Conditions: Cirrhosis
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 23
Healthy volunteers: no

Brief summary:
This Phase 1b/2a, randomized, double-blind, placebo-controlled study was designed to evaluate the safety, tolerability, and pharmacodynamics of SYNB1020 in hepatic insufficiency and cirrhosis patients with hyperammonemia, with dosing of the investigational medicinal product (IMP) administered in an inpatient unit and subsequent outpatient follow-up for SYNB1020 clearance in two study parts.

Detailed description:
In Part 1, a sentinel open-label cohort of subjects with cirrhosis and Model for End-Stage Liver Disease (MELD) score \<12 was admitted to an inpatient facility for a run-in diet, baseline assessments, IMP administration, safety monitoring, and collection of blood, urine, and stool samples for evaluation of safety, tolerability, and pharmacokinetic (PK) and pharmacodynamic (PD) evaluations. Subjects in Part 1 were enrolled sequentially to receive SYNB1020. Once the safety and tolerability were established in Part 1, enrollment was opened to subjects in Part 2.

Part 2 comprised a randomized, double-blind, placebo-controlled study in subjects with cirrhosis and hyperammonemia. Subjects were permitted to be pre-screened for eligibility based on medical history and a single fasting spot venous ammonia measurement. Eligible subjects with elevated fasting spot venous ammonia then underwent full screening within 7 days of pre-screening. Eligible subjects were admitted to an inpatient facility for a run-in diet and 24-hour ammonia profile, and those with an elevated 24-hour ammonia area under the curve (AUC) (\>1.2 × the upper limit of normal \[ULN\]) proceeded with computer-generated randomization in a 1:1 ratio to receive either SYNB1020 or matching placebo. Randomization was followed by IMP administration, safety monitoring, and collection of blood, urine, and stool samples for PK and PD evaluations.

Arms:
arm 1: Part 1 comprised a sentinel open-label cohort of subjects enrolled sequentially to receive SYNB1020, which was administered orally at a dose of 5 × 10\^11 colony-forming units (CFU) 3 times daily (TID) given immediately after meals from Days 1 through 6. arm 2: Subjects randomized to receive SYNB1020 in Part 2 received SYNB1020 administered orally at a dose of 5 × 10\^11 CFU TID given immediately after meals from Days 1 through 6. arm 3: Subjects randomized to receive control in Part 2 received matching placebo (100 mL masking solution) administered orally TID given immediately after meals from Days 1 through 6.

Interventions: intervention 1: SYNB1020 intervention 2: Placebo
Intervention descriptions: intervention 1: SYNB1020 was supplied at a concentration of approximately 1 × 10\^11 CFU/mL in a buffered solution in 5 mL cryovials with a nominal 5 mL fill volume, administered with 100 mL of masking buffer solution. intervention 2: Subjects received placebo orally in a chilled buffered solution (100 mL).

Protocol document:
Prot_001.pdf: 
 
 
 
 
CLINICAL STUDY SYNB1020-CP-002 
 
Protocol Version 6:  27 November 2018 
 
Study Title: 
A Randomized, Double-blind, Placebo-controlled Study to 
Assess the Safety, Tolerability, and Pharmacodynamics of 
SYNB1020 in Hepatic Insufficiency and Cirrhosis Patients  
Study Number: 
SYNB1020-CP-002 
Study Phase: 
Phase 1b/2a 
Product Name: 
SYNB1020 
Indication: 
Cirrhosis Patients 
Sponsor: 
Synlogic, Inc. 
 
 
Synlogic, Inc. 
301 Binney St, Suite 402 
Cambridge, MA 02142 
Phone: (617) 401-9975 
 
CONFIDENTIAL INFORMATION: This protocol contains trade secrets and other confidential information. 
Accordingly, this protocol shall be treated as confidential and restricted to its intended use, namely, the guidance of 
the clinical investigator. This material is the sole property of Synlogic, Inc. and shall not be disclosed or used by 
others except as authorized in writing by Synlogic, Inc. This material may be disclosed to and used by your staff and 
associates only to the extent necessary to conduct the clinical study. 


SYNB1020 
 
Synlogic, Inc. 
Clinical Study SYNB1020-CP-002 
Version 6 
2 
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27 November 2018 
SIGNATURE PAGE (SPONSOR) 
I have read and understand the contents of the clinical protocol for Clinical Study 
SYNB1020-CP-002 Version 6, dated 27 November 2018, and agree to meet all obligations of 
Synlogic, Inc. as detailed in all applicable regulations and guidelines. In addition, I will ensure 
that the Principal Investigators are informed of all relevant information that becomes available 
during the conduct of this study. 
 
______________________________________________  
_____________ 
Aoife Brennan, MB, BCh, BAO, MMSc 
 
 
 
Date 
CMO 
Synlogic, Inc.  


SYNB1020 
 
Synlogic, Inc. 
Clinical Study SYNB1020-CP-002 
Version 6 
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Confidential 
27 November 2018 
PRINCIPAL INVESTIGATOR AGREEMENT 
I have read and understand the contents of the clinical protocol for Clinical Study 
SYNB1020-CP-002 Version 6, dated 27 November 2018, and will adhere to the study 
requirements as presented, including all statements regarding confidentiality. In addition, I will 
conduct the study in accordance with the requirements of this protocol and also protect the rights, 
safety, privacy and well-being of study patients in accordance with the following: 
• International Council for Harmonisation (ICH) of Technical Requirements for 
Pharmaceuticals for Human Use Harmonised Tripartite Guideline for Good Clinical 
Practice (GCP) E6(R1). 
• All applicable laws and regulations, including, without limitation, data privacy laws 
and regulations. 
• Requirements for reporting serious adverse events as defined in Section 9 of this 
protocol. 
• Terms outlined in the Clinical Study Site Agreement. 
My signature also acknowledges that: 
• Neither my sub investigators nor I are members of the Institutional Review Board 
reviewing this protocol, or 
• I and/or my sub investigators are members of the Institutional Review Board, but I/we 
will not participate in the initial review or continuing review of this study. 
 
Name of Principal Investigator 
 
 
Signature of Principal Investigator 
 
 
 
 
 
Date 
 
 
 
 


SYNB1020 
 
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PROTOCOL SYNOPSIS  
Name of Sponsor/Company: 
Synlogic, Inc. 
Name of Investigational Product: 
SYNB1020 
Name of Active Ingredient: 
SYNB1020 
Title of Study: 
A Randomized, Double-blind, Placebo-controlled Study to Assess the Safety,Tolerability, and 
Pharmacodynamics of SYNB1020 in Hepatic Insufficiency and Cirrhosis Patients  
Study Center: Up to 10 centers in the United States 
Objectives and 
Endpoints 
The primary objective is to evaluate the safety and tolerability of SYNB1020 
following multiple doses in hepatic insufficiency and cirrhosis patients. The 
endpoints related to this objective include the nature and frequency of adverse 
events (AEs), measurement of vital signs and laboratory assessments (e.g., blood 
chemistry, complete blood count [CBC], liver function tests, international 
normalized ratio [INR], and urinalysis), and electrocardiograms (ECGs). 
Secondary: 
• 
SYNB1020 kinetics in feces (measured with qualitative and 
quantitative polymerase chain reaction [qPCR] fecal assays) following 
dosing 
• 
Change from baseline of venous ammonia, including fasting and 
24-hour area under the curve (AUC), following SYNB1020 dosing in 
subjects with hyperammonemia 
Exploratory: 
• 
Placebo-adjusted change from baseline in pharmacodynamic effects of 
SYNB1020 on: 
− 
Serum inflammatory markers (serum tumor necrosis factor alpha 
(TNF-α), interleukin (IL)-6, and endotoxin) 
− 
Urinary urea/urinary nitrogen (24 hour) and calculated nitrogen 
balance 
− 
Plasma nitrate and urinary nitrate (24 hour) 
− 
Plasma amino acids including glutamine, arginine, aromatic amino 
acids, and branched chain amino acids 
− 
Psychometric test performance at baseline and following dosing as 
measured by Psychometric Hepatic Encephalopathy Score testing 
(i.e., number connection test A/B, digit symbol test, line tracing 
test, and serial dotting test) 
Design 
This Phase 1b/2a, randomized, double-blinded placebo-controlled study will 
evaluate the safety, tolerability, and pharmacodynamics of SYNB1020 in hepatic 
insufficiency and cirrhosis patients, with dosing of the investigational product (IP) 


SYNB1020 
 
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administered in an inpatient unit and subsequent outpatient follow-up for 
SYNB1020 clearance. This study consists of two parts: 
Part 1: 
A sentinel open-label cohort of subjects with cirrhosis and Model for End-Stage 
Liver Disease (MELD) score <12 will be admitted to an inpatient facility for a 
run-in diet, baseline assessments, IP administration, safety monitoring, and 
collection of blood, urine, and fecal samples for evaluation of safety, tolerability, 
and pharmacokinetics and PD evaluations. Once the safety and tolerability have 
been established in Part 1, enrollment will be opened to subjects in Part 2. 
Part 2:  
Part 2 of the trial will comprise a randomized, double-blinded, placebo-controlled 
study in subjects with cirrhosis and hyperammonemia. Subjects may be 
pre-screened for eligibility based on medical history and a single fasting spot 
venous ammonia measurement. Eligible subjects with elevated fasting spot venous 
ammonia will then undergo full screening within 7 days of pre-screening. Eligible 
subjects will be admitted to an inpatient facility for a run-in diet and 24-hour 
ammonia profile, and those with an elevated 24-hour ammonia AUC (>1.2 × the 
upper limit of normal [ULN]) will proceed with randomization, IP administration, 
safety monitoring, and collection of blood, urine, and fecal samples for 
pharmacokinetic and pharmacodynamic (PD) evaluations. 
Study Regimens 
Subjects will receive a calorie and protein controlled diet during inpatient 
monitoring, with specified macronutrient content based on individual subject food 
preferences. Subjects will be expected to consume the entirety of their meals and 
not consume any external foods for the duration of their inpatient study 
participation. Diet consumption will be recorded.  
In addition, subjects will be administered a histamine-2 receptor (H2) inhibitor to 
reduce stomach acid, comprising oral ranitidine 150 mg administered twice daily 
(BID). Subjects already on a proton pump inhibitor (PPI) will not be switched to 
ranitidine but will continue their prescribed PPI regimen for the duration of the 
study. 
Part 1 is open-label; in Part 2 both subjects and investigators will be blinded to 
randomized IP assignment. 
Inpatient Monitoring 
Part 1: 
Each subject will be admitted to the inpatient unit after an overnight fast on the 
morning of Day -5. Beginning on Day -5, subjects will initiate a controlled diet and 
ranitidine. Days -5 through -1 will be a run-in phase to allow for dietary 
stabilization on the controlled diet. On Day -2, all subjects will undergo baseline 
24-hour ammonia profile with 8 measurements (AUC), baseline clinical laboratory 
sampling and other safety measurements, FibroScan® measurement, and baseline 
hepatic encephalopathy grading and psychometric function testing. Subjects will 
receive up to 5 × 1011 colony-forming units (CFU) of SYNB1020 administered 
3 times per day (TID) immediately after meals from Days 1 through 6 (including 
mid-day and evening doses on Day 6). Fasting ammonia levels will be measured 
daily. On Day 5, subjects will undergo ammonia AUC assessment. On Day 6, 
subjects will undergo ammonia AUC assessment, repeat hepatic encephalopathy 
grading and psychometric function testing, as well as 24-hour urine and blood 
collection. Subjects will be monitored in the inpatient setting for at least 12 hours 


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following completion of dosing, with discharge on Day 7 for clinically stable 
subjects. Stool samples will be collected daily during the inpatient period. 
Part 2: 
Each subject will be admitted to the inpatient unit after an overnight fast on the 
morning of Day -5. Beginning on Day -5, subjects will initiate a controlled diet and 
ranitidine. Days -5 through -1 will be a run-in phase to allow for dietary 
stabilization on the controlled diet. On Day -2, all subjects will undergo baseline 
24-hour ammonia profile with 8 measurements (AUC), baseline clinical laboratory 
sampling and other safety measurements, FibroScan® measurement, and baseline 
hepatic encephalopathy grading and psychometric function testing. Subjects whose 
24-hour AUC venous ammonia measurement is above 1.2 × ULN will undergo 
computer-generated randomization on Day -1 in a 1:1 ratio to receive either up to 
5 × 1011 CFU of SYNB1020 or matching placebo administered TID immediately 
after meals for 6 days. Subjects will receive IP dosing from Days 1 through 
6 (including mid-day and evening doses on Day 6). Fasting ammonia levels will be 
measured daily. On Day 5, subjects will undergo ammonia AUC assessment. On 
Day 6, subjects will undergo ammonia AUC assessment, repeat hepatic 
encephalopathy grading and psychometric function testing, as well as 24-hour 
urine and blood collection. Subjects will be monitored in the inpatient setting for at 
least 12 hours following completion of dosing, with discharge on Day 7 for 
clinically stable subjects. Stool samples will be collected daily during the inpatient 
period. 
Outpatient Follow-up 
One week (7 ± 1 days) after discharge from the inpatient unit, subjects will return 
for clinical evaluation and bring a 1-week post-dose fecal sample. Subjects will be 
provided diary cards for collection of AE data. Feces will continue to be collected 
weekly for the first 6 weeks following discharge and biweekly until a subject has a 
negative SYNB1020 fecal test for up to 10 weeks following completion of study 
dosing. Subjects who remain colonized with SYNB1020 after 10 weeks following 
the last dose will be treated with a 3-day course of oral ciprofloxacin (or other 
antibiotic for subjects allergic to ciprofloxacin). A SYNB1020 fecal test will be 
performed 3 to 5 days following completion of antibiotic treatment solely for 
research purposes and will not guide subject follow-up decision making. An AE 
assessment will be performed by telephone at least 7 days after completion of 
antibiotic treatment. If no AEs are reported, subjects will not require any further 
follow-up and will be considered to have completed the study requirements. If AEs 
are reported, subjects will be advised to return to the inpatient facility for a 
comprehensive physical examination, laboratory assessment, and AE causality 
determination.  
Study 
Population 
This study will comprise adult male and female patients with hepatic insufficiency 
and cirrhosis. 
Duration of 
Study 
Participation 
The maximum time of study participation for a subject is 133 days, including 
pre-screening (up to 7 days), screening (up to 45 days), inpatient monitoring 
(12 days), and outpatient follow-up (up to 70 days after last dose). 
Analytical and 
Statistical 
Methods 
Sample Size Determination 
Part 1: The sample size for the sentinel cohort (6 subjects) is primarily designed for 
empirical evaluation of safety and tolerability in subjects with cirrhosis. 
Part 2: A sample of 24 subjects must complete all 3 ammonia AUC intervals 


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(baseline, Day 5, and Day 6) to detect a 20% reduction in average daily ammonia 
(AUC0-24/24, from a baseline of 70 µmol/L) with an approximate significance level 
of 10% and an approximate power of 90%. Significance and power are 
approximate based on the assumptions below. The number of subjects assumes that 
the standard deviation (SD) of spot ammonia is 36.4 µmol/L, half of the variance is 
between subject, and that the SD for average daily ammonia is as little as half that 
of spot ammonia. The spot ammonia SD is based on a weighted average of SD 
observed in a prior study of ammonia lowering in a similar population 
(Ghabril 2013). The variability of AUC may be 50% lower than spot ammonia 
based on the Food and Drug Administration (FDA) summary basis of approval for 
glycerol phenylbutyrate for urea cycle disorders  (Center for Drug Evaluation and 
Research 2012). 
Due to uncertainty in the assumptions of decrease in variability in AUC of 
ammonia relative to spot measurements in this population, up to 2 interim analyses 
for sample size re-estimation may occur after at least 12 subjects have completed 
all ammonia AUC assessments, and the total number of subjects in Part 2 of the 
study may be increased up to 40 at either point based on the SD of data observed. 
Additional subjects may be enrolled to ensure that at least the planned number of 
evaluable subjects complete the study. 
Safety Analysis 
Safety will be evaluated by continuous monitoring of AEs, vital signs, clinical 
laboratory measurements (including MELD score components), ECGs, and 
physical examinations. After completion of Part 1, a Safety Review Committee 
will conduct a safety evaluation (including any available data on AEs, laboratory 
measurements, vital signs, and ECGs up to 7 days after the last dose of 
SYNB1020) before opening Part 2 for enrollment. In Part 2, the Safety Review 
Committee will conduct blinded safety evaluations at predetermined study time 
points and ad hoc, as needed, and will be defined in the Safety Review Committee 
charter. 
Adverse events will be coded using the Medical Dictionary for Regulatory 
Activities (MedDRA®), and severity of AEs and laboratory abnormalities will be 
graded using the National Cancer Institute (NCI) Common Terminology Criteria 
for Adverse Events (CTCAE), version 4.03. Adverse events will be tabulated by 
system organ class (SOC) and preferred term. Incidence tables of subjects with 
AEs will be presented for all AEs by maximum severity, serious adverse events 
(SAEs), AEs assessed as related to SYNB1020/placebo, and AEs resulting in 
discontinuation of study dosing. 
Microbiotic-Kinetic Analysis 
Feces will be collected daily following dosing through Day 7 for analysis of 
SYNB1020 transit through the gastrointestinal tract. In Part 1, the 24-hour fecal 
sample will be weighed, homogenized, and a sample analyzed for SYNB1020 by 
qPCR. In Part 2, a fecal sample from the first void of the day will be collected 
using a collection kit. The following endpoints will be calculated from the serial 
fecal sampling from each subject, if data support calculation: 
• 
Tlag – the time of the first detectable fecal SYNB1020 
• 
Tss – the time to steady-state 
• 
qmax – the maximum observed fecal SYNB1020 qPCR signal 


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• 
Tlast – the time of the last detectable fecal SYNB1020 
• 
Half-life – the time for the fecal SYNB1020 qPCR signal to decline by 
50% 
A sample will be considered negative for the purposes of SYNB1020 clearance if it 
is below the limit of quantification or estimated to be below 0.1% of the 
administered dose. 
The microbiotic-kinetic parameters of SYNB1020 will be summarized 
descriptively. 
Pharmacodynamic Analysis  
Urine and blood samples will be collected at screening and on study both before 
and after administration of SYNB1020 or placebo. The following laboratory 
measurements will be performed to evaluate the preliminary PD of SYNB1020: 
• 
Venous ammonia (morning fasting and 24-hour profile to calculate 
AUC) 
• 
Inflammatory markers (serum TNF-α, IL-6, endotoxin) 
• 
Urinary urea, urinary nitrogen (24-hour) and nitrogen balance 
• 
Plasma amino acid profile (glutamine, arginine, aromatic amino acids 
and branched chain amino acids) 
• 
Plasma nitrate and urinary (24-hour) nitrate 
• 
Bristol stool form scale (BSFS) for each bowel movement 
• 
Psychometric test performance as measured by Psychometric Hepatic 
Encephalopathy Score testing (i.e., number connection test A/B, digit 
symbol test, line tracing test, and serial dotting test) 
The PD parameters of SYNB1020 will be summarized descriptively. 


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TABLE OF CONTENTS 
SIGNATURE PAGE (SPONSOR) ..................................................................................................2 
PRINCIPAL INVESTIGATOR AGREEMENT .............................................................................3 
PROTOCOL SYNOPSIS ................................................................................................................4 
TABLE OF CONTENTS .................................................................................................................9 
LIST OF ABBREVIATIONS ........................................................................................................14 
1. 
INVESTIGATORS AND STUDY ADMINISTRATIVE STRUCTURE .................18 
2. 
INTRODUCTION ......................................................................................................19 
2.1. 
Background of Hepatic Encephalopathy ....................................................................19 
2.1.1. 
Unmet Medical Need in Hepatic Encephalopathy ......................................................19 
2.2. 
Investigational Medicinal Product ..............................................................................20 
2.2.1. 
Nonclinical Studies .....................................................................................................21 
2.2.1.1. 
Nonclinical Pharmacology ..........................................................................................21 
2.2.1.2. 
In Vitro Characterization of SYNB1010/SYNB1020 ................................................22 
2.2.1.3. 
Nonclinical Toxicology ..............................................................................................23 
2.2.1.4. 
Summary of the Nonclinical Development Program ..................................................23 
2.2.2. 
Clinical Studies ...........................................................................................................23 
2.2.2.1. 
Study SYNB1020-CP-001 ..........................................................................................23 
2.3. 
Rationale for the Study ...............................................................................................25 
3. 
STUDY OBJECTIVES AND ENDPOINTS ..............................................................26 
3.1. 
Primary .......................................................................................................................26 
3.2. 
Secondary ...................................................................................................................26 
3.3. 
Exploratory .................................................................................................................26 
4. 
STUDY DESIGN .......................................................................................................27 
4.1. 
Study Design Overview ..............................................................................................27 
4.2. 
Study Dosing Regimens .............................................................................................27 
4.2.1. 
Dose selection .............................................................................................................27 
4.2.2. 
Dietary Regimen and Study Supplements ..................................................................27 
4.2.3. 
Inpatient Monitoring and Investigational Product Administration .............................28 
4.3. 
Stopping Rules ............................................................................................................28 
4.4. 
Safety Review Committee ..........................................................................................29 


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4.5. 
Duration of Study Participation ..................................................................................29 
4.5.1. 
Pre-screening Period in Part 2 ....................................................................................29 
4.5.2. 
Screening Period .........................................................................................................29 
4.5.3. 
Study Dosing ..............................................................................................................29 
4.5.4. 
Post-dose Follow-up ...................................................................................................30 
5. 
PATIENT POPULATION ..........................................................................................31 
5.1. 
Number of Subjects ....................................................................................................31 
5.2. 
Selection of Subjects ...................................................................................................31 
5.2.1. 
Eligibility Criteria .......................................................................................................31 
5.2.1.1. 
Inclusion Criteria in Part 1 ..........................................................................................31 
5.2.1.2. 
Exclusion Criteria in Part 1 .........................................................................................32 
5.2.1.3. 
Inclusion Criteria in Part 2 ..........................................................................................33 
5.2.1.4. 
Exclusion Criteria in Part 2 .........................................................................................33 
6. 
INVESTIGATIONAL MEDICAL PRODUCT AND CONCOMITANT 
MEDICATIONS .........................................................................................................36 
6.1. 
Investigational Product Supply and Administration ...................................................36 
6.1.1. 
SYNB1020 ..................................................................................................................36 
6.1.1.1. 
Manufacturing and Formulation .................................................................................36 
6.1.1.2. 
Packaging and Labeling ..............................................................................................36 
6.1.1.3. 
Shipping, Storage, and Handling ................................................................................36 
6.1.1.4. 
Dispensing and Administration ..................................................................................36 
6.1.2. 
Placebo ........................................................................................................................37 
6.1.3. 
Ranitidine ....................................................................................................................37 
6.2. 
Accountability and Dosing Compliance .....................................................................37 
6.3. 
Concomitant Medications ...........................................................................................37 
6.3.1. 
Prohibited Therapies and Substances .........................................................................37 
7. 
STUDY PROCEDURES AND ASSESSMENTS......................................................38 
7.1. 
Screening ....................................................................................................................38 
7.1.1. 
Part 1 ...........................................................................................................................38 
7.1.2. 
Part 2 ...........................................................................................................................38 
7.1.2.1. 
Pre-screening ..............................................................................................................38 
7.1.2.2. 
Screening ....................................................................................................................38 
7.2. 
Safety Assessments .....................................................................................................39 


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7.2.1. 
Adverse Events ...........................................................................................................39 
7.2.2. 
Medical History ..........................................................................................................39 
7.2.3. 
Vital Signs ..................................................................................................................39 
7.2.4. 
Physical Examination .................................................................................................39 
7.2.5. 
FibroScan ....................................................................................................................39 
7.2.6. 
Clinical Laboratory Measurements.............................................................................39 
7.2.7. 
Venous Ammonia .......................................................................................................40 
7.2.8. 
Electrocardiograms .....................................................................................................40 
7.2.9. 
Blood and Urine Cultures/Polymerase Chain Reaction..............................................40 
7.2.10. 
Hepatic Encephalopathy Severity Grading .................................................................40 
7.2.11. 
Psychometric Testing ..................................................................................................40 
7.2.12. 
Cirrhosis Severity Grading .........................................................................................41 
7.3. 
Microbiotic-Kinetic Assessments ...............................................................................41 
7.4. 
Pharmacodynamic Assessments .................................................................................41 
7.5. 
Follow-up Assessments ..............................................................................................42 
7.5.1. 
End-of-study/Discharge Visit .....................................................................................42 
7.5.2. 
Follow-up After Completion of Study Dosing ...........................................................42 
7.6. 
Sample Retention for Exploratory Analyses ..............................................................42 
8. 
STUDY DISCONTINUATION .................................................................................47 
8.1. 
Sponsor Discontinuation Criteria ...............................................................................47 
8.2. 
Study Discontinuation for Individual Subjects ...........................................................47 
9. 
ADVERSE EVENTS..................................................................................................49 
9.1. 
Adverse Event .............................................................................................................49 
9.1.1. 
Assessment of Severity ...............................................................................................49 
9.1.2. 
Assessment of Causality .............................................................................................49 
9.2. 
Serious Adverse Event ................................................................................................50 
9.2.1. 
Clarification of Serious Adverse Event Definition .....................................................50 
9.2.2. 
Serious, Unexpected, Suspected Adverse Reactions ..................................................51 
9.3. 
Procedures for Treating and Reporting Special Safety Events ...................................51 
9.3.1. 
Infection (Suspected or Confirmed) ...........................................................................51 
9.3.2. 
Spontaneous Bacterial Peritonitis ...............................................................................52 
9.3.3. 
Pregnancy ...................................................................................................................52 
9.3.4. 
Hepato-Renal Syndrome .............................................................................................52 


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9.4. 
Reporting of Adverse Events ......................................................................................52 
9.5. 
Pregnancy ...................................................................................................................52 
9.6. 
Clinical Laboratory Abnormalities .............................................................................52 
9.7. 
Review of Safety Data ................................................................................................53 
10. 
STATISTICAL METHODS AND DETERMINATION OF SAMPLE SIZE ...........54 
10.1. 
Populations for Analysis .............................................................................................54 
10.2. 
Safety Variables ..........................................................................................................54 
10.3. 
Microbiotic-Kinetic Variables ....................................................................................55 
10.4. 
Pharmacodynamic Variables ......................................................................................55 
10.5. 
Determination of Sample Size ....................................................................................55 
10.6. 
Randomization and Blinding ......................................................................................55 
10.7. 
Changes in the Conduct of the Study or Planned Analysis ........................................56 
11. 
DATA RECORDING, RETENTION AND MONITORING ....................................57 
11.1. 
Case Report Forms .....................................................................................................57 
11.2. 
Data Retention ............................................................................................................57 
11.3. 
Data Monitoring ..........................................................................................................57 
11.4. 
Quality Control and Quality Assurance ......................................................................58 
12. 
REGULATORY, ETHICAL, AND LEGAL OBLIGATIONS .................................59 
12.1. 
Good Clinical Practice ................................................................................................59 
12.2. 
Institutional Review Board Approval .........................................................................59 
12.3. 
Regulatory Authority Approval ..................................................................................59 
12.4. 
Other Required Approvals ..........................................................................................59 
12.5. 
Informed Consent .......................................................................................................59 
12.6. 
Patient Confidentiality ................................................................................................60 
12.7. 
Disclosure of Information ...........................................................................................60 
12.8. 
Publication of Study Data ...........................................................................................60 
12.9. 
Ethical Standards ........................................................................................................61 
13. 
REFERENCES ...........................................................................................................62 
APPENDIX 1. RANITIDINE PACKAGE INSERT ..................................................................64 
APPENDIX 2. WEST HAVEN GRADING SYSTEM ..............................................................65 
APPENDIX 3. PSYCHOMETRIC HEPATIC ENCEPHALOPATHY SCORE 
TESTING ....................................................................................................................66 
APPENDIX 4. CHILD-TURCOTTE-PUGH SCORE TESTING ..............................................72 


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APPENDIX 5. MODEL FOR END-STAGE LIVER DISEASE (MELD) ................................73 
LIST OF TABLES 
Table 1: 
Study Enrollment in Study SYNB1020-CP-001 ........................................................24 
Table 2: 
Laboratory Exclusion Criteria ....................................................................................35 
Table 3: 
Schedule of Events .....................................................................................................43 
Table 4: 
Schedule of Plasma Sampling for 24-Hour Ammonia and Nitrate ............................46 
 
LIST OF FIGURES 
Figure 1: 
Schematic of Key SYNB1020 Components ...............................................................21 


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LIST OF ABBREVIATIONS 
Abbreviation or Specialist Term 
Explanation 
AE 
adverse event 
ALT 
alanine aminotransferase 
aPTT 
activated partial thromboplastin time 
Arg 
arginine 
ArgA 
N-acetylglutamate synthase 
argA 
N-acetylglutamate synthase gene 
argAfbr 
feedback-resistant N-acetylglutamate synthase 
ArgB 
acetylglutamate kinase 
ArgC 
N-acetylglutamylphosphate reductase 
ArgD 
N-acetylornithine aminotransferase 
ArgE 
acetylornithine deacetylase 
ArgFI 
ornithine carbamoyltransferase 
ArgG 
arginosuccinate synthase 
ArgH 
arginosuccinate lyase 
ArgR 
transcriptional repressor 
argR 
arginine repressor gene 
ΔargR 
arginine repressor gene deletion 
AST 
aspartate aminotransferase 
Asp 
aspartic acid 
AUC 
area under the curve 
AUC0-24 
area under the curve from time 0 to 24 hours 
BID 
bis in die (twice daily) 
BSFS 
Bristol stool form scale 
CarAB 
carbamoylphosphate synthetase 
CBC 
complete blood count 
CFU 
colony-forming unit(s) 
CRO 
Contract Research Organization 
CTCAE 
Common Terminology Criteria for Adverse Events 
CV 
curriculum vitae 
DRI 
dietary reference intakes 
ECG 
electrocardiogram 
EcN 
Escherichia coli Nissle 1917 


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Abbreviation or Specialist Term 
Explanation 
E. coli 
Escherichia Coli 
eCRF 
electronic case report form 
EDC 
electronic data capture 
eGFR 
estimated glomerular filtration rate 
EOS 
End of Study 
FDA 
Food and Drug Administration 
FNR 
fumarate and nitrate reductase 
FSH 
follicle-stimulating hormone 
g 
gram(s) 
GCP 
Good Clinical Practice 
GI 
gastrointestinal 
Gln 
Glutamine 
GLP 
Good Laboratory Practice 
Glu 
Glutamate 
h 
hour(s) 
H2 
histamine-2 receptor 
HE 
hepatic encephalopathy 
HCO3 
Bicarbonate 
HIV 
human immunodeficiency virus 
HRS 
hepato-renal syndrome 
ICH 
International Council for Harmonisation of Technical Requirements for 
Pharmaceuticals for Human Use 
IL 
Interleukin 
INR 
international normalized ratio 
IP 
investigational product 
IRB 
Institutional Review Board 
KG 
Ketoglutarate 
kg 
Kilogram 
LFT 
liver function test 
m2 
meter(s) squared 
malEK 
malE = maltose gene, malK = maltose transpose complex, ATP-binding 
subunit gene 
MedDRA® 
Medical Dictionary for Regulatory Activities 
MELD 
Model for End-Stage Liver Disease 
mg 
milligram(s) 


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Abbreviation or Specialist Term 
Explanation 
µg 
microgram(s) 
min 
minute(s) 
MK 
microbiotic-kinetic 
μL 
microliter(s) 
mL 
milliliter(s) 
mM 
millimolar(s) 
μmol 
micromole(s) 
MOPS 
3-(N-morpholino) propanesulfonic acid 
mRNA 
messenger ribonucleic acid 
ms 
millisecond(s) 
MTD 
maximum tolerated dose 
NCI 
National Cancer Institute 
NH3 
ammonia 
NHP 
nonhuman primate 
No. 
number 
O2 
oxygen 
OTC 
ornithine transcarbamylase 
PD 
pharmacodynamics(s) 
PfnrS 
fumarate and nitrate reductase gene S promoter 
PO 
oral administration 
PPI 
proton pump inhibitor 
PT 
prothrombin time 
QD 
quaque die (once daily) 
qmax 
maximum observed fecal SYNB1020 qPCR signal 
(q)PCR 
(quantitative) polymerase chain reaction 
QTc 
QT interval corrected for heart rate 
QTcF 
QT interval corrected for heart rate using Fridericia’s formula 
SAE 
serious adverse event 
SAD 
single ascending dose 
SAF 
safety population 
SAP 
statistical analysis plan 
SBP 
spontaneous bacterial peritonitis 
SD 
standard deviation 
SOC 
system organ class 


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Abbreviation or Specialist Term 
Explanation 
SUSAR 
serious, unexpected, suspected adverse reaction 
TAA 
thioacetamide 
TEAE 
treatment-emergent adverse event 
thyA 
thymidylate synthase 
ΔthyA 
thymidylate synthase deletion 
TID 
ter in die (3 times daily) 
Tlag 
time of the first detectable fecal SYNB1020 
Tlast 
time of the last detectable fecal SYNB1020 
TNF-α 
tumor necrosis factor alpha 
Tss 
time to steady-state 
ULN 
upper limit of normal 
US(A) 
United States (of America) 


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1. 
INVESTIGATORS AND STUDY ADMINISTRATIVE 
STRUCTURE 
The Principal Investigator is the person responsible for the conduct of the study at the 
investigational site. A sub investigator is any member of the clinical study team designated and 
supervised by the Principal Investigator to perform critical study-related procedures and/or to 
make important study-related decisions. 
Prior to study initiation, the Principal Investigator at each site must provide to Synlogic, Inc. 
(also referred to as “Synlogic” or “Sponsor”) a signed protocol signature page, a fully executed 
and signed United States (US) Food and Drug Administration (FDA) Form 1572, a current 
curriculum vitae (CV) and medical license and a financial disclosure form. Financial disclosure 
forms, current CVs and medical licenses must also be provided for all sub investigators listed on 
Form 1572 who will be directly involved in the evaluation of subjects. 
The study will be administered and monitored by employees or representatives of Synlogic in 
accordance with all applicable regulations. Clinical research associates will monitor each site on 
a periodic basis and perform verification of source documentation for each subject. Synlogic or 
designee will be responsible for ensuring timely reporting of expedited serious adverse event 
(SAE) reports to regulatory authorities and Investigators. 


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2. 
INTRODUCTION 
2.1. 
Background of Hepatic Encephalopathy 
The proposed target indication for the clinical development of SYNB1020 is hepatic 
encephalopathy (HE), a potentially life-threatening condition. 
Chronic liver diseases affect approximately 150,000 new patients in the United States (US) each 
year, nearly 20% of whom present with cirrhosis.1 The primary function of the liver is to convert 
toxins, such as ammonia, into nontoxic molecules, such as urea and glutamine to enable safe 
elimination from the body. Thus, patients with impaired liver function are susceptible to an 
excess accumulation of toxins in the blood stream, which presents as HE in approximately 55% 
of patients with chronic liver disease.2 Hepatic encephalopathy comprises a heterogenous group 
of disorders characterized by declining neurocognitive function, and while many HE symptoms 
may be reversible with appropriate treatment, persistent impairment of cognitive function may 
occur.1,3  
The classification of HE severity is based largely on a patient’s mental state at presentation and 
is often graded according to the West Haven criteria, which are based on clinical findings in 
changes of consciousness, intellectual function, and behavior (see Appendix 2). Covert HE, also 
known as minimal HE, is difficult to diagnose and is often observed in patients with cirrhosis 
who show no obvious neurologic symptoms and signs. However, more sensitive psychomotor 
testing reveals subtle changes in attention, psychomotor speed, and executive decision-making, 
which have a profound impact on daily life. Such changes result in difficulty concentrating, 
forgetfulness, changes in personality or behavior, and poor sleep.3,4 The more severe form of HE, 
referred to as overt HE, is associated with obvious mental disorientation and physical symptoms 
(e.g., lethargy, seizures, tremors, organ failure, or brain swelling) that arise suddenly over several 
days or hours and may induce a coma or even death.3,4 Overt HE is associated with a poor 
prognosis, with 1-year survival estimates ranging from 20% to 54%.5 Due to difficulty in 
diagnosis, the prognosis of covert HE is less understood but has been associated with poor 
quality of life, reduced work performance, increased risk of road traffic accidents, and a higher 
risk of developing potentially severe overt HE episodes.4,6,7 
The pathogenesis of HE is believed to be largely attributable to hyperammonia, i.e., the 
inadequate de-toxicification and accumulation of ammonia, as well as the inflammatory process.8 
The metabolism and subsequent renal elimination of ammonia, a highly toxic substance 
produced in the body as a by-product of protein metabolism, is predominantly reliant on a 
functional liver. While ammonia may also be minimally metabolized in the muscle and by the 
brain in patients with impaired liver function, the excessive ammonia levels (i.e., 
hyperammonemia) often overwhelm the metabolic capacity of brain tissue and result in an 
increased risk of brain edema, herniation, and death for patients with HE.3,9 
2.1.1. 
Unmet Medical Need in Hepatic Encephalopathy 
The current standard of care for HE includes reducing blood ammonia levels using lactulose, a 
nonabsorbable disaccharide, administered concurrently with antibiotics to reduce 
inflammation.4,5,8 In addition, rifaximin, a broad spectrum antibiotic, is approved as a treatment 


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to reduce the risk of overt HE recurrence in adults.10 Rifaximin has demonstrated improved 
maintenance of remission, reduced hospitalizations over 6 months, and improved quality of life 
in patients with HE.11 Dietary modification and supplementation may also be implemented for 
patients with HE. While restrictions in dietary protein intake were historically recommended to 
reduce the amount of ammonia required to be metabolized, current recommendations include 
adequate (but not excessive) consumption of protein and other nutrients to avoid muscle wasting, 
as muscles are able to metabolize ammonia and may alleviate the burden of ammonia 
metabolism required by the brain.4 Probiotics may also be administered as a component of HE 
therapy, as they have demonstrated some benefit in the treatment of HE through modulation of 
bacterial growth in the gut.4,8  
When these management approaches fail to control HE, patients with liver disease may be 
candidates for a potentially curative liver transplantation. However, the benefits of 
transplantation must be carefully examined to ensure that they outweigh the added exposure to 
potentially life-threatening risks, such as suppression of the immune system and its 
consequences, including fatal viruses (e.g., Epstein-Barr, cytomegalovirus) and 
lymphoproliferative diseases. Furthermore, many patients succumb to their underlying disease 
while waiting for an eligible liver for transplantation.12 
Morbidity and mortality associated with HE remain high, and hospitalizations for HE impose a 
high burden on community resources.1,8 While overt HE is associated with substantially more 
severe symptoms, adequate management of covert disease is also important in order to maintain 
optimal neurocognitive function and stave off the more severe manifestations of the disease. 
Although rifaximin and lactulose are approved for overt HE, no treatments are currently 
approved for covert disease. 
2.2. 
Investigational Medicinal Product 
SYNB1020 is an orally administered live biotherapeutic product that is a genetically modified 
strain of Escherichia coli Nissle 1917 (EcN) designed to consume ammonia and produce 
arginine in the intestinal tract (Figure 1). Two modifications are made to the EcN chromosome 
within the arginine biosynthetic pathway: deletion of the arginine repressor gene (argR), and 
insertion of a gene that encodes a feedback resistant version of N-acetylglutamate synthase 
(argAfbr) under the control of an inducible promoter. Removing the argR gene prevents 
expression of the transcriptional repressor ArgR. ArgR inhibits the expression of all genes in the 
arginine biosynthetic pathway when arginine is abundant; therefore, a loss of ArgR results in the 
expression of all of the arginine biosynthetic enzymes. In unmodified EcN, the 
N-acetylglutamate synthase (ArgA) enzyme can be inhibited by arginine in order to prevent 
excess arginine production. In contrast, the argAfbr enzyme cannot be inhibited by arginine; 
therefore this enzyme increases arginine production in cells lacking ArgR. In SYNB1020, the 
promoter fumarate and nitrate reductase gene S (PfnrS) controls the expression of the argAfbr gene. 
The PfnrS promoter inhibits expression of the argAfbr gene in aerobic environments, and only 
allows expression of the gene when the cell experiences micro-aerobic or anaerobic 
environments. Thus, excess arginine is only produced in environments where oxygen is limiting, 
such as the mammalian gut. In order to limit unwanted growth of the strain, the thymidylate 
synthase (thyA) gene is deleted. Because of this deletion, SYNB1020 can only grow in 
thymidine-rich environments. 


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SYNB1020 and its parent strain, EcN, were evaluated for antibiotic susceptibility using 
established methodology and interpretive criteria against a panel of 16 common antibiotics  
(see the Investigator’s Brochure). Both strains were susceptible to all antibiotics tested, which is 
consistent with analyses from whole genome sequencing that failed to identify any antibiotic 
resistance elements in these strains. 
Figure 1: 
Schematic of Key SYNB1020 Components 
 
Abbreviations: Arg = arginine; ArgA = N-acetylglutamate synthase; argA = N-acetylglutamate synthase gene; argAfbr = feedback 
resistant N-acetylglutamate synthase gene; ArgB = acetylglutamate kinase; ArgC = N-acetylglutamylphosphate reductase; ArgD 
= N-acetylornithine aminotransferase; ArgE = acetylornithine deacetylase; ArgFI = ornithine carbamoyltransferase; ArgG = 
arginosuccinate synthase; ArgH = arginosuccinate lyase; ΔargR = arginine repressor gene deletion; Asp = aspartic acid ; CarAB 
= carbamoylphosphate synthetase; E. coli = Escherichia coli; FNR = fumarate and nitrate reductase; Gln = glutamine; Glu = 
glutamate;  
HCO3 = bicarbonate; KG = ketoglutarate; malEK = malE = maltose gene, malK = maltose transpose complex, ATP-binding 
subunit gene; NH3 = ammonia; O2 = oxygen; PfnrS = fumarate and nitrate reductase regulator sensor promoter;  
ΔthyA = thymidylate synthase deletion 
The rationale for development of SYNB1020 was to create a commensal strain of EcN that 
would produce excess arginine and continuously absorb excess ammonia where it is naturally 
produced, in the colon, before it can be absorbed into the blood. Because ammonia is produced 
in the gastrointestinal (GI) tract, a gut commensal bacterial strain programmed to consume 
ammonia may be an ideal therapeutic to reduce the levels of excess ammonia in the blood of 
patients with liver disease and therefore reduce hyperammonemia in patients with HE.13-16 The 
endogenous arginine pathway was chosen for augmentation because 4 molecules of ammonia are 
consumed for every molecule of arginine produced, which may result in a more effective channel 
for ammonia assimilation than other amino acid biosynthetic pathways.17  
2.2.1. 
Nonclinical Studies 
2.2.1.1. 
Nonclinical Pharmacology 
The SYNB1020 strain was evaluated with relevant comparators in a series of in vitro assays of 
arginine biosynthesis as well as in vivo studies designed to measure ammonia consumption. 
Nonclinical evaluation included an antibiotic-resistant strain, SYNB1010, that was designed with 
the same gene modifications as SYNB1020 except for the nucleotide changes corresponding to 


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the kanamycin resistance gene. For a comprehensive description of the nonclinical development 
program, refer to the SYNB1020 Investigator’s Brochure.  
2.2.1.2. 
 In Vitro Characterization of SYNB1010/SYNB1020 
As a biocontainment measure, SYNB1020 and precursor strains were engineered with a deletion 
of the thyA gene encoding thymidylate synthase to create a thymidine auxotroph. At least 2 mM 
of thymidine was found to be required for continuous growth of these strains, and at lower 
concentrations the strains grew for a short period of time and then began to die. Concentrations 
of thymidine in intestinal fluid, urine, and blood are very low (< 0.03 mM). Therefore, 
SYNB1020 is not expected to grow in intestinal fluids, blood, or urine. 
2.2.1.2.1. In Vivo Characterization of SYNB1010/SYNB1020 
The distribution of SYNB1010 following oral administration was studied in normal mice. It was 
found that within 1 hour of oral dosing of mice with 1010 colony-forming units (CFU), peak 
levels of bacteria were found in the distal small intestine and cecum. The cecum and colon 
remained the sites of highest concentration of bacterial load over the 30 hours post-dosing. Oral 
dose levels of 107 to 1010 CFU of SYNB1010 on Days 1 and 2 were evaluated for recovery in 
mouse feces from Days 1 to 5. Higher doses were associated with higher levels of recovery, with 
the greatest amounts recovered on Days 2 and 3 after dosing. Complete clearance of detectable 
SYNB1010 requires longer than 3 days following the last oral dose for dose levels greater than 
107 CFUs of bacteria. 
Following oral dosing of mice with 1010 CFU of SYNB1010, SYNB1010 recovered from cecum 
and colonic contents was metabolically active and converted ammonia to arginine in ex vivo 
studies at levels similar to those measured in vitro for the strain. 
2.2.1.2.2. Pharmacodynamic Studies in Thioacetamide Induced Liver Injury Mouse 
Model  
In vivo efficacy of SYNB1020 was studied in thioacetamide (TAA)-treated BALB/c mice 
(N=10/group). Following 4 weeks of TAA administration (150 mg/kg 3 times/week) mice were 
switched to 70% high protein diet for 3 days in order to induce hyperammonemia. SYNB1020 
was orally administrated at increasing doses. Administration of 1 × 109 to 1 × 1010 CFU of 
SYNB1020 lowered blood ammonia levels in a dose dependent manner, with statistically 
significant ammonia reduction at 1 × 1010 CFU of SYNB1020 (from 144 ± 10.9 µg/dL to 84 ± 
11.1 µg/dL).   
2.2.1.2.3. Pharmacodynamic Studies in spfash Mouse Model with Orthinine 
Transcarbamylase Deficiency 
In vivo efficacy of the bacterial strains was studied in the spfash mouse model of ornithine 
transcarbamylase (OTC) deficiency, a subset of hyperammonemia that results from a defect in 
the activity of one of the enzymes of the urea cycle.18 Mice exhibit a deficiency in OTC 
messenger ribonucleic acid (mRNA), and affected mice exhibit increased blood levels of 
ammonia observed upon feeding with an extremely protein-rich diet. Mice dosed with 
SYNB1010 exhibited significantly decreased blood ammonia levels. Hyperammonemic mice 
dosed with SYNB1010 showed an increased survival benefit.  


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2.2.1.3. 
Nonclinical Toxicology 
2.2.1.3.1. Nonhuman Primate Study 
A non-Good Laboratory Practice (non-GLP) repeat-dose safety and tolerability preclinical safety 
study in the nonhuman primate (NHP) was conducted. Female cynomolgus monkeys, 2 to 
8 years of age, were dosed once daily (QD) for 28 days with SYNB1010 at a maximum dose of 
1012 CFU/animal followed by a bicarbonate flush. Control animals received bicarbonate solution 
only. SYNB1010 was well tolerated; no study-compound-related mortality occurred and no test 
article-related effects were identified on clinical observations, body weight, or clinical pathology 
assessments including hematology, coagulation, and clinical chemistry. In the SYNB1010-dosed 
animals, no bacteria were detected in the feces after Day 7 post-dosing. 
2.2.1.3.2. 28-day Good Laboratory Practice Mouse Toxicology Study 
To evaluate the potential toxicity of SYNB1020, a 28-day, GLP toxicology study was conducted 
in male and female CD-1 mice. The study evaluated twice daily (BID) oral gavage (PO) dose 
administration of SYNB1020 for 4 weeks followed by a 2-week recovery period. The maximum 
dose was 1.56 × 1011 CFU/day, which is the highest feasible dose in this species and corresponds 
to a human dose of 2.3 × 1014 based on the human and mouse GI volume ratio of 1100.19 There 
was no test-article-related mortality, no clinical observations, and no pathological findings on 
necropsy. Low levels of SYNB1020 DNA were detectable in stomach ileum and colon 24 hours 
following the last dose but were not detected outside the GI tract (spleen, liver, bladder, gonads) 
in animals correctly dosed by oral gavage. 
2.2.1.4. 
Summary of the Nonclinical Development Program 
In summary, SYNB1020/1010 have been evaluated in a preclinical efficacy and safety program 
that includes in vitro characterization that demonstrates increased ammonia consumption and 
arginine production as well as the growth-limiting effect of thymidine auxotrophy. SYNB1020 
does not contain any antibiotic resistance genetic markers and has demonstrated susceptibility to 
a wide range of antibiotics. In vivo, kinetics in cynomolgus monkeys demonstrated that the strain 
does not colonize and is cleared from the feces within a week following discontinuation of 
dosing. In the nonclinical safety program, SYNB1020 was evaluated in a GLP toxicology study 
in mice and a closely related strain, SYNB1010, was evaluated in a non-GLP toxicology study in 
NHPs. There were no toxicological findings after 28 days of dosing at dose levels of up to 1011 
CFU in mice and 1012 CFU in the NHP. 
2.2.2. 
Clinical Studies 
Study SYNB1020-CP-002 represents the second clinical investigation of SYNB1020. One study 
of SYNB1020 is ongoing in healthy adult male and female volunteers (see Section 2.2.2.1). In 
addition, a large body of evidence is available for the clinical safety and efficacy of E. coli 
Nissle, the parent strain of SYNB1020 (see the SYNB1020 Investigator’s Brochure). 
2.2.2.1. 
Study SYNB1020-CP-001 
Study SYNB1020-CP-001 is an ongoing Phase 1, dose-escalating, randomized, double-blinded 
study designed to determine the maximum tolerated dose (MTD) of single and multiple doses of 


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SYNB1020 in placebo-controlled cohorts of male and female healthy adult subjects. As of 
30 October 2017, 52 subjects (40 male, 12 female) aged between 21 and 64 years were enrolled 
in Study SYNB1020-CP-001 and received at least 1 dose of SYNB1020 or matching placebo 
(Table 1).  
Table 1: 
Study Enrollment in Study SYNB1020-CP-001 
Regimen 
SYNB1020 Dose 
No. Subjects Enrolled 
(SYNB1020/Placebo) 
Single Day 
Single dose of 2 × 109 CFU 
3/1 
Single Day 
Single dose of 2 × 1010 CFU 
3/1 
Single Day 
Single dose of 2 × 1011 CFU 
3/1 
Single Day 
Single dose of 2 × 1012 CFU 
3/1 
Single Day 
Multiple dose of 2 × 1012 CFU TID for 1 day 
3/1 
Single Day 
Multiple dose of 5 × 1011 CFU TID for 1 day 
3/1 
Single Day 
Multiple dose of 1 × 1012 CFU TID for 1 day 
3/1 
Multiple Day 
Multiple dose of 2 × 109 CFU TID for 14 days 
6/2 
Multiple Day 
Multiple dose of 2 × 1011 CFU TID for 14 days 
6/2 
Multiple Day 
Multiple dose of 5 × 1011 CFU TID for 14 days 
6/2 
Abbreviations: CFU = colony-forming unit(s); TID = ter in die (3 times daily) 
Note: These preliminary data are based on a cutoff date of 30 October 2017 and may contain unresolved queries. 
At the time of data cutoff, subjects in all cohorts had completed at least 72 hours of follow-up 
after the last dose of SYNB1020/placebo. Upon discharge from the inpatient unit, all subjects 
continued follow-up as an outpatient until 2 consecutive fecal samples were negative for 
SYNB1020 by polymerase chain reaction (PCR) analysis. At the time of cutoff, 8 subjects were 
continuing to be followed, and 44 subjects had completed the study. All subjects who completed 
study participation had fecal clearance within approximately 4 weeks following the last dose, and 
no subjects required treatment with antibiotics for positive fecal PCR. Four subjects discontinued 
the study prematurely due to mild to moderate treatment-emergent adverse events (TEAEs). 
Adverse Events 
As of 30 October 2017, there were no SAEs or deaths in the study. The most frequently reported 
TEAEs observed to date in Study SYNB1020-CP-001 were mild to moderate GI disorders, 
including nausea and vomiting. SYNB1020 was well tolerated in the single ascending dose 
(SAD) cohorts at single doses up to 5 × 1011 CFU, which was determined to be the MTD. Six 
subjects (21.4%) experienced TEAEs in the SAD part of the study. Three subjects in the SAD 
part discontinued dosing due to TEAEs (moderate nausea and vomiting), including 2 subjects in 
the 2 × 1012 CFU three times daily (TID) cohort and 1 subject in the 1 × 1012 CFU TID cohort. 
Doses up to 5 × 1011 CFU TID were well tolerated for up to 14 days in the MAD cohorts. Seven 
subjects (29.2%) experienced TEAEs in the MAD cohorts. One subject in the 5 × 1011 CFU TID 
cohort discontinued dosing due to TEAEs (mild nausea and vomiting). All TEAEs leading to 
discontinuation in both the SAD and MAD cohorts occurred during the first day of dosing. 
Further safety data are presented in the Investigator’s Brochure.  


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2.3. 
Rationale for the Study 
Study SYNB1020-CP-002 represents the second clinical investigation of SYNB1020. Part 1 of 
the study comprises a sentinel cohort designed to establish the safety and tolerability of 
SYNB1020 in subjects with cirrhosis. Part 2 of the study is designed to evaluate the safety, 
tolerability, and pharmacodynamics (PD) of SYNB1020 compared with placebo in patients with 
hyperammonemia resulting from hepatic insufficiency. The dose of SYNB1020 to be 
administered in this study will be up to 5 × 1011 CFU given TID immediately after meals for 
6 days. The dose level is based on the MTD determined in the Phase 1 single and multiple-
ascending dose study (Study SYNB1020-CP-001) in healthy adult volunteers (see 
Section 2.2.2.1). The duration of dosing (6 days) is based on the estimate that unmodified EcN 
requires 2 to 4 days to reach steady state. 
Because SYNB1020 is a live biotherapeutic and is therefore susceptible to degradation by 
stomach acid, subjects enrolled in the study will receive a concomitant histamine-2 receptor (H2) 
inhibitor. Subjects will receive oral ranitidine (150 mg BID) beginning on Day -5 and for the 
duration of investigational product (IP) dosing (i.e., through Day 6). Ranitidine was selected for 
use as it is a widely used over-the-counter therapeutic with a well characterized safety profile 
and has demonstrated efficacy in increasing gastric pH. Subjects already on a proton pump 
inhibitor (PPI) will not be switched to ranitidine but will continue their prescribed PPI regimen 
for the duration of the study. 


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3. 
STUDY OBJECTIVES AND ENDPOINTS 
3.1. 
Primary 
The primary objective is to evaluate the safety and tolerability of SYNB1020 following multiple 
doses in hepatic insufficiency and cirrhosis patients. The endpoints related to this objective 
include the nature and frequency of adverse events (AEs), measurement of vital signs and 
laboratory assessments (e.g., blood chemistry, complete blood count [CBC], liver function tests, 
international normalized ratio [INR], and urinalysis), and electrocardiograms (ECGs). 
3.2. 
Secondary 
Secondary objectives include evaluation of the following: 
• SYNB1020 kinetics in feces (measured with qualitative and quantitative PCR fecal 
assays) following dosing 
• Change from baseline of venous ammonia, including fasting and 24-hour area under 
the curve (AUC), following SYNB1020 dosing in subjects with hyperammonemia 
3.3. 
Exploratory 
Exploratory objectives include evaluation of the following: 
• Placebo-adjusted change from baseline in pharmacodynamics effects of SYNB1020 
on: 
− Serum inflammatory markers (serum tumor necrosis factor alpha (TNF-α), 
interleukin (IL)-6, and endotoxin) 
− Urinary urea/urinary nitrogen (24 hour) and calculated nitrogen balance 
− Plasma nitrate and urinary nitrate (24-hour) 
− Plasma amino acids including glutamine, arginine, aromatic amino acids, and 
branched chain amino acids 
− Psychometric test performance at baseline and following dosing as measured by 
Psychometric Hepatic Encephalopathy Score testing (i.e., number connection test 
A/B, digit symbol test, line tracing test, and serial dotting test; see Appendix 3) 


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4. 
STUDY DESIGN 
4.1. 
Study Design Overview 
This Phase 1b/2a, randomized, double-blinded placebo-controlled study will evaluate the safety, 
tolerability, and PD of SYNB1020 in hepatic insufficiency and cirrhosis patients, with dosing of 
the IP administered in an inpatient unit and subsequent outpatient follow-up for SYNB1020 
clearance. Part 1 comprises a sentinel open-label cohort of 6 subjects with cirrhosis and Model 
for End-Stage Liver Disease (MELD) score <12 to establish the safety and tolerability of 
SYNB1020. Once the safety and tolerability have been established in Part 1, enrollment will be 
opened to subjects in Part 2, a randomized, double-blinded, placebo-controlled study in subjects 
with cirrhosis and hyperammonemia. Approximately 24 to 40 evaluable subjects are anticipated 
for enrollment in Part 2, with randomization proceeding in a 1:1 ratio to SYNB1020 and placebo. 
In Part 1, eligible subjects will be admitted to an inpatient facility for a run-in diet, baseline 
assessments, IP administration, safety monitoring, and collection of blood, urine, and fecal 
samples for evaluation of safety, tolerability, and pharmacokinetic and PD evaluations. 
In Part 2, subjects may be pre-screened for eligibility based on medical history and a single 
fasting spot venous ammonia measurement. Eligible subjects with elevated fasting spot venous 
ammonia will then undergo full screening within 7 days of pre-screening. Eligible subjects will 
be admitted to an inpatient facility for a run-in diet and 24-hour ammonia profile, and those with 
an elevated 24-hour ammonia AUC (>1.2 × ULN) will proceed with randomization, IP 
administration, safety monitoring, and collection of blood, urine, and fecal samples for 
pharmacokinetic and PD evaluations.  
4.2. 
Study Dosing Regimens 
4.2.1. 
Dose selection 
Dosing is based on data from Study SYNB1020-CP-001, a Phase 1, dose-escalating, randomized, 
double-blinded study designed to determine the MTD of single and multiple doses of SYNB1020 
in placebo-controlled cohorts of male and female healthy adult subjects. The dose administered 
in this study will be at or below the MTD of 5 × 1011 CFU TID determined in Study SYNB1020-
CP-001. The duration of dosing (6 days) was selected to be greater than the time to steady-state 
for unmodified EcN of 2 to 4 days. Preliminary data for the study are presented in 
Section 2.2.2.1. 
4.2.2. 
Dietary Regimen and Study Supplements 
All subjects will be placed on a weighed research diet calculated based on overall body weight 
(kg), age, and gender to meet each subject’s caloric and protein (2 g/kg/day) requirements based 
on the dietary reference intakes (DRI).20 This diet will be based on subject preferences as 
assessed during screening. Diets will include equal caloric and protein content on each day of 
inpatient monitoring. Subjects will be expected to consume the entirety of their meals and not 
consume any external foods for the duration of their inpatient study participation. Specifics 
regarding the diet can be found in the Diet Manual. 


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In addition, subjects will be administered an H2 inhibitor to reduce stomach acid. Oral ranitidine 
(150 mg) will be administered BID, 30 to 60 minutes before breakfast and in the evening, for the 
duration of the inpatient stay (Table 3). Subjects already on a PPI will not be switched to 
ranitidine but will continue their prescribed PPI regimen for the duration of the study. 
4.2.3. 
Inpatient Monitoring and Investigational Product Administration 
Each subject will be admitted to the inpatient unit after an overnight fast on the morning of 
Day -5. Beginning on Day -5, subjects will initiate a controlled diet and ranitidine. Days -5 
through -1 will be a run-in phase to allow for dietary stabilization on the controlled diet. On 
Day -2, all subjects will undergo baseline 24-hour ammonia profile with 8 measurements (AUC), 
baseline clinical laboratory sampling and other safety measurements, FibroScan® measurement, 
and baseline HE grading and psychometric function testing. In Part 1, all subjects will receive  
up to 5 × 1011 CFU of SYNB1020 administered TID immediately after meals from Days 1 
through 6 (including mid-day and evening doses on Day 6). Fasting ammonia level will be 
measured daily. On Day 5, subjects will undergo ammonia AUC assessment. On Day 6, subjects 
will undergo ammonia AUC assessment, repeat HE grading and psychometric function testing, 
as well as 24-hour urine and blood collection. Subjects will be monitored in the inpatient setting 
for at least 12 hours following completion of dosing, with discharge on Day 7 for clinically 
stable subjects. Stool samples will be collected daily during the inpatient period. Subjects will be 
followed for safety and SYNB1020 fecal clearance as outlined in Section 7.5. This cohort is 
open-label. 
In Part 2, only subjects whose screening 24-hour AUC venous ammonia measurement is above 
1.2 × the upper limit of normal (ULN) will undergo computer-generated randomization on 
Day -1 in a 1:1 ratio to receive either up to 5 × 1011 CFU of SYNB1020 or matching placebo 
administered TID immediately after meals for 6 days. Other assessments will be the same as Part 
1. Both subjects and investigators will be blinded to randomized IP assignment. 
4.3. 
Stopping Rules 
The Sponsor may stop this study at any time. Investigators will be notified by the Sponsor or its 
designee if the study is stopped. The occurrence of the following events will require that further 
enrollment in the study be stopped: 
• Any suspected or proven invasive infection (e.g., sepsis or bacteremia) assessed as at 
least possibly related to the IP. 
• Three or more subjects experience the same National Cancer Institute (NCI) Common 
Terminology Criteria for Adverse Events (CTCAE) grade 3 AE assessed as at least 
possibly related to study treatment, or two or more subjects experience the same 
CTCAE grade 4 AE assessed as at least possibly related to the IP. 
• Two or more subjects experience SAEs that are considered by the Investigator to be 
related to the IP. 
• Death occurs at any time during the study and is considered by the Investigator to be 
related to the IP. 


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• Approximately 6% mortality is observed after 30 days following dosing. This is 
defined as ≥2 deaths observed in 24 subjects or ≥3 deaths observed in 40 subjects 
who have received the IP. 
• The Investigator, Medical Monitor, or Sponsor determines that an event or current 
data warrant stopping the study. 
The Sponsor’s medical staff, the Contract Research Organization (CRO)’s pharmacovigilance 
physician and the Lead Investigator will review the data concerning these event(s), along with 
other available data. Based on the results of their investigation, the Sponsor’s medical staff, the 
CRO’s pharmacovigilance physician and the Lead Investigator will determine appropriate 
follow-up and decide whether the study should be stopped. 
If the study is stopped, the events will be investigated, and the study will be closed to further 
enrollment. Subjects already participating in the study at the time the study is stopped will not 
receive further IP and will continue the follow-up schedule. 
4.4. 
Safety Review Committee 
A Safety Review Committee comprising the Lead Investigator, Medical Monitor, 
pharmacovigilance physician, and an external disease expert, as needed, will meet after the 
completion of Part 1 and ad hoc, as needed, to assess the safety and tolerability of SYNB1020 in 
subjects with cirrhosis, and to determine whether Part 2 can be conducted safely. The Part 1 
safety evaluation will include any available data on AEs, laboratory measurements, vital signs, 
and ECGs up to 7 days after the last dose of SYNB1020. In Part 2, the Safety Review Committee 
will meet at predetermined intervals during the study and ad hoc, as needed, to conduct periodic 
reviews of interim safety data (see Section 9.7). Further details will be provided in a separate 
Safety Review Committee charter. 
4.5. 
Duration of Study Participation 
The maximum time of study participation for a subject is approximately 19 weeks (133 days). 
4.5.1. 
Pre-screening Period in Part 2 
The pre-screening period for subjects in Part 2 will be up to 7 days prior to initiation of full 
screening. 
4.5.2. 
Screening Period 
The screening period for all subjects will be up to 45 days prior to admission to the inpatient 
facility (i.e., Days -51 to -6). 
4.5.3. 
Study Dosing 
In Part 1, subjects will be dosed with SYNB1020 TID for 6 days. The total duration of inpatient 
study participation will be 12 days (Days -5 through 7). 
In Part 2, subjects will be dosed with SYNB1020 or placebo TID for 6 days. The total duration 
of inpatient study participation will be 12 days (Days -5 through 7). 


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4.5.4. 
Post-dose Follow-up 
One week (7 ± 1 days) after discharge from the inpatient unit, subjects will return for clinical 
evaluation and bring a 1-week post-dose fecal sample. Feces will continue to be collected weekly 
for the first 6 weeks following discharge and biweekly until a subject has a negative SYNB1020 
fecal test for up to 10 weeks following completion of study dosing. Subjects who remain 
colonized with SYNB1020 after 10 weeks following the last dose will be treated with a 3-day 
course of oral ciprofloxacin (or other antibiotic for subjects allergic to ciprofloxacin), undergo a 
SYNB1020 fecal test 3 to 5 days following completion of antibiotic treatment, and be followed 
for AEs at least 7 days after completion of antibiotic treatment (see Section 7.5.2 for further 
details). 


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5. 
PATIENT POPULATION 
5.1. 
Number of Subjects 
Part 1: 6 evaluable subjects are anticipated for enrollment. 
Part 2: Approximately 24 to 40 evaluable subjects, defined as subjects who complete all 
3 ammonia 24-hour AUC intervals, are anticipated for enrollment. 
5.2. 
Selection of Subjects 
5.2.1. 
Eligibility Criteria 
This study will comprise adult patients with hepatic insufficiency and cirrhosis. Patients will be 
eligible for enrollment regardless of gender or race/ethnicity. 
5.2.1.1. 
Inclusion Criteria in Part 1 
Patients must meet all of the following inclusion criteria to be eligible for Part 1: 
1. Male and female patients aged ≥ 18 to < 75 years. 
2. Diagnosis of chronic (≥ 6 months), stable (no acute episodes of illness within the 
previous 2 months due to deterioration in hepatic function) hepatic insufficiency with 
features of cirrhosis due to any etiology. 
3. MELD score <12 (see Appendix 5). 
4. Able to produce at least 2 bowel movements per week on average without the assistance 
of laxatives. 
5. Able and willing to voluntarily complete the informed consent process (patient or 
patient’s representative). 
6. Available for and agree to all study procedures, including inpatient monitoring and 
frequent blood sampling. 
7. Male subjects who agree to use an acceptable method of contraception after informed 
consent, throughout the study, and for 90 days after the last dose of SYNB1020 or 
placebo. 
8. Female subjects of non-childbearing potential: Either surgically sterile (defined as 
hysterectomy, bilateral oophorectomy, or tubal ligation/occlusion) or postmenopausal 
(defined as 12 months with no menses without an alternative medical cause and 
confirmed by follicle-stimulating hormone [FSH] in the range of menopause) confirmed 
at screening via medical history. 
9. Screening laboratory evaluations within defined acceptable limits (Table 2), or judged to 
be not clinically significant by the investigator. 


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5.2.1.2. 
Exclusion Criteria in Part 1 
Patients meeting any of the following criteria are not eligible for Part 1 study enrollment: 
1. Current or past HE of Grade 2 or higher using the West Haven Grading System (see 
Appendix 2) requiring hospitalization. 
2. Child-Turcotte-Pugh score of > 9 (see Appendix 4). 
3. History of liver transplant. 
4. History of hepatocellular carcinoma; patients must have negative imaging result within 
12 months prior to screening. (Exception: hepatocellular carcinoma that has been 
considered cured or in remission > 6 months prior to screening.) 
5. Poorly controlled diabetes, or diabetes requiring insulin treatment. 
6. Chronic obstructive pulmonary disease requiring the use of supplemental oxygen. 
7. Visual or motor deficits leading to interference on psychometric testing. 
8. Body mass index < 18.5 or ≥ 40 kg/m2. 
9. History of active or chronic passage of 3 or more loose stools per day within 4 weeks 
prior to screening. 
10. Active inflammatory or irritable bowel disorder of any grade. 
11. Active or prior history of GI bleeding within 8 weeks prior to screening, as confirmed by 
hospitalization-related event(s) or medical history of hematemesis or hematochezia. 
12. Prior transjugular intrahepatic portosystemic shunt placement. 
13. Prolonged QT interval corrected for heart rate (QTc) on screening ECG (defined as QTc 
> 500 ms) 
14. Renal insufficiency (defined as serum creatinine >1.5 mg/dL). 
15. Apart from chronic liver disease, any acute or chronic medical, surgical, psychiatric, or 
social condition including history of cerebrovascular disease (stroke, transient ischemic 
attack) or dementia, or laboratory abnormality that may increase the patient risk 
associated with study participation, compromise adherence to study procedures and 
requirements, confound interpretation of the safety, kinetics, or pharmacodynamics 
results, and, in the judgment of the investigator, make the patient inappropriate for 
enrollment. 
16. Current or anticipated treatment with systemic (e.g., oral or intravenous) antibiotic 
(including rifaximin) within 4 weeks prior to screening through the final outpatient 
follow-up for any reason (e.g., infection or planned surgery, hospitalization, dental, or 
interventional studies) (Exception: topical antibiotics are allowed). 
17. Current use of valproate, corticosteroids, or cytotoxic drugs. 
18. Current or anticipated treatment for viral hepatitis within 3 months prior to screening 
through the final outpatient follow-up. 
19. Current or prior use of laxatives (including lactulose) within 4 weeks prior to screening. 


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20. Current or prior use of probiotic supplements (excluding enriched foods such as yogurt) 
within 4 weeks prior to screening. 
21. Any condition, prescription medication, or over-the-counter product that may possibly 
affect absorption of medications or nutrients (e.g., celiac disease, gastrectomy, bypass 
surgery, ileostomy). 
22. Alcohol intake of more than two units per week within 1 month prior to screening. 
23. Dependence on drugs of abuse, use of any opiods (except stable regimen of prescription 
drugs). 
24. Administration or ingestion of an investigational drug within 8 weeks or 5 half-lives, 
whichever is longer, prior to screening or current enrollment in an investigational study. 
25. History of allergy to ranitidine, or intolerance to any of the excipients (glycerol, CVS 
Health Easy Fiber). 
5.2.1.3. 
Inclusion Criteria in Part 2 
Patients must meet all of the following inclusion criteria to be eligible for Part 2: 
1. Male and female patients aged ≥ 18 to < 75 years. 
2. Diagnosis of chronic (≥ 6 months), stable (no acute episodes of illness within the 
previous 2 months due to deterioration in hepatic function) hepatic insufficiency with 
features of cirrhosis due to any etiology. 
3. Fasting venous ammonia > ULN as defined by the site laboratory required for admission; 
ammonia AUC >1.2 × ULN required for randomization. 
4. Able and willing to voluntarily complete the informed consent process (patient or 
patient’s representative). 
5. Available for and agree to all study procedures, including inpatient monitoring and 
frequent blood sampling. 
6. Male subjects who agree to use an acceptable method of contraception after informed 
consent, throughout the study, and for 90 days after the last dose of SYNB1020 or placebo. 
7. Female subjects of non-childbearing potential: Either surgically sterile (defined as 
hysterectomy, bilateral oophorectomy, or tubal ligation/occlusion) or postmenopausal 
(defined as 12 months with no menses without an alternative medical cause and 
confirmed by FSH in the range of menopause) confirmed at screening via medical 
history. 
8. Screening laboratory evaluations within defined acceptable limits (Table 2), or judged to 
be not clinically significant by the investigator. 
5.2.1.4. 
Exclusion Criteria in Part 2  
Patients meeting any of the following criteria are not eligible for Part 2 study enrollment: 
1. Current or past HE of Grade 2 or higher using the West Haven Grading System (see 
Appendix 2) requiring hospitalization. 


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2. Child-Turcotte-Pugh score of > 9 (see Appendix 4). 
3. MELD score >20 (see Appendix 5). 
4. History of liver transplant. 
5. History of hepatocellular carcinoma; patients must have negative imaging result within 
12 months prior to screening. (Exception: hepatocellular carcinoma that has been 
considered cured or in remission > 6 months prior to screening.) Imaging may be 
obtained during the screening period. 
6. Poorly controlled diabetes or significant changes to diabetes medications within 6 weeks 
prior to Day -5. 
7. Chronic obstructive pulmonary disease requiring the use of supplemental oxygen. 
8. Body mass index < 18.5 or ≥ 40 kg/m2. 
9. History of active or chronic passage of 3 or more loose stools per day within 4 weeks 
prior to screening. 
10. Active inflammatory or irritable bowel disorder of any grade. 
11. Active or prior history of GI bleeding within 8 weeks prior to screening, as confirmed by 
hospitalization-related event(s) or medical history of hematemesis or hematochezia. 
12. Prior transjugular intrahepatic portosystemic shunt placement. 
13. Prolonged QTc on screening ECG (defined as QTc > 500 ms). 
14. Renal insufficiency (defined as serum creatinine >1.5 mg/dL). 
15. Apart from chronic liver disease, any acute or chronic medical, surgical, psychiatric, or 
social condition including history of cerebrovascular disease (stroke, transient ischemic 
attack) or dementia, or laboratory abnormality that may increase the patient risk 
associated with study participation, compromise adherence to study procedures and 
requirements, confound interpretation of the safety, kinetics, or pharmacodynamics 
results, and, in the judgment of the investigator, make the patient inappropriate for 
enrollment. 
16. Current or anticipated treatment with systemic (e.g., oral or intravenous) antibiotic 
(including rifaximin) within 4 weeks prior to Day -5 through the final outpatient 
follow-up for any reason (e.g., infection or planned surgery, hospitalization, dental, or 
interventional studies) (Exception: topical antibiotics are allowed). 
17. Current use of valproate, corticosteroids, or cytotoxic drugs. 
18. Current or anticipated treatment for viral hepatitis within 3 months prior to Day -5 
through the final outpatient follow-up. 
19. Current or prior use of lactulose within 2 weeks prior to Day -5. 
20. Current or prior use of probiotic supplements (excluding enriched foods such as yogurt) 
within 4 weeks prior to Day -5. 


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21. Any condition, prescription medication, or over-the-counter product that may possibly 
affect absorption of medications or nutrients (e.g., celiac disease, gastrectomy, bypass 
surgery, ileostomy). 
22. Alcohol intake of more than two units per week within 1 month prior to screening. 
23. Dependence on drugs of abuse, use of any opiods (except stable regimen of prescription 
drugs). 
24. Administration or ingestion of an investigational drug within 8 weeks or 5 half-lives, 
whichever is longer, prior to screening or current enrollment in an investigational study. 
25. History of allergy to ranitidine, or intolerance to any of the excipients (glycerol, CVS 
Health Easy Fiber). 
Table 2: 
Laboratory Exclusion Criteria 
Laboratory measure 
Exclusion criterion 
White blood cell count 
< 1×109/L 
Hemoglobin 
< 8.5 g/dL 
Platelet count 
< 35,000 /µL 
aPTT 
> 1.5 × ULN 
PT  
> 4.0 sec prolongation from ULN  
INR 
> 1.7 
Serum creatinine 
>1.5 mg/dL 
Total bilirubin 
> 50 µmol/L or > 3 mg/dL, unless diagnosed with 
Gilbert’s syndrome   
Serum Albumin 
< 2.8 g/L 
AST and/or ALT 
> 3 × ULN  
P-sodium 
< 132 or > 148 mmol/L 
P-potassium 
< 3.2 or > 5.3 mmol/L  
QTc 
> 500 ms  
Abbreviations: aPTT = activated partial thromboplastin time; ALT = alanine aminotransferase; AST = aspartate 
aminotransferase; INR = international normalized ratio; PT = prothrombin time; QTc = QT interval corrected for 
heart rate; ULN = upper limit of normal 


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6. 
INVESTIGATIONAL MEDICAL PRODUCT AND 
CONCOMITANT MEDICATIONS 
6.1. 
Investigational Product Supply and Administration 
Within this protocol, IP refers to SYNB1020 and/or placebo. 
6.1.1. 
SYNB1020 
6.1.1.1. 
Manufacturing and Formulation 
SYNB1020 investigational drug product is formulated in 15% glycerol/5% trehalose/10 mM 
3-(N-morpholino) propanesulfonic acid (MOPS), pH 7.0. The formulated SYNB1020 drug 
product will be further diluted in a taste-masking solution for PO. 
6.1.1.2. 
Packaging and Labeling 
SYNB1020 is packaged as a frozen liquid suspension in 5-mL cryogenic vials (5-mL/vial). The 
following is a sample vial label (final study label may vary):  
Investigational Protocol Number: SYNB1020-CP-002 
Lot Number: XXXX 
Description: SYNB1020 at 1011 CFU/mL 
Dosing Instructions: Oral dose 
Retest Date: Month Year 
Store at < -65˚C 
“Caution: New Drug – Limited by Federal (or United States) law to investigational use.” 
SYNLOGIC INC 
Manufactured by: Paragon Bioservices 
801 West Baltimore Street, Suite 401 
Baltimore, MD 21201 
Contact Information: XXX-XXX-XXXX 
6.1.1.3. 
Shipping, Storage, and Handling 
Labeled, packaged SYNB1020 will be shipped to the study center(s) by the CRO (Parexel). 
SYNB1020 should be stored at < -65°C. 
Detailed instructions for the storage, handling, and administration of IP will be provided in the 
Pharmacy Manual and may override instructions provided in the following protocol sections. 
6.1.1.4. 
Dispensing and Administration 
The manufactured SYNB1020 drug product will have a pre-specified concentration. To obtain 
appropriate doses, vial(s) of concentrated drug product will be thawed and appropriately diluted 
in a chilled formulation buffer and masking solution.  


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6.1.2. 
Placebo 
Placebo will contain only 100 mL of a chilled masking solution.  
6.1.3. 
Ranitidine  
Ranitidine will be obtained from a commercial source and supplied to study sites as 150-mg 
tablets for oral administration. Ranitidine should be stored in accordance with the prescribing 
information (see Appendix 1). 
6.2. 
Accountability and Dosing Compliance 
All doses of IP will be administered in the clinic by trained study personnel during inpatient 
hospitalization. A temperature mechanism will be included with the IP to ensure the temperature 
has not been compromised. 
6.3. 
Concomitant Medications 
Concomitant medications can be administered at the Investigator’s discretion to conform to 
standard practice during the dosing period. Overall, SYNB1020 has a low likelihood for eliciting 
clinically relevant drug-drug interactions; however, Investigators should use caution when 
prescribing concomitant-medications, as this compound has not been extensively investigated in 
the clinical setting. Investigators should contact the Medical Monitor when they are unsure as to 
whether a drug should be prescribed to a subject. All concomitant medications and dietary 
supplements will be recorded in the electronic case report forms (eCRFs) using generic drug 
names when possible. 
6.3.1. 
Prohibited Therapies and Substances 
Therapies and substances that are prohibited at screening and during study participation are 
outlined in the study eligibility criteria (Section 5.2.1). 


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7. 
STUDY PROCEDURES AND ASSESSMENTS 
Before recruitment of subjects into the study, written Institutional Review Board (IRB) approval 
of the protocol, informed consent, and any additional patient information must be obtained. 
The various assessments that will be conducted during this study are summarized in Table 3 
(Schedule of Events). 
Detailed instructions regarding sample collection and handling, as well as all laboratory 
procedures, will be provided to the study site in separate study manuals. Site-specific handling 
instructions for hematology and clinical chemistry samples should be followed. 
7.1. 
Screening 
Subjects who fail screening due to a transient cause or missing information are eligible for 
re-screening. 
7.1.1. 
Part 1 
Screening procedures must be performed to determine eligibility for enrollment in Part 1 within 
45 days prior to admission to the inpatient facility. If a particular assessment is repeated, the 
results obtained closest to the first dose of IP should be used to assess eligibility. 
During screening, a unique number will be assigned to each subject who signs informed consent 
for the study. Once a subject is enrolled in the study, s/he will only be identified by the assigned 
study number. 
At the site, the Investigator will maintain a log for all screened subjects (including subjects who 
failed screening after signing informed consent) and all enrolled subjects. 
7.1.2. 
Part 2 
7.1.2.1. 
Pre-screening  
Subjects may be pre-screened for eligibility in Part 2 based on medical history and a single spot 
fasting venous ammonia measurement. Subjects passing the pre-screen will undergo full 
evaluation and screening procedures within 7 days. 
7.1.2.2. 
Screening  
Screening procedures must be performed to determine eligibility for enrollment in Part 2 within 
45 days prior to admission to the inpatient facility. If a particular assessment is repeated, the 
results obtained closest to the first dose of IP should be used to assess eligibility, with the 
exception of fasting venous ammonia, which will not be repeated if measured at pre-screening. 
During screening, a unique number will be assigned to each subject who signs informed consent 
for the study. If a subject meets all the inclusion criteria and none of the exclusion criteria, 
including ammonia AUC after the run-in period, and is selected to participate in the study, s/he 
will receive a randomization number. Once a subject is enrolled in the study, s/he will only be 
identified by the assigned randomization number. 


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The Investigator or designee is responsible for verifying that the subject is eligible before 
requesting a randomization number. At the site, the Investigator will maintain a log for all 
screened subjects (including subjects who failed screening after signing informed consent) and 
all enrolled subjects. 
Subjects must be dosed within 3 calendar days of randomization. 
7.2. 
Safety Assessments 
7.2.1. 
Adverse Events 
Adverse events will be assessed continuously by direct observation and subject interviews. The 
severity of AEs will be evaluated using the NCI CTCAE, version 4.03.21 All AEs occurring from 
the time a subject signs informed consent through the safety follow-up period will be recorded, 
regardless of causal assessment to SYNB1020/placebo. After the end of the follow-up period, 
only AEs that are considered related to IP will be recorded, if clinically indicated. 
7.2.2. 
Medical History 
Past and present medical history will be recorded. Any ongoing condition and signs and 
symptoms observed prior to the initiation of study dosing, including bowel habits, should be 
recorded as medical history. 
7.2.3. 
Vital Signs 
Semi-supine vital signs (systolic blood pressure, diastolic blood pressure, pulse, and body 
temperature) will be collected at screening and at pre-specified pre- and post-dose time points 
during inpatient monitoring (see Table 3). Subjects are required to remain in the semi-supine 
position for at least 5 minutes prior to obtaining vital signs. 
7.2.4. 
Physical Examination 
Complete physical examinations will be performed by trained medical personnel during 
screening and baseline (Day -5), with symptom-directed physical examinations performed at 
subsequent time points (see Table 3). Any abnormal findings will be recorded as AEs. 
7.2.5. 
FibroScan 
A FibroScan (vibration-controlled transient elastrography) will be performed at baseline to 
quantify liver fibrosis. 
7.2.6. 
Clinical Laboratory Measurements 
The following clinical safety laboratory tests will be performed at the time points specified in 
Table 3: CBC with differential, prothrombin time (PT)/activated partial thromboplastin time 
(aPTT), serum chemistry including liver function tests (LFTs), and urinalysis. These tests will 
include the parameters necessary for calculation of the MELD score (i.e., serum bilirubin, serum 
creatinine, and INR; see Appendix 5). Screening for infectious diseases and drugs of abuse will 
also be performed prior to study dosing. At screening, if non-childbearing potential status is 
equivocal based on medical history, FSH will be assessed in selected females to confirm post-
menopausal status. 


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Screening results will be assessed by the Investigator for inclusion of subjects in the study. 
Additionally, unscheduled clinical laboratory tests can be obtained at any time during the study 
at the Investigator’s discretion. The diagnosis corresponding to any clinically significant 
abnormality, or abnormality requiring treatment/intervention, must be recorded as an AE. 
7.2.7. 
Venous Ammonia 
Ammonia samples will be analyzed locally. Samples for plasma ammonia will be obtained from 
a free-flowing venous blood sample. Plasma should be separated immediately after collection, 
and analysis must be performed within 60 minutes. Refer to the Laboratory Manual for further 
details.  
The normal reference range of ammonia at each site will be defined based on approximately 
30 healthy volunteers devoid of any chronic or acute medical conditions and medications with 
normal liver enzymes. Ammonia sampling, sample processing and analysis will be performed as 
defined in this protocol and the coordinating laboratory manual. The 95th percentile will be used 
as the ULN for each site. 
7.2.8. 
Electrocardiograms 
A semi-supine single 12-lead ECG will be performed at screening, baseline, on Day 4, and at 
discharge. Electrocardiogram parameters to be evaluated include the RR, QT, QRS, and PR 
intervals. In addition, Fridericia’s formula should be used to calculate the QT interval corrected 
for heart rate (QTcF). 
7.2.9. 
Blood and Urine Cultures/Polymerase Chain Reaction 
Blood and urine cultures and/or PCR will be performed as needed according to standard clinical 
practice if clinical presentation warrants. This may include, but is not limited to, cases in which 
subjects develop persistent (> 24 hours) fever, chills, night sweats, or other symptoms suggestive 
of local or systemic clinical infection. SYNB1020 is a thymidine auxotroph and requires 
sufficient concentration of thymidine in the cell culture media. Refer to the Laboratory Manual 
for further details. 
7.2.10. 
Hepatic Encephalopathy Severity Grading 
The severity of HE on study will be evaluated at the pre- and postdose time points specified in 
Table 3 using the West Haven Grading System, which ranges from Grade 0 to Grade 4 
depending on the extent of a subject’s impairment of consciousness, intellectual function, and/or 
behavior (see Appendix 2). 
7.2.11. 
Psychometric Testing 
Psychometric testing will be performed at the pre- and postdose time points specified in Table 3 
as measured by Psychometric Hepatic Encephalopathy Score testing (i.e., number connection test 
A/B, serial dotting test, line tracing test, and digit symbol test; see Appendix 3). Full details on 
the timing, performance and scoring of psychometric testing will be provided in a separate 
study-specific manual. 


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7.2.12. 
Cirrhosis Severity Grading 
The severity of cirrhosis will be assessed using the Child-Turcotte-Pugh score, which reflects a 
composite score incorporating HE grade, extent of ascites, and bilirubin, albumin, and INR 
values (see Appendix 4) and MELD score calculated based on bilirubin, creatinine, and INR 
values (see Appendix 5). 
7.3. 
Microbiotic-Kinetic Assessments 
Feces from each voiding of the day will be collected at the pre- and postdose time points 
specified in Table 3 for analysis of SYNB1020 transit through the GI tract. In Part 1, the 24-hour 
fecal sample will be weighed, homogenized, and a sample analyzed for SYNB1020 by qPCR. In 
Part 2, a fecal sample from the first void of the day will be collected using a collection kit. For 
full details on sample collection and preparation for shipment, refer to the study-specific 
laboratory manual.  
7.4. 
Pharmacodynamic Assessments 
In addition to the analyses specified in the following sections, serum, plasma, urine, and feces 
will be collected and stored for exploratory analyses, which may include, but are not limited to, 
microbiome analyses, fecal arginine measurements, and an ex vivo fecal ammonia consumption 
studies. For full details on sample collection, refer to the study-specific laboratory manual. A 
single blood sample may be collected at pre-screening. Urine and blood samples will be 
collected at screening and at the pre- and postdose time points specified in Table 3. The 
following laboratory measurements will be performed to evaluate the preliminary 
pharmacodynamics of SYNB1020: 
• Plasma ammonia (morning fasting and 24-hour profile to calculate AUC) 
− Samples for venous ammonia will be drawn relative to meal times (breakfast, 
lunch, and dinner) according to the following schedule. Following an overnight 
fast, subjects will have a baseline sample drawn for ammonia at 0 hours (before 
breakfast) and will be given a high-protein shake for breakfast, followed by IP 
administration on dosing days. Subsequently, samples for venous ammonia will 
be drawn on: 
− 
Day -2: +2 hours, +4 hours (before lunch), +6 hours, +9 hours (before 
dinner), +11 hours, +14 hours, +18 hours, and +24 hours (before breakfast on 
Day -1). 
− 
Day 5: +2 hours, +3 hours, +4 hours (before lunch), +5 hours, +6 hours, 
+9 hours (before dinner), +11 hours, +14 hours, +18 hours, and +24 hours 
(before breakfast on Day 6). 
− 
Day 6: +2 hours, +4 hours (before lunch), +6 hours, +9 hours (before dinner), 
+11 hours, +14 hours, +18 hours, and +24 hours (before breakfast on Day 7). 
• Inflammatory markers (serum TNF-α, IL-6, endotoxin) 
• 24-hour urinary urea, urinary nitrogen, and nitrogen balance 
• Plasma nitrate and urinary nitrate (24 hour) 


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• Plasma amino acid profile (glutamine, arginine, aromatic amino acids and branched 
chain amino acids) 
• Bristol stool form scale (BSFS) for each bowel movement22  
• Psychometric test performance as measured by Psychometric Hepatic 
Encephalopathy Score testing (i.e., number connection test A/B, digit symbol test, 
line tracing test, and serial dotting test)  
7.5. 
Follow-up Assessments 
7.5.1. 
End-of-study/Discharge Visit 
At least 12 hours after completion of the dosing period (i.e., on Day 7 for subjects who continue 
IP dosing per protocol), an EOS/Discharge visit will be performed. For subjects who discontinue 
study dosing prematurely, the EOS/Discharge Visit should be completed within 24 hours of 
discontinuation, and every effort should be made to perform the procedures specified in 
(Table 3). 
7.5.2. 
Follow-up After Completion of Study Dosing 
One week (7 ± 1 days) after discharge from the inpatient unit, subjects will return for clinical 
evaluation and bring a 1-week post-dose fecal sample. Subjects will be provided diary cards for 
collection of AE data. Feces will continue to be collected weekly for the first 6 weeks following 
discharge and biweekly until a subject has a negative SYNB1020 fecal test for up to 10 weeks 
following completion of study dosing. Subjects who remain colonized with SYNB1020 after 
10 weeks following the last dose will be treated with a 3-day course of oral ciprofloxacin. 
Subjects who are allergic to ciprofloxacin can be treated with antibiotics based on reported 
sensitivity at the investigator’s discretion. Refer to the SYNB1020 Investigator’s Brochure for 
further details. A SYNB1020 fecal test will be performed 3 to 5 days following completion of 
antibiotic treatment solely for research purposes and will not guide subject follow-up decision 
making. An AE assessment will be performed by telephone at least 7 days after completion of 
antibiotic treatment. If no AEs are reported, subjects will not require any further follow-up and 
will be considered to have completed the study requirements. If AEs are reported, subjects will 
be advised to return to the inpatient facility for a comprehensive physical examination, 
laboratory assessment, and AE causality determination. 
7.6. 
Sample Retention for Exploratory Analyses 
Blood, urine, and fecal samples may be stored for future analyses related to metabolism and 
microbiome evaluations for up to 5 years. Any unused samples will be destroyed. 


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Table 3: 
Schedule of Events 
Study Period 
Optional 
Pre-
Screening 
Screening 
Inpatient Monitoring 
Follow-upa 
Study Day 
Within 7 
days of 
Screening 
-51 to -6 
-5 
-4 
-3 
-2 
-1 
1 
2-4 5 
6 
7 
EOS/Discharge 
Visitb 
14 
Follow-
up Visit 
Up to 70 
days after 
last dose 
Informed consent for pre-screening 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Informed consent 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history 
 
c 
 
 
 
 
 
 
 
 
 
 
 
 
Detection of elevated portal hypertension 
 
d 
 
 
 
 
 
 
 
 
 
 
 
 
Imaging to rule out hepatocellular carcinoma 
 
d 
 
 
 
 
 
 
 
 
 
 
 
 
Physical examination 
 
 
 
 
 
 
 
 
 
 
 
e 
e 
 
FibroScan 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Screening for drugs of abuse (urine drug screen) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Screening for infectious disease (HIV/Hepatitis B, C serology) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Laboratory tests (CBC, LFTs, blood chemistry, urinalysis, 
PT/aPTT) 
 
 
 
 
 
 
 
 
f 
 
 
 
 
 
FSH (in females if non-childbearing potential status is equivocal 
based on medical history) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Record concomitant medications 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Adverse event reporting 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Vital signsg 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Electrocardiogram 
 
 
 
 
 
 
 
 
f 
 
 
 
 
 
Fasting spot venous ammonia 
 
h 
 
 
 
 
 
 
 
 
 
 
 
 
Admit to inpatient facility (in the morning after an overnight fast) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Required inpatient stay 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Subject randomization (Part 2 only) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Dietary controli 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Study Period 
Optional 
Pre-
Screening 
Screening 
Inpatient Monitoring 
Follow-upa 
Study Day 
Within 7 
days of 
Screening 
-51 to -6 
-5 
-4 
-3 
-2 
-1 
1 
2-4 5 
6 
7 
EOS/Discharge 
Visitb 
14 
Follow-
up Visit 
Up to 70 
days after 
last dose 
Administer H2 inhibitor (ranitidine 150 mg BID 30-60 minutes 
before breakfast and the evening snack)j 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Administer assigned IP (SYNB1020 or placebo, TID) with 
breakfast, lunch, and dinnerk 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
24-hour profile for venous ammonial 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
24-hour urinary urea, urinary nitrogen and creatinine 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inflammatory markers (TNF-α, IL-6, endotoxin) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Plasma amino acid analyses 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Plasma nitratem 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
24-hour urinary nitratem 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Fecal SYNB1020 PCRn 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Psychometric testing 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Hepatic encephalopathy grading (West Haven grade) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Child-Turcotte-Pugh score 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Model for End-Stage Liver Disease score 
 
 
 
 
 
 
 
 
 f 
 
 
 
 
 
BSFS for each bowel movement collected 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Plasma collection for exploratory analyses 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine collection for exploratory analyses 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Review of adverse event diary card 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Abbreviations: aPTT = activated partial thromboplastin time; BID = twice daily; BSFS = Bristol stool form scale; CBC = complete blood count; EOS = End of Study; FSH = follicle-
stimulating hormone; h = hour(s); H2 = histamine-2 receptor; HIV = human immunodeficiency virus; IL = interleukin; IP = investigational product; LFT = liver function test; 
PCR = polymerase chain reaction; PPI = proton pump inhibitor; PT = prothrombin time; TID = 3 times daily; TNF-α = tumor necrosis factor alpha 
a One week (7± 1 days) after discharge from the inpatient unit, subjects will return for clinical evaluation and bring a 1-week post-dose fecal sample. Feces will continue to be 
collected weekly for the first 6 weeks following discharge and biweekly until a subject has a negative SYNB1020 fecal test for up to 10 weeks following completion of study dosing. 
Subjects who remain colonized with SYNB1020 after 10 weeks following the last dose will be treated as described in Section 7.5.2.  


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b At least 12 hours after completion of the dosing period (i.e., on Day 7 for subjects who continue IP dosing per protocol), an EOS/Discharge visit will be performed. For subjects 
who discontinue study dosing prematurely after having received at least one dose of SYNB1020/placebo, the EOS/Discharge Visit should be completed within 24 hours of 
discontinuation. 
c If not obtained during pre-screening. 
d Procedures to detect evidence of elevated portal hypertension (i.e., FibroScan to measure liver stiffness, endoscopy to determine the presence of abdominal or esophageal varices, 
imaging to detect splenomegaly, or ascites) and imaging to rule out hepatocellular carcinoma may be performed during screening if necessary to determine eligibility as outlined in 
Section 5.2.1.3. 
e Symptom-driven physical examinations will be performed on Days 7 and 14. 
f Day 4 only. 
g Vital signs will be collected every 8 hours during the dosing period (Days 1 through 6). 
h Fasting venous ammonia will not be repeated at screening if measured at pre-screening. 
i Meal times for breakfast, lunch, and dinner should be the same every day ± 30 minutes, unless otherwise specified. 
j Subjects already on a PPI regimen will not be switched to ranitidine but will continue their prescribed PPI regimen for the duration of the study. 
k In Part 1, all subjects receive open-label IP. In Part 2, subjects are randomized to either SYNB1020 or placebo. IP will be administered immediately following a meal. 
l Samples for venous ammonia will be drawn relative to meal times  according to Table 4. Following an overnight fast, subjects will have a baseline sample drawn for ammonia at 0h 
(before breakfast) and will be given a high-protein shake for breakfast, followed by IP administration on dosing days. Samples for venous ammonia will be drawn at +2h, +4h (before 
lunch), +6h, +9h (before dinner), +11h, +14h, +18h, and +24h the following morning (before breakfast). Two additional samples will be collected on Day 5 at +3h and +5h 
m Plasma nitrate samples are drawn at the same time as ammonia 24-hour samples on Day -2 and Day 6 at the following time points: 0 hours, +2 hours, +4 hours (before lunch), and 
+6 hours. For details of sample collection refer to the Laboratory Manual and Table 4. 
n One baseline fecal sample is required to be collected prior to SYNB1020 dosing (i.e., any time from Day -5 through Day 1). Feces will continue to be collected weekly for the first 
6 weeks following discharge and biweekly (every other week) until a subject has a negative SYNB1020 fecal test for up to 10 weeks following completion of study dosing. Subjects 
who remain colonized with SYNB1020 after 10 weeks following the last dose will be treated with a 3-day course of oral ciprofloxacin, or other antibiotics. On Day 7, if no void has 
occurred by the time all other assessments have been completed, the subject may be discharged without a fecal sample. 
 
 


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Table 4: 
Schedule of Plasma Sampling for 24-Hour Ammonia and Nitrate 
Study Day 
Activitya 
Time of Day 
Day -2 
0 hours 
+2 hours 
+3 hours 
+4 hours 
+5 hours 
+6 hours 
+9 hours 
+11 hours +14 hours +18 hours +24 hours 
Plasma draw for 24-hour ammonia 
 
 
 
 
 
 
 
 
 
 
 
Plasma draw for plasma nitrate 
 
 
 
 
 
 
 
 
 
 
 
Breakfast 
 
 
 
 
 
 
 
 
 
 
 
Lunch 
 
 
 
 
 
 
 
 
 
 
 
Dinner 
 
 
 
 
 
 
 
 
 
 
 
Day 5 
 
 
 
 
 
 
 
 
 
 
 
Plasma draw for 24-hour ammonia 
 
 
 
 
 
 
 
 
 
 
 
Breakfast 
 
 
 
 
 
 
 
 
 
 
 
Give IP 
 
 
 
 
 
 
 
 
 
 
 
Lunch 
 
 
 
 
 
 
 
 
 
 
 
Dinner 
 
 
 
 
 
 
 
 
 
 
 
Day 6 
 
 
 
 
 
 
 
 
 
 
 
Plasma draw for 24-hour ammonia 
 
 
 
 
 
 
 
 
 
 
 
Plasma draw for plasma nitrate 
 
 
 
 
 
 
 
 
 
 
 
Breakfast 
 
 
 
 
 
 
 
 
 
 
 
Give IP 
 
 
 
 
 
 
 
 
 
 
 
Lunch 
 
 
 
 
 
 
 
 
 
 
 
Dinner 
 
 
 
 
 
 
 
 
 
 
 
Abbreviations: IP = investigational product 
a Plasma draws occur before meals and meals occur before IP administration, as applicable. 
 


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8. 
STUDY DISCONTINUATION 
8.1. 
Sponsor Discontinuation Criteria 
This study may be discontinued at any time due to safety concerns (including, but not limited to, 
the stopping rules described in Section 4.3), failure to meet expected enrollment goals, 
administrative reasons, or at the discretion of the Sponsor. Should the study be terminated 
prematurely, the Sponsor will provide written notification to the Investigator and regulatory 
authorities and will specify the reason(s) for early termination. The Investigator must inform the 
IRB promptly and provide the reason(s) for the termination. 
8.2. 
Study Discontinuation for Individual Subjects 
Subjects must discontinue further IP administration and withdraw from the study for the 
following reasons: 
• Withdrawal of consent; 
• Investigator decision; 
• Significant noncompliance with protocol procedures; 
• >1 missed dose of investigational product 
• Necessity for treatment with systemic antibiotics; 
• Clinically significant allergy or hypersensitivity reaction to SYNB1020; 
• Development of any of the following toxicities: 
− AE of Grade 3 or greater severity according to the NCI CTCAE or 
life-threatening AE if not covered by CTCAE;  
− Episode of clinically overt HE (West Haven grade 2 or higher; see Appendix 2)  
− QTc interval of > 520 msec or an increase from baseline of > 60 msec confirmed 
in a repeat test after at least 20 minute;  
− Any of the following laboratory abnormalities:  
 Grade 3 liver enzyme abnormalities and 10-fold ULN for those with normal 
values at baseline OR a 5-fold increase or 10-fold ULN for those with 
abnormal values at baseline, whichever is lower; 
 Hemoglobin levels of < 8 g/dL confirmed in a repeat test or requiring blood 
transfusion; 
 Platelet count of < 20,000/mL confirmed in a repeat test; 
 eGFR < 30 mL/min/1.73m2  confirmed in a repeat test; 
 PT > 6.0 sec over ULN or INR level of > 2.3 confirmed in a repeat test; 
 Bilirubin > 5 × ULN confirmed in a repeat test; 


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 Any other laboratory abnormality of Grade 3 or great severity not covered 
above and not present at baseline, confirmed in a repeat test. 
Subjects may withdraw their consent at any time for any reason without prejudice to their future 
medical care by the physician or at the institution. If a subject withdraws consent, the date and 
stated reason for consent withdrawal should be documented. Subject data collected up to the date 
of consent withdrawal will be included in the analyses. 
Subjects who withdraw from the study prior to receiving SYNB1020/placebo are not required to 
complete the safety follow-up period. Subjects who receive at least one dose of IP and withdraw 
from the study are required to complete the tests and evaluations for the EOS/Discharge Visit at 
the time of withdrawal. Subjects who discontinue study treatment after the first administration of 
SYNB1020/placebo and withdraw from the study will not be allowed to enroll again at a later 
date. If a subject misses a dose of IP for any reason, the subject should continue with the study 
and take the next dose at the time specified in Table 3. A subject who misses more than one dose 
of IP should be withdrawn from the study. If a subject who withdraws from the study has an 
ongoing AE, every effort will be made to follow the AE until the event has resolved, stabilized, 
or returned to baseline status. The Sponsor must be notified of all study withdrawals within 
24 hours. 


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9. 
ADVERSE EVENTS 
9.1. 
Adverse Event 
An AE is any untoward medical occurrence, including the exacerbation of a pre-existing 
condition, in a subject administered a pharmaceutical product regardless of causality. 
9.1.1. 
Assessment of Severity 
The severity rating of an AE refers to its intensity. The severity of each AE will be categorized 
using the NCI CTCAE, version 4.03.21 For any term that is not specifically listed in the CTCAE 
scale, intensity should be assigned a grade of 1 through 5 using the following CTCAE 
guidelines: 
Grade 1: Mild; asymptomatic or mild symptoms; clinical or diagnostic observations only; 
intervention not indicated 
Grade 2: Moderate; minimal, local or noninvasive intervention indicated; limiting  
age-appropriate instrumental activities of daily living 
Grade 3: Severe or medically significant but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated; disabling; limiting 
self-care activities of daily living 
Grade 4: Life-threatening consequences; urgent intervention indicated 
Grade 5: Death related to AE 
9.1.2. 
Assessment of Causality 
Medical judgment should be used to determine the cause of the AE, considering all relevant 
factors such as (but not limited to) the underlying study indication, coexisting disease, 
concomitant medication, relevant history, pattern of the AE, temporal relationship to 
SYNB1020/placebo, and de-challenge or re-challenge. 
Yes (possibly, probably, or definitely related): there is a reasonable possibility that 
SYNB1020/placebo caused the event; one or more of the following criteria apply: 
• The event follows a reasonable temporal sequence from administration of 
SYNB1020/placebo. 
• The event could not be reasonably attributed to the known characteristics of the 
subject’s clinical state, environmental or toxic factors, or other modes of therapy 
administered to the subject. 
• The event follows a known pattern of response to SYNB1020. 
• The event disappears or decreases on cessation or reduction in dose. (It should be 
noted that in some situations an AE will not disappear or decrease in intensity upon 
discontinuation of study dosing despite other clear indications of relatedness). 


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No (unlikely, probably not related, or definitely not related): there is no reasonable possibility 
that the IP caused the event; one or more of the following criteria apply: 
• The event does not follow a reasonable temporal sequence from administration of 
SYNB1020/placebo  
• The event could be reasonably attributed to the known characteristics of the subject’s 
clinical state, concurrent illness, environmental or toxic factors, or other modes of 
therapy administered to the subject. 
• The event does not follow a known pattern of response to SYNB1020. 
• The event does not disappear or decrease on cessation or reduction in dose(ing), and 
it does not reappear or worsen when dosing is resumed. 
9.2. 
Serious Adverse Event 
An SAE is any untoward medical occurrence that meets any of the following criteria: 
• Results in death. 
• Is immediately life-threatening (refers to an event in which the subject is at risk of 
death at the time of the event; it does not refer to an event, which hypothetically 
might have caused death if it were more severe). 
• Requires inpatient hospitalization or prolongation of existing hospitalization. 
• Results in a persistent or significant incapacity or substantial disruption of the ability 
to conduct normal life functions. 
• Is a congenital anomaly/birth defect. 
• Based on appropriate medical judgment, represents an important medical event that 
may jeopardize the subject or may require intervention to prevent one of the other 
outcomes described above. 
9.2.1. 
Clarification of Serious Adverse Event Definition 
• Death is an outcome of an SAE and not an SAE in itself. When death is an outcome, 
the event(s) resulting in death should be reported (e.g., “pulmonary embolism” with a 
fatal outcome). The appropriate diagnosis or term should be recorded and assigned 
severity Grade 5. 
• In instances of death due ultimately to the underlying disease, the cause of death 
should be indicated as the specific event or condition resulting in death to the extent 
possible. If no appropriate term with a Grade 5 severity in the CTCAE can be 
identified, then a term should be selected from the CTCAE category “Death”. 
• “Life-threatening” means that the subject was at immediate risk of death from the 
event as it occurred. This does not include an event that might have led to death if it 
had occurred with greater severity. Grade 4 events (e.g., thrombocytopenia) are not 
always serious unless they have life-threatening consequences or result in 
hospitalization. 


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• Pre-planned or elective hospitalizations including social and/or convenience 
situations (e.g., respite care) are excluded from SAE reporting. In addition, 
“admissions” under 23-hour Observation or Emergency Room visits are excluded 
from SAE reporting; however, such events should still be reported on the appropriate 
eCRF page. 
• Overdose of either SYNB1020 or concomitant medication without any overdose signs 
or symptoms unless the event meets SAE criteria (e.g., hospitalization) are excluded 
from SAE reporting; however, such events should still be reported on the appropriate 
eCRF page. 
• SAEs that occur within the safety follow-up period but are related to subsequent 
therapies are excluded from SAE reporting: 
− Subjects who have completed study dosing or who terminate from study and then 
undergo subsequent therapies during the safety follow-up period and experience 
an SAE specifically related to the administration of the subsequent therapy will 
not have those events reported as SAEs. This exclusion will include “elective” 
hospitalizations necessary for the administration of such therapies. 
9.2.2. 
Serious, Unexpected, Suspected Adverse Reactions 
In accordance with regulatory requirements, the Sponsor or designee will immediately notify 
regulatory authorities and the Investigators, who will in turn notify their IRB as necessary, of any 
AE associated with IP administration or study procedures that is a serious, unexpected, suspected 
adverse reaction (SUSAR) or any finding from tests in laboratory animals that suggests a 
significant risk for human subjects, including reports of mutagenicity, teratogenicity, or 
carcinogenicity. An AE or suspected adverse reaction is considered “unexpected” if it is not 
listed in the Investigator’s Brochure or is not listed at the specificity or severity that has been 
previously observed. 
9.3. 
Procedures for Treating and Reporting Special Safety Events 
9.3.1. 
Infection (Suspected or Confirmed) 
Subjects who demonstrate any symptoms, signs, and/or laboratory assessments suggestive of 
possible infection during the dosing or follow-up periods should be assessed for infection, 
including blood and urine evaluation (culture and/or PCR, see Section 7.2.9). Samples of all 
available body fluids should be obtained for PCR assessment prior to initiating broad spectrum 
antibiotic treatment guided by the subject’s clinical condition and local standard of care; 
however, administration of antibiotics should not be significantly delayed to allow for collection 
(e.g., of feces). Subjects who require systemic antibiotic treatment should discontinue IP dosing 
and the event should be reported to the Sponsor within 24 hours. SYNB1020 is a Gram-negative 
bacteria with no antibiotic resistance genetic markers. Antibiotic sensitivity testing has been 
performed as described in the Investigator’s Brochure and can be used to guide treatment if 
infection is suspected. 
 


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9.3.2. 
Spontaneous Bacterial Peritonitis  
Spontaneous bacterial peritonitis (SBP) is considered an adverse event of special interest. 
Suspected or confirmed cases of SBP should be diagnosed and treated per local standard of care. 
Samples of all available body fluids should be obtained for PCR assessment prior to initiating 
broad spectrum antibiotic treatment. Subjects who develop SBP should discontinue IP dosing 
and the event should be reported to the Sponsor within 24 hours. 
9.3.3. 
Pregnancy  
If a subject becomes pregnant while participating in this study, information on the pregnancy and 
outcome will be requested. If a partner of a participating subject becomes pregnant while their 
partner is participating in the study or 90 days following their participation, information will be 
requested on the pregnancy and outcome.  
9.3.4. 
Hepato-Renal Syndrome  
Hepato-renal syndrome (HRS) is considered an adverse event of special interest. Suspected or 
confirmed cases of HRS should be diagnosed and treated per local standard of care. Subjects 
who develop HRS should discontinue IP dosing and the event should be reported to the Sponsor 
within 24 hours. 
9.4. 
Reporting of Adverse Events 
All AEs, serious and nonserious, will be fully documented on the appropriate eCRF. For each 
AE, the Investigator must provide its duration (start and end dates or ongoing), intensity, 
assessment of causality, and whether specific action or therapy was required. 
All AEs occurring from the time a subject signs informed consent through the safety follow-up 
period must be recorded on the eCRF. Subjects will be provided with diary cards for recording 
AEs during follow-up period after discharge from the inpatient unit.  
All SAEs, regardless of relationship to SYNB1020/placebo, must be reported to the Sponsor 
within 24 hours of the Investigator’s knowledge. This should be done by faxing the completed 
SAE Report Form to the Sponsor at the number provided on the SAE Report Form. After the 
final follow-up visit, only SYNB1020/placebo-related AEs/SAEs need to be collected and 
reported to the Sponsor, if clinically indicated. 
Investigators must follow subjects with AEs/SAEs until event resolution or stabilization, 
withdrawal of consent, subject loss to follow-up, or death, whichever occurs first. 
9.5. 
Pregnancy 
Only females of non-childbearing potential are eligible for study participation. 
9.6. 
Clinical Laboratory Abnormalities 
It is the responsibility of the Investigator to assess the clinical significance of all abnormal 
laboratory values as defined by the appropriate reference range(s). All abnormal values assessed 
to be of clinical concern and at least possibly related to SYNB1020/placebo or of uncertain 
causality should be repeated. Persistent abnormal values and changes of possible clinical concern 


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that remain within the normal range should be followed at the discretion of the Investigator. Of 
note, due to the underlying condition under study, ammonia values will often fall outside of the 
reference ranges; clinical significance and follow-up of these abnormal values will be determined 
by the treating Investigator on an individual subject basis. 
An abnormal laboratory value that is not already associated with an AE is to be recorded as an 
AE if an action on IP dosing is made as a result of the abnormality, if intervention for 
management of the abnormality is required, or at the discretion of the Investigator. 
9.7. 
Review of Safety Data 
The designated pharmacovigilance physician in collaboration with the Medical Monitor will be 
responsible for the ongoing review and evaluation of safety data, including AEs, clinical 
laboratory data, and any other safety evaluations, for the duration of the study in accordance with 
the medical monitoring plan. A Safety Review Committee comprising the Lead Investigator, 
Medical Monitor, pharmacovigilance physician, and an external disease expert, as needed, will 
meet at predetermined intervals during the study as well as in response to certain clinical 
outcomes. At each meeting, the Safety Review Committee will review blinded data from the 
ongoing study and determine whether the study should continue as planned, be modified, or be 
terminated. (Section 4.4). For details refer to the separate Safety Review Committee charter.   


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10. 
STATISTICAL METHODS AND DETERMINATION OF 
SAMPLE SIZE 
All evaluations and tabulations will be carried out as described in detail in a statistical analysis 
plan (SAP), which will be finalized and approved prior to database lock and unblinding.  
In addition to the analyses described within this section, interim analyses may be conducted. 
10.1. 
Populations for Analysis 
The following populations will be defined: 
Safety Population (SAF): 
All subjects in Part 1 who received at least one dose of 
SYNB1020 and have at least one post-baseline safety 
assessment. All randomized subjects in Part 2 who received at 
least one dose of SYNB1020 or placebo and have at least one 
post-baseline safety assessment. Subjects will be included in 
the analysis according to the dose and IP received. 
Microbiotic-kinetic (MK) 
Population: 
Subjects in the SAF who completed the clinical study and were 
considered evaluable for analysis of the MK data. Subjects will 
be included in the analysis according to the dose and IP 
received. 
Pharmacodynamic (PD) 
Population: 
Subjects in the SAF who completed the clinical study and were 
considered evaluable for analysis of the PD data. Subjects will 
be included in the analysis according to the study part and IP 
received. 
Data will be excluded from the MK and/or PD population in case of any major protocol 
deviation that may influence the MK and/or PD outcome. 
If not indicated otherwise, the analyses of safety endpoints will be conducted based on the SAF 
and all MK/PD analyses will be performed based on the MK/PD population. 
10.2. 
Safety Variables 
Adverse events will be coded using the Medical Dictionary for Regulatory Activities 
(MedDRA), and the severity of AEs and laboratory abnormalities will be graded using the NCI 
CTCAE, version 4.03. AEs will be tabulated by SOC and preferred term. Incidence tables of 
subjects with AEs will be presented for all AEs by maximum severity, SAEs, AEs assessed as 
related to SYNB1020/placebo, and AEs resulting in discontinuation of study dosing. 
Clinical laboratory data, ECGs, and other safety assessments will be presented in by-subject 
listings and will be presented in tabular format, including absolute values and changes from 
baseline (if applicable), by study day. 


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10.3. 
Microbiotic-Kinetic Variables 
The following variables will be calculated from the serial fecal sampling from each subject, if 
data support calculation. Further details on these calculations will be included in the SAP.  
• Tlag – the time of the first detectable fecal SYNB1020 
• Tss – the time to steady-state 
• qmax – the maximum observed fecal SYNB1020 qPCR signal 
• Tlast – the time of the last detectable fecal SYNB1020 
• Half-life – the time for the fecal SYNB1020 qPCR signal to decline by 50% 
A sample will be considered negative for the purposes of SYNB1020 clearance if it is below the 
limit of quantification or estimated to be below 0.1% of the administered dose. 
The microbiotic-kinetic parameters of SYNB1020 will be summarized descriptively.  
10.4. 
Pharmacodynamic Variables 
Pharmacodynamic data will be presented in by-subject listings and will be presented in tabular 
and/or graphical format, including absolute values and changes from baseline, by study day. 
10.5. 
Determination of Sample Size 
Part 1: The sample size for the sentinel cohort (6 subjects) is primarily designed for empirical 
evaluation of safety and tolerability in subjects with cirrhosis. 
Part 2: A sample of 24 subjects must complete all 3 ammonia AUC intervals (baseline, Day 5, 
and Day 6) to detect a 20% reduction in average daily ammonia (AUC0-24/24, from a baseline of 
70 µmol/L) with an approximate significance level of 10% and an approximate power of 90%. 
Significance and power are approximate based on the assumptions below. The number of 
subjects assumes that the standard deviation (SD) of spot ammonia is 36.4 µmol/L, half of the 
variance is between subject, and that the SD for average daily ammonia is as little as half that of 
spot ammonia. The spot ammonia SD is based on a weighted average of SD observed in a prior 
study of ammonia lowering in a similar population.23 The variability of AUC may be 50% lower 
than spot ammonia based on the FDA summary basis of approval for glycerol phenylbutyrate for 
urea cycle disorders.24 
Due to uncertainty in the assumptions of decrease in variability in AUC of ammonia relative to 
spot measurements in this population, up to 2 interim analyses for sample size re-estimation may 
occur after at least 12 subjects have completed all ammonia AUC assessments, and the total 
number of subjects in Part 2 of the study may be increased up to 40 at either point based on the 
SD of data observed. Additional subjects may be enrolled to ensure that at least the planned 
number of evaluable subjects complete the study. 
10.6. 
Randomization and Blinding 
Part 1: Open-label, all subjects will receive SYNB1020. Neither subjects nor investigators are 
blinded.   


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Part 2: Subjects will be randomized in a 1:1 ratio to receive either SYNB1020 or placebo. 
Randomization will be performed using a computer-generated central randomization scheme 
maintained by the Sponsor or designee. Both subjects and investigators will be blinded to study 
compound assignment, as will all other study personnel, with the exception of one unblinded 
pharmacist at the study site(s). In addition, an unblinded observer will be assigned to this study 
and will be independent of the study team. The unblinded observer will be able to review 
unblinded data as needed to aid in study progress, and this individual will be responsible for re-
evaluation of the sample size as defined in Section 10.5. Details of unblinded roles will be 
provided in a separate Unblinding Guidance Document. Upon observation of a safety event that 
requires knowledge of IP assignment to determine event causality and further action and/or 
treatment (e.g., SUSAR), the Sponsor’s Medical Monitor may be unblinded to IP assignment.  A 
procedure for emergency access to a subject’s treatment assignment within the interactive voice 
response system will be provided. The unblinding of any blinded team member will be 
documented.  
10.7. 
Changes in the Conduct of the Study or Planned Analysis 
Only the Sponsor may modify the protocol. Amendments to the protocol will be made only after 
consultation and agreement between the Sponsor and the Investigator. The only exception is 
when the Investigator considers that a subject’s safety is compromised without immediate action. 
In these circumstances, the Investigator should inform the Sponsor and the full IRB within 
1 working day after the emergency occurred. All amendments that have an impact on subject risk 
or the study objectives or require revision of the informed consent document must receive 
approval from the IRB prior to implementation. 


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11. 
DATA RECORDING, RETENTION AND MONITORING 
11.1. 
Case Report Forms 
Data will be collected using source documents through an electronic data capture (EDC) system 
at the clinical site. The Investigator or designee will record data specified in the protocol using 
eCRFs. Changes or corrections to eCRFs will be made by the Investigator or an authorized 
member of the study staff according to the eCRF completion guidelines. It is expected that the 
site will enter all data within 5 days of a subject visit. 
It is the Investigator’s responsibility to ensure eCRFs are complete and accurate, regardless of 
whether this responsibility has been delegated in whole or in part. Following review and 
approval, the Investigator or designee will electronically sign and date the pages, which certifies 
that the Investigator has thoroughly reviewed and confirmed all data on the eCRF.  
11.2. 
Data Retention 
Data retention practices will follow International Council for Harmonisation of Technical 
Requirements for Pharmaceuticals for Human Use (ICH) guidelines, which note that essential 
documents should be retained for a minimum of 2 years after the last approval of a marketing 
application in an ICH region and until there are no pending or contemplated marketing 
applications in an ICH region or at least 2 years have elapsed since the formal discontinuation of 
clinical development of the IP. However, these documents should be retained for a longer period 
if required by the applicable legal requirements.  
11.3. 
Data Monitoring 
This study will be closely monitored by representatives of the Sponsor or designee throughout its 
duration. Monitoring will include personal visits with the Investigator and study staff as well as 
appropriate communications by telephone, fax, mail, email, or use of the EDC system, as 
applicable. It is the monitor’s responsibility to inspect eCRFs at regular intervals throughout the 
study to verify the completeness, accuracy, and consistency of the data and to confirm adherence 
to the study protocol and to Good Clinical Practice (GCP) guidelines. The Investigator agrees to 
cooperate with the monitor to ensure that any problems detected during the course of this study 
are resolved promptly. The Investigator and site will permit study-related monitoring, audits, 
IRB review, and regulatory inspection, including direct access to source documents. 
It is understood that study monitors and any other personnel authorized by the Sponsor may 
contact and visit the Investigator and will be permitted to inspect all study records (including 
eCRFs and other pertinent data) on request, provided that subject confidentiality is maintained 
and that the inspection is conducted in accordance with local regulations. 
Every effort will be made to maintain the anonymity and confidentiality of subjects during this 
study. However, because of the experimental nature of the IP, the Investigator agrees to allow 
representatives of the Sponsor and authorized representatives of regulatory authorities to inspect 
the facilities used in the conduct of this study and to inspect, for purposes of verification, the 
hospital or clinic records of all subjects enrolled in the study. 


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11.4. 
Quality Control and Quality Assurance 
Quality control procedures will be conducted according to the Sponsor and CRO’s internal 
procedures. The study site may be audited by a quality assurance representative of the Sponsor. 
All necessary data and documents will be made available for inspection. 


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12. 
REGULATORY, ETHICAL, AND LEGAL OBLIGATIONS 
12.1. 
Good Clinical Practice 
The study will be performed in accordance with the protocol, guidelines for GCP established by 
the ICH and applicable local regulatory requirements and laws.  
12.2. 
Institutional Review Board Approval 
The Investigator must inform and obtain approval from the IRB for the conduct of the study at 
named sites, the protocol, informed consent documents, and any other written information that 
will be provided to the subjects and any advertisements that will be used. Written approval must 
be obtained prior to recruitment of subjects into the study and shipment of SYNB1020/placebo. 
Proposed amendments to the protocol (see Section 10.7) and aforementioned documents must be 
submitted to the Sponsor for review and approval, then to the IRB. Amendments may be 
implemented only after a copy of the approval letter from the IRB has been transmitted to the 
Sponsor. 
Per GCP guidelines, the Investigator will be responsible for ensuring that an annual update is 
provided to the IRB until the study is completed (i.e., finalization of the clinical study report) to 
facilitate continuing review of the study and that the IRB is informed about the end of the study. 
Copies of the update, subsequent approvals, and final letter must be sent to the Sponsor. 
12.3. 
Regulatory Authority Approval 
The study will be performed in accordance with the requirements of the US FDA and will also 
meet all of the requirements of ICH GCP guidance. Amendments to the protocol will be 
submitted to the FDA prior to implementation in accordance with applicable regulations. 
12.4. 
Other Required Approvals 
In addition to IRB and regulatory authority approval, all other required approvals (e.g., approval 
from the local research and development board or scientific committee) must be obtained prior to 
recruitment of subjects into the study and shipment of SYNB1020/placebo. 
12.5. 
Informed Consent 
Informed consent is a process that is initiated prior to the subject’s agreeing to participate in the 
study and continues throughout the subject’s study participation. It is the Investigator’s 
responsibility (or designee) to obtain written informed consent from each subject after adequate 
explanation of the aims, methods, anticipated benefits, and potential hazards of the study and 
before any study procedures are initiated. Each subject should be given a copy of the informed 
consent document and associated materials. The original copy of the signed and dated informed 
consent document must be retained at the site and is subject to inspection by representatives of 
the Sponsor or regulatory authorities. If any amendments occur throughout the course of the 
study that affect the informed consent form (i.e., when new study procedures or assessments 


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have been added), all active subjects should be reconsented using the same process for the initial 
consent. 
12.6. 
Patient Confidentiality 
The Investigator must ensure that the subject’s privacy is maintained. On eCRFs and other 
documents submitted to the Sponsor, including any required adverse event reporting, subjects 
will be identified by their assigned subject number. Documents that are not submitted to the 
Sponsor (e.g., signed informed consent documents) should be kept in a confidential file by the 
Principal Investigator. 
The Investigator shall permit authorized representatives of the Sponsor, regulatory authorities, 
and Ethics Committees to review the portion of the subject’s medical record that is directly 
related to the study. As part of the required content of informed consent documents, the subject 
must be informed that his/her records will be reviewed in this manner. 
12.7. 
Disclosure of Information 
Information concerning the study, protocol, processes, assessments, investigational product, 
scientific data, or other non-public  information related to the study or the Sponsor and its 
representatives are confidential and remain the property of the Sponsor. The Principal 
Investigator may use this information for the purposes of the study only. 
It is understood by the Principal Investigator that the Sponsor will use information obtained in 
this clinical study in connection with the clinical development program, and therefore may 
disclose it as required to other clinical investigators and to regulatory authorities. In order to 
allow the use of the information derived from this clinical study, the Principal Investigator 
understands that he/she has an obligation to provide complete test results and all data obtained 
during this study to the Sponsor. 
Verbal or written discussion of results prior to study completion and full reporting should only 
be undertaken with written consent from the Sponsor. 
12.8. 
Publication of Study Data 
The Sponsor encourages the scientific publication of data from clinical research studies. 
However, Investigators many not present or publish partial or complete study results individually 
without participation of the Sponsor. The Principal Investigator and the Sponsor may propose 
appropriate scientific manuscripts or abstracts from the study data. All proposed publications 
must be reviewed and commented on by the Sponsor before submission for publication. The 
detailed procedures for the review of publications are set out in the clinical trial agreement 
entered into with the Sponsor in connection with this study. These procedures are in place to 
ensure coordination of study data publication and adequate review of data for publication against 
the validated study database for accuracy.  
Qualification of authorship will follow the requirements of the International Committee of 
Medical Journal Editors (www.icmje.org). The names of Investigators and Sponsor 
representatives responsible for designing the study and analyzing the results will be included in 
the publication(s). This custom can be adjusted upon mutual agreement of the authors and 


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Synlogic. In addition, this clinical trial must be registered with ClinicalTrials.gov, which will be 
done by the Sponsor or its representative. 
12.9. 
Ethical Standards 
We are committed to designing, implementing, conducting, analyzing and reporting these trials 
in compliance with the highest ethical and scientific standards. Protection of subject safety is the 
overriding concern in the design of clinical trials. In all cases, Synlogic clinical trials will be 
conducted in compliance with local and/or national regulations and in accordance with the 
ethical principles that have their origin in the Declaration of Helsinki.  


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13. 
REFERENCES 
1. 
Kim Y, Ejaz A, Tayal A, et al. Temporal trends in population-based death rates 
associated with chronic liver disease and liver cancer in the United States over the last 
30 years. Cancer 2014;120:3058-65. 
2. 
Kalaitzakis E, Bjornsson E. Hepatic encephalopathy in patients with liver cirrhosis: is 
there a role of malnutrition? World J Gastroenterol 2008; 14:3438-39. 
3. 
Aldridge DR, Tranah EJ, Shawcross DL. Pathogenesis of hepatic encephalopathy: role of 
ammonia and systemic inflammation. J Clin Exp Hepatol 2015; 5:S7-20. 
4. 
Bajaj JS, Cordoba J, Mullen KD, et al. Review article: the design of clinical trials in 
hepatic encephalopathy – an International Society for Hepatic Encephalopathy and 
Nitrogen Metabolism (ISHEN) consensus statement. Aliment Pharmacol Ther 2011; 
33:739-47. 
5. 
Suraweera D, Sundaram V, Saab S. Evaluation and management of hepatic 
encephalopathy: current status and future directions. Gut Liver 2016; 10:509-19. 
6. 
Patidar KR, Thacker LR, Wade JB, et al. Covert hepatic encephalopathy is independently 
associated with poor survival and increased risk of hospitalization. Am J Gastroenterol 
2014; 109:1757-63. 
7. 
Bajaj JS, Hafeezullah M, Hoffmann RG, et al. Minimal hepatic encephalopathy: a vehicle 
for accidents and traffic violations. Am J Gastroenterol 2007;102:1903-9. 
8. 
Patidar KR, Bajaj JS. Covert and overt hepatic encephalopathy: diagnosis and 
management. Clin Gastroenterol Hepatol 2015; 13(12)2048-61. 
9. 
Clemmesen J, Larsen F, Kondrup J, Hansen B, Ott P. Cerebral herniation in patients with 
acute liver failure is correlated with arterial ammonia concentration. Hepatology 1999; 
29:648-53. 
10. 
Xifaxan® (rifaximin) US Package Insert. Salix Pharmaceuticals, Inc. November 2015. 
11. 
Bass NM, Mullen KD, Sanyal A, et al. Rifaximin treatment in hepatic encephalopathy. 
N Engl J Med 2010; 362(12):1071-81. 
12. 
Wong RJ, Gish RG, Ahmed A. Hepatic encephalopathy is associated with significantly 
increased mortality among patients awaiting liver transplantation. Liver Transpl 2014; 
20:1454-61. 


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13. 
Wolpert E, Phillips SF, Summerskill WH. Transport of urea and ammonia production in 
the human colon. Lancet 1971; 2(7739):1387-90. 
14. 
Summerskill WH, Wolpert E. Ammonia metabolism in the gut. Am J Clin Nutr 1970; 
23(5):633-9. 
15. 
Savage DC. Microbial ecology of the gastrointestinal tract. Annu Rev Microbiol 1977; 
31:107-33. 
16. 
Romero-Gómez M, Jover M, Galán JJ, Ruiz A. Gut ammonia production and its 
modulation. Metab Brain Dis 2009; 24(1):147-57. 
17. 
Scaglia F, Lee B. Clinical, biochemical, and molecular spectrum of hyperargininemia due 
to arginase I deficiency. Am J Med Genet Part C Semin Med Genet 2006; 142C(2):113-
20. 
18. 
Hodges PE, Rosenberg LE. The spf-ash mouse: a missense mutation in the ornithine 
transcarbamylase gene also causes aberrant mRNA splicing. Proc Natl Acad Sci U S A. 
1989; 86(11):4142-6. 
19. 
Davies B, Morris T. Physiological parameters in laboratory animals and humans. Pharm 
Res 1993; 10(7):1093-5. 
20. 
Amodio P, Bemeur C, Butterworth R, et al. The nutritional management of hepatic 
encephalopathy in patients with cirrhosis: International Society for Hepatic 
Encephalopathy and Nitrogen Metabolism Consensus. Hepatology 2013; 58(1):325-36. 
21. 
US Department of Health and Human Services. Common Terminology Criteria for 
Adverse Events (CTCAE) Version 4.0. Published 28 May 2009. Available at: 
<http://evs.nci.nih.gov/ ftp1/CTCAE/CTCAE_4.03_2010-06-
14_QuickReference_5x7.pdf > Accessed 15 July 2016. 
22. 
Lewis SJ, Heaton KW. Stool form scale as a useful guide to intestinal transit time. Scand 
J Gastroenterol 1997; 32(9):920-4. 
23. 
Ghabril M, Zupanets IA, Vierling J, et al. Glycerol phenylbutyrate in patients with 
cirrhosis and episodic hepatic encephalopathy: a pilot study of safety and effect on 
venous ammonia concentration. Clin Pharmacol Drug Dev 2013;2(3):278-84. 
24. 
Center for Drug Evaluation and Research. Medical review(s). New Drug Application 
number: 203284. November 2012. Available at: 
<https://www.accessdata.fda.gov/drugsatfda_docs/nda/2013/203284orig1s000medr.pdf> 
Accessed 01 August 2017. 


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APPENDIX 1. RANITIDINE PACKAGE INSERT 
https://www.accessdata.fda.gov/drugsatfda_docs/label/2009/018703s068,019675s035,020251s01
9lbl.pdf 


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APPENDIX 2. WEST HAVEN GRADING SYSTEM 
 
Sign/Symptom 
Grade 
Trivial lack of awareness 
Euphoria or anxiety 
Shortened attention span 
Impaired performance of addition 
1 
Lethargy or apathy 
Minimal disorientation for time or place 
Subtle personality change 
Inappropriate behavior 
Impaired performance of subtraction 
2 
Somnolence to semistupor, but responsive to 
verbal stimuli 
Confusion 
Gross disorientation 
3 
Coma (unresponsive to verbal or noxious stimuli) 
4 
Source: Ferenci P, Lockwood A, Mullen K, et al. Hepatic encephalopathy—definition, nomenclature, diagnosis, 
and quantification: final report of the Working Party at the 11th World Congresses of Gastroenterology, Vienna 
1998. Hepatology 2002; 35:716-21. 


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APPENDIX 3. PSYCHOMETRIC HEPATIC ENCEPHALOPATHY 
SCORE TESTING 


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APPENDIX 4. CHILD-TURCOTTE-PUGH SCORE TESTING 
 
Parameter 
Assign 1 Point 
Assign 2 Points 
Assign 3 Points 
Encephalopathy grade 
None 
Grade 1-2 
(or precipitant-induced) 
Grade 3 or 4 
(or chronic) 
Ascites 
None 
Mild/moderate 
(diuretic-responsive) 
Severe 
(diuretic-refractory) 
Serum bilirubin (mg/dL) 
< 2 
2 to 3 
> 3 
Serum albumin (g/dL) 
> 3.5 
2.8 to 3.5 
< 2.8 
PT (sec prolonged) or 
INR 
< 4 
< 1.7 
4 to 6 
1.7 to 2.3 
> 6 
> 2.3 
Abbreviations: INR = international normalized ratio; PT = prothrombin time 
Source: https://www.hepatitis.va.gov/pdf/Child-Pugh-score.pdf 
Child-Turcotte-Pugh scoring is obtained by adding the score for each parameter: 
Child-Turcotte-Pugh class: 
• A = 5 to 6 points 
• B = 7 to 9 points 
• C = 10 to 15 points 
 


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APPENDIX 5. MODEL FOR END-STAGE LIVER DISEASE (MELD) 
 
MELD scores should be calculated using the following formula: 
 
MELD = 3.78×ln[serum bilirubin (mg/dL)] + 11.2×ln[INR] + 9.57×ln[serum creatinine (mg/dL)] + 6.43 
 
Any value less than 1 is given a value of 1 (i.e., if bilirubin is 0.8 a value of 1.0 is used). 
 
Source: Kamath PS, Kim WR, Advanced Liver Disease Study Group. The model for end-stage liver disease 
(MELD). Hepatology 2007; 45 (3):797–805. 
 

SAP_000.pdf: 
Confidentiality 
This document is confidential and proprietary property of Synlogic, Inc. and to be used only as authorized by 
Synlogic, Inc.  No part is to be reproduced, disclosed to others, or quoted without prior written authorization from 
Synlogic, Inc. 
STATISTICAL ANALYSIS PLAN 
Protocol SYNB1020-CP-002 
A Randomized, Double-blind, Placebo-controlled Study to Assess the Safety, 
Tolerability, and Pharmacodynamics of SYNB1020 in Hepatic Insufficiency 
and Cirrhosis Patients 
Protocol Number: 
(Version Date) 
SYNB1020-CP-002  
Name of Test Drug: 
SYNB1020 
Phase: 
1b/2a 
Methodology: 
Randomized, Double-Blind, Placebo-Controlled 
Sponsor: 
Synlogic, Inc. 
301 Binney St, Suite 402 
Cambridge, MA 02142 
(617) 401-9975 
Sponsor Representative: 
Larry Blankstein  
Executive Director, Clinical Operations 
Sponsor Representative: 
 
Marja Puurunen 
Medical Director 
Document Date: 
01 April 2019 
Document Version: 
Version 2.0 
 


 
SIGNATURE PAGE 
Protocol Title: 
A Randomized, Double-blind, Placebo-controlled Study to 
Assess the Safety, Tolerability, and Pharmacodynamics of 
SYNB1020 in Hepatic Insufficiency and Cirrhosis Patients 
Sponsor: 
Synlogic, Inc.  
301 Binney St, Suite 402 
Cambridge, MA 02142 (617) 401-9975 
Protocol Number: 
SYNB1020-CP-002 
Document Date/Version: 
27 Nov 2018/Version 6 
Cytel, Inc. Author: 
Katy Jaffee 
Cytel, Inc. 
675 Massachusetts Avenue 
Cambridge, MA 02139 
 
Signature:____________________________________ 
Date:________________________________________ 
Sponsor Approval 
By signing this document, I acknowledge that I have read the document and approve of the 
planned statistical analyses described herein.  I agree that the planned statistical analyses are 
appropriate for this study, are in accordance with the study objectives, and are consistent with 
the statistical methodology described in the protocol, clinical development plan, and all 
applicable regulatory guidances and guidelines. 
I have discussed any questions I have regarding the contents of this document with the 
biostatistical author. 
I also understand that any subsequent changes to the planned statistical analyses, as described 
herein, may have a regulatory impact and/or result in timeline adjustments.  All changes to the 
planned analyses will be described in the clinical study report (CSR). 
Sponsor Signatory: 
Marja Puurunen 
Synlogic, Inc. 
301 Binney Street, Suite 401 
Cambridge, MA 02142 
 
Signature:____________________________________ 
Date:________________________________________ 
 
 


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Protocol SYNB1020-CP-002 
01 APR 2019 
 
CONFIDENTIAL 
3
TABLE OF CONTENTS 
Section 
   Page 
1. 
INTRODUCTION AND OBJECTIVES OF ANALYSIS ............................................ 7 
1.1. 
Introduction .......................................................................................................... 7 
1.2. 
Objectives of Statistical Analysis ........................................................................ 7 
2. 
STUDY DESIGN .............................................................................................................. 9 
2.1. 
Synopsis of Study Design ..................................................................................... 9 
2.2. 
Randomization Methodology .............................................................................. 9 
2.3. 
Stopping Rules and Unblinding .......................................................................... 9 
2.4. 
Study Procedures ............................................................................................... 10 
2.5. 
Safety, Pharmacokinetic, and Pharmacodynamic Variables ......................... 16 
2.5.1. 
Primary Safety Endpoints ..................................................................... 16 
2.5.2. 
Secondary Microbiotic-Kinetic and PD Endpoints ............................... 16 
2.5.3. 
Exploratory PD Endpoints .................................................................... 16 
3. 
SUBJECT POPULATIONS .......................................................................................... 17 
3.1. 
Population Definitions ....................................................................................... 17 
3.2. 
Protocol Violations ............................................................................................. 17 
4. 
STATISTICAL METHODS ......................................................................................... 19 
4.1. 
Sample Size Justification ................................................................................... 19 
4.2. 
General Statistical Methods and Data Handling ............................................ 19 
4.2.1. 
General Methods ................................................................................... 19 
4.2.2. 
Computing Environment ....................................................................... 20 
4.2.3. 
Methods of Pooling Data ...................................................................... 20 
4.2.4. 
Adjustments for Covariates ................................................................... 20 
4.2.5. 
Multiple Comparisons/Multiplicity ...................................................... 20 
4.2.6. 
Subpopulations ...................................................................................... 20 
4.2.7. 
Withdrawals, Dropouts, Loss to Follow-up .......................................... 21 
4.2.8. 
Missing, Unused, and Spurious Data .................................................... 21 
4.2.9. 
Visit Windows ...................................................................................... 21 
4.3. 
Interim Analyses ................................................................................................ 22 
4.4. 
Subject Disposition ............................................................................................. 24 
4.5. 
Demographic and Baseline Characteristics ..................................................... 24 


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Section 
   Page 
4.6. 
Safety Analyses ................................................................................................... 24 
4.6.1. 
Adverse Events ..................................................................................... 24 
4.6.2. 
Laboratory Data .................................................................................... 25 
4.6.3. 
Vital Signs and Physical Examinations ................................................ 25 
4.6.4. 
Electrocardiogram ................................................................................. 25 
4.6.5. 
FibroScan .............................................................................................. 26 
4.6.6. 
Blood and Urine Cultures/Polymerase Chain Reaction ........................ 26 
4.6.7. 
Hepatic Encephalopathy Severity Grading ........................................... 26 
4.6.8. 
Psychometric Testing ............................................................................ 26 
4.6.9. 
Cirrhosis Severity Grading ................................................................... 26 
4.6.10. 
Concomitant Medications ..................................................................... 27 
4.7. 
Pharmacodynamic Evaluations ........................................................................ 27 
4.7.1. 
Venous Ammonia Area under the Curve .............................................. 27 
4.7.2. 
Fasting Spot Venous Ammonia ............................................................ 28 
4.7.3. 
Urinary Urea/Nitrogen, Plasma/Urinary Nitrate, Plasma 
Amino Acids, Bristol Stool Form Scale, Psychometric Test 
Performance .......................................................................................... 28 
4.8. 
Microbiotic-Kinetic Evaluations ....................................................................... 28 
5. 
CHANGES TO PLANNED ANALYSES .................................................................... 30 
6. 
CLINICAL STUDY REPORT APPENDICES ........................................................... 31 
6.1. 
Statistical Tables to be Generated .................................................................... 31 
6.2. 
Data Listings to be Generated ........................................................................... 33 
6.3. 
Figures to be Generated .................................................................................... 35 
6.4. 
Operating Characteristics of the Interim Analysis ......................................... 36 
 
LIST OF IN-TEXT TABLES 
Table 
Page 
Table 3 
Visit Windows ................................................................................................................. 22 
Table 4 
Microbiotic-Kinetic Parameters for Analysis .................................................................. 29 
 


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ABBREVIATIONS 
Abbreviation 
Definition 
%CV 
Arithmetic percent coefficient of variation  
AE 
Adverse events 
aPTT 
Activated partial thromboplastin time 
ATC 
Anatomic Therapeutic Class 
AUC 
Area under the curve 
AUC0-24 
Area under the concentration versus time curve from time 0 to the end 
of the dosing interval 24 hours later, calculated using linear trapezoid 
rule 
BP 
Blood pressure 
BQL 
Below the quantifiable limit 
BSFS 
Bristol stool form scale 
BUN 
Blood urea nitrogen 
CBC 
Complete blood count 
CRF 
Case report form 
CRO  
Contract research organization  
CSR 
Clinical study report 
CTCAE 
Common Terminology Criteria for Adverse Events 
ECG 
Electrocardiogram 
FDA 
Food and Drug Administration 
FSH 
Follicle-stimulating hormone 
HE 
Hepatic Encephalopathy 
IA 
ICH 
Interim Analysis 
International Conference on Harmonisation 
IL 
Interleukin 
INR 
International normalized ratio 
IP 
Investigational product 
IRT 
Interactive Response Technology 
ITT 
Intent-to-treat 
LFT 
Liver function test  
LLOQ 
Lower limit of quantification 
MedDRA 
Medical Dictionary for Regulatory Activities 
MELD 
Model for End-Stage Liver Disease 
MK 
Microbiotic-kinetic  
NCI 
National Cancer Institute 
PD 
Pharmacodynamic 
PHES 
Psychometric Hepatic Encephalopathy Score 
PK 
Pharmacokinetic 
PPI 
Proton pump inhibitor  


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Abbreviation 
Definition 
PT 
Preferred Term (AEs), or prothrombin time (labs) 
qmax 
Maximum observed fecal SYNB1020 qPCR signal 
qPCR 
Quantitative polymerase chain reaction 
QTcF 
QT interval corrected for heart rate 
SAE 
Serious adverse event 
SAF 
Safety  
SAP 
Statistical analysis plan 
SD 
Standard deviation 
SMC 
Safety Monitoring Committee 
SOC  
System/Organ/Class 
TEAE 
Treatment-emergent adverse event 
Tlag 
Time to first detectable fecal SYNB1020 
Tlast 
Time to last detectable fecal SYNB1020 
Tss 
Time to steady-state 
TNF-α 
Tumor necrosis factor alpha 
ULN 
Upper limit of normal 
WHO 
World Health Organization 


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1. 
INTRODUCTION AND OBJECTIVES OF ANALYSIS 
1.1. 
Introduction 
This document presents the statistical analysis plan (SAP) for Synlogic, Protocol SYNB1020-
CP-002: A Randomized, Double-blind, Placebo-controlled Study to Assess the Safety, 
Tolerability, and Pharmacodynamics of SYNB1020 in Hepatic Insufficiency and Cirrhosis 
Patients. 
This SAP is based on the final protocol (version 6) dated 27 November 2018. 
1.2. 
Objectives of Statistical Analysis 
Primary Objective  
• To evaluate the safety and tolerability of SYNB1020 following multiple doses in hepatic 
insufficiency and cirrhosis patients. The endpoints related to this objective include the 
nature and frequency of adverse events (AEs), measurement of vital signs and laboratory 
assessments (e.g., blood chemistry, complete blood count [CBC], liver function tests 
(LFTs), international normalized ratio [INR], and urinalysis), and electrocardiograms 
(ECGs) 
Secondary Objectives 
• To evaluate SYNB1020 kinetics in feces (measured with qualitative and quantitative 
polymerase chain reaction [qPCR] fecal assays) following dosing 
• To evaluate change from baseline of venous ammonia, including fasting and 24-hour 
area under the curve (AUC), following SYNB1020 dosing in subjects with 
hyperammonemia 
Exploratory Objectives 
• To evaluate placebo-adjusted change from baseline in pharmacodynamics (PD) effects 
of SYNB1020 on: 
o Serum inflammatory markers (serum tumor necrosis factor alpha (TNF-α), 
interleukin (IL)-6, and endotoxin) 
o Urinary urea/urinary nitrogen (24 hour) and calculated nitrogen balance 
o Plasma nitrate and urinary nitrate (24-hour) 
o Plasma amino acids including glutamine, arginine, aromatic amino acids, and 
branched chain amino acids 
o Psychometric test performance at baseline and following dosing as measured by 
Psychometric Hepatic Encephalopathy (HE) Score testing 
This SAP is designed to outline the methods to be used in the analysis of study data in order to 
answer the study objective(s).  Populations for analysis, data handling rules, statistical methods, 
and formats for data presentation are provided.  The statistical analyses and summary 
tabulations described in this SAP will provide the basis for the results sections of the clinical 
study report (CSR) for this trial. 


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This SAP also will outline any differences in the currently planned analytical objectives relative 
to those planned in the study protocol. 


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2. 
STUDY DESIGN 
2.1. 
Synopsis of Study Design 
SYNB1020-CP-002 is a Phase 1b/2a, randomized, double-blinded placebo-controlled study to 
evaluate the safety, tolerability, and PD of SYNB1020 in hepatic insufficiency and cirrhosis 
patients, with dosing of the investigational product (IP) administered in an inpatient unit and 
subsequent outpatient follow-up for SYNB1020 clearance. Part 1 comprises of a sentinel open-
label cohort of 6 subjects with cirrhosis and Model for End-Stage Liver Disease (MELD) score 
<12 to establish the safety and tolerability of SYNB1020. Once the safety and tolerability have 
been established in Part 1, enrollment will be opened to subjects in Part 2, a randomized, 
double-blinded, placebo-controlled study in subjects with cirrhosis and hyperammonemia. 
Approximately 20 to 40 evaluable subjects are anticipated for enrollment in Part 2, with 
randomization proceeding in a 1:1 ratio to SYNB1020 and placebo. 
In Part 1, eligible subjects will be admitted to an inpatient facility for a run-in diet, baseline 
assessments, IP administration, safety monitoring, and collection of blood, urine, and fecal 
samples for evaluation of safety, tolerability, and pharmacokinetic (PK) and PD evaluations.  
In Part 2, subjects may be pre-screened for eligibility based on medical history and a single 
fasting spot venous ammonia measurement. Eligible subjects with elevated fasting spot venous 
ammonia will then undergo full screening within 7 days of pre-screening. Eligible subjects will 
be admitted to an inpatient facility for a run-in diet and 24-hour ammonia profile, and those with 
an elevated 24-hour ammonia AUC (>1.2 × upper limit of normal [ULN]) will proceed with 
randomization, IP administration, safety monitoring, and collection of blood, urine, and fecal 
samples for PK and PD evaluations. 
Due to uncertainty in the assumptions of variability in AUC of ammonia relative to spot 
measurements in this population, Part 2 of the study may recruit up to a total of 40 subjects 
guided by the re-evaluation of the sample size based on the standard deviation (SD) of data 
observed at the IA.  
2.2. 
Randomization Methodology 
There is no randomization in Part 1 of the trial; Part 1 is open-label, single-treatment, single-
sequence.  
In Part 2, all eligible subjects will be randomized via the Interactive Response Technology 
(IRT) to one of the 2 treatment arms. The investigator will contact the IRT after confirming 
eligibility, and the IRT will assign a randomization number to each subject. The randomization 
number links the subject to the treatment arm and specifies a unique medication number for the 
first package of study drug. Eligible subjects will be randomized to SYNB1020 and placebo at a 
ratio of 1:1. Randomization will not be stratified. 
2.3. 
Stopping Rules and Unblinding 
There is no concern for unblinding in Part 1 of the trial.  
Part 2 is a double-blind study. The Investigator, subject, clinical staff and Sponsor’s study 
management team will be blinded to treatment assignments. The pharmacist at the clinical site 
or Investigator’s designee will be responsible for dispensation of study drug according to the 


Synlogic, Inc. 
Protocol SYNB1020-CP-002 
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randomization schedule provided by the Sponsor or designee.  Study drug will be administered 
in a blinded fashion.  
During Part 2, an unblinded interim analysis (IA) will occur once the first 16 subjects finish 
their inpatient visits in order to determine if the trial should be stopped for efficacy or futility, or 
if more subjects should be enrolled. Unblinded data and who will be unblinded prior to and for 
the IA is defined in a separate unblinding guidance document. In addition to the IA, the Safety 
Review Committee (comprised of the Lead Investigator, Medical Monitor, pharmacovigilance 
physician, and as needed an external expert) will meet at predetermined intervals and ad hoc, as 
needed, to conduct periodic interim reviews of safety. Further details are provided in a separate 
Safety Review Committee charter. The Safety Review Committee will also meet at the end of 
Part 1 to examine safety of the study drug.  
The Sponsor may stop either part of this study at any time. Investigators will be notified by the 
Sponsor or its designee if the study is stopped. The occurrence of the following events will 
require that further enrollment in the study be stopped: 
• Any suspected or proven invasive infection (e.g., sepsis or bacteremia) assessed as at 
least possibly related to the IP. 
• Three or more subjects experience the same National Cancer Institute (NCI) 
Common Terminology Criteria for Adverse Events (CTCAE) grade 3 AE assessed as 
at least possibly related to study treatment, or two or more subjects experience the 
same CTCAE grade 4 AE assessed as at least possibly related to the IP. 
• Two or more subjects experience serious AEs (SAEs) that are considered by the 
Investigator to be related to the IP. 
• Death occurs at any time during the study and is considered by the Investigator to be 
related to the IP. 
• Approximately 6% mortality is observed after 30 days following dosing. This is 
defined as ≥2 deaths observed in 24 subjects or ≥3 deaths observed in 40 subjects 
who have received the IP. 
• The Investigator, Medical Monitor, or Sponsor determines that an event or current 
data warrant stopping the study. 
The Sponsor’s medical staff, the Contract Research Organization (CRO)’s pharmacovigilance 
physician and the Lead Investigator will review the data concerning these event(s), along with 
other available data. Based on the results of their investigation, the Sponsor’s medical staff, the 
CRO’s pharmacovigilance physician and the Lead Investigator will determine appropriate 
follow-up and decide whether the study should be stopped. 
If the study is stopped, the events will be investigated, and the study will be closed to further 
enrollment. Subjects already participating in the study at the time the study is stopped will not 
receive further IP and will continue the follow-up schedule. 
2.4. 
Study Procedures 
The schedule of assessments, as outlined in the study protocol, are provided in Tables 1 and 2. 


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Protocol SYNB1020-CP-002 
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Table 1 
Schedule of Assessments 
Study Period 
Optional 
Pre-
Screening 
Screening 
Inpatient Monitoring 
Follow-upa 
Study Day 
Within 7 
days of 
Screening 
-36 to -6 
-5 
-4 
-3 
-2 
-1 
1 
2-4 5 
6 
7 
EOS/Discharge 
Visitb 
14 
Follow-
up Visit 
Up to 70 
days after 
last dose 
Informed consent for pre-screening 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Informed consent 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history 
 
c 
 
 
 
 
 
 
 
 
 
 
 
 
Physical examination 
 
 
 
 
 
 
 
 
 
 
 
d 
d 
 
FibroScan 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Screening for drugs of abuse (urine drug screen) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Screening for infectious disease (HIV/Hepatitis B, C serology) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Laboratory tests (CBC, LFTs, blood chemistry, urinalysis, 
PT/aPTT) 
 
 
 
 
 
 
 
 
e 
 
 
 
 
 
FSH (in females if non-childbearing potential status is equivocal 
based on medical history) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Record concomitant medications 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Adverse event reporting 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Vital signsf 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Electrocardiogram 
 
 
 
 
 
 
 
 
e 
 
 
 
 
 
Fasting spot venous ammonia 
 
g 
 
 
 
 
 
 
 
 
 
 
 
 
Admit to inpatient facility (in the morning after an overnight fast) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Required inpatient stay 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Subject randomization (Part 2 only) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Dietary controlh 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


Synlogic, Inc. 
Protocol SYNB1020-CP-002 
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Study Period 
Optional 
Pre-
Screening 
Screening 
Inpatient Monitoring 
Follow-upa 
Study Day 
Within 7 
days of 
Screening 
-36 to -6 
-5 
-4 
-3 
-2 
-1 
1 
2-4 5 
6 
7 
EOS/Discharge 
Visitb 
14 
Follow-
up Visit 
Up to 70 
days after 
last dose 
Administer H2 inhibitor (ranitidine 150 mg BID 30-60 minutes 
before breakfast and the evening snack)i 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Administer assigned IP (SYNB1020 or placebo, TID) with 
breakfast, lunch, and dinnerj 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
24-hour profile for venous ammoniak 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
24-hour urinary urea, urinary nitrogen and creatinine 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inflammatory markers (TNF-α, IL-6, endotoxin) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Plasma amino acid analyses 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Plasma nitratel 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
24-hour urinary nitratel 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Fecal SYNB1020 PCRm 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Psychometric testing 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Hepatic encephalopathy grading (West Haven grade) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Child-Turcotte-Pugh score 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Model for End-Stage Liver Disease score 
 
 
 
 
 
 
 
 
 e 
 
 
 
 
 
BSFS for each bowel movement collected 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Plasma collection for exploratory analyses 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine collection for exploratory analyses 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Review of adverse event diary card 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Abbreviations: aPTT = activated partial thromboplastin time; BID = twice daily; BSFS = Bristol stool form scale; CBC = complete blood count; EOS = End of Study; FSH = 
follicle-stimulating hormone; h = hour(s); H2 = histamine-2 receptor; HIV = human immunodeficiency virus; IL = interleukin; IP = investigational product; LFT = liver function 
test; PCR = polymerase chain reaction; PPI = proton pump inhibitor; PT = prothrombin time; TID = 3 times daily; TNF-α = tumor necrosis factor alpha 


Synlogic, Inc. 
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a One week (7± 1 days) after discharge from the inpatient unit, subjects will return for clinical evaluation and bring a 1-week post-dose fecal sample. Feces will continue to be 
collected weekly for the first 6 weeks following discharge and biweekly until a subject has a negative SYNB1020 fecal test for up to 10 weeks following completion of study 
dosing. Subjects who remain colonized with SYNB1020 after 10 weeks following the last dose will be treated as described in Section 7.5.2.  
b At least 12 hours after completion of the dosing period (i.e., on Day 7 for subjects who continue IP dosing per protocol), an EOS/Discharge visit will be performed. For subjects 
who discontinue study dosing prematurely after having received at least one dose of SYNB1020/placebo, the EOS/Discharge Visit should be completed within 24 hours of 
discontinuation. 
c If not obtained during pre-screening. 
 
d Symptom-driven physical examinations will be performed on Days 7 and 14. 
e Day 4 only. 
f Vital signs will be collected every 8 hours during the dosing period (Days 1 through 6). 
g Fasting venous ammonia will not be repeated at screening if measured at pre-screening. 
h Meal times for breakfast, lunch, and dinner should be the same every day ± 30 minutes, unless otherwise specified. 
i Subjects already on a PPI regimen will not be switched to ranitidine but will continue their prescribed PPI regimen for the duration of the study. 
j In Part 1, all subjects receive open-label IP. In Part 2, subjects are randomized to either SYNB1020 or placebo. IP will be administered immediately following a meal. 
k Samples for venous ammonia will be drawn relative to meal times  according to Table 4. Following an overnight fast, subjects will have a baseline sample drawn for ammonia at 
0h (before breakfast) and will be given a high-protein shake for breakfast, followed by IP administration on dosing days. Samples for venous ammonia will be drawn at +2h, +4h 
(before lunch), +6h, +9h (before dinner), +11h, +14h, +18h, and +24h the following morning (before breakfast). Two additional samples will be collected on Day 5 at +3h and +5h 
l Plasma nitrate samples are drawn at the same time as ammonia 24-hour samples on Day -2 and Day 6 at the following time points: 0 hours, +2 hours, +4 hours (before lunch), and 
+6 hours. For details of sample collection refer to the Laboratory Manual and Table 4. 
m One baseline fecal sample is required to be collected prior to SYNB1020 dosing (i.e., any time from Day -5 through Day 1). Feces will continue to be collected weekly for the 
first 6 weeks following discharge and biweekly (every other week) until a subject has a negative SYNB1020 fecal test for up to 10 weeks following completion of study dosing. 
Subjects who remain colonized with SYNB1020 after 10 weeks following the last dose will be treated with a 3-day course of oral ciprofloxacin, or other antibiotics. On Day 7, if 
no void has occurred by the time all other assessments have been completed, the subject may be discharged without a fecal sample. 
 
 
 
 
 
 
 
 
 
 


Synlogic, Inc. 
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01 APR 2019 
 
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Table 2 
Schedule of Plasma Sampling for 24-Hour Ammonia and Nitrate 
Study Day 
Activitya 
Time of Day 
Day -2 
0 hours 
+2 hours 
+3 hours 
+4 hours 
+5 hours 
+6 hours 
+9 hours 
+11 hours +14 hours +18 hours +24 hours 
Plasma draw for 24-hour ammonia 
 
 
 
 
 
 
 
 
 
 
 
Plasma draw for plasma nitrate 
 
 
 
 
 
 
 
 
 
 
 
Breakfast 
 
 
 
 
 
 
 
 
 
 
 
Lunch 
 
 
 
 
 
 
 
 
 
 
 
Dinner 
 
 
 
 
 
 
 
 
 
 
 
Day 5 
 
 
 
 
 
 
 
 
 
 
 
Plasma draw for 24-hour ammonia 
 
 
 
 
 
 
 
 
 
 
 
Plasma draw for plasma nitrate 
 
 
 
 
 
 
 
 
 
 
 
Breakfast 
 
 
 
 
 
 
 
 
 
 
 
Give IP 
 
 
 
 
 
 
 
 
 
 
 
Lunch 
 
 
 
 
 
 
 
 
 
 
 
Dinner 
 
 
 
 
 
 
 
 
 
 
 
Day 6 
 
 
 
 
 
 
 
 
 
 
 
Plasma draw for 24-hour ammonia 
 
 
 
 
 
 
 
 
 
 
 
Plasma draw for plasma nitrate 
 
 
 
 
 
 
 
 
 
 
 
Breakfast 
 
 
 
 
 
 
 
 
 
 
 
Give IP 
 
 
 
 
 
 
 
 
 
 
 


Synlogic, Inc. 
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Study Day 
Activitya 
Time of Day 
Day -2 
0 hours 
+2 hours 
+3 hours 
+4 hours 
+5 hours 
+6 hours 
+9 hours 
+11 hours +14 hours +18 hours +24 hours 
Plasma draw for 24-hour ammonia 
 
 
 
 
 
 
 
 
 
 
 
Plasma draw for plasma nitrate 
 
 
 
 
 
 
 
 
 
 
 
Lunch 
 
 
 
 
 
 
 
 
 
 
 
Dinner 
 
 
 
 
 
 
 
 
 
 
 
Abbreviations: IP = investigational product 
a Plasma draws occur before meals and meals occur before IP administration, as applicable. 


Synlogic, Inc. 
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2.5. 
Safety, Pharmacokinetic, and Pharmacodynamic Variables 
2.5.1. 
Primary Safety Endpoints  
The primary endpoint variables will be the following: nature and frequency of AEs, 
measurement of vital signs and laboratory assessments (e.g., blood chemistry, CBC, LFTs, INR, 
and urinalysis) and ECGs. Safety assessments performed during the study include physical 
examinations, measurement of vital signs, a baseline FibroScan, 12-lead ECGs, clinical 
laboratory evaluations including hematology, serum chemistry, and urinalysis, monitoring of 
adverse events, blood and urine cultures/PCR, HE Severity Grading, psychometric testing and 
cirrhosis severity grading. 
2.5.2. 
Secondary Microbiotic-Kinetic and PD Endpoints 
The secondary endpoints are the following: SYNB1020 kinetics in feces and change from 
baseline of venous ammonia, including fasting and 24-hour AUC, following SYNB1020 dosing.  
In Part 1, a 24-hour fecal sample will be weighed, homogenized, and a sample analyzed for 
SYNB1020 by qPCR. In Part 2, a fecal sample from the first void of the day will be collected 
using a collection kit. The following variables will be calculated from the serial fecal sampling 
from each subject, if data support calculation: Tlag, Tss, qmax, Tlast and half-life.  
2.5.3. 
Exploratory PD Endpoints 
Exploratory endpoints specifically examine placebo-adjusted change from baseline in PD 
effects of SYNB1020 on the following: 
o Serum inflammatory markers: serum TNF-α, IL-6 and endotoxin 
o Urinary urea/urinary nitrogen (24 hour) and calculated nitrogen balance 
o Plasma nitrate and urinary nitrate (24-hour) 
o Plasma amino acids including glutamine, arginine, aromatic amino acids, and 
branched chain amino acids 
o Bristol stool form scale (BSFS) for each bowel movement 
o Psychometric test performance at baseline and following dosing as measured by 
Psychometric HE Score testing (i.e., the number connection test A/B, digit 
symbol test, line tracing test and serial dotting test).  


Synlogic, Inc. 
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3. 
SUBJECT POPULATIONS 
3.1. 
Population Definitions 
The following subject populations will be evaluated and used for presentation and analysis of 
the data: 
Safety Population Part 2 
(SAF) and Safety 
Population Expanded 
(SAF2): 
All subjects in Part 1 who received at least one dose of 
SYNB1020 and have at least one post-baseline safety 
assessment. All randomized subjects in Part 2 who received at 
least one dose of SYNB1020 or placebo and have at least one 
post-baseline safety assessment. Subjects will be included in 
the analysis according to the dose and actual treatment 
received. Expanded population includes both Parts 1 and 2. 
Microbiotic-kinetic (MK) 
Population Part 2 and 
Microbiotic-kinetic (MK2) 
Population Expanded: 
Subjects in SAF or SAF2 who completed the clinical study and 
were considered evaluable for analysis of the MK data. 
Subjects will be included in the analysis according to the dose 
and actual treatment received. Expanded population includes 
both Parts 1 and 2. 
Pharmacodynamic (PD) 
Population Part 2 and 
Pharmacodynamic (PD2) 
Population Expanded 
(PDE): 
Subjects in SAF or SAF2 who completed the clinical study and 
were considered evaluable for analysis of the PD data. 
Subjects will be included in the analysis according to the study 
part and actual treatment received. PDE population includes 
both Parts 1 and 2. 
Data will be excluded from the MK/MK2 and/or PD/PD2 Populations in case of any major 
protocol deviation that may influence the MK and/or PD outcome. 
The SAF2 Population will be the primary population for the analysis of all safety parameters for 
both Part 1 and Part 2. All MK/PD analyses will be performed based on the MK/PD Population 
for Part 2 and on the MK2/PD2 Populations for both Parts combined (where Part 1 and both 
Parts combined will be reviewed for sensitivity and Part 2 is the Part for reporting of the 
primary and secondary analyses). 
For on-treatment assessments, subjects will be considered “evaluable for analysis of the PD 
data” if they received all doses during the ammonia AUC interval and for the preceding 2 days 
(i.e. 9 doses). 
3.2. 
Protocol Violations 
At the discretion of the sponsor, major protocol violations as determined by a review of the data 
prior to unblinding of the study results and the conduct of statistical analyses may result in the 
removal of a subject’s data from the MK/PD Populations.  The Sponsor, or designee, will be 
responsible for producing the final protocol violation file (formatted as a Microsoft Excel file), 
in collaboration with the CRO (Cytel) and the data monitoring group as applicable; this file will 
include a description of the protocol violation, and clearly identify whether or not this violation 


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warrants exclusion from either the MK or PD Population.  This file will be finalized prior to 
hard database lock. 
Major protocol violations are defined as those deviations from the protocol that are likely to 
have an impact on the subject’s rights, safety, well-being, and/or on the validity of the data for 
analysis regarding primary and secondary outcomes. The Biostatistician may consider some 
data to be invalid for analysis, which would be defined as a major deviation and would lead to 
exclusion of that subject from the relevant population. 
All protocol violations will be presented in the data listings. 


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4. 
STATISTICAL METHODS 
4.1. 
Sample Size Justification 
Part 1: The sample size for the sentinel cohort (6 subjects) is primarily designed for empirical 
evaluation of safety and tolerability in subjects with cirrhosis. 
Part 2: A sample of 16 subjects must complete all 3 ammonia AUC intervals (baseline, Day 5, 
and Day 6) to detect a 20% reduction in average daily ammonia (AUC0-24/24, from a baseline of 
70 µmol/L) with an approximate significance level of 10% and an approximate power of 90%. 
Significance and power are approximate based on the assumptions below. The number of 
subjects assumes that the SD of spot ammonia is 9 µmol/L, approximately half of the variance is 
between subject, and that the SD for average daily ammonia is as little as half that of spot 
ammonia. The spot ammonia SD is based on a weighted average of SD observed in a prior study 
of ammonia lowering in a similar population. The variability of AUC may be 50% lower than 
spot ammonia based on the FDA summary basis of approval for glycerol phenylbutyrate for 
urea cycle disorders.  
4.2. 
General Statistical Methods and Data Handling 
4.2.1. 
General Methods 
All output will be incorporated into Microsoft Excel or Word files, sorted and labeled according 
to the International Conference on Harmonisation (ICH) recommendations, and formatted to the 
appropriate page size(s). 
Tabulations will be produced for appropriate demographic, baseline, PD, microbiotic-kinetic 
and safety parameters.  For categorical variables, summary tabulations of the number and 
percentage within each category (with a category for missing data) of the parameter will be 
presented.  For continuous variables, the mean, standard deviation, arithmetic percent 
coefficient of variation (%CV), geometric mean (geo mean), geometric percent coefficient of 
variation (geo %CV), median, minimum and maximum values will be presented. For discrete 
numeric variables (e.g. Tlag), the median, minimum, and maximum will be presented. 
Formal statistical hypothesis testing will be performed on all primary, secondary and 
exploratory endpoints with all tests conducted at the 1-sided, 0.10 level of significance.  
Summary statistics will be presented, as well as confidence intervals on selected parameters, as 
described in the sections below. 
Baseline will be defined as the last scheduled measurement prior to the first IP administration 
(i.e., pre-dose). If a sequence of baseline measurements is taken pre-dose on the same day, time-
matched baseline will be used. If multiple measurements are included within a baseline 
measurement (e.g., repeated measures at the same nominal time), the arithmetic mean of the 
multiple samples will be considered the baseline.  Unscheduled measurements will only be used 
as baseline upon consultation with the Sponsor. 
For each local laboratory where normal reference range of ammonia has not been locally 
adequately validated, the normal reference range of ammonia will be defined based on 
approximately 30 healthy volunteers devoid of any chronic or acute medical conditions and 
medications with normal liver enzymes. Ammonia sampling, sample processing and analysis 
will be performed as defined in the SYNB1020-CP-002 protocol and lab manual. The 95th 
percentile will be used as ULN. Outliers based on visual inspection may be excluded on a case-


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by-case basis, and if any outliers are excluded, will be reported in the Changes to Planned 
Analysis section of the CSR with appropriate notes added to tables and figures where outliers 
have been excluded. 
4.2.1.1. 
Handling of Values below the Quantifiable Limit (BQL) 
Results for endogenous compounds below the quantifiable limit (BQL) will be set to ½ the 
lower limit of quantification (LLOQ).  
Results for exogenous compounds (other than SYNB1020) BQL will be set to zero. 
For SYNB1020 fecal copy number, BQL before the first quantifiable measurement will be set to 
0 for calculations; BQL between two quantifiable measurements will be set to ½ LLOQ; and 
BQL after the last quantifiable measurement will be set to 0 for calculations.  For semi-
logarithmic plotting, SYNB1020 fecal copy number BQL will be set to ½ LLOQ for all 
measurements. 
4.2.2. 
Computing Environment 
All descriptive statistical analyses will be performed using SAS statistical software 
(Version 9.4) or R (Version 3.4.0 or greater), unless otherwise noted.  Medical history and AEs 
will be coding using the Medical Dictionary for Regulatory Activities (MedDRA) version 20.1, 
and the severity of AEs and laboratory abnormalities will be graded using the NCI CTCAE, 
version 4.03.  Concomitant medications will be coded using World Health Organization (WHO) 
Drug Version 10DEC01DDE.  
4.2.3. 
Methods of Pooling Data 
Not applicable to the present study. 
4.2.4. 
Adjustments for Covariates 
No formal statistical analysis that adjusts for possible covariate effects is planned. 
4.2.5. 
Multiple Comparisons/Multiplicity 
Multiplicity will be addressed via the Haybittle-Peto stopping boundary method for group 
sequential designs. 
First, the IA will be evaluated against the efficacy p-value of 0.01.  
The overall type I error will be fixed at alpha = 0.10, allowing for unequal p-value boundaries at 
the two looks (IA and final). Further details will be provided in Section 4.6. 
4.2.6. 
Subpopulations 
The following subpopulations will be explored for the primary and key secondary endpoints, 
provided the sample size is adequate for analysis:  
• Ammonia level: subjects with ammonia > 1.5 times ULN versus subjects with ammonia 
≤ 1.5 times ULN 
• Proton pump inhibitor (PPI) use: subjects already on PPI versus subjects given ranitidine 
• Hepatitis status: subjects with a history of Hepatitis B/C versus subjects without a 
history of Hepatitis B/C  


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If subgroup analyses for the primary endpoint indicate important subgroup-by-treatment 
interactions, then further exploratory analyses on subgroups and other endpoints may be taken.  
4.2.7. 
Withdrawals, Dropouts, Loss to Follow-up 
Subjects who withdrew from the study may be replaced as defined in section 10.5 of the 
Protocol (Determination of Sample Size). 
4.2.8. 
Missing, Unused, and Spurious Data 
In general, there will be no substitutions made to accommodate missing data points.  All data 
recorded on the case report forms (CRFs) will be included in data listings that will accompany 
the CSR.   
If the start date of an AE is partially or completely missing, the date will be compared as far as 
possible with the date of the start of administration of study drug. The AE will be assumed to be 
treatment-emergent if it cannot be definitively shown that the AE did not occur or worsen 
during the treatment-emergent period (worst case approach).  
The following general rules will be used: 
• If the start date is complete, an AE will only be excluded as being treatment-emergent if 
the start date is before the date of first study drug administration or if the stop date is 
before first study drug administration.  
• If the start day is missing but the start month and year are complete, an AE will only be 
excluded as being treatment-emergent if the start month/year is before the month/year of 
first study drug administration or if the stop month/year is before first study drug 
administration.  
• If the start day and month are missing but the start year is complete, an AE will only be 
excluded as being treatment-emergent if the start year is before the year of first study 
drug administration or if the stop year is before the year of first study drug 
administration.  
If the start date is completely missing, an AE will be considered treatment-emergent unless the 
stop date is before first study drug administration. 
4.2.9. 
Visit Windows 
Screening procedures must be performed to determine eligibility for enrollment in both Part 1 
and Part 2 within 30 days prior to admission to the inpatient facility.  
During inpatient monitoring, visit windows are not a concern.  
The protocol-specified visit window for the End of Study (EOS)/Discharge Visit to occur is at 
least 12 hours after completion of the last dose (Day 7); subjects who withdraw early should 
have this visit within 24 hours of discontinuation of the study drug. The first follow-up visit is 
specified in the protocol as occurring 7 ± 1 day after the EOS Visit, and subjects will be 
followed for up to 10 weeks after this first follow-up visit occurs. For continuation of follow-up 
visits, subjects will be required to provide weekly fecal samples (7 ± 1 day after the follow-up 
visit) for the first 6 weeks following discharge, and bi-weekly (14 ± 1 day) until the subject has 


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a negative SYNB1020 fecal test (up to 10 weeks after completion of study). However, all data 
will be analyzed regardless of visit windows. 
Table 1 
Visit Windows 
Evaluation 
Protocol-Specified Interval 
Screening 
Day -36 to Day -6 
Part 1 Inpatient 
Day -5 to Day 7 
Part 2 Inpatient  
Day -5 to Day 7 
EOS/Discharge 
Day 7 (12 hours post-dose) 
Follow-Up 
Day 13 to Day 15 
4.3. 
Interim Analyses 
After all N=6 subjects are discharged from the clinic following Part 1, safety data will be 
examined as an informal review (not a formal IA).  
A formal, unblinded IA will be performed on the PD2 population (Part 2 only) after at least 16 
subjects have completed dosing in Part 2 and have been discharged from the unit, while 
enrollment may continue, for a possible final sample size of N=20 (NCP). NCP may be adjusted 
based on actual enrollment just before the IA decision but will not be less than 20.   
It is assumed that baseline average daily ammonia is 70 µmol/L, and the placebo group will 
show a 0 – 10% reduction from baseline, while the desired reduction for the treatment group is 
20% versus placebo. Furthermore, the assumed SD of the difference in the means (desired 
reduction) is 9 µmol/L. Additional assumptions were examined, and the operating 
characteristics at the IA for these more pessimistic possibilities are shown in Section 6.4. 
If the p-value at the IA is < 0.028 the trial could be stopped for efficacy.  Otherwise, conditional 
power (CP) will be computed at the IA for N=16 subjects. This is the probability that the trial 
will yield a statistically significant reduction in AUC of average daily ammonia change from 
baseline greater for the treatment group at the end of the trial if the observed difference at the IA 
is the TRUE underlying difference and the trial is continued to completion with a total of NCP 
subjects. If this probability is less than 30%, the Safety Monitoring Committee (SMC) could 
suggest that the trial be stopped for futility.  Otherwise, if the CP is less than 90% the sample 
size will be boosted to target a conditional power of 90%, up to a maximum of 40 patients.  If 
the CP is greater than 90% and the p-value at the IA is greater than 0.028, the trial will end with 
NCP subjects.   
Conditional power for an active dose versus placebo is derived as follows: 
𝐶𝐶𝑝𝑝(Ɵ෡) = 1 −𝛷𝛷൜𝑐𝑐2 −𝐵𝐵(𝑡𝑡)/𝑡𝑡
√1 −𝑡𝑡
ൠ 
Parameter 
Description 
𝛷𝛷{𝑥𝑥} 
cumulative distribution function for a 𝑁𝑁(0, 1) random variable 
𝑐𝑐2 
Adjusted critical cut-off for the final analysis. Assuming that alpha of 0.028 is 
spent at the interim analysis for one-sided testing, that the total one-sided alpha 


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level is 0.05, and that only one IA occurs at the planned information fraction of 
0.80 (=16/20), the adjusted critical cut-off is 𝑐𝑐2 = −1.904.  
t 
= 𝑛𝑛𝑁𝑁
ൗ , where N is the planned total sample size for the treatment arm and for 
the placebo arm (N=20), and n is the sample size of the IA (16 total). 
𝐵𝐵(𝑡𝑡) 
B value, calculated as 𝑍𝑍𝑛𝑛√𝑡𝑡 , where 
𝑍𝑍𝑛𝑛 is the Z statistic associated with the linear regression model of observed data 
on the treatment group versus placebo group at the IA 
 
Because the total number of subjects could potentially be increased in a data-dependent manner, 
the final analysis does not use the conventional statistic to determine if statistical significance is 
reached.  Instead it uses the weighted statistic proposed by Cui, Hung, and Wang (Cui et al., 
1999) (referred to as the “CHW approach”) in which the independent increments of the Z 
statistic of the 2 stages are combined by pre-specified weights that are based on the planned 
proportion of total number of subjects at which the IA would be taken if there were no change in 
the design. In the present case, the study was designed for 30 subjects with an interim analysis 
when 24 subjects complete the study. Upon further consideration, the interim analysis will be 
performed when 16 subjects complete the study with a planned total sample size of 20 without 
adaptation. Details on the operating characteristics can be found in Section 6. Therefore, the 
planned proportion n is 0.80 (16/20) for stage 1 and 0.2 (4/20) for stage 2 (these values will be 
adjusted for the actual N and NCP at the IA), and the independent incremental Z statistics for the 
2 stages are combined with weights that equal 𝑤𝑤1 = √0.80 for stage 1 and 𝑤𝑤2 = √0.20 for 
stage 2. These weights will remain fixed. They will not be modified based on any changes to the 
final sample size.  The final test statistic under the CHW approach is  
𝑍𝑍𝐶𝐶𝐶𝐶𝐶𝐶= 𝑤𝑤1𝑍𝑍1 + 𝑤𝑤2𝑍𝑍2 
Where 𝑍𝑍1is the Z statistic calculated using the IA PD2 population data (i.e., stage 1 data)and 𝑍𝑍2 
is the Z statistic calculated using PD2 population data from all remaining subjects not included 
in IA (i.e., stage 2 data). .  Statistical significance is achieved at the final analysis if 𝑍𝑍𝐶𝐶𝐶𝐶𝐶𝐶< 𝑐𝑐2.    
 
The model used for the IA will be a log-scale, linear mixed model with repeated measures with 
categorical effects for treatment, time (as a categorical variable), and treatment by time and a 
random effect by subject on the intercept where time will be coded as baseline for pre-dose and 
treated for both post-dose assessments: 
log൫𝐴𝐴𝐴𝐴𝐶𝐶𝑖𝑖,𝑡𝑡൯= 𝛽𝛽𝑡𝑡× 𝛽𝛽𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡+ 𝛽𝛽𝑡𝑡+ 𝛽𝛽𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡+ 𝜂𝜂𝑖𝑖+ 𝜖𝜖 
where: 
• AUCi,t is the ammonia AUC for subject i at time t, 
• βt is the effect of time (baseline or on-treatment), 
• βtreatment is the effect of treatment (placebo or active), 
• ηi is the sample from a normal distribution of the random effect of subject i, and 
• ε is the log-scale, residual variability. 


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4.4. 
Subject Disposition 
A tabulation of subject disposition will be created, including the number screened, the number 
dosed with placebo, the number dosed with SYNB1020, the number in each subject population 
for analysis, the number that withdrew prior to completing the study, and reasons for 
withdrawal.   
A by-subject listing of study completion information, including the reason for premature study 
withdrawal, if applicable, will be presented. 
4.5. 
Demographic and Baseline Characteristics 
Baseline, demographic and medical history information will be summarized for the SAF and 
SAF2 Populations using descriptive statistics.  No formal statistical comparisons will be 
performed.  
Demographic, baseline data and medical history will be provided in data listings.  
Exposure to study drug will be shown in a by-subject listing for the SAF and SAF2 populations.  
 
The demographic, baseline data and medical history data described above will be shown for Part 
1 alone, Part 2 (by treatment), overall by treatment (Parts 1 and 2), and study overall (Parts 1 
and 2 – all treatments) using descriptive statistics.   
4.6. 
Safety Analyses 
Safety analyses will be conducted using the SAF/SAF2 Populations.  
4.6.1. 
Adverse Events 
AEs will be coded using MedDRA and severity will be evaluated using the NCI CTCAE 
version 4.03. These will be displayed in tables and listings using System/Organ/Class (SOC) 
and Preferred Term (PT). AEs will also be presented by maximum severity.  
AEs will be recorded from the time a subject signs informed consent through the safety follow-
up period.  Analyses will focus on any AE recorded, as well as treatment-emergent AEs 
(TEAEs), which are those events that have occurred after the start of the study drug.   
AEs are summarized by subject incidence rates, therefore, in some tabulations, a subject 
contributes only once to the count for a given AE (SOC or PT). Several tabulations also show 
the total number of events, where a subject can contribute more than once. 
The number and percentage of subjects, along with total number of events, with any AE, any 
TEAE, any TEAE assessed by the Investigator as related to treatment (definite, probable, or 
possible relationship), any AE resulting in discontinuation of study dosing and with any SAE 
will be summarized by treatment group and overall.  In these tabulations, each subject will 
contribute only once (i.e., the most related occurrence or the most intense occurrence) to each of 
the incidence rates in the descriptive analysis, regardless of the number of episodes. 
Tabulations will also be presented by MedDRA SOC and PT for the following: all TEAEs, 
related TEAEs, TEAEs by severity and TEAEs leading to permanent study drug withdrawal. 
No formal hypothesis-testing analysis of AE incidence rates will be performed.   
All AEs occurring on study will be listed in subject data listings. 


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By-subject listings also will be provided for the following: subject deaths; SAEs; AEs leading to 
withdrawal and Grade 3 or 4 AEs, and narratives will be shown for these. 
4.6.2. 
Laboratory Data 
Clinical laboratory values will be expressed using SI units and will be measured during 
screening, on Day -2 (Baseline), Day 4, Day 6 and Day 7.  
The following will be performed:  
• Alkaline phosphatase, total bilirubin, direct bilirubin, CO2, chloride, glucose, 
potassium, AST, ALT, sodium, albumin, total protein, phosphorous, calcium, 
activated partial thromboplastin time (aPTT), international normalized ratio (INR), 
prothrombin time (PT), CBC with differential, C-reactive protein, follicle-
stimulating hormone (FSH, for women), blood urea nitrogen (BUN) and creatinine   
• Urinalysis 
The actual value and change from Baseline to each on study evaluation will be summarized for 
each clinical laboratory parameter.  In the event of repeat values, the last non-missing value per 
study day/time will be used. Hematology, chemistry and urinalysis will also be displayed by the 
number and percent of subjects with normal, abnormal and clinically significant abnormal in 
shift from baseline tables at each visit. 
All laboratory data will also be provided in data listings. Clinical significance and abnormality 
will be indicated within these listings.  
4.6.3. 
Vital Signs and Physical Examinations 
Semi-supine vital signs will be collected during screening, every 8 hours during the dosing 
period (Day -5 – Day 6), Day 7 and Day 14. 
The following will be collected: systolic blood pressure (BP), diastolic BP, pulse and body 
temperature. Subjects are required to remain in the semi-supine position for at least 5 minutes 
prior to obtaining vital signs. 
The actual value and time-matched change from Baseline (or Screening) to each on study 
evaluation will be summarized for vital signs. Vitals will also be displayed by the number and 
percent of subjects with normal, abnormal and clinically significant abnormal. 
By-subject listings of vital sign measurements will be presented in data listings. 
Physical examinations will be performed during screening and Day -5; and symptom-driven on 
Day 7 and Day 14. 
Any abnormal physical examination findings will be recorded as AEs and included in AE 
outputs. 
4.6.4. 
Electrocardiogram 
A semi-supine single 12-lead ECG will be performed during screening, Day -2 (Baseline), Day 
4 and Day 7.  
The following will be collected: RR, QT, QRS and PR intervals. Fridericia’s formula should be 
used to calculate the QT interval corrected for heart rate (QTcF). 


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The actual value and change from Baseline (or Screening) to each on study evaluation will be 
summarized for ECGs.   
ECG results will also be summarized descriptively, including the number and percent of 
subjects with normal, abnormal and clinically significant abnormal results at Baseline and each 
study visit.  
All ECG data for each subject will be provided in data listings. 
4.6.5. 
FibroScan 
A FibroScan (vibration-controlled transient elastography) will be performed once at Day -2 
(Baseline) to quantify liver fibrosis.  
FibroScan results for each subject will be provided in data listings. 
4.6.6. 
Blood and Urine Cultures/Polymerase Chain Reaction 
Blood and urine cultures and/or PCR will be performed as needed according to standard clinical 
practice if clinical presentation warrants. This may include, but is not limited to, cases in which 
subjects develop persistent (> 24 hours) fever, chills, night sweats, or other symptoms 
suggestive of local or systemic clinical infection.  
4.6.7. 
Hepatic Encephalopathy Severity Grading  
HE severity grading will be collected via the West Haven Grading System on Day -2 (Baseline) 
and Day 6.   
HE grades will be summarized descriptively, including the number and percent of subjects at 
each grade on Day -2 (Baseline) and Day 6.  
All HE data for each subject will be provided in data listings. 
4.6.8. 
Psychometric Testing 
Psychometric testing will be performed via the Psychometric HE Score (PHES) testing (i.e., 
number connection test A/B, serial dotting test, line tracing test, and digit symbol test) on Day -
2 (Baseline) and Day 6. 
The actual value and change from Baseline (or Screening) to each on study evaluation will be 
summarized for psychometric testing results. Due to the lack of validated American reference 
values, total score for PHES will be calculated using the original German reference tables.  
All psychometric data for each subject will be provided in data listings. 
4.6.9. 
Cirrhosis Severity Grading  
Severity of cirrhosis will be assessed by the Child-Turcotte-Pugh score, which reflects a 
composite score incorporating HE grade, extent of ascites, and bilirubin, albumin, and INR 
values and MELD score calculated based on bilirubin, creatinine, and INR values. This will be 
collected during screening, Day -2 (Baseline) and Day 6. 
Cirrhosis severity results will be summarized descriptively for each class at every time point it 
was collected. The composite Child-Turcotte-Pugh and MELD scores will also be summarized 
by their actual score and change from Baseline to each on study evaluation. 
All cirrhosis severity data for each subject will be provided in data listings. 


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4.6.10. 
Concomitant Medications 
Concomitant medications will be coded using the WHO Drug Dictionary. Results will be 
tabulated by Anatomic Therapeutic Class (ATC) and preferred term. 
The use of concomitant medications will be included in by-subject data listing. 
4.7. 
Pharmacodynamic Evaluations  
PD analyses will be conducted using the PD Population. Urine and blood samples will be 
collected at screening and on study both before and after administration of SYNB1020 or 
placebo for PD measurements.  
All PD parameters of SYNB1020 will be summarized in tables, listings, and figures (Section 6); 
no formal statistical analysis will be performed, with the exception of venous ammonia AUC 
and fasting spot venous ammonia. Additional post-hoc exploratory PD analyses of interest may 
be carried out, in addition to those described below. 
4.7.1. 
Venous Ammonia Area under the Curve 
Samples for venous ammonia will be drawn relative to meal times.  In order to analyze AUC0-24, 
subjects must complete all 3 ammonia AUC intervals (Baseline, Day 5 and Day 6).  
To calculate the AUC over 24 hours (AUC0-24), the linear trapezoidal method will be utilized, 
with the following formula:  
 
𝐴𝐴𝐴𝐴𝐴𝐴0−24 = ෍
(𝑡𝑡𝑖𝑖 − 𝑡𝑡𝑖𝑖−1)(𝐴𝐴𝑖𝑖+ 𝐴𝐴𝑖𝑖−1)
2
 
 
Symbol 
Definition  
∑ 
Summation over all times, i 
ti 
Time point, in hours  
ti-1 
Previous time point, in hours 
Ai 
Venous ammonia measurement at time i 
Ai-1 
Venous ammonia measurement at previous 
time point, i-1 
Once AUC0-24 is calculated, this will be divided by 24 to obtain the average daily ammonia.  
Statistical analysis will be performed on log-transformed venous ammonia AUC0-24 as a mixed-
model with repeated measures with baseline as a covariate, a fixed effect by treatment (Day 5 
and 6 treated as repeated measures on treatment, not differentiated by day), and a random effect 
by subject.  Results will be transformed back to the linear scale for reporting; at the Sponsor’s 
option, results may be normalized to the average (or weighted-average based on number of 
subjects) upper limit of normal across the labs used in the study. 
A bar chart will be generated illustrating the mean change from baseline (average of Day 5 and 
Day 6 minus baseline) in AUC0-24 for ammonia on the y-axis, bar fill color will indicate active 
(black) or placebo (white), and subjects across all treatment groups will be sorted in order from 


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greatest mean change from baseline (left) to least mean change from baseline (right).  The bar 
chart will be generated twice, once for the PD population in Part 2 and once for the PD 
population in the full study (Part 1 and Part 2).  (For clarity, this representation is sometimes 
referred to as a “waterfall plot”.) 
4.7.2. 
Fasting Spot Venous Ammonia 
In addition to tables, listings, and figures for descriptive statistics (Section 6), statistical analysis 
will be performed on log-transformed fasting spot venous ammonia (defined as the ammonia 
measurement during the day) to assess change from baseline and change from placebo.  The 
statistical model for fasting spot venous ammonia will be a mixed-model with repeated 
measures with baseline as a covariate, fixed effects by day and treatment, and a random effect 
by subject.  Results will be transformed back to the linear scale for reporting; at the Sponsor’s 
option, results may be normalized to the average (or weighted-average based on number of 
subjects) upper limit of normal across the labs used in the study. 
4.7.3. 
Urinary Urea/Nitrogen, Plasma/Urinary Nitrate, Plasma Amino Acids, Bristol Stool 
Form Scale, Psychometric Test Performance 
The following laboratory measurements will be performed to evaluate the preliminary PD of 
SYNB1020: 
o Serum inflammatory markers: serum TNF-α, IL-6 and endotoxin 
o Urinary urea/urinary nitrogen (24 hour) and calculated nitrogen balance 
o Plasma nitrate and urinary nitrate (24-hour) 
o Plasma amino acids including glutamine, arginine, aromatic amino acids, and 
branched chain amino acids 
o BSFS for each bowel movement 
o Psychometric test performance at baseline and following dosing as measured by 
PHES testing (i.e., the number connection test A/B, digit symbol test, line tracing 
test and serial dotting test).  
4.8. 
Microbiotic-Kinetic Evaluations 
MK analyses will be conducted using the MK Population. Additional post-hoc exploratory MK 
analyses of interest may be carried out, aside from what is described below. 
MK variables will be calculated from the serial fecal sampling from each subject. One baseline 
fecal sample is required to be collected prior to SYNB1020 dosing (i.e., any time from Day -5 
through Day 1) and daily following dosing through Day 7. Feces will continue to be collected 
weekly for the first 6 weeks following discharge and every other week until a subject has a 
negative SYNB1020 fecal test for up to 10 weeks following completion of study dosing.  
The variables to be determined and the time points of determination are summarized below: 


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Table 2 
Microbiotic-Kinetic Parameters for Analysis 
Parameter 
Description 
Tlag 
The time of the first detectable fecal SYNB1020 
Tss 
The time to steady-state 
qmax 
The maximum observed fecal SYNB1020 qPCR signal 
Tlast 
The time of the last detectable fecal SYNB1020 
Half-life 
The time for the fecal SYNB1020 PCR signal to decline 
by 50% 
A sample will be considered negative for the purposes of SYNB1020 clearance if it is BQL, and 
therefore SYNB1020 clearance is the same as Tlast. 
Missing values will not be imputed, and if sufficient data are missing for a given subject, that 
subject may be considered non-evaluable for MK analysis and would not be included in the MK 
Population. 
All MK parameters of SYNB1020 will be summarized in tables and in listings; no formal 
statistical analysis will be performed.  


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5. 
CHANGES TO PLANNED ANALYSES 
In Section 10.3 of version 4.0 of the protocol, it is specified that a fecal sample will be 
considered negative for the purposes of SYNB1020 clearance if it is below the quantifiable limit 
(BQL) or estimated to be below <0.1% of the administered dose. This is clarified in Section 4.8 
of the SAP: a sample will only be considered negative for the purposes of SYNB1020 clearance 
if it is BQL. Additionally, this is described in as note to file.  
In Section 10.1 of version 4.0 of the protocol, the populations for analysis are described without 
detail of the differences in populations for Part 1 and Part 2. This is expanded upon in Section 
3.1 of the SAP. 
Protocol version 6 amended the number of subjects required for of the interim analysis from 
what was planned in the original protocol, as described in Section 10.5.


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6. 
CLINICAL STUDY REPORT APPENDICES 
6.1. 
Statistical Tables to be Generated 
Table 
Number 
Description 
At Interim 
Analysis 
Table 
14.1.1.1 
Subject Enrollment and Disposition (SAF)  
 
Table 
14.1.1.2 
Subject Demographic and Baseline Characteristics (SAF) 
X 
Table 
14.1.1.3 
Medical History (SAF) 
X 
Table 
14.2.1.1 
Overview of Adverse Events and Treatment-Emergent Adverse Events (SAF) 
X 
Table 
14.2.1.2 
Summary of Treatment-Emergent Adverse Events by System Organ Class, Preferred Term and 
Treatment Group (SAF) 
X 
Table 
14.2.1.3 
Summary of Treatment-Emergent Adverse Events by System Organ Class, Preferred Term, 
Severity and Treatment Group (SAF) 
X 
Table 
14.2.1.4 
Summary of Related Treatment-Emergent Adverse Events by System Organ Class, Preferred 
Term and Treatment Group (SAF) 
X 
Table 
14.2.1.5 
Summary of Treatment-Emergent Adverse Events Leading to Study Drug Withdrawal by 
System Organ Class, Preferred Term and Treatment Group (SAF) 
X 
Table 
14.2.2.1 
Hematology Results: Change from Baseline (SAF) 
 
Table 
14.2.2.2.x 
Hematology: Shifts from Baseline by Visit (SAF) 
 
Table 
14.2.2.3 
Chemistry Results: Change from Baseline (SAF) 
 
Table 
14.2.2.4.x 
Chemistry: Shifts from Baseline by Visit (SAF) 
 
Table 
14.2.2.5 
Urinalysis Results: Change from Baseline (SAF) 
 
Table 
14.2.2.6.x 
Urinalysis: Shifts from Baseline by Visit (SAF) 
 
Table 
14.2.2.7 
Vital Signs: Change from Baseline (SAF) 
 
Table 
14.2.2.8.x 
Vital Signs: Shifts from Baseline by Visit (SAF) 
 
Table 
14.2.2.9 
Electrocardiogram Results: Change from Baseline (SAF) 
 
Table 
14.2.3.0.x 
Electrocardiogram: Shifts from Baseline by Visit (SAF) 
 
Table 
14.2.3.1 
Psychometric Testing Results: Change from Baseline (SAF) 
X 
Table 
14.2.3.2 
Serum Inflammatory Markers: Change from Baseline (SAF) 
X 
Table 
14.2.3.3.x 
Serum Inflammatory Markers: Shifts from Baseline by Visit (SAF) 
X 
Table 
14.2.3.4 
Urinary Urea/Urinary Nitrogen and Calculated Nitrogen Balance: Change from Baseline (SAF) 
 


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Table 
14.2.3.5 
Plasma Nitrate and Urinary Nitrate: Change from Baseline (SAF) 
X 
Table 
14.2.3.6 
Plasma Amino Acids: Change from Baseline (SAF) 
X 
Table 
14.2.3.7 
Severity of Cirrhosis: Child-Turcotte-Pugh Score and Components (SAF) 
X 
Table 
14.2.3.8 
Severity of Cirrhosis: Model for End Stage Liver Disease (MELD) Score and Components 
(SAF) 
X 
Table 
14.3.1.1 
Average Area Under the Curve of Venous Ammonia over 24 Hours, Change from Baseline and 
Change from Baseline/Change from Placebo (PD) 
X 
Table 
14.3.1.2 
Daily Fasting Spot Venous Ammonia Change from Baseline and Change from Baseline/Change 
from Placebo (PD) 
X 
Table 
14.4.1.1 
Microbiotic-Kinetic Parameters from Fecal Sampling (MK) 
 
 


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6.2. 
Data Listings to be Generated 


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Listing 
Number 
Description 
At Interim 
Analysis 
Listing 
16.1.1.1 
Subject Enrollment and Disposition (All Screened) 
X 
Listing 
16.1.1.2 
Demographic and Baseline Characteristics (SAF) 
X 
Listing 
16.1.1.3 
Subject Medical History (SAF) 
 
Listing 
16.1.1.4 
Exposure to Study Drug (SAF) 
X 
Listing 
16.1.1.5 
Concomitant Medications (SAF) 
 
Listing 
16.1.1.6 
Inclusion/Exclusion Criteria Not Met (All Screened) 
 
Listing 
16.1.1.7 
Protocol Deviations (All Subjects)  
X 
Listing 
16.2.1.1 
Adverse Events (SAF) 
X 
Listing 
16.2.1.2 
Serious Adverse Events (SAF) 
X 
Listing 
16.2.1.3 
Treatment-Emergent Adverse Events Leading to Study Drug Withdrawal (SAF) 
X 
Listing 
16.2.1.4 
Adverse Events of Grade 3 or 4 (SAF) 
X 
Listing 
16.2.1.5 
Deaths (SAF) 
X 
Listing 
16.2.2.1 
Hematology Results (SAF) 
 
Listing 
16.2.2.2 
Chemistry Results (SAF) 
 
Listing 
16.2.2.3 
Urinalysis Results (SAF) 
 
Listing 
16.2.2.4 
Vital Signs (SAF) 
 
Listing 
16.2.2.5 
Electrocardiogram Results (SAF) 
 
Listing 
16.2.2.6 
FibroScan Results (SAF) 
 
Listing 
16.2.3.1 
Psychometric Testing Results (SAF) 
X 
Listing 
16.2.3.2 
Serum Inflammatory Markers (PD) 
X 
Listing 
16.2.3.3 
Urinary Urea/Urinary Nitrogen and Calculated Nitrogen Balance (PD) 
 


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Listing 
16.2.3.4 
Plasma Nitrate (PD) 
X 
Listing 
16.2.3.5 
Urinary Nitrate (PD) 
X 
Listing 
16.2.3.6 
Plasma Amino Acids (PD) 
X 
Listing 
16.2.3.7 
Hepatic Encephalopathy Severity Grading (SAF) 
X 
Listing 
16.2.3.8 
Child-Turcotte-Pugh Score and Components (SAF) 
X 
Listing 
16.2.3.9 
Model for End Stage Liver Disease and Components (SAF) 
X 
Listing 
16.3.1.1 
Venous Ammonia 24-Hour Profile (PD)  
X 
Listing 
16.3.1.2 
Daily Fasting Spot Venous Ammonia (PD) 
X 
Listing 
16.3.1.3 
Venous Ammonia Area Under the Curve over 24 Hours (PD) 
X 
Listing 
16.4.1.1 
Bristol Stool Scale Assessment (MK) 
 
Listing 
16.4.1.2 
Fecal Sampling and qPCR Results (MK) 
 
 
6.3. 
Figures to be Generated 
Figure 
Number 
Description 
At Interim 
Analysis 
Figure 
15.1.1.x 
Venous Ammonia: Time Profiles by Subject (PD) 
X 
Figure 
15.1.2.1 
Venous Ammonia Levels: Time Profiles in Part 2 (PD) 
X 
Figure 
15.1.2.2 
Venous Ammonia Levels: Time Profiles in Parts 1 and 2 (PD) 
X 
Figure 
15.1.2.3 
Venous Ammonia Levels: Change from Baseline by Time and Treatment in Part 2 (PD) 
X 
Figure 
15.1.2.4 
Venous Ammonia Levels: Change from Baseline by Time and Treatment in Parts 1 and 2 (PD) 
X 
Figure 
15.1.2.5 
Fasting Venous Ammonia by Patient and Treatment  
X 
Figure 
15.1.2.6 
Fasting Venous Ammonia Levels in Part 2 
X 
Figure 
15.1.2.7 
Fasting Venous Ammonia Levels in Parts 1 and 2 
X 
Figure 
15.1.3.1 
Venous Ammonia Levels: Mean Change from Baseline in AUC0-24 in Part 2 (PD) 
X 
Figure 
15.1.3.2 
Venous Ammonia Levels: Mean Change from Baseline in AUC0-24 in Parts 1 and 2 (PD) 
X 


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Figure 
15.1.4.1 
Serum Inflammatory Markers: TNF-alpha in Part 2 (PD) 
X 
Figure 
15.1.4.2 
Serum Inflammatory Markers: TNF-alpha in Parts 1 and 2 (PD) 
X 
Figure 
15.1.5.1 
Serum Inflammatory Markers: IL-6 in Part 2 (PD) 
X 
Figure 
15.1.5.2 
Serum Inflammatory Markers: IL-6 in Parts 1 and 2 (PD) 
X 
Figure 
15.1.6.1 
Serum Inflammatory Markers: Endotoxin in Part 2 (PD) 
X 
Figure 
15.1.6.2 
Serum Inflammatory Markers: Endotoxin in Parts 1 and 2 (PD) 
X 
Figure 
15.1.7.1 
Urinary Nitrate Levels in Part 2 (PD) 
X 
Figure 
15.1.7.2 
Urinary Nitrate Levels in Parts 1 and 2 (PD) 
X 
Figure 
15.1.8.1 
Plasma Nitrate Levels: Time Profiles in Part 2 (PD) 
X 
Figure 
15.1.8.2 
Plasma Nitrate Levels: Time Profiles in Parts 1 and 2 (PD) 
X 
 
 
6.4. 
Operating Characteristics of the Interim Analysis 
An IA will be carried out at N=12 (with the study ongoing), with a possible sample size re-
estimation to a maximum of N=40 subjects, or if no sample size re-estimation, a final sample size of 
N=16.  
The study design was simulated 10,000 times under the assumption that the true AUC of average 
daily ammonia change from baseline to Day 5/6 shows a 20% reduction (-0.22 on the log scale) in 
the treatment group versus the control group.  
Based on previous data, the standard deviation of average daily ammonia was estimated to possibly 
be as small as 50% of that of spot ammonia (~13 µmol/L on the log scale). For a more conservative 
estimate, we assumed that the standard deviation could also be as high as that of spot ammonia (~26 
µmol/L on the log scale). Additionally, measurements for healthy volunteers in protocol 
SYNB1020-CP-001 showed that the total standard deviation was 25 µmol/L and the standard 
deviation between and within subject was ~20 µmol/L and 15 µmol/L, respectively (64% of the total 
variation). Thus, a more pessimistic standard deviation from this data will be examined as 29 
µmol/L (64% of 36 µmol/L), with a less pessimistic standard deviation as 20 µmol/L. 
Analysis of Stage 1 ammonia data indicated a within-subject, log-scale residual standard deviation 
of 0.1324 from a baseline AUC0-24/24 of 64.8 µmol/L (approximately 9 µmol/L). 
For an IA at N=16, with overall type I error (alpha) set at 0.10 and efficacy p-value set at 0.028, 
conditional power (CP) will be calculated to drive the decision about whether to increase the sample 
size. The following boundaries were set: futility boundary at CP=0.30 and efficacy boundary at < 
0.028 (one-sided). If the trial is not stopped for efficacy or futility, then if CP≥90%, the trial will 
continue to completion with a total of 20 subjects, and if CP <90%, the sample size will be increased 


Synlogic, Inc. 
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to target a CP of 90% up to a maximum sample size 40. The operating characteristics of the design 
can be seen in the below table:   
Pessimistic SD #1 
SD=26 𝜇𝜇𝜇𝜇𝜇𝜇𝜇𝜇/𝐿𝐿 
Pessimistic SD 
#2 SD=29 
𝜇𝜇𝜇𝜇𝜇𝜇𝜇𝜇/𝐿𝐿 
Healthy 
Volunteer SD 
SD=20 𝜇𝜇𝜇𝜇𝜇𝜇𝜇𝜇/𝐿𝐿 
Stage 1 
Observations 
SD=9 µmol/L 
(log-scale SD 
= 0.1324) 
Probability to stop for 
futility at N=16 
41.91% 
47.19% 
25.07% 
0.13% 
Probability to stop for 
efficacy at N=16 
36.62% 
33.03% 
55.32% 
99.12% 
Probability of increasing 
beyond 20 
20 < 𝑁𝑁≤40 
21.47% 
19.78% 
19.61% 
0.75% 
Overall power 
54.73% 
48.77% 
73.48% 
99.87%