis 
Additional serum and plasma samples will be maintained for future analysis.  Exploratory 
measures for MRI/MRE sequence development will be maintained for future analysis.  
8.6.4. Interim Analysis 
 An interim analysis is not planned. 
8.7. 
Sample Size 
The sample size of this Phase 2 proof of concept study is based on clinical assessment. Twenty 
male and female subjects with histologically confirmed NASH and fibrosis stage 3 or 4 
(compensated cirrhosis only) will provide sufficient information to assess the safety and 
tolerability of NTZ 500mg BID over 24 weeks. 
9. 
Responsibilities 
9.1. 
Investigator Responsibilities 


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By signing this protocol, the Investigator agrees to conduct the study in an efficient and 
diligent manner and in conformance with this protocol; generally accepted standards of GCP; 
and all applicable local laws, rules and regulations relating to the conduct of the clinical study.  
The Investigator also agrees to allow monitoring, audits, IRB review and regulatory agency 
inspection of trial-related documents and procedures and provide for direct access to all study 
related source data and documents.  
The Investigator shall prepare and maintain complete and accurate study documentation in 
compliance with GCP standards and applicable local laws, rules and regulations; and, for each 
subject participating in the study, provide all data, and upon completion or termination of the 
clinical study submit any other reports to the Fundor as required by this protocol or as 
otherwise required pursuant to any agreement with the Fundor.  
Study documentation will be promptly and fully disclosed to regulatory authorities by the 
Investigator upon request and also shall be made available at the Investigator’s site upon 
request for inspection, copying, review and audit at reasonable times by representatives any 
regulatory agencies.   
Persons debarred from conducting or working on clinical studies by any court or regulatory 
agency will not be allowed to conduct or work on this study.   
9.2. 
Good Clinical Practice 
The investigator will ensure that this study is conducted in accordance with the principles of 
the Declaration of Helsinki (as amended in Edinburgh, Tokyo, Venice, Hong Kong, and South 
Africa), International Conference on Harmonisation (ICH) guidelines.  These standards are 
consistent with the European Union Clinical Trials Directive 2001/20/EC and Good Clinical 
Practice Directive 2005/28/EC. The investigator will ensure adherence to the basic principles of 
Good Clinical Practice, as outlined in 21CFR312, subpart D, “Responsibilities of Sponsors and 
Investigators,” 21CFR, part50, 1998, and 21CFR, part56, 1998. The investigator and all 
applicable sub investigators will comply with 21CFR, Part54, 1998, providing documentation of 
their financial interest or arrangements with any study fundor, or proprietary interests in the 
investigational drug under study. This documentation must be provided prior to the 


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investigator’s (and any sub investigator’s) participation in the study. The investigator and sub 
investigator agree to notify study fundor of any change in reportable interests during the study 
and for 1 year following completion of the study. Study completion is defined as the date 
when the last subject completes the protocol-defined activities 
9.3. 
Institutional Review Board Review & Approval 
The investigator will submit this protocol, informed consent form, and any accompanying 
material to be provided to the subject (such as advertisements, subject information sheets, or 
descriptions of the study used to obtain informed consent) to an IRB/IEC.  The investigator will 
not begin any study subject activities until approval from the IRB/IEC has been documented 
and provided as a letter to the investigator. Before implementation, the investigator will 
submit to and receive documented approval from the IRB/IEC any modifications made to the 
protocol or any accompanying material to be provided to the subject after initial approval, 
with the exception of those necessary to reduce immediate risk to study subjects. 
9.4. 
Informed Consent 
The investigator is responsible for obtaining written informed consent from each individual 
participating in this study after adequate explanation of the aims, methods, objectives, and 
potential hazards of the study and before undertaking any study-related procedures. The 
investigator must use the most current IRB/IEC approved consent form for documenting 
written informed consent. Each informed consent will be appropriately signed and dated by 
the subject and the person conducting the consent discussion, and also by an impartial witness 
if required by local requirements. 
9.5. 
Confidentiality 
The investigator will assure that subjects’ anonymity will be strictly maintained and that their 
identities are protected from unauthorized parties. Only subject initials, date of birth, another 
unique identifier (as allowed by local law) and an identification code will be recorded on any 
form or biological sample submitted to the study funder, IRB/IEC or laboratory. Laboratory 
specimens will be labeled in such a way as to protect subject identity while allowing the results 
to be recorded to the proper subject. Refer to specific laboratory instructions for further 


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details. NOTE: The investigator will keep a screening log showing codes, names, and addresses 
for all subjects screened and for all subjects enrolled in the trial. Subject data will be processed 
in accordance with all applicable regulations. The investigator agrees that all information 
received from the study funder, including but not limited to the investigator brochure, the 
IMP, and any other study information, remain the sole and exclusive property of the study 
funder during the conduct of the study and thereafter. This information is not to be disclosed 
to any third party (except employees or agents directly involved in the conduct of the study or 
as required by law) without prior written consent from the study funder.  The investigator 
further agrees to take all reasonable precautions to prevent the disclosure by any employee or 
agent of the study site to any third party or otherwise into the public domain. 
9.6. 
Study Files and Retention of Records 
The investigator will maintain adequate and accurate records to enable the conduct of the 
study to be fully documented and the study data to be subsequently verified. These 
documents should be classified into at least the following two categories: (1) investigator’s 
study master file, and (2) subject clinical source documents. The investigator’s study file will 
contain the protocol/amendments, CRF and query forms, IRB/IEC and governmental approval 
with correspondence, informed consent, drug records, staff curriculum vitae and authorization 
forms, and other appropriate documents and correspondence. The required source data 
should include sequential notes containing at least the following information for each subject:  
• 
Subject identification (name, date of birth, gender);  
• 
Documentation that subject meets eligibility criteria, i.e., history, physical 
examination, and confirmation of diagnosis (to support inclusion and exclusion 
criteria); 
• 
Documentation of the reason(s) a consented subject is not enrolled  
• 
Participation in study (including study number);  
• 
Study discussed and date of informed consent;  
• 
Dates of all visits;  


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• 
Documentation that protocol specific procedures were performed;  
• 
Results of efficacy parameters, as required by the protocol;  
• 
Start and end date (including dose regimen) of IMP, including dates of 
dispensing and return;  
• 
Record of all adverse events and other safety parameters (start and end date, 
and including causality and severity);  
• 
Concomitant medication (including start and end date, dose if relevant; dose 
changes); 
• 
Date of study completion and reason for early discontinuation, if it occurs.  
All clinical study documents must be retained by the investigator until at least 2years or 
according to local laws, whichever is longer, after the last approval of a marketing application 
in an ICH region (i.e., United States, Europe, or Japan) and until there are no pending or 
planned marketing applications in an ICH region; or, if no application is filed or if the 
application is not approved for such indication, until 2years after the investigation is 
discontinued and regulatory authorities have been notified. Investigators may be required to 
retain documents longer if specified by regulatory requirements, by local regulations, or by an 
agreement with study funder. The investigator must notify study funder before destroying any 
clinical study records. Should the investigator wish to assign the study records to another party 
or move them to another location, study funder must be notified in advance. If the 
investigator cannot provide for this archiving requirement at the study site for any or all of the 
documents, special arrangements must be made between the investigator and study funder to 
store these records securely away from the site so that they can be returned sealed to the 
investigator in case of an inspection. When source documents are required for the continued 
care of the subject, appropriate copies should be made for storage away from the site. 
9.7. 
Electronic Case Report Forms 
For each subject consented, an eCRF will be completed by an authorized study staff member 
whose training for this function is documented according to study procedures. The eCRF will 


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be completed in a timely manner. Original entries as well as any changes to data fields will be 
stored in the audit trail of the system. At the conclusion of the trial, a read-only archive copy of 
the data entered will be generated. This archive will be stored in accordance with the records 
retention requirements. 
9.8. 
Investigational Medicinal Product Accountability and Return 
The investigator or designee is responsible for ensuring adequate accountability of all used and 
unused study drug.  This includes acknowledgment of receipt of each shipment of study 
product (quantity and condition), subject dispensing records, and returned or destroyed study 
product. Dispensing records will document quantities received from study funder and 
quantities dispensed to subjects, including lot number, date dispensed, subject identifier 
number, subject initials, and the initials of the person dispensing the medication. Drug may be 
returned or destroyed on an ongoing basis during the study, if appropriate.  At the end of the 
study, following final drug inventory reconciliation, the study site will arrange for the return of 
unused investigational medicinal product supplies. All drug supplies and associated 
documentation will be periodically reviewed and verified by the study funder over the course 
of the study. 
9.9. 
Inspections 
The investigator understands that source documents for this trial should be made available to 
appropriately qualified personnel from study funder and its representatives, to IRBs/IECs, or to 
regulatory authority or health authority inspectors. 
9.10. Protocol Compliance 
The investigator is responsible for ensuring the study is conducted in accordance with the 
procedures and evaluations described in this protocol. 
9.11. Protocol Modifications 
Protocol modifications, except those intended to reduce immediate risk to study subjects, may 
be made by the investigator in collaboration with the study funder. All protocol modifications 


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must be submitted to the IRB/IEC in accordance with local requirements. Approval must be 
obtained before changes can be implemented. 
9.12. Study Reports 
A clinical study report (CSR) will be prepared and provided to the regulatory agency (ies). The 
Investigator will ensure that the report meets the standards set out in the ICH Guideline for 
Structure and Content of Clinical Study Reports (ICHE3). Note that an abbreviated report may 
be prepared in certain cases. After conclusion of the study, Investigators in this study may 
communicate, orally present, or publish in scientific journals or other scholarly media only 
after the following conditions have been met:  
• 
The results of the study in their entirety have been publicly disclosed by or with 
the consent of the study funder in an abstract, manuscript, or presentation form. 
9.13. Study Termination 
The Investigator reserves the right to terminate the study at any time. Should this be 
necessary, the investigator will arrange discontinuation procedures and notify the appropriate 
regulatory authorities, IRBs, IECs, and the Fundor. In terminating the study, the investigator 
will assure that adequate consideration is given to the protection of the subjects’ interests. 


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10. References 
 
10.1. 
Literature  
Abd-Elsalam, S., Kobtan, A., El-Kalla, F., Elkhalawany, W., Nawasany, S.E., Saif, S.A., Yousef, M., 
Ali, L.A., Soliman, S., Mansour, L., et al. (2016). A 2-week Nitazoxanide-based quadruple 
treatment as a rescue therapy for Helicobacter pylori eradication: A single center experience. 
Medicine (Baltimore) 95, e3879. 
 
Alinia (nitazoxanide): US Prescribing Information. Tampa, FL. Romark, L.C., 2016 
 
Amorosa, V.K., Luetkemeyer, A., Kang, M., Johnson, V.A., Umbleja, T., Haas, D.W., Yesmin, S., 
Bardin, M.C., Chung, R.T., Alston-Smith, B., et al. (2013). Addition of nitazoxanide to PEG-IFN 
and ribavirin to improve HCV treatment response in HIV-1 and HCV genotype 1 coinfected 
persons naive to HCV therapy: results of the ACTG A5269 trial. HIV Clin Trials 14, 274-283. 
 
Assy, N., Kaita, K., Mymin, D., Levy, C., Rosser, B., and Minuk, G. (2000). Fatty infiltration of 
liver in hyperlipidemic patients. Dig Dis Sci 45, 1929-1934. 
 
Bacon, B.R., Shiffman, M.L., Lim, J.K., Berman, A., Rustgi, V.K., and Keeffe, E.B. (2010). A Phase 
II, Randomized, Double-Blind, Placebo-Controlled Study of Nitazoxanide Plus Peginterferon 
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